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### Review of transcranial photobiomodulation for major depressive disorder: targeting brain metabolism, inflammation, oxidative stress, and neurogenesis

Paolo Cassano; Samuel R. Petrie; Michael R. Hamblin; Theodore A. Henderson; Dan V. Iosifescu; - Neurophotonics, 3(3), 031404 (2016). doi:10.1117/1.NPh.3.3.031404 March 4, 2016 (Publication)
This study shows some of the most detailed parameters (power, wavelenght, dosage) for working with the brain and seems to be unbiased because of the diverse background of authors..
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Abstract
We examined the use of near-infrared and red radiation (photobiomodulation, PBM) for treating major depressive disorder (MDD). While still experimental, preliminary data on the use of PBM for brain disorders are promising. PBM is low-cost with potential for wide dissemination; further research on PBM is sorely needed. We found clinical and preclinical studies via PubMed search (2015), using the following keywords: “near-infrared radiation,” “NIR,” “low-level light therapy,” “low-level laser therapy,” or “LLLT” plus “depression.” We chose clinically focused studies and excluded studies involving near-infrared spectroscopy. In addition, we used PubMed to find articles that examine the link between PBM and relevant biological processes including metabolism, inflammation, oxidative stress, and neurogenesis. Studies suggest the processes aforementioned are potentially effective targets for PBM to treat depression. There is also clinical preliminary evidence suggesting the efficacy of PBM in treating MDD, and comorbid anxiety disorders, suicidal ideation, and traumatic brain injury. Based on the data collected to date, PBM appears to be a promising treatment for depression that is safe and well-tolerated. However, large randomized controlled trials are still needed to establish the safety and effectiveness of this new treatment for MDD.

## Introduction

Infrared (IR) light is ubiquitously present to most life on the earth. Of the total amount of solar energy reaching the human skin, 54% is IR and 30% is IR type A—near-infrared—(NIR; with a wavelength range of 760 to 1440 nm),1 which penetrates through the human skin and reaches deeply into tissue, depending on wavelength and energy.2

NIR is used to treat a variety of conditions such as muscle pain,3 wounds,4 neuropathic pain,5 and headache.6 NIR is also used for wellness and lifestyle purposes such as for cosmetic improvement in peri-orbital wrinkles.7,8 The clinical use of NIR light applied in NIR-spectroscopy dates from the mid-1980s, when it was used for monitoring of the brain in the neonate and the fetus.9

The use of transcranial phototherapy for treating brain disorders started with its application to acute stroke. Numerous preclinical animal studies1011.12 suggested that the application of NIR laser (810 nm) to the head at various times (hours) after induction of an acute stroke had beneficial effects on subsequent neurological performance and reduced lesion size. Evidence was obtained for the anti-inflammatory, anti-apoptotic, and proneurogenesis effects in the brain stimulated by this approach.13,14 These promising animal studies led to the conduction of a series of clinical trials called NeuroThera Effectiveness and Safety Trials (NEST). All together there were three large studies conducted in 1410 stroke patients [NEST-1 (n=120" role="presentation">n=120

), NEST-2 (n=660" role="presentation">n=660), NEST-3 (n=630" role="presentation">n=630

)] that demonstrated that NIR light delivered transcranially with a class-IV laser is safe, with no significant differences in rates of adverse events with NIR, when compared to sham exposure.1516.17 Other preclinical studies and clinical trials have suggested that transcranial photobiomodulation (PBM: laser or light emitting diodes—LED) is safe and effective for acute1822 and chronic2324.25 traumatic brain injury (TBI) and has beneficial effects on neurodegenerative diseases (Alzheimer’s and Parkinson’s).26,27

For the transcranial treatment of major depressive disorder (MDD), both PBM LEDs and lasers have been experimentally tested, although PBM is not FDA-approved for the treatment of MDD. Certain forms of PBM treatment are also referred to as low-level light therapy (LLLT), since it utilizes light at a low power (0.1 to 0.5 W output at the source) to avoid any heating of tissue. The irradiance of the PBM medical devices (or power density) typically ranges from 1 to 10 times the NIR irradiance from sunlight on the skin (33.6  mW/cm2" role="presentation">33.6mW/cm2

at the zenith). However, most PBM medical devices only deliver light energy at one or two selected wavelengths, as opposed to the whole spectrum of IR that is contained in sunlight. To our knowledge and to this date, transcranial PBM treatment has not caused any retinal injury—one of the most likely postulated adverse events, although care is taken routinely in such studies to protect the eyes with goggles or eye covers.28

In this review, we will first discuss the mechanisms of action by which NIR and red light (PBM) might improve symptoms of depression, and then present the clinical evidence for their use as a treatment for MDD and other comorbid psychiatric syndromes.

## Methods

We found clinical and preclinical studies via PubMed search (December 15, 2015), using the following keywords: “near-infrared radiation,” “NIR,” “low-level light therapy,” “low-level laser therapy,” or “LLLT” plus “depression.” We chose studies that had a clinical focus, and we excluded studies involving NIR spectroscopy. We also located studies using the references from the articles found in the PubMed search. As the searched literature encompassed different conditions and disorders frequently comorbid with depression, a specific section of this review was devoted to the effect of PBM on psychiatric comorbidity. In the latter section, the following conditions were included, based on available literature: TBI, anxiety and post-traumatic stress syndromes, insomnia, and suicidal ideation. The literature search for the use of PBM to treat comorbid conditions was neither systematic nor extensive, but rather a secondary focus of this review. The information is presented in an organized fashion to allow the reader to easily grasp the potential applications of PBM for the treatment of depression and of its comorbid conditions. To attain this goal, the authors have allowed a margin of redundancy, by distributing different information derived from any given publication in separate sections of this review. To avoid an artificial inflation of the extant literature on the chosen topic, we referenced the main authors—and when appropriate their affiliation—when referring to the same articles more than once. The reader will find a table summarizing the six key clinical articles reviewed, also to avoid unintended inflation of the literature. The six clinical reports included in this review where extracted from a pool of 58 articles, that were originally identified with the literature search.

In addition, we used PubMed to find articles that examined the link between PBM and each of the various biological processes including metabolism, inflammation, oxidative stress, and neurogenesis.

## Targeting Brain Metabolism

Multiple studies have reported regional and global hypometabolism in MDD, which could be related (either causally or consequentially) to the neurobiology of mood disorders.2932 Positron emission tomography studies have shown abnormalities in glucose consumption rates and in blood flow in several brain regions of subjects with major depression.33 Moreover, metabolic abnormalities in the anterior cingulate, the amygdala-hippocampus complex, the dorsolateral prefrontal cortex (DLPFC), and inferior parietal cortex seem to improve after antidepressant treatment or after recovery.3435.36

With phosphorus magnetic resonance spectroscopy (P31-MRS" role="presentation">31P-MRS

), the baseline pool of nucleotide triphosphate (NTP)—a product of the cellular utilization of glucose and a marker of the cellular energy availability—was low in subjects who subsequently responded to antidepressant treatment.32 Iosifescu et al.32 also demonstrated for the first time with P31-MRS" role="presentation">31P-MRS a correlation between treatment response (to a regimen that combined antidepressants and triiodothyronine) and restoration of a higher NTP pool (with compensatory decrease in phosphocreatine) in the anterior cingulate cortex. This study suggests a pathway to antidepressant response based on restoration of a high cellular energy state. In fact, phosphocreatine represents a long-term storage depot of energy, while NTP and ATP are energy-rich molecules that are readily available to the cell. The same authors replicated the aforementioned findings in MDD subjects treated with standard antidepressants (Iosifescu et al., unpublished). In this cohort, P31-MRS" role="presentation">31P-MRS

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

In experimental and animal models, PBM (NIR and red light) noninvasively delivers energy to the cytochrome c oxidase and by stimulating the mitochondrial respiratory chain leads to increased ATP production (see Fig. 1).3738.39 A study of the effects of NIR on patients with MDD found that a single session of NIR led to a marginally significant increase in regional cerebral blood flow.40 Whether the observed changes in cerebral blood flow resulted from fundamental changes in neuronal metabolism or changes in vascular tone remain to be clarified. Given the correlation of both hypometabolism and abnormal cerebral blood flow with MDD, the beneficial effect of NIR on brain metabolism is one potential mechanism for its antidepressant effect.

## Fig. 1

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

## Targeting Inflammation

Animal and clinical research suggests that the inflammatory arm of the immune system contributes to MDD. Post-mortem gene expression profiling on tissue samples from Brodmann area 10 (BA10—prefrontal cortex) have shown that MDD is characterized by increased inflammation and apoptosis.41 In a case-control study, Simon et al.42 found that antidepressant-naive MDD subjects had significant elevations in the following cytokines and chemokines when compared to healthy controls: MIP-1α" role="presentation">MIP-1α

, IL-1α" role="presentation">IL-1α, IL-1β" role="presentation">IL-1β, IL-6, IL-8, IL-10, Eotaxin, GM-CSF, and IFNγ" role="presentation">IFNγ

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

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

NIR light and red light (600 to 1600 nm) decreased synovial IL-6 gene expression (decreased mRNA levels) in a rat model of rheumatoid arthritis.48 In another study, NIR (810 nm) used as a treatment for pain in patients with rheumatoid arthritis decreased production of the following proinflammatory cytokines: TNF-α" role="presentation">TNF-α

, IL-1β" role="presentation">IL-1β

, and IL-8.49 Khuman et al.50 showed that transcranial NIR improved cognitive function and reduced neuroinflammation as measured by Iba1+ activated microglia in brain sections from mice that had suffered a TBI. Finally, NIR (970 nm) has been found to be an effective treatment for inflammatory-type acne.51 In summary, it is reasonable to predict that transcranial NIR treatment would likewise have an anti-inflammatory effect in patients suffering from MDD.

## Targeting Oxidative Stress

Research has demonstrated a correlation between MDD and vulnerability to oxidative stress.52 For example, depression-induced rats show a significant decrease in glutathione peroxidase (GSH-Px) activity in the cortex.53 Glutathione (GSH) is the most abundant and one of the important antioxidants in the brain; GSH-Px enzymes protect against oxidative stress via reducing hydroperoxides and scavenging free radicals.54 GSH also appears reduced in the brains of MDD subjects.55 Additionally, a study by Sarandol et al.52 demonstrated that MDD patients have higher levels of malondialdehyde, a toxic molecule and a biomarker of oxidative stress.56 Moreover, depressed patients have more red blood cell (RBC) oxidation compared to healthy controls.52 In the same study, the authors found a significant positive correlation between RBC superoxide dismutase (SOD) activity and depression severity. SOD serves to catalyze the removal of the toxic superoxide radical.57 Thus, elevated SOD activity in depressed patients might indicate higher levels of oxidative stress. Finally, catalase activity and nitric oxide (NO) levels have also been shown to be lower in depressed patients than in healthy controls.58 Catalase is an enzyme that protects cells against damaging reactive oxygen species (ROS) via degradation of hydrogen peroxide to water and oxygen.59 NO has protective effects against cell damage, which are likely due to its pleiotropic functions in regulating antioxidant enzymes and many other aspects of cell metabolism.60,61

Oxidative stress may be an effective target for antidepressant treatments. However, successful treatments for MDD vary in regard to their protective effects against oxidative stress.52,53,62 Animal research suggests that PBM may have beneficial effects on oxidative stress. In a rat model of traumatized muscle, NIR (904 nm) blocked the release of harmful ROS and the activation of the transcription factor, nuclear factor κB (NF-κB), both induced by muscle trauma. Trauma activates NF-κB by destroying a specific protein inhibitor of NF-κB called IκB, and this destruction was inhibited by NIR light. Furthermore, NIR reduced the associated overexpression of the inducible form of nitric oxide synthase (iNOS) and reduced the production of collagen.63 This regulation of iNOS is important because excessive levels of iNOS can lead to formation of large amounts of NO that combine with superoxide radicals to form the damaging species peroxynitrite, and can interfere with the protective benefits of other forms of NO synthase.64 These findings suggest that NIR protects against oxidative stress induced by trauma. Finally, an in vitro study of the effects of red light and NIR (700 to 2000 nm) on human RBCs found that NIR significantly protected RBCs against oxidation.65

Original Source: https://www.spiedigitallibrary.org/journals/Neurophotonics/volume-3/issue-03/031404/Review-of-transcranial-photobiomodulation-for-major-depressive-disorder--targeting/10.1117/1.NPh.3.3.031404.full?SSO=1

### Treatments for Traumatic Brain Injury With Emphasis on Transcranial Near-Infrared Laser Phototherapy

Larry D Morries, Paolo Cassano, Theodore A Henderson, - This article was published in Neuropsychiatric Disease and Treatment, 20 August 2015 (Publication)
This exceptional research indicated prefered wavelenghts and dosages for treating patients with traumatic brain injuries. The found some surprising results.
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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

## 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

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

### Shining light on the head: Photobiomodulation for brain disorders

Michael R. Hamblin - 10.1016/j.bbacli.2016.09.002 (Publication)
This is 27 pages of independent analysis of how photobiomodulation effects the brain. Covers wavelengths, dosage, depths and underlying reactions. Amazing.
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Photobiomodulation (PBM) describes the use of red or near-infrared light to stimulate, heal, regenerate, and protect tissue that has either been injured, is degenerating, or else is at risk of dying. One of the organ systems of the human body that is most necessary to life, and whose optimum functioning is most worried about by humankind in general, is the brain. The brain suffers from many different disorders that can be classified into three broad groupings: traumatic events (stroke, traumatic brain injury, and global ischemia), degenerative diseases (dementia, Alzheimer's and Parkinson's), and psychiatric disorders (depression, anxiety, post traumatic stress disorder). There is some evidence that all these seemingly diverse conditions can be beneficially affected by applying light to the head. There is even the possibility that PBM could be used for cognitive enhancement in normal healthy people. In this transcranial PBM (tPBM) application, near-infrared (NIR) light is often applied to the forehead because of the better penetration (no hair, longer wavelength). Some workers have used lasers, but recently the introduction of inexpensive light emitting diode (LED) arrays has allowed the development of light emitting helmets or “brain caps”. This review will cover the mechanisms of action of photobiomodulation to the brain, and summarize some of the key pre-clinical studies and clinical trials that have been undertaken for diverse brain disorders.

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

## 1. Introduction

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

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

## 2. Mechanisms of action of photobiomodulation

### 2.1. Mitochondria and cytochrome c oxidase

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

### 2.2. Reactive oxygen species, nitric oxide, blood flow

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

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

### 2.3. Light sensitive ion channels and calcium

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

### 2.4. Signaling mediators and activation of transcription factors

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

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

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

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

### 2.5. Biphasic dose response and effect of coherence

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

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

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

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

### 3.1. Light penetration into the brain

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

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

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

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

### 3.2. Systemic effects

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

### 3.3. Laser acupuncture

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

### 3.4. Light sources

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

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

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

## 4. PBM for stroke

### 4.1. Animal models

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

Reports of transcranial LLLT used for stroke in animal models.

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

### 4.3. Chronic stroke

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

## 5. PBM for traumatic brain injury (TBI)

### 5.1. Mouse and rat models

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

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

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

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

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

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

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

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

### 5.2. TBI in humans

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

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

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

## 6. PBM for Alzheimer's disease (AD)

### 6.1. Animal models

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

### 6.2. Humans

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

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

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

## 7. Parkinson's disease

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

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

Original Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5066074/

### Photobiomodulation of the Brain

Michael R. Hamblin and Yng-Ying Huang - 2019 (Publication)
This is Hamblin and Huang's best summary of PBM for treating the brain.
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Photobiomodulation (PBM) also known as low-level laser (or light) therapy has been known for over 50 years (since 1967), but it is only relatively recently that it has begun to make the transition into the mainstream. PBM describes the use of red or near-infrared light at levels that do not produce undue heating of the tissue to produce beneficial effects on the human body. The introduction of light-emitting diodes (LEDs) has made this approach more accessible than the previously used laser sources, as LEDs are safer, cheaper, and can easily be used at home. Another factor that has led to PBM becoming more widely accepted is the growing understanding of the mechanisms of action at a molecular and cellular level. The lack of a clear mechanism of action was a deterrent to many biomedical scientists who maintained a healthy level of skepticism. Among the wide range of tissues, organs, diseases, and conditions that can be beneficially affected by PBM, the subject of this book is the brain. The brain is probably the single human organ that engenders the most concern, interest, and expenditure in the 21st century. Brain disorders that cause widespread morbidity, mortality, and loss of quality of life can be divided into four broad categories. Traumatic brain disorders include stroke, traumatic brain injury (TBI), global ischemia, and perinatal difficulties. Neurodegenerative diseases include Alzheimer’s disease, Parkinson’s disease, and a range of dementias. Psychiatric disorders include major depression, anxiety, addiction, and insomnia, among many others. Finally there are neurodevelopmental disorders (autism and ADHD) and the possibility of cognitive enhancement in healthy individuals. Many of these brain disorders are specifically addressed in the present volume. The book is divided into three parts. The first part covers some basic considerations, dosimetry, and devices, and discusses the mechanisms of action at a cellular level and on the brain as a whole organ. The second part includes contributions from researchers who have carried out studies on a variety of animal models in their investigations of brain disorders, stroke, TBI, and Alzheimer’s and Parkinson’s diseases, to name a few. The third part concentrates on human studies, including controlled clinical trials, pilot trials, case series, and clinical experience. Disorders treated include TBI, stroke, Alzheimer’s and Parkinson’s diseases, depression, and others. The book is expected to play a role in stimulating the further increase and acceptance of PBM for brain disorders, which has really started to take off in recent years. It will also act as a resource for researchers and physicians wishing to get a broad overview of the field and who are contemplating entering it themselves. The number of individuals considering obtaining a home-use PBM device is also steadily increasing and this book will act as

### “Quantum Leap” in Photobiomodulation Therapy Ushers in a New Generation of Light-Based Treatments for Cancer and Other Complex Diseases: Perspective and Mini-Review

Luis Santana-Blank, MD, Elizabeth Rodríguez-Santana, MD, Karin E. Santana-Rodríguez, BS, and Heberto Reyes, MD - Photomedicine and Laser Surgery (Publication)
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## AbstractObjective: Set within the context of the 2015 International Year of Light and Light-Based Technologies,and of a growing and aging world population with ever-rising healthcare needs, this perspective and mini-review focuses on photobiomodulation (PBM) therapy as an emerging, cost-effective, treatment option for cancer (i.e., solid tumors) and other complex diseases, particularly, of the eye (e.g., age-related macular degeneration, diabetic retinopathy, glaucoma, retinitis pigmentosa) and the central nervous system (e.g., Alzheimer's and Parkinson's disease). Background data: Over the last decades, primary and secondary mechanisms of PBM have been revealed. These include oxygen-dependent and oxygen-independent structural and functional action pathways. Signal and target characteristics determine biological outcome, which is optimal (or even positive) only within a given set of parameters. Methods: This study was a perspective and nonsystematic literature mini-review. Results: Studies support what we describe as a paradigm shift or “quantum leap” in the understanding and use of light and its interaction with water and other relevant photo-cceptors to restore physiologic function. Conclusions: Based on existing evidence, it is argued that PBM therapy can raise the standard of care and improve the quality of life of patients for a fraction of the cost of many current approaches. PBM therapy can, therefore,benefit large, vulnerable population groups, including the elderly and the poor, whilehaving a major impact on medical practice and public finances.Go to:IntroductionThe United Nations declared 2015 to be the International Year of Light and Light-Based Technologies (IYL 2015) in recognition of the vital role of light-based systems in our daily lives, and their growing importance to meeting the world's challenges in areas as diverse as energy, education, telecommunication, agriculture, and health.1 Although our perception of light is often limited to the visible band of the electromagnetic (EM) spectrum,2 both lower and shorter wavelengths are increasingly used in new medical technologies3 including soft, injectable, and bioresorbable electronics.4 Described as an imperative cross-cutting discipline of in the twenty-first century, light science has already revolutionized the physical sciences and industry. The control of light at the nanoscale has unveiled a plethora of phenomena, leading to powerful new applications and setting high expectations for years to come.5 In particular, light's ability to control materials and transport coded signals forms the bases for many new photonic devices and systems, wherein photons act as tailor-made EM energy packets that can perform various functions.Here, we describe a paradigm shift or “quantum leap” in the understanding and use of light and its interaction with water and other relevant photoacceptors to control biologic function in medicine through photobiomodulation (PBM) therapy. We propose that progress will lead to the imminent inception of PBM therapy as a mainstream treatment for multiple complex diseases, including solid tumors, as well as neurodegenerative diseases (NDs) of the eye and central nervous system (CNS)6–10 (Fig. 1). PBM therapy can raise the standard of care and improve the quality of life of patients at a fraction of the cost of many current approaches. Thus, a “quantum leap” in PBM therapy will benefit large and vulnerable population groups, including the elderly and the poor, while having a major impact on medical practice and public finances.11 This is particularly important because the high price of drug therapies, which can reach hundreds of thousands of dollars per year,12 as well as a growing and aging world population, are putting a severe strain on family and public finances around the world.13

Flow chart illustrating fields of light-based technologies, highlighting photobiomodulation (PBM) therapy applied to complex diseases as a quantum leap in medical therapeutics.

