Introduction

Experimental autoimmune encephalomyelitis (EAE) is the primary animal model of the human disease, multiple sclerosis (MS), sharing clinical characteristics and pathogenic mechanisms with MS [1]. Both are considered autoimmune, neurodegenerative diseases characterized by chronic demyelination of neurons and eventual loss of axons. Myelin specific CD4⁺ T helper cells are important in disease initiation and progression through the secretion of proinflammatory cytokines, including interferon-gamma (IFNy), interleukin (IL)-17 and tumor necrosis factor alpha (TNFa) [1]. Conversely, anti-inflammatory cytokines IL-10 and IL-4 have been reported to be important to recovery from EAE and disease amelioration [1].

More recently, oxidative and nitrosative stress have been implicated in EAE/MS pathogenesis, both early and late in the disease process. Qi et al. showed that oxidative injury to mitochondria evident by increased protein nitrosylation preceded infiltration of the central nervous system (CNS) by peripheral immune cells, suggesting that oxidative and nitrosative stress are early events in EAE [2], [3]. In addition, Dutta et al. reported that markers of oxidative stress related to mitochondrial energy depletion are associated with chronic MS, and may explain the irreversible pathology associated with chronic phase of MS [4], [5]. Furthermore, the currently approved therapeutic agents for the treatment of MS target the immune response and slow disease progression but do not prevent disease, providing further evidence that additional mechanisms are at play in disease progression. These data suggest that successful treatment of MS may depend on the development of therapeutic modalities that remediate oxidative stress and preserve mitochondrial function.

Photobiomodulation (PBM) induced by 670 nm light is an alternative therapy shown effective in soft tissue injuries, wound healing [6]–[8] and neurodegenerative diseases [9]–[16]. Mechanistically, PBM is thought to function through intracellular signaling pathways involving NO-mediated mechanisms. These mechanisms are triggered when near infrared (NIR) photons interact with the mitochondrial photoacceptor molecule, cytochrome c oxidase, culminating in improved cellular mitochondrial energy metabolism, increased production of cytoprotective factors and improved cell survival [17]. Deciphering the role of nitro-oxidative stress and neuroprotective strategies in 670 nm NIR-LED photobiomodulation of MOG_35–55 induced EAE mice are the focus of current research.

Our laboratory previously showed that photobiomodulation induced by 670 nm light reduced clinical severity of EAE with concomitant down-regulation of IFy and TNFa and up-regulation of IL-4 and IL-10 [18]. In other models, PBM has been shown to down-regulate nitrosoxidative stress and up-regulate anti-oxidant mechanisms [17], [19]–[21]. As noted above, nitrosoxidative stress was recently suggested to be important in axonal loss associated with disease progression in MS/EAE. Thus, we hypothesized that modulation of nitrosoxidative stress by 670 nm light may offer neuroprotection in the EAE model. Treatment with 670 nm light down-regulated NO production by antigen-primed lymph node cells in vitro and iNOS gene expression in EAE mice. These changes were associated with decreased CNS apoptosis in treated mice. These findings suggest that 670 nm NIR-LED photobiomodulation ameliorates EAE by coordinated immune modulation and modulation of nitrosoxidative stress and further support previous data indicating that PBM may be an effective neuroprotective therapy for the treatment of MS.

Results

Discussion

EAE is the most studied animal model of human multiple sclerosis (MS), a disease affecting 400,000 Americans, and about 5 million people worldwide. Much of the current understanding of MS pathogenesis and therapeutic advancement in MS management are derived from studies utilizing the EAE model [35]. The current paradigm describes MS as an autoimmune disease mediated by CD4⁺ myelin-reactive T cells that gain access to the CNS and initiate a proinflammatory response leading to the destruction of the myelin sheath by multiple mechanisms [1]. With time, axonal loss accumulates and disease progresses. Most agents for the treatment of MS are effective early in the disease process and act by targeting the autoimmune response [1], resulting in a slower disease progression. However, these agents are ineffective against primary progressive or secondary progressive disease, suggesting non-immune mechanisms are driving disease progression. Previous studies describe nitrosoxidative stress in the CNS over the course of EAE and MS as contributing to disease progression [2], [3], [5]. Published studies demonstrated down-regulation of nitrosoxidative stress by near infrared/far-red light induced photobiomodulation [19], [20], [36], [37]. We previously reported amelioration of clinical EAE associated with immunomodulation induced by 670 nm light in MOG_35–55 induced EAE in C57BL/6 mice [18]. In this report, we demonstrate that the amelioration of clinical EAE by 670 nm light therapy is also associated with the down-regulation of nitrosoxidative stress and reduced CNS apoptosis in the MOG_35–55-induced model of EAE.

