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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.

REFERENCES

[1]

Rumyantsev, P.P. (1977) Interrelations of the prolifera- tion and differentiation processes during cardiac myoge- nesis and regeneration. International Review of Cytology, 51, 186-273. doi:10.1016/S0074-7696(08)60228-4

[2]

Poss, K.D., Wilson, L.G. and Keating, M.T. (2002) Heart regeneration in zebrafish. Science, 298, 2188-2190. doi:10.1126/science.1077857

[3]

Rumyantsev, P.P. (1973) Post-injury DNA synthesis, mi- tosis and ultrastructural reorganization of adult frog car- diac myocytes. An electron microscopic-autoradiographic study. Z Zellforsch Mikrosk Anat, 139, 431-50. doi:10.1007/BF00306596

[4]

Barnett, P. and van den Hoff, M.J.B. (2011) Cardiac re- generation: Different cells same goal. Medical & Biologi- cal Engineering & Computing, 49, 723-732. doi:10.1007/s11517-011-0776-5

[5]

Bollini, S., Smart. N. and Riley, P.R. (2011) Resident car- diac progenitor cells: At the heart of regeneration. Jour- nal of Molecular and Cellular Cardiology, 50, 296-303. doi:10.1016/j.yjmcc.2010.07.006

[6]

Choi, W.Y. and Poss, K.D. (2012) Cardiac regeneration. Current Topics in Developmental Biology, 100, 319-343. doi:10.1016/B978-0-12-387786-4.00010-5

[7]

Laflamme, M.A. and Murry, C.E. (2011) Heart regenera- tion. Nature, 473, 326-335. doi:10.1038/nature10147

[8]

Steinhauser, M.L. and Lee, R.T. (2011) Regeneration of the heart. EMBO Molecular Medicine, 3, 701-712. doi:10.1002/emmm.201100175

[9]

Urbanek, K., Torella, D., Sheikh, F., De Angelis, A., Nur- zynska, D., Silvestri, F., Beltrami, C.A., Bussani, R., Bel- trami, A.P., Quaini, F., Bolli, R., Leri, A., Kajstura. J. and Anversa, P. (2005) Myocardial regeneration by activation of multipotent cardiac stem cells in ischemic heart failure. Proceedings of the National Academy of Sciences of the USA, 102, 8692-8697. doi:10.1073/pnas.0500169102

[10]

Bittner, R.E., Schofer, C., Weipoltshammer, K., Ivanova, S., Streubel, B., Hauser, E., Freilinger, M., Höger, H., Elbe- Bürger, A. and Wachtler, F. (1999) Recruitment of bone- marrow-derived cells by skeletal and cardiac muscle in adult dystrophic mdx mice. Anatomy and Embryology, 199, 391-396. doi:10.1007/s004290050237

[11]

Jackson, K.A., Majka, S.M., Wand, H., Pocius, J., Hart- ley, C.J., Majesky, M.W., Entman, M.L., Michael, L.H., Hirschi, K.K. and Goodell, M.A. (2001) Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. The Journal of Clinical Investigation, 107, 1395-1402. doi:10.1172/JCI12150

[12]

Pfister, O., Mouquet, F., Jain, M., Summer, R., Helmes, M., Fine, A., Colucci, W.S. and Liao, R. (2005) CD3- but not CD31+ cardiac side population cells exhibit functio- nal cardiomyogenic differentiation. Circulation Research, 97, 52-61. doi:10.1161/01.RES.0000173297.53793.fa

[13]

Balsam, L.B., Wagers, A.J., Christensen, J.L., Kofidis, T., Weissman, I.L. and Robbins, R.C. (2004) Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium. Nature, 428, 668-673. doi:10.1038/nature02460

[14]

Hatzistergos, K.E., Quevedo, H., Oskouei. B.N., Hu, Q., Feigenbaum, G.S., Margitich, I.S., Mazhari, R., Boyle, A.J., Zambrano, J.P., Rodriguez, J.E., Dulce, R., Pattany, P.M., Valdes, D., Revilla, C., Heldman, A.W., McNiece, I. and Hare, J.M. (2010) Bone marrow mesenchymal stem cells stimulate cardiac stem cell proliferation and differ- entiation. Circulation Research, 107, 913-922. doi:10.1161/CIRCRESAHA.110.222703

[15]

Porrello, E.R., Mahmoud, A.I., Simpson, E., Hill, J.A., Richardson, J.A., Olson, E.N. and Sadek, H.A. (2011) Transient regenerative potential of the neonatal mouse heart. Science, 331, 1078-1080. doi:10.1126/science.1200708

[16]

Conlan, M.J., Rapley, J.W. and Cobb, C.M. (1996) Bio- stimulation of wound healing by low energy laser irradia- tion. Journal of Clinical Periodontology, 23, 492-496. doi:10.1111/j.1600-051X.1996.tb00580.x

[17]

Karu, T. (2007) Ten lectures on basic science of laser photherapy. Prima Books, Gragesberg.

[18]

Karu, T. (2010) Mitochondrial mechanisms of photobio- modulation in context of new data about multiple roles of ATP. Photomedicine and Laser Surgery, 28, 159-160. doi:10.1089/pho.2010.2789

[19]

Bibikova, A. and Oron, U. (1993) Promotion of muscle regeneration in the toad (Bufo viridis) gastrocnemius mu- scle by low energy laser irradiation. Anatomical Record, 235, 374-380. doi:10.1002/ar.1092350306

[20]

Bibikova, A., Belkin, A. and Oron, U. (1994) Enhancement of angiogenesis in regenerating gastrocnemius muscle of the toad (Bufo viridis) by low energy laser irradiation. Anatomy and Embryology, 190, 597-602. doi:10.1007/BF00190110

[21]

Oron, U. (2006) Photoengineering of tissue repair in ske- letal and cardiac muscles. Photomedicine and Laser Sur- gery, 24, 111-120. doi:10.1089/pho.2006.24.111

[22]

Yaakobi, T., Shoshani, Y., Levkovitz, S., Rubin, O., Ben- Haim, S.A. and Oron, U. (2001) Long term effect of low energy laser irradiation on infarction and reperfusion in- jury in the rat heart. Journal of Applied Physiology, 90, 2411-2441.