Origin, Trajectory and Myriad Relationships in PBM's “Quantum Leap” in Medicine

Concurrent with progress in PBM therapy, a long history of discoveries has put medicine at the brink of a revolution in the use of light–water interactions for the treatment of complex diseases.7,8,10,14 Long ago, Albert Szent-Gyorgyi postulated that water was at the core of energy transfer in biological systems (i.e., quantum biology), and that that explained how energy from biomolecules could be translated into free energy for cells.15–17 Ling further elaborated on the physical state of water in living cells,18 and proposed on theoretical grounds that ordered layers of water could extend infinitely under ideal conditions.19,20Later, Huber proposed a structural basis of light energy and electron transfer in biology.21 More recently, Zewail and others showed that, with rapid laser techniques, it is possible to “see” how atoms in a molecule move during a chemical reaction.22 Light science has now reached microscales at the limit of recordable physical observation (e.g., resonant intermolecular transfer of vibrational energy in water at −100 fs)23,24showing, for example, the memory of persistent correlations in water structures within 50 fs, which is important in stabilizing biological systems.25 These and other tremendous achievements have changed our view of water, from a merely passive medium to an integral active player in the physiology of life, and have opened the gates to both direct measurement and control of physiological processes via light–water interaction.

State of the Art in PBM

In 2016, PBM therapy will be added to the MeSH database as an entry term for records spanning five decades of research.26 As argued by Anders et al., this is a key step, as it distinguishes PBM therapy from light-based devices used for heating of tissues, such as near infrared (NIR) lamps or other applications that rely on thermal effects for all or part of their mechanisms of action.26 In contrast, PBM therapy employs low-level monochromatic or quasimonochromatic light, currently from visible blue (400 nm) to far-infrared (FIR 3200 nm), to induce nonthermal (≤0.01°C) photochemical and photophysical effects. Nonlinear processes through which PBM therapy can stimulate or inhibit; that is, modulate, physiological activity depend upon signal-to-noise rate and target cell/tissue parameters.27–29 Thus, signal and target characteristics determine biological outcome, which is optimal (or even positive) only within a narrow set of parameters.13

Over the last decades, primary and secondary mechanisms of PBM at the tissue, cellular, and molecular levels have been revealed. These include two major structural and functional action pathways. The first, or classic, action pathway relates to oxygen-dependent mechanisms operated by oxidation-reduction enzymes of the respiratory chain, particularly cytochrome c oxidase (CcO), which is partly responsible for light energy absorption and transfer to cells and tissues.30 This pathway is associated to cofactors, pigments, metals, and proteins that act as key redox centers within the body's bioenergetic rack mechanism described by Huber.21 Nitric oxide (NO), as a first-level player, also has an activation and modulation role in the oxygen-dependent pathway.31–33

The second, or oxygen-independent, action pathway centers on the vital role of water not only as the prevalent medium of life but as an active molecule, capable of absorbing radiant energy (e.g., IR light) and transporting/transducing it along extended biological surfaces, from bulk water to confined water in nanoscopic tissue and cell spaces. Light–water dynamics precede/coexist with the classic oxygen-dependent action pathway and complement and facilitate energy transfer for increased adenosine triphosphate (ATP) production.29,34,35 As a point of comparison, correlated internal electron- and proton-transfer reactions have been tracked in real time into the oxidized enzyme (CcO), revealing an overall real time of 3.46 ms.36 This relay is slower by several orders of magnitude than total energy transport through water dynamics from bulk liquid water to confined spaces.34

Oxygen-independent light–water interactions may further power and modulate molecular signaling pathways and gene transcription factors via multiple nonmetabolic pathways.10,35 For examle, the energy of the drive force wave of an infrared pulsed laser device (IPLD) used in our group's previous studies (NIR 0.27 eV) is within the range of the strength of hydrogen bonds,29,37 and the IPLD carrier wave oscillates at a frequency (3x 10e6 Hz) that enters in vibrational resonance with the rate of electron transfer through the DNA double helix.29,37 Theoretical evidence suggests that these wave properties promote the activation of open state dynamics,38,39 allowing the activation of complex chaotic dynamics as well as the regulation of DNA replication and transcription, because the existence of open states in one place of the chain can influence the dynamics of other distant open states.29,34,35 Resulting effects match reported reductions in the frequency of chromosome aberrations induced by that low-energy laser irradiation,40 as well as theoretical,38,39 experimental,27,28 and clinical studies.41–48 These and other oxygen-independent PBM effects are channeled through metabolic control levels to regulate the energy-dependent path from the genotype to the phenotype.49,50

Light–Water Interactions and the Quantum Leap in PBM

We propose that the key to understanding and controlling the biophysics and biochemistry of higher-order organisms stems from their dual aqueous and energy-dependent nature. Water represents 70% by mass of an adult human body, or nearly 99% of total molecules by number, given water's low molecular weight. In addition, high-order organisms, including humans, can be represented as complex electrochemical (semiconducting) systems that comprise a vast array of energy-sensitive materials and machinery, such as ion pumps (e.g., chemically driven electron pumping through molecular wires, such as the D pathway in CcO),34 molecular motors (e.g., ATP synthase and Brownian biomotors), transistors-capacitors (e.g., cell membrane), liquid crystals (e.g., membrane structure), and rechargeable electrolytic biological batteries (e.g., hydrophilic interface in cells/tissues). Life system's double nature, whose two main structural and functional pillars are energy and water joined to biomolecules, has, therefore, tremendous consequences for life and health.

Water's permittivity, calculated considering the system as a plane capacitor, is generally high. Therefore, radiant energy can penetrate and be absorbed by tissues to provide powerful tools in medicine.51 One example is the exclusion zone (EZ) described by Pollack.52 High-energy EZ water forms along hydrophilic surfaces (e.g., tissue interfaces) in response to radiant energy.53 Remarkably, EZ water can separate and store electrical charges, and can release up to 70% of such charges when it is perturbed, such as by injury-induced redox potentials.54 We have argued that supplied energy can power and modulate cellular work and signaling pathways, even when the metabolic energy pathway has been compromised, steering cells toward or away from programmed cell death.34 EZ water may, thus, act as an electrolytic bio-battery,35 which can efficiently and selectively transfer energy to sites expressing redox injury potentials, as found in cancer and other complex diseases, triggering reparative and regenerative mechanisms that can lead to restoring homeostasis/homeokinesis and, ultimately, health.29,34,35

Experimentally, IR energy absorption by water has been recently modeled in a porcine model, confirming that absorption depends upon fluence and wavelength. Further, the higher the concentration of water in tissues, the higher IR energy absorption will be.55 This is consistent with controlled clinical studies in solid tumors and complex ophthalmic and neurologic diseases,9,46,56 as well as molecular, biochemical, biophysical, and metabolic mechanistic support for a quantum leap in medical therapeutics based on the simple, but powerful, idea that properly tailored light can power and modulate physiologically reparative mechanisms.30,57–62

Cancer and Tumor Microenvironments

The bases of our understanding of cancer are constantly being questioned and revised, leading to new treatment goals. In a paradigm-changing editorial, Prendergast recently argued that “disorders in microenvironment and peripheral systems that control cancer might increasingly be viewed as primary rather than secondary factors in the root nature of cancer as a clinical disease.” This constitutes “a crucial and radical distinction from prevailing thought, since it implies that cancer may be a symptom of an underlying clinical disorder, rather than the root problem itself that needs to be addressed.” 6,63

Prendergast further suggested that “effective treatment of cancer may not necessarily entail understanding or addressing this complexity, but mastering the use of tissue or systemic systems that have the inherent ability to do so.” Hence, a common thread linking emerging perspectives in oncology and PBM therapy may well be the restitution of tissue homeostasis-homeokinesis via light-energy supplementation, a microenvironment effect that comprises and extends the Warburg effect previously discussed by our group.57,64–67

Photobiomodulation and Cancer

As far back as 1964–1966, McGuff et al. showed 64,65 that “laser energy has a selective effect on certain malignant tumors, resulting in their progressive regression and ultimate dissolution.” Following years of controversy,66,67 editorials by Karu68 and Lanzafame11,69 now stress evidence supporting the potential anticancer effects of PBM.11,68,69 New data confirm that PBM under certain parameters is safe for use in cancer patients.60 This is in accord with clinical results from our group using the abovementioned proof of concept IPLD.4,44

A phase I trial in patients with advanced neoplasias demonstrated that the IPLD studied was safe for clinical use and improved performance status and quality of life.41 Antitumor activity was observed in 88.23% of patients with 10 years of follow-up.41

In that series, T2-weighted MRI data showed increased water content of tumor heterogeneities42,44 preceding tumor-volume reduction and a therapeutic anticancer effect.42,44 Structural, kinetic, and thermodynamic implications of these changes in water dynamics have been analyzed at the tissue, cell, and interstitial levels.27 In conjunction, selective activation of programmed cellular death [i.e., apoptosis, necrosis, and anoikis (cell death by loss of cell adhesion)] and cytomorphologic modification (e.g., reduced size, increased roundness, increased vacuoles) were documented in neoplastic cells, but not in peripheral tissues.8,42 Modulation of cluster of differentiation (CD)4 CD45RA+, CD25 activated, tumor necrosis factor alpha (TNF-α), and soluble interleukin (IL)-2 receptor (sIL-2R) was further documented.43These hallmark results, supported by independent data,70–72 demonstrate that PBM therapy can modulate antitumor effects,6,8 in sharp contrast with long-held views.45,73,74 This evidence is also consistent with growing experimental and clinical reports from multiple other authors.60,75–82

PBM and Ophthalmic and Neurodegenerative Disorders

Recent evidence underscores common mechanisms between cancer and NDs of the eye and CNS. Research suggests that oxidative proteome damage may be the most likely cause of aging and age-related maladies such as cancer and other complex diseases, including NDs.83 Findings also show “common mechanisms of onset,” with a focus on genes such as DJ-1 and Myc-Modulator 1 (MM-1) and signaling pathways that contribute to the onset and pathogenesis of cancer and NDs such as retinitis pigmentosa (RP), Parkinson disease (PD), and cerebellar atrophy.”84 Finally, both disease groups are profoundly energetic in nature, featuring prominent deterioration of metabolic energy pathways.10

External light energy supplementation has been shown to generate neuroprotective, vasoprotective, baroprotective, immunomodulatory, and regenerative effects (Fig. 2). 47 We have documented that such effects may be activated and modulated locally and/or remotely via oxygen-dependent and oxygen-independent pathways that can encompass extended biologic surfaces and may even reach avascular eye tissues (i.e., cornea, lens, aqueous humor, and vitreous) noninvasively. Although a full elucidation of involved mechanisms escapes the scope of this perspective and mini-review, a very brief discussion of results from multiple authors is given subsequently.

Electromagnetic (light) energy supplementation based on water–light interactions. Upper left side shows classic oxygen (O2) dependent pathways by which light energy generates adenosine triphosphate (ATP)/ guanosine-5′-triphosphate (GTP) and other high-energy molecules. Upper right side shows O2 independent pathways by which photoinduced, nonlinear, oscillations in water provide energy for cellular work, signaling, and gene transcription. Top center shows interfacial exclusion zone (EZ) water, which acts as a selective rechargeable electrolytic bio-battery. Together, these pathways activate and modulate physiologically reparative mechanisms which, at appropriate irradiation parameters, can generate neuroprotective, vasoprotective, baroprotective, immunomodulator, and regenerative effects locally and remotely, promoting homeostasis/homeokinesis through the coupling and synchronization of biophysical, biochemical, biomechanical, and hydrodynamic oscillators, as guided by the second law of thermodynamics. Arrows point to the sequence and direction of events. (Updated from reference 47. Authors retained copyright.)

PBM has shown promise in the treatment of diabetic retinopathy (DR),85,86 age-related macular degeneration (AMD),46 glaucoma,47 RP,87 Stargardt disease,88 Leber's hereditary optic neuropathy,89 Alzheimer's disease (AD), and PD, 90,91 among other conditions.89 Strikingly, although each of these NDs has different etiologies and pathogeneses, “they frequently induce a set of cell signals that lead to well-established and similar morphological and functional changes, including programmed cell death. Furthermore, oxidative stress, activation of apoptotic pathways and inflammatory response, are common features in all these diseases.”92

Remarkably, PBM can modulate apoptosis as well as necrosis.42,45,47 PBM can also be both pro-oxidant in the short term, but antioxidant in the long term,93 thus modulating reactive oxygen species (ROS) generation. We also found clinical evidence of immune regulatory effects over inflammation during treatment of solid tumors with the IPLD, a NIR diode laser pulsed at a frequency of 3 MHz.43 These results are in agreement with the regulating role of the vagal reflex on the inflammatory reflex reported by Tracey, using an electronic device that stimulated nerves to treat inflammation.71,72

In addition, PBM has been shown to protect against retinal dysfunction and photoreceptor cell death in rodent models of retinal injury and retinal degeneration.94 PBM has been further reported to attenuate oxidative stress and inflammation in primary astrocytes induced by amyloid β peptide (Aβ),95 and to reduce Aβ-induced apoptosis,96 which is thought to play a major role in AD. Nevertheless, it has been argued that red to NIR light cannot be transmitted through the scalp to the brain more than a few centimeters,97 which makes it nearly impossible to noninvasively treat AD with PBM 98 using conventional (direct) delivery systems/methods. Similarly, although an absence of adverse effects from 670 and 830 nm PBM applied to the retina in Sprague Dawley albino rats has been reported,94 extreme care must be taken to avoid photodamage of the eye99 from direct PBM procedures.

Conversely, we published an interventional case report of a patient with bilateral geographic atrophic AMD (gaAMD) and associated neurologic disease treated noninvasively, indirectly, and at a distance (i.e., remotely) from ocular structures and the CNS with the above-referenced IPLD/photo-infrared pulsed bio-modulation (PIPBM).46 Results showed neurologic improvement, transitory color vision, enhanced visual acuity, full-field electroretinogram (ERG) modifications toward a normal rhythm, drusen mobilization, decreased lens opacity, and lower intraocular pressure (IOP), in accord with a retrospective noncomparative data analysis from the phase I trial of patients with advanced cancer treated with the IPLD,41 which showed statistically significant evidence of a therapeutic hypotensor effect over IOP,47 and they are consistent with the positive neurological evolution of two trial patients.

Moreover, although trial participants did not develop media opacity, one pre-existing incipient cataract in the right eye of a patient (transitional meningioma) became denser and slightly smaller 3 months post-treatment, and remained unchanged 1 year post-treatment. The left eye lens of the same patient was unaffected. Although the finding could be part of the natural history of the cataract, we stressed that possible deterministic effects related to the initial metabolic or biochemical state of lens opacities should be studied.41

In accordance with the what was described, a robust body of evidence suggests that protein misfolding, insolubility, and aggregation are at the root of both cataracts and other diseases including AD, PD, and Huntington's disease,100 and that external EM energy (light) supplementation can have reparative effect on protein misfolding, activating and modulating metabolic control levels of protein folding/unfolding.10,34In addition, PBM effects on targets such as heat shock proteins (α crystalline), enzymes of the antioxidative system, Na+-K+-ATPase, Ca +2-ATPase, aquaporins (AQPs), and ion pumps have been referred to as part of mechanisms that could have influenced the response observed in the lens on the cases studied.46 We further proposed that, among other effects, PBM can stimulate and/or substitute ATP production via water dynamics, which is vital for the activation and inactivation kinetics in phototransduction.46 PBM can also affect the synthesis of molecules in a liquid crystalline (LC) state (e.g., self-assembly of lipids, water, and other biomolecules such as proteins and sterols, which are sensitive to temperature and/or electric fields) If confirmed, the latter may have multidisciplinary applications in medicine and biology in areas such as photovision, in which LCs are essential functional components.28

A first rapid communication referring to the retina and optic nerve additionally showed first evidence of EZ water as a selective rechargeable bio-battery applicable to PBM, suggesting a new understanding of the eye's energetic environment, which may have deep implications in ocular physiology as well as in the pathophysiology, diagnosis, and treatment of blinding diseases using light-based therapies.48 Therefore, as a promising alternative to drug therapies,101 or in combination with other treatments, PBM therapy may be developed into a viable therapeutic approach with multidisciplinary applications in ophthalmology and neuroscience,46 inducing and modulating physiologically reparative and regenerative effects that can favor homeostasis/homeokinesis27–29 through the coupling and synchronization of biophysical, biochemical, biomechanical, and hydrodynamic oscillators, as guided by thermodynamics.

Treatment Costs and Availability

At the 2015 American Society of Clinical Oncology (ASCO) annual meeting, Dr. Leonard Saltz, chief of gastrointestinal oncology at Memorial Sloan Kettering Cancer Center, discussed the high cost of cancer drugs. He argued that “the unsustainably high prices of cancer drugs is a big problem, and it's our problem,” citing as examples the cost of nivolumab ($28.78/mg) and ipilimumab ($157.46/mg), which is “approximately 4000 times the cost of gold.”102 Previously, >100 oncologists had protested the high price of cancer drugs, also calling them economically “unsustainable.” They noted that, of 12 cancer drugs approved in 2012, 11 were priced > $100,000 per year,103 with multiple drugs often being required for extended periods. Such high prices and their impact on families, governments, and society at large are leading some to propose that cost should be considered a “financial toxicity” to be assessed with other toxicities when treatments are considered by doctors and patients.104 In contrast, although it has been estimated the cost of developing new drug therapies can run up to USD$1.3–\$1.7 billion,105,106 the development cost of new photonics devices can be substantially lower, which can lower therapy costs and increase treatment availability. For the same reasons, PBM can also offer a noninvasive and cost-effective therapeutic option for patients with NDs of the retina, brain, and beyond.5685

Conclusions

The celebration of the IYL 2015 by the United Nations1 is a fitting time to announce what we describe here as a “quantum leap” in PBM therapy. It is also a good opportunity to ensure that policy makers and the medical community become aware of and embrace the immense potential of light-based medical technologies, especially PBM therapy, as an emerging treatment option for cancer and other complex diseases.107 Although not all tissues respond to PBM therapy,69 in vitro and in vivo xenografts and evidence from clinical studies does suggest that it is time to begin considering PBM therapy as a potential drug equivalent.11,108 In addition, PBM therapy may have minimal or no adverse effects, improve quality of life and functional status and raise the current standard of care for many cancer patients when used alone or in combination with other therapies.9 PBM therapy further represents a novel hope for the treatment of numerous eye and neurologic diseases. And as stated, PBM may be developed at a lower cost than many current treatments,8,10 which can help meet the healthcare needs of an increasing and aging world population. As such, this perspective and mini-review focuses on the large potential tangible contributions of light-based therapies for large demographic segments of the population, such as aging “baby boomers” who are expected to face a higher incidence of diseases such as cancer, AMD, DR, glaucoma, RP, AD, and PD, as well as other neurologic diseases in the next 15 years. In light of the growing costs of drugs and their impact on developed and developing countries, we propose that PBM therapy may offer a novel, safe, and effective therapy choice that would be more accessible to large vulnerable groups, such as the poor and the elderly.

Concurrently with the United Nations' declaration of 2015 as the year of light and light-based technologies, PBM therapy stands at the brink of delivering a new generation of treatments for complex diseases. New PBM therapies will preserve quality of life and raise standard of care in an efficient and cost-efficient manner. This will particularly benefit the most vulnerable demographic sectors, such as the elderly and the poor, and reduce the strain of growing healthcare costs in both industrialized and developing countries. We propose that such developments and their imminent impact represent a paradigm shift or “quantum leap” in PBM therapy and medicine at large.

Acknowledgments

We thank Jesús Alberto Santana-Rodríguez for reviewing and editing this article, and Luis Rafael Santana-Rodríguez for design and technical support. This study was supported by Fundalas, Foundation for Interdisciplinary Research and Development.

Author Disclosure Statement

No competing financial interests exist.

References

Original Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4782038/

### Laser Phototherapy Clinical Practice and Scientific Background

Lars Hode and Jan Tunér - 2014 (Book)
This book is one of the most comprehensive resources for European style laser therapy.
View Resource

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
• wavelength and frequency
• photon energy
• the elecromagnetic spectrum
• the optical reigon
• can electromagnetic radiation cause cancer
• protective mechanisms
• light
• the optical spectrum
• 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
• combo treatment
• interaction with medication
• other considerations
• 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?
• 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
• 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
• diabetes
• tatoos
• light sensitivity
• Coherence
• the role of coherence in laser phototherapy
• itroduction
• summary
• Dose and Intensity
• 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
• 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
• 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)
• 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

Original Source: http://www.coldlasers.org/lllt-books/

### Performance Chiropractic and Wellness: The Complete A-Z Manual for Low Level Laser Therapy 5th edition

Jerome Rerucha B.S., C.S.C.S., D.C. - 2015 (Book)
Dr Rerucha is on the cutting edge at documenting how different pulsing frequencies can be used for different stilulatory effects. He works mainly with Erchonia.
View Resource

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
• 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
• bleeding gums
• bloating
• blood pressure (high)
• blood pressure (low)
• blood sugar balance
• boils
• bone
• bowel
• 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
• ganglion cyst
• general musculoskeletal
• gerd
• gingivitis
• glaucoma
• goiter
• gout
• gums
• 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
• lymphatic
• macular degeneration
• memory problems
• meniere's disease
• 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-op
• prostate
• psoriasis
• punctures
• rash
• reflex sympathetic dystrophy (RSD)
• renal problems
• respiratory problems
• restless leg syndrome
• retinitis pigmentosa
• rheumatism
• ringworm
• 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)
• 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

Original Source: http://www.coldlasers.org/lllt-books/

### Low-level laser therapy ameliorates disesase progression in a mouse model of multiple sclerosis.

Elaine D. Goncalves, Priscila S. Souiza, Vicente Lieberknecht, Giulia S. P. Fidelis, Rafael I. Barbosa, Paulo C. L. Silveria, Ricardo A. de Pinho, Rafael C. Dutra - Taylor & Francis Online 12/2015 (Publication)
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Multiple sclerosis (MS) is an autoimmune demyelinating inflammatory disease characterized by recurrent episodes of T cell-mediated immune attack on central nervous system (CNS) myelin, leading to axon damage and progressive disability. The existing therapies for MS are only partially effective and are associated with undesirable side effects. Low-level laser therapy (LLLT) has been clinically used to treat inflammation, and to induce tissue healing and repair processes. However, there are no reports about the effects and mechanisms of LLLT in experimental autoimmune encephalomyelitis (EAE), an established model of MS. Here, we report the effects and underlying mechanisms of action of LLLT (AlGaInP, 660 nm and GaAs, 904 nm) irradiated on the spinal cord during EAE development. EAE was induced in female C57BL/6 mice by immunization with MOG35–55 peptide emulsified in complete Freund’s adjuvant. Our results showed that LLLT consistently reduced the clinical score of EAE and delayed the disease onset, and also prevented weight loss induced by immunization. Furthermore, these beneficial effects of LLLT seem to be associated with the down-regulation of NO levels in the CNS, although the treatment with LLLT failed to inhibit lipid peroxidation and restore antioxidant defense during EAE. Finally, histological analysis showed that LLLT blocked neuroinflammation through a reduction of inflammatory cells in the CNS, especially lymphocytes, as well as preventing demyelination in the spinal cord after EAE induction. Together, our results suggest the use of LLLT as a therapeutic application during autoimmune neuroinflammatory responses, such as MS.