The present studies utilized the Double Treatment Protocol, in which animals are treated for two separate 7 day periods, separated by a 7 day rest during which no light is administered to the animals [18]. This protocol was developed after early experiments demonstrated a temporary treatment effect by 670 nm light when mice were treated with a single 7 day or 10 day treatment protocol [18]. Also noted in previous studies was a worsening of clinical signs in treated animals if treatment periods were extended past 10 days (unpublished data). The biphasic dose-response curve is well-established [38]. However, the worsening of clinical signs with increasing dose observed in the EAE model has not been seen in other model systems and could be related to the autoimmune pathology of MS and dual roles of nitric oxide on the immune response and disease pathogenesis. Thus, the Double Treatment Protocol results in a sustained clinical effect by 670 nm phototherapy while minimizing potential adverse side effects associated with over-treatment.

In this study, in vitro treatment of heterogeneous lymph node populations isolated from MOG_35–55 immunized mice with 670 nm light demonstrated decreased NO generation compared to sham treated cultures in culture supernatants by the Griess reaction. Similar to the effect of in vitro treatment of lymph node cells on the generation of proinflammatory cytokines [18], this effect appeared to be antigen specific, given that similar down-regulation of nitric oxide following 670 nm light treatment was not seen with nonspecific activation with a mitogenic lectin. Thus, it is possible that photobiomodulation induced by 670 nm light could be effective in treatment of antigen-specific autoimmunity, like multiple sclerosis.

The effect of 670 nm light therapy on iNOS gene expression over the course of EAE differed with disease course when administered by the double treatment protocol. In these experiments, no effect on gene expression was noted during the acute episode, while a significant down-regulation was observed during relapse. As demonstrated by Qi et al, evidence of mitochondrial dysfunction and NO generation was evident within the CNS prior to immune infiltration and disease onset [2]. Treatment initiated at disease onset would not inhibit early generation of NO, but would hinder the generation of NO with subsequent relapse. Alternatively, but not mutually exclusive, a significant down-regulation iNOS/NO with relapse would support the role for nitrosoxidative stress in disease progression during the chronic phase, as demonstrated by the work of Dutta et al [4], [5]. Further studies characterizing the mechanism of iNOS/NO down-regulation and the role of NO in disease pathogenesis will be necessary to elucidate these mechanisms.

Previous reports in other model systems demonstrated induction of NO production by NIR light [39], [40], whereas the current work demonstrated down-regulation of NO in the EAE model when utilizing the double-treatment protocol. A comparison of the literature reveals a number of parameters which could contribute to the differential effects of phototherapy on NO production, including treatment parameters, experimental conditions, and the cell populations studied. Treatment parameters, including wavelength and fluence, affect the outcomes of phototherapy [39]–[42]. Those systems that demonstrated up-regulation of NO utilized 780 nm light [39] or broad-band (400–800 nm) visible light [40], whereas our studies, as well as those of Song et al, showing down-regulation of NO utilized 670 nm (current study) or 632.8 nm [41]. Sharma et al. demonstrated down-regulation of inflammatory mediators, including NO, at low fluence but up-regulation of these same mediators at high fluence [42]. Experimental parameters, such as the timing of the assays could also affect the observed results. Other systems characterize the generation of NO immediately following, or shortly after, exposure to NIR light. Our studies focus on NO generation in the days and weeks following NIR light exposure. In addition, the down-regulation of NO observed in the CNS microenvironment could be affected by the complex interaction between the immune response and the CNS microenvironment. This hypothesis is supported by preliminary data from our lab showing a brief up-regulation of iNOS gene expression by lymph node cells within 2 hours of 670 nm light exposure. Furthermore, we noted an initial up-regulation of iNOS gene expression during the preclinical stage of disease when 670 nm light treatment was initiated the day after immunization and continued through the onset of disease. With this same treatment protocol, iNOS gene expression was down-regulated during the acute phase of disease. The observation of divergent effects of NIR light on NO production indicates that careful optimization of treatment parameters will be necessary for phototherapy to be clinically useful. In addition, care in moving forward with the application of phototherapy for the treatment of MS is also prudent: given the relapsing-remitting nature of early disease, it is possible that exacerbation of clinical signs and symptoms may be noted if patients are treated in the period prior to onset of a relapse.

