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### An Hour of Light and Sound a Day Might Keep Alzheimer’s at Bay

Angus Chen - Scientific American March 14, 2019 (Publication)
This is an summary article about the work of Shannon Macauley, a neuroscientist at Wake Forest School of Medicine who found that light and sound has a siginicant impact on the Alzheimer's
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Original Source: https://www.scientificamerican.com/article/an-hour-of-light-and-sound-a-day-might-keep-alzheimers-at-bay/

### 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/

### Mechanisms and applications of the anti-inflammatory effects of photobiomodulation

Michael R Hamblin - PMC 2017 Jul 24 (Publication)
Chronic diseases of the modern age involving systemic inflammation such as type II diabetes, obesity, Alzheimer's disease, cardiovascular disease and endothelial dysfunction are again worth investigating in the context of PBM.
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## Abstract

Photobiomodulation (PBM) also known as low-level level laser therapy is the use of red and near-infrared light to stimulate healing, relieve pain, and reduce inflammation. The primary chromophores have been identified as cytochrome c oxidase in mitochondria, and calcium ion channels (possibly mediated by light absorption by opsins). Secondary effects of photon absorption include increases in ATP, a brief burst of reactive oxygen species, an increase in nitric oxide, and modulation of calcium levels. Tertiary effects include activation of a wide range of transcription factors leading to improved cell survival, increased proliferation and migration, and new protein synthesis. There is a pronounced biphasic dose response whereby low levels of light have stimulating effects, while high levels of light have inhibitory effects. It has been found that PBM can produce ROS in normal cells, but when used in oxidatively stressed cells or in animal models of disease, ROS levels are lowered. PBM is able to up-regulate anti-oxidant defenses and reduce oxidative stress. It was shown that PBM can activate NF-kB in normal quiescent cells, however in activated inflammatory cells, inflammatory markers were decreased. One of the most reproducible effects of PBM is an overall reduction in inflammation, which is particularly important for disorders of the joints, traumatic injuries, lung disorders, and in the brain. PBM has been shown to reduce markers of M1 phenotype in activated macrophages. Many reports have shown reductions in reactive nitrogen species and prostaglandins in various animal models. PBM can reduce inflammation in the brain, abdominal fat, wounds, lungs, spinal cord.

### 2.1. Cytochrome c oxidase in mitochondria

Cytochrome c oxidase (CCO) is unit IV in the mitochondrial electron transport chain. It transfers one electron (from each of four cytochrome c molecules), to a single oxygen molecule, producing two molecules of water. At the same time the four protons required, are translocated across the mitochondrial membrane, producing a proton gradient that the ATP synthase enzyme needs to synthesize ATP. CCO has two heme centers (a and a3) and two copper centers (CuA and CuB). Each of these metal centers can exist in an oxidized or a reduced state, and these have different absorption spectra, meaning CCO can absorb light well into the NIR region (up to 950 nm) [9]. Tiina Karu from Russia was the first to suggest [10,11], that the action spectrum of PBM effects matched the absorption spectrum of CCO, and this observation was confirmed by Wong-Riley et al in Wisconsin [12]. The assumption that CCO is a main target of PBM also explains the wide use of red/NIR wavelengths as these longer wavelengths have much better tissue penetration than say blue or green light which are better absorbed by hemoglobin. The most popular theory to explain exactly why photon absorption by CCO could led to increase of the enzyme activity, increased oxygen consumption, and increased ATP production is based on photodissociation of inhibitory nitric oxide (NO) [13]. Since NO is non-covalently bound to the heme and Cu centers and competitively blocks oxygen at a ratio of 1:10, a relatively low energy photon can kick out the NO and allow a lot of respiration to take place [14].

### 2.2. Light gated ion channels and opsins

More recently it has become apparent that another class of photoreceptors, must be involved in transducing cellular signals, particularly responding to blue and green light. Thee photoreceptors have been proposed to be members of the family of light-sensitive G-protein coupled receptors known as opsins (OPN). Opsins function by photoisomerization of a cis-retinal co-factor leading to a conformational change in the protein. The most well known opsin is rhodopsin (OPN1), which is responsible for mediating vision in the rod and cone photoreceptor cells in the mammalian retina. There are other members of the opsin family (OPN2-5), which are expressed in many other tissues of the body including the brain [15]. One of the best-defined signaling events that occurs after light-activation of opsins, is the opening of light-gated ion channels such as members of the transient receptor potential (TRP) family of calcium channels [16]. TRP channels are now known to be pleiotropic cellular sensors mediating the response to a wide range of external stimuli (heat, cold, pressure, taste, smell), and involved in many different cellular processes [17]. Activation of TRP causes non-selective permeabilization (mainly of the plasma membrane) to calcium, sodium and magnesium [18]. It is now known that TRP channel proteins are conserved throughout evolution and are found in most organisms, tissues, and cell-types. The TRP channel superfamily is now classified into seven related subfamilies: TRPC, TRPM, TRPV, TRPA, TRPP, TRPML, and TRPN [19]. Light-sensitive ion channels are based on an opsin chromophore (isomerization of a cis-retinal molecule to the trans configuration) as illustrated in Drusophila photoreceptors [20].

We have shown that blue or green light (but not red or 810 nm NIR) increased intracellular calcium in adipose derived stem cells, that could be blocked by ion channel inhibitors [5].

### 2.3. Flavins and flavoproteins

There is another well-known family of biological chromophores called cryptochromes. These proteins have some sequence similarity to photolyases [21], which are blue light responsive enzymes that repair DNA damage in bacteria caused by UV exposure [22]. Cryptochromes rely on a flavin (flavin adenine dinucleotide, FAD) or a pterin (5,10-methenyltetrahydrofolic acid) to actually absorb the light (again usually blue or green). Cryptochromes have been studied mainly in plants and insects. Recent evidence has emerged that mammalian cryptochromes are important in regulation of the circadian clock. It is thought that human cryptochromes (CRY1 and CRY2) send signals via part of the optic nerve to the suprachiasmatic nucleus (SCN) in the brain, which is the master regulator of the CLOCK system to entrain biological responses to the light-dark cycle [23]. However the situation is complicated because retinal ganglion cells containing melanopsin (OPN4) are also involved in photoentrainment [24]. Studies are still ongoing to investigate this redundancy [25].

It should be emphasized that compared to CCO and mitochondria, evidence is still emerging concerning the extent to which opsins, cryptochomes and light-gated ion channels (which may be widely expressed in many different cell types) could be responsible for PBM effects. If their role is significant it is likely to be in the blue and green spectral regions. Further research will be necessary to explore their role in anti-inflammatory effects, wound healing and tissue regeneration.

### 2.4. Water as a chromophore and heat-gated ion channels

Since the biological effects of light continue to be observed, as the wavelength increases in the infra-red region (>1000 nm), beyond those known to be absorbed by CCO, it is now thought likely that an alternative chromophore must be responsible. The obvious candidate for this alternative chromophore is water molecules whose absorption spectrum has peaks at 980 nm, and also at most wavelengths longer than 1200 nm. Moreover, water is by the far the most prevalent molecule in biological tissue (particularly considering its low molecule weight = 18). At present the proposed mechanism involves selective absorption of IR photons by structured water layers (also known as interfacial water) [26] or water clusters [27], at power levels that are insufficient to cause any detectable bulk-heating of the tissue. A small increase in vibrational energy by a water cluster formed in or on a sensitive protein such as a heat-gated ion channel, could be sufficient to perturb the tertiary protein structure thus opening the channel and allowing modulation of intracellular calcium levels [28]. Pollack has shown that interfacial water can undergo charge separation when it absorbs visible or NIR light [29]. This charge separation (equivalent to localized pH changes) could affect the conformation of proteins [30]. It has also been suggested that PBM could reduce the viscosity of interfacial water within the mitochondria, and allow the F0F1 ATP synthase, which rotates as a nanomotor to turn faster [31]. It should be noted here that the first regulatory approvals of PBM were gained as a 510 K device “equivalent to an non-heating IR lamp” [32]. While the involvement of water as a chromophore may still be considered hypothetical it is difficult to think of another explanation for the beneficial of PBM at wavelengths between 1000 nm all the way to 10,000 nm (carbon dioxide laser).

