This video was created to support their published research. The authors did research using several lasers and slices of a sheep’s brain to try and determine the best parameter for treating TBI (Traumatic Brain Injury) with a desired fluency of 0.9 to 15 joules/cm2 at a depth of 2 cm. They state that getting the energy through the skull is especially difficult so they test multiple options so test the transfer rate. They started out using a continuous output split 980/810nm system (the only company that makes that type of split system, 80% of the power at 980nm and 20% of the power at 810nm, is LiteCure with their LightForce series). The result was less than 1/2% of the energy reached a depth of 2cm. Then they switched to pulsing and got an increase in the energy transfer. When they switched to a 810nm-only 15 watt system with pulsing the transfer rate increased to 16% of the output energy reached the target depth.

 Here are some rough numbers to review the feasibility of using this system for treatment. If the duty cycle is 70%, the system will deliver 1.68 joules per second at a depth 2cm (15wattS*70%*16%). To get 5 joules/cm2 over 15 x 15 cm treatment area would require a total of 1125 joules at depth. This would take 23 minutes.

This research shows that only class 4 systems can delivery the level of power needed for this kind of therapy in a typical rushed doctor's office. A class 3b system with 1 watt would take 4 - 5 hours per treatment to get the same dosage.

The original research publication is titled " Treatments for traumatic brain injury with emphasis on transcranial near-infrared laser phototherapy"

video length: (9:18)

), NEST-2 ($n=660$), NEST-3 ($n=630$

)] that demonstrated that NIR light delivered transcranially with a class-IV laser is safe, with no significant differences in rates of adverse events with NIR, when compared to sham exposure.¹⁵16.^–17 Other preclinical studies and clinical trials have suggested that transcranial photobiomodulation (PBM: laser or light emitting diodes—LED) is safe and effective for acute¹⁸19.20.21.^–22 and chronic²³24.^–25 traumatic brain injury (TBI) and has beneficial effects on neurodegenerative diseases (Alzheimer’s and Parkinson’s).^26,27

For the transcranial treatment of major depressive disorder (MDD), both PBM LEDs and lasers have been experimentally tested, although PBM is not FDA-approved for the treatment of MDD. Certain forms of PBM treatment are also referred to as low-level light therapy (LLLT), since it utilizes light at a low power (0.1 to 0.5 W output at the source) to avoid any heating of tissue. The irradiance of the PBM medical devices (or power density) typically ranges from 1 to 10 times the NIR irradiance from sunlight on the skin ($33.6\mathrm{mW}/{\mathrm{cm}}^2$

), the baseline pool of nucleotide triphosphate (NTP)—a product of the cellular utilization of glucose and a marker of the cellular energy availability—was low in subjects who subsequently responded to antidepressant treatment.³² Iosifescu et al.³² also demonstrated for the first time with $31P\text{-}\mathrm{MRS}$ a correlation between treatment response (to a regimen that combined antidepressants and triiodothyronine) and restoration of a higher NTP pool (with compensatory decrease in phosphocreatine) in the anterior cingulate cortex. This study suggests a pathway to antidepressant response based on restoration of a high cellular energy state. In fact, phosphocreatine represents a long-term storage depot of energy, while NTP and ATP are energy-rich molecules that are readily available to the cell. The same authors replicated the aforementioned findings in MDD subjects treated with standard antidepressants (Iosifescu et al., unpublished). In this cohort, $31P\text{-}\mathrm{MRS}$

metabolite changes were noted in brain-only voxels of responders, but not in nonresponders to antidepressants.

In experimental and animal models, PBM (NIR and red light) noninvasively delivers energy to the cytochrome c oxidase and by stimulating the mitochondrial respiratory chain leads to increased ATP production (see Fig. 1).³⁷38.^–39 A study of the effects of NIR on patients with MDD found that a single session of NIR led to a marginally significant increase in regional cerebral blood flow.⁴⁰ Whether the observed changes in cerebral blood flow resulted from fundamental changes in neuronal metabolism or changes in vascular tone remain to be clarified. Given the correlation of both hypometabolism and abnormal cerebral blood flow with MDD, the beneficial effect of NIR on brain metabolism is one potential mechanism for its antidepressant effect.

Fig. 1

Cellular targets of NIR radiation mechanisms of transcranial NIR for psychiatric disease. The NIR photons are absorbed by cytochrome c oxidase in the mitochondrial respiratory chain. This mitochondrial stimulation increases production of ATP but also activates signaling pathways by a brief burst of ROS. This signaling activates antioxidant defenses reducing overall oxidative stress. Proinflammatory cytokines and neuroinflammation are reduced. Neurotrophins such as brain-derived neurotrophic factor are upregulated, which in turn activate synaptogenesis (formation of new connections between existing neurons) and neurogenesis (formation of new neurons from neural stem cells).

, $\mathrm{IL}\text{-}1\alpha$, $\mathrm{IL}\text{-}1\beta$, IL-6, IL-8, IL-10, Eotaxin, GM-CSF, and $\mathrm{IFN}\gamma$

. Although IL-10 is an anti-inflammatory cytokine, the results suggested that the elevated levels of this IL-10 were likely induced in response to the overall elevation of proinflammatory cytokine levels. In a review of the research on inflammation in MDD, Raison et al.⁴³ proposed that proinflammatory cytokines might cause brain abnormalities that are characteristic of MDD. Indeed, animal research has shown that IL-1 mediates chronic depression in mice by suppressing hippocampal neurogenesis.⁴⁴

One proinflammatory cytokine that may be of particular relevance to depression is CSF IL-6 (IL6 measured in cerebrospinal fluid). In a recent report, patients with MDD had significantly higher CSF IL-6 levels compared to healthy controls; CSF IL-6 levels were significantly higher than in the serum, and there was no significant correlation between CSF and serum IL-6 levels.⁴⁵ These findings are consistent with a prior report showing a positive correlation between CSF IL-6 levels and the severity of depression and suicide attempts, with the strongest correlation found in violent suicide attempters.⁴⁶ One report in a smaller sample of depressed patients has shown that CSF IL-6⁴⁷ was lower or comparable to healthy controls.

NIR light and red light (600 to 1600 nm) decreased synovial IL-6 gene expression (decreased mRNA levels) in a rat model of rheumatoid arthritis.⁴⁸ In another study, NIR (810 nm) used as a treatment for pain in patients with rheumatoid arthritis decreased production of the following proinflammatory cytokines: $\mathrm{TNF}\text{-}\alpha$

, $\mathrm{IL}\text{-}1\beta$

Abstract:

Traumatic brain injury (TBI) is a growing health concern affecting civilians and military personnel. In this review, treatments for the chronic TBI patient are discussed, including pharmaceuticals, nutraceuticals, cognitive therapy, and hyperbaric oxygen therapy. All available literature suggests a marginal benefit with prolonged treatment courses. An emerging modality of treatment is near-infrared (NIR) light, which has benefit in animal models of stroke, spinal cord injury, optic nerve injury, and TBI, and in human trials for stroke and TBI. The extant literature is confounded by variable degrees of efficacy and a bewildering array of treatment parameters. Some data indicate that diodes emitting low-level NIR energy often have failed to demonstrate therapeutic efficacy, perhaps due to failing to deliver sufficient radiant energy to the necessary depth. As part of this review, we present a retrospective case series using high-power NIR laser phototherapy with a Class IV laser to treat TBI. We demonstrate greater clinical efficacy with higher fluence, in contrast to the bimodal model of efficacy previously proposed. In ten patients with chronic TBI (average time since injury 9.3 years) given ten treatments over the course of 2 months using a high-power NIR laser (13.2 W/0.89 cm2 at 810 nm or 9 W/0.89 cm2 at 810 nm and 980 nm), symptoms of headache, sleep disturbance, cognition, mood dysregulation, anxiety, and irritability improved. Symptoms were monitored by depression scales and a novel patient diary system specifically designed for this study. NIR light in the power range of 10-15 W at 810 nm and 980 nm can safely and effectively treat chronic symptoms of TBI. The clinical benefit and effects of infrared phototherapy on mitochondrial function and secondary molecular events are discussed in the context of adequate radiant energy penetration. Keywords: infrared, traumatic brain injury, TBI, transcranial infrared light therapy, transcranial laser therapy 

INTRODUCTION

Traumatic brain injury (TBI) has recently moved into the limelight due to the recognition of its impact on professional athletes and military personnel. Yet, TBI is neither a new problem nor limited to those two populations. The Centers for Disease Control and Prevention estimated that 1.5 million Americans sustained TBI annually in 2000.1 As of 2006, the estimates had risen to 1.7 million brain injuries annually.2,3 Undoubtedly, these point prevalence proportions will increase as military personnel return home,4 and the problem of repeated mild TBI (mTBI) becomes more recognized in sports.5 Current estimates of the prevalence of TBI among veterans range from 9.6%6 to 20%,7 with an estimated total of more than 300,000 cases of TBI among military personnel since 2000.4 The current estimates of the combined number of sportsrelated concussions and brain injuries in the US are 1.6-3.8 million annually.8-10 TBI results in a wide spectrum of neurological, psychiatric, cognitive, and emotional consequences. In part, the variation is related to the severity of the injury (mild, moderate, severe TBI), which is stratified based on Glasgow Coma score, periods of unconsciousness, and degrees of amnesia. Furthermore, the diversity of sequalae can be related to the areas of the brain that are injured, the severity of the injury (highly variable within the classification of “mild” and “moderate”), and the evolution of the injury over time due to neuroinflammatory processes.11,12 Additional mechanisms thought to underlie the damage of TBI include decreased mitochondrial function, calcium and magnesium dysregulation, excitotoxicity, disruption of neural networks, free radicalinduced damage, excessive nitric oxide, ischemia, and damage to the blood-brain barrier. Together, these can contribute to a progression of the damage over time. Patients with TBI can experience headache, visual disturbances, dizziness, cognitive impairment, loss of  executive skills, memory impairment, fatigue, impulsivity, impaired judgment, emotional outbursts, anxiety, and depression.3,13-23 The situation can be further clouded by secondary and/ or comorbid posttraumatic stress disorder (PTSD), depression, and anxiety,17-25 which can have symptoms that overlap with those described above and appear to be increasingly likely with repetitive concussive or subconcussive brain injury.5,24,26

TREATMENTS FOR TBI

Pharmacological treatments Pharmacological treatment largely targets the neuropsychiatric sequalae of TBI, rather than providing any means of healing or repairing injury. In general, pharmacological treatment is focused on the modulation of major neurotransmitter systems – dopaminergic, serotonergic, noradrenergic, acetylcholinergic, and glutaminergic.20 Disruption of the major neurotransmitter pathways may result from direct injury or excitotoxicity and other cytotoxic mechanisms. The treatment of depression secondary to TBI is often approached with serotonin reuptake inhibitors. Several studies have examined the benefit of sertraline in post- TBI depression.27-29 Other serotonin reuptake inhibitors also have been examined. Tricyclic antidepressants appear to have some use in the treatment of post-TBI depression, although cautious dose titration is required. Patients with TBI are at greater vulnerability to sedation and cholinergic side effects of confusion and memory impairment. With serotonergic agents other than sertraline, cognitive effects also have been reported.30 Similarly, lithium may be a less desirable agent in this  population due to sedation and cognitive impairment. Patients with TBI may respond at lower doses and lower blood levels than expected. Modulation of the dopaminergic system may improve alertness, attention, and cognitive processing speed. The stimulants are most commonly used for this purpose. Methylphenidate facilitates the release of dopamine and slows its reuptake. Dextroamphetamine strongly inhibits reuptake of dopamine, slows down the breakdown of dopamine by monoamine oxidase, and somewhat increases the release of dopamine. These subtle differences are sometimes imperceptible to the patient, but at other times, a patient will do best on one or the other stimulant. Increasing dopamine in the reticular activating system leads to enhanced arousal. Increasing dopamine within the frontal cortex and the striatum leads to enhanced processing speed and attention. Some evidence suggests that the stimulants may enhance neuronal recovery after injury.31-33 There are numerous potential side effects with stimulants, including abnormal heart rhythms, decreased seizure threshold, and death, but these severe side effects are extremely rare. The most common side effects with stimulants are decreased appetite, stomach upset, and headache. These are most severe at the beginning of treatment and improve over time for most patients. Insomnia is another common side effect, which may be more frequent in those with a TBI. Amantadine and bromocriptine may also increase dopamine. Studies of these agents have shown reduced abulia, anergia, and anhedonia in those with TBI.34,35 Amantadine may cause confusion, hallucinations, and hypotension. Small studies have suggested some benefits of bromocriptine in cognitive function.36,37 Arousal-enhancing agents also have found a use in the treatment of the neurocognitive sequalae of TBI. Modafinil is the oldest form of these medications, and armodafinil is an isomer of modafinil with longer activity and less side effects. These medications help to increase alertness and wakefulness. The precise mechanism of action of odafinil is unclear. It appears to increase histamine in parts of the brain involved in controlling the sleep-wake cycle; however, knock-out mice that lack histamine receptors still show increased wakefulness with modafinil.38,39 The picture is also murky  for modafinil’s effect on orexins, which are wakefulness molecules in the hypothalamus.40 Modafinil has been shown to weakly bind to the dopamine transporter – like the stimulants,41 and dopamine transporter knock-out mice show no response to modafinil.42 A number of research studies have examined the benefit of these agents in fatigue associated with multiple sclerosis, TBI, cancer, and other conditions. Cognitive and memory impairments after TBI may reflect disruption of cholinergic function. The impact of anticholinergic agents on cognitive function of those with TBI supports this contention. Donepezil is the safest and most widely used of the cholinesterase inhibitors. Several easonably large studies have shown improved memory and cognitive function.43-45 Donepezil has benefits in memory and cognition even several years after injury.45,46  Anticonvulsants are often prescribed initially after a TBI due to heightened risk for seizures. Post-TBI mania or mood lability may respond well to anticonvulsants, such as carbamazepine or sodium valproate. They are also often used to treat aggression after TBI. The anticonvulsant agent, topiramate, has been shown to adversely affect cognitive function in the TBI patients.47 While insomnia is a significant issue for patients with TBI, affecting between 15% and 84% (mean of 40%),3,13,19,21,23,48,49 little has been published on the treatment of this aspect of TBI. Benzodiazepines may  be effective but carry a risk of disinhibition. Kemp et al48 found that commonly used sleep aid, melatonin, was not effective. Antidepressants, including serotonin reuptake inhibitors and tricyclic antidepressants, are not effective in resolving insomnia in this population.49 No single agent has emerged as a good solution for this symptom. Cognitive rehabilitation Cognitive rehabilitation now takes many forms and is often individualized to the particular needs of the patients. Protocols have been devised to remediate cognitive difficulties often encountered in those with TBI, such as impaired concentration, executive dysfunction,  inattention, visual disturbances, memory dysfunction, and impaired language function. They range from simple strategies (using a planner to aid memory and organization) to specific protocols targeting particular cognitive functions (eg, short-term memory) that can be monitored with sequential neuropsychological testing. These interventions have been extensively reviewed elsewhere.50,51 Comprehensive programs which include psychotherapy and social skills components have been shown to have greater efficacy.50,52,53 Overall, reports of benefits have been mixed.54,55 Behavioral therapies Behavioral remediation strategies to eliminate problematic  behaviors following TBI have met with mixed success, most often in terms of the poor generalization of specific skills to the outside world. Behavioral deficits that create difficulties for those with TBI and their families include poor hygiene, decline in tidying/cleaning habits, social withdrawal, reduced social comprehension, impaired memory, and poor organization. Behavioral excesses that create difficulties for those with TBI and their families include aggression, sleep disruption, and perseverations. These have been reviewed elsewhere.56 Nutritional supplements Nutritional supplements, herbs, and nootropics have been utilized for many years and are increasingly popular among the patient populations. There remains little clinical research on many of these agents, perhaps reflecting a lack of funding more than a lack of efficacy. Acetyl-l-carnitine is an ester of l-carnitine and is thought to protect brain cells after injury when glucose metabolic pathways are compromised. During this period, acetyll- carnitine supports alternative ketogenic pathways for metabolism.57 It is also believed to enhance cholinergic function. While there are several clinical studies on patients with Alzheimer’s disease and preclinical data on animal models of TBI, the clinical literature on TBI remains sparse. Ginkgo biloba is a natural product of the tree by the same name. It has been shown to improve membrane fluidity and increase resistance to free-radical damage. It provides some subtle benefits to cognitive function in clinical studies of stroke, dementia, aging, and hypoxia damage.58 It has not been systematically studied in TBI but is used extensively in clinic, often in combination with meclofenoxate which is an avid scavenger of free radicals.59 S-Adenosylmethionine (SAMe) is a nutritional supplement which improves cell membrane fluidity and promotes the production of glutathione, an antioxidant. The benefit of SAMe has been assessed in a single clinical study of TBI.60 Patients receiving SAMe had a 77% improvement in clinical scores of post-concussive symptoms. Citicholine provides a source of choline which can cross the bloodbrain barrier. It has been used extensively in Europe and Japan as a treatment for TBI, stroke, and dementia. However, two large US studies failed to demonstrate significant benefit.61,62 Piracetam and the related oxiracetam and phenylpiracetam have shown some promise as nootropic agents. In one double-blind, placebo-controlled study, piracetam improved several symptoms of postconcussive syndrome, including headache and vertigo.63 More recent clinical studies have shown marginal benefit.64 Huperzine-A, an extract of Japanese club moss, is a natural acetylcholinesterase inhibitor. It may serve as a natural alternative to donepezil, rivastigmine, or galantamine. Galantamine warrants special mention as it appears to also modulate nicotinic  eceptors and appears to have more persistent benefit in the treatment of Alzheimer’s disease. It appears to modulate neuroimmune responses, in addition to its effects on acetylcholinesterase.65 Cerebrolysin is a polypeptide that purportedly mimics the actions of neurotrophic factors.66,67 Studies have shown that it can reduce beta amyloid and phosphorylated tau protein accumulation. It may promote neurogenesis, synapse formation, and functional recovery.66 In animal models of acute TBI, cerebrolysin-treated rats had more surviving neurons in the area of impact and showed greater functional recovery.67 In a clinical trial of acute TBI, patients were recruited within 24 hours of injury and treated for 3 months with daily intravenous infusion of cerebrolysin. At 3 months, those receiving cerebrolysin performed significantly better on the Cognitive Abilities Screening Instrument.68 It remains unclear if cerebrolysin provides long-term nootropic benefit. The elevation of free radicals in TBI suggests that antioxidants should be beneficial. Clinical trials of pharmacological antioxidants over the past 30 years have not yielded a useful agent in acute TBI.69 Agents, such as tirilazad70 and polyethylene glycol- onjugated superoxide dismutase, have failed to show benefit in acute TBI. Omega-3 fatty acids may enhance brain repair and recovery, based on animal and clinical studies.71 Similarly, vitamin D may offer neuroprotective and restorative benefits72 in the acute TBI setting. In chronic TBI, vitamin D and omega-3 fatty acids may work synergistically, as they both may reduce neuroinflammation, apoptosis, and oxidative stress.73 Other nutritional supplements have been recommended, but prolonged therapy is necessary to possibly see benefits in TBI. A 6-month trial of ginkgo, vinpocetine, acetyl-lcarnitine, huperzine, alpha-lipoic acid, n-acetyl-cysteine, multivitamins, and over 5 g of omega-3 fatty acids daily yielded improved performance in cognitive testing and increased perfusion (function) in single-photon emission computed tomography (SPECT) scan.74 Long-term use of dietary flavanols may improve cognition in mTBI.75 Transcranial magnetic stimulation Transcranial magnetic stimulation (TMS) has shown some promise in animal models of TBI.76 However, a Cochrane review of the clinical application of TMS for depression noted no difference between repetitive TMS (rTMS) and sham rTMS using the Beck Depression Inventory (BDI) or the Hamilton Depression Rating Scale, except during the initial 2-week period.77 The application of TMS in the post-TBI patients is limited by the risk of seizure induction.78 Hyperbaric oxygen Hyperbaric oxygen treatment has been explored as a treatment for TBI.79-91 Hyperbaric oxygen therapy is neither a benign treatment, given the concerns of oxygen toxicity,79 nor a clear treatment in that the placebo condition of moderate hyperbaric room air also effectively improves cognitive function.80,81 The most carefully performed study compared a group in a cross-over design with an interval of both null treatment and hyperbaric oxygen at 100% oxygen and 1.5 atm.82 The study described improvement in many of the symptoms associated with persistent TBI including headache, tinnitus, vision disturbance, memory dysfunction, and impaired cognitive function. Cognitive testing also showed improvement in attention, information processing speed, and a battery of cognitive tests. In an uncontrolled case series of 16 subjects, Harch et al83 demonstrated that an abbreviated series of hyperbaric treatments using 100% oxygen at 1.5 atm could mitigate subjective symptoms of TBI (eg, headache, sleep disruption, irritability), improve cognitive testing scores, and improve cortical function based on SPECT imaging.83 A study of a higher dose (2.4 atm) did not reveal any significant benefit of hyperbaric oxygen therapy compared to a sham-control group treated with 1.3 atm,84 and this result has been extended and confirmed by a related group.85 However, this may reflect an inverse dose- esponse curve, rather than an absence of benefit, in that the low-dose sham group demonstrated significant changes in cognitive testing and symptom frequency.86 Hyperbaric oxygen remains a controversial area in both acute TBI86-89 and chronic TBI.82,83,85,86,90,91 Physical exercise High-energy activities and exercise programs completed through a health club facility or comprehensive rehabilitation program should focus on the same parameters of an age-adjusted and diagnosis-specific program for aerobic conditioning – flexibility, stabilization, and strength. Though it appears safe and is an accepted intervention for TBI, there is a need for further welldesigned studies.92 Exercise was a part of a 6-month study of lifestyle changes described above which yielded improved function based on cognitive testing and perfusion SPECT scans.74

A NEW TREATMENT FOR TBI

Unfortunately, little has been found to reverse the damage of TBI or repetitive concussion which is the root cause of residual cognitive and psychological impairment following TBI.20,93 One potential avenue of treatment for TBI is infrared light, which has shown promising data in a number of applications. Near-infrared (NIR) light has been investigated for its ability to modulate intracellular mechanisms related to healing. The application of NIR light by low-power laser or by light-emitting diode (LED) is also known as laser phototherapy94 or near-infrared photobiomodulation.92 NIR irradiation can facilitate wound healing,95,96 promote muscle repair,95 and stimulate angiogenesis.95,96 NIR phototherapy has been studied and applied clinically in a wide array of ailments, including skin ulcers,97 osteoarthritis,98 peripheral nerve injury,95,96 low back pain,99 myocardial infarction,100 and stem cell induction.101 The finding that NIR light passes relatively efficiently through bone has spurred interest in its application to treating disorders of the brain. Over the past decade, transcranial near-infrared light therapy (NILT)102 has been studied in animal models to understand its ability to repair damaged or dysfunctional brain tissue resulting from stroke and TBI. The first published study of NILT for TBI in humans described two cases of chronic mTBI with significant disability.103 Each patient was treated with an LED device delivering low-level low-level light therapy (LLLT) in the red and NIR range for 6-10 minutes per area daily for several months. Both patients had marked neuropsychological improvement after a minimum of 7-9 months of LLLT treatment. The precise mechanisms underlying photobiomodulation and its therapeutic benefits are not fully understood. The purported effects of NIR are illustrated in Figure 1. Light in the wavelength range of 600-1,200 nm has significant photobiomodulation capability.104 Current data most strongly support that absorption of NIR photons by cytochrome c oxidase in the mitochondrial respiratory chain is the key initiating event in photobiomodulation.95,96,104,105 This induces an increase in cytochrome c oxidase activity which in turn increases adenosine triphosphate (ATP) production. Such an increase in ATP in wounded or underperfused cells may be sufficient to activate cells in areas of injury or metabolic derangement.106 Data from numerous tissue culture and animal studies point to the importance of several secondary molecular and cellular events. For example, NIR photonic energy can modulate reactive oxygen species,95,96,102 activate mitochondrial DNA replication,95,96 increase early-response genes,95 increase growth factor expression, induce cell proliferation, and alter nitric oxide levels.95,96,102 These mechanisms are more fully described in the companion paper.105 When examined in the specific model of neural tissue injury, NIR phototherapy can lead to demonstrable neural repair and recovery. For example, LLLT of a power density of 0.9-36 J/cm2 applied at 24 hours poststroke in a rodent model yielded a 32% reduction in neurological deficits, as well as histochemical evidence of neuron proliferation and migration.106-108 LLLT had similar benefits in a rodent model of TBI.96,109-111 Interestingly, these cellular changes evolved over a period of days after light exposure and persisted for considerably longer than the interval of actual NIR exposure. These findings are consistent with a progressive regeneration cascade set in motion by the NIR light exposure. NILT in stroke NILT, predominately in the form of LLLT, has been investigated in laboratory models of stroke. LLLT applied in a single dose to an ischemic stroke model appeared to induce expression of the growth factor transforming growth factor – beta 1 and suppress the production of peroxynitrite.112 In a rat model of middle cerebral artery occlusion, LLLT at a dose of 0.5-7.5 mW/ cm2 using continuous wavelength light at 808 nm was administered at 24 hours after the acute stroke.108,113 This single application was estimated to deliver 1.8 J/cm2 in total to the cortex surface and resulted in demonstrable neurological improvement. Functional changes were not manifested until approximately 2 weeks after the single treatment. While there was no significant change in the size of the stroke lesion, histochemical evidence of neurogenesis and migrating neurons108 indicate that a cascade of secondary processes was initiated by NILT. A rabbit model of stroke utilizing injection of a blood clot embolus also demonstrated benefit from LLLT.102,114,115 Herein, 808 nm light was applied with an LED delivering 7.5 mW/cm2 and an estimated 0.9-2.6 J/cm2 to the cortical surface. Cortical ATP levels were increased, indicative of increased mitochondrial activity.114 Significant behavioral recovery was also noted; however, neither ATP increased nor neurological function changed at doses less than 0.3-0.7 J/cm2.114,115 At higher doses of 0.9-15 J/cm2, neurological improvement was seen.114,115 The clinical trials of NILT in acute stroke, the Neuro- Thera Effectiveness and Safety Trials 1, 2, and 3 (NEST- 1,-2, -3), were conducted between 2006 and 2009. The Phase II clinical trial (NEST-1) involved 120 patients in a double-blind, placebo- ontrolled study of the effects of NILT within 24 hours of ischemic stroke.116,117 Approximately 60% of the patients experienced clinical benefit, and the safety profile was very good. Thus, NEST-2, a Phase III clinical trial, was undertaken in 2007. A total of 660 patients were enrolled.118 Somewhat surprisingly, the study did not demonstrate statistical clinical improvement using a different outcome measure.119 Post hoc analysis revealed that a portion of the patients who were moderately affected and/or had strokes limited to the cerebral cortex did realize clinically and statistically significant improvement.102 The NEST-3 trial was halted midpoint when it failed to demonstrate statistical benefit on futility analysis.120 A key factor in the interpretation of the results of NEST-3 is that, different from NEST-1, all types of stroke were included as opposed to just cortical strokes. Continuous laser light has a limited depth of penetration (#1 cm into brain tissue) which likely prevents an effect on deeper brain matter. Therefore, the lack of significant benefits from NIR phototherapy in NEST-3 could be related to the fact that ischemic penumbra was not reached by the light (Luis DeTaboada, personal communication, January 2015). While pulsed NIR was not used in the NEST-3 study, it is estimated that pulsed NIR could penetrate up to 3 cm in depth from the cortical surface, therefore possibly extending the therapeutic target to deeper strokes (Luis DeTaboada, personal communication, January 2015). Figure 1 Hypothesized mechanism of action of NiR light therapy. Notes: NiR light (600-980 nm) penetrates tissue to variable depths depending on wavelength, the tissue involved, coherence, and time. A fraction of the photonic energy reaches the mitochondria and is absorbed by cytochrome c oxidase. This activates increased ATP production, increases production of ROS and RNS, and possibly increases NO. Downstream events include increased early-response genes (c-fos and c-jun) and activation of NF-?B, which in turn induces increased transcription of gene products leading to synaptogenesis, neurogenesis, and increased production of inflammatory mediators and growth factors. Abbreviations: NiR, near-infrared; ATP, adenosine triphosphate; ROS, reactive oxygen species; RNS, reactive nitrogen species; NO, nitric oxide; NF-?B, nuclear factor kappa B. NILT in TBi Oron et al109 conducted the first animal studies of NILT for TBI. They found that a single application of NIR light at 808 nm from a 200 mW emitter at 4 hours post-injury resulted in a significant reduction in lesion size by 5 days.109 To date, several groups have studied NILT in animal models, and this material has previously been reviewed.95,121-123 Single applications of 800-810 nm NIR light within 4 hours of injury have been shown to improve neurological function significantly.110,124-126 The same dose of NIR light at 6 hours was less effective125 and at 8 hours had no appreciable benefit.125 NIR photonic energy at other wavelengths was less effective. Wu et al110 examined red light (670 nm) at 4 hours and found a similar improvement in neurological function; however, 730 nm and 980 nm had no neurological benefit. Similar data for lesion volume have been reported. A single dose of 800-810 nm NIR light (fluence of 36 J/cm2) yielded an approximate 50% reduction in the volume of the lesion at 3-4 weeks110,111,124-126 and a possible reduction in the initial spread of neurological injury, based on the marked reduction in lesion volume found at 5 days post-injury.109 Repeated NIR phototherapy treatments appear to have some benefit, but the frequency and number of treatments are critical factors. While a single NIR light application had benefit, daily applications for 3 days yielded much greater neurological benefit126,127 with smaller lesion size,126 fewer degenerating neurons,126 more proliferating cells,126 and greater levels of brain-derived neurotrophic factor (BDNF)127 compared to a single treatment in a mouse model. In contrast, daily treatment for 7 days128 or 14 days126 showed no difference from controls. NIR energy densities in the range of 0.9-36 J/cm2 resulted in significant biochemical and behavioral changes.109-111,124-127 Pulsing of NIR light appears to yield a greater neurological response but only within certain parameters. Pulsing at 10 Hz yielded greater neurological improvement and a significant reduction in lesion size compared to either continuous-wave or pulsed NIR at 100 Hz.111 In the mouse model of moderate TBI, NILT (800-810 nm) improved learning and memory (Morris water maze performance),128 as well as behaviors associated with depression and anxiety (immobility during tail suspension).111,124 The finding that NILT brought about a smaller lesion in the rodent model of TBI compared to untreated mice suggests that decreased apoptosis, reduced spreading lesion penumbra, and/or neurogenesis are induced by NILT. Indeed, NILT can decrease BAX expression, a pro-apoptosis gene,129 increase expression of BCL-2, an anti-apoptosis gene,129 increase nerve growth factor,95 increase BDNF,127 decrease inflammatory markers,130 and decrease numbers of degenerating neurons.126 Together, these mechanisms may reduce the enlargement of the initial lesion during the first day following the lesion.109 Moreover, increased BDNF and nerve growth factor may contribute to synaptogenesis as shown by increased levels of synapsin-1,127 and neurogenesis, as shown by increased numbers of proliferating cells.127 In a double-blind study in healthy volunteers, NILT was beneficial – compared to sham – in memory and attention.131 In this study, the authors shed only one application of NIR light to the right forehead, targeting the right frontal pole of the cerebral cortex (Brodmann’s area 9 and 10). The device was a Class IV laser CG-5000 (Cell Gen Therapeutics, Dallas, TX, USA), and the parameters were as follows: wavelength 1,064 nm, irradiance 250 mW/cm2, fluence 60 J/cm2, and time 4 minutes per site (two sites).131 The subjects who received the NIR treatment had better attention after 2 weeks, measured by the psychomotor vigilance test. They also had better delayed visual memory at the Delayed Match-to-Sample test. This is the only published controlled trial assessing the impact of NILT on cognition; however, other reports have shown the therapeutic effects of NILT in small numbers of TBI patients. In a two-case report in TBI patients,103 NILT (870 nm) improved sustained attention, memory, and executive functions. Both patients were treated with an instrument with three separate LED cluster heads. The parameters used for the treatment were the following: NIR wavelength 870 nm and 633 nm (red light), irradiance 2.2-25.8 mW/cm2, fluence 13.3 J/cm2, and time 10 minutes per site.103 The same group reported on a cohort of eleven subjects with persistent cognitive dysfunction and treated with a similar NILT protocol for chronic mTBI.132 The eleven subjects received NILT with a device with three LED cluster heads (Model 1100; MedX Health, Toronto, ON, Canada). The parameters used for the treatment were the following: NIR wavelength 870 nm and 633 nm (red light), irradiance 22.2 mW/cm2, fluence 13 J/cm2, and approximate time 10 minutes per site. The NIR light was applied three times per week for 6 weeks (18 sessions), on eleven sites for 10 minutes per site (the total duration of each session was 20 minutes).132 The sites on the skull were chosen on the midline, and bilaterally on frontal, parietal, and temporal areas. At the follow-up neuropsychological testing, NILT had a powerful effect on attention, inhibition, and inhibition switching in the Stroop task, and similarly improved verbal learning and memory, as well as enhanced long-delay free recall on the California Verbal Learning Test. Eight subjects, from the same cohort, were identified as having mild, moderate, or severe depression based on the BDI-II total score (range: 15-34).132 The three cases, who entered the study with only mild depression, remained the same after NILT treatment. Results for the five cases with moderate-severe depression were as follows: two moderate cases improved to mild/minimal depression 8 weeks after the end of NILT series, and one severe case improved to moderate depression. Two moderate or severe depression cases remained the same after 8 weeks of follow-up from the last NILT session.132 Dose response and photonic penetration A prevailing theory in photobiomodulation postulates that a bimodal response curve exists for the biological effects of NIR light.95 The so-called Arndt-Schulz curve (a fundamental principle in homeopathic medicine) is frequently used to describe this biphasic dose response. Some data indicate that low levels of light have a much better effect on stimulating and repairing tissues than higher levels of light. Laboratory studies of cells in culture have demonstrated a bimodal dose response to light exposure in lymphocytes133 and fibroblasts.134,135 For example, Chen et al135 found that a range of 0.03-0.3 J/cm2 was beneficial in activating transcription factors in culture, while 3-30 J/cm2 inhibited the activation of these factors. In contrast, an order-of-magnitude greater dose (2 J/cm2) was best at activating fibroblasts in a superficial wound model.136 Furthermore, an order-ofmagnitude greater dose (30 J/cm2) proved to be best in a rodent joint inflammation model.137 Thus, a dosedependent effect for many biological responses to NIR light has been demonstrated,95,137-139 but the critical parameter is dose at the level of the target tissue, rather than at the surface.137,140 The amount of energy that reaches a volume of tissue at depth is determined by the attenuation of the photonic energy as it passes through the overlying tissue. For example, only 2.45% of the energy from a 980 nm laser emitter penetrates to the level of the peroneal nerve.140 Nevertheless, the biphasic dose response does not appear to be universally true. In primary microglial cell culture, a dose-dependent response to NIR was demonstrated with no detrimental effects at doses as high as 30 J/cm2.141 So a critical question in the use of NILT is that of radiant energy penetration. In particular, some authors have challenged the efficacy of low-power LEDs used in LLLT.142-144 In laboratory studies, LLLT radiant energy is almost entirely absorbed in the first 1 mm of skin.145,146 In two unrelated studies, LLLT diode devices proved to be ineffective in the treatment of diabetic neuropathy,142,144 in contrast with prior reports.147 Similarly, laboratory studies of NILT using LLLT transcranially have not consistently yielded positive results. For example, in a rat model of TBI, Giacci et al148 found no benefit from daily 30-minute irradiation with either 670 nm or 830 nm 0.5 W LED emitters for a period of 7 days. Doses at the skin surface were 28.4 J/cm2 and 22.6 J/cm2, respectively.148 Similarly, treatment of a rat model of contusive spinal cord injury with LLLT (830 nm at 22.6 J/cm2 or 670 nm at 28.4 J/cm2) for 30 minutes per day for 5 days resulted in no significant functional improvement and no reduction in lesion size, despite delivering 2.6 J/cm2 to the spinal cord.148 Lapchak102 reported that the physical parameters of NILT in the clinical trials for the treatment of stroke utilized in the NEST-1 and NEST-2 trials116-120 may have delivered insufficient energy to cortical tissues to be effective. Therein, NIR light of 808 nm wavelength with infrared energy densities of 0.9 J/cm2 was applied to the human scalp for a total of 40 minutes with applications at multiple sites during that time.116,118 Recall that animal models of both stroke and TBI suggest that NIR energy densities in the range of 0.9-36 J/cm2 resulted in significant biochemical and behavioral changes.96,106-115,125-127 The concern raised from the NEST studies102 is that current clinical trials testing the effectiveness of lowenergy NIR diodes to treat TBI may yield negative or inaccurate efficacy data, not because of a failure of infrared light to invoke a change but due to a dose error. Doses that are effective when directly applied to cells in a Petri dish149,150 or to 3-5 mm thick rodent brains96,109-111,125,126,128 may be insufficient to penetrate 2-4 cm into the human brain. In a companion paper, our own studies of photonic energy penetration are detailed.105 To summarize, the laboratory tissue studies showed that 0.5 W LED emitters did not penetrate the 2 mm thickness of human skin. No detectable energy from 0.5 W LED NIR light emitters could be detected penetrating a similar thickness (1-2 mm) of sheep skin or 3 cm thick section containing sheep skin, skull, and brain. In contrast, 11% of the photonic energy from a 10 W 810/980 nm coherent NIR laser penetrated 2 mm of human skin. Similarly, 17% of the photonic energy from a 15 W 810 nm coherent NIR laser penetrated the same distance.105 Energy from these more powerful NIR emitters could be detected penetrating 3 cm of sheep skin, skull, and brain with 0.4% of the 10 W 810/980 nm NIR laser’s energy reaching the depth of 3 cm and 2.9% of the 15 W 810 nm NIR laser’s energy traversing the same distance.105 Anders also has demonstrated penetration of 808 nm light to 40 mm in the brain using a 5 W laser emitter (JJ Anders, personal communication, January 2015). Prompted by the mixed results in the literature and the observations by Lapchak,102 Franzen-Korzendorfer et al,144 Wan et al151 and Lavery et al142 we have been utilizing relatively high-power (10- 5 W) lasers at the wavelengths of 810 nm and 980 nm in the clinic to treat patients with TBI. Clinically, the patients have shown excellent responses with resolution of many of their long-standing symptoms of TBI or post-concussive syndrome. Below is a retrospective series of such patients to illustrate the extent and character of response to this modality. Methods Patients in the case series were sequentially treated patients at a clinic which is engaged in ongoing NILT for a number of clinical conditions. The risks, benefits, and current state of research on the use of NILT were explained to each patient. Each patient consented to treatment. Institutional Review Board approval was obtained in a post hoc review, noting that the risk-benefit ratio was acceptable. Between March 16, 2011 and February 20, 2013, sequential new referrals for chronic mild-to-moderate TBI were evaluated for treatment and selected for NILT using Class IV lasers, either the LT1000 (LiteCure, Newark, DE, USA), a 10 W adjustable NIR laser emitter with wavelengths of 810/980 nm capable of delivering continuous or pulsed NIR light, or the Diowave 810 (Diowave, Riviera Beach, FL, USA), an adjustable NIR emitter up to 15 W with a wavelength of 810 nm capable of delivering continuous or pulsed NIR energy. Demographics and laser treatment settings are detailed in Table 1. The fluence delivered to the skin of patients ranged from 55 J/cm2 to 81 J/cm2. No other treatment modalities (medications, exercise regimen, supplements) were added, discontinued, or changed while receiving NILT. Symptoms were monitored clinically. A baseline Quick Inventory of Depressive Symptomatology Self-Report (QIDS-SR)152 was completed for all patients, and the BDI153 was administered to seven of the ten patients before and after the course of treatment. In addition, each patient was instructed on how to create and maintain a patient and spousal diary of symptoms and subjective progress. Each of six patients received a single series of ten treatments with the LT1000 Class IV laser. Three additional patients each received a single series of 20 treatments with the LT1000 Class IV laser. One patient was treated with the Diowave 810 nm Class IV laser device in a series of 20 treatments. The patients and treating clinician wore protective eyewear. There were no incidents of burns or thermal discomfort (Figure 2). The impact of high-watt NILT While the patient group represented a diverse mix (Table 1 presents demographics), some notable commonalities of symptoms emerged. Over 90% of the patients had complaints of anxiety, depression, irritability, and insomnia. Other symptoms included headache (60%), suicidal ideation (50%), cognitive difficulties (50%), attention problems (50%), short-term memory problems (40%), loss of libido (30%), substance abuse (20%), fatigue (20%), and panic attacks (20%). Six of the patients were unemployed prior to treatment. Three of the patients were experiencing severe marital difficulties. All carried or had a confirmed diagnosis of TBI, but other comorbid diagnoses included PTSD, major depressive disorder, generalized anxiety disorder, bipolar disorder, and attention deficit/hyperactivity disorder. The patients’ baseline scores on the BDI were 25.3±12.1 (moderate depression range), and baseline scores on the QIDS-SR were 12.9±4.6 (moderate depression range). During NILT treatments, skin temperature increased no more than 3°C with rapid cooling after removal of the NIR light. A continuous sweeping motion was utilized to minimize skin heating and cover a larger area. After a course of ten treatments of NILT (20 treatments in four patients), each patient experienced significant clinical improvement with resolution of many of their symptoms (Table 2). In addition, the BDI scores dropped to 12±6.5 (nondepressed range). This represented a significant decrease (P,0.01, Student’s t-test, one-tailed, Microsoft Excel). The QIDS-SR scores after treatment were 2.2±2.3 (nondepressed range), and the difference from baseline was highly significant (P,0.00001, Student’s t-test, one-tailed). Patients noted improvement in cognitive function, mood, anxiety, and sleep. None of the patients continued to have suicidal thoughts (50% at baseline). Other symptoms, such as anxiety and irritability, were markedly improved. Most notable were the nonquantifiable changes in patients’ lives. Patients reported improved cognitive ability and a desire to return to meaningful work. Five of the six unemployed patients have returned to work. The two patients who were Iraq/ Afghanistan veterans have found new careers in highly skilled trades. The patients with marital difficulties have reconciled and were purchasing homes or otherwise solidifying their marriages. The clinical change can be attributed to NILT because no changes in medications, supplements, or exercise regimen were permitted during the course of NILT treatment. All patients in the case series experienced significant clinical improvement which supports the conjecture that high-power NIR laser delivers sufficient energy to the human brain for photobiomodulation to occur. Insomnia and suicidal ideation, common symptoms in those with TBI or post-concussive syndromes,3,17-20,24,25 resolved in 100% of cases. Headache, another common symptom for patients following a TBI,6,14,15,23 was reduced or resolved in the six patients so afflicted. Symptoms such as anxiety,14,15,21,24 depression,21,24,25,27-29 and irritability resolved or were dramatically reduced in all patients. Cognitive function appeared to improve based on return to work or improved work performance, although cognitive tests were not performed. The quality of life dramatically improved in all cases, based on the observations of the patients, their family members, and the treating clinician. At follow-up intervals of 6-7 months post-treatment, patients have reported continued improvements in symptoms. The precise areas of brain injury were not elucidated in Figure 2 Treatment parameters per individual, based on area of the skull treated. Notes: Dimensions varied per head/skull size and hair line. Treatment was warm and comfortable for each patient. There were no incidences of discomfort. Areas treated were (A) temporal- ilateral, (B) frontal, and in patients 1-3, 5, and 6 (B) frontal only. Table 1: Infrared light treatment parameters for each of the ten patients in the case series Patient Area treated Sex Mechanism of TBI Interval since TBI Wavelength of Dosage per area Duration before treatment NIR-PT dual wave Scanning technique per area pulsed 10 Hz 1 B, bilateral frontal Male Concussive blast 2 years 810 and 980 nm 2,700 J 10 minutes Fluence – 20.45 J/cm2 2 areas Area – 132 cm2 10 visits 2 B, bilateral frontal Female MVA 18 years 810 and 980 nm 2,400 J 9 minutes Fluence – 18 J/cm2 2 areas Area – 133 cm2 10 visits 3 B, bilateral frontal Female MVA 5 years 810 and 980 nm 2,400 J 8 minutes Abuse Fluence – 18.3 J/ cm2 2 areas Area – 131 cm2 10 visits 4 A–B, bilateral frontal, left temporal Female MVA x2 8 years and 13 years 810 and 980 nm 2,400 J 8 minutes Fluence – 18.3 J/cm2 3 areas Area – 131 cm2 10 visits 5 B, bilateral frontal Male Vietnam Veteran 20+ years 810 and 980 nm 3,000 J 10 minutes Concussion Fluence – 28.3 J/cm2 2 areas Child abuse Area – 106 cm2 10 visits 6 B, bilateral frontal Male Concussion 5+ years 810 and 980 nm 2,400 J 12 minutes Fluence – 14.8 J/cm2 2 areas Area – 162 cm2 10 visits 7 B–A, bilateral frontal, left temporal Male Afghanistan, Iraqi Disability 810 and 980 nm 3,000 J 10 minutes Disability due to TBI 2 years Fluence – 22.7 J/cm2 3 areas Area – 132 cm2 20 visits  B–A, bilateral frontal, bilateral temporal Female Hypoxic encephalopathy Childbirth-related 810 and 980 nm 2,700 J 9 minutes injury, 8 years Fluence – 27.8 J/cm2 3 areas Area – 97 cm2 20 visits 9 B–A, bilateral frontal, bilateral temporal Male MVA-TBI Numerous episodes 810 and 980 nm 3,000 J 10 minutes Concussions Fluence – 22.72 J/cm2 3 areas Area – 132 cm2 20 visits 10 B–A, bilateral frontal, left temporal Female Bicycle vs car >30 days 810 nm single 2,700 J 9 minutes Concussion, amnesia, LOC wavelength – Fluence – 17.1 J/cm2 3 areas different device Area – 158 cm2 20 visits Note: All safety precautions were followed, including metal protective eyewear (laser eye protection). Abbreviation: LOC, loss of consciousness; MvA, motor vehicle accident; TBi, traumatic brain injury. the majority of these cases, so a correlation of symptoms changes and cortical function changes cannot be made; however, perfusion SPECT imaging in other patients has shown significant increases in perfusion in injured areas of the brain and overall improved cortical function following similar courses of high-watt NILT.154 One concern that has been expressed about high-watt NIR lasers is the risk of tissue heating.155 We explored this issue in our companion paper on NIR penetration.105 Temperature change was 1°C-3°C at the skin surface using continuous-wave NIR lasers in the range of 10-15 W. Using pulsed settings, the high-powered lasers showed no significant temperature change in tissue samples. The temperature change on human skin was 1°C or less in the in vivo penetration studies while maintaining continuous movement of the laser probe head.105 Clinically, patients in this case series reported only slight warming of the skin, but no discomfort, using the continuous motion technique. Laboratory studies have largely focused on treatment of acute brain injury. The processes involved in the benefits of NIR light in chronic TBI as seen in this clinical case series may be quite distinct. Nevertheless, Schiffer et al156 found that a single application of LLLT at 810 nm and 250 mW to the forehead over 8 minutes reduced depression and anxiety symptoms in ten patients for approximately 2 weeks. Similarly, the small case series by Naeser et al103 demonstrated some benefit using NIR light, albeit at very low power levels over a prolonged course of several months with only transient benefit. Together with our clinical data, these findings suggest that at least some of the photobiomodulatory effects of NIR energy likely do occur in chronic neurological conditions. Prior presentations on NILT for the treatment of TBI or stroke in humans have focused on getting photonic energy through the skull to the cortex surface which traverses a distance of about 6-10 mm; however, this model is flawed in that the distance to the areas of damage may be far greater. In other words, the cortex immediately subjacent to a portion of the skull may be 10 mm from the surface, but the NIR light energy may need to penetrate 3-7 cm to reach areas of damage. Much of the cortical surface is actually lining the walls and floors of sulci, rather than immediately subjacent to the skull. Analysis of NIR spectroscopy reveals that light propagation through varying media with irregular boundaries is subject to high levels of scatter.157 In addition, review of the neuroimaging literature on TBI has revealed that the most common areas injured in TBI are the orbitofrontal cortex (at the ventral surface of the frontal lobe) and the anterior and medial temporal lobes.158 It is not anatomically possible to position an NIR light emitter immediately exterior to the skull overlying these areas. Indeed, the orbitofrontal cortex positioned immediately above the eyes can only be reached from the forehead by angling the light emitter. Similarly, the temporal lobes are separated from the surface by epidermis, dermis, subcutaneous fat, subcutaneous blood vessels, accessory head of the temporalis muscle, connective tissue, temporalis muscle, skull, and dura mater.159 Each of these structures has different absorption and refraction properties, and each interface between different materials also creates a barrier to transmission of photonic energy.157 Blood flowing in the subcutaneous vessels is believed to create a unique barrier to transmission.160 In summary, effectively targeting the areas most commonly injured in TBI with sufficient photonic energy to initiate reparative processes represents a significant challenge in NILT. This appears to have been overcome with the high-power laser protocol presented here and in a related paper.154 As yet, the mechanism of action of NILT in treating TBI is not entirely clear. Moreover, the neurological benefits are not immediately apparent. Rather, a delay of 1-4 weeks was noted, consistent with a progressive regeneration cascade set in motion by the NILT.96,103,105 ,107,109,121,122,124,127,135 Similarly, most of the patients in the present case series did not notice benefits immediately or within the first few treatments. Instead, they reported benefits emerging over an interval of weeks, and in some cases, continuing after completion of the course of NILT. In addition, the clinical improvement reported by the patients in the above case series is more profound than that reported by patients treated with LLLT or low-powered lasers.103 In fact, we observed that among seven subjects with documented moderate depression, per BDI scores, four had an antidepressant response (≥50% decrease of depression severity). In contrast, Naeser et al132 reported that out of eight subjects with TBI and comorbid depression, only three had a significant improvement in their depressive symptoms (37.5%). Our results may be due to the greater penetration of more powerful, coherent, and pulsed NIR light from a laser source. A unique outcome measure was developed for this protocol (Morries and Henderson, unpublished data, 2015). A patient diary and separate spousal diary provided a weekly update of patient’s response in his or her home environment. This novel approach to capturing the patient treatment experience provided the patient and family with tangible and pertinent documentation of the clinical response. While time consuming, the experiences recorded in these diaries proved to be valuable clinical tools to the treating clinicians.