The observed effects of 670 nm light on NO production and iNOS gene expression would be expected to have a beneficial effect on EAE/MS disease progression. Thus, mice lacking iNOS expression (iNOS^?/?) were immunized with MOG_35–55 for initiation of EAE and received 670 nm light therapy according to the double treatment protocol. Although disease severity was ameliorated in WT mice, we failed to observe a similar effect in the iNOS^?/? mice, demonstrating that the clinical effect of 670 nm light therapy in the EAE model is dependent on iNOS gene expression. This may seem counter-intuitive given that 670 nm light down-regulates iNOS expression and NO generation. However, this could suggest a role for, or compensation by, other forms of NOS, including neuronal (n)NOS or endothelial (e)NOS in EAE pathogenesis or the clinical effect of 670 nm light. Alternatively, this observation could be due to the potential multiple roles of NO in the regulation of EAE/MS pathogenesis, as discussed above.

Down-regulation of oxidative stress protects against MS/EAE disease progression through protection from apoptosis [3], [43] [44], [45]. Indeed, decreased apoptosis within the CNS was noted in animals receiving 670 nm light treatment. While the phenotype of the cells undergoing apoptosis in untreated animals was not determined, cell localization and physical characteristics suggest that early in the disease process (acute disease), these were primarily infiltrating immune cells. With disease progression, apoptotic cells appeared to be resident glial cells.

Apoptosis in the CNS has been linked with a number of factors including cytokines and reactive radicals [46]. Our previous data [18] as well as data presented here suggest that photobiomodulation by 670 nm light therapy could be acting via both mechanisms–down-regulation of proinflammatory cytokines and NO–to protect against disease progression. Protection could be mediated via independent mechanisms and/or through coordinated actions. Free radicals are known to be involved in cytokine signaling [47], [48]. Conversely, cytokine expression, particularly INFy, affects NO generation [49], [50]. Previous work demonstrated an effect of 810 nm light, also within the NIR/FR region of the spectrum and postulated to act through interaction with cytochrome c oxidase, on the activation of the transcription factor, NFB [51]. NFB is known to play a role in the regulation of cytokine signaling and iNOS expression [49], [52]. Interestingly, Espejo et al reported the modulation of oxidative stress by IFNy, particularly by CNS glial cells [53], further contributing to the potential mechanism of protection afforded by 670 nm light on the clinical course of EAE. These intriguing ideas are under further investigation.

In the current study, we demonstrate down-regulation of nitrosative stress and protection against apoptosis within the CNS by 670 nm light mediated photobiomodulation in the EAE model of MS. These findings, together with our previously published report [18] demonstrate that clinical effects of 670 nm light therapy are in the EAE model, and perhaps other applications of light therapy, are due to multiple and complex mechanisms. Our studies document the potential therapeutic value of NIR/FR light therapy for the treatment of MS and suggest that this noninvasive therapy may provide the neuroprotection necessary to have a lasting effect on disease pathogenesis in the MS patient.

Materials and Methods

Acknowledgments

Funding Statement

Funding was provided by Quantum Biomedical; Graduate School, UW-Milwaukee; and the College of Health Sciences, UW-Milwaukee. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

Abstract

Introduction

Multiple sclerosis (MS) is a chronic neurodegenerative disease characterized by demyelination, axonal degeneration and subsequent loss of motor function. MS is considered autoimmune in nature, mediated by myelin-reactive CD4⁺ T cells. Recent studies suggest mitochondrial dysfunction and oxidative stress contribute to chronic MS [1]–[3]. Currently approved therapies, including the ?-Interferons, Copaxone, and monoclonal antibody therapies, target the immune response and are only partially effective [4]–[7]. Until therapeutic strategies addressing the ongoing demyelination and axonal damage are developed, treatment of MS will remain incomplete.

Experimental Autoimmune Encephalomyelitis (EAE) is a well-studied, CD4⁺ T-cell-mediated inflammatory demyelinating disease of the central nervous system (CNS) and serves as the primary animal model for MS [8]. Much of what is understood of the pathogenesis of MS, as well as many therapeutic advances are derived from studies using the EAE model. It is generally accepted that autoreactive, myelin-specific T cells are responsible for disease initiation. However, as has been noted in MS, recent studies suggest that oxidative/nitrosative stress play a key role in the pathogenesis of EAE. Studies by Qi et al. demonstrated nitrosylation of CNS proteins very early in the disease process, prior to any evidence of immune infiltration, and prevention of nitrosoxidative stress ameliorated clinical EAE [9], [10]. Studies by Dutta et al support a central role for oxidative/nitrosative stress in the axonal loss leading to permanent disability [2], [3].