## Introduction

Multiple sclerosis (MS) is an inflammatory chronic autoimmune and neurodegenerative disorder of the human central nervous system (CNS), in which encephalitogenic Th1 and Th17 lymphocytes induce a response against components of myelin [1–3 Sospedra, M., and R. Martin. 2005. Immunology of multiple sclerosis. Ann. Rev. Immunol. 23: 683747
Steinman, L. 2007. A brief history of T(H)17, the first major revision in the T(H)1/T(H)2 hypothesis of T cell-mediated tissue damage. Nat. Med. 13: 139145
Goverman, J. 2009. Autoimmune T cell responses in the central nervous system. Nat. Rev. Immunol. 9: 393407
]. Inflammation, gliosis and axonal injury are additional prominent neuropathological characteristics, as is the clinical evolution from intermittent attacks to slow, steady progressive worsening [4 Ransohoff, R. M., D. A. Hafler, and C. F. Lucchinetti. 2015. Multiple sclerosis – a quiet revolution. Nat. Rev. Neurol. 11: 134142]. Moreover, some evidence points to an important role for nitric oxide (NO) in the pathogenesis of MS and to its contribution to the various facets of the disorder, including inflammation, oligodendrocytes injury, changes in synaptic transmission, axonal degeneration and neuronal death [5 Smith, K. J., and H. Lassmann. 2002. The role of nitric oxide in multiple sclerosis. Lancet Neurol. 1: 232241].

Patients with MS typically present between the ages of 20 and 40 years, with affected women outnumbering men 2:1, and the progressive phase of disease manifests at any time between 5 and 35 years after onset [4 Ransohoff, R. M., D. A. Hafler, and C. F. Lucchinetti. 2015. Multiple sclerosis – a quiet revolution. Nat. Rev. Neurol. 11: 134142]. MS causes a multitude of symptoms, including visual disturbances, spasticity, weakness, impairment of walking, coordination difficulties, tremor/ataxia, sensory problems and bladder disturbances [1 Sospedra, M., and R. Martin. 2005. Immunology of multiple sclerosis. Ann. Rev. Immunol. 23: 683747,6 McFarland, H. F., and R. Martin. 2007. Multiple sclerosis: a complicated picture of autoimmunity. Nat. Immunol. 8: 913919]. Moreover, “invisible” symptoms, such as fatigue, neuropathic pain and cognitive deficits, are also common [7–9 Shi, J., C. B. Zhao, T. L. Vollmer, et al. 2008. APOE epsilon 4 allele is associated with cognitive impairment in patients with multiple sclerosis. Neurology 70: 185190
Rao, S. M., G. J. Leo, L. Bernardin, and F. Unverzagt. 1991. Cognitive dysfunction in multiple sclerosis. I. Frequency, patterns, and prediction. Neurology 41: 685691
Rao, S. M. 1995. Neuropsychology of multiple sclerosis. Curr. Opin. Neurol. 8: 216220
] and are detectable even before a definitive diagnosis of MS is made. These sensorial, cognitive and emotional symptoms related to MS strongly impact on family, social and work activities, as well as their quality of life [10 Engel, C., B. Greim, and U. K. Zettl. 2007. Diagnostics of cognitive dysfunctions in multiple sclerosis. J. Neurol. 254: II30II34]. MS manifests in several forms, like as: (i) clinically isolated syndrome (CIS) is the first manifestation of MS-like signs and symptoms, usually followed by another attack at which a clinical diagnosis of MS is made; (ii) relapsing remitting MS (RRMS), which is characterized by sudden relapses punctuated by short- or long-term remissions; (iii) secondary progressive MS (SPMS), which has a progressive course resulting in severe, irreversible debilitation and (iv) primary progressive MS (PPMS), which is a progressive type of MS without an initial relapsing and remitting period [11 Confavreux, C., and S. Vukusic. 2006. Natural history of multiple sclerosis: a unifying concept. Brain J. Neurol. 129: 606616]. The economic cost of MS associated with relapses and subsequent disability is considerable. For instance, a multicenter study initially carried out in five European countries examined the costs associated with MS, and the annual cost for those with expanded disability status scale (EDSS) ≤3 ranged from E 13.534 to E 22.561 increasing to E 28.524–E 43.984 for EDSS 4–6.5 and E 39.592–E 65.395 for EDSS ≥7 [12 Karampampa, K., A. Gustavsson, C. Miltenburger, and B. Eckert. 2012. Treatment experience, burden and unmet needs (TRIBUNE) in MS study: results from five European countries. Mult. Scler. 18: 715], and loss of earnings was the biggest contributor to indirect costs [13 O'Connell, K., S. B. Kelly, E. Fogarty, et al. 2014. Economic costs associated with an MS relapse. Mult. Scler. Relat. Disord. 3: 678683]. In this context, phototherapy, especially laser, has been widely used in research of different tissues, such as tendons, nerves, skin tissue, bones, muscles and CNS [14–18 Baroni, B. M., R. Rodrigues, B. B. Freire, et al. 2015. Effect of low-level laser therapy on muscle adaptation to knee extensor eccentric training. Eur. J. Appl. Physiol. 115: 639647
Barbosa, R. I., A. M. Marcolino, R. R. de Jesus Guirro, et al. 2010. Comparative effects of wavelengths of low-power laser in regeneration of sciatic nerve in rats following crushing lesion. Lasers Med. Sci. 25: 423430
Batista, J. D., S. Sargenti-Neto, P. Dechichi, et al. 2015. Low-level laser therapy on bone repair: is there any effect outside the irradiated field? Lasers Med. Sci. 30: 15691574
Allahverdi, A., D. Sharifi, M. A. Takhtfooladi, et al. 2015. Evaluation of low-level laser therapy, platelet-rich plasma, and their combination on the healing of Achilles tendon in rabbits. Lasers Med. Sci. 30: 13051313
Hartzell, T. L., R. Rubinstein, and M. Herman. 2012. Therapeutic modalities – an updated review for the hand surgeon. J. Hand Surg. 37: 597621
].

Low-level laser therapy (LLLT) has been considered as an adjuvant clinical treatment [19 Carrasco, T. G., M. O. Mazzetto, R. G. Mazzetto, and W. MestrinerJr. 2008. Low intensity laser therapy in temporomandibular disorder: a phase II double-blind study. Cranio 26: 274281,20 Gavish, L., L. S. Perez, P. Reissman, and S. D. Gertz. 2008. Irradiation with 780 nm diode laser attenuates inflammatory cytokines but upregulates nitric oxide in lipopolysaccharide-stimulated macrophages: implications for the prevention of aneurysm progression. Lasers Surg. Med. 40: 371378], and its photomodulating, analgesic and direct interference effects on the neuroinflammatory process have drawn the attention of many researchers. LLLT can modulate a broad-spectrum of cellular processes, including: (i) protection from cell and tissue death; (ii) stimulation of healing and repair of injuries and (iii) reduction of pain, swelling and inflammation [21 Chung, H., T. Dai, S. K. Sharma, et al. 2012. The nuts and bolts of low-level laser (light) therapy. Ann. Biomed. Eng. 40: 516533]. It has been reported that the effects of laser irradiation – called photobiomodulation – are related to light fluence [22 Wang, F., T. S. Chen, D. Xing, et al. 2005. Measuring dynamics of caspase-3 activity in living cells using FRET technique during apoptosis induced by high fluence low-power laser irradiation. Lasers Surg. Med. 36: 27]. Evidence suggests that red or near-infra-red light (at wavelengths that can penetrate tissue) is absorbed by mitochondrial chromophores, especially cytochrome c oxidase, leading to increased cellular respiration and ATP formation, and modulation of oxidative stress and NO production that together lead to the activation of signaling pathways and gene transcription [23 Chen, A. C., P. R. Arany, Y. Y. Huang, et al. 2011. Low-level laser therapy activates NF-kB via generation of reactive oxygen species in mouse embryonic fibroblasts. PLoS One 6: e22453]. The effect of LLLT on the brain has also been extensively investigated. Transcranially applied LLLT has been shown to have beneficial effects on Alzheimer’s disease (AD) mouse models, and on rats and rabbits post-stroke [24–26 Oron, A., U. Oron, J. Chen, et al. 2006. Low-level laser therapy applied transcranially to rats after induction of stroke significantly reduces long-term neurological deficits. Stroke 37: 26202624
De Taboada, L., J. Yu, S. El-Amouri, et al. 2011. Transcranial laser therapy attenuates amyloid-beta peptide neuropathology in amyloid-beta protein precursor transgenic mice. J. Alzheimers Dis. 23: 521535
Farfara, D., H. Tuby, D. Trudler, et al. 2015. Low-level laser therapy ameliorates disease progression in a mouse model of Alzheimer's disease. J. Mol. Neurosci. 55: 430436
]. Furthermore, LLLT-regulated microglial function through Src kinase – a non-receptor tyrosine kinase that is activated by oxidative events [27 Song, S., F. Zhou, and W. R. Chen. 2012. Low-level laser therapy regulates microglial function through Src-mediated signaling pathways: implications for neurodegenerative diseases. J. Neuroinflamm. 9: 219] – and reduced long-term neurological deficits after traumatic brain injury (TBI) [28 Xuan, W., F. Vatansever, L. Huang, et al. 2013. Transcranial low-level laser therapy improves neurological performance in traumatic brain injury in mice: effect of treatment repetition regimen. PLoS One 8: e53454,29 Oron, A., U. Oron, J. Streeter, et al. 2012. Near infrared transcranial laser therapy applied at various modes to mice following traumatic brain injury significantly reduces long-term neurological deficits. J. Neurotrauma 29: 401407]. However, there have been no reports about the effects of irradiation on the autoimmune neuroinflammatory diseases, such as MS. Therefore, in the present study, we investigated the therapeutic potential of LLLT on experimental autoimmune encephalomyelitis (EAE) disease progression, an established model of MS. Most importantly, we attempted to elucidate some of the mechanisms through which LLLT modulates the pro-inflammatory environment of CNS.

## Methods

### Experimental animals

Experiments were conducted using female C57BL/6 mice (6–10 weeks of age). The mice were kept in groups of four to six animals per cage, maintained under controlled temperature (22 ± 1 °C) with a 12-h light/dark cycle (lights on at 07:00 h), and were given free access to food and water. All procedures used in the present study followed the Guide for the Care and Use of Laboratory Animals (NIH publication no. 85–23) and were approved by the Animal Ethics Committee of the Universidade Federal de Santa Catarina (CEUA-UFSC, protocol number PP00956) and Universidade do Extremo Sul Catarinense (CEUA-UNESC, protocol number 042/2014-1).

### EAE induction and clinical evaluation

Active EAE was induced by subcutaneous immunization with 200 µg of myelin oligodendrocytes glycoprotein (MOG) peptide, amino acids 35–55 and 500 µg Mycobacterium tuberculosis extract H37Ra in complete Freund’s adjuvant oil, as previously described [30 Stromnes, I. M., and J. M. Goverman. 2006. Active induction of experimental allergic encephalomyelitis. Nat. Protoc. 1: 18101819]. All animals were also injected intraperitoneally on days 0 and 2 with 300 ng of Pertussis toxin. Non-immunized (naive) and EAE non-irradiated animals were used as controls. Mice were monitored and scored daily for clinical disease severity according to the standard 0–5 EAE grading scale: 0, unaffected; 1, tail limpness; 2, failure to right upon attempt to roll over; 3, partial paralysis; 4, complete paralysis and 5, moribund. The mean of the clinical scores and body weight (a parameter of health) of all mice within a given treatment group was determined daily, thereby yielding the mean clinical score and body weight change for that treatment group.

### Laser treatment

The animals were randomly divided into four groups: (I) not immunized and untreated – naïve group; (II) immunized and untreated – EAE group; (III) immunized and treated with AlGaInP LLLT (660 nm) and (IV) immunized and treated with GaAs LLLT (904 nm). A two-laser diode (Ibramed™, São Paulo, Brazil) was used with the following parameters: (i) 660-nm wavelength (AsGaInP), mean power of 30 mW, continuous regime and beam area of 0.06 cm2. The laser irradiation was delivered with a fluency of 10 J/cm2 and energy of 0.6 J, with exposure time of 20 s for each position; (ii) 904-nm wavelength (GaAs), peak power of 70 W, pulsed regime (time of pulse 60 ns) and beam area of 0.10 cm2. The laser irradiation was delivered with a fluency of 3 J/cm2. The animals were irradiated during 30 days (starting on day 0 until day 30 post-immunization), with a total of six position of irradiation per day – laser radiation was timed to contact in six points located 0.5 cm distance between the points. The laser focus was positioned on the spinal cord at an angle of 90° to the skin according to a contact-point technique, and the gauging of the laser emission was conducted before and after completion of the experiments.

### Biochemical assays

#### Nitric oxide

NO release was quantified using the Griess assay [31 Pang, Q., X. Hu, X. Li, et al. 2015. Behavioral impairments and changes of nitric oxide and inducible nitric oxide synthase in the brains of molarless KM mice. Behav. Brain Res. 278: 411416]. After EAE induction, the production of NO was determined by an assay for nitrite. Eight mice of each group were euthanized, and the inguinal lymph nodes, spinal cords and spleen were extracted. The lymph node, spinal cord and spleen were rapidly separated on an ice plate and weighed. The samples were incubated with Griess reagent (1% sulfanilamide in 0.1 mol/L HCl and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride) at room temperature for 10 min, and optical density of the assay samples was measured spectrophotometrically at 540 nm.

#### Oxidative damage to lipids

The levels of 2-thiobarbituric acid-reactive species (TBARS) are expressed as malondialdehyde (MDA) equivalents, as previously described [32 Draper, H. H., and M. Hadley. 1990. Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol. 186: 421431]. Briefly, spinal cord and inguinal lymph nodes were mixed with 1 mL of 10% trichloroacetic acid and 1 mL of 0.67% thiobarbituric acid. Subsequently, the mixture was heated in a boiling water bath for 15 min. The amount of TBARS was determined by measuring absorbance at 532 nm, and the results are given in nanomoles of TBARS per milligram of protein.

#### Glutathione (GSH) levels

Samples of spinal cord were collected and maintained at −80 °C for at least 48 h. The sample was homogenized with 200 μL of 0.02 M EDTA. The homogenate was mixed with 25 μL of 10% trichloroacetic acid, and was homogenized three times over 15 min, followed by centrifugation (15 min× 1500g × 4 °C). The supernatant was added to 200 μL of 0.2 M TRIS buffer, pH 7.4 and 500 μM DTNB. Color development resulting from the reaction between DTNB and thiols reached a maximum in 5 min and was stable for more than 30 min. Absorbance was read at 412 nm after 10 min. A standard GSH curve was formed. The results are expressed as GSH per mg of protein [33 Borghi, S. M., A. C. Zarpelon, F. A. Pinho-Ribeiro, et al. 2014. Role of TNF-alpha/TNFR1 in intense acute swimming-induced delayed onset muscle soreness in mice. Physiol. Behav. 128: 277287].

#### Histopathological examination and assessment

For histopathological analysis, 30 days after EAE induction, animals were sacrificed and each portion of the lumbar spinal cord (L3–L5) was removed and fixed immediately in 10% neutral formalin buffer [formalin:phosphate buffer (0.01 M, pH 7.4) = 1:1] for 24 h. The spinal cord portions were subsequently processed by routine paraffin embedding, sectioned (5 -μm thickness) and mounted on glass slides. A deparaffinization protocol was carried out through a xylene-free method as previously described [34 Falkeholm, L., C. A. Grant, A. Magnusson, and E. Moller. 2001. Xylene-free method for histological preparation: a multicentre evaluation. Lab. Invest. 81: 12131221]. Hematoxylin–eosin (H&E)- or luxol fast blue (LFB)-stained slides were observed for immune cell infiltration and demyelination area, respectively. The settings used for image acquisition were identical for both control and experimental tissues, and representative images are presented. Four ocular fields per section (six to nine mice per group) were captured and a threshold optical density that best discriminated the nuclear staining of inflammatory cells (hematoxylin-eosin) or myelin (luxol fast blue) was obtained using NIH ImageJ 1.36 b imaging software (NIH, Bethesda, MD) and applied to all experimental groups The total pixel intensity was determined, and the data are expressed as optical density (O.D.).

#### ELISA assay

Spinal cord segments were homogenized in phosphate buffer containing 0.05% Tween® 20, 0.1 mM phenylmethylsulphonyl fluoride, 0.1 mM benzethonium chloride, 10 mM EDTA and 20 UI aprotinin A. The homogenate was centrifuged at 3000g for 10 min, and the supernatants were stored at −70 °C until further analysis. IFN-γ, IL-17 and IL-1β levels were estimated with ELISA kits from R&D Systems (Minneapolis, MN) according to the manufacturer’s recommendations.

### Drugs and reagents

Pertussis toxin, phosphate-buffered saline (PBS) and complete Freund’s adjuvant oil were all purchased from Sigma Chemical Co. (St. Louis, MO). The MOG35–55 peptide (MEVGWYRSPFSRVVHLYRNGK) was obtained from EZBiolab (Carmel, IN) and the M. tuberculosis extract H37Ra from Difco Laboratories (Detroit, MI). The anti-mouse-IL-17, IFN-γ, IL-1β DuoSet kits were obtained from R&D Systems (Minneapolis, MN). Other reagents were all of analytical grade and were obtained from different commercial sources.

### Statistical analysis

Results are presented as means ± SEM of measurements made on 6–9 mice per group per experiment, and are representative of one/two independent experiments without overlapping samples by evaluating the reproducibility of these results. One-way ANOVA followed by the Newman–Keuls test was used to compare the groups at each time-point when the parameters were measured at different times after the EAE induction. p values less than 0.05 (p < 0.05) were considered significant. The statistical analyses were performed using GraphPad Prism 4 Software (GraphPad Software Inc., San Diego, CA).

## Results

### LLLT alleviates symptoms and delays disease onset in EAE mice

C57BL/6 mice immunized with MOG35–55 developed EAE clinical symptoms after 7 days and reached a maximum mean clinical on day 30, when the incidence of clinical EAE was 100% and the average score was around 3.5 ± 0.5 (Figure 1A and Table 1). To test the prophylactic efficacy of laser during EAE, treatment starts from day 0 of induction. Compared with the untreated EAE group, AlGaInP 10 J/cm2 or GaAs 3 J/cm2 treatment significantly delayed disease onset (p < 0.001; Table 1) and decreased disease severity as measured by the mean maximal clinical score (2.0 ± 0.2 and 2.5 ± 0.5, respectively), with inhibition of 68 ± 2% (AlGaInP 10 J/cm2, Figure 1A and B) and 54 ± 5% (GaAs 3 J/cm2) (p < 0.0001; F = 48.05), based on the area under the curve (AUC), compared with the EAE-untreated group (Figure 1A and B; Table 1).

Figure 1. Low-level laser therapy attenuates the EAE disease process in C57BL/6 mice. Active EAE was induced in C57BL/6 mice by immunization with MOG35–55 on day 0. The clinical score (A), area under the curve (AUC) (B), body weight change (C) and delta (Δ) body weight gain or loss at the peak of disease (day 30 post-induction) (D) were evaluated in the naive group, the control group (EAE), in mice pre-treated with AlGaInP 10 J/cm2 (660 nm) and in mice pre-treated with GaAs 3 J/cm2 (904 nm), 30 days after immunization. The clinical symptoms were scored every day in a blinded manner and are expressed as the mean clinical score or as the AUC. Data points are presented as the mean ± SEM. Values of ##p < 0.001 versus naive group and **p < 0.001 versus EAE group (one-way ANOVA followed by post-hoc Newman–Keuls).