[23]

Oron, U., Yaakobi, T., Oron, A., Hayam, G., Gepstein, L., Wolf, T., Rubin, O. and Ben Haim, S.A. (2001a) Attenu- ation of the formation of scar tissue in rats and dogs post myocardial infarction by low energy laser irradiation. La- sers in Surgery and Medicine, 28, 204-211. doi:10.1002/lsm.1039

[24]

Oron, U., Yaakobi, T., Oron, A., Mordechovitz, D., Shof- ti, R., Hayam, G., Dror, U., Gepstein, L., Wolf, T., Hau- denschild, C. and Ben Haim, S.A. (2001b) Low energy la- ser irradiation reduces formation of scar tissue following myocardial infarction in dogs. Circulation, 103, 296-301.doi:10.1161/01.CIR.103.2.296

[25]

Tuby, H., Maltz, L. and Oron, U. (2006) Modulations of VEGF and iNOS in the rat heart by low energy laser irra- diation are associated with cardioprotection and enhanced angiogenesis. Lasers in Surgery and Medicine, 38, 682- 688. doi:10.1002/lsm.20377

[26]

Oron, U., Ilic, S., De Taboada, L. and Streeter, J. (2007) Ga-As (808 nm) laser irradiation enhance ATP produc- tion in human neuronal cells in culture. Photomedicine and Laser Surgery, 25, 180-182. doi:10.1089/pho.2007.2064

[27]

Tuby, H., Maltz, L. and Oron, U. (2007) Low-level laser irradiation (LLLI) promotes proliferation of mesenchy- mal and cardiac stem cells in culture. Lasers in Surgery and Medicine, 39, 373-378. doi:10.1002/lsm.20492

[28]

Li, W.T., Leu, Y.C. and Wu, J.L. (2010) Red-light light- emitting diode irradiation increases the proliferation and osteogenic differentiation of rat bone marrow mesenchy- mal stem cells. Photomedicine and Laser Surgery, 28, S-157-S-165. doi:10.1089/pho.2009.2540

[29]

Mvula, B., Moore, T.J. and Abrahamse, H. (2010) Effect of low-level laser irradiation and epidermal growth factor on adult human adipose-derived stem cells. Lasers in Medical Science, 25, 33-39. doi:10.1007/s10103-008-0636-1

[30]

Tuby, H., Maltz, L. and Oron, U. (2011) Induction of au- tologous mesenchymal stem cells in the bone marrow by low-level laser therapy has profound beneficial effects on the infarcted rat heart. Lasers in Surgery and Medicine, 43, 401-409. doi:10.1002/lsm.21063

[31]

Oron, U. (2011) Light therapy and stem cells: A thera- peutic intervention of the future. Journal of Interventio- nal Cardiology, 3, 627-629.

[32]

Toma, C., Pittenger, M.F., Cahill, K.S., Byrne, B.J. and Kessler, P.D. (2002) Human mesenchymal stem cells dif- ferentiate to a cardiomyocyte phenotype in the adult mu- rine heart. Circulation, 105, 93-98. doi:10.1161/hc0102.101442

[33]

Oron, U. and Mandelberg, M. (1985) Focal regeneration in the rat myocardium following cold injury. Cell Tissue Research, 241, 459-463. doi:10.1007/BF00217194

[34]

Mummery, C.L., Davis, R.P. and Krieger, J.E. (2010) Challenges in using stem cells for cardiac repair. Science Translational Medicine, 14, 1-5.

[35]

van Amerongen, M.J., Harmsen, M.C., van Rooijen, N., Petersen, A.H. and van Luyn, M.J. (2007) Macrophage dep- letion impairs wound healing and increases left ventricu- lar remodeling after myocardial injury in mice. American Journal of Pathology, 170, 1093-1103. doi:10.2353/ajpath.2007.060547

[36]

Okazaki, T., Ebihara, S., Asada, M., Yamanda, S., Saijo, Y., Shiraishi, Y., Ebihara, T., Niu, K., Mei, H., Arai, H. and Yambe, T. (2007) Macrophage colony-stimulating factor improves cardiac function after ischemic injury by induc- ing vascular endothelial growth factor production and sur- vival of cardiomyocytes. American Journal of Pathology, 171, 1093-1103. doi:10.2353/ajpath.2007.061191

 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

1. Tuner J. and Hode L. Low Level Laser Therapy: Clinical Practice and Scientific Background, Prima Books, Grängesberg, Sweden, 1999.

2. Karu T. The Science of Low Power Laser Therapy, Gordon & Breach, London, 1998.

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

4. Simunovic Z., Ed. Lasers in Medicine and Dentistry, Vitgraf, Rijeka (Croatia), 2000.

5. Zhukov B.N. and Lysov N.A. Laser irradiation in experimental and clinical angiology (in Russian), Samara (Russia), 1996.

6. Kozlov V.I., et al. Bases of laser physio- and reflexo-therapy (in Russian), Zdorovje, Samara (Russia), 1993.

7. Paleev N.R. Ed. Phototherapy (in Russian), Meditsina, Moscow (Russia), 2001.

8. Skobelkin O. K. Ed. Application of low-intensive lasers in clinical practice (in Russian). Moscow, 1997.

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

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.

30. Lataillade JJ, Clay D, Dupuy C, et al. Chemokine SDF-1 enhances circulating CD34+ cell proliferation in synergy with cytokines: possible role in progenitor survival. Blood. 2000;95:756–768.

31. Hattori K, Heissig B, Tashiro K, et al. Plasma elevation of stromal cell-derived factor-1 induces mobilization of mature and immature hematopoietic progenitor and stem cells. Blood. 2001;97:3354–3360.

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.

34. Pyrikova S.I et al. Effect of laser exposure on human seminal fluid (in Russian). Clinical and laboratory diagnosis. 1998;5:15-16.

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 is an excerpt from the book "Low Level Laser Therapy" by Tunér-Hode, chapter 13. You will find the excerpt at the link below or here. This excerpt talks about how not all negative LLLT studies can necessarily say that LLLT does not work. The main problem being that the dose or wavelength was incorrect for the attempted treatment, leaving reasearchers with less than satisfactory results, in some cases laser parameters were not even recorded. While we must take negative studies seriously, it can be seen that once the majority of them have been examined that the attempted LLLT was simply being done incorrectly. You will find the excerpt broken up thusly:

• Are all the negative lllt studies really negative?
• "I heard it through the grapewine"
• Positive from negative
• Negative from negative
• Important parameters
  • A. Wavelength 
  • B. Dose
  • C. Power density
• Typical traditional laser instruments
• Dose development 
• Pitfalls
  • 1. Low outputs 
  • 2. Inclusion criteria 
  • 3. Lack of proper control groups
  • 4. Therapeutic technique
  • 5. Systemic effects
  • 6. Tissue condition
  • 7. Power density
  • 8. Mixed parameters
  • 9. The influence of ambient light
  • 10. Premature conclusions
  • 11. Meta-analyses
• Confusion between groups

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

 In the book "Laser Therapy and Laser Puncture in Dogs and Cats", you will find acupuncture points and meridians for cats and dogs, with accompanying diagrams in the appendix. This book will go over using your laser therapy system, and determining treatment times and plans; as well as protective measures, contraindications, and side effects. The bulk of the book is about Finding the right treatment plan, and is broken into color coded sections thusly:

• Guidelines
• Wounds, Scars, Disturbance Fields
• Pain
• Locomotor System
• Repiratory Tract
• Gastrointestinal Tract
• Bladder, and Kidneys
• Liver
• Spleen, and Pancreas
• Vessels
• Metabolism, and Detoxification
• Skin
• Immune System
• Psyche
• Other Disorders

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

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.