Photobiomodulation (PBM) employing light in the far-red (FR) to near-infrared (NIR) range (630–1000 nm) with low-energy lasers or light-emitting diode (LED) arrays has shown a therapeutic effect in various clinical conditions. Far-red/near-infrared light has been used clinically for 30 years for the treatment of soft tissue injuries and to promote wound healing [11]–[13]. More recent studies have demonstrated the therapeutic potential of photobiomodulation with FR/NIR light in the treatment of chemotherapy or radiation-induced mucositis in bone marrow transplant patients [14], ischemic injury in the heart [15]–[17], and neurodegenerative diseases [18]–[23]. The therapeutic mechanism of photobiomodulation is believed to function through activation of cellular photoacceptors and subsequent activation of transcription factors leading to improved energy metabolism and mitochondrial function [24]–[26]. As MS pathogenesis is due to an interaction of chronic inflammatory processes and mitochondrial dysfunction, we hypothesized that FR/NIR light therapy would ameliorate disease pathogenesis in MOG_35–55 peptide-induced EAE in C57BL/6 (B6) mice.

Previous studies indicate that the photoacceptor through which FR/NIR light mediates the observed effects is cytochrome C oxidase, a key molecule in the electron transport chain leading to production of ATP [26]–[28]. The action spectrum of FR/NIR light overlays the absorption spectrum of cytochrome C oxidase: the biological activity of FR/NIR light is most pronounced around 670 nm and 830 nm with a nadir in both spectra around 728 nm, mirroring the absorption spectrum of cytochrome C oxidase [26]. Based on previous studies characterizing the action spectrum of FR/NIR light and the absorption spectrum of cytochrome c oxidase, we investigated therapeutic potential of photobiomodulation by 670 nm light for the treatment of MS using the EAE model. Treatment with 670 nm light prior to or with the onset of clinical signs resulted in amelioration of clinical EAE. Subsequent studies revealed decreased expression of proinflammatory cytokines and increased expression of anti-inflammatory cytokines by lymph node cells exposed to 670 nm light compared to sham treatment. Similar results were observed in spinal cords isolated from mice over the course of EAE. Hence, our findings support the hypothesis that photobiomodulation induced by 670 nm light ameliorates disease severity in EAE.

Results

Discussion

Multiple Sclerosis (MS) is a leading cause of neurologic disability in young adults. Disease pathogenesis is complex. Based on studies in the EAE model, disease is believed to be initiated by autoreactive CD4+ Th1 and/or Th17 lymphocytes specific for myelin proteins [31]. These cells are activated in the periphery by unknown mechanisms and infiltrate the CNS where they secrete proinflammatory cytokines (e.g., IFN-?, TNF-?, IL-17) to initiate and propagate a proinflammatory response leading to demyelination of CNS axons by multiple mechanisms, including: cytokine-mediated demyelination by TNF? [32]; antibody-mediated mechanisms, ± complement [33]–[35]; macrophage/phagocytic mechanisms [36]–[38]; and oxidative stress [2], [3]. Ongoing insult leads to axonal loss and permanent disability [3], [39]. More recent studies indicate that disease progression is due to non-immune mechanism, suggesting a role for oxidative stress in the axonal loss occurring late in the disease process [2], [3].

The FDA-approved therapeutics for MS act through immunosuppressive or immunomodulatory mechanisms, targeting those processes important to the initiation of clinical activity associated with the relapsing/remitting phase of disease [4]–[6], [40]–[42]. These agents slow disease progression but do not prevent it, and are ineffective in secondary progressive or primary progressive disease, perhaps because they fail to provide a neuroprotective component. Thus, the development of new agents or adjunct therapies preventing demyelination or axonal loss, or promoting remyelination and oligodendrocyte function, is of great interest.

