Photobiomodulation of Cytochrome c Oxidase by Chronic Transcranial Laser in Young and Aged Brains.

In cellular bioenergetics, cytochrome c oxidase (CCO) is the enzyme responsible for oxygen consumption in the mitochondrial electron transport chain, which drives oxidative phosphorylation for adenosine triphosphate (ATP) production. CCO is also the major intracellular acceptor of photons in the light wavelengths used for photobiomodulation (PBM). Brain function is critically dependent on oxygen consumption by CCO for ATP production. Therefore, our objectives were (1) to conduct the first detailed brain mapping study of the effects of PBM on regional CCO activity, and (2) to compare the chronic effects of PBM on young and aged brains. Specifically, we used quantitative CCO histochemistry to map the differences in CCO activity of brain regions in healthy young (4 months old) and aged (20 months old) rats from control groups with sham stimulation and from treated groups with 58 consecutive days of transcranial laser PBM (810 nm wavelength and 100 mW power). We found that aging predominantly decreased regional brain CCO activity and systems-level functional connectivity, while the chronic laser stimulation predominantly reversed these age-related effects. We concluded that chronic PBM modified the effects of aging by causing the CCO activity on brain regions in laser-treated aged rats to reach levels similar to those found in young rats. Given the crucial role of CCO in bioenergetics, PBM may be used to augment brain and behavioral functions of older individuals by improving oxidative energy metabolism.

Methylene Blue Preserves Cytochrome Oxidase Activity and Prevents Neurodegeneration and Memory Impairment in Rats With Chronic Cerebral Hypoperfusion.

Chronic cerebral hypoperfusion in neurocognitive disorders diminishes cytochrome oxidase activity leading to neurodegenerative effects and impairment of learning and memory. Methylene blue at low doses stimulates cytochrome oxidase activity and may thus counteract the adverse effects of cerebral hypoperfusion. However, the effects of methylene blue on cytochrome oxidase activity during chronic cerebral hypoperfusion have not been described before. To test this hypothesis, rats underwent bilateral carotid artery occlusion or sham surgery, received daily 4 mg/kg methylene blue or saline injections, and learned a visual water task. Brain mapping of cytochrome oxidase activity was done by quantitative enzyme histochemistry. Permanent carotid occlusion for 1 month resulted in decreased cytochrome oxidase activity in visual cortex, prefrontal cortex, perirhinal cortex, hippocampus and amygdala, and weaker interregional correlation of cytochrome oxidase activity between these regions. Methylene blue preserved cytochrome oxidase activity in regions affected by carotid occlusion and strengthened their interregional correlations of cytochrome oxidase activity, which prevented neurodegenerative effects and facilitated task-specific learning and memory. Brain-behavior correlations revealed positive correlations between performance and brain regions in which cytochrome oxidase activity was preserved by methylene blue. These results are the first to demonstrate that methylene blue prevents neurodegeneration and memory impairment by preserving cytochrome oxidase activity and interregional correlation of cytochrome oxidase activity in brain regions susceptible to chronic hypoperfusion. This demonstration provides further support for the hypothesis that lower cerebral blood flow results in an Alzheimer's-like syndrome and that stimulating cytochrome oxidase activity with low-dose methylene blue is neuroprotective.

Transcranial laser stimulation improves human cerebral oxygenation.