As previously described, animals with EAE tend to have a reduced body weight as a result of anorexia and deficient fluid uptake, which fit well with the severity of the clinical score [35 Mix, E., H. Meyer-Rienecker, and U. K. Zettl. 2008. Animal models of multiple sclerosis for the development and validation of novel therapies – potential and limitations. J. Neurol. 255: 714]. Next, we evaluated whether LLLT prevents the body weight change that is induced by EAE in mice. As expected, after EAE induction, a significant body weight loss was observed in the EAE mice compared with the naïve group (Figure 1C and D). Interestingly, a significant body weight gain was found in the EAE plus AlGaInP 10 J/cm2 (10 ± 2.5%; Figure 1D) group and the EAE plus GaAs 3 J/cm2 group (11 ± 3.0%; Figure 1D) (p < 0.01; F = 6.3) when compared with the EAE group.

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

Excess amounts of NO are harmful for CNS function and are implicated in the pathophysiology of many neurologic diseases, such as MS, and the EAE model, in which NO is overproduced, mainly by innate immunity cells, such as macrophages and microglia [36–38 Ghasemi, M., and A. Fatemi. 2014. Pathologic role of glial nitric oxide in adult and pediatric neuroinflammatory diseases. Neurosci. Biobehav. Rev. 45: 168182
Das, U. N. 2012. Is multiple sclerosis a proresolution deficiency disorder? Nutrition 28: 951958
Miller, E. 2012. Multiple sclerosis. Adv. Exp. Med. Biol. 724: 222238
]. Thus, we investigated the effect of LLLT on the level of NO in the CNS and secondary lymphoid tissue of EAE-treated and untreated animals. In agreement with clinical signs, the concentration of NO in the spinal cord of the EAE mice was significantly increased (52 ± 25 µmol/mg of protein) compared with the control animals (Figure 2A). In contrast, both AlGaInP 10 J/cm2 and GaAs 3 J/cm2 treatment down-regulated the NO level in the CNS of the EAE-treated animals, with a mean of 10 ± 5 and 15 ± 10 µmol/mg of protein, respectively (Figure 2A; p < 0.01; F = 7.15). Moreover, this upregulation was attenuated with LLLT (AlGaInP 10 J/cm2 and GaAs 3 J/cm2 treatment) in the spleen tissue after EAE induction (p < 0.05 and p < 0.01 versus the healthy group; Figure 2C). However, compared with the untreated EAE group, LLLT did not significantly modulate NO in the lymph node (Figure 2B). In addition, LLLT failed to inhibit lipid peroxidation (Figure 3A and B; p < 0.08; F = 2.80 and p < 0.7; F = 0.38) or to restore the antioxidant defense (Figure 3C and D; p < 0.31; F = 1.28 and p < 0.45; F = 0.91) after EAE induction in the spinal cord and lymph node, respectively.

Figure 2. Low-level laser therapy selectively inhibits NO level in the CNS and peripheral lymphoid tissue of EAE mice. Active EAE was induced in the C57BL/6 mice with MOG35–55/CFA. The spinal lumbar cords (A), inguinal lymph nodes (B) and spleen (C) were obtained from the naive group, the control group (EAE), from mice pre-treated with AlGaInP 10 J/cm2 (660 nm) and from mice pre-treated with GaAs 3 J/cm2 (904 nm), 30 days after immunization. The NO production was analyzed using the Griess assay. Data are presented as means ± SEM of 6–9 mice per group and are representative of two independent experiments. #p < 0.05 versus naïve group and **p < 0.001 versus EAE group (one-way ANOVA with Newman–Keuls post-hoc test).

Figure 3. Low-level laser therapy ameliorates EAE without affecting lipid peroxidation or the antioxidant defense. Animals were immunized with MOG35–55 peptide/CFA and pertussis toxin. Lumbar spinal cord and inguinal lymph node samples were collected from the naive group, the control group (EAE), from mice pre-treated with AlGaInP 10 J/cm2 (660 nm) and from mice pre-treated with GaAs 3 J/cm2 (904 nm), 30 days after EAE induction for the determination of TBARS (panels A and B) and GSH (panels C and D) levels, respectively. Results are presented as means ± SEM of 6–9 mice/group, and are representative of two separate experiments.

### LLLT limits the infiltration of immune cells to the CNS

The hallmark of EAE disease is the infiltration of inflammatory cells into the CNS, leading to neuronal and oligodendrocyte damage [39 Bogie, J. F., P. Stinissen, and J. J. Hendriks. 2014. Macrophage subsets and microglia in multiple sclerosis. Acta Neuropathol. 128: 191213]. Therefore, we aimed to determine the effect of LLLT on the infiltration of inflammatory cells into the CNS after EAE induction. As shown in Figure 4, no inflammatory foci were detected in the naïve lumbar spinal cord; however, the untreated EAE mice showed profound infiltration of immune cells into the CNS, particularly in the white matter region (Figure 4A and B). Interestingly, treatment with AlGaInP 10 J/cm2 significantly reduced the infiltration of these inflammatory cells into the CNS (Figure 4A and B; p < 0.02; F = 4.36). In contrast, treatment with GaAs 3 J/cm2 only resulted in a moderate inhibition (Figure 4).

Figure 4. Low-level laser therapy blocks infiltration of mononuclear cells into the CNS during EAE pathology. Active EAE was induced in the C57BL/6 mice with MOG35–55/CFA plus pertussis toxin. At the peak of disease (day 30), animals were killed and the lumbar spinal cords from the naive group, the control group (EAE), from mice pre-treated with AlGaInP 10 J/cm2 (660 nm) and from mice pre-treated with GaAs 3 J/cm2 (904 nm) were harvested for infiltration studies. Infiltration of mononuclear cells into spinal cords sections was examined by H&E staining (A), with magnification ×40, ×100 and ×400. Graphical representation of the inflammatory cells evaluated in the lumbar spinal cord (B). Specifically, four alternate 5 -µm sections (six to nine animals/group) of the white matter of the lumbar spinal cord were obtained between L4 and L6. Detail: inflammatory foci in the white matter after EAE induction. Data are presented as means ± SEM. #p < 0.05 versus naïve group and *p < 0.05 versus EAE group (one-way ANOVA with Newman–Keuls post-hoc test).

### LLLT reduces the demyelination area

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

Figure 5. Low-level laser therapy inhibits CNS demyelination during EAE development. Active EAE was induced in the C57BL/6 mice with MOG35–55/CFA plus pertussis toxin. At the peak of disease (day 30), animals were killed and the lumbar spinal cords from the naive group, the control group (EAE), from mice pre-treated with AlGaInP 10 J/cm2 (660 nm) and from mice pre-treated with GaAs 3 J/cm2 (904 nm) were harvested for demyelination studies. Demyelination areas in spinal cord sections were examined by luxol fast blue (LFB) staining (A), with magnification ×40 and ×100. Graphical representation of the CNS demyelination in lumbar spinal cord (B). Specifically, four alternate 5 -µm sections (six to nine animals/group) of the white matter of the lumbar spinal cord were obtained between L4 and L6. Detail: CNS demyelination in the white matter after EAE induction. Data are presented as means ± SEM. #p < 0.05 versus naïve group and *p < 0.05 versus EAE group (one-way ANOVA with Newman–Keuls post-hoc test).

### LLLT attenuated production of pro-inflammatory cytokines during EAE pathology

To initiate CNS inflammation, myelin-specific T cells, especially Th17 and Th1 subsets, must be activated in the periphery, gain access to the CNS and then be reactivated by central APCs presenting self-antigen, initiating a cascade of events, including the secretion of cytokines/chemokines, which recruit macrophages to the sites of T-cell activation [3 Goverman, J. 2009. Autoimmune T cell responses in the central nervous system. Nat. Rev. Immunol. 9: 393407]. Moreover, pro-inflammatory mediators secreted by macrophages/microglia, such as IL-1β, are important for both perpetuating inflammation and contributing to CNS tissue damage in EAE [40 Kuchroo, V. K., A. C. Anderson, H. Waldner, et al. 2002. T cell response in experimental autoimmune encephalomyelitis (EAE): role of self and cross-reactive antigens in shaping, tuning, and regulating the autopathogenic T cell repertoire. Ann. Rev. Immunol. 20: 101123]. Here, pronounced increase in IL-17, IFN-γ and IL-1β levels was observed in the spinal cord after EAE-immunization (Figure 6). AlGaInP 10 J/cm2 and GaAs 3 J/cm2 treatment markedly inhibited the upregulation of IL-17 (Figure 6A), IFN-γ (Figure 6B) and IL-1β (Figure 6C) in the CNS after EAE induction.

Figure 6. Low-level laser therapy inhibits production of pro-inflammatory cytokines during EAE pathology. The spinal cord was extracted and processed to estimate the levels of IL-17 (A), IFN-γ (B) and IL-1β (C) by ELISA in the naive group, the control group (EAE), from mice pre-treated with AlGaInP 10 J/cm2 (660 nm) and from mice pre-treated with GaAs 3 J/cm2 (904 nm). Data are presented as means ± SEM of 6–9 mice per group. #p < 0.05 and ##p < 0.01 versus naïve group; *p < 0.05 and **p < 0.001 versus EAE group (one-way ANOVA with Newman–Keuls post-hoc test).

## Discussion

MS is the prototypic autoimmune inflammatory disorder of the CNS for which no cure is presently known. T cells have a pivotal role in orchestrating the complex cascade of events in MS, which include chronic inflammation, primary demyelination and axonal damage. The adverse events associated with the widely used IFN-β, glatiramer acetate, fingolimod, and, more recently, dimethyl fumarate justifying the search for alternative and less detrimental therapies.

Herein, we show that LLLT reduced the clinical score of EAE and delayed the disease onset through down-regulation of NO levels in the CNS and peripheral lymphoid tissue. Interestingly, a significant body weight gain was found in the EAE plus AlGaInP group and the EAE plus GaAs group, when compared with the EAE group, which could be due to the modulation of leptin levels. In fact, recently, Burduli demonstrated that the combined treatment by means of low-intensity laser irradiation is accompanied by the normalization of the plasma leptin level, suppression of the inflammatory process and a significant improvement of the quality of life of the patients suffering from rheumatoid arthritis [41 Burduli, N. N., and N. M. Burduli. 2015. [The influence of intravenous laser irradiation of the blood on the dynamics of leptin levels and the quality of life of the patients presenting with rheumatoid arthritis]. Vopr. Kurortol. Fizioter. Lech. Fiz. Kult. 92: 1113]. Therefore, further experiments are required to confirm whether or not LLLT modulates the leptin pathway during the development of EAE. In addition, these beneficial effects of LLLT seem to be associated with a block of the entry of the inflammatory cells (especially lymphocytes) into the CNS, as well as immune cell migration, the demyelinating process and production of pro-inflammatory cytokines, after EAE induction (see proposed scheme in Figure 7). These results are in accord with studies in rodent models demonstrating that LLLT: (i) improves cognitive functions in the progressive stages of a mouse model of AD [26 Farfara, D., H. Tuby, D. Trudler, et al. 2015. Low-level laser therapy ameliorates disease progression in a mouse model of Alzheimer's disease. J. Mol. Neurosci. 55: 430436]; (ii) recovers short- and long-term (56 days) neurobehavioral functions and reduces brain lesion volume after TBI [29 Oron, A., U. Oron, J. Streeter, et al. 2012. Near infrared transcranial laser therapy applied at various modes to mice following traumatic brain injury significantly reduces long-term neurological deficits. J. Neurotrauma 29: 401407] and (iii) reduces the delayed-type hypersensitivity reaction to ovalbumin by down-regulation of pro-inflammatory mediators [42 Oliveira, R. G., A. P. Ferreira, A. J. Cortes, et al. 2013. Low-level laser reduces the production of TNF-alpha, IFN-gamma, and IL-10 induced by OVA. Lasers Med. Sci. 28: 15191525,43 de Oliveira, R. G., F. M. Aarestrup, C. Miranda, et al. 2010. Low-level laser therapy reduces delayed hypersensitivity reaction to ovalbumin in Balb/C mice. Photomed. Laser Surg. 28: 773777]. Taken together, these studies demonstrate the biological effects of LLLT with different parameters, confirming the ample therapeutic window of LLLT in different clinical conditions. In the literature, there are a large number of experimental studies with LLLT, although few parameters are described in detail, which results in the comparison and consequent understanding of the mechanisms involved being difficult. In the present study, we used two wavelengths – 660 and 904 nm. In agreement with our data, Enwemeka reported that only 30% of published papers using LLLT reveal consistent information to determine the dose, or even reported inaccurate data [44 Enwemeka, C. S. 2008. Standard parameters in laser phototherapy. Photomed. Laser Surg. 26: 411]. Thus, further studies are required to verify the effectiveness of LLLT in MS.

Figure 7. Schematic representation of low-level laser therapy (LLLT) anti-inflammatory and immunosuppressive effects in an experimental model of MS. Preventive treatment with LLLT during the induction phase of EAE, an experimental model of MS, inhibits development and progression of disease, besides neuroinflammation and demyelinating process in the CNS. Together, LLLT immunomodulatory correlates to inhibition of NO and cytokines levels in the spinal cord after EAE induction. LLLT, low-level laser therapy; EAE, experimental autoimmune encephalomyelitis; MS, multiple sclerosis; BBB, blood–brain barrier; CNS, central nervous systems; MOG, myelin oligodendrocytes glycoprotein; Th cell, T helper lymphocyte.

It has been suggested that LLLT may affect the inflammatory system, although the basis for the immunosuppressive effects of LLLT is still unknown. It is possible that LLLT irradiation changes RNA expression at the level of mRNA or protein synthesis of pro-inflammatory mediators, such as IL-2, TNF-α, IFN-γ, ICAM-1 and COX-2, as well as enhancing peripheral endogenous opioid in rats [45–47 Mafra de Lima, F., M. S. Costa, R. Albertini, et al. 2009. Low level laser therapy (LLLT): attenuation of cholinergic hyperreactivity, beta(2)-adrenergic hyporesponsiveness and TNF-alpha mRNA expression in rat bronchi segments in E. coli lipopolysaccharide-induced airway inflammation by a NF-kappaB dependent mechanism. Lasers Surg. Med. 41: 6874
Hagiwara, S., H. Iwasaka, A. Hasegawa, and T. Noguchi. 2008. Pre-Irradiation of blood by gallium aluminum arsenide (830 nm) low-level laser enhances peripheral endogenous opioid analgesia in rats. Anesth. Analg. 107: 10581063
Matsumoto, M. A., R. V. Ferino, G. F. Monteleone, and D. A. Ribeiro. 2009. Low-level laser therapy modulates cyclo-oxygenase-2 expression during bone repair in rats. Lasers Med. Sci. 24: 195201
], from immune cells. In fact, recently, Mozzati et al. demonstrated that superpulsed laser irradiation blocked down-regulation of IL-1β, IL-6, IL-10 and COX-2, and that this was associated with a reduction in the inflammatory process after tooth extraction [48 Mozzati, M., G. Martinasso, N. Cocero, et al. 2011. Influence of superpulsed laser therapy on healing processes following tooth extraction. Photomed. Laser Surg. 29: 565571]. Specific wavelengths of light trigger different inflammatory pathways of immune cells, such as antigen-presenting cells (APCs, e.g. macrophages) [49 Dube, A., H. Bansal, and P. K. Gupta. 2003. Modulation of macrophage structure and function by low level He–Ne laser irradiation. Photochem. Photobiol. Sci. 2: 851855], which leads to increased infiltration into the tissues. The ability of macrophages to act as phagocytes is also modulated by the application of LLLT [49 Dube, A., H. Bansal, and P. K. Gupta. 2003. Modulation of macrophage structure and function by low level He–Ne laser irradiation. Photochem. Photobiol. Sci. 2: 851855]. The ability of LLLT to drain lymphatic cells can be explained by the direct effects of laser light on the production of cytokines, because laser light can penetrate to 50 mm below the tissue surface [50 Uebelhoer, N. S., and E. V. Ross. 2008. Introduction. Update on lasers. Semin. Cutan. Med. Surg. 27: 221226]. Accumulated evidence now suggests that in the induction phase of EAE and MS disease (day 0–day 7), encephalitogenic T cells in the periphery become activated by a viral or another infectious antigen [1 Sospedra, M., and R. Martin. 2005. Immunology of multiple sclerosis. Ann. Rev. Immunol. 23: 683747]. Here, we hypothesize that LLLT applied during the induction phase of EAE increased phagocytic activity, and thus reduced antigen presentation in draining lymphatic cells and consistently inhibited activation of encephalitogenic Th1 and Th17 cells during the presentation of myelin antigens in peripheral lymphoid organs. Consequently, these cells failed to differentiate, proliferate and migrate to the CNS effectively, an effect that abrogated the development of EAE. In agreement with our data, Farfara et al. showed that laser-induced CD11b-positive phagocytotic monocyte cells were associated with a significant reduction of brain amyloid load following a short period of treatment [26 Farfara, D., H. Tuby, D. Trudler, et al. 2015. Low-level laser therapy ameliorates disease progression in a mouse model of Alzheimer's disease. J. Mol. Neurosci. 55: 430436].

After peripheral activation, CD4+ T cells effectively enter the subarachnoid space by crossing the blood-cerebrospinal fluid (CSF) barrier in either the choroid plexus or the meningeal venules [2 Steinman, L. 2007. A brief history of T(H)17, the first major revision in the T(H)1/T(H)2 hypothesis of T cell-mediated tissue damage. Nat. Med. 13: 139145,3 Goverman, J. 2009. Autoimmune T cell responses in the central nervous system. Nat. Rev. Immunol. 9: 393407]. Moreover, inside the CNS, the T cells are re-activated by MHC class II-expressing microglia, which express myelin epitopes [3 Goverman, J. 2009. Autoimmune T cell responses in the central nervous system. Nat. Rev. Immunol. 9: 393407]. These T cells are capable of producing pro-inflammatory mediators, such as cytokines and reactive oxygen species (ROS). The level of ROS, especially NO, is enhanced in MS [51 Koch, M., G. S. Ramsaransing, A. V. Arutjunyan, et al. 2006. Oxidative stress in serum and peripheral blood leukocytes in patients with different disease courses of multiple sclerosis. J. Neurol. 253: 483487] and consequently causes increased permeability of the blood–brain barrier (BBB) [52 Kuhlmann, C. R., R. Tamaki, M. Gamerdinger, et al. 2007. Inhibition of the myosin light chain kinase prevents hypoxia-induced blood–brain barrier disruption. J. Neurochem. 102: 501507]. Similarly, opening of the BBB and oxidative stress are known to be involved in the pathogenesis of EAE, the animal model of MS [53 van Horssen, J., G. Schreibelt, J. Drexhage, et al. 2008. Severe oxidative damage in multiple sclerosis lesions coincides with enhanced antioxidant enzyme expression. Free Radic. Biol. Med. 45: 17291737]. In this study, we extended and enriched these findings by demonstrating that LLLT inhibited the NO level in the CNS and peripheral lymphoid tissue, especially, spleen after EAE induction. On the other hand, LLLT did not modulate the production of NO in the inguinal lymph nodes, which could be due to either the EAE mice having less NO-producing cells or to a decreased NO-producing capability on a per cell basis in the regional lymph nodes. Thus, future studies will need to clarify this hypothesis, as well as to investigate whether LLLT could modulate, directly, these cells in lymph nodes after EAE induction. Interestingly, the beneficial effect of LLLT can be partially explained based on the rapid elevation of ATP content, as previously demonstrated after laser irradiation in the ischemic heart [54 Oron, U., T. Yaakobi, A. Oron, et al. 2001. Low-energy laser irradiation reduces formation of scar tissue after myocardial infarction in rats and dogs. Circulation 103: 296301]. Furthermore, increases in total antioxidants, angiogenesis, heat-shock protein content and anti-apoptotic activity following LLLT were previously found for ischemic heart and skeletal muscles [29 Oron, A., U. Oron, J. Streeter, et al. 2012. Near infrared transcranial laser therapy applied at various modes to mice following traumatic brain injury significantly reduces long-term neurological deficits. J. Neurotrauma 29: 401407,54 Oron, U., T. Yaakobi, A. Oron, et al. 2001. Low-energy laser irradiation reduces formation of scar tissue after myocardial infarction in rats and dogs. Circulation 103: 296301,55 Avni, D., S. Levkovitz, L. Maltz, and U. Oron. 2005. Protection of skeletal muscles from ischemic injury: low-level laser therapy increases antioxidant activity. Photomed. Laser Surg. 23: 273277], and can be suggested as possible processes that are also attenuated by LLLT in the EAE model.

Additionally, much attention has been paid to therapeutic strategies aimed at controlling microglia-mediated neurotoxicity. Recently, it has been debated whether He–Ne (632.8 nm) LLLT can activate a number of signaling pathways, including MAPK/ERK, Src, Akt and RTK/PKCs signaling pathways [56 Zhang, J., D. Xing, and X. Gao. 2008. Low-power laser irradiation activates Src tyrosine kinase through reactive oxygen species-mediated signaling pathway. J. Cell. Physiol. 217: 518528,57 Zhang, L., D. Xing, X. Gao, and S. Wu. 2009. Low-power laser irradiation promotes cell proliferation by activating PI3K/Akt pathway. J. Cell. Physiol. 219: 553562]. A study by Song et al. employed a microglial activation model (BV2 cells plus lipopolysaccharide) and evaluated the LLLT-induced neuroprotective effect. They found that LLLT prevents Toll-like receptor (TLR)-mediated pro-inflammatory responses in microglia, characterized by down-regulation of pro-inflammatory cytokine expression and NO production [27 Song, S., F. Zhou, and W. R. Chen. 2012. Low-level laser therapy regulates microglial function through Src-mediated signaling pathways: implications for neurodegenerative diseases. J. Neuroinflamm. 9: 219]. Results reported here indicate, for the first time to our knowledge, that LLLT preventive treatment produced a marked reduction in inflammatory cell recruitment into the spinal cord and effectively prevented demyelination areas in the EAE mice. According to our data, the neuroinflammatory process results in neuronal injury that may impair function in the CNS, and these results suggest a neuroprotective effect of LLLT, which can be observed in terms of reduced EAE development and severity of clinical scores.