CONCLUSION

To date, there has been little progress in developing effective treatments for chronic mild-to-moderate TBI or repetitive concussions. This area of need has become even more pressing with the return of veterans from military conflicts in Iraq and Afghanistan4,6,7,16,17,19,161 and the recognition of the magnitude of sport-related TBI.5,8-10 In addition, the dramatic growth in the geriatric population with attendant proprioceptive dysfunction has resulted in a rising incidence of fall-related TBI.162 NILT has shown promise as a tool for the treatment of TBI. A critical issue is to assure that adequate photonic energy reaches the injured areas of the brain. The use of high-wattage lasers, as we have demonstrated, results in marked clinical improvement in patients with chronic TBI. Moreover, symptoms consistent with PTSD, anxiety, and/or depression also improved considerably or resolved in this group of patients. Further work in the use of highwattage NILT in the treatment of TBI, depression, and other neurological disorders is encouraged.

ACKNOWLEDGMENTS

The authors would like to acknowledge the technical assistance of Mr Charles Vorwaller (Aspen Lasers) and Lite Cure Corporation. The authors also acknowledge the contribution of Ms. Taylor Tuteur in the artistic creation of Figure 1.

DISCLOSURE

Dr. Larry D Morries is the CEO of Neuro-Laser Foundation, a nonprofit foundation. He has a private practice in Lakewood, CO. Theodore A Henderson is the president of The Synaptic Space, a medical consulting firm. He is Table 2 NiLT case series with demographics, symptoms, and treatment response

PRETREATMENT POSTTREATMENT

Patient # Sex Occupation Mechanism of TBI Diagnoses Sleep Symptoms Suicidal BDI Sleep Symptoms Suicidal BDI 1 M Veteran, Blast – 5 years; TBI, PTSD, MDD Primary and H, S, I, D, X, L, A, M, + – Resolved None, back No – unemployed Iraqi middle C, SL with spouse, insomnia working 2 F Nurse, MVA – 8 years TBI, PTSD Middle and H, F, I, X, C, A, STM, L, + 18 Resolved A and HA – No 15 unemployed terminal HA, SL but mild, insomnia return to work 3 F Unemployed Assault and TBI, PTSD, MDD, Primary and D, X, P, M, L, HA, S, + 23 Resolved HA – mild, No – MVA, 5 years GAD, ADHD middle insomnia, SA, C, N, STM back with Prior nightmares spouse, no SA, working 4 F Unemployed MVA – 3 years, TBI, PTSD, MDD Primary and D, X, HA, I, M, SA, S, N + 23 Resolved None, marriage No 17 assault middle insomnia, improved, numerous violent nightmares no SA, working 5 M Veteran, Blast – 20+ years TBI, MDD, GAD Primary and D, X, I, S, SL + 18 Resolved None No 1 unemployed 1960s; Vietnam middle insomnia 6 M executive Trauma – TBI, GAD, MDD Primary D, X, I, P, HA, A, S – – Resolved HA, X, and P – No – chronic insomnia but improved 7 M Veteran, Multiple blasts TBI, MDD, GAD Primary and S, D, I, X, C, A, S, STM, – 22 Resolved HA and C – No 16 disability (>12); Afghan middle HA mild, new and Iraqi wars insomnia career 8 F Student Childbirth TBI, learning Primary D, I, X, C, A, SL, F, STM – 16 Resolved, STM improved, No 7 disorder insomnia no bads reading .20% dream more animated 9 F Sales MVA and TBI, LOC Primary and HA, SL, N, D, I, X, H, A – 29 Resolved Mild HA, No 9 sports TBI middle insomnia, job nightmares promotion 10 F Physicist Recent car– TBI, LOC, amnesia Primary and D, I, X, neck, knee pain – 51 Resolved No loss No 19 bicycle middle of skills, accident insomnia maintain intellectual job Notes: Demographics for each of the ten patients in this case study is presented. Also presented is their history of mechanism of injury, diagnosis, and related symptoms. Changes in anxiety levels, sleep patterns, depression, and suicidal ideation were important symptoms and outcomes to track. Patients were instructed for no medication changes, with their primary treatment provider’s approval. Cognitive difficulties, attention problems, and short-term memory difficulties were by patient interpretation of their symptomatic improvement and patient diary changes. Symptom occurrence % was as follows: Anxiety – 100%, Depression – 90%, Irritability – 90%, Primary And Middle Insomnia – 90%, Headache – 60%, Sadness – 60%, Suicidal Ideation – 50%, Cognitive Difficulties – 50%, Attention Problems – 50%, Short-Term Memory Problems – 40%, Marital Difficulties – 30%, Loss Of Libido – 30%, Substance Abuse – 20%, Fatigue – 20%, Panic Attacks – 20%. Abbreviations: NILT: Near-Infrared Light Therapy, TBI: Traumatic Brain Injury, PTSD: Post-traumatic Stress Disorder, MDD: Major Depressive Disorder, GAD: General Anxiety Disorder, ADHD: Attention Deficit/Hyperactivity Disorder, H: Hyperarousal, S: Sadness, I: Irritability, D: Depression, X: Anxiety, L: Loss Of Libido, A: Attention Problems, M: Marital Difficulties, C: Cognitive Problems, SL: Sleep Issues, F: Fatigue, STM: Short- erm Memory Problems, HA: Headache, P: Panic Attacks, SA: Substance Abuse, N: Nightmares, BDI: Beck Depression Inventory, LOC: Loss of Consciousness, MVA: Motor Vehicle Accident. the president of Dr. Theodore Henderson, Inc., a clinical service firm. He is the co-owner of Neuro-Luminance, a clinical service organization. He is the president of the International Society of Applied Neuroimaging. He is the CFO of the Neuro-Laser Foundation, a nonprofit foundation. Dr. Paolo Cassano received funding from the Brain and Behavior Research Foundation; Photothera Inc and from the Dupont Warren Fellowship (Harvard Medical School) to conduct research on NIR light for the treatment of major depressive disorder.

ABOUT THE AUTHORS:

Larry D. Morries, DC brings a distinguished 30-year career studying and treating the brain and body through his private practice based in Lakewood, Colorado. As Neuro-Laser Foundation’s co-founder, his chiropractic expertise is complemented with extensive study of near infrared-light therapy applications, clinical radiology, clinical neurology and sports injury and rehabilitation. In practice since 1973, Dr. Morries has contributed extensively to both chiropractic and medical professions throughout his career. He is a recognized expert often called upon for review services, treatment utilizations, and documentation presentations. In recent years, he has guided the Colorado State of Colorado Workers Compensation Board with a review of treatment guidelines for Chronic Pain, and Complex Regional Pain Syndrome, Shoulder Pain, Low Back Pain, Traumatic Brain Injury, and was asked to present in 2016 on Thoracic Outlet Syndrome.

Other professional involvement include:

• Colorado Chiropractic Association, Board member, President in 1982, Chairman in 1984

• Colorado Chiropractic Society, Vice President and Secretary in 1995-2004

• Colorado Chiropractic Journal Club, Chairman,since 2008

Dr. Morries has continued his study of the human body and brain with postgraduate work in Neurodiagnostic testing at the American Academy of Neurology, and Harvard Medical School-Massachusetts General Hospital. He is also educated on Spinal Mechanics at Chicago Rehabilitation Institute. He earned his Doctorate in Chiropractic from Logan Chiropractic College, with recognition as Student Clinical Director, Teaching Assistant in Radiology. Dr. Morries is most proud of his research papers and awards, in America Academy of Pain Medicine, Sciatic and Suprascapular Nerve Blocks with Dr. Steve Gulevich, MD. He was asked to share two Poster presentations at the North American Laser Foundation in 2011on Low Back Pain, plus Polyneuropathy treatment with Laser (NIR) therapy. His Podium Presentation and publication on Hip dysplasia, in American Board of Chiropractic Sports Physicians®. Additionally, he has given presentations abroad at State of Chiropractic Research, Foundation of Chiropractic Education and Research, in Bournemouth England and Vancouver, BC, Canada. Dr. Theodore Henderson has extensive training and experience to the practice of Psychiatry. He trained in Psychiatry at the prestigious Barnes/Jewish Hospitals at Washington University in St. Louis. Dr. Henderson completed a fellowship in Child & Adolescent Psychiatry at the University of Colorado. He also has training in Radiology, Nuclear Medicine, and the genetics of psychiatry. He established his private practice in Centennial Colorado in July of 2000. Dr. Henderson brings a unique blend of expertise in psychopharmacology, neurobiology, and an understanding of human nature to the practice of psychiatry. Dr. Henderson attended medical school at Saint Louis University School of Medicine. While in medical school, he began studying heart pathology under Dr. Vernon Fischer. He earned an American Heart Association Medical Student Research Fellowship. With this fellowship, he spent one year at the University of Washington studying the pathology of atherosclerosis. In 1991, Dr. Henderson founded the Child Abuse Prevention Task Force at Saint Louis University. This program taught children, parents, and teachers about child sexual abuse and how to prevent it. Each year, this program reached over 8,000 children throughout the metro St. Louis area, primarily in the poor inner-city schools. The program was awarded numerous awards, including a Saint Louis University Community Service Award, Commendations from the school districts, and an award from the American Medical Student Association. Dr. Henderson was nominated for a Student Life Leadership Award and earned a Departmental Award from the Department of Community and Family Medicine. He also received a Weis Humanitarian Award recognizing outstanding humanitarian care as a medical student. Dr. Henderson wrote a training manual on this program that was implemented at other medical schools and he cowrote a book chapter in the book, A Parent’s & Teacher’s Handbook on Identifying and Preventing Child Abuse (1998). During graduate school and medical school, Dr. Henderson published numerous research studies. He published 9 articles and 27 abstracts about his research in brain development. He also published a book chapter on brain development in collaboration with his research professor, Dr. Mark Jacquin. His research focused on the role of neural growth factors and impulse activity on the development of brain organization. He collaborated with leading researchers, including Drs. Thomas Woolsey, Eugene Johnson, and Thomas Rhoades. While a medical student, Dr. Henderson wrote two research grants (as part of program project grants). Both were funded. He continued conducting research at Saint Louis University and Washington University throughout his residencies. Dr. Henderson trained for one year in Radiology, focusing on neuroimaging and pediatrics. With this strong base, he then undertook a residency in Psychiatry at Washington University’s program at Barnes/Jewish Hospitals in St. Louis. His residency included extended training in general pediatrics at St. Louis Children’s Hospital. In 1997, He was awarded the National Institute of Mental Health Outstanding Resident Award for his ongoing work in child abuse prevention and his neurobiological research while a resident. Dr. Henderson completed a residency in Adult (or General) Psychiatry and then undertook a fellowship in Child Psychiatry at the University of Colorado. This included additional specialization in Autism and Autism Spectrum Disorders. He compl

 1Department of Zoology, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv, Israel

2Department of Cell Biology and Immunology, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv, Israel

Email: *oronu@post.tau.ac.il

Received 27 May 2013; revised 29 June 2013; accepted 16 July 2013

ABSTRACT

In this study, we investigated the hypothesis that photo- biostimulation by low-energy laser therapy (LLLT) applied to the bone marrow (BM) of myocardial in- farcted rats may attenuate the scarring processes that follow myocardial infarction (MI). Wistar rats under- went experimental MI. LLLT (Ga-Al-As diode laser) was applied to the BM of the exposed tibia at differ- ent time intervals post-MI (4 hrs, 48 hrs and 5 days). Sham-operated infarcted rats served as control. In- farct size was significantly reduced (55%) in the la- ser-treated rats as compared to the control non-treat- ed rats, at 2 weeks post-MI. A significant 3-fold in- crease was observed in the density of desmin immu- nopositive stained cells 14 days post-MI in the infarc- ted area of the laser-treated rats as compared to the non-laser-treated controls. The electron microscopy from the control infarcted rat hearts revealed a typi- cal interphase area between the intact myocardium and the infarcted area, with conspicuous fibroblasts with collagen deposition dispersed among them. In rats that were laser treated (to BM), the interphase zone demonstrated cells with different intracellular struc- tures. There was also a significant increase in the per- centage of c-kit positive cells and macrophages in the circulating blood of the laser treated rats as compar- ed to control non treated ones. In the majority of the cells clusters of myofibrils anchored to well-developed Z-lines and structures resembling the morphological characteristics of mature intact cardiomyocytes were evident. In conclusion, LLLT to the BM of rats post- MI induces cardiogenesis mainly at the borders of the infarcted area in the heart.

Keywords: Low-Level Laser Therapy; Myocardial Infarction; Macrophage; Desmin; Ultrastructure; c-Kit Positive Cells

1. INTRODUCTION

Regenerative capacity and mitotic activity in the heart are confined mainly to the lower vertebrates [1]. Amputation of ~20% of the zebrafish’s ventricular myocardium re- sulted in full regeneration without scarring [2]. In am- phibians, heart injury was associated with increased cell proliferation of myocytes and enhanced regeneration [3]. The adult mammalian heart was traditionally considered to be a post-mitotic organ with terminally differentiated cardiac myocytes. However, this dogma has recently been challenged by several studies and reviews [4-8]. These studies have suggested that cardiac myocytes are replaced throughout the lifespan even in the human heart, and that myocytes can regenerate from resident cardiac progenitor cells (CPC) as well as from bone marrow (BM). Studies in human infarcted hearts have shown evidence of cytoki- nesis of cells in the heart and evidence of cardiac stem cells that are activated in response to ischemic injury. This growth response is attenuated in chronic heart fail- ure [9]. Some studies have reported that cardiac myocyt- es can be derived from BM; specifically, side population precursor cells following induction of myocardial infarc- tion (MI) by left anterior descending artery (LAD) liga- tion [10-12]. Contradicting these findings, other laborato- ries using genetic markers have reported that lineage ne- gative, c-kit+ BM cells did not differentiate into cardio- myocytes [13]. It was also suggested that BM-derived stem cells may stimulate the small population of stem cells in the ischemic heart to proliferate and differentiate to enhance cardiac repair post-MI [14]. In a recent study transient regenerative potential in the mouse heart was demonstrated during the neonatal period [15].

Low-level laser therapy (LLLT) has been found to modulate various biological processes [16,17], such as increasing mitochondrial respiration and ATP synthesis [18], facilitating wound healing and promoting the proc- ess of skeletal muscle regeneration and angiogenesis [19- 21]. In an experimental model of the infarcted heart in rats and dogs, it was demonstrated that LLLT application directly to the infarcted area in the heart at optimal power parameters significantly reduced scar tissue formation [22-24]. This phenomenon was partially attributed to a significant elevation in ATP content, heat shock proteins, vascular endothelial growth factor (VEGF), inducible ni- tric oxide (NO) synthase, and angiogenesis in the ischemic zone of the laser-irradiated rats, as compared to non- irradiated rats [25].

The effect of photobiostimulation on stem cells or pro- genitor cells has not been extensively studied. LLLT ap- plication to normal human neural progenitor cells signi- ficantly increases ATP production in these cells [26]. LLLT delivery to MSCs and cardiac stem cells in vitro caused a significant enhancement in their proliferation rate [27,28]. LLLT has also been shown to increase the proliferation rate of adipose-derived stem cells in vitro [29]. Recently, we demonstrated that LLLT application to autologous BM could induce mesenchymal stem cells (MSCs) in the BM to proliferate and cause their recruit- ment and specific homing in on the infarcted rat heart and not on other organs [30,31]. The laser treatment to the BM also caused a marked and statistically significant reduction of 79% in the scarring and ventricular dilata- tion followed MI as compared to infarcted non-laser- treated rats. The aim of the present study was to investi- gate the possibility that induction of stem cells in the BM of rats by LLLT could also affect cardiogenesis in the in- farcted rat heart.

2. MATERIALS AND METHODS

2.1. Experimental Procedures

A total of 21 Wistar male rats, weighing 200 - 250 gr, that underwent ligation of the LAD artery to induce MI, were used as described by us previously [23]. All the ex- perimental procedures were approved by the animal care committee of Tel-Aviv University. Briefly, rats were anes- thetized with Avertin (1 ml/100 g body weight I.P.) and the lungs were ventilated. Thoractomy was performed by invasion of the intercostals muscles between the 5th and 6th rib to expose the heart. The LAD artery was occluded 2 mm from the origin with 5-0 polypropylene thread (Ethicon Inc., Cincinnati, OH). Following LAD artery occlusion the chest muscles and skin were sutured and the rats were ventilated until they woke up. The infarcted rats were divided randomly into two groups. In one group LLLT was applied directly to the BM 4 hrs, 48 hrs and 5 days post-MI (see below). The second group was non-laser-treated (the rat’s bone was exposed for the same duration as the laser-treated group but the laser was not turned on). Food and water were supplied ad libitum. Rats were sacrificed 14 days post-MI.

2.2. Laser Application

After induction of MI rats were randomly assigned to a laser-treated or control non-laser-treated group. A diode (Ga-Al-As) laser, wavelength 804 nm with a tunable po- wer output of maximum of 400 mW (Lasotronic Inc., Zug, Switzerland) for application to the BM was used. The laser device was equipped with a metal-backed glass fiber optic (1.5 mm diameter). An infrared viewer (Laso- tronic Inc. Zug, Switzerland) and infrared-sensitive de-tecting card (Newport, Inc., Irvine, CA) were used to de- termine the infrared irradiation area. Laser application was done by a 10 mm longitudinal cut in the skin above the medial aspect, and further delicate cleaning of the bone surface was carried out. The tip of the fiber optic (1.5 mm diameter) was placed perpendicularly to the center of the exposed medial aspect of the tibia and power den- sity of 10 mW/cm2 was applied to the BM. The laser was applied for a duration of 100 sec (energy density 1.0 J/cm2). Left or right exposed tibias were chosen at random for LLLT application. In sham-operated infarcted rats that served as control the tibias were exposed and the fi- ber optic was placed as described above but the laser beam was not turned on.

2.3. Histology and Electron Microscopy

A defined cross-section sample (2 mm thick) from the central part of the infarcted area was taken from all hearts for histology. Eight micron paraffin sections were pre- pared from the tissue samples of each heart. Infarct size was determined using Masson’s trichrome staining as described by us previously [23]. Three observers, blinded to control or laser-treated rats, analyzed infarct size. Six microscopic slides from the infarcted area of each heart were chosen at random for determination of infarct size. Infarct size was expressed as the percentage of the total infarcted area relative to the total area of the left ventri- cle (LV) in each section, using image analysis software Sigma Scan Pro (Sigma, St. Louis, MO).

For electron microscopy three tissue samples from each of the control and laser-irradiated rat hearts were taken from the interphase zone between the infarcted and non-infarcted tissue by macroscopic examination. Fixa- tion was performed in 3.5% glutaraldehyde in 0.1 M ca- codylate buffer for 24 hrs followed by embedment in Epon-812. Semi-thin sections (1 micron) were prepared in order to localize the interphase zone. Thin sections were then prepared and stained with uranyl acetate and lead citrate followed by examination with a Jeol electron microscope.

2.4. Immunohistochemistry

The total number of cells immunostained for desmin (bone marrow cells or newly formed) in the infarcted area were determined using a desmin kit (Zytomed Laboratory, Ber- lin, Germany). The procedure was performed at room temperature with anti-mouse (dilution 1:25 - 1:50) primary antibody for 60 min. Following washing, slides were in- cubated with HRP secondary antibody for mouse for 30 min followed by DAB Chromogen system (Covance Inc., Dedham). Slides were rinsed again in wash buffer, stain- ed in Hematoxylin for nuclei detection, mounted and viewed using a Zeiss microscope equipped with a camera and video screen. The total number of desmin immuno- stained cells within the infarcted area was counted and their density expressed as the percentage of the total area of the infarct using SigmaPro software.

2.5. Flow Cytometry Analysis

Blood samples were taken 2 and 7 days post-IR injury for fluorescence-activated cell sorting (FACS) analysis. 100 μl of blood were mixed with different antibodies: anti-mouse CD117 (c-kit) PE (eBioscience San Diego, USA) and rat IgG2b isotype control PE (eBioscience San Diego, USA) and anti-rat macrophage marker PE (eBio- science San Diego, USA) and mouse IgG2a K isotype control PE (eBioscience San Diego, USA), were used for the FACS analysis according to the manufacturer’s guide- lines. Forty five min post incubation of the whole fresh blood with the relevant antibodies, 2 ml of Fix/Lyse so- lution (eBioscience, San Diego, USA) was added. After mixture the suspended cells were left for 60 min in the dark at room temperature. Centrifugation was performed for 10 min, supernatant was removed and washing of the pellet was performed with 2 ml of Flow Cytometry Stain- ing Buffer Solution (eBioscience, San Diego, California, USA). After another centrifugation for 10 minutes the supernatant was decanted. The pellet containing mono- nucleated cells was resuspended in 200 μl of flow stain buffer for FACS analysis.

2.6. Statistical Analysis

The SigmaStat 2.0 (Sigma, St. Luis, USA) software was used for statistical analysis. Tests were performed first for normality distribution, followed by parametric (stu- dent’s t-test) test.

3. RESULTS

Application of LLLT to the infarcted heart caused a sig- nificant (p = 0.049) reduction of 55% in infarct size as compared to control. The present of macrophages and c- kit positive cells in the blood was determined by FACS analysis (Figure 1). It was found that at 5 days post MI there was a statistical significant 2-fold higher concentra- tion of macrophages and significant 1.4-fold higher c-kit positive cells (mesenchymal cells) in the laser treated rats as compared to the infarcted non laser treated rats. Des- min immunostaining of histological sections of the in- farcted zone from laser-treated rats demonstrated a higher density of positively stained cells than in the non laser-treated ones (Figures 2-4). In the interphase zone, cells extending from the myocardium towards the in 

Figure 1. Percent (out of total mononucleated cells) of macro- phages and c-kit positive cells in blood of control and laser treated rats (to the bone marrow) 5 days post MI as revealed by FACS analysis. The results are mean ± S.E.M of 15 rats at each group. Statistical significance *p < 0.05; **p < 0.01.

Figure 2. Representative desmin immunostained light micro- graphs of the infarcted zone of non-laser-treated rats (a, c) and laser-treated rats (to the bone marrow at 4 and 48 hrs and 5 days) (b, d) taken 2 weeks post-MI. Note that the zone in the control non-laser-treated rats contains mainly collageneous mate- rial with a few desmin immunopositive cells in the infarcted area (a, c); while in the laser-treated rats the zone displays posi- tive desmin staining in extended outgrowths (arrow) from the myocardium (MC) in (b), and in the cytoplasm of many cells in the infarcted area in (d). IF, Infarcted area. Bar = 50 μm.

farcted area showed higher immunostaining for desmin in the laser-treated rat hearts as compared to the control non-treated ones (Figure 2). The cell density of desmin immune-positive cells was also determined quantitatively in histological sections of both the infarcted laser-treated rats and infarcted non-laser-treated rats. The cell density was significantly (p < 0.01) 3-fold higher in the infarcted area of the laser-treated rats as compared to the non-la- ser-treated controls (Figure 4).

The electron micrographs of all samples taken from the control non-laser-treated infarcted rat hearts revealed a typical interphase area between intact and infarcted heart (Figure 5(a)). Adjacent to the non-ischemic intact myocardium there were conspicuous fibroblasts with col- lagen deposition dispersed among them (Figure 5(a)). In all samples taken from the laser-irradiated hearts the in- terphase zone between intact and infarcted area demon- strated different characteristics to those of the non-laser- treated infarcted rat hearts. Cells with newly-formed or- ganized contractile myofilaments dispersed in the cyto- plasm were detected in groups of several cells (Figure 5(b)). In these cells numerous mitochondria, clusters of ribosomes, and conspicuous clusters of contractile pro- teins were evident in the cytoplasm (Figures 6-8). Some cells contained dispersed contractile myofilaments in the cytoplasm that were still in an early stage of organization (Figure 6). The organization of newly-formed contractile myofilaments in the cytoplasm was observed in various

Figure 3. Representative desmin immunostained light micro- graphs of the interphase of the infarcted zone of laser-treated rats. Note that desmin positively stained cross-sections of myo- fibers (arrows) intermingled in the infarcted zone in (a). In (b) immunopositively stained cross-sections of myofibers (arrow) are visible in the infarcted area (IF). In (c) newly-formed car- diomyocytes (NC) are seen, with the desmin immunostaining mainly confined to the Z-line. Bar = 50 μm.

Figure 4. Density of desmin positively stained area (relative to total area) in the infarcted areas of control (non-laser-treated) and laser-treated (to the bone marrow) rats at 14 days post-MI. Results are mean+ S.E.M from 6 - 8 rats in each group. **p < 0.01.

Figure 5. Electron micrographs of typical interphase zone be- tween myocardium and infarcted area of control non-laser- treated (a) and laser-treated (b) to bone marrow rats. Note intact myocardium (MY) and adjacent fibroblast (FB) in the infarcted area surrounded by collagen (CL) deposition in (a). In (b) sev- eral newly-formed cardiomyocytes (marked with asterix) with conspicuous well-organized myofilaments (MF) in their cyto- plasm are evident adjacent to blood capillaries (CA). EN, En- dothelial cell.

degrees of maturation in those cells. In some cells the myofilaments were dispersed in the cytoplasm and in others they were organized in clusters anchored to well- developed Z-lines (Figure 7(a)). In certain cells the myo- filaments were organized parallel to the longitudinal di- rection of the cells, resembling the morphological char- acteristics of mature intact cardiomyocytes (Figure 7(b)). Some of the cells were also seen in a process of forma- tion of typical intercalated disc between them (Figure 9).

4. DISCUSSION AND CONCLUSION

The most significant outcome of this study was the ap- pearance of newly-formed cardiomyocytes following laser treatment to the BM, as indicated by light and electron microscopy. There was a 3-fold increase in the density of

Figure 6. Electron micrographs of most probably newly-formed cardiomyocytes at an early stage of organization of contractile myofilaments. Note myofilaments (MF) in the cytoplasm. M, Mitochondrion. Bar = 1 μm.

Figure 7. Electron micrographs of most probably newly-formed cardiomyocytes with early (a) and late (b) stages of the organi- zation of the contractile myofilaments in the cytoplasm. Note contractile myofilaments that are dispersed (DMF) in the cyto- plasm with a few organized in clusters anchored to Z-lines (Z) in (a). In (b) myofilaments (MF) are organized in parallel to the longitudinal axis of the cardiomyocyte, resembling their orga- nization in mature cardiomyocyte. N, Nucleus. Bar = 1 μm.

desmin immunostained cells in the infarcted rat hearts that had been laser treated. Desmin is a protein found in the cytoplasm of developing myocytes and cardiomyo- cytes [32]. The significantly higher occurrence of des- min-positive cells in the infarcted area of the laser- treated hearts may indicate the synthesis of new contrac- tile proteins in the developing new cardiomyocytes, re- sembling the process that takes place during embryonic development. The ultrastructural features of the cells in the interphase between the intact myocardium and the

Figure 8. Electron micrographs of typical interphase zone be- tween myocardium and infarcted area of laser-treated infarcted rat heart. Note numerous mitochondria (M) in the cytoplasm of the cardiomyocytes in (a) and (b). Also note organized contrac- tile myofilament with well-developed Z-lines (Z), some dis- persed myofilaments and clusters of ribosomes (R). Bar = 1 μm.

Figure 9. Electron micrographs of typical intercalated disk formation in the interphase region of the infarcted heart of la- ser-treated rats. Formation of intercalated disks (ID) between cells (marked with asterix) is evident. Note that the most proba- bly newly-formed cardiomyocytes contain clusters of myofila- ments (MF) in the cytoplasm that are conspicuous in their obli- que or cross-sections (arrows). Bar = 1 μm.

infarcted myocardium of the laser-treated rats, as shown in this study, clearly resemble the characteristics of car- diomyocytes during embryonic development of the heart [33]. Furthermore, the clusters of ribosomes and the nu- merous clusters of mitochondria in the cytoplasm of these cells may characterize cells that are active in the synthe- sis of proteins. It was previously demonstrated that direct LLLT to the infarcted hearts of rats, dogs and pigs caus- ed a significant reduction of scarring post-MI [23,24]. It was suggested that part of this reduction could be ex- plained by the regenerative response that takes place in the interphase zone [24].

The results of the present study indicate that the LLLT

applied to autologous BM attenuates the concentration of macrophages and MSC in the circulating blood. We have previously shown that LLLT application to the BM of infarcted rats caused a 2 fold enhancement in the rate of proliferation of MSC in the BM [30]. Those cells that most probably leave the BM to the circulating blood in- deed show a significant elevation of their concentration (as reveled by the FACS analysis in the present paper) at 5 days post MI. Consequently these cells probably home in on the infarcted heart, and even migrate specifically to the infarcted area [30]. These cells may induce cardiac stem cells to differentiate to newly-formed cardiomyo- cytes, as suggested previously by Hatzistergos et al. [14]. Indeed, it was found that endogenous c-kit+ cardiac stem cells were increased by 20-fold in the rat infarcted heart compared to control, following transcardial injection of BM-derived MSCs [14]. Such induction may be enabled due to paracrine secretion of various growth factors by the laser-stimulated MSC that originated from the BM. The possibility that paracrine secretion occurs in im- planted stem cells during cell therapy to the heart post- MI has been suggested previously [34]. Another mecha- nism that may take place after homing of stem cells to the infarcted heart of the laser-stimulated rats is that these cells continue to proliferate in the appropriate mi-lieu of the interphase zone in the infarcted heart and then differentiate to cardiomyocytes [30].

Another possible mechanism that maybe associates with the reduction of infarct size is the significant increase in the concentration of macrophages in the circulation fol- lowing LLLT to the BM as revealed from the FACS analysis in the present study. These findings corroborate with studies indicating that macrophages activity in the infarcted area at early stages post MI cause reduction of scarring post MI [35,36]. Thus, it could be postulated that more macrophages that will eventually home in the infarcted area from the circulating blood in the laser treated rats will also contribute to the reduction of scar- ring.

Although the findings of the present study do not in- dicate the extent of regenerative capacity of the rat in- farcted heart post-laser-irradiation, they do reveal a shift from practically no cardiomyocytes in the tissue samples taken from the non-laser-treated hearts, to the presence of newly-formed cardiomyocytes in all the electron mi- croscope sections taken from the hearts of rats that are laser-treated to the BM.

In conclusion, to the best of our knowledge, this is the first study to demonstrate the appearance of newly-form- ed cardiomyocytes in the infarcted area following LLLT to autologous BM in the infarcted rat heart. The mecha- nisms associated with this phenomenon remain to be elu- cidated in further studies.

5. ACKNOWLEDGEMENTS

This study was partially supported by the Elizabeth and Nicholas Shle- zak Super-center for Cardiac Research and Medical Engineering. The authors wish to acknowledge N. Paz for editing the manuscript and V. Wexler for helping with preparation of the figures.

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 Abstract: The long term effects of low level laser therapy can involve treatment mechanisms connected with activation of stem cells.

In the current study migration of stem cells was tested under the influence of laser light alone as well as in case of combined influence of light and stromal cell-derived factor-1α (SDF-1α). This cytokine plays a role in lymphocyte trafficking, hematopoietic progenitor cell and stem cell homing.

To investigate the light influence on stem cells, we analyzed factor-dependent cell-Patersen (FDCP)-mix multipotent progenitor cells.

Migration of the stem cell line was tested using Transwell system (Corning, NY) under influence of red diode laser (λ=659.6 nm, 19.5 mW) or infrared diode laser (λ=958 nm, 36 mW) during 15 min at continuous wave, as well as in case of applying 150 ng/ml SDF-1α.

Group 1 cells were a group of control, group 2 cells received only red light irradiation, while group 3 cells had IR light irradiation. Group 4 cells were treated with 150 ng/ml SDF-1α. Group 5 cells were irradiated with red laser light in addition to 150 ng/ml SDF-1α, and group 6 cells by IR light and 150 ng/ml SDF-1α.

The count of migrated cells was 1496,5±409 (100%) in case of control. Red and IR laser light increased migration activity of stem cells up to 1892±283 (126%) and 2255,5±510 (151%) accordingly. Influence of SDF-1α was more significant, than effects of light irradiation alone 3365,5±489 (225%). Combined effects of light irradiation and SDF-1α were significantly stronger 5813±1199 (388%) for SDF-1α and red laser light, and 6391,5±540 (427%) for SDF-1α and IR laser light irradiation.

Preliminary study results showed that laser light irradiation can activate stem cell migration in vitro. The results are more reliable in the case of combined application of light and SDF-1α. These results are giving ground to consider that stem cell reactions to light irradiation can be one of the factors of light therapy.

Key words: low level laser irradiation, low level laser therapy, stem cells, SDF-1, stromal cell-derived factor-1

INTRODUCTION

More than 30 years ago first reports about biological effects of low doses of laser light were presented. Currently low level laser therapy (LLLT) is successfully applied in the treatment of numerous diseases and pathological conditions. LLLT exhibits positive effects for the treatment of disorders, having in common failure of blood supply with development of acute or chronic tissue hypoxia, different level of destruction of tissues, following decreased regenerative abilities of tissues and organs, defects in immune system, and altered cell metabolism. At the same time some important mechanisms of influence of laser light on the body are still far to be fully understood [1 - 8].

Recent studies discovered important role of bone marrow hematopoetic stem cell (HSCs) for naturally occurred recovery and regeneration processes, following tissue hypoxia and injury. The three clinically important steps in this natural process are mobilization of stem cells from the bone marrow, homing of these cells to the site of injury, and differentiation of the stem cell into a functional cell of the injured tissue [9]. Different methods of stem cell therapy, the treatment method, based on mobilization and transplantation of stem cells, proves to be effective method of therapy for different disorders.

We proposed a hypothesis that wide range of positive effects following laser therapy can be connected to increased activity of stem cells in damaged tissues. To test that, we examined in vitro the influence of laser light on migration of stem cells in absence and in presence of stromal cell-derived factor-1 (SDF-1), a potent chemoattractor for lymphocytes, monocytes, HSCs, which plays a critical role in the stem cell migration towards areas of tissue injury and hypoxia.

MATERIALS AND METHODS

To investigate the light influence on stem cells, we analyzed factor-dependent cell-Patersen (FDCP)-mix multipotent progenitor cells. The FDCP-mix stem cell line was maintained in ISCOVE’S medium supplemented with 20% horse serum and penicillin/streptomycin in the presence of 20 ng/ml IL-3. The cells were supplied with fresh medium each 5 days. Migration of the stem cell line was tested using Transwell system (Corning, NY). The cells were washed with PBS once and re-suspended in the medium containing 0.1% BSA (2x106/ml). Then, 600 μl of the mixture was irradiated by red diode laser (λ=659.6 nm, 19.5 mW) or infrared diode laser (λ=958 nm, 36 mW) during 15 min at continuous wave. Next, 100 μl of the mixture (2x105 cells) was seeded into upper chambers of the Transwell system, and the filters were placed into the wells containing 600? μl of the medium with or without 150 ng/ml SDF-1α. The plate was incubated for 4 h (37°C, 5% CO2, humidified atmosphere), after which the cells were collected and counted by a FACS sorter (Beckton Dickinson) during 1 min. All samples were performed in duplicate.

Group 1 cells are control group, group 2 cells received only red light irradiation, while group 3 cells – only IR light irradiation. Group 4 cells were treated with 150 ng/ml SDF-1α. Group 5 cells were irradiated with red laser light in addition to 150 ng/ml SDF-1α, and group 6 cells – IR light and 150 ng/ml SDF-1α.

RESULTS

Small amount of stem cells can migrate without SDF-1α or laser light influence. The count of migrated cells in control group was 1496,5±409 (Fig). This amount was considered as 100%. Red and IR laser light at the above mentioned dosage and methods of irradiation increased migration activity of stem cells up to 1892±283 (126%) and 2255,5±510 (151%) accordingly. Influence of SDF-1α was more noticeable, than effects of red or IR laser light irradiation alone - 3365,5±489 (225%). It is important to stress attention on the finding, that rate of stem cell migration towards the filter and SDF-1α containing medium was much higher after laser irradiation of cells - 5813±1199 (388%) for red laser light, and 6391,5±540 (427%) for IR laser light irradiation.

DISCUSSION

The main scientific result of this study is the fact, that red and infrared laser light irradiation can activate migration of stem cells in vitro. Moreover, red and IR laser radiation can up-regulate the rate of stem cell migration towards higher SDF-1α gradient.

How to explain the direct effects of mobility of stem cells in vitro under red and IR laser light irradiation, and use this fact for better understanding the wide range of therapeutic effects of laser therapy?

Modern medical science has accepted that every pathologic condition or disease should be treated according to its clinical stage and symptoms, considering its pathogenesis and etiology. Similar treatment methods can be applied only for the treatment of different diseases, having common pathogenesis.

Not very many examples of successful application of the similar or close therapy method for the treatment of different pathologies are known in modern medicine. Steroid hormone therapy is one of such cases.

Another illustration of successful application of the similar treatment techniques for treatment of different disorders is stem cell therapy, a novel treatment method, which is still under development. Growing data suggests, that transplanted stem cell can successfully and for long period of time improve heart myocardial contractility and other heart functions after myocardial infarction, can support neoangiogenesis in areas of tissue infarction and damage, can replace several cell types in tissues, including β-cells in diabetes models, neurons, cardiomyocytes, hematopoetic cells of different lineages and so on, as well as be useful in the treatment of atherosclerosis [9].

The main principle of stem cell therapy is the idea of replacement of damaged and dead cells in injured tissues and organs with new healthy ones. It is known, that severe stress, tissue hypoxia and damage mobilizes some hematopoetic stem cells (HSCs) from bone marrow to peripheral bloodstream. After that HSCs can migrate towards hypoxic tissues and reach them. Finally they can start to proliferate to the cells types, typical for that damaged tissues. HSCs in the tissues are also able to produce several cytokines, chemokines, growthfactors, improve survival of damaged cells and limit apoptosis. As a result of some tissue regeneration, improvement in the function of a damaged organ can be achieved. Similar and even stronger regeneration and treatment effects can be displayed after transplantation of fetal or adult HSCs to recipient [10-12].

Low laser light irradiation is one other example of application of the same factor for the treatment of number of disorders, which, at first glance, have nothing or very little in common in their pathogenesis. Laser light can accelerate wound and burn healing, improve condition of patients after myocardial infarction and stroke, can support hematopoiesis of bone marrow after X-ray radiation or during cancer chemotherapy, can help for the treatment of diabetic angiopathy and neuropathy, as well as reduce atherosclerotic plaque formation. In cellular and tissue level LLLT exhibits positive effects for the treatment of disorders, having in common failure of blood supply with development of acute or chronic tissue hypoxia, different level of destruction of tissues, following with decreased regenerative abilities of cells, as well as altered cell metabolism [6, 7, 13, 14].

One can see that the therapeutic applications of LLLT and stem cell therapy are very close. So, earlier we proposed the hypotheses that one of the mechanisms of light therapy includes acceleration of tissue repair due to better mobilization of stem cells to the spot of injury after laser light irradiation [15]. That process should include several phases, including activation of stem cell migration towards area of tissue damage and hypoxia.

Stem cells are being investigated for their potential use in regenerative medicine. Stem cells share the following two defining characteristics: the capacity to differentiate into a spectrum of different cell types and the capacity to renew themselves [16]. The biological principle that underlies stem cell therapy is tissue-directed differentiation. For example, adult stem cells isolated from liver tissue and re-injected into liver become hepatocytes, whereas the same cells injected into myocardium become myocytes. [17] Stem cells have been engrafted into a broad spectrum of tissues, including regenerating bone, neural tissue, dystrophic skeletal muscle, and injured skeletal muscle. [18]. Myocardial regeneration is perhaps the most widely studied and debated example of stem cell plasticity. The most promising results have been obtained after transplantation and mobilization of bone marrow cells to the area of infarction.

The three clinically important steps in this natural process are mobilization of stem cells from the bone marrow, homing of these cells to the site of injury, and differentiation of the stem cell into a functional cell of the injured tissue [19].

Stem cell repair of cardiac and vascular tissue is a naturally occurring process after injury [20, 21] Circulating CD34+ mononuclear cell counts and plasma levels of endothelial growth factor are significantly increased in patients with acute myocardial infarction, peaking on day 7 after onset [22]. Due to limitations of the naturally occurring repair process after myocardium infarction and other injuries or pathologies several stem cell transplantation strategies were proposed and tested.

At present, however, enthusiasm for the therapeutic potential of strategies of stem cell transplantation is limited by certain practical considerations. For example, the number of stem cells, required for injection for the treatment of myocardial infarction, can be harvested approximately from 6 l of donor blood [23].

Other important limitation for autologous bone marrow stem/progenitor cell mobilization is a recent finding, that circulating endothelial progenitor cells in patients with coronary heart disease are impaired with respect to number and functional activity. Moreover, Heeschen et al [24] reported that regeneration and functional ability of bone marrow-derived mononuclear cells (BM-MNCs) in patients with chronic ischemic cardiomyopathy (ICMP) are also limited. In spite of the fact that, the number of BM-MNCs isolated from bone marrow aspirates of 18 patients with ICMP and 8 healthy subjects s did not differ, the colony-forming capacity of BM-MNCs from patients with ICMP was significantly lower compared with BM-MNCs from healthy controls. Likewise, the migratory response to SDF-1 and vascular endothelial growth factor (VEGF) was significantly reduced in BM-MNCs derived from patients with ICMP compared with BM-MNCs from healthy controls. The reduced neovascularization capacity in vivo of BM-MNCs derived from patients with ICMP closely correlated with the in vitro assessment of SDF-1-induced migration and colony-forming capacity.

The need for development of new methods for mobilization, as well as for homing of stem cells to the site of injury is therefore evident.

Several growth factors, chemokines and cytokines are involved in the regulation of stem cell mobilization, homing and differentiation. Stromal cell-derived factor-1 (SDF-1) is one of them. SDF-1 is a chemokine playing an important role in the trafficking of hematopoietic stem cells. SDF-1 is expressed on stromal cells of various tissues. CXCR4 is the only known receptor for SDF-1 [25]. SDF-1/CXCR4 interaction is reported to play an important physiological role during embryogenesis in hematopoiesis, vascular development, cardiogenesis, and cerebellar development [26-28].

Recently, several investigators have reported that CD34+ cells, classically considered to be hematopoietic stem cells, expressed CXCR4, and that SDF-1 could induce CD34+ cell migration in vitro [29]. Accordingly, SDF-1 is considered as one of the key regulators of hematopoietic stem cell trafficking between the peripheral circulation and bone marrow. SDF-1 has also been shown to effect CD34+ cell proliferation and mobilization and to induce angiogenesis in vivo [30 -32].

Hattori et al [31] reported that plasma elevation of SDF-1 induced mobilization of mature and immature hematopoietic progenitors and stem cells, including endothelial progenitor cells (EPCs). However, application of granulocyte colony-stimulating factor (G-CSF) for stem cell mobilization is widely accepted nowadays.

Yamaguchi et al [23] studied the effects of SDF-1 on migration and accumulation of EPCs. SDF-1 induced EPCs migration in a dose dependent manner in vitro. The magnitude of migration was similar to that induced by VEGF. Authors also reported that locally (in hind-limb ischemic muscle of experimental animals) administered SDF-1 could augment the local accumulation of transplanted EPCs from peripheral blood, thereby resulting in enhanced neovascularization. As a result, cell transplantation not only improved neovascularization but also reduced adverse biological consequences such as limb necrosis and auto-amputation in the mouse ischemic hind-limb model. These studies also disclosed that systemic EPCs transplantation improved myocardial neovascularization and cardiac function corresponding to reduced left ventricular scarring. Authors concluded that, at least under the experimental conditions used in the study, the effect of SDF-1 on neovascularization appears to result primarily from its ability to enhance the recruitment and incorporation of transplanted EPCs.

Damas at al. [33] reported that SDF-1α, at least in high concentrations, may mediate anti-inflammatory and matrix-stabilizing effects in unstable angina. These effects may promote plaque stabilization, and therapeutic intervention that enhances SDF-1 α activity could potentially be beneficial in acute coronary syndromes. Authors demonstrated significantly altered SDF-1/CXCR4 expression in patients with angina, with particularly marked changes in those with unstable disease, with low SDF-1 levels in plasma and altered expression of its corresponding receptor on peripheral blood mononuclear cells (PBMC). In contrast to the raised plasma levels of inflammatory chemokines in patients with angina plasma levels of SDF-1 and the surface expression of its corresponding receptor (CXCR4) on PBMC appear to be down-regulated in these patients. Thus, although persistent inflammation may involve up-regulation of inflammatory chemokines, recent studies suggest that inflammatory cytokines (eg, TNF-α and IL-1) may decrease the expression of SDF-1 and CXCR4.

Future progress of stem therapy techniques probably will include development of incubation methods for enhancement stem cell mobility and homing ability, as well as for faster proliferation into desire tissue cells. Increasing migration abilities will help to achieve better and faster results.

The ability of laser light to activate migration and mobility of different cells is well known. It was noticed, that irradiation of sperm cells in vitro can increase their mobility and fertility [34]. Moreover, this effect is more pronounced in case of damaged cells with low mobility rate. This gives a ground to assume that laser light irradiation in certain dosage and condition can improve functional abilities of cells. Future experiments are required to ascertain if stem cells respond to the laser light the same way.

The main finding on this study is that red and IR laser light can stimulate stem cell migration in vitro, and especially increase migration towards SDF-1α gradient. Stem cell ability to migrate towards tissues with higher SDF-1 concentration is one of the key mechanisms of stem cell homing. These results are giving ground to speculate that activation of stem cell migration can be one of the mechanisms of low level laser therapy. Taking into consideration that the combined of SDF-1 and laser irradiation had the strongest effect on stem cell homing, it would be reasonable to assume that this combination could be used in not only increasing the activity of stem cells but also in determining the main area of stem cell mobilization and homing. The current study did not aim to study the mechanisms of increased migration ability, which will be study in the future. But it is possible to suggest following explanation: laser irradiation can change the metabolism of stem cells, increase ATP production and so increase the migration, as well as up-regulate CXCR4 receptor expression or syntheses de novo. More studies are required to test if the laser light irradiation in vivo is able to make homing of transplanted stem cells to the area of damage more efficient, to check the influence of laser light on the mobilization rate of stem cells from bone marrow, to investigate if laser light can enhance functional abilities of stem cells. These studies would be desirable for better understanding of the mechanisms of laser therapy and for development of more effective methods of stem cell therapy.

References

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3. Baxter G.D. Therapeutic Lasers: Theory and Practice, Churchill Livingstone, London, 1994.

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10. Orlic D., Hill J., Arai A. Stem Cells for Myocardial Regeneration Circulation Research. 2002;91:1092.

11. Hodgson D., Behfar A., Zingman L.V., Kane G.C., Perez-Terzic C., Alekseev A.E., Puceat M., and Terzic A. Stable benefit of embryonic stem cell therapy in myocardial infarction. Am J Physiol Heart Circ Physiol, August 1, 2004; 287(2): H471 - H479.

12. Ozbaran M., Omay S. B., Nalbantgil S., Kultursay H., Kumanlioglu K., Nart D., and Pektok E. Autologous peripheral stem cell transplantation in patients with congestive heart failure due to ischemic heart disease. Eur. J. Cardiothorac. Surg., March 1, 2004; 25(3): 342 - 350.