Low Level Laser Therapy still has many critics and is not readily accepted as a natural treatment modality in all countries. One main point emphasized by the critics is the lack of scientific documentation. While this was a valid point in the 80s and partly in the beginning of the 90s, is it still a solid argument? There are more than 2000 published studies and the vast majority of these report positive biological effects from Low Level Laser Therapy (LLLT).

The heart of a scientific documentation is the double blind clinical studies. There are some 140 such studies in the field of LLLT and it may come as a suprise to many critics that more than 100 of these are positive. In fact, even most advocaters of LLLT are unaware of this fact. The aim of this Editorial is to disseminate this information to the LLLT community.

Some of the negative double blind studies are well designed and should be taken seriously. Certainly all indications and all parameters cannot work. However, a number of the often quoted negative double blind studies suffer from flaws of several kinds. Some of this is outlined on http://www.laser.nu/lllt/LLLT_critic_on_critics.htm which is a chapter from our recent book "Low Level Laser Therapy - clinical practice and scientific background"

A closer analysis of 100 positive double blind studies will be presented at Laser Florence '99 (October 28-31) and will also appear in the EMLA Millennium laser book.
A weakness in the list is that many double blind studies have only been identified in the abstract form. They may have been published in full at a later stage, but not found by us. 14 studies have only been found as references in reference lists and these have not been found in spite of intensive efforts. For a complete analysis of the 100 positive double blind studies we need the assistance of the visitors of LaserWorld. In the following list abstracts are marked in red and studies not found marked in green. If you have any information about the green studies please contact us. And if you know that an abstract has been published in a journal, please do likewise. The more complete the list is, the better for the LLLT community.

The studies published in journals are listed in full in the book mentioned above.

Atsumi K et al. Biostimulation effect of low-power energy diode laser for pain relief. Lasers Surg Med. 1987; 7: 77.
Barabas K et al. Controlled clinical and experimental examinations on rheumatoid arthritis patients and synovial membranes performed with neodym phosphate glas laser irradiation. Proc. 7th Congr Internat Soc for Laser Surg and Med, Munich June 1987. Abstract no 216a.
Boerner E et al. Double-blind study on the efficacy of the lasertherapy. SPIE Proc. 1996. Vol. 2929: 75-79.
Cheng R. Combined treatments of electrotherapy plus soft laser therapy has synergistic effect in pain relief and disease healing. Surgical and Medical Lasers. 1990; 3 (3): 135
Cieslar G et al. Effect of low-power laser radiation in the treatment of the motional system overloading syndromes. SPIE Proc. Vol 3198. 1997, pp. 76-82.
Emmanoulidis O et al. CW IR low-power laser application significantly accelerates chronic pain relief rehabilitation of professional athletes. A double blind study. Lasers Surg Med. 1986; 6: 173.
Haruki E, Yamaguchi S. Double blind evaluation of low energy laser treatment for painful disease. J Phys Med. 1995; 6: 60-67. (In Japanese with English abstract)
Hopkins G O et al. Double blind cross over study of laser versus placebo in the treatment of tennis elbow. Proc Internat Congr on Lasers, "Laser Bologna". 1985: 210. Monduzzi Editore S.p.A., Bologna. Hoshino H et al. The effect of low reactive level laser therapy in the field of orthopedic surgery. Chronic Pain. 1994; 13: 101-109. (In Japanese with English abstract)
Hoteya K et al. Effects of a 1 W GaAlAs diode laser in the field of orthopedics. In: Meeting Report: The first Congress of the International Association for Laser and Sports Medicine. Tokyo, 1997. Laser Therapy 1997; 9 (4): 185.
Kamikawa K et al. Double blind experiences with mid-Lasers in Japan. 1985. Proc Int Congr on Lasers, "Laser Bologna". 1985: 165-169. Monduzzi Editore S.p.A., Bologna.
Kim J W, Lee J O. Double blind cross-over clinical study of 830 nm diode laser and 5 years clinical experience of biostimulation in plastic & aesthetic surgery in Asians. Lasers Surg Med. 1998; Suppl. 10: 59.
Kinoshita F et al. Clinical evaluation of low-energy, semi-conductor laser therapy in oral surgery - a double blind study. Josai Shika Daigaku Kiyo (Bulletin of Josai Dental University). 1986; 15 (3): 735-742. (in Japanese with English abstract)
Kosaka R et al. Double blind study of low energy diode laser irradiation for chronic pain disorders. J Phys Med. 1993; 4: 156-160.
Kouno A et al. The evaluation of pain therapy with low powerlaser- Comparative study of thermography and double blind test. Biomedical Thermology. 1993; 13: 102-107.
Lonauer G: Controlled double blind study on the efficacy of HeNe-laser beams versus HeNe- plus Infrared-laser beams in the therapy of activated osteoarthritis of finger joints. Clin Experim Rheuma. 1987; 5 (suppl 2) : 39
Lucas C et al. Low level laser therapy bij decubitus statium III. Rapport Hoegschool van Amsterdam. 1994.
Mach E S et al. Helium-Neon (Red Light) Therapy of Arthritis. Rhevmatologia, 1983; 3: 36. (In Russian)
Mester A: Biostimulative effect in wound healing by continous wave 820 nm laser diode. Double-blind randomized cross-over study. Lasers in Med Science, abstract issue July 1988, No 289.
Miyagi K. Double-blind comparative study of the effect of low-energy laser irradiation to rheumatoid arthritis. In: Current awareness of Excerpts Medica. Amsterdam. Elsevier Science Publishers BV. 1989; 25: 315.
Mokhtar B et al. A double blind placebo controlled investigation of the hypoalgesic effects of low intensity laser irradiation of the cervical roots using experimental ischaemic pain. Proc. Second Meeting of the International Laser Therapy Assn., "London Laser", Sept 1992, p 61. Mokhtar B et al. The possible significance of pulse repetition rate in lasermediated analgesia: A double blind placebo controlled investigation using experimental ischaemic pain. Proc. Second Meeting of the International Laser Therapy Assn, "London Laser" Sept 1992. p 62
Neuman I et al. Low energy phototherapy in allergic rhinitis and nasal polyposis. Laser Therapy. 1996. 1: 37.
Palmgren N et al. Low Level Laser Therapy of infected abdominal wounds after surgery. Lasers Surg Med. 1991; Suppl 3:11.
Poliakova A G., Gladkova N D, Triphonova T.D. Laserpuncture in patients with rheumatoids arthritis. Abstracts of ICMART '97 International Medical Acupuncture Symposium, Nicosia, Cyrprus, March 26-29 1997.
Rochkind S et al. Double-blind Randomized Study Using Neurotube and Laser Therapy in the Treatment of Complete Sciatic Nerve Injury of Rats. Proc. 2nd Congr World Assoc. for Laser Therapy, Kansas City, 1998.
Roumeliotis D et al. 820nm 15mW 4J/cm2, laser diode application in sports injuries. A double blind study. Proc. Fifth Annual Congress British Medical Laser Ass. 1987.
Ryo E et al. Double blind test of low energy laser radiation treatment. Evaluation of effectiveness for shoulder stiffness, arthralgia etc. Pain Clinic. 1986; 7: 185-192. (In Japanese with English abstract)
Saeki N et al. Double blind test for biostimulation effects on pain releif by diode laser. 1989. Laser Surgery; 1066: 93-100.
Sasaki K et al. A double-blind controlled study on free amino acid analysis in CO2 laser burn wounds in the mouse model following doses of low incident infrared (830 nm) diode laser energy. Proc. 2nd Meeting if the Internat Laser Therapy Assn., London, 1992, p.4.
Sato K et al. A double blind assessment of low power laser therapy in the treatment of postherpetic neuralgia. Surgical and Medical Lasers. 1990; 3 (3): 134.
Scudds R A et al. A double-blind crossover study of the effects of low-power gallium arsenide laser on the symptoms of fibrositis. Physiotherapy Canada.1989; 41: (suppl 3): 2.
Taghawinejag M et al. Laser-Therapie in der Behandlung kleiner Gelenke bei chronischer Polyarthritis. Z Phys Med Baln Med Klin. 1985; 14.
Tsurko V V et al. Laser therapy of rheumatoid arthritis. A clinical and morphological study. Ter Arkh. 1983; 55 (7) 97-102. (Russian).
Umegaki S et al. Effectiveness of low-power laser therapy on low back pain. Double blind comparative study to evaluate the analgesic effect of low power laser therapy on low-back pain. The Clinical Report. 1989; 23: 2839-2846. (In Japanese with English abstract)
Vélez-Gonzalez M et al. Treatment of relapse in herpes simplex on labial and facial areas and of primary herpes simplex on genital areas and "area pudenda" with low power HeNe-laser or Acyclovir administred orally. SPIE Proc. 1995; Vol. 2630: 43-50
Willner R et al. Low power infrared laser biostimulation of chronic osteoarthritis in hand. Lasers Surg Med. 1985; 5: 149.
Wylie L et al. The hypoalgesic effects of low intensity infrared laser therapy upon mechanical pain threshold. Lasers Surg Med. 1995; Suppl 7: 9.
Yamaguchi M et al. Clinical study on the treatment of hypersensitive dentine by GaAlAs laser diode using the double blind test. Aichi Gakuin Daigaku Shigakkai Shi - Aichi-Gakuin Journal of Dental Science. 1990; 28( 2): 703-707. (in Japanese)
Yoh K et al. A clinical trial for treatment of chronic pain in orthopedic diseases by using 150 mW diode laser system. Result of double blind test. Chronic Pain; 13: 96-100.(In Japanese with English abstract)