The present study also indicates a possible preferable mode of laser use for LLLT application after EAE immunization. The 660-nm wavelength (AsGaInP 10 J/cm2) in continuous-pulse mode demonstrated a better outcome in the percentage of mice showing complete recovery compared with the 904-nm wavelength (GaAs 3 J/cm2). In agreement with our data, Oron et al. (LLLT 808 nm, GaAlAs) described the superiority of the 100-Hz laser compared to the 600-Hz frequency after closed-head injury (CHI), and suggested that this difference may be associated with a resonance effect between pulsed light and brain waves (such as α-waves and θ-waves) [29 Oron, A., U. Oron, J. Streeter, et al. 2012. Near infrared transcranial laser therapy applied at various modes to mice following traumatic brain injury significantly reduces long-term neurological deficits. J. Neurotrauma 29: 401407]. In addition, there is a higher elevation in ATP content in the rabbit brain when laser energy was applied in the 100-Hz mode compared with 600 Hz [58 Lapchak, P. A., and L. De Taboada. 2010. Transcranial near infrared laser treatment (NILT) increases cortical adenosine-5′-triphosphate (ATP) content following embolic strokes in rabbits. Brain Res. 1306: 100105]. Thus, we can propose that the AsGaInP 10-J/cm2 mode is perhaps the preferable mode with which to obtain a beneficial effect after autoimmune and neurodegenerative diseases, such as MS. Finally, in agreement with our data, Hudson et al. used the LLLT at 808 and 980 nm (1 W/cm2), which was projected through bovine tissue samples ranging in thickness from 18 to 95 mm and power density measurements were taken for each wavelength at the various depths. Thus, the authors concluded that 808 nm of light penetrates as much as 54% deeper than 980 nm light in bovine tissue, although we have not found any data with another tissue, such as bone, skin, nerves or MS [59 Hudson, D. E., D. O. Hudson, J. M. Wininger, and B. D. Richardson. 2013. Penetration of laser light at 808 and 980 nm in bovine tissue samples. Photomed. Laser Surg. 31: 163168]. Moreover, Byrnes et al. showed that LLLT at 810 nm can penetrate deep into the body and promote neuronal regeneration and functional recovery for spinal cord injury (SCI) [60 Byrnes, K. R., R. W. Waynant, I. K. Ilev, et al. 2005. Light promotes regeneration and functional recovery and alters the immune response after spinal cord injury. Lasers Surg. Med. 36: 171185]. The noninvasive nature and almost complete absence of side effects encourage further studies in neuroscience. Usually every laser and light source has a therapeutic window, narrow or wide for a particular lesion or condition. In laser treatment side effects can be divided into: (i) immediate effects, which occur either immediately or within a few minutes or hours of laser treatment, occasionally related to improper technique and rarely related to an accident; (ii) late effects, excess fluence leads to epidermal erythema, superficial burn or deep dermal burn with incident scarring depending on the extent of injury and pigmentary changes and (iii) sequelae, which rarely occurs. Importantly, these adverse effects can be prevented or minimised by proper patient and lesion selection, proper parameter selection, test shots and stepping down on fluence [61 Patil, U. A., and L. D. Dhami. 2008. Overview of lasers. Indian J. Plastic Surg. 41: S101S113].

In summary, the present study indicates that LLLT applied daily post-EAE induction to C57BL/6 mice markedly inhibits clinical signs, neuroinflammation and oxidative damage induced by encephalitogenic T lymphocytes and microglia in the CNS. Thus, LLLT may be a promising non-pharmacological disease-modifying therapy for the treatment of autoimmune conditions, such as MS.

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### Low-level laser therapy ameliorates disease progression in a mouse model of Alzheimer's disease.

Farfara D1, Tuby H, Trudler D, Doron-Mandel E, Maltz L, Vassar RJ, Frenkel D, Oron U. - J Mol Neurosci. 2015 Feb;55(2):430-6. doi: 10.1007/s12031-014-0354-z. Epub 2014 Jul 4. (Publication)
PBMT increased conginitive ability in tests on rats.
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Intro: Low-level laser therapy (LLLT) has been used to treat inflammation, tissue healing, and repair processes. We recently reported that LLLT to the bone marrow (BM) led to proliferation of mesenchymal stem cells (MSCs) and their homing in the ischemic heart suggesting its role in regenerative medicine. The aim of the present study was to investigate the ability of LLLT to stimulate MSCs of autologous BM in order to affect neurological behavior and Î²-amyloid burden in progressive stages of Alzheimer's disease (AD) mouse model. MSCs from wild-type mice stimulated with LLLT showed to increase their ability to maturate towards a monocyte lineage and to increase phagocytosis activity towards soluble amyloid beta (AÎ²). Furthermore, weekly LLLT to BM of AD mice for 2 months, starting at 4 months of age (progressive stage of AD), improved cognitive capacity and spatial learning, as compared to sham-treated AD mice. Histology revealed a significant reduction in AÎ² brain burden. Our results suggest the use of LLLT as a therapeutic application in progressive stages of AD and imply its role in mediating MSC therapy in brain amyloidogenic diseases.

Background: Low-level laser therapy (LLLT) has been used to treat inflammation, tissue healing, and repair processes. We recently reported that LLLT to the bone marrow (BM) led to proliferation of mesenchymal stem cells (MSCs) and their homing in the ischemic heart suggesting its role in regenerative medicine. The aim of the present study was to investigate the ability of LLLT to stimulate MSCs of autologous BM in order to affect neurological behavior and Î²-amyloid burden in progressive stages of Alzheimer's disease (AD) mouse model. MSCs from wild-type mice stimulated with LLLT showed to increase their ability to maturate towards a monocyte lineage and to increase phagocytosis activity towards soluble amyloid beta (AÎ²). Furthermore, weekly LLLT to BM of AD mice for 2 months, starting at 4 months of age (progressive stage of AD), improved cognitive capacity and spatial learning, as compared to sham-treated AD mice. Histology revealed a significant reduction in AÎ² brain burden. Our results suggest the use of LLLT as a therapeutic application in progressive stages of AD and imply its role in mediating MSC therapy in brain amyloidogenic diseases.

Abstract: Abstract Low-level laser therapy (LLLT) has been used to treat inflammation, tissue healing, and repair processes. We recently reported that LLLT to the bone marrow (BM) led to proliferation of mesenchymal stem cells (MSCs) and their homing in the ischemic heart suggesting its role in regenerative medicine. The aim of the present study was to investigate the ability of LLLT to stimulate MSCs of autologous BM in order to affect neurological behavior and Î²-amyloid burden in progressive stages of Alzheimer's disease (AD) mouse model. MSCs from wild-type mice stimulated with LLLT showed to increase their ability to maturate towards a monocyte lineage and to increase phagocytosis activity towards soluble amyloid beta (AÎ²). Furthermore, weekly LLLT to BM of AD mice for 2 months, starting at 4 months of age (progressive stage of AD), improved cognitive capacity and spatial learning, as compared to sham-treated AD mice. Histology revealed a significant reduction in AÎ² brain burden. Our results suggest the use of LLLT as a therapeutic application in progressive stages of AD and imply its role in mediating MSC therapy in brain amyloidogenic diseases.

Original Source: http://www.ncbi.nlm.nih.gov/pubmed/24994540

### Mitochondrial respiration as a target for neuroprotection and cognitive enhancement.

Gonzalez-Lima F1, Barksdale BR2, Rojas JC3. - Biochem Pharmacol. 2014 Apr 15;88(4):584-93. doi: 10.1016/j.bcp.2013.11.010. Epub 2013 Dec 4. (Publication)
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Intro: This paper focuses on brain mitochondrial respiration as a therapeutic target for neuroprotection and cognitive enhancement. We propose that improving brain mitochondrial respiration is an important future direction in research and treatment of Alzheimer's disease (AD) and other conditions associated with cognitive impairment and neurodegeneration. The central thesis is that supporting and improving brain mitochondrial respiration constitutes a promising neurotherapeutic principle, with potential applications in AD as well as in a wide variety of neuropsychological conditions. We propose three different interventional approaches to improve brain mitochondrial respiration based on (a) pharmacology, (b) photobiomodulation and (c) nutrition interventions, and provide detailed examples for each type of intervention. First, low-dose USP methylene blue is described as a pharmacological intervention that can successfully increase mitochondrial respiration and result in memory enhancement and neuroprotection. Second, transcranial low-level light/laser therapy with near-infrared light is used to illustrate a photobiomodulation intervention with similar neurometabolic mechanisms of action as low-dose methylene blue. Finally, a nutrition intervention to improve mitochondrial respiration is proposed by increasing ketone bodies in the diet. The evidence discussed for each intervention supports a fundamental neurotherapeutic strategy based on improving oxidative energy metabolism while at the same time reducing the pro-oxidant tendencies of the nervous system. Targeting brain mitochondrial respiration with these three types of interventions is proposed as part of a holistic neurotherapeutic approach to improve brain energy metabolism and antioxidant defenses. This strategy represents a promising new bioenergetics direction for treatment of AD and other neuropsychological disorders featuring cognitive impairment and neurodegeneration.

Background: This paper focuses on brain mitochondrial respiration as a therapeutic target for neuroprotection and cognitive enhancement. We propose that improving brain mitochondrial respiration is an important future direction in research and treatment of Alzheimer's disease (AD) and other conditions associated with cognitive impairment and neurodegeneration. The central thesis is that supporting and improving brain mitochondrial respiration constitutes a promising neurotherapeutic principle, with potential applications in AD as well as in a wide variety of neuropsychological conditions. We propose three different interventional approaches to improve brain mitochondrial respiration based on (a) pharmacology, (b) photobiomodulation and (c) nutrition interventions, and provide detailed examples for each type of intervention. First, low-dose USP methylene blue is described as a pharmacological intervention that can successfully increase mitochondrial respiration and result in memory enhancement and neuroprotection. Second, transcranial low-level light/laser therapy with near-infrared light is used to illustrate a photobiomodulation intervention with similar neurometabolic mechanisms of action as low-dose methylene blue. Finally, a nutrition intervention to improve mitochondrial respiration is proposed by increasing ketone bodies in the diet. The evidence discussed for each intervention supports a fundamental neurotherapeutic strategy based on improving oxidative energy metabolism while at the same time reducing the pro-oxidant tendencies of the nervous system. Targeting brain mitochondrial respiration with these three types of interventions is proposed as part of a holistic neurotherapeutic approach to improve brain energy metabolism and antioxidant defenses. This strategy represents a promising new bioenergetics direction for treatment of AD and other neuropsychological disorders featuring cognitive impairment and neurodegeneration.

Abstract: Abstract This paper focuses on brain mitochondrial respiration as a therapeutic target for neuroprotection and cognitive enhancement. We propose that improving brain mitochondrial respiration is an important future direction in research and treatment of Alzheimer's disease (AD) and other conditions associated with cognitive impairment and neurodegeneration. The central thesis is that supporting and improving brain mitochondrial respiration constitutes a promising neurotherapeutic principle, with potential applications in AD as well as in a wide variety of neuropsychological conditions. We propose three different interventional approaches to improve brain mitochondrial respiration based on (a) pharmacology, (b) photobiomodulation and (c) nutrition interventions, and provide detailed examples for each type of intervention. First, low-dose USP methylene blue is described as a pharmacological intervention that can successfully increase mitochondrial respiration and result in memory enhancement and neuroprotection. Second, transcranial low-level light/laser therapy with near-infrared light is used to illustrate a photobiomodulation intervention with similar neurometabolic mechanisms of action as low-dose methylene blue. Finally, a nutrition intervention to improve mitochondrial respiration is proposed by increasing ketone bodies in the diet. The evidence discussed for each intervention supports a fundamental neurotherapeutic strategy based on improving oxidative energy metabolism while at the same time reducing the pro-oxidant tendencies of the nervous system. Targeting brain mitochondrial respiration with these three types of interventions is proposed as part of a holistic neurotherapeutic approach to improve brain energy metabolism and antioxidant defenses. This strategy represents a promising new bioenergetics direction for treatment of AD and other neuropsychological disorders featuring cognitive impairment and neurodegeneration. Copyright Â© 2013 Elsevier Inc. All rights reserved.

Original Source: http://www.ncbi.nlm.nih.gov/pubmed/24316434

### Alzheimer's Research

Shannon Macauley - Scientific American (Publication)
Combining light with sound show promise for treating Alzheimer's
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There is no cure for Alzheimer’s disease. Although a few drugs manage temporarily certain cognitive symptoms of the illness, none can stop or meaningfully slow its progression. “We really don’t have much to offer people,” says Shannon Macauley, a neuroscientist at Wake Forest School of Medicine. Virtually all new treatments have failed in clinical trials. But new research is looking beyond drugs to see what relief might come from a simple LED light and a speaker. Bathing patients in flashing light and pulsing sounds both tuned to a frequency of 40 hertz might reverse key signs of Alzheimer’s in the brain, according to a paper published in Cell on Thursday. “I think it’s an absolutely fascinating paper to be honest,” says Macauley, who was not involved in this work. “It’s a very provocative idea. It’s noninvasive and easy and low cost, potentially, so if it were to come to fruition in humans—that’s fabulous.” Still, all this is a big if, Macauley acknowledges. The work was done in mice with genetic alterations that doomed them to develop key symptoms and pathology of Alzheimer’s disease. One batch of mice formed neurofibrillary tangles inside their neurons—dysfunctional knots of a protein called tau that can lead to the cell’s death. Another batch of the mice developed amyloid beta plaques—sticky heaps of protein that dam the flow of communication between neurons. All the mice also had a third hallmark of the disease—irregular brain activity in the gamma range of brain waves that oscillate between 30 and 100 times a second. In 2015 neuroscientist Li-Huei Tsai, director at The Picower Institute for Learning and Memory at Massachusetts Institute of Technology, was working on an experiment to manipulate that brain activity by flashing a white light at these mice. Like light strobes, our brains flicker. Brain waves are generated when large groups of neurons oscillate on and off together. Neurons encode our thoughts and actions and senses in this rhythmic electrical flutter. So when Tsai tuned her light to flash 40 times a second, or 40 hertz, and flickered it at the mice, their brains flickered back—generating gamma waves at a corresponding 40 hertz. Then, something unexpected happened. When Tsai dissected the mice brains afterward, the amount of amyloid plaques and tau tangles in the mice that saw the light had plummeted. “It was the most remarkable thing,” Tsai says. “The light flicker stimulation triggers a tremendous microglia response. These are the brain’s immune cells that clear cell debris and toxic waste including amyloid. They’re impaired in Alzheimer’s disease, but [the light] seems to restore their abilities.” This clearing-out process only happened in the visual cortex where the brain processes light information. To get these effects to penetrate deeper into the brain, she added a clicking sound like a dolphin’s chirrup that also had a 40-hertz frequency. When the mice sat in a room with both the flashing light and the droning sound for an hour day, seven days in a row, amyloid plaques and tau tangles began falling in not just the audio and visual cortices but the prefrontal cortex and the hippocampus as well. “This was one of the big jumps in the new paper,” Macauley says. “These are the learning and memory centers of the brain. And there was about a 40 or 50 percent decrease in amyloid and tau levels. It’s an absolutely impressive feat.” That showed when Tsai put the mice through a set of cognitive tests. In one, where the mice were given a familiar and an unfamiliar object to explore, mice that didn’t get the treatment acted as though they’d never seen the familiar object. “That shows some memory problems,” Tsai says. Mice that saw the light and heard the sound spent about two thirds of the time that untreated mice did examining the familiar object. “It was unbelievable,” Tsai says. “This is the first time we’ve seen that this noninvasive stimulation can improve cognitive function. It’s not a drug or an antibody or anything, it’s just light and sound.” One possible explanation for this is brains with Alzheimer’s have irregular, often hyperactive, neurons, says Jorge Palop, a neurologist at the University of California, San Francisco, who did not work on the study. By providing the brains with a steady and regular beat, the repeating light and sound might work as a kind of metronome for brain activity. “This could be like resetting the mice every day and correcting some of this abnormal activity that they have,” he says. “Then downstream of that are all these beneficial effects.”
Original Source: https://www.scientificamerican.com/article/an-hour-of-light-and-sound-a-day-might-keep-alzheimers-at-bay/

### Nanoparticle-emitted light attenuates amyloid-Î²-induced superoxide and inflammation in astrocytes.

Bungart BL1, Dong L1, Sobek D2, Sun GY3, Yao G1, Lee JC4. - Nanomedicine. 2014 Jan;10(1):15-7. doi: 10.1016/j.nano.2013.10.007. Epub 2013 Nov 4. (Publication)
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## Abstract

Alzheimer’s disease (AD) is the sixth leading cause of age-related death with no effective intervention yet available. Our previous studies have demonstrated the potential efficacy of Low Level Laser Therapy (LLLT) in AD cell models by mitigating amyloid-β peptide (Aβ)-induced oxidative stress and inflammation. However, the penetration depth of light is still the major challenge for implementing LLLT in animal models and in the clinical settings. In this study, we present the potential of applying Bioluminescence Resonance Energy Transfer to Quantum Dots (BRET-Qdots) as an alternative near infrared (NIR) light source for LLLT. Our results show that BRET-Qdot-emitted NIR suppresses Aβ-induced oxidative stress and inflammatory responses in primary rat astrocytes. These data provide a proof of concept for a nanomedicine platform for LLLT.

Keywords: Alzheimer’s disease, light nanomedicine, inflammation

## Background

Many diseases, including AD, are characterized with aberrant cellular activities resulting from increased oxidative stress and inflammation (1, 2). In AD, Aβ when aggregated to its oligomeric form has the most cytotoxic effects (3). Toxic Aβ increases oxidative stress and triggers inflammatory responses in glial cells through increased expression of inflammatory proteins, such as interleukin-1β (IL-1β) and inducible nitric oxide synthase (iNOS) (4).

Low level light with wavelengths ranging from 632.8 to 904 nm have been reported to attenuate inflammation and oxidative stress in a number of models (5, 6). For example, we have previously demonstrated that a low level energy laser at 632.8 nm attenuated oxidative stress and inflammation in primary astrocytes induced by Aβ (5). However, the use of laser or LED light as a clinical therapy for the treatment of AD brains is limited due to the low penetration of light in tissues (7).

In this study, we demonstrate a new light delivery method by using a recently engineered nanoparticle, BRET-Qdot. This light delivery nanoparticle has been used in a number of in vivo applications (8) and exploits the F?ster resonance energy transfer from an engineered enzyme, Renilla Reniformis luciferase (Luc8) (9). When activated with its substrate, coelenterazine-h (coel-h), Luc8 is known to emit at 480 nm to a CdSe or CdTe quantum dot, which then re-emits the light at 800 nm. A typical BRET-Qdot construct incorporates 3 to 6 Luc8 molecules conjugated to the surface of CdTe quantum dots (Figure 1). Conceptually, this light delivery approach could potentially resolve difficulties in providing light from external sources to the central areas of the brain.

BRET-Qdot light delivery system to cultured tissues for protective, anti-inflammatory effects and anti-oxidative benefits

## Methods

To test the efficacy of BRET-Qdots (Zymera, San Jose, CA, USA) as proposed, we used primary cortical rat astrocytes (Invitrogen, Grand Island, NY, USA) as in our previous study (5). The maximum fluency and duration of the BRET-Qdots’ emitted light was observed using a PIXIS 512 CCD camera (Princeton Instruments, Trenton, NJ, USA) in conjunction with a cold mirror with a 0° AOI (Edmund Optics Inc., Barrington, NJ, USA) to reflect >90% visible light. Then, the BRET-Qdots were used as the pretreatment prior to a 5 μM Aβ (American Peptide, Sunnyvale, CA, USA) exposure to astrocyte, followed by the characterizations of superoxide anion accumulation using fluorescence microscopy of dihydroethidium (DHE), and the expressions of IL-1β and iNOS using Western blot analysis. Detailed methodological information can be found in the Supplementary Materials.

## Results

### BRET-Qdot with coel-h attenuates Aβ-induced superoxide anion production

We test if the superoxide anion induced by Aβ can be reduced by 800 nm-emitting BRET-Qdots which preceded Aβ treatment. Figure 2 shows that exposing astrocytes to 5 μM Aβ for 2 h increased superoxide anion production by 40% as compared to the untreated control. Light pre-treatment using 66.7 pM BRET-Qdots activated with 2.5 μM coel-h reduces Aβ-induced superoxide anion production to untreated control levels. Either BRET-Qdots or coel-h individually reduces superoxide anion production on average compared to Aβ-treated cultures, but not statistically significant.

BRET-Qdot with coel-h pretreatment on Aβ-induced superoxide anions production. (A) Aβ treatment increased the fluorescent intensity of DHE by 40% as compared to control. The pretreatment of BRET-Qdot (66.7 pM) and ceol-h (2.5 μM) ...