### 3.1. PBM increases ROS in normal cells

When PBM stimulates CCO activity in normal healthy cells, the resulting increase in mitochondrial membrane potential (MMP) above normal baseline levels, leads to a brief and rather modest increase in generation of reactive oxygen species (ROS) [33]. However this brief burst of ROS caused by 3 J/cm2 of 810 nm laser (Figure 2A) was shown to be sufficient to activate the redox-sensitive transcription factor, NF-kB in embryonic fibroblasts [34] (Figure 2B). Addition of the anti-oxidant N-acetyl-cysteine to the cells could block the NK-kB activation (Figure 2C), but not the increase in cellular ATP caused by the mitochondrial stimulation (Figure 2D). In primary cultured cortical neurons [35], 810 nm laser produced a biphasic dose response in ATP production (Figure 3A) and MMP (Figure 3B) with a maximum at 3 J/cm2. At a high dose (30 J/cm2) the MMP was actually lowered below baseline. Interestingly the dose-response curve between fluence (J/cm2) and ROS production showed two different maxima (Figure 3C). One of these maxima occurred at 3 J/cm2 where the MMP showed its maximum increase. The second maximum in ROS production occurred at 30 J/cm2 where the MMP had been reduced below baseline. At a value between these two fluences (10 J/cm2) a dose at which the MMP was approximately back to baseline, there was not much ROS generation. These data are very good examples of the “biphasic dose response” or “Arndt-Schulz curve” which is often discussed in the PBM literature [7,8].

Thus it appears that ROS can be generated within mitochondria when the MMP is increased above normal values and also when it is decreased below normal values. It remains to be seen whether these two kinds of PBM-generated ROS are identical or not. One intriguing possibility is that whether the ROS generated by PBM is beneficial or detrimental may depend on the rate at which it is generated. If superoxide is generated in mitochondria at a rate that allows superoxide dismutase (SOD) to detoxify it to hydrogen peroxide, then the uncharged H2O2 can diffuse out of the mitochondria to activate beneficial signaling pathways, while if superoxide is generated at a rate or at levels beyond the ability of SOD to deal with it, then the charged superoxide may build up inside mitochondria and damage them.

### 3.2. PBM reduces ROS in oxidative stressed cells and tissues

Notwithstanding, the ability of PBM to produce a burst of ROS in normal cells, it is well-accepted that PBM when as a treatment for tissue injury or muscle damage is able to reduce markers of oxidative stress [36,37,38]. How can these apparently contradictory findings be reconciled? A study attempted to answer this question [39]. Primary cultured cortical neurons were treated with one of three different interventions, all of which were chosen from literature methods of artificially inducing oxidative stress in cell culture. The first was cobalt chloride (CoCl2), which is used as a mimetic for hypoxia and works by a Fenton reaction producing hydroxyl radicals [40]. The second was direct treatment with hydrogen peroxide. The third was treatment with the mitochondrial complex I inhibitor, rotenone [41]. All three of these different treatments increased the intracellular mitochondrial ROS as judged by Cell-Rox Red (Figure 4A), and at the same time lowered the MMP as measured by tetramethyl-rhodamine methyl ester (TMRM) (Figure 4B). PBM (3 J/cm2 of 810 nm laser) raised the MMP back towards baseline, while simultaneously reducing the generation of ROS in oxidatively stressed cells (while slightly increasing ROS in normal cells). In control cells (no oxidative stress), PBM increased MMP above baseline and still produced a modest increase in ROS.

Since most laboratory studies of PBM as a therapy have looked at various animal models of disease or injury, it is not surprising that most workers have measured reduction in tissue markers of oxidative stress (TBARS) after PBM [36,42]. There have been a lot of studies looking at muscles. In humans, especially in athletes, high-level exercise produces effects in muscles characterized by delayed-onset muscle soreness, markers of muscle damage (creatine kinase), inflammation and oxidative stress.

One cellular study by Macedo et al [43] used muscle cells isolated from muscular dystrophy mice (mdx LA 24) and found that 5 J/cm2 of 830 nm increased the expression levels of myosin heavy chain, and intracellular [Ca2+]i. PBM decreased H2O2 production and 4-HNE levels and also GSH levels and GR and SOD activities. The mdx cells showed significant increase in the TNF-α and NFκB levels, which were reduced by PBM.

While it is highly likely that the effects of PBM in modulating ROS are involved in the anti-inflammatory effects of PBM, it would be dangerous to conclude that that is the only explanation. Other signaling pathways (nitric oxide, cyclic AMP, calcium) are also likely to be involved in reduction of inflammation.

As mentioned above we found [34] that PBM (3 J/cm2 of 810 nm laser) activated NF-kB in embryonic fibroblasts isolated from mice that had been genetically engineered to express firefly luciferase under control of an NF-kB promoter. Although it is well-known that NF-kB functions as a pro-inflammatory transcription factor, but on the other hand it is also well known that in clinical practice or in laboratory animal studies) PBM has a profound anti-inflammatory effect in vivo. This gives rise to another apparent contradiction that must be satisfactorily resolved.

### 4.2. PBM reduces levels of pro-inflammatory cytokines in activated inflammatory cells

Part of the answer to the apparent contradiction highlighted above, was addressed in a subsequent paper [44]. We isolated primary bone marrow-derived dendritic cells (DCs) from the mouse femur and cultured them with GM-CSF. When these cells were activated with the classical toll-like receptor (TLR) agonists, LPS (TLR4) and CpG oligodeoxynucleotide (TLR9), they showed upregulation of cell-surface markers of activation and maturation such as MHC class II, CD86 and CD11c as measured by flow cytometry. Moreover IL12 was secreted by CpG-stimulated DCs. PBM (0.3 or 3 J/cm2 of 810 nm laser) reduced all the markers of activation and also the IL12 secretion. Figure 5.

Yamaura et al [45] tested PBM (810 nm, 5 or 25 J/cm2) on synoviocytes isolated from rheumatoid arthritis patients. They applied PBM before or after addition of tumor necrosis factor-α (TNF-α). mRNA and protein levels of TNF-α and interleukins (IL)-1beta, and IL-8 were reduced (especially by 25 J/cm2).

Hwang et al [46] incubated human annulus fibrosus cells with conditioned medium obtained from macrophages (THP-1 cells) containing proinflammatory cytokines IL1β, IL6, IL8 and TNF-α. They compared 405, 532 and 650 nm at doses up to 1.6 J/cm2. They found that all wavelengths reduced IL8 expression and 405 nm also reduced IL6.

The “Super-Lizer” is a Japanese device that emits linear polarized infrared light. Imaoka et al [47] tested it against a rat model of rheumatoid arthritis involving immunizing the rats with bovine type II collagen, after which they develop autoimmune inflammation in multiple joints. The found reductions in IL20 expression in histological sections taken from the PBM-treated joints and also in human rheumatoid fibroblast-like synoviocyte (MH7A) stimulated with IL1β.

Lim et al [48] studied human gingival fibroblasts (HGF) treated with lipopolysaccharides (LPS) isolated from Porphyromonas gingivalis. They used PBM mediated by a 635 nm LED and irradiated the cells + LPS directly or indirectly (transferring medium from PBM treated cells to other cells with LPS). Both direct and indirect protocols showed reductions in inflammatory markers (cyclooxygenase-2 (COX2), prostaglandin E2 (PGE2), granulocyte colony-stimulating factor (GCSF), regulated on activated normal T-cell expressed and secreted (RANTES), and CXCL11). In the indirect irradiation group, phosphorylation of C-Raf and Erk1/2 increased. In another study [49] the same group used a similar system (direct PBM on HGF + LPS) and showed that 635 nm PBM reduced IL6, IL8, p38 phosphorylation, and increased JNK phosphorylation. They explained the activation of JNK by the growth promoting effects of PBM. Sakurai et al reported [50] similar findings using HGF treated with Campylobacter rectus LPS and PBM (830 nm up to 6.3 J/cm2) to reduce levels of COX2 and PGE2. In another study [51] the same group showed a reduction in IL1β in the same system.