13. Brill A.G., Shenkman B., Brill G.E. et al. Blood irradiation by He-Ne laser induces a decrease in platelet responses to physiological agonists and an increase in platelet cyclic GMP. Platelets. 2000. Vol. 11. P. 87-93.

14. Mester A. Biostimulative effect in wound healing by continuous wave 820 nm laser diode. Lasers in Med Science, abstract issue July 1988, No. 289.

15. Gasparyan L.V. Stem cells and therapeutic effect of light irradiation (in Russian). Collection of abstracts of the 10th International Conference of Quantum Medicine, Moscow, 2003, pp. 43-44.

16. Graf T. Differentiation plasticity of hematopoietic cells. Blood. 2002;99:3089–3101.

17. Malouf NN, Coleman WB, Girsham JW, et al. Adult-derived stem cells from the liver become myocytes in the heart in vivo. Am J Pathol. 2001;158:1929–1935.

18. Donovan PJ, Gearhart J. The end of the beginning for pluripotent stem cells. Nature. 2001;414:92–97.

19. Forrester J, Price M, Makkar R. Stem Cell Repair of Infarcted Myocardium. An Overview for Clinicians. Circulation. 2003;108:1139–1145.

20. Beltrami AP, Urbanek K, Kajstura J, et al. Evidence that human cardiac myocytes divide after myocardial infarction. N Engl J Med. 2001;344:1750–1757.

21. Gill M, Dias S, Hattori K, et al. Vascular trauma induces rapid but transient mobilization of VEGFR2(+)AC133(+) endothelial precursor cells. Circ Res. 2001;88:167–174.

22. Shintani S, Murohara T, Ikeda H, et al. Mobilization of endothelial progenitor cells in patients with acute myocardial infarction. Circulation. 2001;103:2776–2779.

23. Yamaguchi J, Kusano K, Masuo O, at al. Stromal Cell–Derived Factor-1 Effects on Ex Vivo Expanded Endothelial Progenitor Cell Recruitment for Ischemic Neovascularization. Circulation. 2003;107: 1322–1328.

24. Heeschen C, Lehmann R, Honold J, Assmus B, Aicher A, Walter DH, Martin H, Zeiher AM, Dimmeler S. Profoundly reduced neovascularization capacity of bone marrow mononuclear cells derived from patients with chronic ischemic heart disease. Circulation. 2004;109(13):1615-22.

25. Bleul CC, Farzan M, Choe H, et al. The lymphocyte chemoattractant SDF-1 is a ligand for LESTR/fusin and blocks HIV-1 entry. Nature. 1996;382:829–833.

26. Nagasawa T, Hirota S, Tachibana K, et al. Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1. Nature. 1996;382:635–638.

27. Tachibana K, Hirota S, Iizasa H, et al. The chemokine receptor CXCR4 is essential for vascularization of the gastrointestinal tract. Nature. 1998; 393:591–594.

28. Zou YR, Kottmann AH, Kuroda M, et al. Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development. Nature. 1998;393:595–599.

29. Mohle R, Bautz F, Rafii S, et al. The chemokine receptor CXCR-4 is expressed on CD34+ hematopoietic progenitors and leukemic cells and mediates transendothelial migration induced by stromal cell-derived factor-1. Blood. 1998;91:4523–4530.

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32. Salcedo R, Wasserman K, Young HA, et al. Vascular endothelial growth factor and basic fibroblast growth factor induce expression of CXCR4 on human endothelial cells: in vivo neovascularization induced by stromal derived factor-1α. Am J Pathol. 1999;154:1125–1135.

33. Damas J, Wæhre T, Yndestad A, et al. Stromal Cell–Derived Factor-1a in Unstable Angina. Circulation. 2002;106:36-42.

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There is a notable lack of therapeutic alternatives for what is fast becoming a global epidemic of traumatic brain injury (TBI). Photobiomodulation (PBM) employs red or near-infrared (NIR) light (600-1100nm) to stimulate healing, protect tissue from dying, increase mitochondrial function, improve blood flow and tissue oxygenation. PBM can also act to reduce swelling, increase antioxidants, decrease inflammation, protect against apoptosis, and modulate microglial activation state. All these mechanisms of action strongly suggest that PBM delivered to the head should be beneficial in cases of both acute and chronic TBI. Most reports have used NIR light either from lasers or from light-emitting diodes (LEDs). Many studies in small animal models of acute TBI have found positive effects on neurological function, learning and memory, and reduced inflammation and cell death, in the brain. There is evidence that PBM can help the brain to repair itself by stimulating neurogenesis, upregulating BDNF synthesis, and encouraging synaptogenesis. In healthy human volunteers (including students and healthy elderly women) PBM has been shown to increase regional cerebral blood flow, tissue oxygenation and improve memory, mood and cognitive function. Clinical studies have been conducted in patients suffering from the chronic effects of TBI. There have been reports of improvements in executive function, working memory, and improved sleep. Functional magnetic resonance imaging has shown modulation of activation in intrinsic brain networks likely to be damaged in TBI (default mode network and salience network).

Keywords: photobiomodulation, low-level laser therapy, traumatic brain injury, stroke, chromophores, animal studies, clinical trials, human studies

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1. Introduction

Photobiomodulation (PBM) formerly known as low-level laser (light) therapy (LLLT) is approaching its 50th anniversary, after being discovered by Endre Mester working in Hungary in 1967 (Hamblin et al. 2016). Originally thought to be a property of red lasers (600-700 nm), PBM has broadened to include near-infrared (NIR) wavelengths 760-1200 nm, and even blue and green wavelengths. Moreover the advent of inexpensive and safe light emitting diodes (LEDs) has supplanted the use of expensive lasers in many indications. The better tissue penetration properties of NIR light, together with its good efficacy, has made it the most popular wavelength range overall. The best-known medical applications of PBM have been for indications such as stimulation of wound healing (Hopkins et al. 2004; Kovacs et al. 1974), reduction of pain and inflammation in orthopedic and musculoskeletal conditions (Aimbire et al. 2006; Gam et al. 1993), and mitigation of cancer therapy side-effects (Zecha et al. 2016a; Zecha et al. 2016b). However in recent years there has been growing interest in the use of PBM in various brain disorders (Hamblin 2016b; Hennessy and Hamblin 2016; Naeser and Hamblin 2011; Naeser and Hamblin 2015). The almost complete lack of any adverse side-effects of PBM, coupled with growing disillusion with pharmaceutical drugs that affect brain function, have combined together to suggest an alternative physical therapy approach to improving brain function.

Traumatic brain injury (TBI) is caused by some type of trauma to the head, often resulting from road traffic accidents, assaults, falls, sports injuries, or blast injuries suffered in military conflict. TBI is classified as mild (loss of consciousness 0-30 minutes; altered mental state <24 hours; post-trauma amnesia <1 day); moderate (loss of consciousness 30 minutes to 24 hours; altered mental state >24 hours; post-trauma amnesia >1-7 days), or severe (loss of consciousness >24 hours; altered mental state >24 hours; post-trauma amnesia >7 days) (Blennow et al. 2016). There are three cases of TBI sustained each minute in the US (Faul et al. 2010). Repeated mild episodes of TBI (also known as concussions) even without loss of consciousness, may have devastating cumulative effects (Kamins and Giza 2016). Chronic traumatic encephalopathy is a recently recognized condition resulting from repeated head trauma, found in boxers, football players, and military personnel (McKee et al. 2016; Safinia et al. 2016). There is presently no accepted treatment for TBI, although some investigational approaches are being tested in both the acute (neuroprotection) and chronic (neurorehabilitation) settings (Loane and Faden 2010). One of these novel approaches is PBM or LLLT (Hamblin 2016a; Hamblin 2016b; Huang et al. 2012; Thunshelle and Hamblin 2016).

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2. Mechanisms of action

Uncertainties about the mechanism of action of PBM at the molecular and cellular levels, have undoubtedly held back its acceptance in the wider biomedical community. However in recent years substantial progress has been made in this regard (de Freitas and Hamblin 2016). In the following section the state-of-the-art knowledge about the mechanisms of PBM is summarized. Figure 1 shows a graphical representation of the cellular and molecular mechanisms of PBM.

Figure 1

Molecular mechanisms of tPBM

Light passes through the scalp and skull, where depending on the wavelength it is absorbed by two different chromophores. Red and NIR (up to 940nm) is primarily absorbed by cytochrome c oxidase in the mitochondrial respiratory chain of the cortical neurons. Longer wavelength NIR light (980nm, 1064nm) is primarily absorbed by heat and light-sensitive transient receptor potential ion channels. In both cases cell signaling and messenger molecules are upregulated as a result of stimulated mitochondrial activity, including reactive oxygen species (ROS), and adenosine triphosphate (ATP). hv is light, TRPV is transient receptor potential vanilloid (ion channels).

2.1 Chromophores

The first law of photobiology states that a photon must be absorbed by some molecule within the tissue to have any biological effect. The identity of these chromophores has been the subject of much scientific investigation and speculation. Largely due to the efforts of Tiina Karu in Russia, the enzyme cytochrome c oxidase (CCO) has been identified as a major chromophore of red/NIR light (Karu 1999; Karu and Kolyakov 2005; Karu et al. 2004a; Karu et al. 2004b). CCO is unit IV in the mitochondrial respiratory chain and has absorption peaks reaching well into the NIR spectral region (up to 900 nm) as well as in the red and blue regions. The most discussed hypothesis to explain exactly how photon absorption can stimulate the activity of CCO involves the photodissociation of inhibitory nitric oxide (NO) that can bind to the copper and heme centers in the enzyme and prevent oxygen from gaining access to the active sites (Lane 2006). In experimental models (such as isolated mitochondria) oxygen consumption and ATP production are increased, and the mitochondrial membrane potential is raised (Passarella et al. 1984).

A less well-appreciated mechanism involves light and heat-gated ion channels. These cation ion channels are thought to be members of the transient receptor potential (TRP) superfamily consisting of over 28 distinct members organized into six subfamilies, based on their primary amino acid structures (Caterina and Pang 2016). TRPV (vanilloid sub-family) members including TRPV1 (capsaicin receptor) have been shown to be activated by various wavelengths of light including green, red and NIR.

2.2 Cellular mechanisms

After the primary photon absorption event occurs, whether that the photons are absorbed by CCO, or by TRP ion channels a series of secondary events occurs. One of these events is the generation of reactive oxygen species (ROS), which are thought to be produced inside the mitochondria due to an increase in electron transport, and a rise in the mitochondrial membrane potential above the baseline levels (Suski et al. 2012). It should be noted that mitochondrial ROS can be produced when MMP is raised above normal, and also when ROS is reduced below normal. It is thought that the ROS produced when MMP is lowered (mitochondrial dysfunction) are more damaging than ROS produced when MMP is raised (mitochondrial stimulation). Nitric oxide is produced after PBM (Hamblin 2008), possibly by photodissociation from CCO where it inhibits oxygen consumption and electron transport (Lane 2006). Cyclic adenosine monophosphate (cAMP) (Gao and Xing 2009) and intracellular calcium are increased (Alexandratou et al. 2002). Many of these secondary mediators in the signaling pathways triggered by PBM, can induce activation of transcription factors, that go on to upregulate or downregulate expression levels of a large number of genes. One of the best-known transcription factors is NF-kB that can regulate expression of over one hundred genes including proteins with antioxidant, anti-apoptotic, pro-proliferation, and pro-migration functions. PBM (810 nm 3J/cm2) was shown to activate NF-kB in mouse embryonic fibroblasts via ROS production (Chen et al. 2011a). Since NF-kB is known to be a pro-inflammatory transcription factor, it might be thought that PBM would be pro-inflammatory. However it was shown that NF-KB was decreased in already activated (treated with Toll-like receptor ligands) inflammatory dendritic cells by PBM (810 nm 3J/cm2) (Chen et al. 2011b).

2.3 Tissue mechanisms

The changes in expression levels of proteins involved in antioxidant and redox-regulation, anti-apoptotic and pro-survival, cellular proliferation, etc mean that distinct changes in tissue homeostasis, healing and regeneration can be expected after PBM. For instance, structural proteins such as collagen are newly synthesized in order to repair tissue damage (Tatmatsu-Rocha et al. 2016). Cells at risk of dying in tissue that has been subjected to ischemic or other insults are protected (Sussai et al. 2010). Stem cells are activated to leave their niche, proliferate and differentiate (Oron and Oron 2016; Zhang et al. 2016). Pain and inflammation are reduced (Chow et al. 2009). Blood flow is increased (Samoilova et al. 2008) (possibly as a result of the release of NO (Mitchell and Mack 2013)), which also stimulates lymphatic drainage thereby reducing edema (Dirican et al. 2011).

2.4 Brain specific mechanisms

In addition to the foregoing, there are some PBM tissue mechanisms that are specific to the brain. One of the most important is an increase in cerebral blood flow often reported after transcranial photobiomodualtion (tPBM) (Salgado et al. 2015), leading to increased tissue oxygenation, and more oxidized CCO as measured by NIR spectroscopy (Rojas and Gonzalez-Lima 2013). tPBM has been shown to reduce activated microglia in the brains of TBI mice as measured by IBA1 (ionized calcium-binding adapter molecule-1) expression thus demonstrating reduced neuroinflammation (Khuman et al. 2012). tPBM has been shown to increase neurogenesis (formation of new brain cells derived from neuroprogenitor cells) (Xuan et al. 2014), and synaptogenesis (formation of new connections between existing brain cells) (Xuan et al. 2015) both in TBI mice. Figure 2 shows a graphical representation of a variety of these brain-specific tissue mechanisms.

Figure 2

Brain-specific mechanisms of tPBM

The gene transcription process described in Figure 1 can lead to decreases in neuronal apoptosis and excitotoxicity and lessening of inflammation and reduction of edema due to increased lymphatic flow, which together with protective factors such as antioxidants, will all help to reduce progressive brain damage. Increases in angiogenesis, expression of neurotrophins leading to activation of neural progenitor cells and more cell migration, and increased synaptogenesis may all contribute to the brain repairing itself from damage sustained in the trauma. AUC is area under the curve.

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3. Transcranial photobiomodulation

Transcranial PBM is a growing approach to many different brain disorders that may be classified as sudden onset (stroke, TBI, global ischemia), neurodegenerative (Alzheimer's, Parkinson's, dementia), or psychiatric (depression, anxiety, posttraumatic stress disorder)(Hamblin 2016b; Hennessy and Hamblin 2016; Thunshelle and Hamblin 2016). In the following section some issues concerning where the light should be delivered, and the effects of PBM on uninjured mice and humans are addressed.

3.1 Light penetration

Several laboratories working in the field of tissue optics, have investigated the penetration of light of different wavelengths though the scalp and the skull, and to what depths into the parenchyma of the brain this light can penetrate. Answering the question “can light shone on the head sufficiently penetrate to reach the brain?” is difficult. The main reason is that at present it is unclear exactly what threshold of power density is necessary (expressed in mW/cm2) at some depth inside the brain to have a biological effect. There clearly must be a minimum value below which, the light can be delivered for an infinite time without having any effect, but whether this threshold is in the region of μW/cm2 or mW/cm2 is unknown at present.

Haeussinger et al. estimated that the mean penetration depth (5% remaining intensity) of NIR light through the scalp and skull was 23.6 + 0:7 mm (Haeussinger et al. 2011). Other studies have found comparable results with some variations depending on the precise location on the head and the precise wavelength studied (Okada and Delpy 2003; Strangman et al. 2014).

Jagdeo et al. (Jagdeo et al. 2012) used human cadaver heads (skull with intact soft tissue) to measure penetration of 830 nm light, and found that penetration depended on the anatomical region of the skull (0.9% at the temporal region, 2.1% at the frontal region, and 11.7% at the occipital region). Tedord et al. (Tedford et al. 2015) also used human cadaver heads to compare penetration of 660 nm, 808 nm, and 940 nm light. They found that 808 nm light penetrated best, and could reach a depth in the brain of 40–50 mm. Lapchak et al. compared the transmission of 810 nm light through the skulls (no soft tissue) of four different species, and found the mouse skull transmitted 40%, while for rat it was 21%, for rabbit it was 11.3 and for the human skull it was only 4.2% (Lapchak et al. 2015). Pitzschke and colleagues compared penetration of 670 nm and 810 nm light into the brain when delivered by a transcranial or a transphenoidal approach, and found that the best combination was 810 nm delivered transphenoidally (Pitzschke et al. 2015). Yaroslavsky et al. examined light penetration of different wavelengths through different parts of the brain tissue (white brain matter, gray brain matter, cerebellum, and brainstem tissues, pons, thalamus). Best penetration was found with wavelengths between 1000 and 1100 nm (Yaroslavsky et al. 2002). Henderson and Morries found that between 0.45% and 2.90% of 810 nm or 980 nm light penetrated through 3 cm of scalp, skull and brain tissue in ex vivo lamb heads (Henderson and Morries 2015a).

3.2 Local vs systemic effects of light

It is possible that the beneficial effects of PBM on the brain cannot be entirely explained by penetration of light through the scalp and skull into the brain itself, at a sufficient intensity to have an effect on the brain cells. The surface power density that can be safely applied to the head, is limited by heating of the skin. Perceptible heating of the skin starts to be felt when the power density is over about 500 mW/cm2, and can become severe at 1 W/cm2.

There has been one study that explicitly addressed whether direct transcranial PBM or indirect PBM is best for the brain. In a study of PBM for Parkinson's disease in a mouse model, Mitrofanis and colleagues compared the direct delivery of light to the mouse head, and they also covered up the head with aluminum foil so that the light was delivered to the remainder of the mouse body. They found that there was a highly beneficial effect on brain histology with light delivered to the head, but nevertheless there was also a statistically significant although less pronounced benefit (referred to as an “abscopal effect”) when the head was shielded from light. Moreover Oron and co-workers (Farfara et al. 2015) have shown that delivering NIR light to the mouse tibia (using either surface illumination or a fiber optic) resulted in improvements in memory and spatial learning in a transgenic mouse model of Alzheimer's disease. They proposed the mechanism involved PBM stimulating c-kit-positive mesenchymal stem cells (MSCs) that were normally resident in autologous bone marrow. These MSCs were proposed to be able to infiltrate the brain, and clear β-amyloid plaques (Oron and Oron 2016). It should be noted in general that the calvarial bone marrow of the skull contains substantial numbers of stem cells (Iwashita et al. 2003).

3.3 PBM for brain in uninjured animals

Several laboratories have reported that shining light onto the head of uninjured healthy mice or rats can improve various cognitive and emotional parameters. The first study reported that exposure of the middle aged (12 months) CD1 female mice to 1072 nm LED arrays (Michalikova et al. 2008) produced improved performance in a 3D maze compared to sham treated age-matched controls. Gonzalez-Lima and coworkers (Gonzalez-Lima and Barrett 2014) showed that transcranial PBM (9 mW/cm2 with a 660 nm LED array) delivered to rats induced dose-dependent increases in oxygen consumption (5% after 1 J/cm2 and 16% after 5 J/cm2) [113]. They also found that tPBM reduced fear renewal and prevented the reemergence of the extinguished conditioned fear-responses (Rojas et al. 2012).

3.4 PBM for enhancement of brain function in uninjured human volunteers

Gonzalez-Lima et al delivered transcranial PBM (1064 nm laser, 60 J/cm2 at 250 mW/cm2) to the forehead in uninjured human volunteers in a placebo-controlled, randomized study. The goal was to improve performance of cognitive tasks related to the prefrontal cortex, including a psychomotor vigilance task (PVT), a delayed match-to-sample (DMS) memory task, and improved mood as measured by the positive and negative affect schedule (PANAS-X) (Barrett and Gonzalez-Lima 2013). Subsequent studies in uninjured humans showed that tPBM with 1064 nm laser could improve performance in the Wisconsin Card Sorting Task (considered the gold standard test for executive function) (Blanco et al. 2015). They also showed that tPBM to the right forehead (but not the left forehead) could improve attention bias modification (ABM) in humans with depression (Disner et al. 2016).

Salgado et al. applied transcranial LED to enhance cerebral blood flow in healthy elderly women, as measured by transcranial Doppler ultrasound (TCD) of the right and left middle cerebral artery and basilar artery. Twenty-five non-institutionalized elderly women (mean age 72 years), with cognitive status > 24, were assessed using TCD before and after transcranial LED therapy. tPBM (627 nm, 70 mW/cm2, 10 J/cm2) was performed at four points of the frontal and parietal region for 30 s each twice a week for 4 weeks. There was a significant increase in the systolic and diastolic velocity of the left middle cerebral artery (25 and 30%, respectively) and the basilar artery (up to 17 and 25%), as well as a decrease in the pulsatility index and resistance index values of the three cerebral arteries analyzed (Salgado et al. 2015).

3.5 PBM for acute stroke

Transcranial PBM delivered to the head, has been investigated as a possible treatment for acute stroke (Lapchak 2010). Animal models such as rats and rabbits, were first used as laboratory models, and these animals had experimental strokes induced by a variety of methods and were then treated with light (usually 810 nm laser) within 24 h of stroke onset (Lampl 2007). In these studies intervention by tLLLT within 24 h had meaningful beneficial effects.

Treatment of acute stroke in human patients was then addressed in a series of three clinical trials called “Neurothera Effectiveness and Safety Trials” (NEST-1 (Lampl et al. 2007), NEST-2 (Huisa et al. 2013), and NEST-3 (Zivin et al. 2014)). The protocol used an 810 nm laser applied to the shaved head (20 separate points in the 10/20 EEG system) within 24 h of patients suffering an ischemic stroke. The first study, NEST-1, enrolled 120 patients between the ages of 40 to 85 years of age and found a significantly improved outcome (p < 0.05 real vs sham, NIH Stroke Severity Scale) 5 days after a single laser treatment had been administered (Lampl et al. 2007). This significantly improved status was still present 90 days post-stroke in 70% of the PBM patients (but only 51% of the sham patients). The second clinical trial, NEST-2, enrolled 660 patients, aged 40 to 90, who were randomly assigned to one of two groups (331 to PBM, 327 to sham) (Zivin et al. 2009). Significant improvements (p < 0.04) were found in the moderate and moderate-severe (but not for the severe) stroke patients. The last clinical trial, NEST-3, was planned for 1000 patients enrolled, but the study was prematurely terminated by the DSMB for futility (an expected lack of statistical significance) (Lapchak and Boitano 2016). Many commentators have asked how tPBM could work so well in the first trial, yet fail in the third trial. Insufficient light penetration, too long an interval between stroke onset and PBM, inappropriate stroke severity measurement scale, use of only one single tPBM treatment, and failure to illuminate different specific areas of the brain for individual patients, have all been suggested as contributory reasons (Hamblin 2016b). It is undoubtedly the case that the failure of NEST-3 has cast a cloud over the whole application of PBMT for TBI as well as for stroke. Many commentators have asked “Why are you testing PBMT for TBI, if it has been shown not to work for stroke?” The failure of the investigators not to take into account the anatomical location of the stroke (and also whether it was deep or superficial) was also likely to have played a role in the failure of NEST-3. It is logical that light should be applied to the same side of the head where the lesion was located, not both sides of the head (Naeser et al. 2012). In my opinion the use of a single application of PBMT also bore some of the responsibility. Although a single application of PBM to the head works very well for experimental animals (mice, rats, rabbits) who have suffered a stroke or a TBI, the same may not apply to humans.

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4. Animal studies of PBM in acute TBI models

4.1 Studies from the Oron laboratory

Oron's group was the first (Oron et al. 2007) to demonstrate that a single exposure of the head of a mouse a few hours after creation of a TBI lesion using a NIR laser (808 nm) could improve neurological performance and reduce the size of the brain lesion. A weight-drop device was used to induce a closed-head TBI in the mice. An 808 nm diode laser with two energy densities calculated at the surface of the brain (1.2-2.4 J/cm2 delivered by 2 minutes of irradiation with 200mW laser power to the scalp) was delivered to the head 4 hours after TBI was induced. Neurobehavioral function was assessed by the neurological severity score (NSS). There was no significant difference between the control and laser-treated group in NSS between the power densities (10 vs 20 mW/cm2), and no significant difference at early time points (24 and 48 hours) post TBI. However, there was a significant improvement (27% lower NSS score) in the PBM group at times between 5 days and 4 weeks. The laser treated group also showed a smaller loss of cortical tissue than the sham group (Oron et al.). In another study (Oron et al. 2012) they varied the pulse parameters (CW, 100Hz, or 600Hz) and tested whether the tPBM was equally effective when delivered at 4, 6, or 8 hours post-TBI. They first established that a calculated dose to the cortical surface of 1.2 J/cm2 of 808nm laser at 200mW applied to the head, was more effective when delivered at 6 hours post TBI than at 8 hours. They then selected an even shorter time post-TBI (4 hours) and compared CW with 100Hz and 600Hz. At 56 days, more mice in the 100Hz group (compared to the CW and 600 Hz groups) had fully recovered. The 600Hz group had lower NSS scores than the CW and 100Hz groups up to 20 days. Magnetic resonance imaging (MRI) analysis demonstrated significantly smaller lesion volumes in PBM-treated mice compared to controls.

4.2 Studies from the Hamblin laboratory

Wu et al. (Wu et al. 2012) first explored the effect of varying the laser wavelengths of PBM had on closed-head TBI in mice. Mice were randomly assigned to a PBM treatment group with a particular wavelength, or to a sham treatment group as a control. Closed-head injury (CHI) was induced via a weight- drop apparatus. To analyze the severity of the TBI, the neurological severity score (NSS) was measured and recorded. The injured mice were then treated with varying wavelengths of laser light (665, 730, 810 or 980 nm) at an energy density of 36 J/cm2 directed onto the scalp at 4 hours post-TBI. The 665 nm and 810 nm laser groups showed significant improvement in NSS when compared to the control group between days 5 to 28. By contrast, the 730 nm and 980 nm laser groups did not show any significant improvement in NSS (Wu et al. 2012) (Figure 3). The tissue chromophore cytochrome c oxidase (CCO) is proposed to be responsible for the underlying photon absorption process that underlies many PBM effects. CCO has absorption bands around 665 nm and 810 nm while it has a low absorption region at the wavelength of 730 nm (Karu et al.). It should be noted that this particular study (Wu et al. 2012) found that the 980 nm did not produce the same positive effects as the 665 nm and 810 nm wavelengths did; nevertheless previous studies did find that the 980 nm wavelength was an active one for PBM (Anders et al. 2014). Wu et al. suggested that these dissimilar results may be due to differences in the energy density, irradiance etc. between the other studies and the Wu study (Wu et al. 2012). In particular a much lower dose of 980 nm might have been effective had it been tested (Wang et al. 2016). Ando et al. (Ando et al. 2011) next used the 810 nm wavelength produced by a Ga-Al-As diode laser delivered at parameters used in the Wu study, and varied the pulse modes of the laser. These modes consisted of either pulsed wave at 10 Hz or at 100 Hz (50% duty cycle) or continuous wave laser. They used a different mouse model of TBI induced with a controlled cortical impact device directly inflicting a lesion on the cortex via an open craniotomy. A single treatment with a power density of 50 mW/m2 and an energy density of 36 J/cm2 (duration of 12 minutes) was given via tLLLT to the closed head in mice at 4 hours post CCI. At 48 hours to 28 days post TBI, all laser treated groups had significant decreases in the measured neurological severity score (NSS) when compared to the controls. Although all laser treated groups had similar NSS improvement rates up to day 7, the PW 10 Hz group began to show even greater improvement beyond this point as seen in Figure 4. At day 28, the forced swim test for depression and anxiety was used and showed a significant decrease in the immobility time for the PW 10 Hz group. In the tail suspension test, which measures depression and anxiety, there was also a significant decrease in the immobility time at day 28, and also at day 1, in the PW 10 Hz group.

Figure 3

Effect of different laser wavelengths of tPBM in closed-head TBI in mice

(A) Sham-treated control versus 665 nm laser. (B) Sham-treated control versus 730 nm laser. (C) Sham-treated control versus 810 nm laser. (D) Sham-treated control versus 980 nm laser. Points are means of 8–12 mice and bars are SD. *P < 0.05; **P < 0.01; ***P < 0.001 (one-way ANOVA). Reprinted with permission from (Wu et al. 2012)

Figure 4

Effects of pulsing in tPBM for CCI-TBI in mice

(A) Time course of neurological severity score (NSS) of mice with TBI receiving either control (no laser-treatment), or 810 nm laser (36 J/cm2 delivered at 50 mW/cm2 with a spot size of 0.78 cm2 in either CW, PW 10 Hz or PW 100 Hz modes. Results are expressed as mean +/- S.E.M ***P < 0.001 vs. the other conditions. (B) Mean areas under the NSS-time curves in the two-dimensional coordinate system over the 28-day study for the 4 groups of mice. Results are means +/- SD (n = 10). Reprinted from (Ando et al. 2011) (open access).

Studies using immunofluorescence staining of sections cut from mouse brains showed that tPBM increased neuroprogenitor cells (incorporating BrdU) in the dentate gyrus (DG) of the hippocampus and the subventricular zone (SVZ) at 7 days after the treatment (Xuan et al. 2014). The neurotrophin known as brain derived neurotrophic factor (BDNF) was also increased in the DG and SVZ at 7 days, while the protein marker (synapsin-1) for synaptogenesis and neuroplasticity was increased in the cortex at 28 days but not in the DG, SVZ or in any location at 7 days (Xuan et al. 2015). Learning and memory as measured by the Morris water maze was also improved by tPBM (Xuan et al. 2014).

4.3 Studies from the Wu laboratory

Zhang et al. (Zhang et al. 2014) first showed that secondary brain injury occurred to a worse degree in mice that had been genetically engineered to lack “Immediate Early Response” gene X-1 (IEX-1). When these mice were exposed to a gentle head impact (thought to closely resemble mild TBI in humans) they had a worse NSS than uninjured mice with the same TBI. Exposure of IEX-1 knockout mice to PBM (150 mW/cm2, 4 min, and 36 J/cm2) delivered at 4 hours post injury, restored the NSS to almost baseline levels, suppressed proinflammatory cytokine expression of interleukin (IL)-Iβ and IL-6, but upregulated TNF-α. The original lack of IEX-1 decreased ATP production, but exposing the injured brain to LLLT elevated ATP production back to near normal levels.

Dong et al. (Dong et al. 2015) asked whether the beneficial effects of PBM on TBI in mice could be enhanced by combining PBM with administration of metabolic substrates such as pyruvate and/or lactate. The goal was to even further improve mitochondrial function in the brain. This combinatorial treatment was able to reverse memory and learning deficits in TBI injured mice back to normal levels as well as leaving the hippocampal region completely protected from tissue loss; a stark contrast to control TBI mice that exhibited severe tissue loss from secondary brain injury.

4.4 Studies from the Whalen laboratory

Khuman et al (Khuman et al. 2012) delivered PBM (800nm) either directly to the injured brain tissue (through the craniotomy) or transcranially in mice beginning 60-80 min after CCI TBI. At a dose of 60J/cm2 (500mW/cm2) the mice showed increased performance in the Morris water maze (latency to the hidden platform, p<0.05, and probe trial, p<0.01) compared to non-treated controls. When PBM was delivered via open craniotomy there was reduced microgliosis at 48h (IbA-1+ cells, p<0.05). Little or no effect of tPBM on post-injury cognitive function was observed using lower or higher doses, a 4-h administration time point or 60J/cm2 at 7-days post-TBI.

4.5 Studies from the Whelan laboratory

Quirk et al (Quirk et al. 2012) studied Sprague-Dawley rats who had received a severe CCI TBI and were divided into three groups: real TBI, sham surgery, and anesthetization only. Each group received either real or sham PBM consisting of 670nm LED treatments of 15J/cm2, 50mW/cm2, 5min, given two times per day for 3 days (chemical analysis) or 10 days (behavioral analysis using a TruScan nose-poke device). Significant differences in task entries, repeat entries, and task errors were seen in the TBI rats treated with PBM vs untreated TBI mice, and in sham surgery mice treated with PBM vs untreated sham surgery mice. A statistically significant decrease was found in the pro-apoptotic marker Bax, and increases in the anti-apoptotic marker Bcl-2 and reduced glutathione (GSH) levels in tPBM TBI mice.

4.6 Studies from the Marques laboratory

Moreira et al used a different model of TBI (Moreira et al. 2009). Wistar rats received a craniotomy and a copper probe cooled in liquid nitrogen was applied to the surface of the brain to create a standardized cryogenic injury. They treated the rats with either a 780nm or 660nm laser at one of two different doses (3J/cm2 or 5J/cm2) twice (once immediately after the injury and again 3 hours later). Rats were sacrificed 6h and 24h after the injury. The 780nm laser was better at reducing levels of pro-inflammatory cytokines (TNFα, IL1β, IL6) particularly at early timepoints (Moreira et al. 2009). In a follow-up study using 3 J/cm2 (Moreira et al. 2011) these workers reported on the healing of the injuries in these rats at timepoints 6h, 1, 7 and 14 days after the last irradiation. Cryogenic injury created focal lesions in the cortex characterized by necrosis, edema, hemorrhage and inflammatory infiltrate. The most striking findings were: PBM-treated lesions showed less tissue loss than control lesions at 6h. During the first 24h the amount of viable neurons was significantly higher in the PBM groups. PBM reduced the amount of GFAP (glial fibrillary acidic protein, a marker of astrogliosis) and the numbers of leukocytes and lymphocytes, thus demonstrating its anti-inflammatory effect.

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5. Patients with chronic TBI

The majority of studies of PBM for TBI in laboratory animals have been conducted in the acute setting, while the majority of human studies of PBM for TBI have been conducted in patients who have suffered head injuries at various times in the past (sometimes quite a long time ago).

5.1 Naeser case reports

In 2011 Naeser, Saltmarche et al., published the first report describing two chronic, TBI cases treated with tPBM (Naeser et al. 2011). A 500 mW CW LED source (mixture of 660 nm red and 870 nm NIR LEDs) with a power density of 22.2 mW/cm2 (area of 22.48 cm2), was applied all over the head, for 10 minutes at each placement location (13.3 J/cm2). In the first case study the patient reported that she could concentrate on tasks for a longer period of time (the time able to work at a computer increased from 20 minutes to 3 hours). She had a better ability to remember what she read, decreased sensitivity when receiving haircuts in the spots where PBM was applied, and improved mathematical skills after undergoing PBM. The second patient had statistically significant improvements compared to prior neuropsychological tests after 9 months of treatment. The patient had a 2 standard deviation (SD) increase on tests of inhibition and inhibition accuracy (9th percentile to 63rd percentile on the Stroop test for executive function and a 1 SD increase on the Wechsler Memory scale test for the logical memory test (83rd percentile to 99th percentile) (Naeser et al. 2011).

5.2 Naeser case series

Naeser et al then went on to report a case series containing a further eleven patients (Naeser et al. 2014). This was an open protocol study that examined whether scalp application of red and NIR LED could improve cognition in patients with chronic, mild TBI (mTBI). This study enrolled 11 participants ranging in age from 26 to 62 years (6 males, 5 females) who suffered from persistent cognitive dysfunction after mTBI. The injuries in the participants had been caused by motor vehicle accidents, sports related events and for one participant, an improvised explosive device (IED) blast. tPBM consisted of 18 sessions (Monday, Wednesday, and Friday for 6 weeks) and was started anywhere from 10 months to 8 years post-TBI. A total of 11 LED cluster heads (5.25 cm in diameter, 500 mW, 22.2 mW/cm2, 13 J/cm2) were applied for 10 minutes per set (5 or 6 LED placements per set, Set A and then Set B, in each session). Neuropsychological testing was performed pre-LED application and 1 week, 1 month and 2 months after the final treatment. They found that there was a significant positive linear trend for the Stroop Test for executive function, in trial 3 inhibition (p = 0.004); Stroop, trial 4 inhibition switching (p = 0.003); California Verbal Learning Test (CVLT)-II, total trials 1-5 (p = 0.003); CVLT-II, long delay free recall (p = 0.006). Improved sleep and fewer post-traumatic stress disorder (PTSD) symptoms, if present beforehand, were observed after treatment. Participants and family members also reported better social function and a better ability to perform interpersonal and occupational activities. Although these results were significant, the authors suggested that further placebo-controlled studies would be needed to ensure the reliability of this approach (Naeser et al. 2014).

Naeser has proposed (Naeser et al. 2016; Naeser et al. 2014) that specific scalp placements of the LED cluster heads may affect specific cortical nodes in the intrinsic networks of the brain, such as the default mode network (DMN), the salience network (SN), and the central executive network (CEN). These intrinsic networks are often dysregulated after TBI (Sharp et al. 2014). Naeser proposed that the specific areas of the head to receive light, to target cortical nodes in these networks were as follows:

1. For the DMN, placement of the LED cluster head on the midline of face, centered on the upper forehead and the front hairline, targeted the left and right mesial prefrontal cortex; and on a midline, scalp location half-way between the occipital protuberance and the vertex of the head, targeted the precuneus; and on left and right LED placements superior to the tip of each ear and posterior to each ear, targeted the inferior parietal cortex/angular gyrus areas.

2. For the SN, placement of LED cluster heads on the left and right temple areas, to target the anterior insula (but due to depth of insula, unknown if the photons reached the target); midline of the vertex of the head, to target the left and right presupplementary motor areas; and the LED cluster head placed on the midline of face, centered on the upper forehead and the front hairline, also targeted the left and right dorsal anterior cingulate cortex.

3. For the CEN, left and right scalp LED placements immediately posterior to the front hairline (on a line directly superior from the pupils of the eyes), targeted the dorso-lateral prefrontal cortex areas; and the left and right LED placements superior to the tip of each ear and posterior to each ear, also targeted the posterolateral inferior parietal cortex/angular gyrus areas (also treated as part of the DMN).

Further studies from Naeser and colleagues (Naeser et al. 2016) tested an intranasal LED (iLED) device. Two small iLEDs (one red and the other NIR) were clipped into each nostril and used at the same time for 25 min. The parameters were as follows: red, 633nm, 8mW CW, 1 cm2, energy density 12 J/cm2 (25 min); NIR 810nm, 14.2mW, pulsed 10Hz, 1cm2, 21.3J/cm2. The first mTBI participant (24-year old female) who had sustained four sports-related concussions (two during snowboarding and two during field hockey), received iLED PBM three times per week for 6 weeks. Significant improvements were observed in tasks measuring executive function and verbal memory as well as attention and verbal fluency. At 1 week after the 18th iLED treatment, the average total time asleep had increased by 61 min per night and her sleep efficiency (total sleep time divided by total time in bed) had increased by 11%. At 12 weeks after the last iLED treatment, she was able to discontinue all sleep medications that she had previously been using. The second, mTBI participant who received the intranasal only, LED treatment series is a 49 Yr. M (non-Veteran) who sustained mTBI in a MVA, 30 years prior to receiving the intranasal LED treatment series. He showed significant improvement on the Controlled Oral Word Association-FAS Test post- the iLED treatment series, improving by +1.3 SD and +1.5 SD at 1 and 2 months post- the 18th iLED treatment. His sleep data indicated he was already a good sleeper, at entry.

5.3 Bogdanova and Naeser studies

Bogdanova reported (Bogdanova et al. 2014) a case report of two patients (1 female) with moderate TBI (medical records and clinical evaluation) and persistent cognitive dysfunction (as measured by neuropsychological tests of executive function and memory). Patients received 18 sessions of transcranial LED therapy (3×/week for 6 weeks) using the mixed red/NIR cluster described above (Naeser et al. 2011).

Standardized neuropsychological tests for executive function, memory, depression, PTSD and sleep measures (PSQI, actigraphy) were administered to participants pre-(T1), mid-(T2), and one week (T3) post-PBM treatment. Both PBM treated cases (P1 and P2) showed marked improvement in sleep (actigraphy total sleep) 1 week post-LED treatment (T3), as compared to pre-treatment (T1). P1 also improved in executive function, verbal memory, and sleep efficiency; while P2 significantly improved on measures of PTSD (PCL-M) and depression. No adverse events were reported.

5.4 Studies from Henderson and Morries

Henderson and Morries (Henderson and Morries 2015b) used a high-power NIR laser (10-15 W at 810 and 980 nm) and applied it to the head to treat a patient with moderate TBI. The patient received 20 NIR applications over a 2-month period. They carried out anatomical magnetic resonance imaging (MRI) and perfusion single-photon emission computed tomography (SPECT). The patient showed decreased depression, anxiety, headache, and insomnia, whereas cognition and quality of life improved, accompanied by changes in the SPECT imaging.

They next reported (Morries et al. 2015) a series of ten patients with chronic TBI (average time since injury 9.3 years) where each patient received ten treatments over the course of 2 months using a high-power NIR laser (13.2 W/0.89 cm2 equivalent to 14.6 W/cm2 at 810nm; or 9 W/0.89 cm2 equivalent to 10.11 W/cm2 at 980nm). A continuous sweeping motion over the forehead was utilized to minimize skin heating and cover a larger area. Skin temperature increased no more than 3°C. Overall symptoms of headache, sleep disturbance, cognition, mood dysregulation, anxiety, and irritability improved. Symptoms were monitored by depression scales and a novel patient diary system specifically designed for this study. These authors have proposed that high power lasers are preferable for tPBM treatments because the photons can better reach the brain (Henderson and Morries 2015a).

5.5 Case study from Nawashiro

Nawashiro et al (Nawashiro et al. 2012) treated a single patient who had suffered a severe TBI. The patient survived but was left in a persistent vegetative state for 8 months after the accident. He showed no spontaneous movement of limbs and a CT scan of the head 8 months after the accident showed a focal low-density area in the right frontal lobe. The device had 23 individual 850nm LEDs (13mW each; total power 299mW, total area 57cm2). A treatment time of 30 min per session delivered 20.5 J/cm2 over the left and right forehead areas repeated twice daily (6h apart), for 73 days. Five days after beginning the PBM (after 10 treatments), the patient began to spontaneously move his left arm and hand, which had not occurred during the previous 8 months. Single-photon emission computed tomography with N-isopropyl-[123I]p-iodoamphetamine (IMP-SPECT) was performed twice. The IMP-SPECT scans showed a focal increase (20% higher) in cerebral blood flow in the uninjured left anterior frontal lobe 30 min after the last (146th) PBM treatment, compared to before PBM began.

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6. Conclusion and future prospects

As was mentioned above, one of the most important questions to be answered when contemplating clinical treatment of TBI patients with tPBM, is what is the best time to administer the treatment? All the available reports of studies using PBM in laboratory animal models of TBI and stroke, and also in patients treated for stroke, have been in the acute phase where the overall goal of the intervention can be best described as neuroprotection. Not only that but there are several reports (Lapchak et al. 2007; Oron et al. 2012) that PBM for both TBI and stroke is most effective when it is delivered as soon as possible after the actual event (head impact or ischemic stroke). The protocols for the series of NEST clinical trials specified that patients should be treated with PBM within 24 hours of the stroke occurring. By contrast, all the clinical trials of PBM for patients with TBI, that have so far been carried out, have been with chronic TBI, after varying periods of time having elapsed after the original head injury, sometimes as long as 8 years. Although it would be generally supposed that tPBM would be effective when delivered to acute TBI patients, this has not yet been actually tested. If tPBM were to be used for acute TBI patients, then presumably the PBM should be delivered perhaps beginning at 4 to 6 hours post-TBI, for a limited number of times after the injury; perhaps once a day for 7 days?

The dosimetry and optimum delivery apparatus of tPBM is still uncertain. Although there is some consensus that wavelengths in the region of 800-900nm will penetrate the scalp and skull, other workers have used longer NIR wavelengths, 980nm, 1064nm, or 1072nm. Pulsing or CW is another unresolved question. The exact locations on the head that should receive the light are still unknown. Naeser has proposed (Naeser et al. 2016) some interesting considerations regarding the scalp placements of the tLEDS, and their effect on various intrinsic cortical networks of the brain. Targeted LED placements could promote better neuromodulation (activation/deactivation) in specific cortical nodes. It is possible that communication between nodes within one single network, and/or across networks could be improved. Moreover preliminary data indicate that intranasal, red plus near-infrared LEDs can also benefit TBI patients, although the degree to which light incident on the nasal mucosa, and possibly delivered transsphenoidally (Pitzschke et al. 2015) can penetrate directly into the brain, remains to be determined.

An advantage of intranasal and/or transcranial LED PBM therapy is that it can be performed in the home, for long-term use (Naeser et al. 2011). Also, 5 chronic, mild to moderately-severe dementia cases recently showed significant improvement on the Mini-Mental State Examination (p<0.003), and on the Alzheimer's Disease Assessment Scale-Cognitive subscale (p<0.023) after 12 weeks of daily, at-home, intranasal, near-infrared LED PBM treatments (810nm, pulsed at 10 Hz), and once-a-week in-office, tLED treatments applied to the cortical nodes of the Default Mode Network (Saltmarche et al. 2017). Anecdotally, there was also improved sleep, fewer angry outbursts, and less wandering. When all LED treatments were withdrawn after 12 weeks of active LED PBM treatment, there was precipitous decline in cognition and behavior. Thus, at-home, long-term use of iLED plus tLED PBM offers a potential therapy to mitigate the sequelae of Alzheimer's disease and possibly other neurodegenerative disorders, as well as TBI and stroke.

One highly distressing aspect of TBI symptomatology that has not so far been addressed by PBM, is that of post-traumatic epilepsy (PTE). TBI is the most significant cause of symptomatic epilepsy in people from 15 to 24 years of age. The frontal and temporal lobes are the most frequently affected regions, but imaging (MRI) often fails to show the precise cause. During PTE seizures there is an abnormal electrical discharge in the brain, with staring and unresponsiveness, stiffening or shaking of the body, legs, arms or head; strange sounds, tastes, visual images, feelings or smells; inability to speak or understand, etc (Cotter et al. 2017). Epilepsy has traditionally been considered to be a contra-indication for PBMT (Navratil and Kymplova 2002). However the knowledge that has recently been gained concerning the beneficial effects of PBMT on the damaged brain, suggests that this view may need to be critically revisited.

Moreover there is also potential of tPBM to treat a wide range of brain disorders only loosely associated with TBI, including Parkinson's disease (Purushothuman et al. 2013), depression, anxiety, post-traumatic stress disorder, autism spectrum disorder and so on (Hamblin 2016b).

The ongoing and accelerating clinical research efforts in testing PBM for TBI, are expected to lead to the answering of many of these questions in the coming years.

INTRODUCTION

Since the introduction of low-level laser (light) therapy in 1967, over two hundred randomized, double-blinded, and placebo-controlled phase III clinical trials have been published from over a dozen countries. Whereas there is some degree of consensus as to the best wavelengths of light and acceptable dosages to be used, there is no agreement on whether continuous wave (CW) or pulsed wave (PW) light is more suitable for the various applications of LLLT. This review will raise (but not necessarily answer) several questions. How does pulsed light differ from CW on the cellular and molecular level, and how is the outcome of LLLT affected? If pulsing is more efficacious, then at what pulse parameters is the optimal outcome achieved? In particular, what is the ideal pulse repetition rate or frequency to use?

PULSE PARAMETERS AND LIGHT SOURCES

There are five parameters that could be specified for pulsed light sources. The pulse width or duration or ON time (PD) and the pulse Interval or OFF time (PI) are measured in seconds. Pulse repetition rate or frequency (F) is measured in Hz. The duty cycle (DC) is a unitless fractional number or %. The peak power and average power are measured in Watts.

Pulse duration, pulse repetition rate, and duty cycle are related by the simple equation:

DC=F×PD

Peak power is a measure of light intensity during the pulse duration, and related to the average power (measured in Watts) by:

Average power=Peak power×F×PD

Alternatively,

Peak power=Average powerDC

In all cases, it is necessary to specify any two out of three of: PD, F, and DC, and either the peak or average power for the pulse parameters to be fully defined.

Figure 1 graphically shows the relationship between peak power and pulse duration.

Fig. 1

Conceptual diagram comparing the structure of CW with pulsed light of various pulse durations.

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TYPES OF PULSED LIGHT SOURCES

Five major types of pulsed lasers (or other light sources) are commonly utilized: (1) Q-switched, (2) Gain-switched, (3) Mode-locked, (4) Superpulsed, and (5) Chopped or gated. Each utilizes a different mechanism to generate light in a pulsed as opposed to continuous manner, and vary in terms of pulse repetition rates, energies, and durations. However the first three classes of “truly” pulsed lasers mentioned above are in general not used for LLLT; instead superpulsed or gated lasers are mainly used. The concept of super-pulsing was originally developed for the carbon dioxide laser used in high power tissue ablative procedures. The idea was that by generating relatively short pulses (µsecond) the laser media could be excited to higher levels than those normally allowed in CW mode where heat dissipation constraints limit the maximum amounts of energy that can be used to excite the lasing media. With the original carbon dioxide superpulsed lasers, the short pulses would confine the thermal energy in the tissue (by making the pulse duration less than the thermal diffusion time) reducing collateral thermal damage to normal tissue.