### BRET-Qdot with coel-h pretreatment attenuates Aβ-induced inflammatory marker expression

Similarly to the superoxide anion experiment, we investigate if the light pretreatment with 66.7 pM BRET-Qdot activated with 2.5 μM coel-h reduce the inflammation contributed by 5 μM Aβ as measured by IL-1β and iNOS expression levels (Figure 3). Incubating astrocytes with 5 μM Aβ for 18 h increased IL-1β and iNOS expression by 30 and 50%, respectively. BRET-Qdot-based light pre-treatment lowered Aβ-induced expression of IL-1β to that of the untreated control, while Aβ-induced iNOS expression was reduced to below untreated control levels. Neither BRET-Qdot nor coel-h alone showed the same reduction in IL-1β or iNOS expression.

Inflammation induced by Aβ is attenuated by BRET-Qdot with Coel-h pretreatment. Western blots representing the expression of (A) iNOS and (B) IL-1β. Aβ increased iNOS by 50% as compared to control, while the pretreatment of BRET-Qdot ...

## Discussion

Our previous study with astrocytes showed that application of 632.8 nm laser caused a decrease in oxidative stress and inflammation (5). However, red to NIR light cannot be transmitted through the scalp to the brain more than a few centimeters (7). This makes non-invasive LLLT for AD nearly impossible.

In this present study, we demonstrated the ability of a light-generating nanoparticle to attenuate Aβ-induced oxidative stress and inflammatory responses in astrocytes. The application of this light delivery method based on light-generating nanoparticles demonstrated a novel method for applying LLLT.

Some studies on the application of quantum dots in biological systems may consider the possible toxic effects of quantum dots due to their heavy metal content, nanoparticulate nature, or coating composition . Nevertheless, investigations of quantum dots in vivo have shown retention of the material in tissues from days to weeks with no major complications (10). Here we also show that exposing astrocytes to BRET-Qdots with coel-h for 2 and 12 h did not significantly lower cell viability as compared with control. (see Supplementary Materials, Fig. S2)

Results here show the advantages of light-generating nanoparticles as a concept to be further developed for in vivo light-delivery for LLLT. CdSe/CdTe quantum dots may be used further in animal models as non-toxic, NIR-emitting nanoparticles become available. In addition, even though this study only covered Aβ-induced inflammation and oxidative stress, as studied in the AD pathology, the LLLT delivered by light-generating BRET-dots may also be applied to other cellular systems and diseases involving oxidative stress and inflammation, such as diabetes (6). A light-generating nanoparticle-based therapy could potentially create the ability to offer LLLT in an in vivo setting to reduce the onset of these other pernicious illnesses.

Intro: Alzheimer's disease (AD) is the sixth leading cause of age-related death with no effective intervention yet available. Our previous studies have demonstrated the potential efficacy of Low Level Laser Therapy (LLLT) in AD cell models by mitigating amyloid-Î² peptide (AÎ²)-induced oxidative stress and inflammation. However, the penetration depth of light is still the major challenge for implementing LLLT in animal models and in the clinical settings. In this study, we present the potential of applying Bioluminescence Resonance Energy Transfer to Quantum Dots (BRET-Qdots) as an alternative near infrared (NIR) light source for LLLT. Our results show that BRET-Qdot-emitted NIR suppresses AÎ²-induced oxidative stress and inflammatory responses in primary rat astrocytes. These data provide a proof of concept for a nanomedicine platform for LLLT.

Background: Alzheimer's disease (AD) is the sixth leading cause of age-related death with no effective intervention yet available. Our previous studies have demonstrated the potential efficacy of Low Level Laser Therapy (LLLT) in AD cell models by mitigating amyloid-Î² peptide (AÎ²)-induced oxidative stress and inflammation. However, the penetration depth of light is still the major challenge for implementing LLLT in animal models and in the clinical settings. In this study, we present the potential of applying Bioluminescence Resonance Energy Transfer to Quantum Dots (BRET-Qdots) as an alternative near infrared (NIR) light source for LLLT. Our results show that BRET-Qdot-emitted NIR suppresses AÎ²-induced oxidative stress and inflammatory responses in primary rat astrocytes. These data provide a proof of concept for a nanomedicine platform for LLLT.

Abstract: Abstract Alzheimer's disease (AD) is the sixth leading cause of age-related death with no effective intervention yet available. Our previous studies have demonstrated the potential efficacy of Low Level Laser Therapy (LLLT) in AD cell models by mitigating amyloid-Î² peptide (AÎ²)-induced oxidative stress and inflammation. However, the penetration depth of light is still the major challenge for implementing LLLT in animal models and in the clinical settings. In this study, we present the potential of applying Bioluminescence Resonance Energy Transfer to Quantum Dots (BRET-Qdots) as an alternative near infrared (NIR) light source for LLLT. Our results show that BRET-Qdot-emitted NIR suppresses AÎ²-induced oxidative stress and inflammatory responses in primary rat astrocytes. These data provide a proof of concept for a nanomedicine platform for LLLT. FROM THE CLINICAL EDITOR: Low Level Laser Therapy has already been demonstrated to mitigate amyloid-Î² peptide induced oxidative stress and inflammation, a key driver of Alzheimer's disease. The major issue in moving this forward from cell cultures to live animals and potentially to human subjects is light penetration depth. In this novel study, BRET-Qdots were used as an alternative near infrared light source with good efficacy, paving the way to the development of a nanomedicine platform. Copyright Â© 2014 Elsevier Inc. All rights reserved.

Methods: Low Level Laser Therapy has already been demonstrated to mitigate amyloid-Î² peptide induced oxidative stress and inflammation, a key driver of Alzheimer's disease. The major issue in moving this forward from cell cultures to live animals and potentially to human subjects is light penetration depth. In this novel study, BRET-Qdots were used as an alternative near infrared light source with good efficacy, paving the way to the development of a nanomedicine platform.

Original Source: http://www.ncbi.nlm.nih.gov/pubmed/24200521

### Quantitative In Vivo Imaging of Tissue Absorption, Scattering, and Hemoglobin Concentration in Rat Cortex Using Spatially Modulated Structured Light

David J. Cuccia, David Abookasis, Ron D. Frostig, and Bruce J. Tromberg. - 2009 (Book)
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## 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 []. 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 []. 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 [] (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 [,]. The slower absorption factors have been demonstrated to correlate with the changes in metabolic demand and subsequent hemodynamic cascades following neuronal activation [,,].

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) []. Researchers have also used ISOI to locate and quantify the spatial extent of the stroke injury, including ischemic core, penumbra, and healthy tissue zones [,]. 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 [,].

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 [,].

Quantitative diffuse optical methods [] 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 [,,]. DOS and DOT utilize the near-infrared spectral region (600–1000 nm) to separate and quantify the multispectral absorption (μa) and reduced scattering coefficients (μs′), 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 []. 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) [], 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.

## 12.2. METHODS AND INSTRUMENTATION

### 12.2.1. Modulated Imaging Spectroscopy

The MI instrument platform was introduced originally by Cuccia et al. [] 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 []. In this work, we modeled diffuse reflectance using a transport-based White Monte Carlo (WMC) method [,]. 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 []. Due to the moderate albedo of brain tissue (μs′/μa ~ 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 []. 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)=2.303i=13ci?i(λ),
12.1

where ci 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 (StO2) can then be calculated as HbT = HbR + HbO2 and StO2= 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 []. 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 [], we expect the resulting image resolution to scale directly with the transport length, l* = (μa + μs′)− 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. [] 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 []. In Figure 12.2a, we depict this differential sensitivity using diffuse reflectance (MTF) predictions versus frequency, increasing μa by 100% from 0.02 (black line) to 0.04mm−1 (gray line). This is done for two values of μs′, 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 (ΔRd = 0.56 μs′ versus 0.024 * 10−3 for μa). 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 fx = 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.

## 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 μa and μs′ 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 μa (left) and μs′ (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 (μs ′ = 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 [], we believe this effect is primarily due to frequency-dependent probing volumes in the presence of depth-heterogeneous structures.

#### FIGURE 12.4

(a) Average μa (left) and μs′(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 StO2 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 μa 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 StO2 image, highlighting the effect of tissue oxygen extraction. Conversely, notice that the high albedo regions with less structural detail are highly oxygenated, with StO2 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 (StO2) 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 12.712.9 (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 fx = 0 mm−1 (DC, top) and fx = 0.26 mm−1 (AC, bottom). In part (b), we plot the recovered Δμa (top) and Δμs′ (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 μs′ trends tracking directly with the measured AC reflectance. As expected, μa 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 fx = 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, Δμs′, is calculated to be −0.03 mm−1. In order for this change to instead be due to an absorption-only event, μa 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 Rd (fx = 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 12.712.9) 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 [], 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. [] 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 [,,]. 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 StO2. This similarity is not clear in the DC traces of Figures 12.712.9, 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 StO2 (3) a dramatic spike in both StO2 (3–10%) and HbT (2–4 μM) with rise and decay times of approximately 1 minute each. For each region, the final StO2 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 []. From left to right, we show HbO2, HbR, HbT, StO2, and μs′. 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 StO2 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 []. Interestingly, the scattering depolarization wave is clearly followed in space and time by the increase in deoxyhemoglobin (HbR), decrease in saturation (StO2), 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

Original Source: https://www.ncbi.nlm.nih.gov/books/NBK20233/

### Low-level laser therapy rescues dendrite atrophy via upregulating BDNF expression: implications for Alzheimer's disease.

Meng C1, He Z, Xing D. - J Neurosci. 2013 Aug 14;33(33):13505-17. doi: 10.1523/JNEUROSCI.0918-13.2013. ()
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Intro: Downregulation of brain-derived neurotrophic factor (BDNF) in the hippocampus occurs early in the progression of Alzheimer's disease (AD). Since BDNF plays a critical role in neuronal survival and dendrite growth, BDNF upregulation may contribute to rescue dendrite atrophy and cell loss in AD. Low-level laser therapy (LLLT) has been demonstrated to regulate neuronal function both in vitro and in vivo. In the present study, we found that LLLT rescued neurons loss and dendritic atrophy via upregulation of BDNF in both AÎ²-treated hippocampal neurons and cultured APP/PS1 mouse hippocampal neurons. Photoactivation of transcription factor CRE-binding protein (CREB) increased both BDNF mRNA and protein expression, since knockdown CREB blocked the effects of LLLT. Furthermore, CREB-regulated transcription was in an ERK-dependent manner. Inhibition of ERK attenuated the DNA-binding efficiency of CREB to BDNF promoter. In addition, dendrite growth was improved after LLLT, characterized by upregulation of Rac1 activity and PSD-95 expression, and the increase in length, branching, and spine density of dendrites in hippocampal neurons. Together, these studies suggest that upregulation of BDNF with LLLT by activation of ERK/CREB pathway can ameliorate AÎ²-induced neurons loss and dendritic atrophy, thus identifying a novel pathway by which LLLT protects against AÎ²-induced neurotoxicity. Our research may provide a feasible therapeutic approach to control the progression of AD.

Background: Downregulation of brain-derived neurotrophic factor (BDNF) in the hippocampus occurs early in the progression of Alzheimer's disease (AD). Since BDNF plays a critical role in neuronal survival and dendrite growth, BDNF upregulation may contribute to rescue dendrite atrophy and cell loss in AD. Low-level laser therapy (LLLT) has been demonstrated to regulate neuronal function both in vitro and in vivo. In the present study, we found that LLLT rescued neurons loss and dendritic atrophy via upregulation of BDNF in both AÎ²-treated hippocampal neurons and cultured APP/PS1 mouse hippocampal neurons. Photoactivation of transcription factor CRE-binding protein (CREB) increased both BDNF mRNA and protein expression, since knockdown CREB blocked the effects of LLLT. Furthermore, CREB-regulated transcription was in an ERK-dependent manner. Inhibition of ERK attenuated the DNA-binding efficiency of CREB to BDNF promoter. In addition, dendrite growth was improved after LLLT, characterized by upregulation of Rac1 activity and PSD-95 expression, and the increase in length, branching, and spine density of dendrites in hippocampal neurons. Together, these studies suggest that upregulation of BDNF with LLLT by activation of ERK/CREB pathway can ameliorate AÎ²-induced neurons loss and dendritic atrophy, thus identifying a novel pathway by which LLLT protects against AÎ²-induced neurotoxicity. Our research may provide a feasible therapeutic approach to control the progression of AD.

Abstract: Abstract Downregulation of brain-derived neurotrophic factor (BDNF) in the hippocampus occurs early in the progression of Alzheimer's disease (AD). Since BDNF plays a critical role in neuronal survival and dendrite growth, BDNF upregulation may contribute to rescue dendrite atrophy and cell loss in AD. Low-level laser therapy (LLLT) has been demonstrated to regulate neuronal function both in vitro and in vivo. In the present study, we found that LLLT rescued neurons loss and dendritic atrophy via upregulation of BDNF in both AÎ²-treated hippocampal neurons and cultured APP/PS1 mouse hippocampal neurons. Photoactivation of transcription factor CRE-binding protein (CREB) increased both BDNF mRNA and protein expression, since knockdown CREB blocked the effects of LLLT. Furthermore, CREB-regulated transcription was in an ERK-dependent manner. Inhibition of ERK attenuated the DNA-binding efficiency of CREB to BDNF promoter. In addition, dendrite growth was improved after LLLT, characterized by upregulation of Rac1 activity and PSD-95 expression, and the increase in length, branching, and spine density of dendrites in hippocampal neurons. Together, these studies suggest that upregulation of BDNF with LLLT by activation of ERK/CREB pathway can ameliorate AÎ²-induced neurons loss and dendritic atrophy, thus identifying a novel pathway by which LLLT protects against AÎ²-induced neurotoxicity. Our research may provide a feasible therapeutic approach to control the progression of AD.

Original Source: http://www.ncbi.nlm.nih.gov/pubmed/23946409

### Photobiomodulation by low-power laser irradiation attenuates AÎ²-induced cell apoptosis through the Akt/GSK3Î²/Î²-catenin pathway.

Liang J1, Liu L, Xing D. - Free Radic Biol Med. 2012 Oct 1;53(7):1459-67. doi: 10.1016/j.freeradbiomed.2012.08.003. Epub 2012 Aug 11. ()
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Intro: Apoptosis induced by amyloid Î² peptide (AÎ²) is thought to associate with the pathogenesis of Alzheimer disease (AD). Accumulating evidence shows that low-power laser irradiation (LPLI) is capable of reducing AÎ²-induced apoptosis. However, the underlying mechanisms remain unclear. In this study, we report a novel molecular mechanism by which LPLI attenuates AÎ²(25-35)-induced apoptosis through the Akt/GSK3Î²/Î²-catenin pathway. We found that Akt activated by LPLI interacted with GSK3Î² and phosphorylated it on Ser9 in the presence of AÎ²(25-35), which resulted in the inhibition of GSK3Î². Furthermore, LPLI increased the nuclear translocation of Î²-catenin and enhanced its T cell factor/lymphocyte enhancer factor-dependent transcriptional activity via the Akt/GSK3Î² pathway to promote cell survival upon treatment with AÎ²(25-35.) Our data demonstrate that LPLI has a prosurvival effect on AÎ²-induced apoptosis and may be an effective therapeutic strategy in treating AD by targeting GSK3Î².

Background: Apoptosis induced by amyloid Î² peptide (AÎ²) is thought to associate with the pathogenesis of Alzheimer disease (AD). Accumulating evidence shows that low-power laser irradiation (LPLI) is capable of reducing AÎ²-induced apoptosis. However, the underlying mechanisms remain unclear. In this study, we report a novel molecular mechanism by which LPLI attenuates AÎ²(25-35)-induced apoptosis through the Akt/GSK3Î²/Î²-catenin pathway. We found that Akt activated by LPLI interacted with GSK3Î² and phosphorylated it on Ser9 in the presence of AÎ²(25-35), which resulted in the inhibition of GSK3Î². Furthermore, LPLI increased the nuclear translocation of Î²-catenin and enhanced its T cell factor/lymphocyte enhancer factor-dependent transcriptional activity via the Akt/GSK3Î² pathway to promote cell survival upon treatment with AÎ²(25-35.) Our data demonstrate that LPLI has a prosurvival effect on AÎ²-induced apoptosis and may be an effective therapeutic strategy in treating AD by targeting GSK3Î².

Abstract: Abstract Apoptosis induced by amyloid Î² peptide (AÎ²) is thought to associate with the pathogenesis of Alzheimer disease (AD). Accumulating evidence shows that low-power laser irradiation (LPLI) is capable of reducing AÎ²-induced apoptosis. However, the underlying mechanisms remain unclear. In this study, we report a novel molecular mechanism by which LPLI attenuates AÎ²(25-35)-induced apoptosis through the Akt/GSK3Î²/Î²-catenin pathway. We found that Akt activated by LPLI interacted with GSK3Î² and phosphorylated it on Ser9 in the presence of AÎ²(25-35), which resulted in the inhibition of GSK3Î². Furthermore, LPLI increased the nuclear translocation of Î²-catenin and enhanced its T cell factor/lymphocyte enhancer factor-dependent transcriptional activity via the Akt/GSK3Î² pathway to promote cell survival upon treatment with AÎ²(25-35.) Our data demonstrate that LPLI has a prosurvival effect on AÎ²-induced apoptosis and may be an effective therapeutic strategy in treating AD by targeting GSK3Î². Copyright Â© 2012 Elsevier Inc. All rights reserved.

Original Source: http://www.ncbi.nlm.nih.gov/pubmed/22917976

### Low-level light therapy improves cortical metabolic capacity and memory retention.

Rojas JC1, Bruchey AK, Gonzalez-Lima F. - J Alzheimers Dis. 2012;32(3):741-52. doi: 10.3233/JAD-2012-120817. ()
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Intro: Cerebral hypometabolism characterizes mild cognitive impairment and Alzheimer's disease. Low-level light therapy (LLLT) enhances the metabolic capacity of neurons in culture through photostimulation of cytochrome oxidase, the mitochondrial enzyme that catalyzes oxygen consumption in cellular respiration. Growing evidence supports that neuronal metabolic enhancement by LLLT positively impacts neuronal function in vitro and in vivo. Based on its effects on energy metabolism, it is proposed that LLLT will also affect the cerebral cortex in vivo and modulate higher-order cognitive functions such as memory. In vivo effects of LLLT on brain and behavior are poorly characterized. We tested the hypothesis that in vivo LLLT facilitates cortical oxygenation and metabolic energy capacity and thereby improves memory retention. Specifically, we tested this hypothesis in rats using fear extinction memory, a form of memory modulated by prefrontal cortex activation. Effects of LLLT on brain metabolism were determined through measurement of prefrontal cortex oxygen concentration with fluorescent quenching oximetry and by quantitative cytochrome oxidase histochemistry. Experiment 1 verified that LLLT increased the rate of oxygen consumption in the prefrontal cortex in vivo. Experiment 2 showed that LLLT-treated rats had an enhanced extinction memory as compared to controls. Experiment 3 showed that LLLT reduced fear renewal and prevented the reemergence of extinguished conditioned fear responses. Experiment 4 showed that LLLT induced hormetic dose-response effects on the metabolic capacity of the prefrontal cortex. These data suggest that LLLT can enhance cortical metabolic capacity and retention of extinction memories, and implicate LLLT as a novel intervention to improve memory.

Background: Cerebral hypometabolism characterizes mild cognitive impairment and Alzheimer's disease. Low-level light therapy (LLLT) enhances the metabolic capacity of neurons in culture through photostimulation of cytochrome oxidase, the mitochondrial enzyme that catalyzes oxygen consumption in cellular respiration. Growing evidence supports that neuronal metabolic enhancement by LLLT positively impacts neuronal function in vitro and in vivo. Based on its effects on energy metabolism, it is proposed that LLLT will also affect the cerebral cortex in vivo and modulate higher-order cognitive functions such as memory. In vivo effects of LLLT on brain and behavior are poorly characterized. We tested the hypothesis that in vivo LLLT facilitates cortical oxygenation and metabolic energy capacity and thereby improves memory retention. Specifically, we tested this hypothesis in rats using fear extinction memory, a form of memory modulated by prefrontal cortex activation. Effects of LLLT on brain metabolism were determined through measurement of prefrontal cortex oxygen concentration with fluorescent quenching oximetry and by quantitative cytochrome oxidase histochemistry. Experiment 1 verified that LLLT increased the rate of oxygen consumption in the prefrontal cortex in vivo. Experiment 2 showed that LLLT-treated rats had an enhanced extinction memory as compared to controls. Experiment 3 showed that LLLT reduced fear renewal and prevented the reemergence of extinguished conditioned fear responses. Experiment 4 showed that LLLT induced hormetic dose-response effects on the metabolic capacity of the prefrontal cortex. These data suggest that LLLT can enhance cortical metabolic capacity and retention of extinction memories, and implicate LLLT as a novel intervention to improve memory.

Abstract: Abstract Cerebral hypometabolism characterizes mild cognitive impairment and Alzheimer's disease. Low-level light therapy (LLLT) enhances the metabolic capacity of neurons in culture through photostimulation of cytochrome oxidase, the mitochondrial enzyme that catalyzes oxygen consumption in cellular respiration. Growing evidence supports that neuronal metabolic enhancement by LLLT positively impacts neuronal function in vitro and in vivo. Based on its effects on energy metabolism, it is proposed that LLLT will also affect the cerebral cortex in vivo and modulate higher-order cognitive functions such as memory. In vivo effects of LLLT on brain and behavior are poorly characterized. We tested the hypothesis that in vivo LLLT facilitates cortical oxygenation and metabolic energy capacity and thereby improves memory retention. Specifically, we tested this hypothesis in rats using fear extinction memory, a form of memory modulated by prefrontal cortex activation. Effects of LLLT on brain metabolism were determined through measurement of prefrontal cortex oxygen concentration with fluorescent quenching oximetry and by quantitative cytochrome oxidase histochemistry. Experiment 1 verified that LLLT increased the rate of oxygen consumption in the prefrontal cortex in vivo. Experiment 2 showed that LLLT-treated rats had an enhanced extinction memory as compared to controls. Experiment 3 showed that LLLT reduced fear renewal and prevented the reemergence of extinguished conditioned fear responses. Experiment 4 showed that LLLT induced hormetic dose-response effects on the metabolic capacity of the prefrontal cortex. These data suggest that LLLT can enhance cortical metabolic capacity and retention of extinction memories, and implicate LLLT as a novel intervention to improve memory.