### 4.3. Effects of PBM on macrophage phenotype

Another very interesting property of PBM is its ability to change the phenotype of activated cells of the monocyte or macrophage lineage. These cells can display two very different phenotypes depending on which pathological situation the cells are faced with. The M1 phenotype (classically activated) applies to macrophages that are faced with a situation in which bacteria or other pathogens need to be killed, or alternatively tumor cells need to be destroyed. Inducible nitric oxide synthase is a hallmark of the M1 phenotype and nitric oxide secretion is often measured. On the other hand the M2 phenotype (alternatively activated) applies to macrophages that are involved in disposal of cellular or protein debris and stimulation of healing by angiogenesis. The M2 phenotype produces arginase, an enzyme that inhibits NO production and allows them to produce ornithine, a precursor of hydroxyproline and polyamines [52]. The markers of these two phenotypes of activated macrophage have some aspects in common, but also show many aspects that are very different [53]. It should be noted that this concept of M1 and M2 activation states, applies to other specialized macrophage type cells that are resident in different tissues, such as microglia in brain [54], alveolar macrophages in lung [55], Kuppfer cells in liver [56], etc.

Fernandes et al used J774 macrophage-like cells activated with interferon-γ and LPS to produce a MI phenotype and compared 660 nm and 780 nm laser. They found that both wavelengths reduced TNF-α, COX-2 and iNOS expression, with the 780 nm being somewhat better [57]. Silva et al used RAW264.7 macrophages to test two wavelengths (660 nm and 808 nm) at a range of fluences (11-214 J/cm2) [58]. They found increases in NO release with 660 nm at the higher fluences. von Leden et al carried out an interesting study looking at the effects of PBM on microglia and their interaction with cortical neurons [59]. They used both primary microglia isolated from mouse brains and the BV2 mouse microglial cell line and compared four fluences (0.2, 4, 10, and 30 J/cm2, at 808 nm. Fluences between 4 and 30 J/cm2 induced expression of M1 markers in microglia. Markers of the M2 phenotype, including CD206 and TIMP1, were observed at lower energy densities of 0.2–10 J/cm2. In addition, co-culture of PBM or control-treated microglia with primary neuronal cultures demonstrated a dose-dependent effect of PBM on microglial-induced neuronal growth and neurite extension. This suggests that the benefits of PBM on neuroinflammation may be more pronounced at lower overall doses. The same group went on to show that M1 activated macrophages receiving PBM (660 nm laser) showed significant decreases in CCL3, CXCL2 and TNFα mRNA expression 4 h after irradiation [60]. However, 24 h after irradiation, M1 macrophages showed increased expression of CXCL2 and TNFα genes. M1 activated macrophages irradiated with 780 nm showed a significant decrease in CCL3 gene expression 4h after irradiation. These data could explain the anti-inflammatory effects of LLLT in wound repair.

This section will cover some of the most important medical indications where PBM has been shown in laboratory studies to be effective (at least partly) by its pronounced anti-inflammatory effects. Figure 6 shows a graphical summary of the anti-inflammatory applications of PBM in experimental animal models.

### 5.1. Wound healing

Many papers have demonstrated the efficacy of PBM in stimulating wound healing. In animal models these studies have generally been on acute wounds [61], while in clinical trials they are often been concerned with chronic non-healing wounds such as diabetic ulcers [62]. Gupta et al [63] tested PBM using a superpulsed 904 nm laser on burn wounds in rats. They found faster healing, reduced inflammation (histology), decreased expression of TNF-α and NF-kB, and up-regulated expression of VEGF, FGFR-1, HSP-60, HSP-90, HIF-1α and matrix metalloproteinases-2 and 9 compared to controls. It is intriguing to speculate that the effects of PBM on wound healing (especially the use of for chronic non-healing wounds) could involve both pro-inflammatory effects and anti-inflammatory effects. This seemingly contradictory statement may be possible due to the recent discovery of resolvins and protectins, which are multifunctional lipid mediators derived from omega-3 polyunsaturated fatty acids [64]. If resolvins were produced as a result of the brief acute inflammation induced by application of PBM to chronic wounds, then it has been already shown that resolvins can hasten the healing of diabetic wounds in mice [65]. Resolvins have been shown to reduce tumor necrosis factor-α, interleukin-1β, and neutrophil platelet-endothelial cell adhesion molecule-1 in a mouse burn wound model [66].

### 5.2. Arthritis

In humans, arthritis is most often caused by a degenerative process occurring in osteoarthritis, or an autoimmune process occurring in rheumatoid arthritis. Both are characterized by pronounced inflammatory changes in the joint and even systemically. Different animal models are produced to mimic these diseases, but a common approach is to inject the sterile preparation of yeast cell walls known as zymosan into the knee joints of rats.

Castano et al [67] used this zymosan-induced arthritis model to study the effects of two different fluences of 810 nm laser (3 and 30 J/cm2) delivered at two different power densities (5 and 50 mW/cm2). PBM was delivered once a day for 5 days commencing after zymosan injection, and the swelling in the knee was measured daily. Prostagladin E2 (PGE2) was measured in the serum. They found that 3 out of the 4 sets of parameters were approximately equally effective in reducing swelling and PGE2, but the ineffective set of parameters was 3 J/cm2 delivered at 50 mW/cm2 which only took 1 min of illumination time. The conclusion was, that the illumination time was important in PBM, and if that time was too short, then the treatment could be ineffective.

Moriyama et al [68] used a transgenic mouse strain (FVB/N-Tg(iNOS-luc) that had been engineered to express luciferase under control of the inducible nitric oxide synthase promoter, to allow bioluminescence imaging of PBM of the zymosal-induced arthritis model in mice knees. They compared the same fluence of 635, 660, 690, and 905 nm (CW0 and 905 nm (short pulse). Animals younger than 15 weeks showed mostly reduction of iNOS expression, while older animals showed increased iNOS expression. Pulsed 905 nm also increased iNOS expression.

Pallotta et al [69] used a model where carageenan was injected into the rat knee and tested 810 nm laser at 1, 3, 6 or 10 J/cm2. Rats were sacrificed after 6 or 12 hours and the joint tissue removed. PBM was able to significantly inhibit the total number of leukocytes, as well as the myeloperoxidase activity. Vascular extravasation was significantly inhibited at the higher dose of energy of 10 J. Gene expression of both COX-1 and 2 were significantly enhanced by laser irradiation while PGE2 production was inhibited. These apparently contradictory results require more study to fully explain.

### 5.3. Muscles

One of the most robust applications of PBM, is its effects on muscles [70,71]. PBM can potentiate muscular performance especially when applied to the muscles 3 hours before exercise [72]. PBM can also make exercise-training regimens more effective. It is not therefore surprising that PBM can also help to heal muscle injuries, not to mention reducing muscle pain and soreness after excessive exercise. Many of the animal studies that have been done have looked at markers of inflammation and oxidative stress in muscle tissue removed from sacrificed animals. For instance, Silveira et al [73] caused a traumatic muscle injury by a single blunt-impact to the rat gastrocnemius muscle. PBM (850 nm, 3 or 5 J/cm2) was initiated 2, 12, and 24  h after muscle trauma, and repeated for five days. The locomotion and muscle function was improved by PBM. TBARS, protein carbonyls, superoxide dismutase, glutathione peroxidase, and catalase, were increased after muscle injury, these increases were prevented by PBM. PBM prevented increases in IL-6 and IL-10 and reversed the trauma-induced reduction in BDNF and VEGF.