Another type of laser that particularly benefited from super-pulsing is the gallium-arsenide (GaAs) diode laser. This laser has a wavelength in the region of 904-nm and pulse duration usually in the range of 100–200 nanoseconds. Another semiconductor laser amenable to superpulsing is the indium-gallium-arsenide (In-Ga-As) diode laser. It emits light at a similar wavelength (904–905-nm) as the GaAs diode laser, producing very brief pulses (200 nanoseconds) of high frequencies (in the range of kilohertz). These pulses are of very high peak powers (1–50 W) and an average power of 60 mW. Theoretically, the super-pulsed GaAs and In-Ga-As lasers allow for deep penetration without the unwelcome effects of CW (such as thermal damage), as well as allowing for shorter treatment times.

The other major class of pulsed light sources used in LLLT are simply CW lasers (usually diode lasers) that have a pulsed power supply generated by a laser driver containing a pulse generator. This technology is described as “chopped” or “gated.” It is also equally feasible to use pulse generator technology to pulse LEDs or LED arrays [1].

WHY COULD PULSING BE IMPORTANT IN LLLT?

Pulsed light offers numerous potential benefits. Because there are “quench periods” (pulse OFF times) following the pulse ON times, pulsed lasers can generate less tissue heating. In instances where it is desirable to deliver light to deeper tissues increased powers are needed to provide adequate energy at the target tissue. This increased power can cause tissue heating at the surface layers and in this instance pulsed light could be very useful. Whereas CW causes an increase in temperature of the intervening and target tissues or organ, pulsed light has been shown to cause no measurable change in the temperature of the irradiated area for the same delivered energy density. Anders et al. administered pulsed light to pig craniums, and found no significant change in temperature of the scalp or skull tissue (J.J. Anders, personal communication). Ilic et al. [2] found that pulsed light (peak power densities of 750 mW/cm2) administered for 120 seconds produced no neurological or tissue damage, whereas an equal power density delivered by CW (for the same number of seconds) caused marked neurological deficits.

Aside from safety advantages, pulsed light might simply be more effective than CW. The “quench period” (pulse OFF times) reduces tissue heating, thereby allowing the use of potentially much higher peak power densities than those that could be safely used in CW. For example, when CW power densities at the skin of ≥2 W/cm2 are used, doubling the CW power density would only marginally increase the treatment depth while potentially significantly increasing the risk of thermal damage; in contrast, peak powers of ≥5 W/cm2 pulsed using appropriate ON and OFF times might produce little, or no tissue heating. The higher peak powers that can be safely used by pulsing light can overcome tissue heating problems and improve the ability of the laser to penetrate deep tissues achieving greater treatment depths.

There may be other biological reasons for the improved efficacy of pulsed light (PW) over CW. The majority of the pulsed light sources used for LLLT have frequencies in the 2.5–10,000 Hz range and pulse durations are commonly in the range of a few millisecond. This observation suggests that if there is a biological explanation of the improved effects of pulsed light it is either due to some fundamental frequency that exists in biological systems in the range of tens to hundreds of Hz, or alternatively due to some biological process that has a time scale of a few milliseconds. Two possibilities for what these biological processes could actually be occur to us. Firstly, it is known that mammalian brains have waves that have specific frequencies [3]. Electroencephalography studies have identified four distinct classes of brain waves [4,5]. Alpha waves (8–13 Hz) occur in adults who have their eyes closed or who are relaxed [6]. Beta waves (14–40 Hz) mainly occur in adults who are awake, alert or focused [7]. Delta waves (1–3 Hz) occur mainly in infants, adults in deep sleep, or adults with brain tumors [8]. Theta waves (4–7 Hz) occur mainly in children ages 2–5 years old and in adults in the twilight state between sleeping and waking or in meditation [9]. The possibility of resonance occurring between the frequency of the light pulses and the frequency of the brain waves may explain some of the results with transcranial LLLT using pulsed light.

Secondly, there are several lines of evidence that ion channels are involved in the subcellular effects of LLLT. Some channels permit the passage of ions based solely on their charge of positive (cationic) or negative (anionic) while others are selective for specific species of ion, such as sodium or potassium. These ions move through the channel pore single file nearly as quickly as the ions move through free fluid. In some ion channels, passage through the pore is governed by a “gate,” which may be opened or closed by chemical or electrical signals, temperature, or mechanical force, depending on the variety of channel. Ion channels are especially prominent components of the nervous system. Voltage-activated ion channels underlie the nerve impulse and while transmitter-activated or ligand-gated channels mediate conduction across the synapses.

There is a lot of literature on the kinetics of various classes of ion channels but in broad summary it can be claimed that the time scale or kinetics for opening and closing of ion channels is of the order of a few milliseconds. For instance Gilboa et al. [10] used pulses having a width 10 milliseconds and a period of 40 milliseconds (25 Hz). Other reports on diverse types of ion channels have given kinetics with timescales of 160 milliseconds [11], 3 milliseconds [12] and one paper giving three values of 0.1, 4 and 100 milliseconds [13]. Potassium and calcium ion channels in the mitochondria and the sarcolemma may be involved in the cellular response to LLLT [14–16].

Thirdly there is the possibility that one mechanism of action of LLLT on a cellular level is the photodissociation of nitric oxide from a protein binding site (heme or copper center) such as those found in cyctochrome c oxidase [17]. If this process occurs it is likely that the NO would rebind to the same site even in the presence of continuous light. Therefore if the light was pulsed multiple photodissociation events could occur, while in CW mode the number of dissociations may be much smaller.

PENETRATION DEPTH

The most important parameter that governs the depth of penetration of laser light into tissue is wavelength. Both the absorption and scattering coefficients of living tissues are higher at lower wavelength so near-infrared light penetrates more deeply that red and so on. It is often claimed that pulsed lasers penetrate more deeply into tissue than CW lasers with the same average power. Why exactly should this be so? Let us suppose that at a certain wavelength (for instance 810-nm) the depth of tissue at which the intensity of a laser is reduced to 10% of its value at the surface of the skin is 1-cm. Therefore if we are using a laser with a power density (irradiance) of 100 mW/cm2 at the skin, the power density remaining at 1 cm below the skin is 10 mW/cm2 and at 2-cm deep is 1 mW/cm2. Now let us suppose that a certain threshold power density (minimum number of photons per unit area per unit time) at the target tissue is necessary to have a biological effect and that this value is 10 mW/cm2. The effective penetration depth at CW may be said to be 1-cm. Now let us suppose that the laser is instead pulsed with a 10-milliseconds pulse duration at a frequency of 1 Hz (DC = 1 Hz×0.010 seconds = 0.010) and the same average power. The peak power and peak power densities are now 100 times higher (peak power = average power/DC = average power×100). With a peak power density of 10 W/cm2 at the skin, the tissue depth—at which this peak power density is attenuated to the threshold level of 10 mW/cm2—is now 3-cm rather than 1-cm in CW mode. But what we have to consider is that the laser is only on for 1% of the time so the total fluence delivered to the 3-cm depth in pulsed mode is 100 times less than that delivered to 1-cm depth in CW mode. However it would be possible to increase the illumination time by a factor of 100 to reach the supposed threshold of fluence as well as the threshold of power at the 3-cm depth. In reality the increase in effective penetration depth obtained with pulsed lasers is more modest than simple calculations might suggest. Many applications of LLLT do not require deep penetration such as tendinopathies and joint pain.

Similarly, deep penetration is often not required to alleviate joint pain. The target tissue in such cases is the synovia; with the exception of back, neck, and hip, most joints have readily accessible synovia. Bjordal et al. [19] conducted a review of literature and concluded that “superpulsed” lasers (904 nm) were not significantly more effective than CW lasers (810–830 nm); both types of laser achieved similar results, but half the energy was needed to be used for superpulsed lasers. On the other hand, deeper penetrance is needed to reach back, neck, and hip joints. If power densities greater than a few mW/cm2 are to be safely delivered to target tissues >5 cm below the skin, it appears likely that this can only be done by using pulsed lasers. It is postulated that successful LLLT treatments in such joints bring benefit not by reaching the deep target tissue but by inhibiting superficial nociceptors. In other words, they bring relief primarily by attenuating pain perception, as opposed to decreasing inflammation. Does deeper penetration via pulsed lasers offer any significant benefit over CW? It is quite possible that a relatively higher fluence is necessary to attenuate pain, whereas a lower fluence decreases inflammation. If this is indeed the case, for musculo-skeletal applications achieving higher doses at the level of the target tissue may not be ideal. Further studies must be done to confirm this hypothesis, as well as to determine if there is any real benefit to the deeper penetration attained by pulsed lasers. Muscles such as the biceps and rectus femoris are not small organs, and have quite deep target tissue. Yet, various studies have shown significant improvement with CW lasers and CW LED. It remains to be seen whether or not pulsed lasers offer any additional advantage. Similarly, depression [20] and stroke studies [21] using LLLT have demonstrated that CW LED’s and CW lasers (respectively) produce a beneficial therapeutic effect. There are reports from Anders’ laboratory that fluences as low as 0.1–0.2 J/cm2 may be optimal for cells in the brain [22]. However, further studies must be done to determine whether pulsed light, with higher peak power densities deeper into the brain tissues, might increase the effectiveness of these therapies.

STUDIES COMPARING CW AND PW

In this review thirty-three studies involving pulsed LLLT were examined. Of these studies, nine of them directly compared continuous wave (CW) with pulsed wave (PW) light, as recorded in Table 1. Six of these nine studies found PW to be more effective than CW. One study comparing CW and PW found both modes of operation to be equally effective, with no statistically significant difference between the two. Only two of the nine articles reported better results with CW than PW, although in both of these studies PW treated subjects were found to have better outcomes than placebo groups. One of the recurring limitations of the papers in this review was that like for like irradiation parameters were not used. For instance, Gigo-Benato et al. [23] found CW superior to PW in nerve regeneration, but is this because of the mode of operation (CW or PW) or because the CW laser used 808 nm and the pulsed laser used 905 nm?

TABLE 1

Studies Comparing CW and PW

Of the six studies that found PW to be more effective than CW, four of them involved the use of LLLT to cure the following pathologies in vivo: wound healing, pain, and ischemic stroke. The two remaining studies reported pulsing to be beneficial in vitro; in the first such study, PW promoted bone stimulation more so than CW. The other in vitro study comparing CW and PW found the latter mode of operation better able to penetrate through melanin filters, indicating that pulsing may be beneficial in reaching deep target tissue in dark-skinned patients.

In the wound healing study, Kymplova et al. [24] used a large sample size of women to study the effects of phototherapy on wound repair following surgical episiotomies (one of the most common surgical procedures in women). A pulsed laser emitted light (wavelength of 670 nm) at various frequencies (10, 25, and 50 Hz). The pulsed laser promoted wound repair and healing more so than the CW light source.

In the pain study, Sushko et al. [25] investigated the role of pulsed LLLT to attenuate pain in white male mice. The same wavelength of light was used as in Kymplova et al.’s study (670 nm), with the frequencies of 10, 600, and 8,000 Hz. Both modes of delivery (CW and PW) reduced the behavioral manifestations of somatic pain as compared to controls, but pulsed light (10 and 8,000 Hz in particular) was more effective.

The two studies involving pulsed LLLT and stroke were both done by Lapchak et al. [26]. Ischemic strokes were induced in rabbits, and a pulsed laser with a wavelength of 808 nm was used. In the first study, two frequencies of pulsed light were used (100 and 1,000 Hz), both of which reduced neurological deficits more so than CW. Accordingly, pulsed LLLT may play a major role in the management of stroke patients. Lapchak et al.’s second study attempted to prove the hypothesis that LLLT’s neuroprotective effect following stroke was a result of enhanced mitochondrial energy production (increased ATP synthesis) [27]. As with the previous study, LLLT was administered following stroke induction. CW radiation raised cortical ATP levels but was unable to bring them back to baseline. PW radiation, on the other hand, not only mitigated the effects of stroke on cortical ATP levels, but was able to raise cortical ATP levels to higher than those found in healthy rabbits (those in which stroke was not induced). This study provides valuable insight into one of the potential cellular and molecular mechanisms behind the enhanced neurogenesis (and improved clinical outcomes) observed in subjects receiving transcranial LLLT following stroke.

One of the nine studies reviewed found CW and PW to be equally effective in the promotion of wound healing. This study compared the effects of a CW laser (632.8 nm) and a PW laser (904 nm) on the promotion of wound healing in rabbits. Both lasers improved tensile strength during wound healing, but did not significantly improve wound-healing rates. A combined laser (CW+PW) was also tested. All three of the laser regimens improved tensile strength to a similar extent.

As mentioned earlier, there were nine studies that compared CW and PW, only two of which found CW to be more effective. These two studies involved wound healing and nerve regeneration respectively. Al-Watban and Zhang [28] study involved rats that were inflicted with aseptic wounds. The rats were divided into three groups: a control group, those irradiated with continuous wave light, and those irradiated with pulsed light at various repetition rates (100, 200, 300, 400, and 500 Hz). Of the pulse repetition rates administered, 100 Hz was the most efficacious and 500 Hz the least. Both CW and PW (635 nm) promoted wound healing, but CW was more efficacious. These results conflict with earlier studies that found pulsed light to be more beneficial in the promotion of wound healing. However, it should be noted that the difference between CW and PW treated subjects was small (a relative wound healing rate of 4.81 as compared to 4.32).

The second study that found CW to be more effective than PW involved nerve regeneration. There were three articles involving nerve regeneration, all of which found pulsed LLLT to be ineffective, as discussed in the section below entitled “Studies Involving Nerve Conduction and Regeneration.” Of these three, only Gigo-Benato et al. [23] compared CW (808 nm) and PW (905 nm). This study involved rats in which the left median nerve was completely transected and then repaired by end-to-end neurorrhaphy. The CW laser (808 nm) promoted faster nerve and muscle recovery than the pulsed laser (905 nm). However, Gigo-Benato also tested a combination of the CW and pulsed lasers, finding this to be the most effective of all. In other words, seven of the nine studies comparing CW and PW found pulsing to play a beneficial role. Only one of the nine studies found no role of PW, and even in this study the benefit of CW over PW was minimal.

STUDIES INVOLVING THE USE OF COMBINED LASERS (CW+PW)

We reviewed three studies, as recorded in Table 2, which investigated the role of a combined laser (using both CW and PW). Of these, only Gigo-Benato’s study compared the combined laser to stand alone CW or PW. This study has been discussed in the above section: the combined laser was found to be effective in stimulating nerve regeneration, more so than CW or PW alone.

TABLE 2

Studies Involving the Use of Combined Lasers (CW + PW)

The two other studies used a combined laser (CW and PW) to administer laser acupuncture, along with Transcutaneous Electrical Nerve Stimulation (TENS), to patients with symptoms of pain. Naeser et al. [29] administered this “triple therapy” to patients suffering from carpal tunnel syndrome (CTS). Eleven patients with mild-to-moderate symptoms of CTS were selected, all of who had failed to respond to standard medical or surgical treatment regimens. Subjects were divided into two groups, one of which received sham irradiation and the other that received a combined treatment of LLLT (CW and pulsed) and TENS. As compared to controls, the treated group experienced statistically significant improvement and remained stable for 1–3 years. The results of this study are promising, and indicate a possible role of LLLT and TENS in the conservative management of CTS.

Ceccherelli et al. [30] administered laser acupuncture to patients suffering from myofascial pain. In this double-blinded placebo controlled trial, patients received either the same “triple therapy” as in the Naeser et al. study (CW, PW, and TENS) or sham irradiation, every other day over the course of 24 days. Results were encouraging, with the treatment group experiencing a significant improvement in symptoms, both immediately after the treatment regimen and at a 3-month follow up visit.

In both preceding studies, the combined regimen of CW, PW, and TENS was compared to untreated controls, and found to be effective. However, neither study compared CW and PW or administered CW, PW, or TENS individually. As such, it is difficult to determine whether standalone CW or PW would have produced similar results, or if the combined regimen (along with TENS) was necessary.

STUDIES EVALUATING THE USE OF PULSED LASERS

Of the 33 studies reviewed, 21 of them compared PW treated subjects with untreated controls, as reported in Table 3. Of these, fourteen studies found pulsed LLLT to be effective, whereas seven of them found PW treated subjects to have no benefit over untreated controls. Only one study found PW to have a worse outcome than controls. Of the fourteen studies that found pulsed LLLT to be effective, seven involved the promotion of wound healing, four involved the attenuation of pain, two involved the promotion of bone and cartilage growth respectively, and one involved the treatment of a very rare condition (hyperphagic syndrome caused by traumatic brain injury). Of the seven studies that found no benefit to pulsed light, three involved the promotion of nerve conduction, two involved the promotion of nerve regeneration, and the remaining two involved the attenuation of pain.

TABLE 3

Studies Evaluating the Use of Pulsed Lasers

Studies Comparing Various Pulse Repetition Rates

If pulsed LLLT is effective (or ineffective), then what pulse repetition rates are to be used (or avoided)? Ten of the 33 articles reviewed tested and compared various repetition rates, as reported in Table 4. Four of these studies involved the use of pulsed LLLT to promote wound healing. Longo et al. [31] used the pulse repetition rates of 1,500 and 3,000 Hz, and found only the latter setting to promote wound healing. Korolev et al. [32] similarly used two pulse repetition rates, 500 and 3,000 Hz. In this case, both were found to be effective but 500 Hz was more so. Al-Watban and Zhang [28] compared five different pulse repetition rates (100, 200, 300, 400 and 500 Hz), finding 100 Hz to be the most effective and 500 Hz the least. el Sayed and Dyson [33] compared four different pulse repetition rates (2.5, 20, 292, and 20,000 Hz), and found only the two middle values (20 and 292 Hz) beneficial. The more effective pulse repetition rates in these four studies were very disparate, including 20, 100, 292, 500, and 3,000 Hz (a range of 20–3,000 Hz).

TABLE 4

Studies Comparing Various Pulse Repetition Rates

Two studies compared the role of various pulse repetition rates in the attenuation of pain. Ponnudurai et al. [34] used laser photobiostimulation to decrease pain levels in rats, and investigated the effect of using various pulsing frequencies (4, 60, and 200 Hz). The rat tail-flick test was utilized, and tail-flick latencies were measured at five intervals between 30 minutes and 7 days following irradiation. The pulsing frequency of 4 Hz increased pain threshold rapidly but very transiently, whereas 60 Hz produced a delayed but longer lasting effect. On the other hand, 200 Hz failed to produce any hypoalgesic effect whatsoever. Sushko et al. [25] conducted a similar experiment, using mice instead of rats. The center of pain was irradiated (610–910 nm) for 10 minutes with either CW or pulsed light (10, 600, and 8,000 Hz). Both modes of delivery (CW and pulsed) reduced the behavioral manifestations of somatic pain as compared to controls, but pulsed light was more effective. In particular, 10 and 8,000 Hz produced the best effect. The more effective pulse repetition rates from these two studies (involving pain attenuation) included 4, 10, 60, and 8,000 Hz (a range of 4–8,000 Hz), and the less effective pulse repetition rates included 200 and 600 Hz.

Lapchak et al. [26] not only compared CW and PW, but also pulsed light at two different repetition rates, P1 (1,000 Hz) and P2 (100 Hz). Ischemic strokes were induced in rabbits, and the neuroprotective effects of LLLT were assessed via behavioral analysis 48 hours post-stroke. Both P1 (1,000 Hz) and P2 (100 Hz) produced a similar effect (superior to CW).

Rezvani et al. [35] studied the use of low level light therapy to prevent X-ray induced late dermal necrosis. An X-ray dose of 23.4 Gy is known to invariably cause dermal necrosis after 10–16 weeks. This dose was delivered to pigs, which were then treated with LLLT for several weeks using various wavelengths (660, 820, 880, and 950 nm) pulsed at either 2.5 or 5,000 Hz. Light pulsed at 2.5 Hz did not reduce the incidence of dermal necrosis. On the other hand, light pulsed at 5,000 Hz significantly reduced (P = 0.001) the incidence to 52% when given 6–16 weeks after irradiation.

Of the 10 articles reviewed that compared various pulse repetition rates, two of them involved in vitro experiments. Brondon et al. [36] undertook a study to determine if pulsing light would overcome the filtering effects of melanin. Melanin filters were placed in front of human HEP-2 cells, which were then irradiated for 72 hours (670 nm wavelength) with either CW or pulsed light at various repetition rates (6, 18, 36, 100, and 600 Hz). Both cell proliferation and oxidative burst activity, were increased in the group treated with pulsed light, indicating that pulsed light is indeed better able to penetrate melanin rich skin. Specifically, cell proliferation was maximal at 100 Hz at 48 and 72 hours (n = 4, P≤0.05), and oxidative burst was maximal at 600 Hz (n = 4, P≤0.05).

Ueda and Shimizu [37] studied the effects of pulsed low-level light on bone formation in vitro. Osteoblast-like cells were isolated from fetal rat calvariae; one group was not irradiated at all, another was irradiated with continuous wave light, and the third group with pulsed light at three repetition rates (1, 2, and 8 Hz). As compared to the control group, both CW and PW light resulted in increased cellular proliferation, bone nodule formation, alkaline phosphatase (ALP) gene expression, and ALP activity. Pulsed light at 2 Hz stimulated these factors the most.

Out of all 10 articles that compared various pulse repetition rates, the following pulse repetition rates were found to be beneficial: 2, 10, 20, 100, 292, 500, 600, 1,000, 3,000, 5,000, and 8,000 Hz. In this wide range of frequencies (2–8,000 Hz), no particular frequencies stood out as being particularly more or less useful than others.

STUDIED INVOLVING WOUND HEALING

Ten studies out of the 33 involved LLLT’s role in the promotion of wound healing, as recorded in Table 5. Only two of these studies compared CW and PW. Kymplova et al. [24] found pulsed LLLT to promote wound healing over CW, whereas Al-Watban and Zhang [28] found CW to be slightly more effective than PW. Both studies used light of a similar wavelength (670 vs. 635 nm), although the pulse repetition rates used by Kymplova et al. were lower (10–50 Hz vs. 100–500 Hz in Al-Watban et al.’s study). The energy densities applied were also different (2 J/cm2 vs. 1 J/cm2).

TABLE 5

Studied Involving Wound Healing

Every study reviewed found pulsed LLLT effective in promoting wound healing (as compared to untreated controls), including the Al-Watban et al. study. Six of these studies used light in the wavelength range of 820–956 nm, and four in the range of 632.8–670 nm. Once again, a wide range of frequencies were used (2.5–20,000 Hz), most of which were found to promote wound healing. (Tested frequencies included 2.5, 5, 8.58, 10, 15.6, 20, 25, 31.2, 50, 78, 80, 287, 292, 500, 700, 3,000, 4,672, 9,000, and 20,000 Hz). Most of these articles also reported energy densities, usually in the range of 1–2 J/cm2.

STUDIES INVOLVING NERVE CONDUCTION AND REGENERATION

We reviewed three articles evaluating the role of pulsed LLLT in the promotion of nerve conduction, and another three involving nerve regeneration, as reported in Table 6. Unlike the studies involving wound healing where positive outcomes were reported, all six of these studies reported negative outcomes with pulsed light. Five of these studies found PW to have no statistically significant effect on outcome, whereas one of them found PW to have a deleterious effect. There was no study that directly compared CW and PW in regards to nerve conduction. Walsh et al. [38] conducted a study with 32 human volunteers to determine if pulsed LLLT would influence nerve conduction in the superficial radial nerve. Action potentials were measured pre- and post-irradiation (at 5, 10, and 15 minutes). No significant difference was appreciated between control and treatment groups, indicating that LLLT with those particular pulsing parameters and dosimetry had no specific neurophysiologic effects on nerve conduction. Bagis et al. [39] and Comelekoglu et al. [40] obtained similar negative results using frog nerves. Walsh et al. used a wavelength of 820 nm, whereas Bagis et al. used a 904 nm laser. All three studies tested pulse repetition rates within the range of 1–128 Hz.

TABLE 6

Studies Involving Nerve Conduction and Regeneration

Similarly, the nerve regeneration studies reviewed reported negative outcomes. Chen et al. [41] found PW to have a counterproductive effect, reducing nerve regeneration as compared to untreated controls. Only one study compared CW with PW, and found the former to be superior to the latter. However, the combined laser (CW+PW) was superior to CW alone, indicating that there might in fact be a role of pulsing in nerve regeneration.

STUDIES INVOLVING PAIN ATTENUATION

Nine of the thirty-three studies involved pulsed LLLT’s role in the attenuation of pain, as reported in Table 7. Of these, only one of them directly compared CW and PW. This study was conducted by Sushko et al. [25] and found that although both CW and PW decreased pain levels, PW was more effective. This study also determined that pulse repetition rates of 10 and 8,000 Hz were more effective than 600 Hz. Ponnudurai et al. [34] similarly compared various pulse repetition rates (4, 60, and 200 Hz). A rapid but transient analgesic effect was exhibited with 4 Hz, whereas a delayed but longer lasting effect was achieved with 60 Hz. On the other hand, 200 Hz failed to produce any analgesic effect whatsoever.

TABLE 7

Studies Involving Pain Attenuation

Two of the studies used a combined laser (CW+PW) along with TENS; both found the combined regimen to be effective. The five remaining studies compared pulsed LLLT with untreated controls. Three of these studies found pulsed LLLT to be effective, whereas two did not. Of the nine total studies on pain attenuation, seven found pulsed LLLT to be effective in its role of attenuating pain. Only two studies found no statistically significant effect. However, it should be noted that both of these involved pain of a different nature than commonly tested in pulsed LLLT studies. The first of these was by Craig et al. [42] and involved the use of pulsed LLLT to relieve the symptoms of delayed-onset muscle soreness (DOMS). DOMS refers to the feeling of pain and muscle stiffness that can result 1–3 days after intense sporting activity such as weightlifting. This pain is duller in quality than that tested in the other studies. The second study that showed no benefit to pulsed LLLT, published by de Bie et al. [43], involved the treatment of lateral ankle sprains.

STUDIES INVOLVING ISCHEMIC STROKE

Table 8 records the two studies that involved pulsed LLLT and stroke. In the first study, PW but not CW decreased neurological deficits when delivered six hours post-stroke. Two pulse repetition rates were tested (100 and 1,000 Hz) and found to be equally effective. On the other hand, both CW and PW produced no benefit if delivered 12 hours post-stroke, indicating that timely administration of LLLT is essential.

TABLE 8

Studies Involving Stroke

The second study investigated the possible mechanisms behind the neuroprotective effect of LLLT. It was postulated that LLLT enhances mitochondrial energy production (and ATP synthesis), which allows for enhanced neurogenesis. This hypothesis was tested using the rabbit small clot embolic stroke model (RSCEM). Four groups of rabbits were used: (1) a naïve control group which was neither embolized or irradiated, (2) a placebo group which was embolized and sham irradiated, (3) an embolized group which was irradiated with CW (808 nm), and (4) an embolized group which was irradiated with pulsed light (808 nm) at two different frequencies. Forty-five percent less cortical ATP was measured in the second group (placebo) as compared to the first (naïve), confirming the hypothesis that ischemic strokes decrease cortical mitochondrial energy. All laser irradiated groups were able to mitigate this effect. CW radiation managed to raise the cortical ATP levels by 41%, whereas PW administration raised these levels by over 150%. Surprisingly, this was even higher than the cortical ATP content measured in naïve rabbits that had never suffered stroke.

OTHER APPLICATIONS OF PULSED MODALITIES IN BIOMEDICINE

Many of the modalities of treatment employed in biomedicine and physical therapy are used in pulsed format [44]. Electricity, electromagnetic fields and ultrasound are applied with particular pulse structures. It may be possible to gain some insight into the effect of pulsing structures in LLLT by a brief review of the other pulsed modalities. Transcutaneous electrical neural stimulation (TENS) is the application of pulses of electric current to the skin [45]. This application stimulates the brain and has been used for the treatment of various psychological and neurological conditions, including Parkinson’s, epilepsy, chronic pain, depression, and neuromuscular rehabilitation. Frequencies usually fall between 5 and 25 Hz, but may range from 2 to 80 Hz [46]. Deep brain stimulation (DBS) is a surgical treatment involving the implantation of a brain pacemaker, a medical device that sends electrical impulses to specific parts of the brain. DBS has the potential to provide substantial benefit to patients suffering from a variety of neurological conditions, including epilepsy, Parkinson’s disease, dystonia, Tourette’s syndrome, and depression [47]. The Food and Drug Administration (FDA) approved DBS at 130 Hz as a treatment for essential tremor in 1997, for Parkinson’s disease in 2002, and dystonia in 2003. Pulsed electromagnetic field (PEMF) therapy has been used for a wide range of conditions, including bone healing and regeneration [48], osteoporosis [49], arthritis [50] wound healing and pain [51], carpal tunnel syndrome [52], spinal cord injury [53], nerve regeneration [54], soft tissue injuries [55], and cancer [56]. Frequencies used for these conditions range from 1 Hz (“low”) to 200 Hz (“high”). Transcranial magnetic stimulation (TMS) is a noninvasive method used to excite neurons in the brain. Weak electric currents are induced by butterfly coils positioned above the head. TMS has been approved for the treatment of resistant depression in several countries and is under investigation for migraine [57], aphasia [58], and tinnitus [59]. Low-intensity pulsed ultrasound (LIPUS) utilizes a non-thermal mechanism of action, which can be used to promote bone healing by inducing the expression of growth factors and prostaglandins, which stimulate osteoblasts, chondrocytes and fibroblasts [60].

CONCLUSION

There has been remarkably little information available in the peer-reviewed literature on the rationale for using pulsed lasers or pulsed light in LLLT rather than CW. Moreover there is no consensus on the effects of different frequencies and pulse parameters on the physiology and therapeutic response of the various disease states that are often treated with laser therapy. This has allowed manufacturers to claim advantages of pulsing without hard evidence to back up their claims.

CW light is the gold standard and has been used for all LLLT applications. However, this review of the literature indicates that overall pulsed light may be superior to CW light with everything else being equal. This seemed to be particularly true for wound healing and post-stroke management. On the other hand, PW as a solo treatment may be less beneficial than CW in patients requiring nerve regeneration. This could possibly be explained by the mechanism of action LLLT that can either cause cell stimulation or cell inhibition or both stimulation and inhibition at the same time on different cell types. It is possible that stimulation in neurons is desired to promote neurogenesis following stroke (increased mitochondrial synthesis of ATP results in more energy for neurons to regenerate themselves), whereas inhibition of inflammatory cells, inhibition of immune response or inhibition of the glial scar may also occur at the same time. The logic in favor of PW is that cells may need periods of rest, without which they can no longer be stimulated further.

Considering that the biology of LLLT is known to be complex, it is likely that there may several optimal sets of pulse parameters and that these may relate to the specific wavelengths and chromophores and may well also be affected by other optical properties of tissues.

It was impossible to draw any meaningful correlations between pulse frequency and pathological condition, due to the wide-ranging and disparate data. As for other pulse parameters, these were in general poorly and inconsistentl

There were 5 groups of rats in the study. Groups 2 through 5 were injured in a controlled operation. Groups 3 through 5 were given different dosage of laser therapy.

Group 1: No damage
Group 2: Damaged and then area treated with placebo
Group 3: Damaged and given non-optimal therapy
Group 4: Damaged and optimal dosage
Group 5: Damaged and non-optimal therapy

Any laser therapy showed a significant improvement over no laser and the optimal laser (Group 4 with a total dosage of 4 j/cm^2) showed no difference from the uninjured tissue. The image below show the group 2 and group 4 tissue under a polarization microscope.

Photobiomodulation (PBM) describes the use of red or near-infrared light to stimulate, heal, regenerate, and protect tissue that has either been injured, is degenerating, or else is at risk of dying. One of the organ systems of the human body that is most necessary to life, and whose optimum functioning is most worried about by humankind in general, is the brain. The brain suffers from many different disorders that can be classified into three broad groupings: traumatic events (stroke, traumatic brain injury, and global ischemia), degenerative diseases (dementia, Alzheimer's and Parkinson's), and psychiatric disorders (depression, anxiety, post traumatic stress disorder). There is some evidence that all these seemingly diverse conditions can be beneficially affected by applying light to the head. There is even the possibility that PBM could be used for cognitive enhancement in normal healthy people. In this transcranial PBM (tPBM) application, near-infrared (NIR) light is often applied to the forehead because of the better penetration (no hair, longer wavelength). Some workers have used lasers, but recently the introduction of inexpensive light emitting diode (LED) arrays has allowed the development of light emitting helmets or “brain caps”. This review will cover the mechanisms of action of photobiomodulation to the brain, and summarize some of the key pre-clinical studies and clinical trials that have been undertaken for diverse brain disorders.

Keywords: Photobiomodulation, Low level laser (light) therapy, Ischemic stroke, Traumatic brain injury, Alzheimer's disease, Parkinson's disease, Major depression, Cognitive enhancement

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Graphical abstract

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1. Introduction

Photobiomodulation (PBM) as it is known today (the beneficial health benefits of light therapy had been known for some time before), was accidently discovered in 1967, when Endre Mester from Hungary attempted to repeat an experiment recently published by McGuff in Boston, USA [1]. McGuff had used a beam from the recently discovered ruby laser [2], to destroy a cancerous tumor that had been experimentally implanted into a laboratory rat. However (unbeknownst to Mester) the ruby laser that had been built for him, was only a tiny fraction of the power of the laser that had previously been used by McGuff. However, instead of curing the experimental tumors with his low-powered laser, Mester succeeded in stimulating hair regrowth and wound healing in the rats, in the sites where the tumors had been implanted [3], [4]. This discovery led to a series of papers describing what Mester called “laser biostimulation”, and soon became known as “low level laser therapy” (LLLT) [5], [6], [7].

LLLT was initially primarily studied for stimulation of wound healing, and reduction of pain and inflammation in various orthopedic conditions such as tendonitis, neck pain, and carpal tunnel syndrome [8]. The advent of light emitting diodes (LED) led to LLLT being renamed as “low level light therapy”, as it became more accepted that the use of coherent lasers was not absolutely necessary, and a second renaming occurred recently [9] when the term PBM was adopted due to uncertainties in the exact meaning of “low level”.

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2. Mechanisms of action of photobiomodulation

2.1. Mitochondria and cytochrome c oxidase

The most well studied mechanism of action of PBM centers around cytochrome c oxidase (CCO), which is unit four of the mitochondrial respiratory chain, responsible for the final reduction of oxygen to water using the electrons generated from glucose metabolism [10]. The theory is that CCO enzyme activity may be inhibited by nitric oxide (NO) (especially in hypoxic or damaged cells). This inhibitory NO can be dissociated by photons of light that are absorbed by CCO (which contains two heme and two copper centers with different absorption spectra) [11]. These absorption peaks are mainly in the red (600–700 nm) and near-infrared (760–940 nm) spectral regions. When NO is dissociated, the mitochondrial membrane potential is increased, more oxygen is consumed, more glucose is metabolized and more ATP is produced by the mitochondria.

2.2. Reactive oxygen species, nitric oxide, blood flow

It has been shown that there is a brief increase in reactive oxygen species (ROS) produced in the mitochondria when they absorb the photons delivered during PBM. The idea is that this burst of ROS may trigger some mitochondrial signaling pathways leading to cytoprotective, anti-oxidant and anti-apoptotic effects in the cells [12]. The NO that is released by photodissociation acts as a vasodilator as well as a dilator of lymphatic flow. Moreover NO is also a potent signaling molecule and can activate a number of beneficial cellular pathways [13]. Fig. 2 illustrates these mechanisms.

Fig. 2

Tissue specific processes that occur after PBM and benefit a range of brain disorders. BDNF, brain-derived neurotrophic factor; LLLT, low level light therapy; NGF, nerve growth factor; NT-3, neurotrophin 3; PBM, photobiomodulation; SOD, superoxide dismutase. ...

2.3. Light sensitive ion channels and calcium

It is quite clear that there must be some other type of photoacceptor, in addition to CCO, as is clearly demonstrated by the fact that wavelengths substantially longer than the red/NIR wavelengths discussed above, can also produce beneficial effects is some biological scenarios. Wavelengths such as 980 nm [14], [15], 1064 nm laser [16], and 1072 nm LED [17], and even broad band IR light [18] have all been reported to carry out PBM type effects. Although the photoacceptor for these wavelengths has by no means been conclusively identified, the leading hypothesis is that it is primarily water (perhaps nanostructured water) located in heat or light sensitive ion channels. Clear changes in intracellular calcium can be observed, that could be explained by light-mediated opening of calcium ion channels, such as members of the transient receptor potential (TRP) super-family [19]. TRP describes a large family of ion channels typified by TRPV1, recently identified as the biological receptor for capsaicin (the active ingredient in hot chili peppers) [20]. The biological roles of TRP channels are multifarious, but many TRP channels are involved in heat sensing and thermoregulation [21].

2.4. Signaling mediators and activation of transcription factors

Most authors suggest that the beneficial effects of tPBM on the brain can be explained by increases in cerebral blood flow, greater oxygen availability and oxygen consumption, improved ATP production and mitochondrial activity [22], [23], [24]. However there are many reports that a brief exposure to light (especially in the case of experimental animals that have suffered some kind of acute injury or traumatic insult) can have effects lasting days, weeks or even months [25]. This long-lasting effect of light can only be explained by activation of signaling pathways and transcription factors that cause changes in protein expression that last for some considerable time. The effects of PBM on stimulating mitochondrial activity and blood flow is of itself, unlikely to explain long-lasting effects. A recent review listed no less than fourteen different transcription factors and signaling mediators, that have been reported to be activated after light exposure [10].

Fig. 1 illustrates two of the most important molecular photoreceptors or chromophores (cytochrome c oxidase and heat-gated ion channels) inside neuronal cells that absorb photons that penetrate into the brain. The signaling pathways and activation of transcription factors lead to the eventual effects of PBM in the brain.

Fig. 1

Molecular and intracellular mechanisms of transcranial low level laser (light) or photobiomodulation. AP1, activator protein 1; ATP, adenosine triphosphate; Ca2 +, calcium ions; cAMP, cyclic adenosine monophosphate; NF-kB, nuclear factor kappa ...

Fig. 2 illustrates some more tissue specific mechanisms that lead on from the initial photon absorption effects explained in Fig. 1. A wide variety of processes can occur that can benefit a correspondingly wide range of brain disorders. These processes can be divided into short-term stimulation (ATP, blood flow, lymphatic flow, cerebral oxygenation, less edema). Another group of processes center around neuroprotection (upregulation of anti-apoptotic proteins, less excitotoxity, more antioxidants, less inflammation). Finally a group of processes that can be grouped under “help the brain to repair itself” (neurotrophins, neurogenesis and synaptogenesis).

2.5. Biphasic dose response and effect of coherence

The biphasic dose response (otherwise known as hormesis, and reviewed extensively by Calabrese et al. [26]) is a fundamental biological law describing how different biological systems can be activated or stimulated by low doses of any physical insult or chemical substance, no matter how toxic or damaging this insult may be in large doses. The most well studied example of hormesis is that of ionizing radiation, where protective mechanisms are induced by very low exposures, that can not only protect against subsequent large doses of ionizing radiation, but can even have beneficial effects against diseases such as cancer using whole body irradiation [27].

There are many reports of PBM following a biphasic dose response (sometimes called obeying the Arndt-Schulz curve [28], [29]. A low dose of light is beneficial, but raising the dose produces progressively less benefit until eventually a damaging effect can be produced at very high light [30]. It is often said in this context that “more does not mean more”.

Another question that arises in the field of PBM is whether the coherent monochromatic lasers that were used in the original discovery of the effect, and whose use continued for many years, are superior to the rather recent introduction of LEDs, that are non-coherent and have a wider band-spread (generally 30 nm full-width half-maximum). Although there are one or two authors who continue to believe that coherent lasers are superior [31], most commentators feel that other parameters such as wavelength, power density, energy density and total energy are the most important determinants of efficacy [8].

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3. Tissue optics, direct versus systemic effects, light sources

3.1. Light penetration into the brain

Due to the growing interest in PBM of the brain, several tissue optics laboratories have investigated the penetration of light of different wavelengths through the scalp and the skull, and to what depths into the brain this light can penetrate. This is an intriguing question to consider, because at present it is unclear exactly what threshold of power density in mW/cm2 is required in the b5rain to have a biological effect. There clearly must be a minimum value below which the light can be delivered for an infinite time without doing anything, but whether this is in the region of μW/cm2 or mW/cm2 is unknown at present.

Functional near-infrared spectroscopy (fNIRS) using 700–900 nm light has been established as a brain imaging technique that can be compared to functional magnetic resonance imaging (fMRI) [32]. Haeussinger et al. estimated that the mean penetration depth (5% remaining intensity) of NIR light through the scalp and skull was 23:6 + 0:7 mm [33]. Other studies have found comparable results with variations depending on the precise location on the head and wavelength [34], [35].

Jagdeo et al. [36] used human cadaver heads (skull with intact soft tissue) to measure penetration of 830 nm light, and found that penetration depended on the anatomical region of the skull (0.9% at the temporal region, 2.1% at the frontal region, and 11.7% at the occipital region). Red light (633 nm) hardly penetrated at all. Tedord et al. [37] also used human cadaver heads to compare penetration of 660 nm, 808 nm, and 940 nm light. They found that 808 nm light was best and could reach a depth in the brain of 40–50 mm. Lapchak et al. compared the transmission of 810 nm light through the skulls of four different species, and found mouse transmitted 40%, while for rat it was 21%, rabbit it was 11.3 and for human skulls it was only 4.2% [38]. Pitzschke and colleagues compared penetration of 670 nm and 810 nm light into the brain when delivered by a transcranial or a transphenoidal approach, and found that the best combination was 810 nm delivered transphenoidally [39]. In a subsequent study these authors compared the effects of storage and processing (frozen or formalin-fixed) on the tissue optical properties of rabbit heads [40]. Yaroslavsky et al. examined light penetration of different wavelengths through different parts of the brain tissue (white brain matter, gray brain matter, cerebellum, and brainstem tissues, pons, thalamus). Best penetration was found with wavelengths between 1000 and 1100 nm [41].

Henderson and Morries found that between 0.45% and 2.90% of 810 nm or 980 nm light penetrated through 3 cm of scalp, skull and brain tissue in ex vivo lamb heads [42].

3.2. Systemic effects

It is in fact very likely that the beneficial effects of PBM on the brain cannot be entirely explained by penetration of photons through the scalp and skull into the brain itself. There have been some studies that have explicitly addressed this exact issue. In a study of PBM for Parkinson's disease in a mouse model [43]. Mitrofanis and colleagues compared delivering light to the mouse head, and also covered up the head with aluminum foil so that they delivered light to the remainder of the mouse body. They found that there was a highly beneficial effect on neurocognitive behavior with irradiation to the head, but nevertheless there was also a statistically significant (although less pronounced benefit, referred to by these authors as an ‘abscopal effect”) when the head was shielded from light [44]. Moreover Oron and co-workers [45] have shown that delivering NIR light to the mouse tibia (using either surface illumination or a fiber optic) resulted in improvement in a transgenic mouse model of Alzheimer's disease (AD). Light was delivered weekly for 2 months, starting at 4 months of age (progressive stage of AD). They showed improved cognitive capacity and spatial learning, as compared to sham-treated AD mice. They proposed that the mechanism of this effect was to stimulate c-kit-positive mesenchymal stem cells (MSCs) in autologous bone marrow (BM) to enhance the capacity of MSCs to infiltrate the brain, and clear β-amyloid plaques [46]. It should be noted that the calvarial bone marrow of the skull contains substantial numbers of stem cells [47].

3.3. Laser acupuncture

Laser acupuncture is often used as an alternative or as an addition to traditional Chinese acupuncture using needles [48]. Many of the applications of laser acupuncture have been for conditions that affect the brain [49] such as Alzheimer's disease [50] and autism [51] that have all been investigated in animal models. Moreover laser acupuncture has been tested clinically [52].

3.4. Light sources

A wide array of different light sources (lasers and LEDs) have been employed for tPBM. One of the most controversial questions which remains to be conclusively settled, is whether a coherent monochromatic laser is superior to non-coherent LEDs typically having a 30 nm band-pass (full width half maximum). Although wavelengths in the NIR region (800–1100 nm) have been the most often used, red wavelengths have sometimes been used either alone, or in combination with NIR. Power levels have also varied markedly from Class IV lasers with total power outputs in the region of 10 W [53], to lasers with more modest power levels (circa 1 W). LEDs can also have widely varying total power levels depending on the size of the array and the number and power of the individual diodes. Power densities can also vary quite substantially from the Photothera laser [54] and other class IV lasers , which required active cooling (~ 700 mW/cm2) to LEDs in the region of 10–30 mW/cm2.

3.5. Usefulness of animal models when testing tPBM for brain disorders

One question that is always asked in biomedical research, is how closely do the laboratory models of disease (which are usually mice or rats) mimic the human disease for which new treatments are being sought? This is no less critical a question when the areas being studied include brain disorders and neurology. There now exist a plethora of transgenic mouse models of neurological disease [55], [56]. However in the present case, where the proposed treatment is almost completely free of any safety concerns, or any reported adverse side effects, it can be validly questioned as to why the use of laboratory animal models should be encouraged. Animal models undoubtedly have disadvantages such as failure to replicate all the biological pathways found in human disease, difficulty in accurately measuring varied forms of cognitive performance, small size of mice and rats compared to humans, short lifespan affecting the development of age related diseases, and lack of lifestyle factors that adversely affect human diseases. Nevertheless, small animal models are less expensive, and require much less time and effort to obtain results than human clinical trials, so it is likely they will continue to be used to test tPBM for the foreseeable future.

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4. PBM for stroke

4.1. Animal models

Perhaps the most well-investigated application of PBM to the brain, lies in its possible use as a treatment for acute stroke [57]. Animal models such as rats and rabbits, were first used as laboratory models, and these animals had experimental strokes induced by a variety of methods and were then treated with light (usually 810 nm laser) within 24 h of stroke onset [58]. In these studies intervention by tLLLT within 24 h had meaningful beneficial effects. For the rat models, stroke was induced by middle cerebral artery occlusion (MCAO) via an insertion of a filament into the carotid artery or via craniotomy [59], [60]. Stroke induction in the “rabbit small clot embolic model” (RSCEM) was by injection of a preparation of small blood clots (made from blood taken from a second donor rabbit) into a catheter placed in the right internal carotid artery [61]. These studies and the treatments and results are listed in Table 1.

Table 1

Reports of transcranial LLLT used for stroke in animal models.

CW, continuous wave; LLLT, low level light therapy; MCAO, middle cerebral artery occlusion; NOS, nitric oxide synthase; RSCEM, rabbit small clot embolic model; TGFβ1, transforming growth factor β1.

4.2. Clinical trials for acute stroke

Treatment of acute stroke was addressed in a series of three clinical trials called “Neurothera Effectiveness and Safety Trials” (NEST-1 [65], NEST-2 [66], and NEST-3 [67]) using an 810 nm laser applied to the shaved head within 24 h of patients suffering an ischemic stroke. The first study, NEST-1, enrolled 120 patients between the ages of 40 to 85 years of age with a diagnosis of ischemic stroke involving a neurological deficit that could be measured. The purpose of this first clinical trial was to demonstrate the safety and effectiveness of laser therapy for stroke within 24 h [65]. tPBM significantly improved outcome in human stroke patients, when applied at ~ 18 h post-stroke, over the entire surface of the head (20 points in the 10/20 EEG system) regardless of stroke [65]. Only one laser treatment was administered, and 5 days later, there was significantly greater improvement in the Real- but not in the Sham-treated group (p < 0.05, NIH Stroke Severity Scale). This significantly greater improvement was still present at 90 days post-stroke, where 70% of the patients treated with Real-LLLT had a successful outcome, while only 51% of Sham-controls did. The second clinical trial, NEST-2, enrolled 660 patients, aged 40 to 90, who were randomly assigned to one of two groups (331 to LLLT, 327 to sham) [68]. Beneficial results (p < 0.04) were found for the moderate and moderate-severe (but not for the severe) stroke patients, who received the Real laser protocol [68]. These results suggested that the overall severity of the individual stroke should be taken into consideration in future studies, and very severe patients are unlikely to recover with any kind of treatment. The last clinical trial, NEST-3, was planned for 1000 patients enrolled. Patients in this study were not to receive tissue plasminogen activator, but the study was prematurely terminated by the DSMB for futility (an expected lack of statistical significance) [67]. NEST-1 was considered successful, even though as a phase 1 trial, it was not designed to show efficacy. NEST-2 was partially successful when the patients were stratified, to exclude very severe strokes or strokes deep within the brain [66]. There has been considerable discussion in the scientific literature on precisely why the NEST-3 trial failed [69]. Many commentators have wondered how could tPBM work so well in the first trial, in a sub-group in the second trial, and fail in the third trial. Lapchak's opinion is that the much thicker skull of humans compared to that of the other animals discussed above (mouse, rat and rabbit), meant that therapeutically effective amounts of light were unlikely to reach the brain [69]. Moreover the time between the occurrence of a stroke and initiation of the PBMT may be an important factor. There are reports in the literature that neuroprotection must be administered as soon as possible after a stroke [70], [71]. Furthermore, stroke trials in particular should adhere to the RIGOR (rigorous research) guidelines and STAIR (stroke therapy academic industry roundtable) criteria [72]. Other contributory causes to the failure of NEST-3 may have been included the decision to use only one single tPBM treatment, instead of a series of treatments. Moreover, the optimum brain areas to be treated in acute stroke remain to be determined. It is possible that certain areas of the brain that have sustained ischemic damage should be preferentially illuminated and not others.