Original Source: http://www.ncbi.nlm.nih.gov/pubmed/22850314

### 670 nm laser light and EGCG complementarily reduce amyloid-Î² aggregates in human neuroblastoma cells: basis for treatment of Alzheimer's disease?

Sommer AP1, Bieschke J, Friedrich RP, Zhu D, Wanker EE, Fecht HJ, Mereles D, Hunstein W. - Photomed Laser Surg. 2012 Jan;30(1):54-60. doi: 10.1089/pho.2011.3073. Epub 2011 Oct 26. ()
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Intro: The aim of the present study is to present the results of in vitro experiments with possible relevance in the treatment of Alzheimer's disease (AD).

Background: The aim of the present study is to present the results of in vitro experiments with possible relevance in the treatment of Alzheimer's disease (AD).

Methods: Despite intensive research efforts, there is no treatment for AD. One root cause of AD is the extra- and intracellular deposition of amyloid-beta (AÎ²) fibrils in the brain. Recently, it was shown that extracellular AÎ² can enter brain cells, resulting in neurotoxicity.

Results: After internalization of AÎ²(42) into human neuroblastoma (SH-EP) cells, they were irradiated with moderately intense 670-nm laser light (1000 Wm(-2)) and/or treated with epigallocatechin gallate (EGCG).

Conclusions: In irradiated cells, AÎ²(42) aggregate amounts were significantly lower than in nonirradiated cells. Likewise, in EGCG-treated cells, AÎ²(42) aggregate amounts were significantly lower than in non-EGCG-treated cells. Except for the cells simultaneously laden with AÎ²(42) and EGCG, there was a significant increase in cell numbers in response to laser irradiation. EGCG alone had no effect on cell proliferation. Laser irradiation significantly increased ATP levels in AÎ²(42)-free cells, when compared to nonirradiated cells. Laser-induced clearance of AÎ²(42) aggregates occurred at the expense of cellular ATP.

Original Source: http://www.ncbi.nlm.nih.gov/pubmed/22029866

### Inhibition of AÎ²(25-35)-induced cell apoptosis by low-power-laser-irradiation (LPLI) through promoting Akt-dependent YAP cytoplasmic translocation.

Zhang H1, Wu S, Xing D. - Cell Signal. 2012 Jan;24(1):224-32. doi: 10.1016/j.cellsig.2011.09.004. Epub 2011 Sep 14. ()
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Intro: Deposition of amyloid-Î²-peptide (AÎ²) in the brain is considered a pathological hallmark of Alzheimer's disease (AD). Our previous studies show that Yes-associated protein (YAP) is involved in the regulation of apoptosis induced by AÎ²(25-35) through YAP nuclear translocation and its pro-apoptotic function is mediated by its interaction with p73. In the present study, we first found that Low-power laser irradiation (LPLI) promoted YAP cytoplasmic translocation and inhibited AÎ²(25-35)-induced YAP nuclear translocation. Moreover, the cytoplasmic translocation was in an Akt-dependent manner. Activated Akt by LPLI phosphorylated YAP on ser127 (S127) and resulted in decreasing the interaction between YAP and p73, and in suppressing the proapoptotic gene bax expression following AÎ²(25-35) treatment. Inhibition of Akt expression by siRNA significantly abolished the effect of LPLI. More importantly, LPLI could inhibit AÎ²(25-35)-induced cell apoptosis through activation of Akt/YAP/p73 signaling pathway. Therefore, our findings first suggest that YAP may be a therapeutic target and these results directly point to a potential therapeutic strategy for the treatment of AD through Akt/YAP/p73 signaling pathway with LPLI.

Background: Deposition of amyloid-Î²-peptide (AÎ²) in the brain is considered a pathological hallmark of Alzheimer's disease (AD). Our previous studies show that Yes-associated protein (YAP) is involved in the regulation of apoptosis induced by AÎ²(25-35) through YAP nuclear translocation and its pro-apoptotic function is mediated by its interaction with p73. In the present study, we first found that Low-power laser irradiation (LPLI) promoted YAP cytoplasmic translocation and inhibited AÎ²(25-35)-induced YAP nuclear translocation. Moreover, the cytoplasmic translocation was in an Akt-dependent manner. Activated Akt by LPLI phosphorylated YAP on ser127 (S127) and resulted in decreasing the interaction between YAP and p73, and in suppressing the proapoptotic gene bax expression following AÎ²(25-35) treatment. Inhibition of Akt expression by siRNA significantly abolished the effect of LPLI. More importantly, LPLI could inhibit AÎ²(25-35)-induced cell apoptosis through activation of Akt/YAP/p73 signaling pathway. Therefore, our findings first suggest that YAP may be a therapeutic target and these results directly point to a potential therapeutic strategy for the treatment of AD through Akt/YAP/p73 signaling pathway with LPLI.

Abstract: Abstract Deposition of amyloid-Î²-peptide (AÎ²) in the brain is considered a pathological hallmark of Alzheimer's disease (AD). Our previous studies show that Yes-associated protein (YAP) is involved in the regulation of apoptosis induced by AÎ²(25-35) through YAP nuclear translocation and its pro-apoptotic function is mediated by its interaction with p73. In the present study, we first found that Low-power laser irradiation (LPLI) promoted YAP cytoplasmic translocation and inhibited AÎ²(25-35)-induced YAP nuclear translocation. Moreover, the cytoplasmic translocation was in an Akt-dependent manner. Activated Akt by LPLI phosphorylated YAP on ser127 (S127) and resulted in decreasing the interaction between YAP and p73, and in suppressing the proapoptotic gene bax expression following AÎ²(25-35) treatment. Inhibition of Akt expression by siRNA significantly abolished the effect of LPLI. More importantly, LPLI could inhibit AÎ²(25-35)-induced cell apoptosis through activation of Akt/YAP/p73 signaling pathway. Therefore, our findings first suggest that YAP may be a therapeutic target and these results directly point to a potential therapeutic strategy for the treatment of AD through Akt/YAP/p73 signaling pathway with LPLI. Copyright Â© 2011 Elsevier Inc. All rights reserved.

Original Source: http://www.ncbi.nlm.nih.gov/pubmed/21945154

### Long-term safety of single and multiple infrared transcranial laser treatments in Sprague-Dawley rats.

McCarthy TJ1, De Taboada L, Hildebrandt PK, Ziemer EL, Richieri SP, Streeter J. - Photomed Laser Surg. 2010 Oct;28(5):663-7. doi: 10.1089/pho.2009.2581. ()
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Intro: Growing interest exists in the use of near-infrared laser therapies for the treatment of numerous neurologic conditions, including acute ischemic stroke, traumatic brain injury, Parkinson's disease, and Alzheimer's disease. In consideration of these trends, the objective of this study was to evaluate the long-term safety of transcranial laser therapy with continuous-wave (CW) near-infrared laser light (wavelength, 808 Â± 10 nm, 2-mm diameter) with a nominal radiant power of 70 mW; power density, 2,230 mW/cm(2), and energy density, 268 J/cm(2) at the scalp (10 mW/cm(2) and 1.2 J/cm(2) at the cerebral cortical surface) in healthy Sprague-Dawley rats.

Background: Growing interest exists in the use of near-infrared laser therapies for the treatment of numerous neurologic conditions, including acute ischemic stroke, traumatic brain injury, Parkinson's disease, and Alzheimer's disease. In consideration of these trends, the objective of this study was to evaluate the long-term safety of transcranial laser therapy with continuous-wave (CW) near-infrared laser light (wavelength, 808 Â± 10 nm, 2-mm diameter) with a nominal radiant power of 70 mW; power density, 2,230 mW/cm(2), and energy density, 268 J/cm(2) at the scalp (10 mW/cm(2) and 1.2 J/cm(2) at the cerebral cortical surface) in healthy Sprague-Dawley rats.

Abstract: Abstract BACKGROUND AND OBJECTIVE: Growing interest exists in the use of near-infrared laser therapies for the treatment of numerous neurologic conditions, including acute ischemic stroke, traumatic brain injury, Parkinson's disease, and Alzheimer's disease. In consideration of these trends, the objective of this study was to evaluate the long-term safety of transcranial laser therapy with continuous-wave (CW) near-infrared laser light (wavelength, 808 Â± 10 nm, 2-mm diameter) with a nominal radiant power of 70 mW; power density, 2,230 mW/cm(2), and energy density, 268 J/cm(2) at the scalp (10 mW/cm(2) and 1.2 J/cm(2) at the cerebral cortical surface) in healthy Sprague-Dawley rats. MATERIALS AND METHODS: In this study, 120 anesthetized rats received sequential transcranial laser treatments to the right and left parietal areas of the head on the same day (minimum of 5 min between irradiation of each side), on either Day 1 or on each of Days 1, 3, and 5. Sixty anesthetized rats served as sham controls. Rats were evaluated 1 year after treatment for abnormalities in clinical hematology and brain and pituitary gland histopathology. RESULTS: No toxicologically important differences were found in the clinical hematology results between sham-control and laser-treated rats for any hematologic parameters examined. All values fell within historic control reference ranges for aged Sprague-Dawley rats. Similarly, brain and pituitary gland histopathology showed no treatment-related abnormalities or induced neoplasia. CONCLUSIONS: Single and multiple applications of transcranial laser therapy with 808-nm CW laser light at a nominal power density of 10 mW/cm(2) at the surface of the cerebral cortex appears to be safe in Sprague-Dawley rats 1 year after treatment.

Methods: In this study, 120 anesthetized rats received sequential transcranial laser treatments to the right and left parietal areas of the head on the same day (minimum of 5 min between irradiation of each side), on either Day 1 or on each of Days 1, 3, and 5. Sixty anesthetized rats served as sham controls. Rats were evaluated 1 year after treatment for abnormalities in clinical hematology and brain and pituitary gland histopathology.

Results: No toxicologically important differences were found in the clinical hematology results between sham-control and laser-treated rats for any hematologic parameters examined. All values fell within historic control reference ranges for aged Sprague-Dawley rats. Similarly, brain and pituitary gland histopathology showed no treatment-related abnormalities or induced neoplasia.

Conclusions: Single and multiple applications of transcranial laser therapy with 808-nm CW laser light at a nominal power density of 10 mW/cm(2) at the surface of the cerebral cortex appears to be safe in Sprague-Dawley rats 1 year after treatment.

Original Source: http://www.ncbi.nlm.nih.gov/pubmed/20961232

### Low energy laser light (632.8 nm) suppresses amyloid-Î² peptide-induced oxidative and inflammatory responses in astrocytes.

Yang X1, Askarova S, Sheng W, Chen JK, Sun AY, Sun GY, Yao G, Lee JC. - Neuroscience. 2010 Dec 15;171(3):859-68. doi: 10.1016/j.neuroscience.2010.09.025. Epub 2010 Sep 25. ()
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Intro: Oxidative stress and inflammation are important processes in the progression of Alzheimer's disease (AD). Recent studies have implicated the role of amyloid Î²-peptides (AÎ²) in mediating these processes. In astrocytes, oligomeric AÎ² induces the assembly of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complexes resulting in its activation to produce anionic superoxide. AÎ² also promotes production of pro-inflammatory factors in astrocytes. Since low energy laser has previously been reported to attenuate oxidative stress and inflammation in biological systems, the objective of this study was to examine whether this type of laser light was able to abrogate the oxidative and inflammatory responses induced by AÎ². Primary rat astrocytes were exposed to Helium-Neon laser (Î»=632.8 nm), followed by the treatment with oligomeric AÎ². Primary rat astrocytes were used to measure AÎ²-induced production of superoxide anions using fluorescence microscopy of dihydroethidium (DHE), assembly of NADPH oxidase subunits by the colocalization between the cytosolic p47(phox) subunit and the membrane gp91(phox) subunit using fluorescent confocal microscopy, phosphorylation of cytosolic phospholipase A(2) cPLA(2) and expressions of pro-inflammatory factors including interleukin-1Î² (IL-1Î²) and inducible nitric-oxide synthase (iNOS) using Western blot Analysis. Our data showed that laser light at 632.8 nm suppressed AÎ²-induced superoxide production, colocalization between NADPH oxidase gp91(phox) and p47(phox) subunits, phosphorylation of cPLA(2,) and the expressions of IL-1Î² and iNOS in primary astrocytes. We demonstrated for the first time that 632.8 nm laser was capable of suppressing cellular pathways of oxidative stress and inflammatory responses critical in the pathogenesis in AD. This study should prove to provide the groundwork for further investigations for the potential use of laser therapy as a treatment for AD.

Background: Oxidative stress and inflammation are important processes in the progression of Alzheimer's disease (AD). Recent studies have implicated the role of amyloid Î²-peptides (AÎ²) in mediating these processes. In astrocytes, oligomeric AÎ² induces the assembly of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complexes resulting in its activation to produce anionic superoxide. AÎ² also promotes production of pro-inflammatory factors in astrocytes. Since low energy laser has previously been reported to attenuate oxidative stress and inflammation in biological systems, the objective of this study was to examine whether this type of laser light was able to abrogate the oxidative and inflammatory responses induced by AÎ². Primary rat astrocytes were exposed to Helium-Neon laser (Î»=632.8 nm), followed by the treatment with oligomeric AÎ². Primary rat astrocytes were used to measure AÎ²-induced production of superoxide anions using fluorescence microscopy of dihydroethidium (DHE), assembly of NADPH oxidase subunits by the colocalization between the cytosolic p47(phox) subunit and the membrane gp91(phox) subunit using fluorescent confocal microscopy, phosphorylation of cytosolic phospholipase A(2) cPLA(2) and expressions of pro-inflammatory factors including interleukin-1Î² (IL-1Î²) and inducible nitric-oxide synthase (iNOS) using Western blot Analysis. Our data showed that laser light at 632.8 nm suppressed AÎ²-induced superoxide production, colocalization between NADPH oxidase gp91(phox) and p47(phox) subunits, phosphorylation of cPLA(2,) and the expressions of IL-1Î² and iNOS in primary astrocytes. We demonstrated for the first time that 632.8 nm laser was capable of suppressing cellular pathways of oxidative stress and inflammatory responses critical in the pathogenesis in AD. This study should prove to provide the groundwork for further investigations for the potential use of laser therapy as a treatment for AD.

Original Source: http://www.ncbi.nlm.nih.gov/pubmed/20884337

### Low-power laser irradiation inhibiting Abeta25-35-induced PC12 cell apoptosis via PKC activation.

Zhang L1, Xing D, Zhu D, Chen Q. - Cell Physiol Biochem. 2008;22(1-4):215-22. doi: 10.1159/000149799. Epub 2008 Jul 25. ()
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Intro: Apoptosis is a contributing pathophysiological mechanism of Alzheimer's disease (AD). Recently, low-power laser irradiation (LPLI) has been applied to moderate AD, but the underlying mechanism remains unknown. In this study, the techniques of fluorescence resonance energy transfer (FRET) and real-time quantitative RT-PCR were used to investigate the anti-apoptotic mechanism of LPLI. Rat pheochromocytoma (PC12) cells were treated with amyloid beta 25-35 (Abeta(25-35)) for induction of apoptosis before LPLI treatment. The cell viability assays and morphological examinations show that low fluence of LPLI (0.156 J/cm(2)-0.624 J/cm(2)) could inhibit the cells apoptosis. An increase of PKC activation was dynamically monitored in the cells treated with PMA (specific activator of PKC), LPLI only or Abeta(25-35) followed by 5 min LPLI treatment, respectively. However, the effect of LPLI activating PKC could be inhibited by Go 6983 (specific inhibitor of PKC). Similar results were obtained by using Western blot analysis. Furthermore, LPLI involved an increase in mRNA of the cell survival member bcl-xl and a decrease in the up-regulation of cell death member bax mRNA caused by Abeta(25-35). Further data show that low fluence of LPLI could reverse the increased level of bax/bcl-xl mRNA ratio caused by Abeta(25-35) treatment. In addition, Go 6983 could inhibit the decreased level of bax/bcl-xl mRNA ratio. Taken together, these data clearly indicate that LPLI inhibited Abeta(25-35)-induced PC12 cell apoptosis via PKC-mediated regulation of bax/bcl-xl mRNA ratio.

Background: Apoptosis is a contributing pathophysiological mechanism of Alzheimer's disease (AD). Recently, low-power laser irradiation (LPLI) has been applied to moderate AD, but the underlying mechanism remains unknown. In this study, the techniques of fluorescence resonance energy transfer (FRET) and real-time quantitative RT-PCR were used to investigate the anti-apoptotic mechanism of LPLI. Rat pheochromocytoma (PC12) cells were treated with amyloid beta 25-35 (Abeta(25-35)) for induction of apoptosis before LPLI treatment. The cell viability assays and morphological examinations show that low fluence of LPLI (0.156 J/cm(2)-0.624 J/cm(2)) could inhibit the cells apoptosis. An increase of PKC activation was dynamically monitored in the cells treated with PMA (specific activator of PKC), LPLI only or Abeta(25-35) followed by 5 min LPLI treatment, respectively. However, the effect of LPLI activating PKC could be inhibited by Go 6983 (specific inhibitor of PKC). Similar results were obtained by using Western blot analysis. Furthermore, LPLI involved an increase in mRNA of the cell survival member bcl-xl and a decrease in the up-regulation of cell death member bax mRNA caused by Abeta(25-35). Further data show that low fluence of LPLI could reverse the increased level of bax/bcl-xl mRNA ratio caused by Abeta(25-35) treatment. In addition, Go 6983 could inhibit the decreased level of bax/bcl-xl mRNA ratio. Taken together, these data clearly indicate that LPLI inhibited Abeta(25-35)-induced PC12 cell apoptosis via PKC-mediated regulation of bax/bcl-xl mRNA ratio.

Abstract: Abstract Apoptosis is a contributing pathophysiological mechanism of Alzheimer's disease (AD). Recently, low-power laser irradiation (LPLI) has been applied to moderate AD, but the underlying mechanism remains unknown. In this study, the techniques of fluorescence resonance energy transfer (FRET) and real-time quantitative RT-PCR were used to investigate the anti-apoptotic mechanism of LPLI. Rat pheochromocytoma (PC12) cells were treated with amyloid beta 25-35 (Abeta(25-35)) for induction of apoptosis before LPLI treatment. The cell viability assays and morphological examinations show that low fluence of LPLI (0.156 J/cm(2)-0.624 J/cm(2)) could inhibit the cells apoptosis. An increase of PKC activation was dynamically monitored in the cells treated with PMA (specific activator of PKC), LPLI only or Abeta(25-35) followed by 5 min LPLI treatment, respectively. However, the effect of LPLI activating PKC could be inhibited by Go 6983 (specific inhibitor of PKC). Similar results were obtained by using Western blot analysis. Furthermore, LPLI involved an increase in mRNA of the cell survival member bcl-xl and a decrease in the up-regulation of cell death member bax mRNA caused by Abeta(25-35). Further data show that low fluence of LPLI could reverse the increased level of bax/bcl-xl mRNA ratio caused by Abeta(25-35) treatment. In addition, Go 6983 could inhibit the decreased level of bax/bcl-xl mRNA ratio. Taken together, these data clearly indicate that LPLI inhibited Abeta(25-35)-induced PC12 cell apoptosis via PKC-mediated regulation of bax/bcl-xl mRNA ratio. Copyright 2008 S. Karger AG, Basel.

Methods: Copyright 2008 S. Karger AG, Basel.

Original Source: http://www.ncbi.nlm.nih.gov/pubmed/18769048

### Preliminary results of highly localized plantar irradiation with low incident levels of mid-infrared energy which contributes to the prevention of dementia associated with underlying diabetes mellitus.

Ryotokuji K, Ishimaru K, Kihara K, Nakajima T, Otani S, Namiki Y - (Publication)
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Stress-free Therapy Research Center, Ryotokuji University.

BACKGROUND AND AIMS: The incidence of vascular dementia (VD) and Alzheimer's disease (AD) has recently increased and the prevention of progression of these diseases is very difficult.

RESULTS: The application of pinpoint plantar long-wavelength infrared light irradiation (PP-LILI) to a patient's sole, at the point where the line drawn between the first and second metatarsal heads intersects with the vertical line from the medial malleolus, was effective in increasing blood flow to the facial artery, elevating high- density lipoprotein cholesterol (HDL-C) levels, and reducing insulin resistance.

CONCLUSIONS: We found that these effects of PP-LILI might be helpful for preventing VD and AD, conditions that are becoming a social problem in an aging Japanese society.

Original Source: https://www.ncbi.nlm.nih.gov/pubmed/?term=25941422

### Low-Level Laser Therapy Ameliorates Disease Progression in a Mouse Model of Alzheimer's Disease.

Farfara D, Tuby H, Trudler D, Doron-Mandel E, Maltz L, Vassar RJ, Frenkel D, Oron U - (Publication)
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Department of Neurobiology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.