### 5.4. Inflammatory pain

There have been many studies that have looked at the effects of PBM on pain in animal models. Some studies have looked at sensitivity to pain [74] using the von Frey filaments (a graded set of fibers of increasing stiffness and when the animal feels the pressure it withdraws its foot [75]).

Some studies have looked at animal models of neuropathic pain such as the “spared nerve injury” [76]. This involves ligating two out of three branches of the sciatic nerve in rats and causes long lasting (>6 months) mechanical allodynia [77]. Kobelia Ketz et al found improvements in pain scores with PBM (980  nm applied to affected hind paw 1 W, 20 s, 41 cm above skin, power density 43.25  mW/cm2, dose 20 J). They also found lower expression of the proinflammatory marker (Iba1) in microglia in the dorsal root ganglion, gracile nucleus, dorsal column and dorsal horn. The M1/M2 balance of the macrophage phenotype was switched from M1 to M2 by PBM, as judged by relative staining with anti-CD86 (M1) and anti-CD206 (M2).

Martins et al looked at the effect of PBM on a model of inflammatory pain [42]. This involved injecting complete Freund's adjuvant (CFA) into the mouse paw, and produces hyperalgesia and elevated cytokine levels (TNF-α, IL-1β, IL-10). They found that LEDT (950-nm, 80 mW/cm2, 1, 2 or 4 J/cm2) applied to the plantar aspect of the right hind limb, reduced pain, increased the levels of IL-10 prevented TBARS increase in both acute and chronic phases, reduced protein carbonyl levels and increased SOD and CAT activity in the acute phase only.

### 5.5. Lung inflammation

Aimbire and his laboratory in Brazil have carried out several studies on the use of PBM to reduce acute lung inflammation (ALI) in various animal models. In a mouse model of lung inflammation caused either by inhalation of lipolysaccharide or intranasal administration of TNFα they analyzed the bronchoalveolar lavage fluid (BALF). PBM (660 nm, 4.5 J/cm2) was administered to the skin over the right upper bronchus 15 min after ALI induction. PBM attenuated the neutrophil influx and lowered TNFα in BALF. In alveolar macrophages, PBM increased cAMP and reduced TNFα mRNA.

They also studied a different model of ALI caused by intestinal ischemia and reperfusion (I/R), that produces an analogue of acute respiratory distress syndrome (ARDS) [78]. Rats were subjected to superior mesenteric artery occlusion (45 min) and received PBM (660 nm, 7.5 J/cm2) carried out by irradiating the rats on the skin over the right upper bronchus for 15 and 30 min, and rats were euthanized 30 min, 2, or 4 h later. PBM reduced lung edema, myeloperoxisdase activity, TNF-α and iNOS, LLLT increased IL-10 in the lungs of animals subjected to I/R.

A third animal model was related to asthma [79]. Mice were sensitized to ovalbumin (OVA), and then challenged by a single 15-min exposure to aerosolized OVA. PBM was applied as above (660 nm, 30 mW, 5.4 J). Bronchial hyper-responsiveness (as measured by dose response curves to acetylcholine) was reduced by PBM as well as reductions in eosinophils and eotaxin. PBM also diminished expression of intracellular adhesion molecule and Th2 cytokines, as well as signal transducer and activator of transduction 6 (STAT6) levels in lungs from challenged mice. Recently Rigonato-Oliveira et al. presented a study that concluded that the reduced lung inflammation and the positive effects of PBM on the airways appear to be mediated by increased secretion of the anti-inflammatory cytokine IL-10, and reduction of mucus in the airway [80].

### 5.6. Traumatic brain injury

In recent years the use of PBM as a treatment for traumatic brain injury [81,82], and other brain disorders including stroke, neurodegenerative diseases and even psychiatric disorders has increased markedly [83]. It is thought that the actions of NIR light shone on the head and penetrating into the brain are multi-factorial, but one clear effect is the anti-inflammatory action of transcranial PBM. This was shown by a series of mouse experiments conducted by Khuman et al [84]. They used the controlled cortical impact model of TBI and delivered PBM (800  nm) was applied directly to the contused parenchyma or transcranially in mice beginning 60–80 min after CCI. Injured mice treated with 60 J/cm2 (500  mW/cm2 × 2  min) had improved latency to the hidden platform and probe trial performance in the Morris water maze. PBM in open craniotomy mice reduced the number of activated microglia in the brain at 48  h (21.8 ± 2.3 versus 39.2 ± 4.2 IbA-1 + cells/field).

### 5.7. Spinal cord injury

Spinal cord injury (SCI) is another promising area of central nervous system injury that could be benefited by PBM. Veronez et al [85] used a rat model of SCI involving a contusion produced by a mechanical impactor (between the ninth and tenth thoracic vertebrae), with a pressure of 150 kdyn. Three different doses of PBM (808-nm laser) were tested: 500 J/cm2, 750 J/cm2 and 1000 J/cm2 delivered daily for seven days. Functional preformance and tactile sensitivity were improved after PBM, at 1000 J/cm2. PBM at 750 and 1000 J/cm2 reduced the lesion volume and also reduced markers of inflammation (lower CD-68 protein expression).

### 5.8. Autoimmune diseases

Experimental autoimmune encephalomyelitis (EAE) is the most commonly studied animal model of multiple sclerosis (MS), a chronic autoimmune demyelinating disorder of the central nervous system. Immunomodulatory and immunosuppressive therapies currently approved for the treatment of MS slow disease progression, but do not prevent it. Lyons et al [86] studied a mouse model of EAE involving immunization with myelin oligodendrocyte glycoprotein (MOG35-55). They treated the female C57BL/6 mice with PBM (670 nm) for several days in different regimens. In addition to improved muscular function, they found down-regulation of inducible nitric oxide synthase (iNOS) gene expression in the spinal cords of mice as well as an up-regulation of the Bcl-2 anti-apoptosis gene, an increased Bcl-2:Bax ratio, and reduced apoptosis within the spinal cord of animals over the course of disease. 670 nm light therapy failed to ameliorate MOG-induced EAE in mice deficient in iNOS, confirming a role for remediation of nitrosative stress in the amelioration of MOG-induced EAE by 670 nm mediated photobiomodulation.

### 5.1. Wound healing

Many papers have demonstrated the efficacy of PBM in stimulating wound healing. In animal models these studies have generally been on acute wounds [61], while in clinical trials they are often been concerned with chronic non-healing wounds such as diabetic ulcers [62]. Gupta et al [63] tested PBM using a superpulsed 904 nm laser on burn wounds in rats. They found faster healing, reduced inflammation (histology), decreased expression of TNF-α and NF-kB, and up-regulated expression of VEGF, FGFR-1, HSP-60, HSP-90, HIF-1α and matrix metalloproteinases-2 and 9 compared to controls. It is intriguing to speculate that the effects of PBM on wound healing (especially the use of for chronic non-healing wounds) could involve both pro-inflammatory effects and anti-inflammatory effects. This seemingly contradictory statement may be possible due to the recent discovery of resolvins and protectins, which are multifunctional lipid mediators derived from omega-3 polyunsaturated fatty acids [64]. If resolvins were produced as a result of the brief acute inflammation induced by application of PBM to chronic wounds, then it has been already shown that resolvins can hasten the healing of diabetic wounds in mice [65]. Resolvins have been shown to reduce tumor necrosis factor-α, interleukin-1β, and neutrophil platelet-endothelial cell adhesion molecule-1 in a mouse burn wound model [66].

### 5.2. Arthritis

In humans, arthritis is most often caused by a degenerative process occurring in osteoarthritis, or an autoimmune process occurring in rheumatoid arthritis. Both are characterized by pronounced inflammatory changes in the joint and even systemically. Different animal models are produced to mimic these diseases, but a common approach is to inject the sterile preparation of yeast cell walls known as zymosan into the knee joints of rats.