The therapeutic application of light to elicit a biological response has been studied using different modalities and different wavelengths. Various approaches, all involving introduction of an exogenous photosensitizer which is then activated to elicit a clinical effect, have been employed in a variety of systems, including the EAE model [43]–[47]. The biological response of ultraviolet light is well recognized [48]–[53], and has been studied in the EAE model [54]. Photobiomodulation, also referred to as Low Level Light Therapy (LLLT), as employed in the current studies, differs from these previous modalities in the use of FR/NIR light delivered in the absence of an exogenous photosensitive moiety (reviewed in [25]). Photobiomodulation has recently attracted increased attention. In particular, photobiomodulation performed with FR/NIR light has demonstrated efficacy in chronic wound healing in clinical and experimental systems [12], [13], [30] and for the treatment of neurodegenerative diseases [18]–[20], [22], including Parkinson’s disease [22], [23], [55], retinitis pigmentosa [20], and stroke [56], [57]. As MS pathogenesis is proinflammatory and neurodegenerative, we investigated the therapeutic potential of FR/NIR light using the EAE model.

Data presented demonstrate that photobiomodulation by 670 nm light led to amelioration of the clinical severity of MOG_35–55 induced EAE compared to sham-treated animals when treatment was initiated following immunization but prior to onset of clinical signs (e.g., suppression protocol) and by a single 7 day treatment protocol initiated on the day of disease onset (e.g., treatment protocol). Interestingly, the effect noted with these 2 protocols (e.g., decreased severity of the acute phase and increased duration of recovery) was similar. Furthermore, no effect on the onset of clinical signs was noted with the suppression protocol. These data suggest that there is no effect on the induction of the autoimmune response, but rather on the pathogenic mechanisms affecting the disease process.

The efficacy of the clinical effect of 670 nm light improved when a second treatment period was added (e.g., double-treatment protocol), leading to sustained clinical improvement in affected animals. The improved efficacy of the double-treatment protocol is explained by the current understanding of disease pathogenesis and the therapeutic mechanism of photobiomodulation. MS and EAE are, by nature, relapsing-remitting diseases, with initial attacks due to immunoregulatory mechanisms. While the B6 model of EAE employed here displays a more chronic phenotype, mice do often times recover partially from peak disease scores attained during the acute phase. Disease progression or sustained neurological deficit is thought to be due to oxidative damage to CNS axons. Photobiomodulation mediated by FR/NIR light is hypothesized to induce gene transcription, mediated by interaction of FR/NIR light with the cytochrome c oxidase photoacceptor [26]. Cytochrome c oxidase is an important participant in the electron transport chain, critical for the maintenance of mitochondrial function. The molecule binds, interchangeably, molecular oxygen and nitric oxide. Nitric oxide is known to mediate intracellular signal transduction cascades and communication between the mitochondrion and the nucleus [58]. Thus, interaction of FR/NIR light with cytochrome c oxidase is thought to lead to the release of nitric oxide, initiating signaling transduction cascades culminating in activation of transcription and translation of nuclear-encoded proteins, leading to the observed effects of photobiomodulation [17], [59]–[61]. A recent study by Chen et al suggests that the observed effects are at least in part mediated by the transcription factor, NF?B, and an increase in cellular ATP synthesis [62]. In the EAE model, these effects are apparent as down-regulation of pro-inflammatory mechanisms and up-regulation of anti-inflammatory mechanisms. Thus, gene induction by suppression and treatment protocols would be expected to be similar. However, with the cessation of the suppression protocol at or around the day of onset, gene transcription would eventually return to pre-treatment levels, thus abrogating the protective effect of FR/NIR light. On the other hand, the double-treatment protocol would be expected to sustain gene transcription and improve the clinical effect.

The structure of the double treatment protocol, with inclusion of a 7 day rest period, was developed with insight provided by other protocols not presented here. For example, early studies employed a protocol in which treatment was initiated the day after immunization and continued through the peak of disease. This protocol resulted in a worsening of clinical signs in 670 nm-treated animals compared to sham-treated mice. This could be due to the dual role of iNOS/nitric oxide in regulation of EAE and the immune response [63]–[68]. These studies indicated that nitric oxide is important in regulating T cell activation to neuroantigens during disease initiation [63] while playing a pathogenic role in ongoing disease [2], [64]. The 7 day rest period typically occurs during the remission/recovery phase of disease when the immune response responsible for relapse is being primed, and treatment is again initiated at or around the day of the first indications of relapse. Thus, this 7 day rest period may be providing an opportunity for important immunomodulatory functions of nitric oxide to occur, preventing excessive activation of autoreactive T cells, while subsequent treatment is down-regulating the pathogenic contribution of nitric oxide to disease progression. In support of this, more recent studies incorporating a third treatment period following a second 7 day rest demonstrated continued suppression of clinical signs in treated animals.