4.3. Chronic stroke

Somewhat surprisingly, there have not as yet been many trials of PBM for rehabilitation of stroke patients with only the occasional report to date. Naeser reported in an abstract the use of tPBM to treat chronic aphasia in post-stroke patients [73]. Boonswang et al. [74] reported a single patient case in which PBM was used in conjunction with physical therapy to rehabilitate chronic stroke damage. However the findings that PBM can stimulate synaptogenesis in mice with TBI, does suggest that tPBM may have particular benefits in rehabilitation of stroke patients. Norman Doidge, in Toronto, Canada has described the use of PBM as a component of a neuroplasticity approach to rehabilitate chronic stroke patients [75].

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5. PBM for traumatic brain injury (TBI)

5.1. Mouse and rat models

There have been a number of studies looking at the effects of PBM in animal models of TBI. Oron's group was the first [76] to demonstrate that a single exposure of the mouse head to a NIR laser (808 nm) a few hours after creation of a TBI lesion could improve neurological performance and reduce the size of the brain lesion. A weight-drop device was used to induce a closed-head injury in the mice. An 808 nm diode laser with two energy densities (1.2–2.4 J/cm2 over 2 min of irradiation with 10 and 20 mW/cm2) was delivered to the head 4 h after TBI was induced. Neurobehavioral function was assessed by the neurological severity score (NSS). There were no significant difference in NSS between the power densities (10 vs 20 mW/cm2) or significant differentiation between the control and laser treated group at early time points (24 and 48 h) post TBI. However, there was a significant improvement (27% lower NSS score) in the PBM group at times of 5 days to 4 weeks. The laser treated group also showed a smaller loss of cortical tissue than the sham group [76].

Hamblin's laboratory then went on (in a series of papers [76]) to show that 810 nm laser (and 660 nm laser) could benefit experimental TBI both in a closed head weight drop model [77], and also in controlled cortical impact model in mice [25]. Wu et al. [77] explored the effect that varying the laser wavelengths of LLLT had on closed-head TBI in mice. Mice were randomly assigned to LLLT treated group or to sham group as a control. Closed-head injury (CHI) was induced via a weight drop apparatus. To analyze the severity of the TBI, the neurological severity score (NSS) was measured and recorded. The injured mice were then treated with varying wavelengths of laser (665, 730, 810 or 980 nm) at an energy level of 36 J/cm2 at 4 h directed onto the scalp. The 665 nm and 810 nm groups showed significant improvement in NSS when compared to the control group at day 5 to day 28. Results are shown in Fig. 3. Conversely, the 730 and 980 nm groups did not show a significant improvement in NSS and these wavelengths did not produce similar beneficial effects as in the 665 nm and 810 nm LLLT groups [77]. The tissue chromophore cytochrome c oxidase (CCO) is proposed to be responsible for the underlying mechanism that produces the many PBM effects that are the byproduct of LLLT. COO has absorption bands around 665 nm and 810 nm while it has low absorption bands at the wavelength of 730 nm [78]. It should be noted that this particular study found that the 980 nm did not produce the same positive effects as the 665 nm and 810 nm wavelengths did; nevertheless previous studies did find that the 980 nm wavelength was an active one for LLLT. Wu et al. proposed that these dissimilar results may be due to the variance in the energy level, irradiance, etc. between the other studies and this particular study [77].

Fig. 3

tPBM for TBI in a mouse model. Mice received a closed head injury and 4 hours later a single exposure of the head to one of four different lasers (36 J/cm2 delivered at 150 mW/cm2 over 4 min with spot size 1-cm diameter) ...

Ando et al. [25] used the 810 nm wavelength laser parameters from the previous study and varied the pulse modes of the laser in a mouse model of TBI. These modes consisted of either pulsed wave at 10 Hz or at 100 Hz (50% duty cycle) or continuous wave laser. For the mice, TBI was induced with a controlled cortical impact device via open craniotomy. A single treatment with an 810 nm Ga-Al-As diode laser with a power density of 50 mW/m2 and an energy density of 36 J/cm2 was given via tLLLT to the closed head in mice for a duration of 12 min at 4 h post CCI. At 48 h to 28 days post TBI, all laser treated groups had significant decreases in the measured neurological severity score (NSS) when compared to the control (Fig. 4A). Although all laser treated groups had similar NSS improvement rates up to day 7, the PW 10 Hz group began to show greater improvement beyond this point as seen in Fig. 4. At day 28, the forced swim test for depression and anxiety was used and showed a significant decrease in the immobility time for the PW 10 Hz group. In the tail suspension test which measures depression and anxiety, there was also a significant decrease in the immobility time at day 28, and this time also at day 1, in the PW 10 Hz group.

Fig. 4

tPBM for controlled cortical impact TBI in a mouse model. (A) Mice received a single exposure (810 nm laser, 36 J/cm2 delivered at 50 mW/cm2 over 12 min) [121]. (B) Mice received 3 daily exposures starting 4 h post-TBI ...

Studies using immunofluorescence of mouse brains showed that tPBM increased neuroprogenitor cells in the dentate gyrus (DG) and subventricular zone at 7 days after the treatment [79]. The neurotrophin called brain derived neurotrophic factor (BDNF) was also increased in the DG and SVZ at 7 days , while the marker (synapsin-1) for synaptogenesis and neuroplasticity was increased in the cortex at 28 days but not in the DG, SVZ or at 7 days [80] (Fig. 4B). Learning and memory as measured by the Morris water maze was also improved by tPBM [81]. Whalen's laboratory [82] and Whelan's laboratory [83] also successfully demonstrated therapeutic benefits of tPBM for TBI in mice and rats respectively.

Zhang et al. [84] showed that secondary brain injury occurred to a worse degree in mice that had been genetically engineered to lack “Immediate Early Response” gene X-1 (IEX-1) when exposed to a gentle head impact (this injury is thought to closely resemble mild TBI in humans). Exposing IEX-1 knockout mice to LLLT 4 h post injury, suppressed proinflammatory cytokine expression of interleukin (IL)-Iβ and IL-6, but upregulated TNF-α. The lack of IEX-1 decreased ATP production, but exposing the injured brain to LLLT elevated ATP production back to near normal levels.

Dong et al. [85] even further improved the beneficial effects of PBM on TBI in mice, by combining the treatment with metabolic substrates such as pyruvate and/or lactate. The goal was to even further improve mitochondrial function. This combinatorial treatment was able to reverse memory and learning deficits in TBI mice back to normal levels, as well as leaving the hippocampal region completely protected from tissue loss; a stark contrast to that found in control TBI mice that exhibited severe tissue loss from secondary brain injury.

5.2. TBI in humans

Margaret Naeser and collaborators have tested PBM in human subjects who had suffered TBI in the past [86]. Many sufferers from severe or even moderate TBI, have very long lasting and even life-changing sequelae (headaches, cognitive impairment, and difficulty sleeping) that prevent them working or living any kind or normal life. These individuals may have been high achievers before the accident that caused damage to their brain [87]. Initially Naeser published a report [88] describing two cases she treated with PBM applied to the forehead twice a week. A 500 mW continuous wave LED source (mixture of 660 nm red and 830 nm NIR LEDs) with a power density of 22.2 mW/cm2 (area of 22.48 cm2), was applied to the forehead for a typical duration of 10 min (13.3 J/cm2). In the first case study the patient reported that she could concentrate on tasks for a longer period of time (the time able to work at a computer increased from 30 min to 3 h). She had a better ability to remember what she read, decreased sensitivity when receiving haircuts in the spots where LLLT was applied, and improved mathematical skills after undergoing LLLT. The second patient had statistically significant improvements compared to prior neuropsychological tests after 9 months of treatment. The patient had a 2 standard deviation (SD) increase on tests of inhibition and inhibition accuracy (9th percentile to 63rd percentile on the Stroop test for executive function and a 1 SD increase on the Wechsler Memory scale test for the logical memory test (83rd percentile to 99th percentile) [89].

Naeser et al. then went on to report a case series of a further eleven patients [90]. This was an open protocol study that examined whether scalp application of red and near infrared (NIR) light could improve cognition in patients with chronic, mild traumatic brain injury (mTBI). This study had 11 participants ranging in age from 26 to 62 (6 males, 5 females) who suffered from persistent cognitive dysfunction after mTBI. The participants' injuries were caused by motor vehicle accidents, sports related events and for one participant, an improvised explosive device (IED) blast. tLLLT consisted of 18 sessions (Monday, Wednesday, and Friday for 6 weeks) and commenced anywhere from 10 months to 8 years post-TBI. A total of 11 LED clusters (5.25 cm in diameter, 500 mW, 22.2 mW/cm2, 13 J/cm2) were applied for about 10 min per session (5 or 6 LED placements per set, Set A and then Set B, in each session). Neuropsychological testing was performed pre-LED application and 1 week, 1 month and 2 months after the final treatment. Naeser and colleagues found that there was a significant positive linear trend observed for the Stroop Test for executive function, in trial 2 inhibition (p = 0.004); Stroop, trial 4 inhibition switching (p = 0.003); California Verbal Learning Test (CVLT)-II, total trials 1–5 (p = 0.003); CVLT-II, long delay free recall (p = 0.006). Improved sleep and fewer post-traumatic stress disorder (PTSD) symptoms, if present beforehand, were observed after treatment. Participants and family members also reported better social function and a better ability to perform interpersonal and occupational activities. Although these results were significant, further placebo-controlled studies will be needed to ensure the reliability of this these data [90].

Henderson and Morries [91] used a high-power NIR laser (10–15 W at 810 and 980 nm) applied to the head to treat a patient with moderate TBI. The patient received 20 NIR applications over a 2-month period. They carried out anatomical magnetic resonance imaging (MRI) and perfusion single-photon emission computed tomography (SPECT). The patient showed decreased depression, anxiety, headache, and insomnia, whereas cognition and quality of life improved, accompanied by changes in the SPECT imaging.

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6. PBM for Alzheimer's disease (AD)

6.1. Animal models

There was a convincing study [92] carried out in an AβPP transgenic mouse of AD. tPBM (810 nm laser) was administered at different doses 3 times/week for 6 months starting at 3 months of age. The numbers of Aβ plaques were significantly reduced in the brain with administration of tPBM in a dose-dependent fashion. tPBM mitigated the behavioral effects seen with advanced amyloid deposition and reduced the expression of inflammatory markers in the transgenic mice. In addition, TLT showed an increase in ATP levels, mitochondrial function, and c-fos expression suggesting that there was an overall improvement in neurological function.

6.2. Humans

There has been a group of investigators in Northern England who have used a helmet built with 1072 nm LEDs to treat AD, but somewhat surprisingly no peer-reviewed publications have described this approach [93]. However a small pilot study (19 patients) that took the form of a randomized placebo-controlled trial investigated the effect of the Vielight Neuro system (see Fig. 5A) (a combination of tPBM and intranasal PBM) on patients with dementia and mild cognitive impairment [94]. This was a controlled single blind pilot study in humans to investigate the effects of PBM on memory and cognition. 19 participants with impaired memory/cognition were randomized into active and sham treatments over 12 weeks with a 4-week no-treatment follow-up period. They were assessed with MMSE and ADAS-cog scales. The protocol involved in-clinic use of a combined transcranial-intranasal PBM device; and at-home use of an intranasal-only PBM device and participants/ caregivers noted daily experiences in a journal. Active participants with moderate to severe impairment (MMSE scores 5–24) showed significant improvements (5-points MMSE score) after 12 weeks. There was also a significant improvement in ADAS-cog scores (see Fig. 5B). They also reported better sleep, fewer angry outbursts and decreased anxiety and wandering. Declines were noted during the 4-week no-treatment follow-up period. Participants with mild impairment to normal (MMSE scores of 25 to 30) in both the active and sham sub-groups showed improvements. No related adverse events were reported.

Fig. 5

tPBM for Alzheimer's disease. (A) Nineteen patients were randomized to receive real or sham tPBM (810 nm LED, 24.6 J/cm2 at 41 mW/cm2). (B) Significant decline in ADAS-cog (improved cognitive performance) in real but not sham (unpublished ...

An interesting paper from Russia [95] described the use of intravascular PBM to treat 89 patients with AD who received PBM (46 patients) or standard treatment with memantine and rivastigmine (43 patients). The PBM consisted of threading a fiber-optic through a cathéter in the fémoral artery and advancing it to the distal site of the anterior and middle cerebral arteries and delivering 20 mW of red laser for 20–40 min. The PBM group had improvement in cerebral microcirculation leading to permanent (from 1 to 7 years) reduction in dementia and cognitive recovery.

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7. Parkinson's disease

The majority of studies on PBM for Parkinson's disease have been in animal models and have come from the laboratory of John Mitrofanis in Australia [96]. Two basic models of Parkinson's disease were used. The first employed administration of the small molecule (MPTP or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) to mice [97]. MPTP was discovered as an impurity in an illegal recreational drug to cause Parkinson's like symptoms (loss of substantia nigra cells) in young people who had taken this drug [98]. Mice were treated with tPBM (670-nm LED, 40 mW/cm2, 3.6 J/cm2) 15 min after each MPTP injection repeated 4 times over 30 h. There were significantly more (35%–45%) dopaminergic cells in the brains of the tPBM treated mice [97]. A subsequent study showed similar results in a chronic mouse model of MPTP-induced Parkinson's disease [99]. They repeated their studies in another mouse model of Parkinson's disease, the tau transgenic mouse strain (K3) that has a progressive degeneration of dopaminergic cells in the substantia nigra pars compacta (SNc) [100]. They went on to test a surgically implanted intracranial fiber designed to deliver either 670 nm LED (0.16 mW) or 670 nm laser (67 mW) into the lateral ventricle of the brain in MPTP-treated mice [101]. Both low power LED and high power laser were effective in preserving SNc cells, but the laser was considered to be unsuitable for long-term use (6 days) due to excessive heat production. As mentioned above, these authors also reported a protective effect of abscopal light exposure (head shielded) in this mouse model [43]. Recently this group has tested their implanted fiber approach in a model of Parkinson's disease in adult Macaque monkeys treated with MPTP [102]. Clinical evaluation of Parkinson's symptoms (posture, general activity, bradykinesia, and facial expression) in the monkeys were improved at low doses of light (24 J or 35 J) compared to high doses (125 J) [103].

The only clinical report of PBM for Parkinson's disease in humans was an abstract presented in 2010 [104]

 Abstract

Laser radiation provides a means to control the fields of temperature and thermo mechanical stress, mass transfer, and modification of fine structure of the cartilage matrix. The aim of this outlook paper is to review physical and biological aspects of laser-induced regeneration of cartilage and to discuss the possibilities and prospects of its clinical applications. The problems and the pathways of tissue regeneration, the types and features of cartilage will be introduced first. Then we will review various actual and prospective approaches for cartilage repair; consider possible mechanisms of laser-induced regeneration. Finally, we present the results in laser regeneration of joints and spine disks cartilages and discuss some future applications of lasers in regenerative medicine.

1.

Introduction

Cartilage is a kind of highly specialized connective tissue. The structural variety of the cartilage provides its unique biomechanical capacity to bear different kinds of static and dynamic loads over a wide range of intensity. Biological role of cartilage structures stems from their critical significance for growth and development as well as for all kinds of body movements. The exceptional importance of cartilage elements for individual survival is, probably, due to mechanisms of natural selection, resulting in limited reparative potential of this tissue. Scanty cellular sources and low metabolic rate along with avascularity of cartilage contribute to its decreased regeneration ability. As a result of these strong limitations, the injuries of cartilage caused by inflammation, traumas, degeneration, and aging usually become chronic and recalcitrant to any kind of medical treatment. In the USA, according to tentative estimations, the prevalence of all forms of arthritis has been calculated in order of 40 million people; and the annual medical care costs were about 65 billion USD.1Degenerative spine diseases are a major cause of back pain that deteriorates the quality of life of patients and often leads to disability. Direct and indirect medical expenses are estimated as more than 90 billion per year.2

High prevalence and incidence, as well as the social and economic significance of cartilage pathology, attract great interest to this problem. Considerable efforts have been devoted to study various approaches to restore cartilage structures and to stimulate intrinsic capabilities of the tissue to regeneration. There are several treatment modalities of cartilage restoration suggested for clinical use (see Ref. 3 and referred literature): 1. surgical techniques; 2. controllable cell delivery to the lesion; and 3. tissue engineering applications of biodegradable materials (scaffolds) with cell-seeding and modification of cartilage reparative response by different growth factors and cytokines. Although there is a wealth of information regarding the substitution of lost cartilage by the mentioned approaches, the problem of cartilage repair is still unsolved. The long term results show no completed cartilage regeneration; in many cases, the new growing tissue materially differs from the well organized original cartilage. The reasons of insufficient cartilage reparation are connected with its structural and functional organization and with the difficulties of the precise control of the external physical and chemical effects.4, 5 Regeneration of cartilage may be realized in accordance with the natural genetic program of the cells. The efficacy of any approach aimed to control the regeneration process depends on the solution of three tasks: 1. the ability to reproduce the normal cell differentiation sequence from the progenitor cells to mature chondrocytes, 2. stimulation of the specific subpopulations of the resident cells to proliferation and/or new matrix production, and (c) achievement of adequate spatial organization of the new growing tissue. Probably, the most important feature of the laser-based treatment is the involvement and activation of the intrinsic mechanisms of cartilage repair. Many papers are devoted to the effect of low-intensive lasers on cartilage functional state and reparative ability. However, the effectiveness, as well as the placebo-versus-treatment ratio for low level laser therapy, is still under considerable dispute. A more detailed discussion of this issue may be found elsewhere.6 This paper is mainly limited with a consideration of the effect of nonablative laser radiation on the cartilaginous cells through their matrix microenvironment to provide natural and optimal conditions for regeneration. Wide ranges of wavelengths, precise localization of the irradiated area, and temporal and spatial modulation of laser radiation are the main advantages of the laser technologies, which may result in specific tissue response. In particular, the laser-induced modification of the cartilage extracellular matrix (ECM) seems to be of great significance in view of some new data on the developmental roles of the matrix molecules and mechanical loads. Although the evidence of laser irradiation morphogenetic effects is still largely circumstantial, we consider the available observations to address some possible perspectives of the controlled regeneration of cartilage using nonablative laser treatment. So, the aim of this paper is to review physical and biological aspects of laser-induced regeneration of cartilage, to discuss the possibilities and prospects of its clinical applications. The problems and the ways of tissue regeneration and the types and features of cartilage will be introduced first. Then we will review various actual and prospective approaches to cartilage repair, consider possible mechanisms of laser-induced regeneration, present the results in laser regeneration of joints and spine disks cartilages, and finally, discuss some future medical applications of laser regeneration.

2.

Cartilage as a Subject of Regeneration

There are a number of detailed reviews describing the structure and vital functions of cartilages.3, 7, 8 The main components of cartilage are cells (chondrocytes) and ECM consisting of water (70 to 80%), collagens, proteoglycans (PGs), hyaluronic acid (HA), and glycoproteins (GP). The PGs consist of glycosaminoglycanes (chondroitin sulphate and keratan sulphate) linked to the core-protein, which, in turn, is bound with HA threads interweaving between collagen fibrils (Fig. 1). PGs have a lot of negative charged groups; and the electrical neutrality of cartilage is due to the presence of positive ions (K+, Na+, H+, Ca2+, Mg2+). There are three types of cartilage tissue: hyaline cartilage (costal, nasal septum, articular cartilage of the joints), fibrous cartilage (annulus fibrosis of the spine disks, Eustachian tube), and elastic cartilage (auricle, epiglottis). Hyaline cartilage first forms in embryos and later transforms into other types of cartilage and bone tissues. The distinguishing features of the ECM of hyaline cartilage are having a very high content of glycosaminoglycanes and the prevalence of collagen type II fibrils.9, 10, 11, 12, 13 Fibrous cartilage is characterized by predominance of collagen type I.14, 15, 16 Matrix of the elastic cartilage possesses elastic fibers. Nasal and some other cartilages are covered with a perichondrium playing an important role in nutrition and growth of the avascular tissue. Articular cartilage has no perichondrium; it gets nutrition from synovial liquid and subchondral bone. An articular cartilage surface is covered by a cell-free lamina splendens (LS) consisting mainly of the HA and phospholipids.17 An important structural and metabolic unit of articular cartilage is a chondron.11 It includes a chondrocyte and its pericellular matrix (PM) bordered with a pericellular capsule (PC). The chondron is surrounded by territorial and interterritorial matrices. The chondrons and their matrix environment have different mechanical properties.3, 11 The PM is enriched with HA, sulphated PGs, biglycan, and GPs, including link protein and laminin. The PC is predominantly composed of compact thin fibrils of collagen type VI and fibronectin. It is suggested that the PM and PC provide hydrodynamic protection for the chondrocyte against pressure loading and take a part in control of spatial and temporal distribution of newly synthesized macromolecules as well as in the cell-matrix interaction.11 Territorial and interterritorial matrices are characterized by different degrees of the PGs maturity and with a different proportion of the chondroitin sulphate and keratan sulphate. The heteropolymeric fibrils of collagen types II, IX, and XI (HCF) emerging in the territorial matrice become the major load-bearing element in the interterritorial matrice.16 These fibrils are in charge of the tissue protection against multidirectional tensions.

Fig. 1

Cartilage components and structure. PG –proteolycanes; HA – hyaluronic acid, GP – glycoproteins; M – morphogenes; R -molecular receptors of chondrocyte's membrane; (+) ions (K+, Na+, H+, Ca2+, Mg2+).

A number of molecules that possess signal roles in morphogenetic processes, including chondrogenesis from embryonic development to regeneration, may interact with the receptors of the cellular membrane of chondrocyte. Binding of such morphogenes to the membrane receptors triggers various intracellular signaling cascades to result in regulation of the expression of genes. Hydrostatic pressures and fluid flows as well as multidirectional tensions contribute to tissue water displacement leading to changes of local concentrations of ions and morphogens. The GP molecules (integrins, fibronectin, laminin, etc.) distributing over the ECM serve as important mediators of the signaling molecules. They play an important role in the cell-matrix interactions and operate on the growth of cartilage tissue.

Hyaline cartilage has a zonal structure:11, 18, 19 the superficial layer contains fibroblast-like chondrocytes of type I. It is characterized by a decreased level of the PG aggregates (aggrecanes) and by a high content of small leucine-rich PGs (decorin and biglycan). The cells in the middle layer are chondrocytes of type II. They form multicellular clones and keep a certain ability of proliferation. A smaller subpopulation of the middle layer cells is presented by the chondrocytes type III covered with lacunas. These nonproliferating cells are also presented in the deep layer of cartilage. Type IV cells belong to a degrading cell group. Chondrocytes synthesize and degrade all components of cartilage matrix through specialized enzymes (prolyl hydroxylase, lysil oxidase, collagenases, aggrecanases etc.).16, 20 Metabolic activity of the chondrocytes in cartilage is controlled by hormones, various cytokines, growth factors, and vitamins (A, C, and D).21, 22, 23, 24 Ultimately, the biosynthetic and catabolic activities of cartilage cells, as well as the kinetics of the cellular population are governed by the local concentrations of the humoral and insoluble morphogens near the external membranes of chondrocytes.

The main mechanism of cartilage nutrition is diffusion of water carrying low-molecular substances (ions, glucose, amino acids, etc.). As the chondrocytes kinetics are under conditions of hypoxia, their metabolism is generally realized by the anaerobic glycolysis pathway. That, in combination with the chondrocytes paucity, determines a low level of cartilage metabolism. Half life period is three or four years for aggrecans, and about 10 years for collagen.25 All types of cartilage, especially articular cartage and intervertebral disks, have low repair potential. There is a lot of literature on this topic. 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 Extra-articular cartilage is usually repaired by the means of proliferation and chondrogenic differentiation of the perichondrial cells. The defects of hyaline cartilage and the extensive defects of costal and auricular cartilages are usually filled up with fibrous connective tissue or fibrous cartilage, which both do not have adequate functional properties; that determines persistent attempts to find new possibilities for cartilage regeneration. The healing of cartilage defects can be improved with mechanical stimulation, intra-articular application of HA, hormone therapy,38, 39, 40, 41 and also with the use of osteochondral or cartilaginous implants, in particular together with cultivated chondrocytes.42, 43 One of the current leading approaches is in vitro growth of the tissue engineering constructs followed with their implantation into cartilage lesion. Autologous chondrocyte implantation (ACI) resulted in the formation of hyaline-like tissue with a quite stable clinical outcome.44, 45 But according to the histological data, only 39% of the defects treated with ACI were filled with hyaline cartilage, while 43% were filled with fibrocartilage, and 18% did not show any healing response at all.46

Regeneration process is associated with embryonic chondrogenesis mechanisms and partial dedifferentiation of mature cells. Figure 2 shows possible pathways of regeneration-related dedifferentiation of the cells in cartilage. Mesenchymal stem cells (MSC) can differentiate into cartilage cells of various types, including immature and mature chondrocytes, and notochordal and chondrocyte-like cells of the intervertebral disks. These processes are under multilevel control of signaling molecules and mechanical factors. Our main hypothesis is that differentiation and dedifferentiation of cartilage cells, as well as their metabolic activity, may be controlled by direct action of laser radiation on the cells and through laser-induced modification of the ECM.

Fig. 2

Differentiation of cartilage cells and possible pathways of their regeneration-related dedifferentiation. 1 – MSC, 2 – pre-chondrocytes, 3 – early chondrocytes (chondroblasts), 4 – columnar chondrocytes, 5 – hypertrophic chondrocytes, 6 – chondrocytes of fibrous cartilage, 7 – chondrocytes of hyaline cartilage, 8 – differentiation pathways, 9 – pathways of limited dedifferentiation, 10 – additional pathways of cellular differentiation (following the enchondral osteogenesis differentiation mechanism).

3.

Targets for Laser Effect. Possible Types of Cartilage Response on Laser Radiation

To discuss possible ways of using lasers for cartilage regeneration, it is important to know what effect laser parameters have on (a) different types of the cells; (b) different components of the ECM; (c) signaling molecules produced by the cells and accumulated in the ECM; (d) intercellular and cell-matrix interactions; (e) differentiation and dedifferentiation of the cells, their migration and biosynthesis activity. Feasible pathways promoting cartilage regeneration include: 1. additional cellular supply from bone marrow and blood; 2. biosynthesis amplification of the ECM components, 3. stimulation of the motility of mature chondrocytes, and 4. activation of resident adult stem cells toward their proliferation, differentiation, and ECM production. The main reasons of the low regeneration potential of cartilage are advanced differentiation of the resident chondrocytes and relatively slow metabolism of the tissue. The nonablative laser radiation may provide controllable thermal and mechanical effects (as on the cells, as on the matrix) resulting in activation of the cellular biosynthesis. In particular, nonuniform laser heating of cartilage induces heterogeneous thermal expansion, stress, and also the movement of the interstitial water and ions (see Fig. 3 and Sec. 4).

Fig. 3

Targets and mechanisms of the laser-induced regeneration of cartilage. Thin straight arrows show the direct laser influence on the components of cartilage. White thick arrows represent the most important biological responses to laser action. Dark thick arrows show the processes leading directly to regeneration.

One of the major obstacles for regeneration of cartilage, including partial-thickness defect of articular cartilage, is its avascularity, which hampers the progenitor cell movement from the blood and marrow to the damaged areas of the tissue. Preventing an entry of unspecialized cells and diminishing the rate of cartilage repair that slow regeneration, nevertheless, may have its good point, as it may potentially result in the growth of well organized tissue of the hyaline cartilage. Rapid repair of the full-thickness cartilage defects usually leads to undesirable growth of fibrous connective tissue or fibrous cartilage due to the impact of blood and bone-marrow-derived cells. It can be better understood by the following analogy. It is known that skin wound healing resulting in a fibrous scar is going through emergency regeneration due to swift proliferation of unspecialized fibroblasts. Their sources are the precursor cells coming into the wound via blood. These cells have nonspecific genetic program and form scar.47 In a similar manner, the bone-marrow cells coming to the full-thickness defect of articular cartilage differentiate into the fibroblasts of nonspecific connective tissue or into the chondrocytes of the fibrous cartilage. This provides quick filling of the defect, but fails in functionality of the novel tissue. One of the possible ways to promote growth of the hyaline cartilage in the full-thickness defects of articular cartilage plates can be laser-induced coagulation of the bottom of the defect. This may prevent access of unspecialized precursor cells from the blood or bone-marrow in order to develop more specific, i.e., hyaline cartilage.

It is known that in the course of embryogenesis, the hyaline cartilage forms in the zones undergoing compression load (articular cartilage), whereas, the fibrous cartilage (meniscus, annulus fibrosis of the intervertebral disk) usually develops in the stretched or torsioned zones. Spatial and temporal modulation of laser radiation allows controlling the actual distribution of stretched and compressed zones in cartilage. The mechanical loads are important factors governing an orchestra of chondrogenesis, including the processes of cellular differentiation. Therefore, the nonablative laser treatment may play a triggering role in the differentiation of immature cartilage cells. Laser radiation may probably be responsible for the reverse process of dedifferentiation of the mature chondrocytes leading to the recovery of their ability to divide. Existing natural pathways of cells dedifferentiation (see Fig. 2) open possibilities for tissue correction, in particular, replacement of abnormally grown fibrous tissue by hyaline cartilage possessing adequate mechanical and functional properties (Fig. 3).

Laser radiation can also be used to stimulate proliferation and acquiring the specialized phenotype by resident stem cells or MSC coming through synovial liquid in order to promote their transformation into mature hyaline-like chondrocytes. This approach is critically significant for healing of the partial-thickness defects of articular cartilage. At the same time, as the cellular population in full-thickness cartilage defect is highly heterogenic, laser irradiation may effect the proliferation of different kinds of cells. Thereafter, the additional controlling factor of the ECM architecture should be taken into account. Laser modification of the fine structure of ECM does not change its general organization. This provides natural environments for chondrocytes and promotes restoration of the hyaline type cartilage. One of the important factors is cell movement velocity, which correlates with the alignment of the matrix fibrillar components.48, 49 Nonablative laser irradiation allows structure modification and diffusion properties of ECM. This may support cell movement and favor tissue regeneration.

Laser-induced growth of hyaline cartilage in elastic cartilage was established in the course of in vivoexperiments on laser reshaping of porcine ears.50 The effects of laser irradiation on gene expression of chondrocytes and collagen of ECM have been studied for rabbit septal cartilage using laser settings typical for laser reshaping procedure.51 It was shown that laser irradiation of cartilage does not result in the detection of collagen type I. Only collagen type II was observed after laser irradiation in the corresponding cell culture in vitro. This fact indicates that cartilage cellular response to nonablative laser irradiation differs from the reaction of conventional wound healing. Laser irradiation of cartilage can leave intact collagen and preserve general matrix architecture, which favors chondrocyte survival and promotes new tissue growth. Evidence of hyaline cartilage development in laser-irradiated intervertebral disks was revealed in the animal experiments (see Sec. 5.2). The advantage of the laser effect on chondrocytes proliferation compared to other thermal, mechanical, and chemical effects was demonstrated in Ref. 52. No evidence of chondrocyte DNA replication was observed in tissues heated using nonlaser methods, grown in TGF-β-contained media, or mechanically traumatized. In contrast, for laser irradiated chondrocytes, flow cytometry provided evidence that laser irradiation causes a proliferative response in cho...

Abstract

INTRODUCTION

It was not long after the discovery of the first lasers (the ruby laser in 1960 and the helium-neon [HeNe] laser in 1961) that they began to be used in medical applications. In 1967, Endre Mester in Hungary noticed the ability of the HeNe laser to increase hair growth [1] and stimulate wound healing in mice [2], and, shortly afterward, he began to use lasers to treat patients with nonhealing skin ulcers [3]. Since those early days, the use of low-power lasers (as opposed to high-power lasers that can destroy tissue by a photothermal effect) has steadily increased in diverse areas of medical practice that require healing, prevention of tissue death, pain relief, reduction of inflammation, and regenerative medicine. Some of the different organ systems, diseases, and injuries that have been effectively treated with low-level laser therapy (LLLT) are schematically shown in Figure 1.

Figure 1

Diagram of the various medical applications of low-level light therapy.

Nevertheless, this modality, which is variously known as LLLT or photobiomodulation, remains controversial. The reasons for this lack of general acceptance among both the medical community and the general public at large are 2-fold. First, widespread uncertainty and confusion exists about the mechanisms of action of LLLT at the molecular, cellular, and tissue levels. Second, a large number of parameters (eg, wavelength, fluence, irradiance, treatment timing and repetition, pulsing, and polarization) can be chosen in designing LLLT protocols. Furthermore, a biphasic dose response exists in laser therapy [4], which describes the observation that increasing the overall “dose” of the laser either by increasing the power density or by increasing the illumination time may have a counter-productive effect compared with the benefit obtained with lower doses. Taken together, these considerations may explain why a number of negative studies have been published; however, this should not be taken to imply that LLLT in general does not work but rather that the laser parameters used in those particular studies were ineffective.

In recent years, the development of light-emitting diodes (LEDs) as alternative light sources for LLLT has added to the confusion. These devices produce light with wavelengths similar to those of lasers, but they have broader output peaks (ie, they are less monochromatic) and lack the coherence that is a particular feature of laser light. LEDs have the advantage of being significantly less expensive than laser diodes (by a factor of approximately 100 on a milliwatt basis), and the LLLT community is engaged in a vigorous ongoing debate about their respective benefits.

This review covers the mechanisms that are thought to operate at molecular and cellular levels in LLLT. Many of the most compelling applications of LLLT are in the field of neurology (both central and peripheral). Many serious brain diseases and injuries can be successfully treated with noninvasive transcranial laser therapy. Furthermore, in the peripheral nervous system, LLLT can be used effectively for nerve regeneration and pain relief.

CELLULAR AND MOLECULAR MECHANISMS OF LLLT

LLLT uses low-powered laser light in the range of 1-1000 mW, at wavelengths from 632-1064 nm, to stimulate a biological response. These lasers emit no heat, sound, or vibration. Instead of generating a thermal effect, LLLT acts by inducing a photochemical reaction in the cell, a process referred to as biostimulation or photobiomodulation. Photo-biology works on the principle that, when light hits certain molecules called chromophores, the photon energy causes electrons to be excited and jump from low-energy orbits to higher-energy orbits. In nature, this stored energy can be used by the system to perform various cellular tasks, such as photosynthesis and photomorphogenesis. Numerous examples of chromophores exist in nature, such as chlorophyll in plants, bacteriochlorophyll in blue-green algae, flavoproteins, and hemoglobin found in red blood cells. The respective colors of chromophores are determined by the part of the spectrum of light they absorb: chlorophyll is green, flavoprotein is yellow, and hemoglobin is red [5].

Mitochondria are considered the power generators of the eukaryotic cell, converting oxygen and nutrients through the oxidative phosphorylation process and electron transport chain into adenosine triphosphate (ATP), as shown in Figure 2. The basic idea behind cellular respiration is that high-energy electrons are passed from electron carriers, such as reduced nicotinamide adenine dinucleotide (NADH) and the reduced form of flavin adenine dinucleotide (FADH₂), through a series of transmembrane complexes (including cytochrome c oxidase [CCO]) to the final electron acceptor, generating a proton gradient. The gradient is used by F_OF₁ ATP synthase to produce ATP. Various in vitro experiments, such as those that use rat liver isolates, found that cellular respiration was upregulated when mitochondria were exposed to an HeNe laser or other forms of illumination. Laser irradiation caused an increase in mitochondrial products (such as ATP [6], NADH, protein, ribonucleic acid [RNA] [7]) and a reciprocal augmentation in oxygen consumption. A similar effect is produced when tissue that contains mitochondria is exposed to low-level radiation. Visible and near-infrared (NIR) light is absorbed by the organelle, and an upregulation of cellular respiration is observed [8].

Figure 2

Illustration of mitochondrion, as well as of the electron transport chain and oxidative metabolism.

Once it was observed that LLLT's mechanism of action is at the level of the mitochondria, it remained to be determined what specific structure within the mitochondria acted as the chromophore. Four membrane-bound complexes have been identified in mitochondria, each constituting an extremely complex transmembrane structure embedded in the inner membrane. Complex IV, also known as CCO, is a large transmembrane protein complex found in mitochondria, which is a component of the respiratory electron transport chain (Figure 3). CCO appears to absorb the same spectrum of light as that observed for the action spectra for the biological response to light in the NIR range. Thus it is reasonable to assume that CCO acts as an important chromophore in LLLT [9]. CCO consists of 2 copper centers and 2 heme-iron centers that are capable of absorbing light over a wide range, including NIR.

Figure 3

Complex IV (cytochrome c oxidase) is the principal chromophore involved in low-level light therapy. It has 2 copper centers and 2 heme prosthetic groups. Cytochrome c is oxidized and oxygen is reduced to water during respiration.

The next reasonable question to consider is: What action does CCO modulate once it absorbs the energy from light? On the cellular level, LLLT may cause photodissociation of nitric oxide (NO) from CCO. In a stressed cell, NO produced by mitochondrial NO synthase displaces oxygen from CCO, which results in a downregulation of cellular respiration and a subsequent decrease in the production of energy-storing compounds, such as ATP. By dissociating NO from CCO, LLLT prevents the displacement of oxygen from CCO and thereby promotes unhindered cellular respiration [10] (see Figure 4). Increased CCO enzyme activity can be measured [11]; increased ATP production [12] and increased electron transport [13] also have been reported. The basic idea behind cellular respiration is that high-energy electrons are passed from electron carriers, such as NADH and FADH₂, through a series of transmembrane complexes (including CCO) to the final electron acceptor. Increased cellular ATP produced by LLLT may contribute to the positive effects, both by raising cellular energy levels and by upregulating the cyclic AMP molecule (biochemically formed from ATP) that is involved in many signaling pathways.

Figure 4

Nitric oxide can bind to copper (or heme) centers in cytochrome c oxidase and inhibit respiration. The nitric oxide may be photodissociated by absorption of red or near infrared light, allowing oxygen to return and sharply increasing respiration and adenosine ...

Oxygen acts as the final electron acceptor and is, in the process, converted to water. Part of the oxygen that is metabolized produces reactive oxygen species (ROS) as a natural by-product. ROS (eg, superoxide and hydrogen peroxide) are chemically active molecules that play an important role in cell signaling, regulation of cell cycle progression, enzyme activation, and nucleic acid and protein synthesis [14]. Because LLLT promotes the metabolism of oxygen, it also acts to increase ROS production. In turn, ROS activates certain redox-sensitive transcription factors such as nuclear factor-κB [NF-κB] and activator protein 1, which leads to the upregulation of various stimulatory and protective genes. The ultimate effect of LLLT is likely to be produced by transcription factor activation, which modulates the host's downstream cellular and tissue responses (see Figure 5).

Figure 5

Diagram that illustrates the mechanism of low-level light therapy (LLLT) on the cellular and molecular level. Near infrared light, absorbed by the mitochondria, causes upregulation of the cellular respiratory chain. A host of downstream cellular responses ...

Almost certainly, other mechanisms through which LLLT produces its effects are at play in addition to the one just described. For example, NO is a potent vasodilator via its effect on cyclic guanine monophosphate production. Cyclic guanine monophosphate is also involved in many other signaling pathways. LLLT may cause the photodissociation of NO from intracellular stores (ie, nitrosylated forms of both hemoglobin and myoglobin, in addition to CCO) [15]. LLLT promotes the synthesis of deoxyribonucleic acid (DNA) and RNA [16] and increases the production of proteins [17]. It also modulates enzymatic activity [18], affects intracellular and extracellular pH [17,18], and accelerates cell metabolism [18,19]. The expression of multiple genes related to cellular proliferation, migration, and the production of cytokines and growth factors also have been shown to be stimulated by low-level light [20].

Light is a powerful force and has a myriad of effects. The specific mechanisms of action may vary among various clinical applications of LLL and will be discussed in the respective sections below. Furthermore, in spite of a great number of studies that explored how LLLT works, the exact mechanism of action remains to be fully elucidated.

STROKE

Transcranial LLLT (808 nm) has significantly improved recovery after ischemic stroke in rats when they received one treatment 24 hours after sustaining a stroke [21,22]. Stroke was induced in rats by 2 different methods: (1) permanent occlusion of the middle cerebral artery through a craniotomy or (2) insertion of a filament. The laser was used transcranially on the exposed (shaved skin) skull by placing the tip of the 4-mm diameter fiber optic onto the skin at 2 locations on the head (3 mm dorsal to the eye and 2 mm anterior to the ear) on the contralateral hemisphere to the stroke. These locations had been determined from prior measurements to be sufficient to illuminate 1 brain hemisphere as a result of dispersion of the laser beam by the skin and the skull. Results of previous studies had shown that LLLT of the contralateral, or both hemispheres, demonstrated no difference in functional outcome [23]. An NIR gallium arsenic diode laser was used transcranially to illuminate the hemisphere contralateral to the stroke at a power density of 7.5 mW/cm² to the brain tissue [22]. In both models of stroke, the neurologic deficits at 3 weeks after stroke were significantly reduced (by 32%) (P < .01) in the laser-treated rats compared with control subjects.

In this study, the number of newly formed neuronal cells, assessed by double immunoreactivity to bromodeoxyuridine and tubulin isotype III, as well as migrating cells (double Cortin immunoreactivity), was significantly elevated in the subventricular zone of the hemisphere ipsilateral to the induction of stroke when treated by LLLT [21,22]. No significant difference in the stroke lesion area was found between control and laser-irradiated rats. The researchers suggested that an underlying mechanism for the functional benefit after LLLT in this study was possible induction of neurogenesis. Results of other studies also suggested that, because improvement in neurologic outcome may not be evident for 2-4 weeks in the poststroke rat model, delayed benefits may in part be due to induction of neurogenesis and migration of neurons [24,25]. In addition, transcranial LLLT may prevent apoptosis and improve outcomes by exerting a neuroprotective effect, although these exact mechanisms are poorly understood [26].

Other studies in rat and rabbit models also have observed that transcranial LLLT improves functional outcome after stroke [25,27,28]. A recent rabbit study combined transcranial LLLT with thrombolytic therapy by using tissue plasminogen activator, with no increase in bleeding and good safety [29].

In the aforementioned studies, it has long been hypothesized that increased mitochondrial function (ie, increased ATP production) in brain cells irradiated with NIR LLLT was one of the major mechanisms involved with the beneficial behavioral effects observed after LLLT treatment. A recent animal study with rabbits has shown a direct relationship between the level of cortical fluence (energy density) delivered (in J/cm²) and cortical ATP content in embolized rabbits [30]. Five minutes after embolization (right carotid), the rabbits were exposed to 2 minutes of NIR transcranial LLLT with use of an 808-nm laser source (continuous wave [CW] or pulsed wave [PW] at 100 Hz or at 1000 Hz on the skin surface, posterior to bregma at midline). Three hours after embolization, the cerebral cortex was excised and processed for measurement of ATP content. Embolization decreased cortical ATP content in ischemic cortex by 45% compared with naive rabbits.A linear relationship up to 4.5 J/cm² in fluence delivered, was observed for the relationship between cortical fluence (in J/cm²) verus percent increase in cortical ATP content (over sham-treated embolized rabbits). This linear relationship was observed with a power density of 7.5 mW/cm² CW (0.9 J/cm²), where an increase of 41% in cortical ATP was observed; and with a power density of 37.5 mW/cm² PW (100 Hz, 4.5 J/cm²), where an increase of 157% in cortical ATP was observed. An increase in cortical ATP of 221% was observed with fluence of 31.5 J/cm², delivered with a power density of 262.5 mW/cm² PW, 1000 Hz. This suggests that a near-plateau effect was present regarding the fluence level delivered above 4.5 J/cm². It was surprising, however, that the increased cortical ATP levels of 157% and 221%, were higher than those measured in naive rabbits that had never suffered stroke. Because the authors observed that the PW modes (100 Hz and 1000 Hz) were more effective than the CW mode to increase cortical ATP, they hypothesized that in future stroke studies in animals and in humans, even greater improvement in clinical rating scores might be achieved by optimizing the method of NIR transcranial LLLT delivery, including the length of treatment and the mode of treatment (PW).

Transcranial LLLT has been shown to significantly improve outcome in acute human stroke patients when applied approximately 18 hours after the stroke occurs over the entire surface of the head (20 points in the 10/20 electroencephalographic system), regardless of the stroke location [31]. Only one LLLT treatment was administered, and, 5 days later, significantly greater improvement was found in the real-treated group but not in the sham-treated group (P < .05, National Institutes of Health Stroke Severity Scale). This significantly greater improvement was still present 90 days after –the stroke occurred, at which time 70% of the patients treated with real LLLT had a successful outcome compared with only 51% of control subjects. An NIR (808 nm) laser was used, which delivered a fluence of 0.9 J/cm² over the entire surface (2 minutes per each of the 20 points; power density of 7.5 mW/cm²).

In a second, similar study with the same transcranial LLLT protocol, an additional 658 acute stroke patients were randomly assigned to receive real or sham treatments of transcranial LLLT. Similar significant beneficial results (P < .04) were observed for the patients who had a moderate or moderate to severe stroke (n = 434) and received the real laser protocol but not for the patients who had a severe stroke [32]. When all 656 cases were included in the data analysis (including the severe stroke cases), no significant real versus sham LLLT effect was seen. When data for both stroke studies were pooled (n = 778 [120 plus 658]) [31,32], a highly significant beneficial effect was seen for the real transcranial LLLT group (P = .003) compared with those who received the sham laser treatment [33].

Lampl et al [31] wrote that “Although the mechanism of action of infrared laser therapy for stroke is not completely understood . . . infrared laser therapy is a physical process that can produce biochemical changes at the tissue level. The putative mechanism . . . involves stimulation of ATP formation by mitochondria and may also involve prevention of apoptosis in the ischemic penumbra and enhancement of neurorecovery mechanisms.”

To date, no studies have been conducted to examine transcranial LLLT treatment of chronic stroke patients. Naeser et al [34] studied the application of LLLT-laser acupuncture (instead of needles) to stimulate acupuncture points on the body in chronic stroke patients with paralysis. Seven stroke patients (range, 48-71 years; 5 men) were treated, 5 of whom had single left hemisphere stroke, and 2 of whom had single right hemisphere stroke. Five patients were treated for hemiplegia, including severely reduced or no voluntary isolated finger movement, and 2 patients had hand paresis only. Six of the 7 patients received laser acupuncture during the chronic phase after the stroke had occurred (10 months to 6.5 years after stroke onset), clearly beyond the spontaneous recovery phase, which is considered to be up to 6 months after the stroke occurs [35,36]. The patients served as their own controls; no sham LLLT was administered. One patient (who had hand paresis) received LLLT during the acute phase after the stroke occurred (1 month after the stroke occurred). The patients did not receive any physical therapy or occupational therapy treatments while participating in this study.

A 20-mW gallium aluminum arsenide (780 nm) NIR CW laser with a 1-mm-diameter aperture was used (Unilaser, Copenhagen, Denmark). (At the time of this study, more powerful red or NIR lasers were not yet available.) Treatment consisted of stimulation of shallow acupuncture points (located on the hands and face) for 20 seconds per point (51 J/cm²). Deeper acupuncture points (located on the arms and legs) were treated for 40 seconds per point (103 J/cm²). Acupuncture points were treated on both the paralyzed side (arm, leg, and/or face) and on the nonparalyzed side by using primarily acupuncture meridians of the large intestine, triple warmer, gall bladder, liver, small intestine, and stomach [34]. The patients were treated 2-3 times per week for 3-4 months. They received a total of 20, 40, or 60 treatments (based on patient availability and transportation). Within a few days before the first treatment and a few days after the last treatment, physical therapy and/or occupational therapy testing was performed by therapists blinded to the acupuncture treatment program to which the patient had been assigned: LLLT, real or sham needle, or no acupuncture. Overall, 5 of 7 of the patients (71.4%) showed improvement.