Castano et al [67] used this zymosan-induced arthritis model to study the effects of two different fluences of 810 nm laser (3 and 30 J/cm2) delivered at two different power densities (5 and 50 mW/cm2). PBM was delivered once a day for 5 days commencing after zymosan injection, and the swelling in the knee was measured daily. Prostagladin E2 (PGE2) was measured in the serum. They found that 3 out of the 4 sets of parameters were approximately equally effective in reducing swelling and PGE2, but the ineffective set of parameters was 3 J/cm2 delivered at 50 mW/cm2 which only took 1 min of illumination time. The conclusion was, that the illumination time was important in PBM, and if that time was too short, then the treatment could be ineffective.

Moriyama et al [68] used a transgenic mouse strain (FVB/N-Tg(iNOS-luc) that had been engineered to express luciferase under control of the inducible nitric oxide synthase promoter, to allow bioluminescence imaging of PBM of the zymosal-induced arthritis model in mice knees. They compared the same fluence of 635, 660, 690, and 905 nm (CW0 and 905 nm (short pulse). Animals younger than 15 weeks showed mostly reduction of iNOS expression, while older animals showed increased iNOS expression. Pulsed 905 nm also increased iNOS expression.

Pallotta et al [69] used a model where carageenan was injected into the rat knee and tested 810 nm laser at 1, 3, 6 or 10 J/cm2. Rats were sacrificed after 6 or 12 hours and the joint tissue removed. PBM was able to significantly inhibit the total number of leukocytes, as well as the myeloperoxidase activity. Vascular extravasation was significantly inhibited at the higher dose of energy of 10 J. Gene expression of both COX-1 and 2 were significantly enhanced by laser irradiation while PGE2 production was inhibited. These apparently contradictory results require more study to fully explain.

### 5.3. Muscles

One of the most robust applications of PBM, is its effects on muscles [70,71]. PBM can potentiate muscular performance especially when applied to the muscles 3 hours before exercise [72]. PBM can also make exercise-training regimens more effective. It is not therefore surprising that PBM can also help to heal muscle injuries, not to mention reducing muscle pain and soreness after excessive exercise. Many of the animal studies that have been done have looked at markers of inflammation and oxidative stress in muscle tissue removed from sacrificed animals. For instance, Silveira et al [73] caused a traumatic muscle injury by a single blunt-impact to the rat gastrocnemius muscle. PBM (850 nm, 3 or 5 J/cm2) was initiated 2, 12, and 24  h after muscle trauma, and repeated for five days. The locomotion and muscle function was improved by PBM. TBARS, protein carbonyls, superoxide dismutase, glutathione peroxidase, and catalase, were increased after muscle injury, these increases were prevented by PBM. PBM prevented increases in IL-6 and IL-10 and reversed the trauma-induced reduction in BDNF and VEGF.

### 5.4. Inflammatory pain

There have been many studies that have looked at the effects of PBM on pain in animal models. Some studies have looked at sensitivity to pain [74] using the von Frey filaments (a graded set of fibers of increasing stiffness and when the animal feels the pressure it withdraws its foot [75]).

Some studies have looked at animal models of neuropathic pain such as the “spared nerve injury” [76]. This involves ligating two out of three branches of the sciatic nerve in rats and causes long lasting (>6 months) mechanical allodynia [77]. Kobelia Ketz et al found improvements in pain scores with PBM (980  nm applied to affected hind paw 1 W, 20 s, 41 cm above skin, power density 43.25  mW/cm2, dose 20 J). They also found lower expression of the proinflammatory marker (Iba1) in microglia in the dorsal root ganglion, gracile nucleus, dorsal column and dorsal horn. The M1/M2 balance of the macrophage phenotype was switched from M1 to M2 by PBM, as judged by relative staining with anti-CD86 (M1) and anti-CD206 (M2).

Martins et al looked at the effect of PBM on a model of inflammatory pain [42]. This involved injecting complete Freund's adjuvant (CFA) into the mouse paw, and produces hyperalgesia and elevated cytokine levels (TNF-α, IL-1β, IL-10). They found that LEDT (950-nm, 80 mW/cm2, 1, 2 or 4 J/cm2) applied to the plantar aspect of the right hind limb, reduced pain, increased the levels of IL-10 prevented TBARS increase in both acute and chronic phases, reduced protein carbonyl levels and increased SOD and CAT activity in the acute phase only.

### 5.5. Lung inflammation

Aimbire and his laboratory in Brazil have carried out several studies on the use of PBM to reduce acute lung inflammation (ALI) in various animal models. In a mouse model of lung inflammation caused either by inhalation of lipolysaccharide or intranasal administration of TNFα they analyzed the bronchoalveolar lavage fluid (BALF). PBM (660 nm, 4.5 J/cm2) was administered to the skin over the right upper bronchus 15 min after ALI induction. PBM attenuated the neutrophil influx and lowered TNFα in BALF. In alveolar macrophages, PBM increased cAMP and reduced TNFα mRNA.

They also studied a different model of ALI caused by intestinal ischemia and reperfusion (I/R), that produces an analogue of acute respiratory distress syndrome (ARDS) [78]. Rats were subjected to superior mesenteric artery occlusion (45 min) and received PBM (660 nm, 7.5 J/cm2) carried out by irradiating the rats on the skin over the right upper bronchus for 15 and 30 min, and rats were euthanized 30 min, 2, or 4 h later. PBM reduced lung edema, myeloperoxisdase activity, TNF-α and iNOS, LLLT increased IL-10 in the lungs of animals subjected to I/R.

A third animal model was related to asthma [79]. Mice were sensitized to ovalbumin (OVA), and then challenged by a single 15-min exposure to aerosolized OVA. PBM was applied as above (660 nm, 30 mW, 5.4 J). Bronchial hyper-responsiveness (as measured by dose response curves to acetylcholine) was reduced by PBM as well as reductions in eosinophils and eotaxin. PBM also diminished expression of intracellular adhesion molecule and Th2 cytokines, as well as signal transducer and activator of transduction 6 (STAT6) levels in lungs from challenged mice. Recently Rigonato-Oliveira et al. presented a study that concluded that the reduced lung inflammation and the positive effects of PBM on the airways appear to be mediated by increased secretion of the anti-inflammatory cytokine IL-10, and reduction of mucus in the airway [80].

### 5.6. Traumatic brain injury

In recent years the use of PBM as a treatment for traumatic brain injury [81,82], and other brain disorders including stroke, neurodegenerative diseases and even psychiatric disorders has increased markedly [83]. It is thought that the actions of NIR light shone on the head and penetrating into the brain are multi-factorial, but one clear effect is the anti-inflammatory action of transcranial PBM. This was shown by a series of mouse experiments conducted by Khuman et al [84]. They used the controlled cortical impact model of TBI and delivered PBM (800  nm) was applied directly to the contused parenchyma or transcranially in mice beginning 60–80 min after CCI. Injured mice treated with 60 J/cm2 (500  mW/cm2 × 2  min) had improved latency to the hidden platform and probe trial performance in the Morris water maze. PBM in open craniotomy mice reduced the number of activated microglia in the brain at 48  h (21.8 ± 2.3 versus 39.2 ± 4.2 IbA-1 + cells/field).

### 5.7. Spinal cord injury

Spinal cord injury (SCI) is another promising area of central nervous system injury that could be benefited by PBM. Veronez et al [85] used a rat model of SCI involving a contusion produced by a mechanical impactor (between the ninth and tenth thoracic vertebrae), with a pressure of 150 kdyn. Three different doses of PBM (808-nm laser) were tested: 500 J/cm2, 750 J/cm2 and 1000 J/cm2 delivered daily for seven days. Functional preformance and tactile sensitivity were improved after PBM, at 1000 J/cm2. PBM at 750 and 1000 J/cm2 reduced the lesion volume and also reduced markers of inflammation (lower CD-68 protein expression).