12.1. INTRODUCTION

Significant changes in blood flow or in the integrity of cerebral vessels are believed to cause cerebrovascular disease (CVD) and to contribute to dementias including Alzheimer’s disease [1]. Stroke, the most serious form of CVD, is one of the leading causes of death and adult disability worldwide. Acute treatments for stroke, however, are severely limited. Neuroprotective drugs under development show promise at halting the ischemic cascade, but as yet, no such compound has received federal approval in the United States. One of the biggest limitations to this development is the lack of understanding of the mechanisms by which cerebral vessels react to factors such as ischemia, inflammation, blood pressure changes, metabolic demands, and trauma [2]. In order to address these fundamental questions, functional brain imaging techniques such as fMRI and intrinsic signal optical imaging (ISOI) have emerged as tools to visualize and quantify cerebral hemodynamics.

In the neuroscience community, ISOI has long been used to study the organization and functional architecture of different cortical regions in animals and humans [3–5] (see other chapters in this book). Three sources of ISOI signals that affect the intensity of diffusely reflected light derive from characteristic physiologic changes in the cortex. For functional neuronal activation, these have been observed to occur over a range of timescales, including (1) light scattering changes, both fast (over 10 s of milliseconds) and slow (i.e., > ~0.5 s) (2) early (~0.5–2.5 s) absorption changes from alterations in chromophore redox status, i.e., the oxy/deoxy-hemoglobin ratio (known as the “initial dip” period), and (3), slower (~2–10 s) absorption changes due to blood volume increase (correlated with the fMRI BOLD signal). Light scattering changes have been attributed to interstitial volume changes resulting from cellular swelling, organelle swelling due to ion and water movement, capillary expansion, and neurotransmitter release [6,7]. The slower absorption factors have been demonstrated to correlate with the changes in metabolic demand and subsequent hemodynamic cascades following neuronal activation [4,8,9].

Using animal models of acute and chronic brain injury, ISOI has been used to quantify the acute hemodynamic events in response to stroke, including focal ischemia and cortical spreading depression (CSD) [10–21]. Researchers have also used ISOI to locate and quantify the spatial extent of the stroke injury, including ischemic core, penumbra, and healthy tissue zones [18,22]. CSD also plays a key role in migraine headache, and recent laser speckle imaging studies have revealed the neurovascular coupling mechanism to the transmission of headache pain [23,24].

To fully understand the underlying mechanisms in vascular changes associated with cerebrovascular diseases such as stroke, an optical imaging technique that has the capability to rapidly separate absorption from scattering effects can enhance the information content of traditional ISOI, enabling (1) more accurate quantitation of hemodynamic function, (2) isolation of the electro-chemical changes characterized by light scattering, and (3) longitudinal chronic injury studies of function where structural reorganization due to neovascularization can cause significant alterations in scattering [25,26].

Quantitative diffuse optical methods [27] such as spatially-resolved reflectance, diffuse optical spectroscopy (DOS), and tomography (DOT), and diffuse correlation spectroscopy (DCS) possess exquisite sensitivity to these functional and structural alterations associated with brain injury, and have been applied to the study of CSD [11,15,28]. DOS and DOT utilize the near-infrared spectral region (600–1000 nm) to separate and quantify the multispectral absorption (μ_a) and reduced scattering coefficients (μ_s′), providing quantitative determination of several important biological chromophores such as deoxy-hemoglobin (HbR), oxy-hemoglobin (HbO₂), water (H₂O), and lipids. Concentrations of these chromophores represent the direct metrics of tissue function such as blood volume fraction, tissue oxygenation, and edema. Additionally, the scattering coefficient contains important structural information about the size and density of scatterers and can be used to assess tissue composition (exctracellular matrix proteins, cell nuclei, mitochondria) as well as follow the process of tissue remodeling (wound healing, cancer progression). DOS utilizes a limited number of source-detector positions, e.g., 1–2, but often employs broadband content in temporal and spectral domains [29]. In contrast, DOT typically utilizes a limited number of optical wavelengths (e.g., 2–6) and a narrow temporal bandwidth, but forms higher resolution images of subsurface structures by sampling a large number of source-detector “views.” To achieve maximal spatial resolution, the ideal DOT design would employ thousands of source-detector pairs and wavelengths. However, several engineering considerations including measurement time and instrument complexity currently limit the practicality of this approach.