Low-level laser therapy (LLLT) has been used to treat inflammation, tissue healing, and repair processes. We recently reported that LLLT to the bone marrow (BM) led to proliferation of mesenchymal stem cells (MSCs) and their homing in the ischemic heart suggesting its role in regenerative medicine. The aim of the present study was to investigate the ability of LLLT to stimulate MSCs of autologous BM in order to affect neurological behavior and beta-amyloid burden in progressive stages of Alzheimer's disease (AD) mouse model. MSCs from wild-type mice stimulated with LLLT showed to increase their ability to maturate towards a monocyte lineage and to increase phagocytosis activity towards soluble amyloid beta (Abeta). Furthermore, weekly LLLT to BM of AD mice for 2 months, starting at 4 months of age (progressive stage of AD), improved cognitive capacity and spatial learning, as compared to sham-treated AD mice. Histology revealed a significant reduction in Abeta brain burden. Our results suggest the use of LLLT as a therapeutic application in progressive stages of AD and imply its role in mediating MSC therapy in brain amyloidogenic diseases.

Original Source: https://www.ncbi.nlm.nih.gov/pubmed/?term=24994540

### Photobiomodulation with near infrared light mitigates Alzheimer's disease- related pathology in cerebral cortex - evidence from two transgenic mouse models.

Purushothuman S, Johnstone DM, Nandasena C, Mitrofanis J, Stone J - (Publication)
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Bosch Institute, University of Sydney NSW 2006, Australia. Daniel.Johnstone@sydney.edu.au

INTRODUCTION: Previous work has demonstrated the efficacy of irradiating tissue with red to infrared light in mitigating cerebral pathology and degeneration in animal models of stroke, traumatic brain injury, parkinsonism and Alzheimer's disease (AD). Using mouse models, we explored the neuroprotective effect of near infrared light (NIr) treatment, delivered at an age when substantial pathology is already present in the cerebral cortex.

METHODS: We studied two mouse models with AD- related pathologies: the K369I tau transgenic model (K3), engineered to develop neurofibrillary tangles, and the APPswe/PSEN1dE9 transgenic model (APP/PS1), engineered to develop amyloid plaques. Mice were treated with NIr 20 times over a four-week period and histochemistry was used to quantify AD- related pathological hallmarks and other markers of cell damage in the neocortex and hippocampus.

RESULTS: In the K3 mice, NIr treatment was associated with a reduction in hyperphosphorylated tau, neurofibrillary tangles and oxidative stress markers (4-hydroxynonenal and 8-hydroxy-2'- deoxyguanosine) to near wildtype levels in the neocortex and hippocampus, and with a restoration of expression of the mitochondrial marker cytochrome c oxidase in surviving neurons. In the APP/PS1 mice, NIr treatment was associated with a reduction in the size and number of amyloid-beta plaques in the neocortex and hippocampus.

CONCLUSIONS: Our results, in two transgenic mouse models, suggest that NIr may have potential as an effective, minimally-invasive intervention for mitigating, and even reversing, progressive cerebral degenerations.

Original Source: https://www.ncbi.nlm.nih.gov/pubmed/?term=24387311

### Laser acupuncture improves memory impairment in an animal model of Alzheimer's disease.

Sutalangka C, Wattanathorn J, Muchimapura S, Thukham-Mee W, Wannanon P, Tong-Un T - (Publication)
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Department of Physiology (Neuroscience Program), Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; Integrative Complementary Alternative Medicine Research and Development Group, Khon Kaen University, Khon Kaen, Thailand.

The burden of Alzheimer's disease is continually rising globally, especially in the Asia-Pacific region. Unfortunately, the efficacy of the therapeutic strategy is still very limited. Because the effect of acupuncture at HT7 can improve learning and memory, the beneficial effect of laser acupuncture, a noninvasive form of acupuncture, at HT7 on memory improvement in patients with Alzheimer's disease has been a focus of research. To elucidate this issue, we used AF64A, a cholinotoxin, to induce memory impairment in male Wistar rats, which weighed 180-220 g. Then, the animals were treated with laser acupuncture either at HT7 or at a sham acupoint once daily for 10 minutes for a period of 14 days.

Spatial memory assessments were performed at 1, 7, and 14 days after AF64A administration and at the end of the experiment, and the changes in the malondialdehyde (MDA) level and in the superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and acetylcholinesterase (AChE) activities in the hippocampus were recorded. The results showed that laser acupuncture significantly suppressed AChE activity in the hippocampus. Although laser acupuncture enhanced SOD and CAT activities, no reduction in MDA level in this area was observed. Therefore, laser acupuncture at HT7 is a potential strategy to attenuate memory impairment in patients with Alzheimer's disease. However, further research, especially on the toxicity of laser acupuncture following repetitive exposure, is essential.

Original Source: https://www.ncbi.nlm.nih.gov/pubmed/?term=24139462

### Low-Level Laser Therapy Rescues Dendrite Atrophy via Upregulating BDNF Expression: Implications for Alzheimer's Disease.

Meng C, He Z, Xing D - (Publication)
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MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China.

Downregulation of brain-derived neurotrophic factor (BDNF) in the hippocampus occurs early in the progression of Alzheimer's disease (AD). Since BDNF plays a critical role in neuronal survival and dendrite growth, BDNF upregulation may contribute to rescue dendrite atrophy and cell loss in AD. Low- level laser therapy (LLLT) has been demonstrated to regulate neuronal function both in vitro and in vivo. In the present study, we found that LLLT rescued neurons loss and dendritic atrophy via upregulation of BDNF in both Abeta-treated hippocampal neurons and cultured APP/PS1 mouse hippocampal neurons. Photoactivation of transcription factor CRE-binding protein (CREB) increased both BDNF mRNA and protein expression, since knockdown CREB blocked the effects of LLLT. Furthermore, CREB-regulated transcription was in an ERK-dependent manner. Inhibition of ERK attenuated the DNA-binding efficiency of CREB to BDNF promoter. In addition, dendrite growth was improved after LLLT, characterized by upregulation of Rac1 activity and PSD-95 expression, and the increase in length, branching, and spine density of dendrites in hippocampal neurons. Together, these studies suggest that upregulation of BDNF with LLLT by activation of ERK/CREB pathway can ameliorate Abeta-induced neurons loss and dendritic atrophy, thus identifying a novel pathway by which LLLT protects against Abeta-induced neurotoxicity.

Our research may provide a feasible therapeutic approach to control the progression of AD. J Neurosci 2013 Aug 14 33(33) 13505-17

Original Source: https://www.ncbi.nlm.nih.gov/pubmed/?term=23946409

### 670 nm Laser Light and EGCG Complementarily Reduce Amyloid-beta Aggregates in Human Neuroblastoma Cells: Basis for Treatment of Alzheimer's Disease?

Sommer AP, Bieschke J, Friedrich RP, Zhu D, Wanker EE, Fecht HJ, Mereles D, Hunstein W - (Publication)
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1 Institute of Micro and Nanomaterials, Nanobionic Laboratory, University of Ulm , Ulm, Germany .

Abstract Objective: The aim of the present study is to present the results of in vitro experiments with possible relevance in the treatment of Alzheimer's disease (AD).

Background Data: Despite intensive research efforts, there is no treatment for AD. One root cause of AD is the extra- and intracellular deposition of amyloid-beta (Abeta) fibrils in the brain. Recently, it was shown that extracellular Abeta can enter brain cells, resulting in neurotoxicity.

Methods: After internalization of Abeta(42) into human neuroblastoma (SH-EP) cells, they were irradiated with moderately intense 670-nm laser light (1000 Wm (-2)) and/or treated with epigallocatechin gallate (EGCG).

Results: In irradiated cells, Abeta(42) aggregate amounts were significantly lower than in nonirradiated cells. Likewise, in EGCG-treated cells, Abeta(42) aggregate amounts were significantly lower than in non-EGCG-treated cells. Except for the cells simultaneously laden with Abeta(42) and EGCG, there was a significant increase in cell numbers in response to laser irradiation. EGCG alone had no effect on cell proliferation. Laser irradiation significantly increased ATP levels in Abeta(42)-free cells, when compared to nonirradiated cells. Laser- induced clearance of Abeta(42) aggregates occurred at the expense of cellular ATP.

Conclusions: Irradiation with moderate levels of 670-nm light and EGCG supplementation complementarily reduces Abeta aggregates in SH-EP cells. Transcranial penetration of moderate levels of red to near-infrared (NIR) light has already been amply exploited in the treatment of patients with acute stroke; the blood-brain barrier (BBB) penetration of EGCG has been demonstrated in animals. We hope that our approach will inspire a practical therapy for AD.

Original Source: https://www.ncbi.nlm.nih.gov/pubmed/?term=22029866

### Light Therapy to Stem Cells: A New Therapeutic Approach in Regenerative Medicine – A Mini Review

Amir Oron1 and Uri Oron2* - 15 April 2015 (Publication)
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Abstract

Regenerative capacity following injury or an ischemic event is confined to non mammalian vertebrates. Mammals have a limited capacity to restore organs

following injury to organs like the liver and skeletal muscles but practically no ability to regenerate organs like the heart or brain following an ischemic event

or injury. We tried a new approach in cell based therapy to improve regeneration in various organs following ischemic injury. Low-level laser therapy (LLLT)

which has photobiostimulating effects on cells was delivered to autologous bone marrow (BM) that is enriched with stem cells and various progenitor cells, in

order to induce the cells in the BM for the benefit of the injured /ischemic organs. In a model of induced myocardial infarction (MI) in rats laser application

to the BM caused a marked and significant decrease (79%) in infarct size (scarring) 3 weeks post-MI. It was also found that a significantly higher density of

c-kit positive cells (a marker of mesenchymal stem cells) in the myocardium of laser-treated rats relative to non-treated rat’s post-MI. The novel approach

presented in this study, of the use of stem cells for cell therapy to the infracted heart, avoids the need to isolate millions of stem cells, to grow them in vitro and

to inject them back into the patient. In the same line of rationale we tried to find whether LLLT to the BM could be beneficial also to kidney impairment after

ischemic reperfusion injury (IRI) to the rat kidney. C-kit positive cell density in kidneys post-IRI and laser-treatment was significantly (p=0.05) 2.4-fold higher

compared to the non laser treated group. Creatinine, blood urea nitrogen, and cystatin-C levels were significantly lower in the laser-treated rats as compared

to non-treated ones. The effect of LLLT delivery to BM was also tested on Alzheimer’s disease (AD) mice in their late stage of the disease. Mice were given

multiple (every 10 days) LLLT to BM from age 4 to 6 months. It was found that in the treated AD-mice neurological tests (Fear and Cognitive tests) revealed

a significantly (p<0.05) better neurological performance and cognitive capacity compared to the non-treated AD mice. Furthermore, concomitantly with the

improved neurological performance, ß-amyloid density in the hippocampal region of the brains was revealed to be significantly less in the laser-treated mice

as compared to control. In conclusion, a novel approach, of applying LLLT to autologeous BM in order to induce stem cells that are consequently recruited to the

injured/ischemic organ leading to a marked beneficial effect post-ischemic event or degenerative process is presented. This approach is novel in the respect

that it is stimulating the patient’s own abilities to initiate a regenerative response in an organ by the utilization of light. The possibility that this approach can

also be applied to other ischemic/injured organs or organs undergoing degenerative processes (i.e. neurodegenerative diseases), with consequent beneficial

effects, cannot be ruled out

CELL THERAPY FOR INJURED/ISCHEMIC ORGANS

Clinical trials have lately been implemented in a growing

abundance due to the extensive research and new approaches of

cell based therapies for the reconstruction of impaired organs.

Regenerative capacity following injury or an ischemic event

is confined to non mammalian vertebrates. In particular, fish

and primitive amphibians can regenerate organs like the heart,

brain and limbs. However, mammals have a limited capacity

to restore organs following injury to organs like the liver and

skeletal muscles but practically no ability to regenerate organs

like the heart or brain following an ischemic event or injury.

The mammalian heart, including the human heart, for example,

has a very limited capacity to regenerate following damage or

an acute ischemic event like myocardial infarction (MI). This is

due to the very low level of cardiomyocyte proliferation and the

limited number of cells expressing stem-cell marker proteins.

Stem-cell-based therapy was suggested as a potential solution

to the above situation. In recent years, cell-based therapy for

cardiac repair in particular has undergone a rapid transition

from basic science research to clinical reality [1-3]. The general

outcome of the clinical trials was that the procedures and longterm

outcome post-stem-cell implantation to the heart via the

coronary arteries are safe. However, improvement in long-term

functional performance of the heart was either not achieved or

was marginal [1-3].

There are several central issues pertaining to the use of cell

implantation in stem-cell therapy: the number of implanted stem

cells has to be high since there is massive cell death following

implantation or injection of cells into the heart or the blood

circulation. Another central issue in stem-cell implantation for

organ repair is the creation of a receptive cell environment in the

ischemic organ. Several factors (e.g. inhibition of inflammation

and apoptosis, secretion of cell growth factors etc.) are necessary

for optimal cell implantation [4]. The injected cells may have to

migrate from the circulating blood to the ischemic niche. They

can then remain active and secrete growth factors, exerting a paracrine effect on the ischemic tissue [5]. Alternatively, they

may stimulate the small population of stem cells in the ischemic

organ (such as the heart), to proliferate and differentiate so as to

enhance cardiac repair post-MI [6]. Another issue is the timing

of injection of the stem cells to the infarcted heart and effect of

MI (inflammatory phase) on the BM [7]. Photobiostimulation

of cells in the bone marrow (BM), that is enriched with various

progenitor cells, by low level laser therapy (LLLT) may suggest a

new approach that may overcome some of the above limitation.

This new approach will be discussed in the present mini review

[8-11].

LOW LEVEL LASER THERAPY FOR THE ISCHEMIC

HEART

In general LLLT has been found to modulate various biological

processes, such as increasing mitochondrial respiration and ATP

synthesis, facilitating wound healing, and promoting the process

of skeletal muscle regeneration and angiogenesis [12,13]. It

was previously shown that LLLT can enhance skeletal muscle

regeneration following partial excision in the rat hind limb

muscles when the laser was delivered directly to the injured

organ multiple times (for 2 min each time) following injury [14].

This phenomenon was even more prominent following cold

injury to the frog skeletal muscles indicating that enhancement

of regeneration by LLLT is probably a general phenomenon in

vertebrates and maybe more effective in cold blooded animals

which innately have a lower metabolic rate in their cells [13-15].

In an experimental model of the infarcted heart in rats and dogs, it

was demonstrated that LLLT (Diode –Ga-Al-As 810nm at a power

density of 5 mW/cm2 for 120sec duration of laser exposure

comprising 0.6 J/cm2), application directly to the infarcted area

in the heart at optimal power parameters significantly reduces

infarct size (scar tissue formation) [16,17]. This phenomenon

was partially attributed to a significant elevation in ATP content,

heat shock proteins, vascular endothelial growth factor (VEGF),

and angiogenesis in the ischemic zone of the laser-irradiated

rats, as compared to non-irradiated rats [16,17]. The mechanism

associated with the photobiostimulation by LLLT is not yet

clearly understood [12]. There is evidence that cytochrome c

oxidase and perhaps also plasma membranes in cells function as

photoacceptors of the photons, and thereafter a cascade of events

occur in the mitochondria, leading to effects on various processes

like ATP production, up-regulation of VEGF, etc [12].

The effect of photobiostimulation on stem cells or progenitor

cells has not been extensively studied [18-21]. It was previously

shown that laser application (Diode laser at 50mW/cm2 for

100sec, energy density 0.5 mW/cm2) to the mesenchymal stem

cells isolated from bone marrow or cardiac stem cells causes a

significant increase in their proliferation in vitro [20]. Based on

previous studies that showed an increase in cytoprotective effect

on the ischemic heart following LLLT, a new approach was taken

to apply laser irradiation to stem cells grown in culture prior

to their implantation to the infarcted heart as a cell therapy for

heart repair [21]. In that study it was demonstrated that MSCs

that were laser treated prior to their implantation to the rat

infarcted heart caused a significant reduction in infarct size

as compared to MSCs that were injected to the heart without

prior laser treatment. This phenomenon was also associated

with significant elevation of vascular endothelial growth factor

(VEGF) in the myocardium of the rats that received the lasertreated

MSCs. In a recent study [8] the possibility of recruiting

autologous stem cells stimulated by LLLT in the BM to the

infarcted heart was addressed. The rationale behind the attempt

to use LLLT to induce the “crude” BM in the bone was, and still is,

that one cannot significantly affect the complex process post-MI

or ischemic injury to the kidney with a single type of stem cell. The

native BM is known for its many types and subtypes of stem cells,

which are defined by their reactivity to various antibodies. The

BM also contains many progenitor cells (i.e. monocytes) that can

further differentiate, for example to macrophages. Macrophages

have been shown recently to have a crucial role in the scarring

process post-MI. Thus LLLT may induce concomitantly in the BM

various types of cells that will increase in number in the blood

circulation following their enhanced proliferation in the BM.

These cells will probably, eventually, and to a certain extent and

under certain circumstances, home in on the ischemic zone in the

ischemic organ (heart, kidney etc.). In this study [8] it was found

that when LLLT was applied in vivo to the BM, and MSCs were

isolated from that BM 3 and 6 weeks later and grown in vitro, they

grew at a higher rate of proliferation relative to MSCs isolated

from non-laser-treated BM. This indicated that the MSCs when

in the BM, following LLLT application in vivo can be induced to

proliferate to a higher rate than non-treated MSCs. Furthermore,

laser application (Diode laser 808nm at power density of 10mW/

cm2 for 100 sec comprising 1J/cm2 energy density) to the BM (at

about 20 min post-MI) caused a marked and significant decrease

(79%) in infarct size 3 weeks post-MI. This extent of infarct size

reduction was even more effective in reducing scarring than that

of laser application directly to the infarcted heart, as also found

in previous studies with infarcted rat and dog hearts [10]. Even

when laser was applied 4 hours post-MI to the BM of infarcted

rats, a marked and significant reduction in the infarcted area

was observed in the laser-treated rats compared to control.

We also found a significantly higher density of c-kit+ (a marker

of MSCs) cells in the myocardium of laser-treated rats relative

to non-treated rat’s post-MI. Moreover, it was demonstrated in

this study that c-kit+ cells post-laser application to the BM of MIinduced

rats, homed specifically in on the infarcted heart and

not on uninjured organs (i.e. liver, kidney) in the same rat [16].

It can be hypothesized that the increased number of c-kit+ cells

found in the myocardium came from proliferating MSCs in the

BM that had migrated to the circulating blood and homed onto

the infarcted heart. Another finding of this study was that of

the preferred homing of the recruited or endogenous c-kit+ cells

in on the infarcted area, rather than their random deposition

throughout the left ventricle in the heart. Indeed, at 3-weeks

post-MI the density of c-kit+ cells in the infarcted area was 27-

fold higher in the rats whose BM had been treated with LLLT as

compared to control rats. Similarly, Hatzistergos et al. [6] found

that endogenous c-kit+ cardiac stem cells increased by 20-fold

in the porcine infarcted heart as compared to control following

### Home Search Introduction

Ken Teegardin - (Video)
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Welcome to the laser-therapy.us research tool. This tool is a searchable collection of technical publications, books, videos and other resources about the use of lasers for photobiomodulation. This tool includes almost the entire U.S. library of medicine research papers on LLLT, videos from Youtube associated with therapy lasers and the tables of contents from laser therapy books. This allows users to search for a keyword or condition and see resources about using lasers to treat that condition. All the resources include links to the original source so we are not making any statement about the use of lasers for treating non-FDA cleared application, we are simple summarizing what others have said.  Where every possible, we have included a link to the orginal publication.

Here are some of our favorite queries:

This tool uses a broad match query so:

• It does not correct spelling and searches only cold laser related subjects so do not use LLLT, cold or laser in the search bar
• It works better with shorter search terms or even parts of search terms
• It searches all the available fields so you can enter a body part, author, condition or laser brand.
• Where ever possible, the detailed section about the resource will link to the sources.
• This system is only for photobiomodulation or cold laser therapy research (including LLLT, laser acupuncture and high power laser therapy) only. It does NOT include photodynamic laser therapy (where the laser is used to react with a pharmaceutical), hot surgery lasers or cosmetic lasers. It does include some resources on weight loss and smoking cessation.

The results of the search are sorted based on 3 quality factors on a scale of 1 to 10 with 10 being the best score. Originally all the resources were given a 5-5-5 until they could be individually evaluated. These scores are purely opinion and are only used to simplify the rank of the results from more valuable to least valuable. This should not be considered a critique of any work. This system was created to help researchers (including ourselves) find the most usable resources for any cold laser therapy research. The resources are assigned values based on the following 3 factors:

• Efficacy: The resource (especially research papers) should show a significant improvement in the condition being treated. Resources that show better results are given a higher quality score.
• Detail: The source must give enough information that the results can be duplicated. If a resource lacks too many details that it cannot be recreated, it is given a lower detail score.
• Lack of Bias: Many resources are created to try and show that one device is superior to its competition. Many manufacturers have staff that crank out biased papers on a regular basis on the hope that this will make their product look superior. If the author of the resource is paid by a manufacturer of the resource appears to be biased towards one device and not one technology, the resource has much less value.

Over the past few years of working with research, we found that a majority of the published resources are lacking in one of these three ranking factors.
The original goal of this research tool was to tie published resources to the protocols in the laser-therapy.us library. This connection allows users to trace each protocol back to a list of resources so the protocol can be researched and improved.