### 5.8. Autoimmune diseases

Experimental autoimmune encephalomyelitis (EAE) is the most commonly studied animal model of multiple sclerosis (MS), a chronic autoimmune demyelinating disorder of the central nervous system. Immunomodulatory and immunosuppressive therapies currently approved for the treatment of MS slow disease progression, but do not prevent it. Lyons et al [86] studied a mouse model of EAE involving immunization with myelin oligodendrocyte glycoprotein (MOG35-55). They treated the female C57BL/6 mice with PBM (670 nm) for several days in different regimens. In addition to improved muscular function, they found down-regulation of inducible nitric oxide synthase (iNOS) gene expression in the spinal cords of mice as well as an up-regulation of the Bcl-2 anti-apoptosis gene, an increased Bcl-2:Bax ratio, and reduced apoptosis within the spinal cord of animals over the course of disease. 670 nm light therapy failed to ameliorate MOG-induced EAE in mice deficient in iNOS, confirming a role for remediation of nitrosative stress in the amelioration of MOG-induced EAE by 670 nm mediated photobiomodulation.

### 5.1. Wound healing

Many papers have demonstrated the efficacy of PBM in stimulating wound healing. In animal models these studies have generally been on acute wounds [61], while in clinical trials they are often been concerned with chronic non-healing wounds such as diabetic ulcers [62]. Gupta et al [63] tested PBM using a superpulsed 904 nm laser on burn wounds in rats. They found faster healing, reduced inflammation (histology), decreased expression of TNF-α and NF-kB, and up-regulated expression of VEGF, FGFR-1, HSP-60, HSP-90, HIF-1α and matrix metalloproteinases-2 and 9 compared to controls. It is intriguing to speculate that the effects of PBM on wound healing (especially the use of for chronic non-healing wounds) could involve both pro-inflammatory effects and anti-inflammatory effects. This seemingly contradictory statement may be possible due to the recent discovery of resolvins and protectins, which are multifunctional lipid mediators derived from omega-3 polyunsaturated fatty acids [64]. If resolvins were produced as a result of the brief acute inflammation induced by application of PBM to chronic wounds, then it has been already shown that resolvins can hasten the healing of diabetic wounds in mice [65]. Resolvins have been shown to reduce tumor necrosis factor-α, interleukin-1β, and neutrophil platelet-endothelial cell adhesion molecule-1 in a mouse burn wound model [66].

### 5.2. Arthritis

In humans, arthritis is most often caused by a degenerative process occurring in osteoarthritis, or an autoimmune process occurring in rheumatoid arthritis. Both are characterized by pronounced inflammatory changes in the joint and even systemically. Different animal models are produced to mimic these diseases, but a common approach is to inject the sterile preparation of yeast cell walls known as zymosan into the knee joints of rats.

Castano et al [67] used this zymosan-induced arthritis model to study the effects of two different fluences of 810 nm laser (3 and 30 J/cm2) delivered at two different power densities (5 and 50 mW/cm2). PBM was delivered once a day for 5 days commencing after zymosan injection, and the swelling in the knee was measured daily. Prostagladin E2 (PGE2) was measured in the serum. They found that 3 out of the 4 sets of parameters were approximately equally effective in reducing swelling and PGE2, but the ineffective set of parameters was 3 J/cm2 delivered at 50 mW/cm2 which only took 1 min of illumination time. The conclusion was, that the illumination time was important in PBM, and if that time was too short, then the treatment could be ineffective.

Moriyama et al [68] used a transgenic mouse strain (FVB/N-Tg(iNOS-luc) that had been engineered to express luciferase under control of the inducible nitric oxide synthase promoter, to allow bioluminescence imaging of PBM of the zymosal-induced arthritis model in mice knees. They compared the same fluence of 635, 660, 690, and 905 nm (CW0 and 905 nm (short pulse). Animals younger than 15 weeks showed mostly reduction of iNOS expression, while older animals showed increased iNOS expression. Pulsed 905 nm also increased iNOS expression.

Pallotta et al [69] used a model where carageenan was injected into the rat knee and tested 810 nm laser at 1, 3, 6 or 10 J/cm2. Rats were sacrificed after 6 or 12 hours and the joint tissue removed. PBM was able to significantly inhibit the total number of leukocytes, as well as the myeloperoxidase activity. Vascular extravasation was significantly inhibited at the higher dose of energy of 10 J. Gene expression of both COX-1 and 2 were significantly enhanced by laser irradiation while PGE2 production was inhibited. These apparently contradictory results require more study to fully explain.

### 5.3. Muscles

One of the most robust applications of PBM, is its effects on muscles [70,71]. PBM can potentiate muscular performance especially when applied to the muscles 3 hours before exercise [72]. PBM can also make exercise-training regimens more effective. It is not therefore surprising that PBM can also help to heal muscle injuries, not to mention reducing muscle pain and soreness after excessive exercise. Many of the animal studies that have been done have looked at markers of inflammation and oxidative stress in muscle tissue removed from sacrificed animals. For instance, Silveira et al [73] caused a traumatic muscle injury by a single blunt-impact to the rat gastrocnemius muscle. PBM (850 nm, 3 or 5 J/cm2) was initiated 2, 12, and 24  h after muscle trauma, and repeated for five days. The locomotion and muscle function was improved by PBM. TBARS, protein carbonyls, superoxide dismutase, glutathione peroxidase, and catalase, were increased after muscle injury, these increases were prevented by PBM. PBM prevented increases in IL-6 and IL-10 and reversed the trauma-induced reduction in BDNF and VEGF.

### 5.4. Inflammatory pain

There have been many studies that have looked at the effects of PBM on pain in animal models. Some studies have looked at sensitivity to pain [74] using the von Frey filaments (a graded set of fibers of increasing stiffness and when the animal feels the pressure it withdraws its foot [75]).

Some studies have looked at animal models of neuropathic pain such as the “spared nerve injury” [76]. This involves ligating two out of three branches of the sciatic nerve in rats and causes long lasting (>6 months) mechanical allodynia [77]. Kobelia Ketz et al found improvements in pain scores with PBM (980  nm applied to affected hind paw 1 W, 20 s, 41 cm above skin, power density 43.25  mW/cm2, dose 20 J). They also found lower expression of the proinflammatory marker (Iba1) in microglia in the dorsal root ganglion, gracile nucleus, dorsal column and dorsal horn. The M1/M2 balance of the macrophage phenotype was switched from M1 to M2 by PBM, as judged by relative staining with anti-CD86 (M1) and anti-CD206 (M2).

Martins et al looked at the effect of PBM on a model of inflammatory pain [42]. This involved injecting complete Freund's adjuvant (CFA) into the mouse paw, and produces hyperalgesia and elevated cytokine levels (TNF-α, IL-1β, IL-10). They found that LEDT (950-nm, 80 mW/cm2, 1, 2 or 4 J/cm2) applied to the plantar aspect of the right hind limb, reduced pain, increased the levels of IL-10 prevented TBARS increase in both acute and chronic phases, reduced protein carbonyl levels and increased SOD and CAT activity in the acute phase only.

### 5.5. Lung inflammation

Aimbire and his laboratory in Brazil have carried out several studies on the use of PBM to reduce acute lung inflammation (ALI) in various animal models. In a mouse model of lung inflammation caused either by inhalation of lipolysaccharide or intranasal administration of TNFα they analyzed the bronchoalveolar lavage fluid (BALF). PBM (660 nm, 4.5 J/cm2) was administered to the skin over the right upper bronchus 15 min after ALI induction. PBM attenuated the neutrophil influx and lowered TNFα in BALF. In alveolar macrophages, PBM increased cAMP and reduced TNFα mRNA.