BACKGROUND AND OBJECTIVE: Transcranial laser stimulation of the brain with near-infrared light is a novel form of non-invasive photobiomodulation or low-level laser therapy (LLLT) that has shown therapeutic potential in a variety of neurological and psychological conditions. Understanding of its neurophysiological effects is essential for mechanistic study and treatment evaluation. This study investigated how transcranial laser stimulation influences cerebral hemodynamics and oxygenation in the human brain in vivo using functional near-infrared spectroscopy (fNIRS). MATERIALS AND METHODS: Two separate experiments were conducted in which 1,064-nm laser stimulation was administered at (1) the center and (2) the right side of the forehead, respectively. The laser emitted at a power of 3.4 W and in an area of 13.6 cm2, corresponding to 0.25 W/cm2 irradiance. Stimulation duration was 10 minutes. Nine healthy male and female human participants of any ethnic background, in an age range of 18-40 years old were included in each experiment. RESULTS: In both experiments, transcranial laser stimulation induced an increase of oxygenated hemoglobin concentration (Δ[HbO2 ]) and a decrease of deoxygenated hemoglobin concentration (Δ[Hb]) in both cerebral hemispheres. Improvements in cerebral oxygenation were indicated by a significant increase of differential hemoglobin concentration (Δ[HbD] = Δ[HbO2 ] - Δ[Hb]). These effects increased in a dose-dependent manner over time during laser stimulation (10 minutes) and persisted after laser stimulation (6 minutes). The total hemoglobin concentration (Δ[HbT] = Δ[HbO2] + Δ[Hb]) remained nearly unchanged in most cases. CONCLUSION: Near-infrared laser stimulation applied to the forehead can transcranially improve cerebral oxygenation in healthy humans.

Mitochondrial respiration as a target for neuroprotection and cognitive enhancement.

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

Neurological and psychological applications of transcranial lasers and LEDs.

Transcranial brain stimulation with low-level light/laser therapy (LLLT) is the use of directional low-power and high-fluency monochromatic or quasimonochromatic light from lasers or LEDs in the red-to-near-infrared wavelengths to modulate a neurobiological function or induce a neurotherapeutic effect in a nondestructive and non-thermal manner. The mechanism of action of LLLT is based on photon energy absorption by cytochrome oxidase, the terminal enzyme in the mitochondrial respiratory chain. Cytochrome oxidase has a key role in neuronal physiology, as it serves as an interface between oxidative energy metabolism and cell survival signaling pathways. Cytochrome oxidase is an ideal target for cognitive enhancement, as its expression reflects the changes in metabolic capacity underlying higher-order brain functions. This review provides an update on new findings on the neurotherapeutic applications of LLLT. The photochemical mechanisms supporting its cognitive-enhancing and brain-stimulatory effects in animal models and humans are discussed. LLLT is a potential non-invasive treatment for cognitive impairment and other deficits associated with chronic neurological conditions, such as large vessel and lacunar hypoperfusion or neurodegeneration. Brain photobiomodulation with LLLT is paralleled by pharmacological effects of low-dose USP methylene blue, a non-photic electron donor with the ability to stimulate cytochrome oxidase activity, redox and free radical processes. Both interventions provide neuroprotection and cognitive enhancement by facilitating mitochondrial respiration, with hormetic dose-response effects and brain region activational specificity. This evidence supports enhancement of mitochondrial respiratory function as a generalizable therapeutic principle relevant to highly adaptable systems that are exquisitely sensitive to energy availability such as the nervous system.

Transcranial infrared laser stimulation produces beneficial cognitive and emotional effects in humans.

This is the first controlled study demonstrating the beneficial effects of transcranial laser stimulation on cognitive and emotional functions in humans. Photobiomodulation with red to near-infrared light is a novel intervention shown to regulate neuronal function in cell cultures, animal models, and clinical conditions. Light that intersects with the absorption spectrum of cytochrome oxidase was applied to the forehead of healthy volunteers using the laser diode CG-5000, which maximizes tissue penetration and has been used in humans for other indications. We tested whether low-level laser stimulation produces beneficial effects on frontal cortex measures of attention, memory and mood. Reaction time in a sustained-attention psychomotor vigilance task (PVT) was significantly improved in the treated (n=20) vs. placebo control (n=20) groups, especially in high novelty-seeking subjects. Performance in a delayed match-to-sample (DMS) memory task showed also a significant improvement in treated vs. control groups as measured by memory retrieval latency and number of correct trials. The Positive and Negative Affect Schedule (PANAS-X), which tracks self-reported positive and negative affective (emotional) states over time, was administered immediately before treatment and 2 weeks after treatment. The PANAS showed that while participants generally reported more positive affective states than negative, overall affect improved significantly in the treated group due to more sustained positive emotional states as compared to the placebo control group. These data imply that transcranial laser stimulation could be used as a non-invasive and efficacious approach to increase brain functions such as those related to cognitive and emotional dimensions. Transcranial infrared laser stimulation has also been proven to be safe and successful at improving neurological outcome in humans in controlled clinical trials of stroke. This innovative approach could lead to the development of non-invasive, performance-enhancing interventions in healthy humans and in those in need of neuropsychological rehabilitation.