In this chapter we present the basic principles of a new, noncontact quantitative optical imaging technology, modulated imaging (MI) [30–32], and provide examples of MI performance in 2 rat models of brain injury, cortical spreading depression (CSD) and stroke. MI enables both DOS and DOT concepts with high spatial (<1 mm) and temporal resolution (<1 s) in a simple, scan-free platform. MI is capable of both separating and spatially-resolving optical absorption and scattering parameters, allowing wide-field quantitative mapping of tissue optical properties. While compatible with time-modulation methods, MI alternatively uses spatially modulated illumination for imaging of tissue constituents. Periodic illumination patterns of various spatial frequencies are projected over a large area of a sample. The diffusely reflected image is modified from the illumination pattern due to the turbidity of the sample. Typically, sine-wave illumination patterns are used. The demodulation of these spatially modulated waves characterizes the modulation transfer function (MTF) of the material, and embodies the sample optical property information.

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12.2. METHODS AND INSTRUMENTATION

12.2.1. Modulated Imaging Spectroscopy

The MI instrument platform was introduced originally by Cuccia et al. [30] Based on this design, we have developed a custom multispectral near-infrared (NIR) MI spectroscopy system capable of imaging between 650 and 1000 nm. A diagram of this system is shown in Figure 12.1.

FIGURE 12.1

Modulated imaging platform. QTH—quartz tungsten halogen; L1—aspheric condenser; H—hybrid hot mirror; DMD—digital micromirror device; L2—projection lens; L3—camera lens; LCTF—liquid crystal tunable (more...)

Broadband NIR illumination is provided by an intensity-stabilized 250 W quartz-tungsten-halogen (QTH) lamp (Oriel QTH Source with Light Intensity Controller, Newport Corporation-Oriel Instruments, Stratford, Connecticut). Light is collimated and refocused with a pair of aspheric F/#0.7 optical lens systems (Oriel Aspherab). A custom-sized 3.5 in square hybrid hot mirror (Reynard Corporation, i.e., R00670-00) was placed between the lenses to limit the illumination to wavelengths below 1000 nm. Light engine optics taken from a digital projector (NEC HT1000) serve to homogenize and direct the light onto a 0.7 in digital micromirror device (DMD Discovery™ 1100 with ALP Accessory Package, ViALUX, Germany). Grayscale spatial sinusoid patterns are projected at 400 Hz using the ViALUX software development toolkit, which generates the necessary pulse-width modulation of binary sub-frames to produce a specified grayscale bit-depth (1–8 bits). Finally, a fixed focal length (f = 100 mm) projection lens illuminates the tissue at a slight angle from normal with a 15 × 25 mm illumination field. Detection was performed at normal incidence using a CRI Nuance™ camera system, which combines a 12-bit CCD camera and a liquid crystal tunable filter (LCTF; λ = 650–1100 nm, Δλ = 10 nm). To avoid specular reflection, crossed linear polarizers are used in the illumination and detection arms. For this system, the former is a 1.5 in diameter NIR linear polarizer (Meadowlark Optics, VLM-200-IR-R) placed immediately after the projection lens, and the first stage of the Nuance LCTF serves as the latter. The DMD, CCD, and LCTF are controlled via USB by a laptop computer, and synchronized using LabVIEW software (LabVIEW 8, National Instruments), enabling fast acquisition of a series of patterns with various spatial frequencies.

12.2.2. SFD Measurement, Calibration, and Modeling

A detailed description of SFD measurement, calibration, and diffusion modeling is provided by Cuccia [32]. In this work, we modeled diffuse reflectance using a transport-based White Monte Carlo (WMC) method [33,34]. Previously, we have found that compared with Monte Carlo, (1) diffusion predictions over- and underestimate low- and high-frequency diffuse reflectance, respectively, and (2) the quantitative accuracy of diffusion degrades with decreasing albedo [32]. Due to the moderate albedo of brain tissue (μ_s′/μ_a ~ 10–20), we chose to analyze all brain data with the WMC approach. This homogeneous tissue model is a significant simplification of the multilayered rat brain, and more work is necessary to accurately model this complex system. We discuss further the consequences of our simple model in Section 12.2.5.

12.2.3. Optical Property Inversion Methods

In this chapter, we use two inversion methods to calculate the absorption and reduced scattering from measurements of diffuse reflectance. When high measurement precision is desired, we use a “sweep” in spatial frequency space, producing an overdetermined set of diffuse reflectance measurements, which can be fitted to our WMC forward model predictions using least-squares minimization. This method is performed for all spatially averaged region analysis of optical properties and chromophores. When increased acquisition and/or processing speed is desired, we alternatively use a rapid two-frequency lookup table method based on cubic spline interpolation [32]. This data can be achieved with a minimal 3-phase, single frequency image set (by demodulating and averaging the images to obtain AC and DC amplitude maps, respectively). On typical personal computers this approach is capable of millions of inverse lookup calculations per second, and is therefore used to calculate all high-resolution images including time sequences. The signal-to-noise ratio (and thus the measurement precision) of either approach is limited by the data sampling, with the two-frequency method having a lower precision with the tradeoff of higher acquisition and processing speed.

12.2.4. Spectral Analysis-Chromophore Calculation

The quantitative absorption coefficient is assumed to be a linear (Beer’s law) summation of individual chromophore absorption contributions:

μa(λ)=2.303∑i=13ci?i(λ),

12.1

where c_i and ?_i(λ) represent chromophore concentrations and molar extinction coefficients, respectively. Using reported extinction coefficients of HbO₂/HbR35 and H₂O,36 we can invert Equation 12.1 and calculate tissue chromophore concentration separately at each pixel by linear least-squares fitting to the multispectral absorption images. Total hemoglobin (HbT) and oxygen saturation (S_tO₂) can then be calculated as HbT = HbR + HbO₂ and S_tO₂= HbO ₂/(HbR + HbO₂) * 100, respectively.

12.2.5. Optical Property Mapping: Resolution Versus Quantitation

On a pixel-by-pixel basis, diffuse reflectance versus spatial frequency is fitted to the WMC forward model to extract the local absorption and reduced scattering optical property contrast. This process is repeated for each wavelength, resulting in multi-spectral absorption and scattering spectra at each pixel. The measured contrast from discrete absorbers and scatterers on millimeter and submillimeter spatial scales, however, will possess partial volume effects in all three spatial dimensions. This is due to the physical light transport length scales in tissue, limiting the true x-y resolution of optical property contrast to many detector pixels [32]. This phenomenon is not unique to MI, but present in all planar reflectance imaging measurements of turbid media. Absorption and scattering are calculated using a homogeneous reflectance model, extracting a locally averaged sampling of optical property contrast. Based on simulations of the tissue MTF for varying optical properties [32], we expect the resulting image resolution to scale directly with the transport length, l* = (μ_a + μ_s′)^{− 1}, and the spatial frequency of illumination. In this chapter, we place quantitative emphasis on average optical properties and chromophores measured over a field of view that is greater than l*. Spatial maps and videos of these parameters are displayed and referred to as “contrast maps,” with the caveat that high resolution features will exhibit degraded quantitative accuracy.