They also studied a different model of ALI caused by intestinal ischemia and reperfusion (I/R), that produces an analogue of acute respiratory distress syndrome (ARDS) [78]. Rats were subjected to superior mesenteric artery occlusion (45 min) and received PBM (660 nm, 7.5 J/cm2) carried out by irradiating the rats on the skin over the right upper bronchus for 15 and 30 min, and rats were euthanized 30 min, 2, or 4 h later. PBM reduced lung edema, myeloperoxisdase activity, TNF-α and iNOS, LLLT increased IL-10 in the lungs of animals subjected to I/R.

A third animal model was related to asthma [79]. Mice were sensitized to ovalbumin (OVA), and then challenged by a single 15-min exposure to aerosolized OVA. PBM was applied as above (660 nm, 30 mW, 5.4 J). Bronchial hyper-responsiveness (as measured by dose response curves to acetylcholine) was reduced by PBM as well as reductions in eosinophils and eotaxin. PBM also diminished expression of intracellular adhesion molecule and Th2 cytokines, as well as signal transducer and activator of transduction 6 (STAT6) levels in lungs from challenged mice. Recently Rigonato-Oliveira et al. presented a study that concluded that the reduced lung inflammation and the positive effects of PBM on the airways appear to be mediated by increased secretion of the anti-inflammatory cytokine IL-10, and reduction of mucus in the airway [80].

### 5.6. Traumatic brain injury

In recent years the use of PBM as a treatment for traumatic brain injury [81,82], and other brain disorders including stroke, neurodegenerative diseases and even psychiatric disorders has increased markedly [83]. It is thought that the actions of NIR light shone on the head and penetrating into the brain are multi-factorial, but one clear effect is the anti-inflammatory action of transcranial PBM. This was shown by a series of mouse experiments conducted by Khuman et al [84]. They used the controlled cortical impact model of TBI and delivered PBM (800  nm) was applied directly to the contused parenchyma or transcranially in mice beginning 60–80 min after CCI. Injured mice treated with 60 J/cm2 (500  mW/cm2 × 2  min) had improved latency to the hidden platform and probe trial performance in the Morris water maze. PBM in open craniotomy mice reduced the number of activated microglia in the brain at 48  h (21.8 ± 2.3 versus 39.2 ± 4.2 IbA-1 + cells/field).

### 5.7. Spinal cord injury

Spinal cord injury (SCI) is another promising area of central nervous system injury that could be benefited by PBM. Veronez et al [85] used a rat model of SCI involving a contusion produced by a mechanical impactor (between the ninth and tenth thoracic vertebrae), with a pressure of 150 kdyn. Three different doses of PBM (808-nm laser) were tested: 500 J/cm2, 750 J/cm2 and 1000 J/cm2 delivered daily for seven days. Functional preformance and tactile sensitivity were improved after PBM, at 1000 J/cm2. PBM at 750 and 1000 J/cm2 reduced the lesion volume and also reduced markers of inflammation (lower CD-68 protein expression).

### 5.8. Autoimmune diseases

Experimental autoimmune encephalomyelitis (EAE) is the most commonly studied animal model of multiple sclerosis (MS), a chronic autoimmune demyelinating disorder of the central nervous system. Immunomodulatory and immunosuppressive therapies currently approved for the treatment of MS slow disease progression, but do not prevent it. Lyons et al [86] studied a mouse model of EAE involving immunization with myelin oligodendrocyte glycoprotein (MOG35-55). They treated the female C57BL/6 mice with PBM (670 nm) for several days in different regimens. In addition to improved muscular function, they found down-regulation of inducible nitric oxide synthase (iNOS) gene expression in the spinal cords of mice as well as an up-regulation of the Bcl-2 anti-apoptosis gene, an increased Bcl-2:Bax ratio, and reduced apoptosis within the spinal cord of animals over the course of disease. 670 nm light therapy failed to ameliorate MOG-induced EAE in mice deficient in iNOS, confirming a role for remediation of nitrosative stress in the amelioration of MOG-induced EAE by 670 nm mediated photobiomodulation.

### 5.9. Abdominal fat

Yoshimura et al [87] looked at a mouse model of obesity and type 2 diabetes [87]. Four weeks old male adult C57BL/6 mice were fed a hypercaloric high-fat diet (40% calories derived from fat) for eight weeks to induce obesity and hyperglycemia. Over a period of four weeks mice were exposed to six irradiation sessions using an 843 nm LED (5.7 J cm−2, 19 mW cm−2). Non-irradiated control mice had areas of inflammation in their abdominal fat almost five times greater than the PBM group. The PBM group had significantly lower blood glucose levels 24 hours after the last session.

Amongst the many hundreds of reports of clinical applications of PBMT, we will highlight a few here, which seem to be especially relevant to inflammation, and inflammatory disorders.

### 6.1. Achilles tendinopathy

Bjordal et al in Norway carried out a randomized, placebo controlled trial of PBM (904 nm, 5.4 J per point, 20 mW/cm2) for activated Achilles tendinitis [88]. In addition to clinical assessment, they used microdialysis measurement of peritendinous prostaglandin E2 concentrations. Doppler ultrasonography measurements at baseline showed minor inflammation shown by increased intratendinous blood flow, and a measurable resistive index. PGE2 concentrations were significantly reduced with PBM vs placebo. The pressure pain threshold also increased significantly.

### 6.2. Thyroiditis

Chavantes and Chammas in Brazil have studied PBM for chronic autoimmune thyroiditis. An initial pilot trial [89] used 10 applications of PBM (830 nm, 50 mW, 38–108 J/cm2), twice a week, using either the punctual technique (8 patients) or the sweep technique (7 patients). Patients required a lower dosage of levothyroxine, and showed an increased echogenicity by ultrasound. The next study [90] was a randomized, placebo-controlled trial of 43 patients with a 9-month follow-up. In addition to improved thyroid function they found reduced autoimmunity evidenced by lower thyroid peroxidase antibodies (TPOAb), and thyroglobulin antibodies (TgAb). A third study [91] used color Doppler ultrasound to show improved normal vascualrization in the thyroid parenchyma. Finally [92] they showed a statistically significant increase in serum TGF-β1 levels 30 days post-intervention in the PBM group, thus confirming the anti-inflammatory effect. Recently a long-term follow up study of these thyroiditis patients (6 years later) was presented showing that PBM was safe in the long term and demonstrated lasting benefits [93].

### 6.3. Muscles

PBM for muscles aims to benefit athletic performance and training, to reduce delayed onset muscle soreness (DOMS), as well as to ameliorate signs of muscle damage (creatine kinase) after intense or prolonged exercise. Moreover PBM can also be used to treat frank muscle damage caused by muscle strains or trauma. The International Olympic Committee and the World Anti-Doping Agency cannot ban light therapy for athletes considering (1) the intensity is similar to sunlight, and (2) there is no forensic test for light exposure. There have been several clinical trials carried out in Brazil in athletes such as elite runners [94], volleyball players [95] and rugby players [96]. Ferraresi et al conducted a case-controlled study in a pair of identical twins [97]. They used a flexible LED array (850 nm, 75 J, 15 sec) applied to both quadriceps femoris muscles (real to one twin and sham to the other) immediately after each strength training session (3 times/wk for 12 weeks) consisting of leg press and leg extension exercises with load of 80% and 50% of the 1-repetition maximum test, respectively. PBM increased the maximal load in exercise and reduced fatigue, creatine kinase, and visual analog scale (DOMS) compared to sham. Muscle biopsies were taken before and after the training program and showed that PBM decreased inflammatory markers such as interleukin 1β and muscle atrophy (myostatin). Protein synthesis (mammalian target of rapamycin) and oxidative stress defense (SOD2, mitochondrial superoxide dismutase) were up-regulated.