In vivo low-level light therapy increases cytochrome oxidase in skeletal muscle.

Low-level light therapy (LLLT) increases survival of cultured cells, improves behavioral recovery from neurodegeneration and speeds wound healing. These beneficial effects are thought to be mediated by upregulation of mitochondrial proteins, especially the respiratory enzyme cytochrome oxidase. However, the effects of in vivo LLLT on cytochrome oxidase in intact skeletal muscle have not been previously investigated. We used a sensitive method for enzyme histochemistry of cytochrome oxidase to examine the rat temporalis muscle 24 h after in vivo LLLT. The findings showed for the first time that in vivo LLLT induced a dose- and fiber type-dependent increase in cytochrome oxidase in muscle fibers. LLLT was particularly effective at enhancing the aerobic capacity of intermediate and red fibers. The findings suggest that LLLT may enhance the oxidative energy metabolic capacity of different types of muscle fibers, and that LLLT may be used to enhance the aerobic potential of skeletal muscle.

Neuroprotective effects of near-infrared light in an in vivo model of mitochondrial optic neuropathy.

Near-infrared light (NIL) promotes a wide range of biological effects including enhancement of energy production, gene expression and prevention of cell death. This is the first report of the in vivo neuroprotective effects of NIL against optic neuropathy induced by mitochondrial complex I inhibition. Subjects were pigmented rats that received single bilateral intravitreal doses of rotenone, a mitochondrial complex I inhibitor, or rotenone plus one of three different doses of NIL. Treatment effects were evaluated at behavioral, structural and neurochemical levels. Rotenone induced a decrease in visual function, as determined by changes in the dark-adapted illuminance sensitivity threshold, escape latency and rate of successful trials in a two-choice visual task, compared with vehicle-treated controls. Behavioral impairment correlated with a decrease in retinal and visual pathway metabolic activity, retinal nerve fiber layer thickness and ganglion cell layer cell density. These changes were prevented by NIL treatments in a dose-dependent manner. Whole-brain cytochrome oxidase and superoxide dismutase activities were also increased in NIL-treated subjects in a dose-dependent manner, suggesting an in vivo transcranial effect of NIL. In whole-brain membrane isolates, NIL prevented the rotenone-induced decrease in cell respiration. The results show that NIL treatment can effectively prevent the neurotoxic effects of rotenone and that it might be used in the treatment of neurodegenerative disorders associated with mitochondrial dysfunction.

Maturation of extinction behavior in infant rats: large-scale regional interactions with medial prefrontal cortex, orbitofrontal cortex, and anterior cingulate cortex.

The ability to express a behavior during the postnatal period may be related to developmental changes in the recruitment of particular neural systems. Here, we show that developmental changes in the functional interactions involving three cortical regions (the medial prefrontal cortex, orbitofrontal cortex, and anterior cingulate cortex) are associated with maturation of extinction behavior in infant rats. Postnatal day 17 (P17) and P12 pups were trained in a straight-alley runway on an alternating schedule of reward and nonreward [patterned single alternation (PSA)] or on a pseudorandom schedule of partial reinforcement (PRF); the pups were then injected with fluorodeoxyglucose (FDG) and shifted to continuous nonreward (extinction). Handled control groups exposed to the same training environment but not trained on a particular schedule were included. Among P17 pups, extinction proceeded faster in PSA pups relative to PRF pups. No differences were found between P12 groups. FDG uptake, an index of acute changes in functional activity, was quantified in the three cortical regions and 27 other brain regions of interest. A multivariate covariance analysis, seed partial least squares, revealed that functional relationships involving the three cortical regions and large-scale systems of regions throughout the rostrocaudal extent of the brain changed with training in P17 pups. The cortical regions were primarily uncoupled in the younger group. The data suggest that functional maturation of the frontal cortical regions and their interactions with other brain systems are related to the maturational shift in behavior.

Extinction of behavior in infant rats: development of functional coupling between septal, hippocampal, and ventral tegmental regions.