12.2.6. In Vivo Rat CSD Experiments

12.2.6.1. Animal Preparation

MI spectroscopy measurements were performed on an in vivo Wistar rat model with a thinned-skull preparation. All procedures were performed in accordance with approved IACUC protocol guidelines. The animals were anesthetized, placed in a stereotaxic frame, their skulls thinned and glass coverslip applied. This preparation is described in detail by Masino et al. [37] The resulting thinned skulls allowed direct imaging of the cortex over a 5 × 7 mm field-of-view (whisker barrel cortex, centered at the C2 location). In order to investigate the sensitivity of MI toward studying acute cortical injury, we induced cortical spreading depression (CSD) by applying 1 M KCl solution to the surface of the cortex through a perforated section of skull and dura, located approximately 3 mm above the camera’s imaging field.

12.2.6.2. MI Measurement Protocol

For each of three animals, our MI measurement protocol was twofold. Prior to CSD induction, baseline spatial modulation data were acquired at 6 spatial frequencies (3-phase projections each) from 0 to 0.26 mm⁻¹, at 10 nm intervals over the entire range between 650 and 980 nm. Depending on the wavelength, image acquisition times ranged from 200 ms to 4 s, with total spectral imaging time of approximately 30 s per spatial pattern. The entire measurement (34 wavelengths, 3 phases, 6 frequencies) was repeated three times for statistical averaging yielding an entire measurement time of approximately 30 min.

Next, rapid dynamic measurements were performed, beginning 1 min prior to K⁺Cl⁻ administration. Here, a significantly reduced data set was chosen in order to achieve high temporal resolution. Two spatial frequencies (0 and 0.26 mm⁻¹) were acquired with three phase projection images, as described in Section 12.2.2, at each of four wavelengths (680, 730, 780, and 830 nm). The resulting 12 images took in total 6 s, permitting a repetition rate of 10 measurements per minute. The animals were followed for a period of 10 min for rats 1 and 2, and a period of 30 min for rat 3.

All images in this study were smoothed by 2D convolution with a Gaussian filter function (FWHM = 3 pixels), and baseline repetitions were averaged prior to data processing. Additionally, time-series data were post-processed by smoothing slightly in time (Gaussian FWHM of 2 timepoints = 12 s).

12.2.6.3. Spatial Frequency Sensitivity Analysis

Because of the differential absorption sensitivity at low and high frequencies, optimal optical property separation is achieved when a large range of frequencies is used [31]. In Figure 12.2a, we depict this differential sensitivity using diffuse reflectance (MTF) predictions versus frequency, increasing μ_a by 100% from 0.02 (black line) to 0.04mm⁻¹ (gray line). This is done for two values of μ_s′, 0.6 (solid lines) to 1.2mm⁻¹ (dashed lines), simulating a 100% change in scattering. Notice that the low frequencies have a significant reflectance change due to absorption, while high frequency reflectance remains nearly unchanged. Conversely, reflectance changes due to scattering are observed at all spatial frequencies. In Figure 12.2b, we further visualize this by plotting the reflectance sensitivity to 1% changes in absorption and scattering. Whereas DC reflectance is equivalently sensitive to a fractional change in either absorption or scattering, at high spatial frequencies absorption contrast is lost while scattering contrast is retained. For instance, notice that at our maximum measurement frequency of 0.26 mm⁻¹ the reflectance is roughly 24 times more sensitive to scattering compared to absorption (ΔR_d = 0.56 μ_s′ versus 0.024 * 10⁻³ for μ_a). This plays an important role in Section 12.3.2 during our discussion of dynamic scattering measurement.

FIGURE 12.2

(a) Reflectance contrast in absorption and scattering covering a typical range of brain optical properties. (b) The frequency-dependent sensitivity to absorption (black line) and scattering (gray line), respectively. Reflectance at fx = 0.26 mm⁻¹ (more...)

In realistic heterogeneous tissues, a tradeoff exists between maximizing the frequency range for optical property accuracy and obtaining similar sampling volumes. As tissue is a low-pass spatial filter, high frequencies are attenuated quickly with depth. Using diffusion-based forward modeling, we have estimated mean sampling depths at 650 nm using measured average background optical properties of brain tissue. This was done by predicting the depth sensitivity to contrast from a planar perturbation in absorption, given a background fluence profile from spatial frequencies 0 and 0.26 mm⁻¹. Based on these results, we observe qualitatively similar depth sampling, with mean depth sampling ranging between 2.5 mm and 1.2 mm (for f_x = 0 and 0.26 mm⁻¹, respectively). In all cases maximal sensitivity was found in the first 1–2 mm, where cortical hemodynamic changes occur.

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12.3. RESULTS AND DISCUSSION

12.3.1. Baseline MI Spectroscopy

In Figure 12.3a we show a grayscale planar reflectance image of the cortical region of rat 1 at 650 nm. A dotted-line box denotes the region-of-interest (ROI) used for analysis, selected for its uniform illumination and the absence of cerebral bruising. The Monte Carlo-model fitting of spatial frequency data allows calculation of the absorption and reduced scattering coefficients. In Figure 12.3b we show the spatially averaged diffuse reflectance at 650 nm and the corresponding multi-frequency fit. Excellent agreement is observed between measurement data and the model-based fit, with derived μ_a and μ_s′ coefficients of 0.033 and 0.70 mm⁻¹, respectively.

FIGURE 12.3

(a) Reflectance map for rat 1, showing the 3.8 × 5.9 mm region chosen for quantitative analysis. (b) Sample MTF reflectance data (squares) and fit (solid line) at 650 nm. (c) Recovered optical property maps (above) and corresponding image histogram (more...)

Analysis of multifrequency reflectance data separately at each pixel results in spatial maps of absorption and reduced scattering contrast. In Figure 12.3c, we plot the μa and μs′ maps recovered at 650 nm for rat 1. Note the strong absorption in the vein region, due to a large absorption by HbR at this wavelength. Below the images, we show histogram distributions of the corresponding quantitative maps above, indicating the degree of spatial variation in recovered optical properties. The mean and standard deviation for the pixel-wise μa and μs ′ were 0.030 ± 0.007 mm⁻¹ and 0.63 ± 0.13 mm⁻¹, respectively. These statistical results are in good agreement with the spatially averaged reflectance fit from Figure 12.3b, suggesting that our simple pixel-wise fitting approach yields optical properties similar to that calculated using a global analysis.

By mapping the absorption coefficient at multiple wavelengths, we can perform quantitative spectral imaging of tissue. In Figure 12.4, we summarize the baseline spectroscopy results for all three animals. In Figure 12.4a we show the μ_a (left) and μ_s′ (right) coefficients versus wavelength (circles) recovered from spatially averaged fitting. Data for rat 1 is shown in black (rat 2 in dark gray; rat 3 in light gray). Note the distinct spectral features in absorption, resulting from oxy- and deoxy-hemoglobin (HbO₂, HbR), and water (H₂O) absorption. The calculated scattering coefficient generally decays with increasing wavelength, and the results from a power law (μ_s ′ = A·λ(nm) ^−b, solid lines) fit are shown. A small residual coupling is observed between measured scattering and absorption spectral features. In particular, the scattering at the shortest and longest wavelengths appears to be underestimated by 5–10%, occurring where the corresponding absorption is highest (due to HbR and H₂O, absorption features, respectively). Based on our experiments in layered tissue phantoms [38], we believe this effect is primarily due to frequency-dependent probing volumes in the presence of depth-heterogeneous structures.