### 6.4. Psoriasis

Psoriasis is a chronic autoimmune skin disease. Psoriasis is characterized by the abnormally excessive and rapid growth of keratinocytes (instead of being replaced every 28–30 days as in normal skin, in psoriatic skin they are replaced every 3–5 days). This hyperproliferation is caused by an inflammatory cascade in the dermis involving dendritic cells, macrophages, and T cells secreting TNF-α, IL-1β, IL-6, IL-17, IL-22, and IL-36γ [98]. PBM has been used for psoriasis because of its anti-inflammatory effects, which is a different approach from UV phototherapy which tends to kill circulating T-cells. Ablon [99] tested PBM using LEDs (830 nm, 60 J/cm2 and 633 nm, 126 J/cm2) in two 20-min sessions over 4 or 5 weeks, with 48 h between sessions in 9 patients with chronic treatment-resistant psoriasis. Clearance rates at the end of the follow-up period ranged from 60% to 100%. Satisfaction was universally very high.

Choi et al [100] tested PBM in case report of a patient with another inflammatory skin disease called acrodermatitis continua, who also had a 10-yr history of plaque psoriasis on her knees and elbows. As she was pregnant and not suited for pharmacological therapy, she received treatment with PBM (broad-band polarized light, 480–3,400 nm, 10 J/cm2). In two weeks (after only 4 treatments), the clinical resolution was impressive and no pustules were found. Topical methylprednisolone aceponate steroid cream was switched to a moisturizer, and she was treated twice or once a week with PBM until a healthy baby was delivered.

### 6.5. Arthritis

As can be seen from the animal studies section, arthritis is one of the most important clinical indications for PBM [101,102]. The two most common forms of arthritis are osteoarthritis (degenerative joint disease that mostly affects the fingers, knees, and hips) and rheumatoid arthritis (autoimmune joint inflammation that often affects the hands and feet). Osteoarthritis (OA) affects more than 3.8% of the population while rheumatoid arthritis (RA) affects about 0.24%. Both types have been successfully treated with PBM. Cochrane systematic reviews found for good evidence for its effectiveness in RA [103], and some evidence in the case of OA [104]. Most clinical studies have used pain scales and range of movement scores to test the effectiveness, rather than measures of inflammation which are difficult to carry out in human subjects.

Barabas and coworkers [105] made an attempt by testing PBM on ex vivo samples of synovial tissue surgically removed from patients receiving knee joint replacement. Synovial membrane samples received exposure to PBM (810 nm, 448 mW, 25 J/cm2, 1 cm2 area). PBM caused an increase in mitochondrial heat shock protein 1 60 kD, and decreases in calpain small subunit 1, tubulin alpha-1C, beta 2,vimentin variant 3, annexin A1, annexin A5, cofilin 1,transgelin, and collagen type VI alpha 2 chain precursor all significantly decreased compared to the control

### 6.6. Alopecia areata

Alopecia areata (AA) is one of the three common types of hair loss, the other two being androgenetic alopecia (AGA, male pattern baldness) and chemotherapy induced alopecia. AA is a common autoimmune disease resulting from damage caused to the hair follicles (HFs) by T cells. Evidence of autoantibodies to anagen stage HF structures is found in affected humans and experimental mouse models. Biopsy specimens from affected individuals demonstrate a characteristic peri- and intrafollicular inflammatory infiltrate around anagen-stage HFs consisting of activated CD4 and CD8 T lymphocytes [106]. PBM is an excellent treatment for hair loss in general and AGA in particular [107,108]. Yamazaki et al [109] reported the use of the “Super-Lizer” delivering linear-polarized light between 600–1600 nm at a power of 1.26 W to the areas of hair loss on the scalp (4-s pulses delivered at 1-s intervals for 3 min every 1 or 2 weeks until hair growth was observed). Regrowth of vellus hairs was achieved on more than 50% ofthe involved areas in all 15 cases. The frequency of irradiation until regrowth ranged from one to 14 times and the duration of SL treatment was 2 weeks to 5 months.

## 7. Conclusion and Future Studies

The clinical applications of PBM have been increasing apace in recent years. The recent adoption of inexpensive large area LED arrays, that have replaced costly, small area laser beams with a risk of eye damage, has accelerated this increase in popularity. Advances in understanding of PBM mechanisms of action at a molecular and cellular level, have provided a scientific rationale for its use for multiple diseases. Many patients have become disillusioned with traditional pharmaceutical approaches to a range of chronic conditions, with their accompanying distressing side-effects and have turned to complementary and alternative medicine for more natural remedies. PBM has an almost complete lack of reported adverse effects, provided the parameters are understood at least at a basic level. The remarkable range of medical benefits provided by PBM, has led some to suggest that it may be “too good to be true”. However one of the most general benefits of PBM that has recently emerged, is its pronounced anti-inflammatory effects. While the exact cellular signaling pathways responsible for this anti-inflammatory action are not yet completely understood, it is becoming clear that both local and systemic mechanisms are operating. The local reduction of edema, and reductions in markers of oxidative stress and pro-inflammatory cytokines are well established. However there also appears to be a systemic effect whereby light delivered to the body, can positively benefit distant tissues and organs.

There is a lot of scope for further work on PBM and inflammation. The intriguing benefits of PBM on some autoimmune diseases, suggests that this area may present a fertile area for researchers. There may be some overlap between the ability of PBM to activate and mobilize stem cells and progenitor cells, and its anti-inflammatory action, considering that one of the main benefits of exogenous stem cell therapy has been found to be its anti-inflammatory effect. The versatile benefits of PBM on the brain and the central nervous system, encourages further study of its ability to reduce neuroinflammation. Chronic diseases of the modern age involving systemic inflammation such as type II diabetes, obesity, Alzheimer's disease, cardiovascular disease and endothelial dysfunction are again worth investigating in the context of PBM.

Original Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5523874/

### Treatment of Neurodegeneration: Integrating Photobiomodulation and Neurofeedback in Alzheimer's Dementia and Parkinson's: A Review

Marvin H Berman, Trent W Nichols - (Publication)
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Objective: A review of photobiomodulation (PBM) in Alzheimer's dementia is submitted. The addition of PBM in neurodegenerative diseases is a dual modality that is at present gaining traction as it is safe, antiviral, and anti-inflammatory for treating neurodegeneration with photons that stimulate mitochondria increasing adenosine triphosphate and proteasomes increasing misfolded protein removal. Neurofeedback provides neural plasticity with an increase in brain-derived nerve factor mRNA and an increase in dendrite production and density in the hippocampus coupled with overall growth in dendrites, density, and neuronal survival. Background: Alzheimer's disease pathophysiology is the accumulation of hyperphosphorylated tau protein neurofibrillary tangles and subsequently amyloid-beta plaques. PBM and neurobiofeedback (NBF)address the multiple gene expression and upregulation of multiple pathogenic pathway inflammation, reactive oxidative stress, mitochondrial disorders, insulin resistance, methylation defects, regulation of neuroprotective factors, and regional hypoperfusion of the brain. There is no human evidence to suggest a clinical therapeutic benefit from using consistent light sources while significantly increasing safety concerns. Methods: A PBM test with early- to mid-Alzheimer's was reported in 2017, consisting of a double-blind, placebo-controlled trial in a small pilot group of early- to mid-dementia subjects under Institutional Review Board (IRB)-approved Food and Drug Administration (FDA) Clinical Trial. Results: PBM-treated subjects showed that active treatment subjects tended to show greater improvement in the functioning of the executive: clock drawing, immediate recall, practical memory, and visual attention and task switching (Trails A&B). A larger study using the CerebroLite helmet in Temple Texas again of subjects in a double-blind, placebo-controlled IRB-approved FDA Clinical Trial demonstrated gain in memory and cognition by increased clock drawing. Conclusions: Next-generation trials with the Cognitolite for Parkinson's disease subjects will incorporate the insights regarding significant bilateral occipital hypocoherence deficits gained from the quantitative EEG analyses. Future applications will integrate noninvasive stimulation delivery, including full-body and transcranial and infrared light with pulsed electromagnetic frequencies.
Original Source: https://pubmed.ncbi.nlm.nih.gov/31647776/

### 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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