The 2 patients who showed no improvement had severe paralysis. We have observed that severity of paralysis and potential for improvement after LLLT-laser acupuncture (or needle acupuncture) is related to lesion location on chronic computed tomography (CT) scan acquired at least 3 months poststroke onset. Patients with lesion in more than half of the “periventricular white matter area” (PVWM) (adjacent to the body of the lateral ventricle, superior to the posterior limb, internal capsule), an area containing multiple efferent and afferent pathways (eg, thalamocortical, occipitofrontal, pathways from SMA/cingulate gyrus to the body of caudate, medial subcallosal fasciculus, and others), had severe paralysis which did not improve following LLLT-laser acupuncture (or needle) acupuncture treatments [34,37,38]. This area is diagrammed in Figure 6. The CT scan for a chronic stroke patient who had good response after LLLT-laser acupuncture treatments [34,37,38]. This area is diagrammed in Figure 7.

Figure 6

Location of periventricular white matter (PVWM) area (black arrow), adjacent to the body of the lateral ventricle, located immediately superior to the posterior limb, internal capsule (computed tomography slice angulation, coronal and axial views). An ...

Figure 7

(a.) Computed tomography (CT) scan of a 65-year-old woman obtained 5 months after stroke onset shows sparing of the most posterior portion of the periventricular white matter (PVWM) (white arrow), that is, likely sparing of some of the leg fibers. This ...

The 3 chronic stroke patients with hemiplegia who showed improvement after LLLT had an increase of 11%-28% in isolated, active range of motion for shoulder abduction, knee flexion, and/or knee extension (mean, 15.8%; SD, 7.1). This percentage increase after LLLT-laser acupuncture was similar to that observed after a series of 20 or 40 needle acupuncture treatments [37,38]. The person with hand paresis who was treated with LLLT at 33 months after stroke onset showed an increase of 2-6 lb in grip strength, 3-jaw chuck, tip pinch, and lateral pinch in the affected hand. These results are similar to those obtained with needle acupuncture [39]. These findings are intriguing and suggest that some recovery of motor function can occur with needle acupuncture or LLLT acupuncture applied to body acupuncture points in chronic stroke patients.

A reduction in hand spasticity also has been observed when chronic stroke patients are treated with a combination of red-beam laser applied to hand acupuncture points plus microamps transcutaneous electrical nerve stimulation (TENS). Figure 8 shows an immediate reduction in hand spasticity after the first hand treatment when LLLT-laser acupuncture and microamps TENS were used with 2 chronic stroke patients. This LLLT and microamps TENS hand treatment program also may be used with patients who have hand spasticity related to other etiologies, including, for example, traumatic brain injury (TBI), “stiff man syndrome,” and spinal cord injury (SCI) (personal observation, M.A.N., 2001). Similar to red and NIR LLLT, microamps TENS increases ATP levels when applied to the skin [40]. However, Cheng et al [40] observed that when stronger milliamps TENS was used (eg, similar to conventional TENS), the ATP levels were decreased. Hence when microamps TENS is used (as shown in Figure 8) [41], it is advisable to keep the sensation below threshold for the patient to increase ATP (not decrease ATP).

Figure 8

(a.) Before the first low-level laser therapy (LLLT) and microamps transcutaneous electrical nerve stimulation (TENS) acupuncture treatment. It was 1.5 years after stroke onset and the patient still had right hand spasticity and was unable to extend her ...

TRAUMATIC BRAIN INJURY

Each year in the United States, more than 1.4 million new cases of TBI occur, and more than 80,000 persons are left with permanent disability [42]. Mild TBI (mTBI) from single and multiple events is the most frequent type of head injury experienced by military personnel deployed to Iraq and Afghanistan [43]. TBI is known to cause damage that ranges from observable to microscopic throughout the gray and white matter of the brain. Diffuse axonal injury [44] is often observed in the anterior corona radiata and frontotemporal regions [45]. Two regions highly susceptible to damage within the frontal lobes are the prefrontal cortex and the anterior cingulate gyrus. Cognitive processing problems result from tissue damage and inefficient cellular function in these brain regions. The prefrontal cortex is involved with maintaining, monitoring, and manipulating information in working memory [46] and particularly in sustained attention [47,48].

In the first reported study of the use of transcranial LLLT to treat traumatic brain injury, an animal model was used [49]. Mice were subjected to closed-head injury (CHI) by using a weight-drop procedure, and 4 hours after CHI, either sham or real NIR LLLT (808 nm) was administered transcranially. The control group received no laser therapy (n = 8); the laser-treated group (n = 16) received 1 transcranial LLLT treatment by using a 200-mW, 808-nm NIR laser with a 3-mm-diameter probe tip (Photothera Inc, Carlsbad, CA). Either 10 or 20 mW/cm² was administered. A single point was treated on the skull (a skin incision was made) that was located 4 mm caudal to the coronal suture line on the midline. The point was treated for 2 minutes (1.2-2.4 J/cm²). At 24 and 48 hours after CHI, no significant difference in motor behavior was seen between mice in the laser-treated and control groups. After 5 days, the motor behavior was significantly better (P < .05) in the laser-treated group; in addition, the neurobehavioral scores were 26%-27% better (lower scores indicated better motor behavior). At 28 days after CHI, the brain-tissue volume was examined for mice in each group. The mean lesion size of 1.4% in the laser-treated group (SD 0.1) was significantly smaller (P < .001) than in the control group (12.1%, SD 1.3). No difference in lesion size or behavior was observed in the mice treated with 10 mW/cm² and those treated with 20 mW/cm². The researchers suggested various possible mechanisms, including an increase in ATP, total antioxidants, angiogenesis, neurogenesis, heat shock proteins content, and an antiapoptotic effect, similar to observations reported after LLLT treatment of ischemic heart skeletal muscles [50-54].

Moreira et al [55] conducted a study in 2009 using phototherapy with low-intensity lasers and observed the effect on local and systemic immunomodulation after cryogenic brain injury in rats. Brain and blood samples were analyzed by enzyme-linked immunosorbent assay for the production of cytokines interleukin (IL)-6 , IL-10, IL-1b, and tumor necrosis factor (TNF)-α. The study concluded that laser phototherapy could positively affect the balance of IL-1b, TNF-α, and IL-6 in rats and thereby prevent cell death after TBI.

Wu et al [56] reported another mouse study of LLLT mediated by transcranial laser therapy. A nonfocal (diffuse) TBI was produced by a CHI caused by a calibrated weight-drop device. A neurologic severity score for each mouse was determined based on 10 standardized performance tests (involving beam balancing and maze exiting) administered at specified times. Mice with a neurologic severity score of 7-8 (moderately severe brain injury) were used in the study. Mice were given a single treatment to the top of the head with 36 J/cm² of a 665-nm, 810-nm, or 980-nm laser 4 hours after the closed head TBI. Both 665-nm and 810-nm lasers were highly effective in improving the neurologic performance of the mice during the succeeding 4 weeks. The 980-nm wavelength was ineffective (negative control). We believe that this difference in results can be explained by the absorption spectrum of the different chromophores; CCO has peaks at 660 nm and 810 nm, whereas water has a peak at 980 nm.

In humans, 2 persons with chronic mTBI recently have been reported to have improved cognition after a series of treatments with transcranial, red, and NIR LEDs [57,58]. The LED cluster heads were applied to the forehead and scalp areas (the hair was not shaved off but was parted underneath each 2-inch-diameter LED cluster head). Each cluster head had 61 diodes (9 red 633-nm diodes and 52 NIR 870-nm diodes). Each diode was 12-15 mW, and the total power output was 500 mW. The LED cluster heads were applied to bilateral frontal, parietal, and temporal areas and to the mid-sagittal suture line.

Each LED cluster head was applied for 10 minutes per placement. With the device used here (parameters described above), 1 joule per cm² (J/cm²) energy density was produced during every 45 seconds of exposure time. The energy density dose at the forehead-scalp was 13.3 J/ cm²; the power density was 22.2 mW/cm² (±20%). The power density refers to the mW of power applied per cm². The ± refers to the range of fluctuation (plus or minus 20%) on the power density per cm². This power density is well below that used in other transcranial laser or LED studies to treat acute stroke cases or severe depression cases (225 mW/cm²) [59]. It is estimated that only approximately 3% of the photons delivered to the forehead-scalp surface will reach 1 cm, to the cortex [60]. The dose of 13.3 J/cm² per placement area was estimated to deliver only 0.4 J/cm² to the brain cortex. No sensation of heat or pain was reported during the LED application to the skin or scalp. These LED cluster heads (MedX Health Corp, Mississauga, Ontario, Canada) are approved by the U.S. Food and Drug Administration for treatment of musculoskeletal pain; they were used off-label for treatment of cognition in the mTBI cases. No potential existed for ocular damage because the LEDs produce noncoherent light. These LED cluster heads also have been approved by the Food and Drug Administration for home treatment.

A 66-year-old woman (case 1) began transcranial LED treatments 7 years after a motor vehicle–related TBI. Before LED treatment, she could focus on her computer for only 20 minutes. After 8 weekly LED treatments, her focused computer time increased to 3 hours. She has treated herself nightly at home for 5.5 years, with transcranial LED. She maintains her improved cognition at age 72 years.

Case 2 involved a 52-year-old retired, high-ranking female military officer who had a history of multiple TBIs. Her brain MRI showed frontoparietal atrophy. She was medically disabled for 5 months before beginning nightly transcranial LED treatments at home (see Figure 9, A and B). After 4 months of nightly LED treatments, she returned to work full time as an executive consultant for an international technology consulting firm and discontinued medical disability. Neuropsychological tests performed after 9 months of transcranial LED showed significant improvement in cognition (see Figure 9, C). After LED treatments, she improved on tests of executive function (inhibition and inhibition accuracy, +2 SD) and on memory (immediate and delayed recall +1, +2 SD). The improvement of +1 or +2 standard deviations on her scores refers to the degree of improvement on her scores after 9 months of LED treatments (versus before LED treatments). The SDs are provided with the test materials, and they are based on the published norms for each test.

Figure 9

(a.) Red and near-infrared (NIR) light-emitting diode (LED) cluster head (2-inch diameter) for transcranial LED treatments. (b.) Sample placement location on right forehead for one of the LED cluster heads during transcranial LED treatment. (c.) Graph ...

Both patients with TBI reported that they needed to continue with home treatments. If they stop treatment for 1 or 2 weeks, then their cognitive problems started to return. Both patients with TBI reported improved sleep. The second patient with TBI reported a decrease in her posttraumatic stress disorder symptoms after a few months of using the transcranial LEDs, and Schiffer et al [59] also reported a reduction in posttraumatic stress disorder symptoms in 3 of 10 patients with major depression who were treated with transcranial LED.

Several possible mechanisms may be associated with the improved cognition in the mTBI cases treated with transcranial LEDs [58]. Mitochondria display a significant amount of dysfunction after TBI [61-63]. The primary mechanism for improvement posited in one study with human acute stroke patients was an increase in ATP, with photons being used by CCO in the mitochondria to increase ATP, especially in the cortex [64].

An increase in ATP after red and/or NIR LED treatments in patients with chronic TBI would have beneficial effects, including an increase in cellular respiration and oxygenation. Oxidative stress plays a role in the damage present after TBI [65]. One hypothesis is that LLLT produces low levels of ROS in mitochondria of illuminated cells and that these ROS cause NF-κB activation via the redox sensitive sensor enzyme protein kinase D1, which results in upregulation of the mitochondrial superoxide dismutase [66]. A single exposure of LLLT-LED in vitro with fibroblasts has been observed to increase NF-κB in the short term [67]. In stimulated dendritic cells in the longer term, however, NF-κB and pro-inflammatory cytokines were reduced [68]. Thus, in the long term, repeated LED treatments are hypothesized to decrease inflammation (less NF-κB) and upregulate gene products that are cytoprotective, such as superoxide dismutase, glutathione peroxidase, and heat shock protein 70 [54,69]. It is hypothesized that an overall protective response occurs with repeated LED treatments and that major ROS-mediated damage and chronic inflammation that occur in the brain after TBI may actually be reduced.

Acupuncture points located on the scalp were treated with the red-NIR LEDs [57]. This includes points along the Governing Vessel (GV) acupuncture meridian, located on the midline of the skull (including, in part, the mid-sagittal suture line). Some acupuncture points located on the GV meridian have been used historically to help treat patients in coma [70] and stroke [71], for example, GV 16 (inferior to occipital protuberance), GV 20 (vertex), and GV 24 (near center-front hairline); these points were treated in both patients with TBI reported in this study.

Transcranial red-NIR LED may have irradiated the blood via the valveless, emissary veins located on the scalp surface but interconnecting with veins in the superior sagittal sinus (M. Dyson, oral personal communication, June 2009). If red-NIR photons penetrate deeply enough to reach the cortex, then it also is possible they are entering small vessels located between the arachnoid and the pia mater, including those that supply arterial blood to superficial areas of the cortex. Direct in vitro blood irradiation with a red-beam laser has been observed to improve erythrocyte deformability (flexibility) and rheology [72,73]. A beneficial effect from direct-laser blood irradiation in vivo has been observed during stenting procedures where a low-level, red-beam laser (10 mW, 650 nm) was used, with the beam placed directly into a coronary artery [74]. The restenosis rate was reduced and no adverse effects or complications were noted. Thus blood irradiation at the scalp may have affected local intracerebral blood and circulation; however; whether this effect occurred is unknown and would require further study.

An increase in regional cerebral blood flow may have occurred, specifically to the frontal lobes. The second TBI case showed significant improvement on objective, neuro-psychological testing for executive function (inhibition) after administration of LED. These results suggest improved function in the prefrontal cortex and anterior cingulate gyrus regions. Significant improvement on “inhibition” on the Stroop test particularly suggests improved function of the medial prefrontal cortex, anterior cingulate gyrus area [75]. It is possible that this medial prefrontal cortex area could have been treated with NIR photons, especially when the LED cluster head was placed over the midline, front hairline area. The dorsolateral prefrontal cortex also was likely irradiated when the LEDs were placed on the left and right high-frontal areas of the scalp. Increased regional cerebral blood flow also could have occurred in frontal pole areas with the TBI cases, as was observed in the recent transcranial LED study to treat major depression [59]. Additional controlled studies with real and sham transcranial LLLT and LED are recommended to investigate whether these methods can be applied to improve cognition and reduce symptom severity in persons with acute and chronic TBI. The LED technology is not expensive ($1400 for a single LED cluster head and approximately $4000 to $5000 for a unit with 3 LED cluster heads). The transcranial LED treatment protocol can be used in the home.

This video shows before and after treatment footage of a patients with advanced Parkinsons. Treatment lasts about 2 weeks. Dr Riner is using the brain and neurostim setting on the brain, C5 Nerve Root and the Ulnar nerve in the elbow.

The TheraLazr is the prototype for the Avant LZ30 series of lasers.

video length: (2:03)

This book is like a bible for laser acupuncture. It is the most detailed book on the subject that we have been able to find. It can be a little hard to get out because the publisher is in Germany. Dr. Weber operates a large clinic in Germany where he treat a wide variety of conditions. He also does training classes for acupuncturist and is a leader in the field of laser acupuncture. In addition to having a detailed explanation of how lasers stimulate the body, her provide some great general guidelines on the use of lasers and his book includes beautifully detailed protocols. Chapters in the book include 3 major sections: High-Tech Acupuncture with Laser Light, Practical Guidelines and Treatment Concepts. Within the treatment concepts are group of protocols for Orthopedics, Neurology, Psychosomatic disorders, Throat, Nose and Ear, Internal Medicine, Dermatology, Pediatrics, Gynecology ,Dental Medicine and Ophthalmology.

This book introduces you to the science of Laser Therapy, starting with the history and basic physics of laser radiation, including things like:

• Lasers vs. LED's
• Measuring Wavelength
• The Electromagnetic Spectrum
• Depth of Laser Penetration
• Types of Laser Diodes
• Classification of Diodes
• Light Energy in Joules
• Pulsing or Frequency
• Treatment Parameters
• Light Absorbed
• Laser Safety
• Contraindications of Light Therapy
• Optimal Dose
• Calculating Output
• Dose and Time for Different Physical Qualities

You will find suggested treatments, and accompanying diagrams for the syndromes listed below:

Head and Face:

• Bell's Palsy
• Migraine Headache
• Sinusitis
• Temporomandibular Joint Syndrome (TMJ)
• Tension Headache
• Trigeminal Neuralgia
• Wrinkles

Spine and Pelvis:

• Cervicall Disc Herniation
• Cervical Stenosis
• Cervical, Thoracic, Lumbar Sprain/Strain and Neuritis
• Coccydynia
• Costochondritis
• Herniated Lumbar Disc or Annular Tear
• Lumbar Stenosis
• Pubic Symphysis Sprain
• Sacroiliac Sprain or Strain
• Spinal Hypermobility Syndrome

Systemic:

• Addiction to Cigarettes or Other Substances
• Ankylosing Spondylitis
• Arthritis
• Complex Regional Pain Syndrome or Reflex Sympathetic Dystrophy
• Fibromyalgia Syndrome (FMS)
• Herpes Zoster/Shingles and Post Herpetic Neuralgia
• Post Surgical Pain
• Wounds (Slow or Non-Healing)

Upper Body:

• Acromioclavicular (AC) Sprain or Laxity
• Biceps Tendinitis
• Biceps Tendon Strain
• Carpal Tunnel Syndrome
• DeQuervain's Tendinitis
• Dislocated Finger or Thumb
• Fractured Carpal, Metacarpal, or Phalange
• Fractured Clavicle
• Fractured Distal Radius or Casted Forearm or Hand
• Frozen Shoulder
• Ganglion Cyst of the Wrist
• Olecranon Bursitis
• Radial or Ulnar Neuritis
• Rotator Cuff Strain
• Shoulder Rheumatoid and Oseoarthritis
•  Subacromial Bursitis
• Tennis and Golfer's Elbow
• Thumb or Finger Sprain
• Triceps Strain
• Wrist Flexor or Extensor Tendinitis

Lower Body:

• Achilles Tendinitis and Rupture
• Adductor Strain
• Anterior (ACP) and Posterior Compartment Pain (PCP)
• Anterior and Posterior Cruciate Ligament Injury
• Calcaneal Bursitis
• Calf Strain
• Dislocated Patella
• Hallux Valgus and Rigidus
• Hamstring or Ischiogluteal Bursitis and tendinitis
• Hamstring Strain
• Hip Sprain
• Interdigital Neuritis - Metatarsalgia - Morton's Neuroma
• Knee Contusion, Housemaid's Knee, Prepatellar Bursitis
• March or Stress Fracture
• Medial and Lateral Collateral Ligament Injury
• Meniscus Sprain/Strain
• Metatarsalgia - Thinning of the Fat Pad
• Osgood Schlatter Syndrome
• Osteochrondritis Dissecans
• Patellar Teninitis and Quadriceps Insertion Strain
• Patellofemoral Syndrome
• Peripheral Neuropathy (PN)
• Piriformis Syndrome
• Plantar Fasciitis
• Posterior Knee Swelling - Baker's Cyst
• Quadriceps Strain
• Restless Leg Syndrome or Leg Cramps
• Sesamoiditis
• Shin Splints
• Sprained Ankle
• Tarsal Tunnel Syndrome
• Tensor Fascia Lata and Iliotibial Band Syndrome
• Tibial or Fiula Stress Ftacture
• Trochanteric Bursitis

This book consists mostly of diagrams and charts explaining the different points and meridians on the human body.

Five Elements and the Chakras

• five elements
• the nurturing cycle
• the control cycle
• beyond the five elements
• 24-hour horary cycle
• chakra system in meridian therapy

The Meridians

• the meridian network
• the relation between meridians and their organs
• chapter 2 overview
  • heart
  • small intestine
  • bladder
  • kidney
  • pericardium
  • triple heater
  • gallbladder
  • liver
  • lung
  • large intestine
  • stomach
  • spleen
  • governor vessel
  • conception vessel

Point Group Use in Diagnosis and Treatment

• tonification and sedation points
• master points
• source points
• luo points
• accumulation points
• command points
• alarm points
• bladder association points
• ifluential points
• transporting points
  • the jing-well points
  • the spring points
  • the stream points
  • the river points
  • the sea points
  • lower he-sea points
• meeting points
• ma dan-yang points
• vitality collapse points
• entry-exit points
• gohst points
• pulse points
• mensturation points
• pregnancy points
• musculo-tendendo points

Key Points on the Meridians

• heart meridian
• small intestine meridian
• bladder meridian
• kidney meridian
• pericardium meridian
• triple heater meridian
• gallbladder meridian
• liver meridian
• lung meridian
• large intestine meridian
• stomach meridian
• spleen meridian
• governor vessel
• conception vessel

Point Location

• heart
• small intestine
• bladder
• kidney
• pericadium
• triple heater
• gallbladder
• liver
• lung
• large intestine
• stomach
• spleen
• governor vessel
• conception vessel

 This book covers an astonishing amount of information in its near thousand pages, everthing from basic laser physics to dental, and veteranary useage. Here are some of its contents:

• Basic Laser Physics
  • physics
  • energy
  • radiation
  • wavelength and frequency
  • photon energy
  • the elecromagnetic spectrum
  • the optical reigon
  • radiation risks
  • can electromagnetic radiation cause cancer
  • protective mechanisms
  • light
  • the optical spectrum
  • light sources
  • various sources of radiation
  • natural sources of radiation
  • man-made light sources
  • the light emmiting diode (LED)
  • flash lamps
  • the laser
  • laser design
  • practical lasers
  • the properties of laser light coherence
  • interference
  • laser beam characteristics
  • polarisation
  • output power
  • continuous and pulsed lasers
  • the peak power value
  • average power output
  • power density
  • light distribution
  • beam divergence
  • collimation
  • risk of eye injury
  • decisive factors in the risk of eye injury
  • the laser instrument
  • properties of some laser types
  • description of common surgical laser types
  • the CO₂ laser (carbon dioxide laser)
  • carbon dioxide lasers in surgery
  • carbon dioxide lasers in dental applications
  • the Nd:YAG laser
  • Nd:YAG lasers in surgery
  • Nd:YAG lasers in dentistry
  • erbium lasers in dentistry
  • "strong" diode lasers in dentistry
  • the KTP laser
  • Q-switching
• Theraputic Lasers
  • the first generation 1975-85
  • the second generation 1985-95
  • the third generation 1995-2005
  • the fourth generation 2005 and onwards
  • what is a good laser therapy instrument
  • the basic instrument
  • sales tricks
  • high power-low power
  • laser or LED
  • high or low price
  • penetration of light into tissue
  • "a story of a young scientist"
  • the wavelength
  • how deep does light penetrate into tissue?
• Biostimulation
  • history
  • a few words on mechanisms
  • photoreceptors
  • what parameters to use
  • laser parameters
  • whitch wavelength?
  • output power
  • average output power
  • power density
  • energy density
  • the dose
  • treatment dose
  • calculation of doses
  • dose ranges
  • calculation of treatment time for a desired dose
  • "reay reckoner"
  • dose per point
  • pulsed or continuous light
  • pulse repetition rate (PRP)
  • patient parameters
  • treatment area
  • treatment intervals
  • pre- or postoperative treatment
  • treatment method parameters
  • local treatment
  • shallow problems
  • deeper problems
  • treating inside the body
  • systemic treatments
  • acccupuncture
  • trigger points
  • spinal processes
  • dermatome
  • blood irradiation
  • irradiation of lymph nodes
  • irradiation of ganglions
  • combo treatment
  • interaction with medication
  • other considerations
  • what about collimation?
  • depth of penetration, greatest active depth
  • factors that reduce penetration
  • tissue compression
  • how deep does the light penetrate?
  • laser light irradiation through clothes
  • the importance of tissue and cell condition
  • the importance of ambient light
  • in vitro/ in vivo
  • laser therapy with high output lasers
  • laser therapy with carbon dioxide lasers
  • laser therapy with Nd:YAG lasers
  • laser therapy with ruby lasers
  • laser therapy with Er:YAG lasers
  • laser therapy with surgical diode lasers
  • risks and side effects
  • the importance of correct diagnose
  • cancer
  • cytogentic effects?
  • a false picture of health
  • tiredness
  • pain reaction
  • do high doses of laser therapy damage tissue?
  • is it only an effect of temperature?
  • protection against radiation injury
  • how to measure effects of laser therapy
  • thermography
  • magnetic resonance imaging
  • high resolution digitized ultrasound B-scan
  • tensile strength
  • other objective methods
  • does it have to be a laser?
  • FDA (Food and Drug Administration)
  • how well documented?
  • confused?
  • the funding research
  • as time goes by
• Medical indications
  • who and what can be treated?
  • acne
  • allergy
  • antibiotic resistance
  • arteriosclerosis
  • arthritis
  • asthma
  • blood preservation
  • blood pressure
  • bone regeneration
  • burning mouth syndrome
  • cancer
  • cardiac conditions
  • carpal tunnel syndrome
  • cerebral palsy
  • crural and venous ulcers
  • delayed onset muscular soreness (DOMS)
  • depression, psychosomatic problems
  • diabetes
  • duodenal/gastric ulcer
  • epicondylitis
  • erythema multiform major
  • fibrositis/fribomyalgia
  • headache/migraine
  • heamorrhoids
  • herpes simplex
  • immune system modulation
  • inflammation
  • inner ear conditions
  • laryngitis
  • lichen
  • low back pain
  • mastitis
  • microcirculation
  • morbus sluder
  • mucositis
  • muscle regeneration
  • mycosis
  • nerve conduction
  • nerve regeneration and function
  • oedema
  • ophthalmic problems
  • pain
  • periostitis
  • plantar fasciitis
  • salivary glands
  • sinuitis
  • spinal cord injuries
  • snake bites
  • sports injuries
  • stem cells
  • stroke, irradiation of the brain
  • tendinopathies
  • tinnitus, vertigo, meniere's disease
  • tonsillitis
  • trigeminal neuralgia
  • thrombophlebitis
  • tuberculosis
  • urology
  • warts
  • wiplash-assosiated dissorders
  • vitiligo
  • womens' health
  • wound healing
  • zoster
  • idications in the pipeline
  • alzheimer's disease
  • botox failures
  • cellulites
  • cholesterol reduction
  • complex reigonal pain syndrom (CRPS)
  • eczema
  • erectile dysfunction
  • familiar amyotrophic lateral sclerosis (FALS)
  • glomerulonephritis
  • obesity
  • orofacial granulomatosis
  • Parkinson's disease
  • post-mestrual stress
  • pemphigus vulgaris
  • sleeping disorders
  • withdrawal periods
  • wrinkles
  • consumer lasers
• Dental LPT
  • the dental laser literature
  • on which patients can LPT be used?
  • dental indications
  • alveolitis
  • anaesthetics
  • aphthae
  • bleeding
  • bisphosphonate related osteonecrosis of the jaw
  • caries
  • dentitio dificilis (pericoronitis)
  • endodontics
  • extraction
  • gingivitus
  • herpes zoster
  • hypersensitive dentine
  • implantology
  • leukoplakia
  • lingua geographica (glossitis)
  • lip wounds
  • nausea
  • nerve injury
  • orthodontics
  • mild dental pain
  • paediatric dental treatment
  • periodontics
  • prosthetics
  • root fractures
  • secondary dentine formations
  • temperature caveats
  • toemporo-mandibular disorders (TMD)
  • TMD and endodontics
  • other dental laser applications
  • dental pohoto dynamic therapy
  • composite curing
  • deminerallisation
  • tooth bleaching
  • caries detection
  • lasers as a diagnostic tool
  • case reports
• Non Coherent Light Sources
• Veterinary Use
  • case reports
• Contra Idications
  • pacemakers
  • pregnancy
  • epilepsy
  • thyroid gland
  • children
  • cancer
  • haemophilia
  • irradiation of the brain
  • radiation therapy patients
  • diabetes
  • tatoos
  • light sensitivity
• Coherence
  • the role of coherence in laser phototherapy
  • itroduction
  • summary
• Dose and Intensity
  • basics about energy
  • output power
  • power density
  • the laser beam
  • the laser probe
  • pulsed lasers
  • energy density
  • treatment dose
  • the dose does not demend on the intensity
  • dose per point
  • more about treatment technique
• The Mechanisms
  • are biostimulative effects laser specific?
  • is it possible to prove that laser therapy doesn't work?
  • comparisons between coherent and non-coherent light
  • what is the importance of the length of coherence
  • hode's hamburger
  • hode's big burger
  • abrahamson's apple
  • moonlight
  • how deep does light penetrate tissue?
  • bright light phototherapy
  • similarities and differences
  • possible primary mechanisms
  • polarisation effects
  • what characterises the light in a laser speckle
  • porphyrins and polarised light
  • cell cultures and tissue have different optical properties
  • tthe effect of heat development in the tissue
  • macroscopic heating
  • the microscopic heat effect
  • mechanical forces
  • excitation effects
  • primary reactions due to excitation
  • secondary reactions due to cell signaling
  • flourescence-luminescence
  • multi-photon effects
  • llasting effects in tissue
  • non-linear optical effects
  • opto-acoustic waves
  • secondary mechanisms
  • effects on pain
  • effects on blood circulation
  • stimulatory and regulatory mechanisms
  • effects on the immune system
  • other interesting possibilities
  • summary of mechanisms
  • diagnostics with therapeutic lasers
  • photodynamic therapy - PDT
  • other medical uses of lasers
• A Guide for Scientific Work
  • methodology of a trial
  • parameters
  • technical parameters
  • treatment parameters
  • medical parameters
  • closer description of the technical parameters
  • name of instrument (producer)
  • laser type and wavelength
  • laser beam characteristics
  • number of sources
  • beam delivery system
  • output power
  • power density at probe aperture
  • calibration of the instrument
  • closer description of the treatment parameters
  • treatment area
  • dose: energy density
  • dose per treatment and total dose
  • intensity: power density
  • treatment method
  • treatment distance (spot size), type of movement, scanning
  • sites of treatment
  • number of treatment sessions
  • frequency of treatment sessions
  • closer description of the medical parameters
  • description of the problem to be treated
  • patients (number, age, sex)
  • exclusion criteria
  • inclusion criteria
  • condition of patient
  • pre-, parallel-, or post-medication
  • treated with other methods before
  • drop-out rates
  • follow up
  • outcome measures
  • statistical analysis
  • economy
  • gallium-alluminium and all that
  • recommendations of WALT - the world assosiation for laser therapy
• The Laser Phototherapy Literature
  
  • the importance of reporting all laser parameters - even in the abstract
  • diclofenac, dexamethasone or laser phototherapy?
  • another pithole in LPT research
  • database of abstracts of reviews of effects (DARE)
  • the wound healing contradiction
  • wikipedia
  • poor documentation - compared to what?
  • LPT equipment and the future
  • english language books od LPT:
  • books in other languages, with ISBN
  • laser phototherapy journals
  • information for your patient

High-Tech Acupuncture with Laser Light

• an equisite light therapy
• biostimulation
  • light can heal
  • primary stimulation effects
  • secondary simulation effects
  • is ther optimum stimulation
• laser acupuncture
  • high-tech and tradition
  • laser ear acupuncture
  • is there an optimum dose
• resonance therapy
  • what is resonance therapy
  • explanatory models
  • laser frequencies and those who discovered them
  • resonance theapy on the ear
• additional methods and synergisms
  • suplementary acupuncture methods
  • special applications
  • synergisms
• laser types
  • laser types by wavelength
  • laser types by type of signal
  • laser types by form of application
  • laser classes

Practical Guidelines

• point localization
• selecting the frequency
• recommended doses
  • laser acupuncture: doses and treatment time with laser pen and laser needle
  • area therapy: dose and treatment time with laser shower and dermaspot
• important information regarding therapy plans
• containdications and side effects

Treatment Concepts

• orthopedics
  • achillodynia
  • arthitis, idiopathic juvenile (pediatric rheumatism)
  • arthritis, rheumatoid (chronic polyarthritis)
  • arthritis urica (gouty arthritis)
  • aseptic osteonecrosis
  • bakers cyst (popliteal cyst)
  • slipped disk (spinal disk herniation)
  • bursitis
  • chrondophathia patellae
  • coxarthosis (arthosis of th hip)
  • CRPS (complex regional pain syndrome, Sudeck's disease, reflex dystrophy)
  • epicondylitis humeri
  • exostosis (bony outgrowth)
  • heel spur (calcaneal spur)
  • fibromyalgia
  • gonarthosis (arthosis of the knee joint)
  • hallux valgus (hallux rigidus, bunion)
  • cervical spine syndrome
  • sacroiliac joint blockage (SIJ blockage)
  • capsular ligament injury
  • lumbosciatica (sciatica syndrome, irritation of the nerve root)
  • lymphatic edema, postoperative
  • metataralgia
  • muscle fiber rupture (traumatic myopathy)
  • Myofascial pain syndrome
  • shoulder-arm syndrome
  • spinal canal stenosis
  • wound healing disorder
• neurology
  • carple tunnel syndrome CTS (median nerve compression syndrome)
  • cephalgia
  • facial paresis
  • migraine
  • multiple sclerosis MS (encephalomyelitis disseminata)
  • paresis (incomplete paralysis)
  • Parkinson's syndrome (Parkinson's disease)
  • phantom pain
  • polyneuropathy
  • restless leg syndrome RLS
  • transient ischemic attack TIA (stroke)
• psychosomatic disorders
  • anorexia nervosa
  • burnout syndrome
  • depression
  • jet lag (dysrhythmia)
  • concentration disorders
  • addictions - alcohol abuse
  • addictions - nicotine abuse
• throat, nose and ear
  • otitis media (inflammation of the middle ear)
  • parotitis
  • acute sinusitis
  • chronic sinusitis
  • tinnitus
  • tonsillitus (angina tonsillaris)
• internal medicine
  • allergic disorders - basic laser desensitization
  • allergic disorders - allergic exanthema
  • allergic disorders - hay fever
  • allergic disorders - food allergies
  • angiopathies - chronic venous insufficiency CVI
  • angiopathies - hemorrhoids
  • angiopathies - raynaud's disease
  • angiopathies - thrombophlebitis
  • gastrointestinal disorders - ulcerative colitis
  • gastrointestinal disorders - gastritis
  • gastrointestinal disorders - hepatitis
  • gastrointestinal disorders - crohn's disease
  • lung disorders - bronchial asthma
  • lung disorders - acute bronchitis
  • lung disorders - chronic bronchitis
  • lung disorders - COPD (chronic obstructive pulmonary disease)
  • metabolic disorders - diabetes mellitus
• dematology
  • acne (acne simplex)
  • atopic eczema / neurodermatitis
  • hyperhidrosis
  • psoriasis
  • seborrjeic eczema
• pediatrics
  • adenoids (adenoid vegetations, polps, palatine tonsil)
  • attention deficit hyperactivity syndrome ADHS
  • attention deficit syndrome (concentration disorder)
  • abdominal pain, functional
  • chronic bronchitits
  • three months' colic (regulation disorder / infant crying)
  • enuresis nocturna (bedwetting)
  • whooping cough (petussis)
  • tympanic effusion (tubal catarrh)
  • obesity (adipositas)
  • underweight (growth disorder)
  • cerebral paresis (cerbral palsy)
• gynaecology
  • mastitis (inflammation of the mammary glands)
  • PMS (postmenstral syndrome)
  • morning sickness (hyperemesis gravidarium)
• dental medicine
  • stomatitis/gingivitis/aphtea
  • tooth extractions
  • bleeding gums
  • toothache
• ophthalmology
  • age-related macular degeneration AMD
  • central serous chorioretinopathy (central serous retinitis)
  • glaucoma
  • conjuctivitis
  • retinitis pigmentosa
  • dry eyes (sicca syndrome)

History and Fundamentals

• introduction: historical vignettes from the feild of photomedicine
• history and fundamentals of lasers and light sources in photomedicine
• light-tissue interactions
• history and fundamentals of photodynamic therapy
• history and fundamentals of low-level laser therapy

Diseases Caused by Light

• uv effects on the skin
• photocarcinogenesis nonmelenoma skin cancer
• autoimmune photodermatoses
• photoaging
• uvr-induced immunosurpression
• the porphyrias
• photoprotection
• botanical antioxidants for photochemoprevention
• reversal of DNA damage to the skin with DNA repair liposomes
• climate change and ultraviolet radiation exposure
• photochemistry and photobiology of vitamin D

Ultraviolet Phototherapy

• phototherapy for psoriasis
• PUVA therapy
• extracorporeal photopheresis
• ultraviolet C therapy for infections

Photodynamic Therapy (PDT)

• recent advances in developing improved agents for photodynamic therapy
• 5-aminolevulinic acid and its derivatives
• genetically encoded photosensitizers: structure, photosensitization mechanisms, and potential application to photodynamic therapy
• light dosimetry for photodynamic therapy: basic concepts
• multimodality dosimetry
• cell death and PDT-based photooxidative (phox) stress
• vascular and cellular targeted PDT
• photodynamic therapy for increased delivery of anticancer drugs
• targeting strategies in photodynamic therapy for cancer treatment
• enhancing photodynamic treatment of cancer with mechanism-based combination stratagies
• nanoparticles for photodynamic cancer therapy
• drug delivery stratagies for photodynamic therapy
• antimicrobial PDT fo clinical infectious diseases
• PDT and the immune system
• detection of bladder cancer by fluorescence cystocopy: from bench to bedside the hexvix story
• photochemical internalization: from bench to bedside with a novel technology for targeted macromolecule therapy
• the story of tookad: from bench to bedside
• photodynamic therapy in ophthalmology
• photodynamic therapy in dermatology
• photodynamic therapy in the gastrointestinal tract
• photodynamic application in brain tumors
• photodynamic therapy for malignant pleural disease
• clinical photodynamic therapy in the Chinese region
• photodynamic therapy and fluorescent diagnostics in the Russian federation

Low-Level Laser (Light) Therapy (LLLT)

• chromophores (photoacceptors) for low-level laser therapy
• low-level laser therapy signaling pathways
• irradiation parameters, dose response, and devices
• low-level laser therapy: clearly a new paradigm in the management of cancer therapy- induced mucositis
• low-level laser therapy for wound healing
• low-level laser therapy in the treatment of pain
• low-level laser therapy in arthritis and tendinopathies
• low-level laser therapy and LED therapy on muscle tissue: preformance, fatigue, and repair
• low-level laser therapy for stroke and brain disease
• low-level light therapy for nerve and spinal cord regeneration
• low-level laser therapy in dentistry
• low-level laser therapy and stem cells
• low-level light therapy for cosmetics and dermatology

Surgical Laser Therapy

• laser and intense pulsed light treatment of skin
• therapeutic uses of lasers in eye care
• lasers used in dentistry
• lasers used in urology
• lasers used in otolaryngology
• laser treatment to nanoparticles for theranostic applications
• laser imminutherapy
• tissue repair by photochemical cross-linking

Other Phototherapies an Future Outlook

• optical guidance for cance interventions
• phototherapy for newborn jaundice
• biological evidence of the efficacy of light therapy in psychiatric disorders
• future developments in photomedicine and photodynamic therapy

We can talk to the cells, but  we must learn their language.”
Tiina Karu

This challenging statement has been met with enthusiasm as well as with incredulity. Taking command of the cells by the use of light is still
not part of mainstream medicine, in spite of strong scientific evidence. It is now obvious that we can indeed talk to the cells even though we are still rather poor in understanding their language The skepticism about this method has many explanations. In this article, we will focus on one of them – marketing tricks.

The collected evidence about the many advantages of laser phototherapy is rapidly increasing. The knowledge about the basic mechanisms as well as about the optimal dosage intervals has improved dramatically in recent years. It should be easy to sell laser equipment to all kinds of therapists just using the available scientific knowledge – which is truly amazing in and of itself. But this is not always the case. Too many manufacturers deliver poor equipment and training, and too many of them use sales gimmicks in order to make their equipment look unique. It is not that the devices they’re selling are incapable of producing therapeutic effects. They are; even a $10 lecture pointer has some therapeutic potential. It is that they are simply NOT capable of delivering upon many (in some cases, most) of the claims that are made about them, whether those claims be about the range of treatable indications, therapeutic outcomes, depth of penetration, speed of treatment, method of application, or patented waveforms, etc.. Such sales techniques and outright dishonesty are confusing for consumers and risk draining the therapy of the credibility it deserves. Let us look at some examples!

“An extraordinary claim requires extraordinary proof.”
Marcello Truzzi

Sales trick 1: Soliton waves
One laser manufacturer in the USA claims that their lasers produce “soliton waves” by “piggy-backing one wavelength upon another”, and that these “penetrate deeper into the body than is possible with any other type of laser”. This sounds impressive and unique, but it is a sales trick, no more, no less. No therapeutic laser on the market produces solitons. And, even if it were possible and financially viable to do so, what evidence is there to support this manufacturer’s claims of therapeutic benefit?

Sales trick 2: Scalar waves
The husband-and-wife “inventors” of the Scalar Wave Laser claim to have developed the “most advanced low level laser technology with state of the art quantum scalar waves” that supposedly employs a “unique approach to accessing the quantum neutral unified field state” to “dissolve cellular memory, normalize body systems, optimize anti-aging capabilities, and activate the glands and higher dimensional subtle body that yogis and mystics have tapped into throughout the ages”.
This is, of course, a complete fabrication, a crackpot theory. No laser equipment designed for laser phototherapy is producing scalar waves and again, even if such waves existed, there is no evidence whatsoever that they should have a positive or negative effect of cell functions.

Penetration
For many indications, some degree of light penetration through tissue is an advantage. The penetration of laser light into different types of tissue is surprisingly poorly investigated, but enough is certainly known to refute the claims of some manufacturers. There are two extremes oft found in the marketing claims, one that photons can penetrate clothes and even the entire body at very low powers, the other that very high power output is needed to reach very deep-lying targets. Both claims are characterized by gross exaggeration, demonstrating either complete ignorance or deliberate misapplication of the science of optics.

Sales trick 3: Treating through clothes
One particular manufacturer claims that their device, emitting a very low intensity thin line of red laser light, can be used to treat patients effectively through their clothing. Yet it is obvious to anyone who wears a shirt in the sun that clothes are a very effective blocker of light. And the skin barrier in itself reduces the amount of light going below the dermis. A simple experiment on the penetration of 650 nm 20 mW red laser light through different types of textiles can be watched on the following Youtube presentation:

http://www.youtube.com/watch?v=MkGJvvWD1vw

Representatives of this company also claim that these photons go right through our bodies. Whilst it is possible for very high-energy particles such as neutrinos and for x-rays, being very different waves, to penetrate through our bodies, the low energy photons produced by therapeutic lasers are physically incapable of penetrating through that much tissue.
Recent research is hinting that low power and long exposure is better than high power and short time for tissue regeneration, and, seemingly underlining this statement, this same company has presented research papers showing success using their lasers in the clinical setting (without clothes).
Serious users of this approach report treatment times in excess of 15-20 minutes, which may produce a systemic effect by irradiating blood through superficial blood vessels. Well enough, but this does not involve photons penetrating the body, and certainly will not work through clothes. Mixing science with pseudoscience is pseudoscience.

Sales trick 4: Class IV laser therapy
The international system of laser classification is concerned only with the risk for eye injury and, at higher powers, skin damage. It has nothing at all to do with suitability for laser treatment, nor does it mean a generational change nor ensure any improvement in efficacy. Many different parameters are considered in eye risk evaluation (laser wavelength, beam diameter, beam divergence, exposure time, pulsing vs continuous emission, type of pulsing and more). Actually there are Class I lasers that are higher powered than many Class IV instruments! So, there is no sense in or reason for, other than deception, the term “Class IV laser therapy”.
For example, some manufacturers claim that their Class IV lasers (e.g. 10-60 W, 980 nm laser) offer superb penetration through tissue (from 6-to-9 inches according to one manufacturer), and that the so-called “weak” class IIIB lasers (e.g. 500 mW, 808 nm laser) hardly penetrate the surface skin barrier at all. However, in the chosen example below, the very opposite is the truth! Due primarily to its absorption by water in the tissue, 980 nm penetrates less than 808 nm, and this is not compensated by the higher power. At around 808 nm we actually have the best penetration into tissue, and increasing power only increases the depth of penetration marginally. With the higher superficial absorbance of the 980 nm laser there will be considerable heating, and, while heat is fine for many conditions, it is not of what photomedicine is constituted. The picture to the left supposedly illustrates the superiority of a Class IV laser. Although the illustrations and explanations vary, there is more than one laser company using the same flawed argument to promote high-powered lasers.
It is also interesting to note the use of the term “Class IV technology”. There is no specific “technology” that enables a manufacturer to choose a laser emitter that produces more than 500 mW, thus the term “Class IV technology” is simply used to infer a differential benefit that does not exist. Apart from power, the only differences between Class IIIB and IV lasers are the potential hazards and, usually, the price.
For more detailed information about the penetration of laser light, we recommend that you read our article “Penetration of light” in Laser World (www.laser.nu).

Sales trick 5: Claimed output vs. actual output
Two recent papers have considered the same thing:
The power of therapeutic lasers in use. Both studies are from Brazil and the outcome is alarming, although don't think that this is a problem only in Brazil! Certainly, many laser manufacturers are responsible and are producing equipment of a high standard. But too many are not! Read the abstracts below, and take heed!
Photomed Laser Surg. 2009;27(4):633-639. Radiant power determination of low-level laser therapy equipment and characterization of its clinical use procedures.
Guirro RR, Weis LC.
Department of Biomechanics, Medicine and Rehabilitation of the Locomotor System, School of Medicine of Ribeirão Preto, University São Paulo, Ribeirão Preto, SP, Brazil. rguirro@fmrp.usp.br

The main objectives of this study were to characterize low-level laser therapy (LLLT) and the physical therapy clinical procedures for its use. There are few scientific studies that characterize the calibration of LLLT equipment. Forty lasers at 36 physical therapy clinics were selected. The equipment was characterized through data collected from the owner manuals, direct consultation with the manufacturers, and a questionnaire answered by the users.
A digital potency analyzer was used to calibrate released mean potency. Qualitative data were presented throughout the descriptive statistics and quantitative data were analyzedby the Wilcoxon/Kruskal-Wallis and Fisher tests (significance, p < 0.05).

RESULTS: The laser equipment was either GaAs (70.5%) or HeNe (23.5%), and 60% was analog and acquired over 5 years ago. The majority of the equipment was used 10-15 times per week and the most frequent density level used was 2 to 4 J/cm(2). Protective goggles were available in only 19.4% of the clinics evaluated. The association between the analyzed categories demonstrated that a lower mean potency was correlated both with equipment acquired over 5 years ago and analog technology. The determined mean potency was lower than the one
claimed by the manufacturer (p < 0.05). In 30 cases, the analyzed equipment presented a potency between 3 microW and 5.6 mW; in three cases, the potency was >25 mW; and in seven cases, potency was nonexistent. CONCLUSION: The analyzed equipment was out-dated and periodical maintenance was not conducted, which was reflected in the low irradiated potency.

Other laser Our laser
Rev Bras Fisioter. 2010;14(4):303-308. Calibration of low-level laser therapy equipment.Fukuda TY, Jesus JF, Santos MG, Cazarini Junior C, Tanji MM, Plapler H. Physical Therapy Sector, Irmandade Santa Casa de Misericórdia de São Paulo (ISCMSP), São Paulo (SP), Brazil. tfukuda10@yahoo.com.br

Despite the increase in the use of low-level laser therapy (LLLT), there is still a lack of consensus in the literature regarding how often the equipment must be calibrated. For the evaluation, a LaserCheck power meter designed to calibrate continuous equipment was used. The power meter was programmed with data related to the laser's wavelength to gauge the real average power being emitted. The LLLT devices were evaluated in two ways: first with the device cooled down and then with the device warmed up for 10 minutes. For each condition, three tests were performed. The laser probe was aligned with the power meter, which provided the real average power being emitted by the LLLT device. All of the data and information related to the laser application were collected with the use of a questionnaire filled in by the supervising therapists. RESULTS: The 60 devices evaluated showed deficit in real average power in the cooled-down and warmed-up condition. The statistical analysis (ANOVA) showed a significant decrease (p<0.05) in the real average power measured in relation to the manufacturer's average power. On average, the most common dose in  the clinics was 4 J/cm², and the most desired effects were healing and anti-inflammatory effects.
According to the World Association for Laser Therapy (WALT), 1 to 4 J of final energy are necessary to achieve these effects, however only one device was able to reach the recommended therapeutic window.

CONCLUSION: The LLLT devices showed a deficit in real average power that emphasized a lack of order in the application of this tool. The present study also showed the need for periodical calibration of LLLT equipment and a better technical knowledge of the therapists involved.