FIGURE 12.4

(a) Average μ_a (left) and μ_s′(right) spectra over entire ROI (circles). HbO ₂, HbR, and H₂O concentrations are determined by subsequent least-squares fitting (solid lines) of molar extinction coefficients to the absorption. Data (more...)

Simultaneous linear fitting of the absorption to known extinction coefficients yields measures of chromophore concentration. Shown in Figure 12.4a, multispectral fitting (solid line) for rat 1 yields HbO₂, HbR, H₂O, HbT and S_tO₂ values of 56.3 μM, 33.2 μM, 63.9%, 89.6 μM, and 56.3%, respectively. Tabulated results of chromophore values for all three animals are shown in Figure 12.4b. Lipid absorption near 930 nm was not apparent in the μ_a spectrum, and when included in the spectral analysis was not found to significantly affect the results. The small absorption “bump” at 900–910 nm is an artifact of imperfect phantom calibration due to the presence of a sharp, strong silicone absorption peak that is present in the phantom.

We note that the solution for chromophore concentration is well-determined when the number of wavelengths is at least equal to the number of chromophores. Therefore, as few as two wavelengths can be used to separate HbO₂ and HbR (if a constant value of H₂O is assumed). Repeating the above analysis with 780 and 830 nm only (assuming H₂O = 65%) yields results for HbO₂ and HbR within 10% of those from full spectral fitting. Repeating the above analyses using a simple diffusion-based model provided qualitatively similar results for absorption and scattering spectra, but in general was found to overestimate the absorption coefficient by 10–25%.

Absorption spectra at each pixel can be separately analyzed to yield spatial maps of local HbO₂, HbR, and H₂O distribution, shown in Figure 12.5. Notice the high concentration of HbR over the large superficial draining vessel (venous) regions, also reflected in the S_tO₂ image, highlighting the effect of tissue oxygen extraction. Conversely, notice that the high albedo regions with less structural detail are highly oxygenated, with S_tO₂ levels between 60 and 70%. Lastly, the H₂O map reveals a relatively homogeneous distribution of water.

FIGURE 12.5

Chromophore fits to absorption spectra at each pixel yield maps of local HbO₂, HbR, and H₂O concentration (left). Total hemoglobin (HbT) and oxygen saturation (S_tO₂) maps can then be calculated from HbO₂ and HbR.

12.3.2. Dynamic MI Spectroscopy of CSD

We performed measurements of CSD in each of the three rats, as described in Section 12.2.3. The results are presented as follows. We first present data for a single animal, choosing rat 3 for its long observation period of 30 minutes. Three ROIs are selected for analysis, and baseline MI spectroscopy results are reported for each of these regions. Next, the observed dynamic time courses of diffuse reflectance, optical properties, and chromophore concentrations are shown for each ROI. We then present the full spatio-temporal dynamic contrast data for rat 3 (2D + time) in the form of “snapshot” images.

Figure 12.6 summarizes the baseline spectroscopy measurements for rat 3. In Figure 12.6a, we show three regions of interest superimposed on the DC reflectance map, chosen to highlight three different characteristic temporal profiles observed within the field of view. In Figure 12.6b we show the baseline spectral fits for each of these regions, and in Figure 12.6c we tabulate the resulting calculated chromophore concentrations. In general, Region A (black) is a high albedo region lacking any large blood vessels, whereas Regions B (dark gray) and C (light gray) include high-absorption blood vessels and mild cerebral bruising from surgery. These differences are apparent in their recovered absorption spectra and fits, with on average 27% higher HbT, and 32% lower saturation in the vascular regions. Also, 7% higher H₂O is found in Regions B and C, which may indicate increased edema due to bruising.

FIGURE 12.6

Regionwise spectral analysis of rat 1 baseline data including the respective (A) ROIs, (B) spectral absorption data (circles) and fit (lines), and (C) tabulated recovered chromophore data for each region

In Figures 12.7–12.9 (for regions A–C, respectively), we present the temporal dynamics of CSD in each ROI of rat 3 as measured by MI. In part (a) of each figure, we plot the multispectral diffuse reflectance changes at f_x = 0 mm⁻¹ (DC, top) and f_x = 0.26 mm⁻¹ (AC, bottom). In part (b), we plot the recovered Δμ_a (top) and Δμ_s′ (bottom) optical properties at each wavelength. While absolute values of diffuse reflectance and optical properties are measured separately at each time point, for visualization purposes all data are displayed as a change from that prior to KCl administration. Absolute optical property values at t = 0 (not shown) demonstrate excellent agreement (~5–10%) with full multifrequency baseline data.

FIGURE 12.7

(A) Multispectral diffuse reflectance at DC (fx = 0 mm⁻¹, top) and DC (fx =.26 mm⁻¹, bottom) for Region A of rat 3 over approximately 30 min. (B) Corresponding recovered multispectral absorption (top) and reduced scattering (bottom) coefficients. (more...)

FIGURE 12.8

(A) Multispectral diffuse reflectance at DC (fx = 0 mm⁻¹, top) and DC (fx = 0.26 mm⁻¹, bottom) for Region B of rat 3 over approximately 30 min. (B) Corresponding recovered multispectral absorption (top) and reduced scattering (bottom) (more...)

FIGURE 12.9

(A) Multispectral diffuse reflectance at DC (fx = 0 mm⁻¹, top) and DC (fx = 0.26 mm⁻¹, bottom) for Region C of rat 3 over approximately 30 min. (B) Corresponding recovered multispectral absorption (top) and reduced scattering (bottom) (more...)

Looking first at the reflectance time courses of Figure 12.7a (Region A), we see in general a series of three CSD events over the 30 minutes, with each transient event occurring for approximately 4.3 minutes. The first event occurs at minute 2.9 after KCl application, indicating an initial latency between the insult and the first resulting spreading depression wave. Reflectance contrast is present in both DC and AC frequency components, but with markedly different signatures. Generally, the DC time course shows a slow, gradual decay, punctuated by sharp, wavelength-dependent spikes/dips (for short/long wavelengths, respectively). Alternatively, the AC signature contains three sets of transient dips consistent across all wavelengths, with final values leveling off progressively lower than baseline. Discussed in detail in the following paragraph, we believe these AC changes are due primarily a result of optical scattering and may be related to neuronal depolarization. The corresponding derived optical properties in Figure 12.7b reflects this, with μ_s′ trends tracking directly with the measured AC reflectance. As expected, μ_a trends reveal similar wavelength-dependence of the DC reflectance (with opposite polarity), reflecting changes in HbO₂ and HbR.

