The technique of photobiomodulation is based on the phenomenon that exposure to low-level laser light can alter the function of biological cells. The observed physiologic effects of such treatment include increased rates of tissue regeneration as well as inflammation and pain relief. The exact cellular mechanisms underlying photobiomodulation, however, are not entirely understood and are actively being investigated.M
Our group previously reported that near-IR light treatment can prevent cell death (apoptosis) in cultured neuronal (brain) cells. As seen in Figure 1, control cultures had relatively few dead cells (4.75% of total cell population). On the other hand, cultures subjected to 300μM potassium cyanide (KCN) for 28h showed a profusion of neurons with highly condensed DNA (83.6%), indicative of cell death. Similar cultures that underwent 10min of near-IR light treatment demonstrated substantially reduced numbers of neurons exhibiting cell death (43.5%), supporting the role of photobiomodulation in promoting cell survival.1
Figure 1. Near-IR (670nm) light treatment attenuates KCN-induced apoptosis (i.e., programmed cell death) in primary cultured neurons. Primary visual cortical neurons subdivided into three groups: (A) control, (B) exposed to 300μM KCN for 28h, and (C) pretreatment with 670nm light for 10min before exposure to 300μM KCN for 28h. Cultures were then stained with propidium iodide for neurons undergoing cell death (arrows).
Figure 2. Cytochrome oxidase and the correlation between the near-IR absorption spectrum of the protein, ATP (adenosine triphosphate) content, and cytochrome oxidase activity in cultured primary neuronal cells subjected to metabolic inhibition and near-IR light treatment. P side: Positive side of the membrane. N side: Negative side of the membrane.
Figure 3. Light treatment (670nm) attenuates MPP+ cytotoxicity in human dopaminergic cells expressing A30P mutant α-synuclein. Results expressed as mean values ± SE (standard error) of replicated culture wells within an experiment. Differences between groups determined by one-way analysis of variance. ** p<0.05. MPP+: 1-Methyl-4-phenylpyridinium.
Work by us and others ascribes the ability of specific wavelengths of light to promote cellular proliferation to the activation of mitochondria, the energy-producing organelles within the cell. Indeed, a growing body of evidence suggests that cytochrome oxidase, a mitochondrial protein with four redox active metal centers, is a key photoacceptor of light in the far-red to near-IR spectral range (see Figure 2).2–5 We have shown that the action spectrum for the stimulation of cytochrome oxidase activity and cellular energy stores (so-called ATP content) parallels the near-IR absorption spectrum of cytochrome oxidase (see Figure 2). Moreover, irradiation at 660–680nm increases the activity of purified protein and the energy production rates of isolated mitochondria,3 and upregulates cytochrome oxidase activity in cultured neuronal cells.4 These results indicate that cytochrome oxidase, and thus mitochondria energy production, are cellular targets influenced by near-IR light treatment.
The evidence that near-IR treatment can augment mitochondrial function and stimulate antioxidant protective pathways comes from photobiomodulation experiments carried out using a laboratory model of Parkinson's disease (PD). Cultures of human dopaminergic neuronal cells engineered to stably overexpress the PD mutant form of α-synuclein were exposed to increasing concentrations of the dopaminergic toxin MPP+ (1-methyl-4-phenylpyridinium) for 14h (see Figure 3). Cell proliferation (protein concentration), mitochondrial function (mitochondrial dehydrogenase activity), oxidative stress (hydrogen peroxide production), and cell viability (lactate dehydrogenase release) were assessed 12h later. Exposure to MPP+ produced a concentration-dependent decrease in these parameters accompanied by a concentration-dependent increase in the production of reactive oxygen species. Three 670nm light (8J/cm2) treatments significantly attenuated the cytotoxic actions of MPP+.
Near-IR treatment can also augment mitochondrial function and stimulate antioxidant protective pathways in specific neurons that offer protection against degeneration in a mouse model of PD. Mammals treated with MPTP, a metabolic precursor of MPP+, develop many of the cardinal features of PD, manifested predominately as a marked reduction in locomotor activity hours after administration of the toxin. The rapid onset of the Parkinsonian syndrome following acute MPTP intoxication thus provides an excellent paradigm for initial assessment of the therapeutic potential of near-IR photon therapy. MPTP has the added advantage that it poisons the very process thought to account for the beneficial actions of near-IR light, namely, mitochondrial energy production.
Figure 4. Light pretreatment (670nm) ameliorates the toxicity of the Parkinsonian drug MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydroxypyridine). NIR: Near-IR.
To investigate the ability of near-IR light to ameliorate the acute toxicity of the neurotoxin MPTP, mice were either pretreated with 670nm photon irradiation or were treated after MPTP exposure. Each animal was tested for locomotor activity from 0–12, 23–24, 47–48, and 71–72h post-injection. Administration of MPTP alone caused a profound depression of all of the locomotor parameters measured, which was sustained for at least 48h (see Figure 4). A single 670nm light treatment (10min, 60J/cm2) administered following MPTP did not alter the locomotor behavior brought about by MPTP (data not shown). Thus, 670nm light treatment was not able to reverse the effects of MPTP when applied after the toxin. Conversely, 670nm light pretreatment for 5min (30J/cm2) twice a day over 3 days attenuated the deficits in locomotor behavior (length of time spent moving, the number of movements made, distance moved, and velocity) induced by a single injection of MPTP (see Figure 4). Furthermore, by 48h the behaviors were essentially restored to control levels.
Taken together, these results show the potential clinical applications of near-IR photobiomodulation. Identifying the key mitochondrial component involved in this process, cytochrome oxidase, suggests a plausible mechanism of action for promoting cell survival. In addition, the ability of near-IR light treatment to attenuate cell death in a laboratory model of PD and the clear therapeutic benefit against the acute toxicity of MPTP indicates the potential benefit of this therapy for patients suffering with PD and possibly other neurodegenerative diseases.
Harry Whelan, Ellen Buchmann, Michele Henry, Margaret Wong-Riley
Medical College of Wisconsin
Harry Whelan is professor of neurology, pediatrics, and hyperbaric medicine at the Medical College of Wisconsin, where he has over 20 years' experience conducting studies on the use of near-IR light for photodynamic therapy of cancer and, in combination with hyperbaric oxygen, for stimulation of human tissue growth and wound healing.
Kristina Desmet, Janis Eells
University of Wisconsin Milwaukee
University of Wisconsin Madison
1. M. T. Wong-Riley, H. L. Liang, J. T. Eells, B. Chance, M. M. Henry, E. Buchmann, M. Kane, H. T. Whelan, Photobiomodulation directly benefits primary neurons functionally inactivated by toxins: role of cytochrome oxidase, J. Biol. Chem. 280, no. 6, pp. 4761-4771, 2005.