Pulsing
There are principally two types of pulsing in laser phototherapy – chopped (switched) or super pulsed. A chopped beam is a continuous beam that is electronically (or mechanically) switched between on and off. During the moments when it is on it has typically the same output power as in continuous mode, but as it is not on all the time, the average output power is less than when it is continuous. The average power is a function of the continuous wave power and the duty cycle (the ratio of the “on” time of the beam to the total emission (“on” + “off”) time, usually expressed as a percentage). Typical laser types are most of the gas lasers (such as the HeNe laser) and all semiconductor (diode) lasers (except the GaAs laser).
The GaAs laser was the first semiconductor laser in the world. In order to generate laser light, the current density in the GaAs semiconductor crystal had to be extremely high. As a consequence of the high electric current the output power of this semiconductor laser is very high. Typical peak power is in the order of many watts. However, when an electric current is conducted through a material heat is generated, and with the necessary high current in this laser the crystal will burn up immediately unless the time of current conduction is extremely short, i.e., super-pulsed GaAs lasers cannot work continuously. The maximal pulse time for this laser is in the order of 100 to 200 nanoseconds and, after each such pulse, a long cooling time is needed, usually about a thousand times longer than said pulse time. This form of pulsing is called super pulsing and, although the peak power is very high, the average output of super-pulsed lasers is comparatively low. Typically
the GaAs laser produces its maximum emission at 904 nm.

Sales trick 6: The 904 nm trick
Restating the above, even though the peak power of the super-pulsed GaAs laser may be very high, it lasts for an extremely short time compared to the pulse cycle, resulting in an average output power that is usually a thousand times lower than the peak power. For clinical use, it is the average Power that counts. The energy (dose) delivered from pulsed lasers is always the average output power multiplied by the exposure time. The average power is the important output of the laser.
Some manufacturers preferto label these lasers as “very strong” and state only the peak power which then can be in the order of 100 watts. This sounds impressive, but typically these lasers emit 10-100 mW average power, and this is what counts for the treatment. The GaAs lasers are quite useful in physiotherapy, but care has to be taken.
In some super-pulsed lasers the average output changes with the set pulse frequency, so that low pulse repetition rates deliver very low average outputs. This means that with such lasers, with low frequency settings, the treatment time may be impractically long in order to deliver a reasonable dose. One manufacturer, for example, promotes its super-pulsed lasers as having 25,000 mW or 50,000 mW of power, and offers the user a small number of preset ‘programs’ which, essentially, only adjust the pulse frequency and, therefore, the average output power. One of these ‘programs’ sets a frequency of 5 Hz. To calculate the average power one must only know the Peak Power, the Pulse Frequency and the Pulse Duration. As mentioned previously, the pulse duration (i.e., the ‘width’ of each pulse of energy) of most GaAs devices is 100-200 nanoseconds (0.0000001 – 0.0000002 sec). If we use the manufacturer’s ‘highest’ power option (50,000 mW), select their 5 Hz program, and assume the longest possible pulse duration (0.0000002 sec) for our calculation, we arrive at an Average Output Power of only 0.050 mW, or fifty millionths of one Watt. With this very low average power it will take twenty thousand seconds (5.6 hours) for this manufacturer’s laser to deliver one Joule. Impractically long, perhaps? Other super-pulsed lasers employ “pulse trains”, which enable the average output to be maintained at a constant level over all frequencies. The importance of checking upon this is obvious when it comes to acquiring a GaAs laser.

Sales trick 7: False super pulsing
One manufacturer claims that its dual-wavelength (800 nm and 970 nm) high-powered Class IV laser has better penetration due touts ‘Intense Super Pulse’ emission. However, these diode lasers are not super pulsed, they are “chopped”, and chopping does not offer increased penetration. In this case chopping the output simply reduces the tissue-heating effect of the high power laser by both reducing the average power and also allowing time for the tissue to thermally relax (i.e., dissipate heat) between each pulse of light.

Frequencies
The biological differences between super-pulsed and chopped emissions are likely to be fundamental. Is pulsing then of interest? The in vitro studies by e.g. Tiina Karu clearly show that the type of pulsing is of importance. However, in these situations one type of cell and one type of reaction is studied. In the clinical situation, many types of cells are irradiated and a multitude of events happen. So is pulsing then of any clinical importance? The answer is that we do not know.
This is well presented in the recent literature review by Hashmi et al, http://www.ncbi.nlm.nih.gov/pubmed/20662021
Some lasers are pulsed to allow for heat dissipation, but that has nothing to do with biostimulation. Chopping is an option in some continuous lasers and users should be aware of the fact that suggested pulse repetition rates are only setting options; we do not know if the different pulse repetition rates provide different biological results. Many “recommended” frequencies employed in therapeutic lasers are, in fact, carried over from other fields and modalities, especially electrical stimulation. Nogier’s frequencies, for example, are often incorporated into laser therapy protocols for both humans and animals; yet their original application was in humans only, specifically auricular therapy delivered by electrical stimulation. Due largely to the impact of pulse frequency upon the average power of the first
therapeutic diode laser, the GaAs, Nogier’s original frequencies (there are seven, ranging from 1.14
Hz to 146 Hz) are even presented at a higher “harmonic” so as to achieve a higher average output power, further increasing the disparity between their original intended application and their current use. Despite this, and the fact that there have been no studies undertaken to compare or confirm the efficacy of the original or higher-harmonic laser-delivered frequencies in humans or animals, these and other frequencies are provided as an integral part of many different therapeutic laser devices and their pre-programmed protocols.

Sales trick 8: Pre-programmed machines
There are many variations of so called pre-programmed lasers on the market. Some offer ‘starter’ protocols that employ simple variations of power, frequency and time, making these parameters known to the user and even affording them the option of changing them as their knowledge and
experience improves. Others, however, provide the user with nothing more than a choice of letters or numbers that represent different “proprietary programs”, ensuring that the user is kept completely in the dark as to what they’re actually doing. Such programs may consist of various frequencies and exposure times, often in automatically-changing combinations of such; for instance, 20 seconds of 500 Hz + 40 second of 120 Hz + 10 second of 1500 Hz. The user is informed only that that “program” is supposed to be the best for e.g. headache, and that another program and time/frequency combination is the best for arthritis, etc. The buyer of such an instrument trusts that the constructor of the instrument knows that this is a fact. However, there are no such optimal time/frequency combinations scientifically proved to be better than others. Also - how can a setting for “arthritis”, for example, be the same for a finger joint as well as for a knee? Who can verify the pulse repetition rates recommended? Such preset protocols will generate nothing more than vaguely satisfactory outcomes, at best; neither what your patients expect of you, nor what you should expect of a clinical tool that has, most likely, cost you thousands of dollars.
One particular manufacturer has corrupted the use of the terms ‘Optical Window’ and ‘Therapeutic Window’, well-known to many within the phototherapy field, to label their preset programs as so-called ‘Therapeutic Optical Windows’ that, supposedly, deliver optimal combinations of the many different parameters that influence clinical outcomes. As an exercise, let’s consider the various device and treatment parameters and patient characteristics that affect variations in phototherapy outcomes, and determine how many iterations of these must be clinically tested and validated before one could claim, with even a hint of honesty, to have determined the optimal “Therapeutic Optical Windows” for even a handful of indications.
First we take the various parameters of, say, a switched continuous wave device (e.g., output power, spot size, wavelength, pulse frequency, duty cycle). Then we add the irradiation duration, treatment technique, number of points to be treated or the area of affected tissue, and the target tissue depth. Next, toss in a handful of such patient characteristics as skin colour and tissue type and whether their condition is acute, sub-acute and chronic. Finally, consider some desirable clinical outcomes such as analgesia, reduction of inflammation, enhanced tissue repair and/or nerve tissue regeneration. Although this gives us a very simplified set of factors, we are still left with potentially billions of combinations of variables that must be subjected to clinical testing in order to support this manufacturer’s claims. In forty-something years of research into phototherapy, by hundreds of researchers, we have barely even scratched the surface in terms of determining upper and lower activity thresholds of irradiation duration and intensity, and yet we’re now supposed to believe that one company only has considered and tested every possible iteration and distilled them into nine optimal “Optical Therapeutic Windows”? Even the most credulous among us must baulk at that ...
We recommend, instead, availing yourself of high-quality research published peer-reviewed journals, informative manuals and qualified seminars, rather than automatic settings. Use palpation, your own physiologic knowledge, your patients’ feedback and your experience to guide you in your choice of parameters.

High power – low power
There are two extremes on the market – those promoting very low power output and those promoting very high power output. Which is best?
The answer is: none of them. There is no “one size fits all” laser. Each one has its limitation. There is an increased awareness about the necessity to deliver fairly low doses over longer time to optimize anti-inflammatory results (Castano et al 2007, http://www.ncbi.nlm.nih.gov/pubmed/17659584as one example). This means that, at least for healing processes, low power over long time is more effective than high power over short time, even if the total energy is the same. The same goes for stimulation of cell proliferation. For temporary analgesia of painful conditions, high power over short time can give a better momentary effect, subject to certain minimum-time and maximum-power thresholds. The optimal dose windows for musculo-skeletal indications, based upon the current scientific evidence, can be found at www.walt.nu
Conclusion: very high powered lasers are useful for treating large areas in short time and to obtain pain inhibition, but seemingly less effective for basic cell stimulation. And they do not penetrate much deeper due to the high output– in fact, the very act of making a high power laser ‘safe’ for long-duration exposures may make it less capable of penetrating as deeply as a lower-powered laser that can e.g. be applied in contact and with slight pressure to the skin. All types of medical lasers are useful within their own limitations, but the very high powered lasers are still lacking scientific documentation in spite of their increasing popularity with salesmen and their less-informed customers.
And – N.B. – high power does not mean that a laser instrument has to be in laser class IV. Let us assume that the probe has 10 laser diodes, placed at
some distance from each other, each having an output of 450 mW, i.e. class III. This instrument is then a less-hazardous (by definition) class III instrument with an output of 4.5 W (4,500 mW).

Laser or LED
You will find many different configurations of phototherapy instruments in the market, some offering laser output only, some offering only LEDs , and – excluding LEDs that are provided for indication only – other devices combining both lasers and LEDs as active therapeutic components.  The two latter types are sometimes deceptively called “laser” with no reference made to other emitter types; this is inaccurate, at best. Often the buyer is unaware of the distinction, thinking they have bought a true laser device. The primary reason for replacing laser sources with LED sources,
or to add such, is not that LEDs are better or more efficient, but simply that they are cheaper to buy and to drive electrically. Although LED instruments can also elicit good clinical results, they are not lasers and it is technically and ethically incorrect to call them such; doing so serves only to benefit the manufacturer and/or marketer of the device, not the purchaser.

High or low price
If you are in the process of buying a laser instrument without experience of the market, you are vulnerable to the sweet arguments of the salesmen.
One aspect is the price. Is high price indicating high quality and good treatment results? No. Not necessarily the opposite either. We can recommend that you acquire a power meter (separate or built-in). Also find out the service level of the company – what happens when it breaks?

Bottom line
Laser phototherapy is a wonderful tool in medicine and useful for just about any medical practitioner. The scientific evidence is considerable but differs from one indication to the other.
What is already known is sufficient for piquing the interest of anyone with an open mind. So why use sales tricks when the plain truth is good enough?

The Biological Basics of Low Level Laser Light Therapy

• summary
• introduction
• Alexander Gurwitsch: cells emit light
• non-linear dynamics
• introducing quantum physics
• itroduction to quantum biology
• quantum coherence in biology
• biological coherence and the sensitivity of living systems
• Fritz Albert Popp: biophotons
• Guenther Albreecht-Buehler: cells respont to light
• Mae-Wan Ho: visualizing coherence
• conclusions

Therapeutic Laser Applications

• how does low level laser therapy work?
• what are the advantages over other modes of therapy?
• cliniclal use of low level laser therapy
• abstract submitted to laser and surgury medicine
  • background and objective
  • methods
  • results
  • conclusion
  • safety considerations
  • eye considerations
  • pace makers and other implanted devices
  • pregnancy
  • excessive toxicity
  • preface to treatment section

Nerver Roots

• flexion and extension
• lateral flexion
• rotation
• MRT (muscle response testing) through ROM of cervical spine
• shoulder
• neurological level
  • C5
  • C6
  • C7
  • C8
  • T1
  • S1
  • L5
  • L4
  • L3
  • L3-L5
  • L2-L4
  • L1-L3
• low back

Top Ten Laser Protocols

• organ / glands / tissue
• acute injury (shock)
• pain
• lymphatic protocol
• detox protocol
• immune protocol
• hormone protocol
• basic cranial nerve
• tissue memory
• trauma preparation protocol

A-Z Laser Protocols

• abdominal cramping
• abdominal inflammation/pain
• abrasions
• abscess
• achilles tear / strain (partial only; not rupture)
• acidosis (hyperacidity
• acid reflux
• acne
• acute injury
• adenoids
• (ADD) atention deficit disorder and hyperactivity disorder (ADHD)
• Addiction
• addison's disease
• adhesions
• adhesive capsulitis
• adrenal
• aids
• allergies
• alopecia
• alpha waves
• alzheimer's
• amenorrhea
• amoebas
• amyotrophic lateral sclerosis / lou gehrig's disease / motor neuron
• amnesia
• anemia
• anger
• angina
• anosmia (loss of smell)
• anxiety appendicitis
• arrhythmias
• arteries / arteriosclerosis
• arthritis
• asthma
• ataxia
• athlete's foot
• atrophy
• backache / back pain
• bacteria
• bed sores
• bedwetting
• bell's palsy
• beta waves
• bites
• bladder
• bleeding gums
• bloating
• blood pressure (high)
• blood pressure (low)
• blood sugar balance
• boils
• bone
• bowel
• bradycardia
• brain
• breast augmentation
• bronchitis
• bruises
• buerger's disease
• bunions
• burns
• burns (second degree)
• bursitis
• calcium deposits or formations
• candida
• canker sores
• capsulitis
• carpal tunnel syndrome
• cartilage
• cataracts
• chemical peels / resurfacing
• chest pain
• chicken pox (herpes zoster / varicella)
• cholecystitis
• cholelithiasis
• chronic fatigue
• chronic pain
• circulation
• cirrhosis
• cold sores (herpes simplex 1)
• colds and flu
• colitis
• concussion
• confusion
• congestion
• congestive heart falure (CHF)
• conjunctivitis (pink eye)
• costipation
• cramps (muscle)
• cranial nerves (general)
• cranial nerves VIII
• crepitus
• crohn's disease
• cuts
• cushing's syndrome
• cytomegalovirus (herpes syndrome V)
• deer tick
• delta waves
• depression
• dermatitis
• detoxification
•  diabetes
• diabetic neuropathy
• diabetic ulcers
• digestion
• dim vision
• disc herniation
• dizziness
• dupuytren's contracture
• dyslexia
• ear ache
• ear infection
• eczema
• edema
• emotional stress
• emphysema
• emulsification of fat
• endometriosis
• epistaxis
• epstein - barr virus
• esophagitis
• exercise recovery
• eye conditions
• facet syndrome
• facial paralysis
• fever
• fever blisters
• fibromyalgia
• flu
• food intolerance
• food poisoning
• foot fungus
• fracture
• fungus
• gait
• gallbladder (general)
• gallbladder (stones)
• ganglion cyst
• general musculoskeletal
• gerd
• gingivitis
• glaucoma
• goiter
• gout
• gums
• headache
• heart
• heartburn
• hearing difficulty
• hemorrhoids
• hepatitis A
• hepatitis B
• hepatitis C
• hernia
• herpes simplex
• herpes zoster (chickenpox / varicella)
• HIV
• hives
• hoarseness
• hormone balance
• hot flashes
• human papilloma virus (HPV)
• hyperactivity
• hyper/hypo-tension
• hyper/hypo-thyroid
• hyper/hypo-gycemia
• impotence
• immune enhancement
• incontinence
• indigestion
• infection
• inflammatory bowel disease
• inflammation
• influenza
• injuries
• insect bites
• irritable bowel syndrome
• ischemia
• jaundice
• joints
• keloid
• kidney
• kidey stones
• large intestine
• laryngitis
• ligament
• liposuction
• liver (balace and support)
• loss of smell (anosmia)
• loss of taste
• low back pain
• lungs
• lyme disease
• lymphadentis
• lymphatic
• macular degeneration
• memory problems
• meniere's disease
• meniscus sprain (grade 1)
• menopause
• mensturation
• mental fatigue
• meridian balance 15
• migraine
• motion sickness
• multiple sclerosis
• muscle
• muscle spasm
• myocardial inrarction
• nerve root
• neurogenic inflammation
• neuropathy
• nervousness
• nose bleed
• numbness
• nystagmus
• ocular motility disorders
• ocular nerve
• olfactory nerve
• osgood-schlatter disease
• otitis
• pain
• pain (chronic)
• pain (general)
• injury related pain (localized)
• pain (acute injury)
• pancreas
• parasite
• parasympathetic facilitazation
• paresthesia (numbness)
• periodontal disease
• pink eye (conjunctivitis)
• plantar fasciitis
• pneumonia
• polycystic kidney diseases
• polycystic ovary
• post operative scar revision
• post operative wound healing / pain
• post traumatic stress disorder (PTSD)
• postnasal drip
• premenstral syndrome (PMS)
• pre set head PL-touch
• pre-op
• prostate
• psoriasis
• punctures
• rash
• reflex sympathetic dystrophy (RSD)
• renal problems
• respiratory problems
• restless leg syndrome
• retinitis pigmentosa
• rheumatism
• ringworm
• road rash
• scar tissue
• sciatica
• sedation
• seizures
• shingles
• sinusitis
• skin
• sleep apnea
• small intesine
• smell - lack of
• sore throat
• soreness
• spasm
• spider veins
• spleen
• sprains
• spurs
• standars (neurological) setting
• stanard (up-regulation) setting
• staph infection
• stings
• stomach ulcer
• strep infections
• stress
• stroke
• sty
• subluxation
• sunburns
• swimmer's ear
• swollen ankles
• sympathetic calming
• tachycardia
• taste - lack of
• teeth
• tendonmyopathy (tendonitis)
• tension headaches
• theta waves
• thoratic outlet syndrome
• throat
• thrush
• thyroid (hyper)
• thyroid (hypo)
• tinnitus
• TMJ
• toenail fungus
• tonsilitis
• toothache
• ulcer
• ulcerative colotis
• up-regulation
• urinary tract infection
• varicose veins
• veins
• venereal warts
• viral infections
• voice
• vomiting
• water retention
• watery discharge from eye
• warts
• wounds
• yeast

More Lies and Subterfuge from the World of Class IV Laser Therapy

By Jan Tunér

The US laser manufacturer LiteCure (a.k.a. Companion/Pegasus for veterinary version) belongs to a group of laser manufacturers that confuse customers and let consumers pay a high price for something that they do not need. LaserAnnals has previously addressed the so-called Class IV lasers for LPT in general and in a few cases mentioned this particular culprit LiteCure. In this article, we will make a closer check on the credibility and ethics of this company.

Marketing is generally a way of stretching the truth or at least highlighting potential benefits of a product without mentioning the drawbacks. Not very ethical but more or less what consumers expect. Sheer lying is a bit different, and LiteCure uses blatant lies in its marketing. Let us see the first lie:

Lie #1. LiteCure originally claimed that 980 nm has a much better penetration than 808 nm, and that the very high output of their lasers improves the penetration. The illustration below is from their early attempts at marketing the supposed benefits of their device:

Anyone with some basic knowledge about tissue optics knows that 980 nm has a poor penetration due to absorption by water and lipids, and that 808 nm (the illustration actually states 880 nm, but this is not a commonly-used laser wavelength so we assume this was another error…) actually is in an optical window where penetration through skin is optimal. Using very high power with 980 nm doesn’t increase penetration considerably, but instead causes more light to be absorbed superficially more quickly, leading to heat generation. And LPT is not based upon heat but upon stimulation!

Knowledgeable scientists, experienced clinicians and other manufacturers were quick to criticise, however, and to call LiteCure out on this lie, and over time LiteCure has responded by adding the deeper-penetrating 810 nm wavelength to their products, and by modifying the image, as follows:

Although a step in the right direction, even this illustration is still misleading and, basically, incorrect: The effective depth of laser irradiation does not increase over time.

Further to that, the “effortless” non-contact technique causes considerable energy loss by reflection and backscatter – together, remittance, which has been measured at upwards of 80% from bare skin (Al Watban, 1996) – and up to 100% energy loss due to absorption within animal hair/fur.  This is hardly “efficient”!

The truth is the opposite to what their sales claims try to tell: A 0.5 W 808-810 nm Class 3B laser actually has a superior ability to penetrate into the body, whereas a 10.0 W 980 nm Class 4 has limited ability and also causes more problems with regards to heat generation. And, as the lower-powered Class 3B device may be applied in contact with the skin directly over the pathological tissue, and held steady for the necessary time to deliver the appropriate amount of energy, it is also significantly more efficient, accurate and safe.

The problem is that their consumer group is rather ignorant about LPT basics and swallow the bait. Fortunately for LiteCure, very high energies are bio-inhibitory and have a temporary pain relieving effect. This is an impressing effect when demonstrated. The downside of the procedure is that the needed reduction of an inflammatory process in inhibited and so is the body’s ability to regenerate itself. This is what is called “a sales trick”.

Lie #2. In its advertising material the LiteCure company writes: “World renowned Laser Therapy Experts, Jan Tunér and Lars Hode have indicated the advantages of high power laser therapy. The (research) literature supports the hypothesis that higher power density yields better clinical results.”

This is similar to the way the devil reads the bible. The above conclusion follows a part of our book where the remarkably low powered lasers on the Canadian market in the ‘90s is discussed. The vast majority of the lasers used were HeNe 1-2 mW and GaAlAs 5-30 mW. So the 400 mW lasers that had just arrived on the market at that time seemed to have a new potential – and they had.

Continued reading of our book reveals that high energies probably will have a better effect on pain conditions but probably not on superficial conditions such as wound healing. In fact, the discussion following the text about “high power” strongly modulates their usefulness.

This text appeared initially in the 2002 book “Low level laser therapy – clinical practice and scientific background”. In following versions of this book, the text has been modified and becomes more critical of extreme energies. And believe me, the next one will be even more critical, to avoid any misunderstandings.

Read my lips: “Tunér and Hode do not recommend 15 W Class IV lasers, not even 5 W!”  

An appropriately configured and applied Class 3B device can do all that we need, and if you want to reach deep targets the 904 nm superpulsed GaAs is the best tool!

LiteCure type of science

Recently a LiteCure research paper on fibromylaglia (FM) was published:

Panton L, Simonavice E, Williams K, Mojock C, Kim JS, Kingsley JD, McMillan V, Mathis R. Effects of Class IV laser therapy on fibromyalgia impact and function in women with fibromyalgia. J Altern Complement Med. 2013 May;19(5):445-52.

FM is a devastating condition and LPT is probably a viable option to use, especially since other therapies are rather ineffective and life-long intake of painkillers not a viable option, with the side effects in mind. The study by Panton is obviously performed by a competent team of medical experts, but it seems they have “been taken for a ride” by the LiteCure company. The overall effect of the laser treatment was modest, but had some effects.

So let us have a look on this paper…

For the laser group, treatment was rendered utilizing a LCT-1000 (LiteCure LLC, Newark, DE) solid-state GaAlAs laser delivering a continuous-wave, dual-wavelength laser with 20% 810 nm, and 80% 980nm at 10 W. Each 56.45 cm2 treatment point was treated with laser at 10.63 J/cm2 and warm air utilizing a grid scanning technique to avoid overheating tissue. Participants were instructed to expect some warmth but that the treatment should not burn and to provide verbal cues if the treatment spots became excessively warm. Each treatment point was treated for exactly 60 seconds for a total of 600 J per point, for a total daily treatment dose of 4200 J. The dual wavelength was used for two reasons: (1) this is what is commercially available and (2) two wavelengths allow for treatment in patients with different skin colours since different melanin concentrations will absorb light differently. Both wavelengths are in the accepted therapeutic window. The sham treatment consisted of 60 seconds of warm air alone over the seven tender points.

Now, let us try to make some sense about this study:

a. The cause of FM is not known, but it is manifested by painful bodily points. If pain were a separate biological unit, smashing it with a sledge hammer might be useful. But there is probably more to it, like peripheral neural sensitisation and inflammation. 600 J (!) is given to each point and this is a very high and quite inhibitive energy. And a “point” is declared to be 56.45 cm2. This is rather an area. But by spreading out the light over a large area, the dose becomes 10.63 J/cm2. Such a dose appears to be reasonable, but the energy is not.

b. The paper says: Like the IIIB lasers, recently developed Class IV therapeutic lasers use diffuse light at wavelengths in a therapeutic window that allow penetration of the light deep into the tissue. True, but these lasers do not penetrate deeper than the Class IIIB/3B lasers, so this is a deliberately misleading statement. Further, Class IV/4 therapeutic lasers are not exactly “recently developed”: The defocused beams of Class IV/4 surgical lasers have been used for therapy for equally as long as Class IIIB/3B devices. And the first commercially-available dedicated Class IV/4 therapeutic lasers came on the market in Europe during the ‘90s – which, of course, contradicts the claims by LiteCure and others that Class IV/4 laser therapy is new improvement of Class IIIB/3B. As they are now, these earlier Class IV/4 therapeutic lasers  were very expensive and inefficient, and proved no more effective than the already-available lower-powered lasers, so their use did not flourish until the marketing machine took hold in the USA.

c. The paper says: This development has led to the use of Class IV lasers to treat a variety of conditions including skin lesions(24,25), acute soft-tissue injuries (26), and chronic pain syndromes (27) such as FM. In fact, the references 24-27 are not related to the use of “Class IV” LPT lasers at all! This is a technique used often by LiteCure and other marketers of high-powered Class IV therapeutic lasers, banking on the fact that the casual reader will not follow through and actually read the referenced studies.

d. The paper says: There are only a few studies that have used laser therapy to treat pain (16,17,27,37,38). What about 125 published RCTs? If changed to “FM pain”, this is a more valid statement. And one of the most frequently quoted papers on FM and LPT (Gür et al.) used 2 J per point and with better results.

e. The paper says: Studies suggest that Class IV lasers have a beneficial analgesic and anti-inflammatory effect in humans (47-50). No, they don’t! All four papers to which they’ve referred are on Class 3B!

f. Previous studies on FM and LPT have been using considerably lower energies, so the reason for increasing these by a factor 100 seems to have but one background: To prove the superiority of the manufacturer’s product. However, the clinical outcome of this paper was not better than those where is Class 3B lasers have been used.

And let’s address another niggling falsehood: There is no such thing as “Class IV technology”!! 499 mW is Class 3B, 501 mW is Class IV. This is no “technology”. Laser classification is simply related to the relative risk posed by the power, wavelength and distribution of the laser emission!

The manufacturers of the Class IV lasers used in LPT have sponsored a small number of clinical studies. They all contain considerable flaws and even lies and are far from convincing. But they do contribute to the general confusion and are an obstacle in the general acceptance of laser phototherapy.

As mentioned previously, a typical trick of the Class IV vendor is to make reference to Class 3B papers, with proper documentation of their own products lacking. This was the old trick of LED vendors in the ’90s. The LEDs have, in the meantime, created their own scientific groundwork and do not have to use sales tricks any longer.

You can stop reading here, but if you like, here is the actual text from the book that is supposed to recommend Class IV lasers:

Stronger = better?

The power output of therapeutic lasers has increased radically during the nineties. McKibbin reports that there were about 1800 therapeutic laser units in Canada in 1990. 22% of them were HeNe lasers with an output of 1 mW or less, 35% HeNe lasers with 1-2 mW, 13% 830 nm units with an output up to 5 mW, 3% 830 nm units with an output up to 30 mW, 26% GaAs units with an output of 5 mW or less, and 1% units in the 760-780 range nm with an output up to 30 mW.

Now in 2009, the situation is quite different. HeNe units are being replaced by stronger InGaAlP lasers up to 500 mW, GaAlAs units of 7 000 mW are on the market, and GaAs units of 100 mW and more are available.

Even though it is possible to attain some effects with a 1-2 mW laser, there is no doubt that with a laser 100 times stronger, it is much easier to achieve biostimulating effects, at least if one intends to use treatment periods of the same length. Power density is also very important!

The authors used to have certain misgivings about an “inflation” with respect to the output power of therapeutic lasers. One misgiving was, and still is, the obvious risk of eye damage. The need for protective glasses has previously been exaggerated, but is now becoming more important. Another misgiving is the lack of research in the field of “high-power” therapeutic lasers. So far, insufficient data have been published on these powerful lasers. For the moment, we must rely primarily on our own clinical experience. That experience, however, is so encouraging that it cannot be ignored, even with the lack of scientific support. It would appear that “high-powered” therapeutic lasers will be able to further expand the scope of laser therapy, especially in pain therapy.

The doses previously recommended for laser therapy still hold true, in a way. However, much of what we know about dosage is based upon wound healing studies. This is the field in which both stimulating and inhibiting doses have generally been observed. But a wound is superficial, and the superficial tissue will absorb most of the laser energy. So treating a condition in the inner ear through the bone behind the ear is quite a different matter. The dense bone behind the ear absorbs some 90% of the light energy. Skin and blood absorb another 5%. Thus, 100 J in contact mode means only some 5 J or less in the inner ear. For pain and inflammation in large joints, such as the knee, quite a few joules may be required on the surface before the actual target receives the energy needed.

Using the same amount of energy but with different energy densities will not necessarily trigger the same biological response. Kim [545] used 1.2 J in plastic and aesthetic surgery. The energy was delivered either by a 1000 mW or a 60 mW 830 nm laser (1000 mW × 1.2 sec or 60 mW × 200 sec). Both were effective, but the 60 mW laser was more effective in the initial period of wound healing, while the 1000 mW laser was more effective in the late period.

Are strong lasers better than weaker ones?

YES and NO. Output power should not be too low for its purpose. If the power is too low, it causes unnecessarily long treatment time in order to achieve the required total dose (see more about the dose in the next chapter). Also, if output power is too low, it could result in the power density being too low which is an important parameter in treatment. Nor should output power be too high for its purpose. If the power is too high, the light could burn tanned, coloured skin, tattoos or skin with dark hair. Furthermore, in most countries, there is a power limit of 500 mW (= 0.5 watt), above which the laser may be a Class 4 laser. If so, it usually means that it requires oversight by an MD or DDS, more safety measures, and significantly more regulatory control. Also, if the power is too high, it can result in unintentionally high doses which can give less good treatment results than necessary (see the Arndt-Schulz curve in the next chapter). And finally, time is also an important treatment parameter. Administering a certain number of joules over a certain area using a certain laser power during a certain time, may not give the same result as using a ten times stronger laser during one tenth of the time with unchanged optical configuration. Another way to say this is that the rule of reciprocity is not valid. Some laser companies claim that a Class 4 laser ‘by default’ is better than a Class 3B laser (4 is higher than 3, so it has to be better… right?). This is simply not true. The classification of lasers is a measure of eye hazard, nothing else. While defocused Class 4 lasers may well be used successfully in laser therapy, this does not have anything to do with the laser classification.

The following is an except from a USA today article. Please visit the link at the bottom so see the entire article.

What if a cavity could fill itself, a broken tooth regrow? That's the promise of work published today in the journal Science Translational Medicine.

By shining light from a low-powered laser – about the brightness of a sunlit day – researchers were able to turn on a natural healing program and regrow dentin, the material inside a tooth. So far, they can only do this in rodents, but they could receive approval to test it in people within a year.

If it succeeds, the approach might also work for regrowing heart tissue, fighting inflammation and repairing bone and wounds, the researchers say.

"There's potential for this to be broadly useful," said David Mooney, the Harvard University bioengineer, who was the paper's senior author.

The promise is fantastic, said Harold Slavkin, a molecular biologist and professor of dentistry at the Ostrow School of Dentistry at the University of Southern California in Los Angeles. By mimicking a process already found in nature, Mooney's work has the potential to eventually transform medical care, enabling people someday to regrow their own livers, hearts or kidneys, he said.

"Twenty or 30 years from now people may say, 'Isn't it ridiculous that they used to transplant organs from one person to the other,'" Slavkin said.

Co-author Praveen Arany, a dentist and pathologist, said he got interested in the potential healing power of light after hearing anecdotes about light's ability to repair wounds and regrow hair. Laser light at very low frequencies does nothing, and at higher frequencies is commonly used to cut and cauterize tissue, so the dose of light has to be carefully delivered, said Arany, who initiated the research while a student in Mooney's lab.

He spent years carefully calibrating light levels to discover an optimal dose.

At appropriate levels, the light appears to trigger a chemical reaction that releases reactive oxygen species, a potentially damaging type of molecule.

In response to the reactive oxygen, the body's natural healing process activates a protein called Transforming Growth Factor (or TGF)-beta, which plays crucial roles in embryonic development, wound healing and the immune system. The TGF-beta stimulates production of new dentin, the material at the center of the tooth.

Arany and Mooney demonstrated that they can trigger this cascade of events and produce dentin by shining a low-powered laser on a rodent's tooth.

What they can't do yet is stimulate an entire tooth to regrow – the new dentin lacks the structure of a tooth, Mooney said. But Arany, now with the National Institute of Dental and Craniofacial Research, is hopeful of finding a way to get the body to rebuild structures, too.

"If we can figure out a way of activating those (processes), that would be really cool," he said.

Anne George, an endowed professor at the University of Illinois at Chicago, College of Dentistry, praised the work as impressive and important.

"If it works in a clinical trial setting, I think it will be great," she said.

Abstract

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Results

RUNX2, OCN, OSX expression in culture after 420 nm, 540 nm, 660 nm and 810 nm photobiomodulation

An analysis for evaluating the mRNA levels of RUNX2, OCN, and OSX was performed with or without photobiomodulation (PBM). The expression of RUNX2 demonstrated that hASCs differentiate into osteoblasts in culture. For RUNX2 gene expression, we examined mRNA level at 7 days, 14 days and 21 days. PBM was used every two days, so for 7 days group PBM was used 4 times, while 14 days and 21 days groups we used PBM 5 times. We found that the RUNX2 level of the green light group at all three time points were higher than red, near infrared and OM groups. The blue light group was higher than red light, near-infrared and OM group at 7 days (Fig. 1A). For OSX gene expression, the green and blue PBM groups had better effects than the red, near infrared and OM groups at 21 days (Fig. 1B). For OSX gene expression, at 21 days, we found that the green light PBM group was better than the red and OM groups, and the blue light group was better than OM group (Fig. 1C).

Figure 1

Quantitative evaluation of mRNA levels via real-time PCR of RUNX2 (A), OSX (B) and OCN (C) after 4 different wavelengths (420, 540, 660 and 810 nm) PBM. Data are expressed as mean ± SD. Experiments were carried out using two dishes each in three experiments (n = 6). ^#p < 0.05, ^##p < 0.01, ^###p < 0.001.

The activation of 420 nm and 540 nm to promote osteogenic differentiation could be abrogated by TRPV1 and TRPC channel inhibitors

We performed Alizarin red (AR-S) staining as a mineralization assay in osteogenic medium with or without addition of TRP channel antagonists CPZ(5 μM) and SKF(5 μM) incubating for 10 minutes before photobiomodulation. There was a significant difference between OM and 420 nm, 540 nm, 810 nm groups. ***(P < 0.001) for 420 nm and 540 nm groups, and *(P < 0.05) for 810 nm group. There was no significant difference between the OM and 660 nm groups. Compared with 810 nm group, 420 nm (^#P < 0.05) and 540 nm (^###P < 0.001) had better effects in the ARS assay (Fig. 2A–C). The increase in the mineralization level in response to 420 nm and 540 nm groups was abrogated by the TRP channel antagonists CPZ and SKF (Fig. 2A–D). These results imply that TRP calcium channels play a role in blue and green light-enhancement of osteoblast differentiation. The AR staining after red light (660 nm) was partially abrogated by the TRP inhibitors. NIR light-mediated enhancement of osteogenic differentiation was not abrogated by TRP inhibitors, and therefore appears to occur via a different mechanism.

Figure 2

(A) Alizarin red stain was added into cell cultures in osteogenic medium after photobiomodulation at a dose of 3 J/cm² five times (every two days) with or without CPZ or SKF pretreatment. The alizarin red staining was measured after 21 days to determine the level of mineralization. Pre-incubation with CPZ (5 μM) and SKF (5 μM) for 10 minutes before photobiomodulation reduced the effect of photobiomodulation in 420 nm and 540 nm groups, to a lesser extent in the 660 nm group, but not in the 810 nm group. (B) Images of alizarin red staining taken by microscope. A higher intensity of alizarin red after 420 nm and 540 nm groups, while the intensity of 420 nm and 540 nm +CPZ/SKF groups was similar to the control group. (C,D) Quantitative evaluation of calcium deposits using Alizarin red staining. hASCs were treated or not with the TRP channel inhibitors CPZ (5 μM) and SKF (5 μM) for 10 minutes before each application of photobiomodulation. Data are expressed as mean ± SD. Experiments have been carried out for 3 times (n = 8). *,^#P < 0.05, ***,^###P < 0.001.

420 nm and 540 nm photobiomodulation increase osteogenic relative gene expression through TRP/calcium signaling pathway

The expression of osteogenic genes Runx2, OCN and OSX could be regulated by intracellular calcium, which could in turn be elevated by blue and green light. In order to investigate whether intracellular calcium was elevated by blue and green light, hASCs were pretreated with CPZ (5 μM) or SKF (5 μM) 10 min before photobiomodulation. Fluo-4 was used as a fluorescent indicator to measure calcium levels immediately after light and RT-PCR was used to measure osteogenic gene expression after 21 days. We found that 540 nm laser irradiation at 3 J/cm² gave the highest increase in intracellular calcium concentration followed by 420 nm. 660 nm and 810 nm wavelengths did not significantly increase calcium (Fig. 3A). The increase in calcium occurred within 1 min after cessation of 540 nm illumination (Fig. 3B).

Figure 3

CPZ and SKF blocked the increase of intracellular calcium in hASCs cultured in OM caused by 420 nm or 540 nm.

(A) Effects of four different wavelengths on intracellular calcium measured immediately. (B) Time course of intracellular calcium after 540 nm with or without CPZ or SKF. (C) Quantitative analysis for intracellular calcium with or without CPZ (5 μM) or SKF (5 μM) pretreated before photobiomodulation using all four wavelengths.

The increase of intracellular calcium in response to 420 nm and 540 nm groups was abrogated by TRP channel antagonists CPZ and SKF (Fig. 3B,C). SKF also reduced calcium in hASCs in OM alone (no light) but this was not significant. In 660 nm and 810 nm groups there were no significant differences between photobiomodulation group and CPZ or SKF pre-treated groups with intracellular calcium (Fig. 3C).

Incubation with CPZ (5  μM) or SKF (5 μM) before each individual application of 420 nm and 540 nm photobiomodulation delivered 5 times over 21 days, significantly decreased RUNX2, OSX, and OCN expression levels as compared to the control group (OM alone) (Fig. 4A–C). In the 660 nm and 810 nm groups the relative gene expression levels showed no differences in the CPZ or SKF pretreated groups compared to OM alone (Data not shown).

Figure 4

Effects of TRP inhibitors on osteogenic gene expression stimulated by 420 nm or 540 nm photobiomodulation.

(A) Quantitative analysis for gene expression level of RUNX2. The data of RUNX2 expression are shown at day 21; data at days 7 and 14 are not shown. (B) Quantitative analysis for gene expression level of OSX at day 21. (C) Quantitative analysis for gene expression level of OCN at day 21. Data represent means ± SD of the number of determinations (n = 4 or 6, *P <0 .05, **P < 0.01, ***P < 0.001).

Abstract

Light-emitting diode therapy (LEDT) applied over the leg, gluteus and lower-back muscles of mice using a LED cluster (630 nm and 850 nm, 80 mW/cm2, 7.2 J/cm2) increased muscle performance (repetitive climbing of a ladder carrying a water-filled tube attached to the tail), ATP and mitochondrial metabolism; oxidative stress and proliferative myocyte markers in mice subjected to acute and progressive strength training. Six bi-daily training sessions LEDT-After and LEDT-Before-After regimens more than doubled muscle performance and increased ATP more than tenfold. The effectiveness of LEDT on improving muscle performance and recovery suggest applicability for high performance sports and in training programs.

Positioning of the mice and light-emitting diode therapy (LEDT) applied on mouse legs, gluteus and lower-back muscles without contact.

Introduction

Low-level laser (light) therapy has several applications in medicine such as treatment of pain 1, 2, tendinopathies 3 and acceleration of tissue repair 2, 4. Since the 1960s when the first laser (Light Amplification by Stimulated Emission of Radiation) devices were constructed, many applications of this therapy and its mechanisms of action have been investigated around the world 5.

Light therapy can be delivered by different light sources such as diode lasers or light emitting diodes (LEDs). These light sources differ in monochromaticity and coherence, since diode lasers are coherent with a tiny spectral bandwidth and less divergence of the light beams compared to the light emitted by LEDs 5. The spectral regions generally used for light therapy range between red (600 nm) to near infrared (1,000 nm) with total power in range of 1 mW–500 mW and power density (irradiance) in the range of range 1 mW–5 W/cm2 5. These lasers and LEDs are considered to produce equivalent effects on the tissue if the dose of light delivered/applied is in accordance with the possible biphasic dose?response previously reported 5-7. The light?tissue interaction depends on light absorption by specific structures in the cells that are known as chromophores 8-11.

Recently light therapy using lasers and LEDs has been used to increase muscle performance in exercises involving strength 12 or fatigue resistance 13-15; and light therapy may have a role to play in preparing athletes competing in high performance sports. Recent reviews have reported positive effects of light therapy on muscle performance, highlighting protection from exercise?induced muscle damage 16; an increased number of repetitions in maximum exertion tests 17; increased workload, torque and muscle fatigue resistance in training programs; as well as an overview of the main possible mechanisms of action of the light therapy on muscle tissue 18.

Several biological factors govern success or optimum performance in sports that involve high?intensity exercise, or alternatively involve endurance exercise, that both require muscle adaptation during pre?competition training programs. Among these factors are the depletion of the energy supply for muscle contraction which comprises adenosine triphosphate (ATP) and glycogen; accumulation of possibly deleterious metabolites from energy metabolism such as lactate, adenosine diphosphate (ADP), adenosine monophosphate (AMP), ions Ca2+ and H+; production of reactive oxygen species (ROS) 19-22; and the recovery process from microlesions or muscle damage 23. Light therapy seems to be able to benefit all these ”limitations” since its mechanism of action involves the improvement of mitochondrial metabolism and increased ATP synthesis 24, 25 owing to increased activity of cytochrome c oxidase (COX) in the electron transport chain (ETC) 9, 25, 26; reduction of reactive oxygen species (ROS) or improvement of oxidative stress defense 27, 28; and can stimulate faster muscle repair due to an increased proliferation and differentiation of muscle cells 29.

Experimental and clinical trials with different methodologies have reported the benefits of light therapy on muscle performance when applied before 15, 30, 31 or after exercise 12, 13, 32. However there is no consensus about the best time regimen for use of light therapy 18. The best wavelength (red or infrared) to stimulate muscle cells and increase muscle performance is also unclear.

In the current study we used an experimental model of mice exercising on a ladder similar to that reported in a previous study 33, in order to simulate a clinical strength training program that would allow us to identify which light therapy regimen would be better to increase muscle performance. Four different regimens of light therapy were applied to the mouse leg, gluteus and lower?back muscles during a training program: sham; before; before?after; and after each training session. Light therapy was delivered from LEDs (LEDT) with two simultaneous wavelengths (red and infrared). Assessment of muscle performance (load, number of repetitions, muscle work and power), markers of cellular energy and metabolism (ATP, glycogen and COX), oxidative stress markers (protein carbonyls, glutathione, catalase activity, lipid peroxidation, protein thiols) and muscle cell proliferation (BrdU – 5?bromo?2′?deoxyuridine) and adult myonuclei (DAPI – 4′,6?diamidino?2?phenylindole) were carried out.

Materials and methods

Animals

This study was performed with 8 week?old male Balb/c mice, weighing on average 22.22 g (SEM 0.24), housed at five mice per cage and kept on a 12 hour light 12 hour dark cycle. The 22 animals were provided by Charles River Inc and were provided with water and fed ad libitum at the animal facility of Massachusetts General Hospital. All procedures were approved by the IACUC of Massachusetts General Hospital (protocol #2014N000055) and met the guidelines of the National Institutes of Health.

Experimental groups

Twenty?two animals were randomly allocated into 4 exercise groups with 5 animals in each group, and 2 animals were allocated into an ”absolute” control group:

• LEDT?Sham group: animals were treated with sham LEDT (LEDT device in placebo mode) over both legs, gluteus and lower?back muscles 5 minutes before each training session on ladder.

• LEDT?Before: animals were treated with real LEDT over both legs, gluteus and lower?back muscles 5 minutes before each training session on ladder.

• LEDT?Before?After: animals were treated with real LEDT over both legs, gluteus and lower?back muscles 5 minutes before and 5 minutes after each training session on ladder.

• LEDT?After: animals were treated with real LEDT over both legs, gluteus and lower?back muscles 5 minutes after each training session on ladder.

• Control: animals were not subjected to any LEDT or exercise or muscle performance assessment.

Ladder

An inclined ladder (80°) with dimensions of 100 cm × 9 cm (length and width, respectively) with bars spaced at 0.5 cm intervals was used in this study as reported in a previous study 33 (Figure 1).

Figure 1

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Ladder. Inclined ladder (80°) with 100 cm × 9 cm (length and width, respectively) used for the training program and muscle performance assessments. Falcon tube filled with water and attached to the mouse tail.

Load

A Falcon tube (50 ml) was filled with measured volumes of water and weighed using a precise scale. The target load was achieved adding or removing water from the tube and then this tube was attached to the mouse tail using adhesive tape (Figure 1). All loads were calculated in grams.

Procedures

The schedule of the various exercise procedures is described in Table 1.

Table 1. Schedule for exercise procedures

• a : body weight
• b : average load carried during 3RM baseline measurement

Familiarization with ladder?climbing

All experimental groups, except Control group, were familiarized with climbing the ladder one day before the start of muscle performance assessment and training. The familiarization procedure was 4 sets of 10 climbs on the ladder (repetitions) with rest periods of 2 minutes between individual sets. No load was attached to the mouse tail during this procedure.

Three repetitions maximum load (3RM)

This test was the first evaluation of muscle performance and was set as the average of the maximum load carried by each animal during 3 consecutive full climbs of the inclined ladder (3RM). Slight pressure with tweezers was applied on mouse tail if the animal stopped during a climb. The test was stopped when mice were not able to climb or lost their grip on the ladder due to failure of concentric muscle contraction. The first attempt included a load corresponding to 200% of the individual mouse body weight. A maximum of 3 climb attempts was applied. If a mouse finished the climb the load was increased by 10% for the next climb, while if the mouse failed to finish a climb, the load was decreased by 10% for the next climb. The 3RM evaluation was performed twice; the first time was 24 h after familiarization procedure (baseline) and the second time was 24 h after the last training session (final).

Acute strength training protocol

After 24 h from initial 3RM baseline assessment, all experimental groups, except Control, were subjected to 6 training sessions carried out on alternate days (every 48 h). Each training session consisted of 5 sets of 10 repetitions (climbs) on the ladder with a rest period of 2 minutes between each set. If the animal could not complete a set or failed during a climb, the distance climbed (in cm) was measured and the rest period was started immediately. During some repetitions, a slight pressure on the mouse tail was performed with tweezers to stimulate the animal to climb and complete the exercise. If after three applications of gentle pressures the mouse could not resume climbing, and stopped or lost its grip on the ladder, the set of repetitions was stopped and the rest interval was started.

The number of repetitions in each set was measured as well as the time spent to complete the exercise. These data were used to calculate the muscle work and muscle power in each training session. The load of each training session was progressively increased and calculated as percentages of the 3RM (in grams) measured at baseline as follows: first training (80%), second training (90%), third training (100%), fourth training (110%), fifth training (120%) and sixth training (130%).

Light?emitting diode therapy (LEDT)

A non?commercial cluster of 40 LEDs (20 red – 630 ± 10 nm; 20 infrared – 850 ± 20 nm) with diameter of 76 mm was used in this study. A complete description of the LEDT parameters is presented in Table 2. The optical power reaching the surface of the mouse skin was measured with an optical energy meter PM100D Thorlabs® fitted with a sensor S142C (area of 1.13 cm2). All mice (except mice in Control) were shaved and fixed on a plastic plate using adhesive tapes. Afterwards, in accordance with experimental group, these animals were treated with LEDT over both legs, gluteus and lower?back muscles at a distance of 45 mm (without contact) (Figure 2). Irradiation lasted 90 s per session with fixed parameters as described in Table 1. LEDT placebo had no energy (0 J) and no power (0 mW) applied over the targeted muscles. The light dose was based on the possible biphasic dose response reported previously 5, 6. Moreover, dual wavelengths were chosen to function at the same time in this study based on specificities of the chromophores in the cells and therefore optimizing the effects of the light therapy (LEDT) by a double band of absorption 8-11.