In Section 12.2.3.3 we noted that the diffuse reflectance at f_x = 0.26 mm⁻¹ is 23 times more sensitive to scattering changes compared to absorption. In this context, we propose that the observed magnitude of the CSD-induced AC reflectance changes can only be explained by changes in optical scattering. To concretely illustrate this point, we pick as an example the observed 780 nm AC diffuse reflectance dip in Figure 12.7a at t = 3.7 min of -0.003. Here, the corresponding change in reduced scattering in Figure 12.7b, Δμ_s′, is calculated to be −0.03 mm⁻¹. In order for this change to instead be due to an absorption-only event, μ_a would need to increase by 121% from baseline (from 0.038 to 0.084 mm⁻¹). This increase would also need to be accompanied by a drop in R_d (f_x = 0 mm⁻¹) of 0.12 (33%), whereas the actual observed DC reflectance only drops by 0.008 (<1%) and thus cannot explain the change. Secondly, we note that the three sets of AC reflectance dips occur consistently across all four wavelengths. While an approximate 120% increase in HbT could induce this decrease at high frequency, it would also require a large broad-wavelength decrease in the DC reflectance. We instead observe during these events that the DC increases at short wavelengths while the DC decreases at long wavelengths, suggesting primarily an exchange between HbO₂ and HbR volume fractions, as opposed to a dramatic HbT change.

Regions A–C (Figures 12.7–12.9) were chosen to highlight three different time signatures observed in the field of view during the CSD dynamics. The most contrasting feature between all three regions is the measured AC reflectance and the derived scattering coefficient. In Region B (Figure 12.8), each CSD event appears to cause a biphasic scattering change, with a sharp increase and then decrease, whereas a monophasic dip was observed in Region A (Figure 12.7). Region C (Figure 12.9) appears even more complex with a triphasic rise-dip-rise temporal profile. We observe that Regions A to C are located with increasing proximity to the CSD induction point (3 mm above the imaging field).

Because fractional changes in scattering and absorption have an equal (and opposite) effect on DC reflectance (see Section 12.2.3.3), any scattering (i.e., pathlength) changes measured here could be misinterpreted as absorption events with traditional ISOI analyses (i.e., DC reflectance only). In our observations, the measured scattering change of up to −0.05 mm⁻¹ would be interpreted as an increase in absorption of up to +0.005 mm⁻¹, more than the maximum measured absorption change for wavelengths 730, 780, or 830 nm in any of the three regions. In order to account for differential pathlength changes, Kohl et al. proposed a multispectral model [39], which they used to differentiate dynamic scattering and absorption changes using ISOI. This approach improves ISOI accuracy, and has been generally adopted as the method of choice for quantitative functional imaging. For dynamic measurements, we see MI as an improvement over this approach as it alternatively uses frequency domain measurements at a single wavelength to derive absolute scattering and absorption coefficients. This potentially provides a simplified single-wavelength measurement apparatus for detection of scattering, and also avoids potential mis-estimation of background optical properties.

Light scattering changes induced by spreading depression have been reported previously, and a comprehensive review is provided by Somjen. With in vivo spatially resolved reflectance measurements, Kohl et al. [11] separated absorption from scattering and observed a biphasic scattering response similar to that of Region A. With simultaneous laser scattering and electrophysiological measurements, both Jarvis et al. and Tao et al. found a strong correlation between electrical and optical scattering changes [12,13,40]. Tao et al. noted spatial heterogeneity in the dynamic spreading depression (SD) waveform related to the proximity to the SD induction site, similar to our results.

Using linear spectral analysis of absorption at all four wavelengths, we calculated the time-dependent chromophore concentration for Regions A, B, and C, presented in Figure 12.10A,B,C, respectively. In each region, the calculated baseline concentrations of H₂O were assumed to be constant. All three regions exhibit remarkably similar trends in HbR, HbO₂, HbT, and S_tO₂. This similarity is not clear in the DC traces of Figures 12.7–12.9, further highlighting the benefit of accurate separation of μa and μs′. Focusing on the first CSD event, there is a very consistent signature of: (1) a 2-minute latency post-KCl administration, (2) a 30-second period of decreasing S_tO₂ (3) a dramatic spike in both S_tO₂ (3–10%) and HbT (2–4 μM) with rise and decay times of approximately 1 minute each. For each region, the final S_tO₂ is approximately 5–10% lower than baseline, while the HbT restores to baseline values. This process repeats again twice more, except that the phase (2) desaturation appears to be absent. Additionally, in the “vessel” Region 3, we observe a gradual increase in HbT over the 30 minutes, indicating chronic blood pooling.

FIGURE 12.10

Recovered HbR, HbO₂, HbT, and STO2, for ROIs A, B, and C (top, middle, and bottom), recovered by analysis of the multispectral absorption coefficients from Figures 12.7–12.9b (top).

We show in Figure 12.11 the spatio-temporal evolution of both chromophore concentration and scattering changes from the first SD wave in rat 3. These are depicted in the form of a time derivative, i.e., (C(tn + 1) − C(tn))/(tn + 1 − tn), where C represents concentration/saturation/scattering values and tn represents time of acquisition for data point n. This visualization is appealing as it highlights the changes with high contrast [18]. From left to right, we show HbO₂, HbR, HbT, S_tO₂, and μ_s′. Notice the wave in scattering which propagates from top right to bottom left, at a rate of approximately 3 mm/min. An increase, or “spike” in scattering is observed initially in the top right hand corner, in close proximity to the location of KCl administration. Note the large spikes in HbT and S_tO₂ due to vascular activity from depression wave propagation through the measurement field. We observe a transient increase in saturation and blood volume. Over the longer time periods, however, we observe a slow, sustained trend toward hypoxia in the vein regions.

FIGURE 12.11

Spatio-temporal evolution of the hemodynamic and neural scattering response during a single spontaneous CSD event in rat 3. For visualization, a time derivative of the image sequence is displayed to highlight changes.

The spatio-temporal evolution of the scattering coefficient in Figure 12.11 reveals a spatially defined scattering wave (reduction in μs′) that precedes hemodynamic changes. The scattering drop is presumed to be a consequence of neuronal depolarization accompanying CSD. This observed wave pattern has been shown previously with reflectance ISOI and attributed to blood volume changes [18]. Interestingly, the scattering depolarization wave is clearly followed in space and time by the increase in deoxyhemoglobin (HbR), decrease in saturation (S_tO₂), and drop in oxyhemoglobin (HbO₂); changes that are consistent with depolarization-induced neural tissue oxygen consumption.

12.3.3. Dynamic MI Spectroscopy of Stroke

In order to assess the sensitivity of MI to stroke, we conducted preliminary studies in a rat middle cerebral artery occlusion (MCAo) model, the most commonly involved artery in ischemic strokes. The left MCA was surgically cauterized using monopolar cautery or ligated to produce a permanent stroke. Figure 12.12 shows pre-versus post-MCAo results for a representative animal. Data were acquired at 5 wavelengt