Learning of a behavior at a particular age during the postnatal period presumably occurs when the functional brain circuit mediating the behavior matures. The inability to express a learned behavior, such as inhibition, may be accounted for by the functional dissociation of brain regions comprising the circuit. In this study we tested this hypothesis by measuring brain metabolic activity, as revealed by fluorodeoxyglucose (FDG) autoradiography, during behavioral extinction in 12- and 17-d-old rat pups. Subjects were first trained on a straight alley runway task known as patterned single alternation (PSA), wherein reward and nonreward trials alternate successively. They were then injected with FDG and given 50 trials of continuous nonreward (i.e., extinction). Pups at postnatal day 12 (P12) demonstrated significantly slower extinction rates compared to their P17 counterparts, despite the fact that both reliably demonstrated the PSA effect, i.e., both age groups distinguished between reward and nonreward trials during acquisition. Covariance analysis revealed that the dentate gyrus, hippocampal fields CA1-3, subiculum, and lateral septal area were significantly correlated in P17 but not P12 pups. Significant correlations were also found between the lateral septal area, ventral tegmental area, and the medial septal nucleus in P17 pups. Similar correlative patterns were not found in P12 and P17 handled control animals. Taken together, these results suggest that septal, hippocampal, and mesencephalic regions may be functionally dissociated at P12, and the subsequent maturation of functional connectivity between these regions allows for the more rapid expression of behavioral inhibition during extinction at P17.

Extinction after regular and irregular reward schedules in the infant rat: influence of age and training duration.

Greater persistence in extinction is observed following inconsistent reward compared to that observed following consistent reward, an effect termed the partial reinforcement extinction effect (PREE). We report three experiments in which the extinction rates of random partially reinforced (PRF) or continuously reinforced (CRF) infant rat pups were compared to the extinction rate of pups trained with an alternative and regular schedule of partial reinforcement, known as patterned single alternation (PSA). In PSA, subjects learn to alternate speed of responding in anticipation of the regular alternation of reward and nonreward trials in the straight alley runway. In Experiment 1, 17-day-old PSA subjects showed CRF-like extinction rates; whereas in Experiment 2, in which extinction was initiated early in training prior to the onset of the PSA discrimination, PSA subjects showed prolonged, PRF-like extinction curves. In contrast, 12-day-old pups in Experiment 3 showed no reward-schedule-related differences in extinction, despite differences in behavior during acquisition. These results prompt a modification of Amsel's (1962) model of discrimination learning, and suggest the existence of a dissociation between different types of reward-related expectancies in the younger subjects.

Reduced cytochrome oxidase and memory dysfunction after chronic brain ischemia in aged rats.

The effects of chronic cerebrovascular ischemia on memory function and cytochrome oxidase (CO) activity were investigated. Cerebrovascular insufficiency was induced by permanent bilateral carotid artery ligation (2-VO) in 19 month old rats. Sham surgery in no-vessel occlusion (no-VO) rats were used for controls. Memory function was tested 1 week prior to surgery and then weekly for 21 days using the Morris water maze. Regional brain activity of CO was measured 4 weeks after surgery by quantitative histochemistry. Histologic examination of brain slices was used to evaluate any neuropathology present. Results showed that 2-VO rats were significantly impaired in the water maze task at each testing period with respect to no-VO controls. In addition, CO activity in 2-VO rats was markedly reduced only in the dorsal CA1 region of the hippocampus and in the posterior parietal cortex. These brain regions are involved in visuo-spatial memory mechanisms. Analysis of other brain regions in 2-VO rats did not reveal further CO activity changes. There were no damaged or loss of neurons in 2-VO or no-VO groups in any region examined, including CA1 and posterior parietal cortex. The CA1 region however, is known to undergo neuronal loss 25 weeks after chronic 2-VO suggesting that this vascular insult can induce a slowly-evolving cascade consisting of neuronal damage, atrophy and death. The present findings indicate that reduced CO activity in CA1 and posterior parietal regions can predict neural damage and atrophy prior to structural perikaryal pathology following chronic brain ischemia. In addition, the data shows that neuronal energy metabolic deficiency may initiate visuo-spatial memory impairment in this aging rat model.