Figure 2

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LEDT. Positioning of the mice and light?emitting diode therapy (LEDT) applied on mouse legs, gluteus and lower?back muscles without contact.

Muscle performance

The 3RM test was the first evaluation for muscle performance. This test measured the maximum load (in grams) carried by each animal during 3 consecutive full climbs on the inclined ladder.

During each training session the load, number of repetitions (rep), distance climbed and time spent to complete each repetition were recorded. These data were used to calculate muscle work and power.

Although the ladder had a total length of 100 cm available the maximum distance available to climb was set at 70 cm in order to avoid the load touching the floor. Thereby the muscle work was calculated as follows:

Work (J) = mgh

where ”m” is mass of the load (grams converted to kilogram) in each training session plus mouse body mass (values converted to kilogram); ”g” is acceleration due to gravity and ”h” is the distance climbed (converted to meters). Results were obtained in Joules (J) and presented as average ± standard error of mean (SEM) for each group at each training session.

Muscle power was calculated from results of muscle work (J) and time spent (s) to perform all repetitions of each set at all training sessions as follows:

Power (mW) = J/s

where ”J” is Joule and represents the muscle work performed and ”s” is time in seconds. Result were obtained in milliwatts (mW) and presented as average ± standard error of mean (SEM) per each group at each training session.

Muscular ATP

The gastrocnemius muscle from one leg of each animal was used for analysis of muscular ATP. Muscle samples were thawed in ice for 5 min, homogenized at a proportion of 3–4 mg of tissue to 500 µl of 10% perchloric acid (HClO4) following procedures previously published 34. Afterwards, an aliquot of 10 µl of the muscle homogenate plus 40 µl of CellTiter Glo Luminescent Cell Viability Assay mix (Promega), totaling 50 µl, were placed in the well microplate (CostarTM 96?Well White Clear?Bottom Plates). Luminescence signals were measured in a SpectraMax M5 Multi?Mode Microplate Reader (Molecular Devices, Sunnyvale, CA) with integration time of 5 s to increase low signals 34. A standard curve was prepared using ATP standard (Sigma) according to manufacturer's guidelines and then ATP concentration was calculated in nanomol (nmol) per milligram (mg) of protein. An aliquot of muscle homogenate was used to quantify the total protein by QuantiProTM BCA Assay kit (Sigma?Aldrich) following manufacturer's guidelines.

Muscular glycogen

Quadriceps femoris muscles were thawed in ice for 30 min and muscular glycogen was measured in 50 mg of quadriceps femoris tissue homogenized with 6 N NaOH at a proportion of 50 mg/ml. A standard curve was prepared using absolute ethanol (100%), K2SO4 (10%), phenol (4.1%) and 1 mM of glucose (2%) according to Dubois et al. 35. Optical density was read at 480 nm in spectrophotometer (EvolutionTM 300 UV?Vis, software VISPRO – Thermo Scientific). Data were normalized per mg of muscle tissue.

Oxidative stress markers

Protein carbonyl: Quadriceps femoris muscles were homogenized in deionized water (dH2O) at a proportion of 10 mg/200 µl. Protein carbonyl content was quantified using Protein Carbonyl Content Assay kit (Biovision) with the colorimetric method and following manufacturer's guidelines. All results were normalized per total protein quantified by QuantiProTM BCA Assay kit (Sigma?Aldrich) following manufacturer's guidelines.

Glutathione: Quadriceps femoris muscles were homogenized in 100 mM ice cold phosphate buffer (pH = 7.4) at a proportion of 10 mg/250 µl. Phosphate buffer was prepared with dibasic (Na2HPO4) and monobasic (NaH2PO4) sodium phosphate at equal proportions. Total and oxidized glutathione analysis was carried out with Glutathione Colorimetric Assay kit (ARBOR Assays) following manufacturer's guidelines. In addition, all results were normalized per total protein of the samples using QuantiProTM BCA Assay kit (Sigma?Aldrich) following manufacturer's guidelines.

Catalase activity: Quadriceps femoris muscles were homogenized in cold assay buffer provided in a Catalase Activity Assay kit (Biovision) at a proportion of 50 mg/100 µl. This analysis used the colorimetric method and followed manufacture's guidelines.

Lipid peroxidation using TBARS (Thiobarbituric Acid Reactive Substances): Quadriceps femoris muscles were homogenized with RIPA Buffer (Sigma?Aldrich) at a proportion of 25 mg/250 µl. Next, TBARS Colorimetric Assay kit (Cayman Chemical) was used following manufacturer's guidelines.

Protein Thiols: Quadriceps femoris muscles were homogenized in ice cold 100 mM phosphate buffer at a proportion of 10 mg/250 µl. Next, a Fluorescent Protein Thiol Detectiont kit (ARBOR Assays) was used following manufacturer's guidelines. In addition, all results were normalized per total protein quantified by QuantiProTM BCA Assay kit (Sigma?Aldrich) following manufacturer's guidelines.

Immunofluorescence analyses

5?bromo?2′?deoxyuridine (BrdU): BrdU reagent (Sigma?Aldrich) was diluted in saline solution (PBS) at a concentration of 10 mg/ml. Next, during the last 8 days of the experiment all animals (including Control group) received a single daily intra peritoneal injection (50 mg/kg) of BrdU. Mice were anesthetized and submitted to surgical procedures described previously. Gastrocnemius muscles were embedded in paraffin, cut in axial slices of 5 µm thickness from the muscle belly region by a microtome and mounted on slides for immunohistochemical procedures. Briefly, slides were deparaffinized with graded ethanol and then passed through antigen retrieval solution in a water bath pre?heated at 98 °C for 30 min. Afterwards slides were washed and incubated for 15 min at room temperature with 0.1% Triton X?100 TBS for cell membrane permeabilization, washed again and incubated for 30 min in protein blocking solution consisting of 3% BSA (Bovine Serum Albumin – Sigma) and 10% goat serum in TBS. Next, slides were immunostained with sheep anti?BrdU (Ab1893 – Abcam, Cambridge, MA) at 1 : 50 working concentration and selected anti?sheep (Alexa Fluor® 647 – Invitrogen) fluorescent secondary antibody matched to the primary antibody to stain at 1 : 200 working concentration. Finally, slides were cover?slipped with mounting media containing DAPI (4′,6?diamidino?2?phenylindole) (Invitrogen). Cells positively stained for BrdU were imaged using confocal microscope (Olympus America Inc. Center Valley, PA, USA) from three random fields. BrdU and DAPI staining were quantified using software Image J (NIH, Bethesda, MD).

Cytochrome c oxidase subunit IV (COX IV): Gastrocnemius muscles were subjected to the same procedures described for BrdU staining. Slides were immunostained with rabbit anti?COX IV (Cell Signaling Technology®) at 1 : 500 working concentration and selected anti?rabbit (Alexa Fluor® 680 – Invitrogen) secondary antibody matched with primary antibody to stain at 1 : 200 working concentration. Cells positively stained for COX IV were imaged using confocal microscopy as above and then the red channel of the exported images was changed to yellow.

Statistical analysis

Shapiro?Wilk's W test verified the normal distribution of the data. All experimental groups subjected to training protocols were compared at each training session for number of repetitions, muscle work and muscle power using one?way analysis of variance (ANOVA) and Tukey HSD post?hoc test. The load of 3RM among these same groups was compared by Two?way ANOVA with repeated measures (baseline versus final) and Tukey HSD post?hoc test. For muscular ATP, glycogen, oxidative stress markers and immunofluorescence stains, all experimental groups were compared by one?way ANOVA and Tukey's HSD post?hoc test. Significance was set at p < 0.05.

Results

Muscle performance

3RM: The final load 3RM was significantly higher (p < 0.05) in all experimental groups at the end of the experiment period compared to baseline. The final load of LEDT?After (92.28 g, SEM 0.82) was higher than LEDT?Sham (59.58 g, SEM 5.28; p < 0.001) and LEDT?Before (78.98 g, SEM 1.96; p = 0.020). In addition, LEDT?Sham had a significantly lower final load (p < 0.001) compared to LEDT?Before as well as LEDT?Before/After (83.91 g, SEM 1.49) (Figure 4A).

Figure 4

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Muscle performance (n = 5 animals per group). (A) Baseline and Final test of 3 repetitions maximum (3RM) measuring the total load carried by mice during this test. * statistical significance (p < 0.05) comparing the final 3RM load between groups. (B) Number of repetitions or climbs performed by each group treated with different regimens of LEDT during the progressive training program. (C) Muscle power developed by each group treated with different regimens of LEDT during the progressive training program. (D) Muscle work developed by each group treated with different regimens of LEDT during the progressive training program. * statistical significance (p < 0.05) compared to LEDT?Sham. # statistical significance (p < 0.05) compared to LEDT?After. & statistical significance (p < 0.05) compared to LEDT?Before. Abbreviations: LEDT = light?emitting diode therapy; LEDT?Sham (Sham – S) = LEDT placebo (LEDT device in placebo mode) on muscles immediately before (5 minutes) each training session on ladder; LEDT?Before (Before – B) = LEDT applied on muscles immediately before (5 minutes) each training session on ladder; LEDT?Before?After (Before?After – A?B) = LEDT applied on muscles immediately before (5 minutes) and immediately after (5 minutes) each training session on ladder; LEDT?After (After – A) = LEDT applied on muscles immediately after (5 minutes) each training session on ladder. The load of 3RM at baseline versus final was analyzed by Two?way analysis of variance (ANOVA) with repeated measures. Number of repetitions, muscle work and power were analyzed by One?way ANOVA.

Number of repetitions: There were significantly differences (p < 0.05) between all groups in each training session (Figure 4B). At 80% of 3RM (first session): animals in LEDT?Before and LEDT?Before?After groups performed more repetitions compared to animals in LEDT?Sham and LEDT?After (p < 0.01) groups. At 90% of 3RM (second session): animals in LED?Sham group performed fewer repetitions than animals in LEDT?Before, LEDT?Before?After and LEDT?After groups (p < 0.001). At 100% of 3RM (third session): animals in LEDT?Sham group performed fewer repetitions compared to animals in LEDT?Before (p = 0.014), LED?Before?After (p = 0.010) and LEDT?After (p = 0.002) groups. At 110% of 3RM (fourth session): animals in LEDT?Sham group performed fewer repetitions than animals in LEDT?Before?After (p = 0.013) and LEDT?After (p = 0.009) groups. At 120% of 3RM (fifth session): animals in LEDT?After group performed more repetitions than animals in LEDT?Before (p = 0.022) and LEDT?Sham (p < 0.001) groups. In addition, animals in LEDT?Sham performed fewer repetitions than animals in LEDT?Before (p = 0.022), LEDT?Before?After and LEDT?After (p < 0.001) groups. At 130% of 3RM (sixth session): animals in LEDT?Before?After and LEDT?After groups performed more repetitions than animals in LEDT?Sham (p < 0.001) and LEDT?Before (p < 0.01) groups.

Muscle Power: At 80% of 3RM there were no significant differences among all groups (p > 0.05). At 90% of 3RM: animals in LEDT?Sham group had lower muscle power compared to animals in LEDT?Before, LEDT?Before?After and LEDT?After (p < 0.01) groups. At 100% of 3RM: animals in LEDT?Sham group had lower muscle power than animals in LEDT?Before?After (p = 0.025) and LEDT?After (p = 0.007) groups. At 110% of 3RM: animals in LEDT?Before?After group developed more muscle power than animals in LEDT?Sham (p < 0.001) and LEDT?Before (p = 0.013) groups. In addition, animals in LEDT?After group had more muscle power than animals in LEDT?Sham (p = 0.002) group. At 120% of 3RM: animals in LEDT?Before?After and LEDT?After groups developed more muscle power than animals in LEDT?Sham and LEDT?Before (p < 0.001) groups. At 130% of 3RM: animals in LEDT?Before?After group developed more muscle power than animals in LEDT?Sham and LEDT?Before (p < 0.001) as well as LEDT?After (p = 0.001) groups. In addition, animals in LEDT?After group had more muscle power than animals in LEDT?Sham (p < 0.001) and LEDT?Before (p = 0.004) groups. Finally, animals in LEDT?Before group had major muscle power than animals in LEDT?Sham (p = 0.020) group (Figure 4C).

Muscle Work: Similar to results presented in Figure 4B, at 80% of 3RM only animals in LEDT?Before and LEDT?Before?After groups performed more muscle work compared to LEDT?Sham (p < 0.05) group (Figure 4D). At 90% of 3RM: animals in LEDT?Sham group performed less muscle work than animals in LEDT?Before, LEDT?Before?After and LEDT?After (p < 0.001) groups. These results were similar at 100% of 3RM (p < 0.001). At 110% of 3RM: animals in LEDT?Sham group had lower muscle work compared to animals in LEDT?Before?After (p = 0.015) and LEDT?After (p = 0.011) groups. At 120% of 3RM: animals in LEDT?Sham group performed lower muscle work compared to animals in LEDT?Before (p = 0.027) and LEDT?Before?After and LEDT?After (p < 0.001) groups. In addition, animals in LEDT?After group performed more muscle work than animals in LEDT?Before (p = 0.026) group. At 130% of 3RM: animals in LEDT?Before?After and LEDT?After groups performed more muscle work than animals in LEDT?Sham (p < 0.001) and LEDT?Before (p < 0.01) groups (Figure 4D).

Muscle ATP content

Animals in LEDT?After group had significantly (p < 0.001) more ATP concentration (1,367.64 nmol/ mg protein, SEM 105.30) compared to animals in LEDT?Sham (15.85 nmol/mg protein, SEM 5.14), LEDT?Before (81.00 nmol/ mg protein, SEM 10.11), LEDT?Before?After (687.62 nmol/ mg protein, SEM 11.76) and Control (17.53 nmol/mg protein, SEM 7.47) groups. In addition, animals in LEDT?Before?After group had also major contents of ATP compared to animals in LEDT?Before, LEDT?Sham and Control (p < 0.001) groups (Figure 5A).

Figure 5

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Muscular ATP and glycogen contents (n = 5 animals per group). (A) Adenosine triphosphate (ATP) contents in gastrocnemius muscle after the training program. (B) Glycogen contents in quadriceps femoris muscles after the training program. * statistical significance (p < 0.05). Abbreviations: LEDT = light?emitting diode therapy; LEDT?Sham (Sham – S) = LEDT placebo (LEDT device in placebo mode) on muscles immediately before (5 minutes) each training session on ladder; LEDT?Before (Before – B) = LEDT applied on muscles immediately before (5 minutes) each training session on ladder; LEDT?Before?After (Before?After – A?B) = LEDT applied on muscles immediately before (5 minutes) and immediately after (5 minutes) each training session on ladder; LEDT?After (After – A) = LEDT applied on muscles immediately after (5 minutes) each training session on ladder. Control (C) = no exercise or muscle performance assessment. Comparisons among all groups were conducted using One?way analysis of variance (ANOVA).

Muscle glycogen content

Animals in LEDT?After (137.76 nmol/mg tissue, SEM 11.40) and LEDT?Before?After (144.44 nmol/ mg tissue, SEM 16.23) groups had significantly higher concentrations of glycogen in quadriceps femoris muscles (p < 0.001) compared to animals in LEDT?Sham (31.36 nmol/mg tissue, SEM 7.45), LEDT?Before (52.76 nmol/mg tissue, SEM 6.53) and Control (58.78 nmol/ mg tissue, SEM 7.17) groups (Figure 5B).

Oxidative stress markers

Total glutathione: Animals in Control group (1.33 µM/µg protein, SEM 0.11) had a significantly higher concentration of total glutathione compared to animals in LEDT?Sham (0.097 µM/µg protein, SEM 0.046; p = 0.005) and LEDT?Before (1.00 µM/µg protein, SEM 0.02; p = 0.010) groups (Figure 6A).

Figure 6

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Oxidative stress markers (n = 5 animals per group) in quadriceps femoris muscles. (A) Total Glutathione (reduced glutathione – GSH). (B) Oxidized Glutathione (GSSG). (C) Protein Carbonyl. (D) Catalase activity. (E) Lipid peroxidation using TBARS (Thiobarbituric Acid Reactive Substances). (F) Protein Thiol. * statistical significance (p < 0.05). Abbreviations: LEDT = light?emitting diode therapy; LEDT?Sham (Sham – S) = LEDT placebo (LEDT device in placebo mode) on muscles immediately before (5 minutes) each training session on ladder; LEDT?Before (Before – B) = LEDT applied on muscles immediately before (5 minutes) each training session on ladder; LEDT?Before?After (Before?After – A?B) = LEDT applied on muscles immediately before (5 minutes) and immediately after (5 minutes) each training session on ladder; LEDT?After (After – A) = LEDT applied on muscles immediately after (5 minutes) each training session on ladder. Control (C) = no exercise or muscle performance assessment. Comparisons among all groups were conducted using One?way analysis of variance (ANOVA).

Oxidized glutathione: Animals in LEDT?Sham group (0.005 µM/µg protein, SEM 0.001) had significantly minor concentration of glutathione oxidized compared to animals in LEDT?Before (0.20 µM/µg protein, SEM 0.002; p = 0.015), LEDT?Before?After (0.035 µM/µg protein, SEM 0.003; p < 0.001), LEDT?After (0.041 µM/µg protein, SEM 0.003; p < 0.001) and Control (0.027 µM/µg protein, SEM 0.007; p = 0.006) groups. In addition, animals in LEDT?Before group had significantly minor concentration of oxidized glutathione compared to animals in LEDT?After (p < 0.001) and LEDT?Before?After (p = 0.024) groups (Figure 6B).

Protein carbonyl: Animals in LEDT?After group (1.40 nmol/µg protein, SEM 0.15) had significantly lower concentrations of protein carbonyls compared to animals in LEDT?Sham (6.31 nmol/µg protein, SEM 1.09; p = 0.030), LEDT?Before (6.81 nmol/µg protein, SEM 1.21; p = 0.040) and LEDT?Before?After (8.27 nmol/µg protein, SEM 2.35; p = 0.008) groups (Figure 6C).

Catalase activity: Animals in LEDT?Sham group (2.11 nmol/min/ml, SEM 0.10) had significantly lower catalase activity (p < 0.01) compared to animals in LEDT?Before?After (4.33 nmol/min/ml, SEM 0.62), LEDT?After (4.22 nmol/min/ml, SEM 0.37) and Control (4.47 nmol/min/ml, SEM 0.52) groups (Figure 6D).

Lipid peroxidation using TBARS: There were no significant differences between any of the groups (p > 0.05) assessed. Animals in Control group had a concentration of 21.29 µM (SEM 1.13); animals in LEDT?Sham had 21.12 µM (SEM 2.86); animals in LEDT?Before had 23.87 µM (SEM 1.13); animals in LEDT?Before?After had 19.19 µM (SEM 1.01) and animals in LEDT?After had 19.55 µM (SEM 1.24) (Figure 6E).

Protein Thiols: There were no sig

This video discusses the basics of Low Level Laser Therapy. You will learn a little bit about lasers and laser history, and what makes a cold laser a cold laser. It also talks about the difference between lasers and LED's and why the latter may be less effective for medical therapy.

You'll find information on the treatment parameters of LLLT which are:

• wavelength
• power
• duty cycle (continueous or pulsed)
• energy density (dosage)
• treatment duration

It also goes over what indications the FDA has approved LLLT and infrared light for.

 video length (12:13)

Low-level laser (light) therapy (LLLT) has been known since 1967 but still remains controversial due to incomplete understanding of the basic mechanisms and the selection of inappropriate dosimetric parameters that led to negative studies. The biphasic dose-response or Arndt-Schulz curve in LLLT has been shown both in vitro studies and in animal experiments. This review will provide an update to our previous (Huang et al. 2009) coverage of this topic. In vitro mediators of LLLT such as adenosine triphosphate (ATP) and mitochondrial membrane potential show biphasic patterns, while others such as mitochondrial reactive oxygen species show a triphasic dose-response with two distinct peaks. The Janus nature of reactive oxygen species (ROS) that may act as a beneficial signaling molecule at low concentrations and a harmful cytotoxic agent at high concentrations, may partly explain the observed responses in vivo. Transcranial LLLT for traumatic brain injury (TBI) in mice shows a distinct biphasic pattern with peaks in beneficial neurological effects observed when the number of treatments is varied, and when the energy density of an individual treatment is varied. Further understanding of the extent to which biphasic dose responses apply in LLLT will be necessary to optimize clinical treatments.

Keywords: low level laser therapy, photobiomodulation, biphasic dose response, reactive oxygen species, nitric oxide, traumatic brain injury

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INTRODUCTION

Low level laser (light) therapy (LLLT) employs visible (generally red) or near-infrared light generated from a laser or light emitting diode (LED) system to treat diverse injuries or pathologies in humans or animals. The light is typically of narrow spectral width between 600nm – 1000nm. The fluence (energy density) used is generally between 1 and 20 J/cm2 while the irradiance (power density) can vary widely depending on the actual light source and spot size; values from 5 to 50 mW/cm2 are common for stimulation and healing, while much higher irradiances (up to W/cm2) can be used for nerve inhibition and pain relief. LLLT is typically used to promote tissue regeneration, reduce swelling and inflammation and relieve pain and is often applied to the injury for 30 seconds to a few minutes or so, a few times a week for several weeks. Unlike other medical laser procedures, LLLT is not an ablative or thermal mechanism, but rather a photochemical effect comparable to photosynthesis in plants whereby the light is absorbed and exerts a chemical change.

Within a decade of the introduction of LLLT in the 1970s it was realized that more does not necessarily mean better. The demonstration of the biphasic dose response curve in LLLT has been hampered by disagreement about exactly what constitutes a “dose”. Many practitioners concentrate on fluence as the principle metric of dose, while others prefer irradiance or illumination time. The use of very small spot sizes by some practitioners has led to the assertion that they delivered hundreds of mW/cm2 from a 50 mW laser. While this statement is mathematically correct it can give the impression that much higher doses of light were given than actually were delivered.

Two years ago we reviewed (Huang et al. 2009) the biphasic dose response in LLLT and found many reports in the literature concerning biphasic dose responses observed in cell cultures, some in animal experiments but no clinical reports. We now believe that the time is right to revisit this interesting topic for two reasons. Firstly because we have found more instances in our laboratory both in vitro with cultured cortical neurons, and in vivo with LLLT of traumatic brain injuries in mouse models. Secondly because advances have been made in mechanistic understanding of how LLLT works at a cellular level that may explain why a little light may be beneficial and at the same time a lot of light might be harmful.

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MECHANISMS OF LOW LEVEL LIGHT THERAPY

Basic photobiophysics and photochemistry

According to the First Law of Photochemistry, the photons of light must be absorbed by some molecular photoacceptors or chromophores for photochemistry to occur (Sutherland 2002).The mechanism of LLLT at the cellular level has been attributed to the absorption of monochromatic visible and near infrared (NIR) radiation by components of the cellular respiratory chain (Karu 1989). Phototherapy is characterized by its ability to induce photobiological processes in cells. The effective tissue penetration of light and the specific wavelength of light absorbed by photoacceptors are two of the major parameters to be considered in light therapy. In tissue there is an “optical window” that runs approximately from 650 nm to 1200 nm where the effective tissue penetration of light is maximized. Therefore the use of LLLT in animals and patients almost exclusively involves red and near-infrared light (600–1100-nm) (Karu and Afanas’eva 1995). The action spectrum (a plot of biological effect against wavelength) shows which specific wavelengths of light are most effectively used for biological endpoints as well as for further investigations into cellular mechanisms of phototherapy (Karu and Kolyakov 2005). Fluence (J/cm2) is often referred to as “dose”, though many authors and practitioners of LLLT also refer to energy (Joules) as dose. Not only is this confusing to the novice student of LLLT but it also assumes that the product of power and time (and more importantly power density and time) is the goal rather than the right combination of individual values. This lack of reciprocity has been shown many times before and since our first paper on biphasic dose response and several more authors have reported finding these effects since. Examples of recently published “dose-rate” effects are also reviewed later in this article.

Mitochondrial Respiration and Cytochrome c oxidase

Mitochondria play an important role in energy generation and metabolism and are involved in current research about the mechanism of LLLT effects. The absorption of monochromatic visible and NIR radiation by components of the cellular respiratory chain has been considered as the primary mechanism of LLLT at the cellular level (Karu 1989). Cytochrome c oxidase (Cco) is proposed to be the primary photoacceptor for the red-NIR light range in mammalian cells. Absorption spectra obtained for biological responses to light were found to be very similar to the absorption spectra of Cco in different oxidation states (Karu and Kolyakov 2005).LLLT on isolated mitochondria increased proton electrochemical potential, ATP synthesis (Passarella et al. 1984), increased RNA and protein synthesis (Greco et al. 1989) and increases in oxygen consumption, mitochondrial membrane potential, and enhanced synthesis of NADH and ATP.

ROS release and Redox signaling pathway

Mitochondria are an important source of reactive oxygen species (ROS) within most mammalian cells. Mitochondrial ROS may act as a modulatable redox signal, reversibly affecting the activity of a range of functions in the mitochondria, cytosol and nucleus. ROS are very small molecules that include oxygen ions such as superoxide, free radicals such as hydroxyl radical, hydrogen peroxide, and organic peroxides. ROS are highly reactive with biological molecules such as proteins, nucleic acids and unsaturated lipids. ROS are also involved in the signaling pathways from mitochondria to nuclei. It is thought that cells have ROS or redox sensors whose function is to detect potentially harmful levels of ROS that may cause cell damage, and then induce expression of anti-oxidant defenses such as superoxide dismutase and catalase.

LLLT was reported to produce a shift in overall cell redox potential in the direction of greater oxidation (Karu 1999) and increased ROS generation and cell redox activity have been demonstrated (Lubart et al. 2005). These cytosolic responses may in turn induce transcriptional changes. Several transcription factors are regulated by changes in cellular redox state, but the most important one is nuclear factor κB (NF-κB). Figure 1 graphically illustrates some of the intracellular signaling pathways that are proposed to occur after LLLT.

FIG. 1.

Schematic depiction of the cellular signaling pathways triggered by LLLT. After photons are absorbed by chromophores in the mitochondria, respiration and ATP is increased but in addition signaling molecules such as reactive oxygen species (ROS) and nitric oxide (NO) are also produced.

NO release and NO signaling

There have been reports of the production and/or release of NO from cells after in vitro LLLT. It is possible that the delivery of low fluences of red/NIR light produces a small amount of NO from mitochondria by dissociation from intracellular stores (Shiva and Gladwin 2009), such as nitrosothiols (Borutaite et al. 2000), NO bound to hemoglobin or myoglobin (Lohr et al. 2009; Zhang et al. 2009) or by dissociation of NO from Cco (Lane 2006) as depicted in Figure 2. A second mechanism for NO production is by light-mediated increase of the nitrite reductase activity of cytochrome c oxidase (Lane 2006). A third possibility is that light can cause increase of the activity of an isoform of nitric oxide synthase (Poyton and Ball 2011), possibly by increasing intracellular calcium levels. This low concentration of NO produced by illumination is proposed to be beneficial through cell-signaling pathways (Ball et al. 2011).

FIG. 2.

One possible theory that can explain the simultaneous increase in respiration an production of nitric oxide is the photodissociation of bound NO that is inhibiting cytochrome c oxidase by displacing oxygen.

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BIPHASIC DOSE RESPONSES IN LLLT

Many reports of biphasic dose responses in LLLT were reviewed in our previous contribution and for convenience we have assembled these reports into Tables. Table 1 lists reports on cultured cells in vitro, Table 2 lists those reports in animal models in vivo, while Table 3 contains the only report of biphasic dose response in clinical studies.

TABLE 1.

Biphasic dose response studies of LLLT in vitro.

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TABLE 2.

Biphasic dose response studies of LLLT in vivo (animal models).

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TABLE 3.

Biphasic dose response studies of LLLT in clinical studies.

Figure 3 shows a 3D depiction of the Arndt Schulz model to illustrate a possible dose “sweet spot” at the target tissue. This graph suggests that insufficient power density or too short a time will have no effect on the pathology, that too much power density and / or time may have inhibitory effects and that there may be an optimal balance between power density and time that produces a maximal beneficial effect. There even may be a (low) power density for which infinite irradiation time would only have positive effects and no inhibitory effect. We believe that the absolute figures will be different at different wavelengths, tissue types, redox states, and may be affected further by different pulse parameters.

FIG. 3.

Three-dimensional model of the Arndt-Schulz curve illustrating how either irradiance or illumination time (fluence) can have biphasic dose response effects in LLLT.

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CURRENT BIPHASIC DOSE RESPONSE STUDIES IN LLLT

In this section we cover the new reports of biphasic dose responses in LLLT that have been published in the last two years since our previous review.

In an oral mucositis hamster model Lopes and coworkers (Lopes et al. 2009) delivered 660-nm laser at two different irradiances (55 mW/cm2 for 16 seconds per point or 155 mW/cm2 for 6 seconds per point). Both regimens delivered 0.9 J/cm2 per point. On day 7, 11 and 15 the authors reported reduced severity of clinical mucositis and lower levels of COX-2 staining in the 55 mW/cm2 group and that the 155 mW/cm2 had no significant differences when compared with controls. This data is summarized in Figure 4.

FIG. 4.

Mean grading of oral mucositis (OM) in a hamster cheek pouch model treated with 0.9 J/cm2 of 660-nm laser at two different irradiances (55 mW/cm2 for 16 seconds per point or 155 mW/cm2 for 6 seconds per point). Graph redrawn from data contained in (Lopes, Plapler et al. 2009).

Gal et al (Gal et al. 2009) compared the effects of delivering 5 J/cm2 of 670-nm laser at different power densities on wound tensile strength in a rat model. They found (Figure 5) that 670 nm laser achieved a significant effect using 4mW/cm2 applied for 1,250 seconds (20 mins 50 seconds) but that this effect was lost if the same 5J/cm2 fluence was delivered at 15 mW/cm2 for 333 seconds (5 mins 33 seconds).

FIG. 5.

Mean wound tensile strength obtained after delivering 5 J/cm2 of 670-nm laser at different power densities (4mW/cm2 applied for 1,250 seconds or 15 mW/cm2 for 333 seconds). Graph redrawn from data contained in (Gal, Mokry et al. 2009).

(Skopin and Molitor 2009) studied the effects of different influences of 980 nm laser on a human fibroblast in vitro model of wound healing. A small pipette was used to induce a wound in fibroblast cell cultures, which were exposed to a range of laser doses (1.5–66 J/cm2). Exposure to low- and medium-dose laser light accelerated cell growth, whereas high-intensity light negated the beneficial effects of laser exposure as shown in Figure 6.

FIG. 6.

Mean percentage of healing induced in a scratch wounded culture of human fibroblasts using different fluences (constant time, increasing irradiance) of 980-nm laser. Graph redrawn from data contained in (Gal, Mokry et al. 2009).

(Prabhu et al. 2010) performed a dose response study by applying a 7 mW HeNe (632.8-nm) laser with a power density of 4 mW/cm2 to 15×15 mm excisional wounds on Swiss albino mice for a range of irradiation times from 249 seconds (4.15 mins) up to 2,290 seconds (41.46 mins). As Figure 7 shows, there was a clear biphasic response (including a possible inhibitory effect) with changes in irradiation time and therefore fluence.

FIG. 7.

Mean area under the curve of wound area over time in a mouse excisional wound healing model treated with a 7 mW (power density of 4 mW/cm2) HeNe (632.8-nm) laser for times ranging from 249 to 2,290 seconds. Graph redrawn from data contained in (Prabhu, Rao et al. 2010).

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BIPHASIC LLLT DOSE RESPONSE STUDIES IN CULTURED NEURONS AND TRAUMATIC BRAIN INJURY MODELS IN MICE

LLLT studies on cultured cortical neurons

In order to elucidate the mechanism responsible for the beneficial effect reported by LLLT for brain related disorders, we carried out studies to look into effects of 810 nm laser on different cellular signaling molecules in primary cortical neurons. The primary cortical neurons were isolated from brains taken from embryonic mice. We irradiated the neurons with different fluences of 0.03, 0.3, 3, 10 or 30 J/cm2 delivered at a constant irradiance of 25 mW/cm2, and subsequently the intracellular levels of ROS, mitochondrial membrane potential (MMP) and ATP was measured. The changes in mitochondrial function were studied in terms of ATP and MMP. Low-level light was found to induce a significant increase in ATP and MMP at lower fluences and a decrease at higher fluence. ROS was induced significantly by light at all light doses but there was a distinctive pattern of a double peak with the first peak coinciding with the other peaks of ATP and MMP at 3 J/cm2 (Figure 8). However in contrast to ATP and MMP there was a second larger rise in ROS at 30 J/cm2 that coincided with the reduction in MMP below baseline. The results of the this study suggested that LLLT at lower fluences is capable of inducing mediators of cell signaling process which in turn may be responsible for the biomodulatory effects of the low level laser. Conversely at higher fluences beneficial mediators are reduced but potentially harmful mediators are increased. Thus this study offered an explanation for the biphasic dose response induced by LLLT.

FIG. 8.

Mean expression levels of reactive oxygen species (ROS, measured by MitoSox red fluorescence), mitochondrial membrane potential (MMP, measured by red/green fluorescence ration of JC1 dye) and ATP (measured by firefly luciferase assay) in primary mouse cortical neurons treated with various fluences of 810-laser delivered at 25 mW/cm2 over times varying from 1.2 to 1200 seconds.

LLLT in a mouse model of traumatic brain injury

We have been studying the effect of transcranial laser (810-nm) on mouse models of traumatic brain injury. The model involves a controlled cortical impact using a pneumatic piston device through a craniotomy followed by closure of the head. This injury can be adjusted in severity to produce a neurological severity score (NSS based on a panel of standardized behavioral tests) of 7–8 on a scale of 0 (normal mice) to 10 (severe brain injury that causes death). The basic finding was that delivering a single dose of 36 J/cm2 810-nm laser delivered at 50 mW/cm2 (12 minutes illumination time) in a spot of 1-cm diameter centered on the top of the mouse head at a time point of 4 hours post-TBI was highly effective in ameliorating the neurological symptoms suffered by the mice (Figure 9A). When we delivered 10 times as much 810-nm laser (360 J/cm2 at 500 mW/cm2) also taking 12 minutes the beneficial effect totally disappeared, and at early time points (1–6 days) the high fluence appeared to be worse than no treatment (Figure 9B).

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FIG. 9.

Transcranial laser therapy (36 J/cm2 of 810-nm laser delivered at 50 mW/cm2 (12 minutes illumination time) in a spot of 1-cm diameter centered on the top of the mouse head) was used to treat mice with controlled cortical impact TBI four hours after injury. (A) Significant improvement in neurological severity score continuing for 4 weeks after a single treatment. (B) Delivering ten times more light by increasing irradiance tenfold (500 mW/cm2) loses all therapeutic benefit, and produces worse performance soon after laser. (C) Repeating beneficial laser treatment daily for 14 days loses benefit in performance after 5 days.

When we repeated the effective laser treatments 14 times (36 J/cm2 delivered at 50-mW/cm2 once a day for 14 days starting 4 hours post-TB) we found a very interesting result (Figure 9C). For the first 4 days the improvement in NSS in the repeated laser group was marginally better than the single treatment. However on day 5 the gradual improvement ceased and as the laser was repeated the NSS got closer to that of untreated TBI mice until at day 14 it actually crossed over. Although the differences were not statistically significant it appeared that from day 16 until day 28 the mice that received 14 laser treatments did worse than those that received no treatment at all.

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POSSIBLE EXPLANATIONS FOR BIPHASIC DOSE RESPONSE IN LLLT

The triphasic dose response we have observed for ROS production in cultured cortical neurons (see Fig 7) suggests an explanation for the biphasic dose response. The hypothesis is that there are two kinds of ROS. Good ROS are produced at fairly low fluences of light. The reason for the production of good ROS is likely to be connected with stimulation of mitochondrial electron transport as shown by increases in MMP and increases in ATP production. These good ROS can initiate beneficial cell signaling pathwas leading to activation of redox sensitive transcription factors such as NF-κB (Chandel et al. 2000; Groeger et al. 2009). NF-κB activation induces expression of a large number of gene products related to cell proliferation and survival (Karin and Lin 2002; Brea-Calvo et al. 2009). As the fluence of light is increased the beneficial ROS production in the mitochondria decreases in tandem with reductions in MMP and a drop-off in ATP production. Then when even more light is delivered there is a second peak in ROS production, which we will call bad ROS. Bad ROS can damage the mitochondria leading to a drop in MMP below baseline levels and presumably can lead to initiation of apoptosis by the mitochondrial pathway including cytochrome c release. It remains to be seen whether the good and bad ROS are identical species and just differ in amount, or whether they are chemically different species. For instance it may be hypothesized that the good ROS consists mainly of superoxide while the bad ROS consists of more damaging ROS such as hydroxyl radicals and peroxynitrite. In Figure 7 we used just one type of fluorescent ROS indicator (mitoSOX red), which is commonly supposed to be specific for superoxide but will likely also be activated by hydroxyl radicals and peroxynitrite.

There have been several studies showing that relatively high doses of light can induce apoptosis in various cell types via ROS-mediated signaling pathways (Huang et al. 2011). Meanwhile, there is an important proapoptotic signaling pathway has been identified which involv

Results

LLLT alleviates symptoms and delays disease onset in EAE mice

C57BL/6 mice immunized with MOG_35–55 developed EAE clinical symptoms after 7 days and reached a maximum mean clinical on day 30, when the incidence of clinical EAE was 100% and the average score was around 3.5 ± 0.5 (Figure 1A and Table 1). To test the prophylactic efficacy of laser during EAE, treatment starts from day 0 of induction. Compared with the untreated EAE group, AlGaInP 10 J/cm² or GaAs 3 J/cm² treatment significantly delayed disease onset (p < 0.001; Table 1) and decreased disease severity as measured by the mean maximal clinical score (2.0 ± 0.2 and 2.5 ± 0.5, respectively), with inhibition of 68 ± 2% (AlGaInP 10 J/cm², Figure 1A and B) and 54 ± 5% (GaAs 3 J/cm²) (p < 0.0001; F = 48.05), based on the area under the curve (AUC), compared with the EAE-untreated group (Figure 1A and B; Table 1).

Figure 1. Low-level laser therapy attenuates the EAE disease process in C57BL/6 mice. Active EAE was induced in C57BL/6 mice by immunization with MOG_35–55 on day 0. The clinical score (A), area under the curve (AUC) (B), body weight change (C) and delta (Δ) body weight gain or loss at the peak of disease (day 30 post-induction) (D) were evaluated in the naive group, the control group (EAE), in mice pre-treated with AlGaInP 10 J/cm² (660 nm) and in mice pre-treated with GaAs 3 J/cm² (904 nm), 30 days after immunization. The clinical symptoms were scored every day in a blinded manner and are expressed as the mean clinical score or as the AUC. Data points are presented as the mean ± SEM. Values of ##p < 0.001 versus naive group and **p < 0.001 versus EAE group (one-way ANOVA followed by post-hoc Newman–Keuls).

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As previously described, animals with EAE tend to have a reduced body weight as a result of anorexia and deficient fluid uptake, which fit well with the severity of the clinical score [35 Mix, E., H. Meyer-Rienecker, and U. K. Zettl. 2008. Animal models of multiple sclerosis for the development and validation of novel therapies – potential and limitations. J. Neurol. 255: 7–14[Crossref], [PubMed], [Web of Science ®], [Google Scholar]]. Next, we evaluated whether LLLT prevents the body weight change that is induced by EAE in mice. As expected, after EAE induction, a significant body weight loss was observed in the EAE mice compared with the naïve group (Figure 1C and D). Interestingly, a significant body weight gain was found in the EAE plus AlGaInP 10 J/cm² (10 ± 2.5%; Figure 1D) group and the EAE plus GaAs 3 J/cm² group (11 ± 3.0%; Figure 1D) (p < 0.01; F = 6.3) when compared with the EAE group.

LLLT down-regulates NO levels in the CNS and peripheral lymphoid tissue without affecting lipid peroxidation or the antioxidant defense during EAE

Excess amounts of NO are harmful for CNS function and are implicated in the pathophysiology of many neurologic diseases, such as MS, and the EAE model, in which NO is overproduced, mainly by innate immunity cells, such as macrophages and microglia [36–38 Ghasemi, M., and A. Fatemi. 2014. Pathologic role of glial nitric oxide in adult and pediatric neuroinflammatory diseases. Neurosci. Biobehav. Rev. 45: 168–182
Das, U. N. 2012. Is multiple sclerosis a proresolution deficiency disorder? Nutrition 28: 951–958
Miller, E. 2012. Multiple sclerosis. Adv. Exp. Med. Biol. 724: 222–238 ]. Thus, we investigated the effect of LLLT on the level of NO in the CNS and secondary lymphoid tissue of EAE-treated and untreated animals. In agreement with clinical signs, the concentration of NO in the spinal cord of the EAE mice was significantly increased (52 ± 25 µmol/mg of protein) compared with the control animals (Figure 2A). In contrast, both AlGaInP 10 J/cm² and GaAs 3 J/cm² treatment down-regulated the NO level in the CNS of the EAE-treated animals, with a mean of 10 ± 5 and 15 ± 10 µmol/mg of protein, respectively (Figure 2A; p < 0.01; F = 7.15). Moreover, this upregulation was attenuated with LLLT (AlGaInP 10 J/cm² and GaAs 3 J/cm² treatment) in the spleen tissue after EAE induction (p < 0.05 and p < 0.01 versus the healthy group; Figure 2C). However, compared with the untreated EAE group, LLLT did not significantly modulate NO in the lymph node (Figure 2B). In addition, LLLT failed to inhibit lipid peroxidation (Figure 3A and B; p < 0.08; F = 2.80 and p < 0.7; F = 0.38) or to restore the antioxidant defense (Figure 3C and D; p < 0.31; F = 1.28 and p < 0.45; F = 0.91) after EAE induction in the spinal cord and lymph node, respectively.

Figure 2. Low-level laser therapy selectively inhibits NO level in the CNS and peripheral lymphoid tissue of EAE mice. Active EAE was induced in the C57BL/6 mice with MOG_35–55/CFA. The spinal lumbar cords (A), inguinal lymph nodes (B) and spleen (C) were obtained from the naive group, the control group (EAE), from mice pre-treated with AlGaInP 10 J/cm² (660 nm) and from mice pre-treated with GaAs 3 J/cm² (904 nm), 30 days after immunization. The NO production was analyzed using the Griess assay. Data are presented as means ± SEM of 6–9 mice per group and are representative of two independent experiments. #p < 0.05 versus naïve group and **p < 0.001 versus EAE group (one-way ANOVA with Newman–Keuls post-hoc test).

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Figure 3. Low-level laser therapy ameliorates EAE without affecting lipid peroxidation or the antioxidant defense. Animals were immunized with MOG_35–55 peptide/CFA and pertussis toxin. Lumbar spinal cord and inguinal lymph node samples were collected from the naive group, the control group (EAE), from mice pre-treated with AlGaInP 10 J/cm² (660 nm) and from mice pre-treated with GaAs 3 J/cm² (904 nm), 30 days after EAE induction for the determination of TBARS (panels A and B) and GSH (panels C and D) levels, respectively. Results are presented as means ± SEM of 6–9 mice/group, and are representative of two separate experiments.

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LLLT limits the infiltration of immune cells to the CNS

The hallmark of EAE disease is the infiltration of inflammatory cells into the CNS, leading to neuronal and oligodendrocyte damage [39 Bogie, J. F., P. Stinissen, and J. J. Hendriks. 2014. Macrophage subsets and microglia in multiple sclerosis. Acta Neuropathol. 128: 191–213[Crossref], [PubMed], [Web of Science ®], [Google Scholar]]. Therefore, we aimed to determine the effect of LLLT on the infiltration of inflammatory cells into the CNS after EAE induction. As shown in Figure 4, no inflammatory foci were detected in the naïve lumbar spinal cord; however, the untreated EAE mice showed profound infiltration of immune cells into the CNS, particularly in the white matter region (Figure 4A and B). Interestingly, treatment with AlGaInP 10 J/cm² significantly reduced the infiltration of these inflammatory cells into the CNS (Figure 4A and B; p < 0.02; F = 4.36). In contrast, treatment with GaAs 3 J/cm² only resulted in a moderate inhibition (Figure 4).

Figure 4. Low-level laser therapy blocks infiltration of mononuclear cells into the CNS during EAE pathology. Active EAE was induced in the C57BL/6 mice with MOG_35–55/CFA plus pertussis toxin. At the peak of disease (day 30), animals were killed and the lumbar spinal cords from the naive group, the control group (EAE), from mice pre-treated with AlGaInP 10 J/cm² (660 nm) and from mice pre-treated with GaAs 3 J/cm² (904 nm) were harvested for infiltration studies. Infiltration of mononuclear cells into spinal cords sections was examined by H&E staining (A), with magnification ×40, ×100 and ×400. Graphical representation of the inflammatory cells evaluated in the lumbar spinal cord (B). Specifically, four alternate 5 -µm sections (six to nine animals/group) of the white matter of the lumbar spinal cord were obtained between L4 and L6. Detail: inflammatory foci in the white matter after EAE induction. Data are presented as means ± SEM. #p < 0.05 versus naïve group and *p < 0.05 versus EAE group (one-way ANOVA with Newman–Keuls post-hoc test).

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LLLT reduces the demyelination area

To investigate whether clinical improvement was accompanied by decreased neuropathology, we examined the demyelination area in longitudinal sections of the lumbar region of spinal cords by LFB staining 30 days post-immunization. Histological analysis of the spinal cord tissue sections from the healthy control mice showed an intact myelin sheath (Figure 5), whereas typical demyelination was observed in the EAE mice (Figure 5A and B). Again, AlGaInP 10 J/cm² treatment remarkably attenuated CNS demyelination in the EAE mice (Figure 5A and B), while GaAs 3 J/cm² failed to inhibit the demyelination area induced by EAE (Figure 5A and B). These data suggest the clinical relevance of LLLT, especially AlGaInP 10 J/cm², in reducing EAE severity.

Figure 5. Low-level laser therapy inhibits CNS demyelination during EAE development. Active EAE was induced in the C57BL/6 mice with MOG_35–55/CFA plus pertussis toxin. At the peak of disease (day 30), animals were killed and the lumbar spinal cords from the naive group, the control group (EAE), from mice pre-treated with AlGaInP 10 J/cm² (660 nm) and from mice pre-treated with GaAs 3 J/cm² (904 nm) were harvested for demyelination studies. Demyelination areas in spinal cord sections were examined by luxol fast blue (LFB) staining (A), with magnification ×40 and ×100. Graphical representation of the CNS demyelination in lumbar spinal cord (B). Specifically, four alternate 5 -µm sections (six to nine animals/group) of the white matter of the lumbar spinal cord were obtained between L4 and L6. Detail: CNS demyelination in the white matter after EAE induction. Data are presented as means ± SEM. #p < 0.05 versus naïve group and *p < 0.05 versus EAE group (one-way ANOVA with Newman–Keuls post-hoc test).

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LLLT attenuated production of pro-inflammatory cytokines during EAE pathology

To initiate CNS inflammation, myelin-specific T cells, especially Th17 and Th1 subsets, must be activated in the periphery, gain access to the CNS and then be reactivated by central APCs presenting self-antigen, initiating a cascade of events, including the secretion of cytokines/chemokines, which recruit macrophages to the sites of T-cell activation [3 Goverman, J. 2009. Autoimmune T cell responses in the central nervous system. Nat. Rev. Immunol. 9: 393–407[Crossref], [PubMed], [Web of Science ®], [Google Scholar]]. Moreover, pro-inflammatory mediators secreted by macrophages/microglia, such as IL-1β, are important for both perpetuating inflammation and contributing to CNS tissue damage in EAE [40 Kuchroo, V. K., A. C. Anderson, H. Waldner, et al. 2002. T cell response in experimental autoimmune encephalomyelitis (EAE): role of self and cross-reactive antigens in shaping, tuning, and regulating the autopathogenic T cell repertoire. Ann. Rev. Immunol. 20: 101–123[Crossref], [PubMed], [Web of Science ®], [Google Scholar]]. Here, pronounced increase in IL-17, IFN-γ and IL-1β levels was observed in the spinal cord after EAE-immunization (Figure 6). AlGaInP 10 J/cm² and GaAs 3 J/cm² treatment markedly inhibited the upregulation of IL-17 (Figure 6A), IFN-γ (Figure 6B) and IL-1β (Figure 6C) in the CNS after EAE induction.