In vitro and in vivo experiments demonstrate modulation of pro-inflammatory vs. anti-inflammatory cytokines by 670 nm light in a manner consistent with the observed effects on disease course. It is interesting to note that immunomodulation appears to require signaling through antigen-specific mechanisms (i.e., the T cell receptor; TcR), as evidenced by a lack of cytokine modulation when cells are stimulated with ConA. ConA is a mitogenic lectin that activates T cells in a polyclonal manner via cross-linking of cell-surface ?-glycans. It is generally used as a positive control for cell viability and the ability to respond to appropriate stimulatory signals. It does not interact with nor signal through the antigen-specific TcR and therefore activates different signal transduction cascades than would antigen-specific activation through the TcR and requisite co-stimulatory pathways (e.g., CD28). This observation could not only have relevance to the clinical application of 670 nm-mediated photobiomodulation but could also provide insight into the mechanisms behind the observed clinical effects.

The neuroprotection afforded by FR/NIR light noted in other systems is thought to be due to down-regulation of oxidative stress and improved cytoprotective mechanisms [25], [59], [69]. Indeed, ongoing studies suggest that similar mechanisms are involved in the observed effect of 670 nm light on the EAE disease course. However, given the autoimmune nature of MS and the described complications associated with monoclonal antibody therapies [70], as well as the known effects on chronic inflammation, it is important to understand the effect of FR/NIR light on the immune response. Understanding of the effect of FR/NIR light on immunity is limited [30], [71], [72]. Data from in vitro and ex vivo studies presented here demonstrate an immunomodulatory effect of 670 nm light on antigen-specific cytokine production, with down-regulation of proinflammatory cytokines, IFN-? and TNF-?, and up-regulation of anti-inflammatory cytokines, IL-4 and IL-10. These cytokine changes would be expected to have a beneficial effect in the EAE model and on the clinical course of MS [4], [31], [73], [74].

Ongoing studies seek to develop photobiomodulation as an adjunct therapy for the treatment of MS, used in conjunction with the currently approved immunomodulatory agents. However, several questions need to be answered before translation into human system will be possible, including optimization of treatment wavelength, the nature of the treatment device, and the penetration of light to affected structures. A recent comprehensive review indicates photobiomodulation may achieve different effector functions, therapeutic or disease aggravation depending on choice of photobiomodulation parameters. These parameters include; 1) the optical properties of tissues, and 2) dositometry (i.e irradiance and dosing) [60]. A primary question that needs to be addressed before these questions can be answered is whether the observed effects are due to systemic effects or targeted (e.g., within the CNS) effects of light on the system. The total body irradiation employed here leaves this up to debate. The total body irradiation used here could result in systemic, transcutaneous effects as well as effects mediated by transmission of light through the optic nerve. On the one hand, the immunomodulation noted in the current studies may support a systemic effect, or at least an effect in the periphery as opposed to the CNS, as the disease-inducing T-cells must be activated in the periphery in order to traverse the blood brain barrier and cause disease [75], [76]. In addition, the immunomodulatory effects noted in the suppression protocol would have been primed in the lymph nodes. On the other hand, light would also be transmitted via the optic nerve or could directly penetrate the spinal cord. Ongoing studies demonstrate decreased oxidative stress and apoptosis within the spinal cord of treated animals. Furthermore, recent studies utilizing a similar LED device demonstrated that transmission of light via the optic nerve preserved CNS function following transection of the optic nerve [77]. These will be challenging questions to answer, and it is possible that both systemic and targeted mechanisms contribute to the observed clinical effects. Further studies and procurement of additional instrumentation will be necessary to address these questions, and the answers to these questions will be important to the translation of these studies to the human population.

Several studies have established the role of immune system in EAE and MS [31]. Recent studies have implicated other pathogenic mechanisms, including mitochondrial dysfunction and oxidative/nitrosative stress in EAE and MS [2], [3], [9], [10], [62]. Data presented here show that treatment with 670 nm light ameliorates disease severity in EAE, in part through immunomodulatory mechanisms. Ongoing studies further investigate the observed immune modulation and address the role of FR/NIR light in preservation of mitochondrial function and remediation of oxidative stress in the EAE model. These experiments suggest a combination of both mechanisms in the amelioration of EAE, indicating that photobiomodulation may be

Materials and Methods

Acknowledgments

Footnotes

References