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In this study, we explored the effects of a longer term application, up to 12 weeks, of photobiomodulation in normal, naïve macaque monkeys. Monkeys (n = 5) were implanted intracranially with an optical fibre device delivering photobiomodulation (red light, 670 nm) to a midline midbrain region. Animals were then aldehyde-fixed and their brains were processed for immunohistochemistry. In general, our results showed that longer term intracranial application of photobiomodulation had no adverse effects on the surrounding brain parenchyma or on the nearby dopaminergic cell system. We found no evidence for photobiomodulation generating an inflammatory glial response or neuronal degeneration near the implant site; further, photobiomodulation did not induce an abnormal activation or mitochondrial stress in nearby cells, nor did it cause an abnormal arrangement of the surrounding vasculature (endothelial basement membrane). Finally, because of our interest in Parkinson's disease, we noted that photobiomodulation had no impact on the number of midbrain dopaminergic cells and the density of their terminations in the striatum. In summary, we found no histological basis for any major biosafety concerns associated with photobiomodulation delivered by our intracranial approach and our findings set a key template for progress onto clinical trial on patients with Parkinson's disease.

We have reported previously that intracranial application of near-infrared light (NIr) - when delivered at the lower doses of 25J and 35J - reduces clinical signs and offers neuroprotection in a subacute MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) monkey model of Parkinson's disease. In this study, we explored whether a higher NIr dose (125J) generated beneficial effects in the same MPTP monkey model (n=15). We implanted an NIr (670nm) optical fibre device within a midline region of the midbrain in macaque monkeys, close to the substantia nigra of both sides. MPTP injections (1.8-2.1mg/kg) were made over a five day period, during which time the NIr device was turned on and left on continuously throughout the ensuing three week survival period. Monkeys were evaluated clinically and their brains processed for immunohistochemistry and stereology. Our results showed that the higher NIr dose did not have any toxic impact on cells at the midbrain implant site. Further, this NIr dose resulted in a higher number of nigral tyrosine hydroxylase immunoreactive cells when compared to the MPTP group. However, the higher NIr dose monkeys showed little evidence for an increase in mean clinical score, number of nigral Nissl-stained cells and density of striatal tyrosine hydroxylase terminations. In summary, the higher NIr dose of 125J was not as beneficial to MPTP-treated monkeys as compared to the lower doses of 25J and 35J, boding well for strategies of NIr dose delivery and device energy consumption in a future clinical trial.

We have reported previously that intracranial application of near-infrared light (NIr) reduces clinical signs and offers neuroprotection in a subacute MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) monkey model of Parkinson's disease. In this study, we explored whether NIr reduces the gliosis in this animal model. Sections of midbrain (containing the substantia nigra pars compacta; SNc) and striatum were processed for glial fibrillary acidic protein (to label astrocytes; GFAP) and ionised calcium-binding adaptor molecule 1 (to label microglia; IBA1) immunohistochemistry. Cell counts were undertaken using stereology, and cell body sizes were measured using ImageJ. Our results showed that NIr treatment reduced dramatically (~75 %) MPTP-induced astrogliosis in both the SNc and striatum. Among microglia, however, NIr had a more limited impact in both nuclei; although there was a reduction in overall cell size, there were no changes in the number of microglia in the MPTP-treated monkeys after NIr treatment. In summary, we showed that NIr treatment influenced the glial response, particularly that of the astrocytes, in our monkey MPTP model of Parkinson's disease. Our findings raise the possibility of glial cells as a future therapeutic target using NIr.

Objective To examine whether near-infrared light (NIr) treatment reduces clinical signs and/or offers neuroprotection in a subacute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) monkey model of Parkinson disease. Methods We implanted an optical fiber device that delivered NIr (670nm) to the midbrain of macaque monkeys, close to the substantia nigra of both sides. MPTP injections (1.5–2.1mg/kg) were made over a 5- to 7-day period, during which time the NIr device was turned on. This was then followed by a 3-week survival period. Monkeys were evaluated clinically (eg, posture, bradykinesia) and behaviorally (open field test), and their brains were processed for immunohistochemistry and stereology. Results All monkeys in the MPTP group developed severe clinical and behavioral impairment (mean clinical scores = 21–34; n = 11). By contrast, the MPTP-NIr group developed much less clinical and behavioral impairment (n = 9); some monkeys developed moderate clinical signs (mean scores = 11–15; n = 3), whereas the majority—quite remarkably—developed few clinical signs (mean scores = 1–6; n = 6). The monkeys that developed moderate clinical signs had hematic fluid in their optical fibers at postmortem, presumably limiting NIr exposure and overall clinical improvement. NIr was not toxic to brain tissue and offered neuroprotection to dopaminergic cells and their terminations against MPTP insult, particularly in animals that developed few clinical signs. Interpretation Our findings indicate NIr to be an effective therapeutic agent in a primate model of the disease and create the template for translation into clinical trials. ANN NEUROL 2016;79:59–75

Following central nervous system lesion, the ability of injured axons to regrowth may depend on the level and duration of the injured cell body response (CBR). Therefore, to investigate whether axotomized brainstem neurons maintain a durable growth-competent state after spinal cord injury, we studied the effect of a chronic C2 hemisection in rats on the expression of various CBR markers involved in axon regeneration, such as c-Jun, ATF-3, HSP27, NO synthase (NOS), and also of the neural mature phenotype marker NeuN, in the bulbospinal respiratory neurons as compared to the gigantocellularis nucleus. Both at 7 and 30 days post-lesion (DPL), c-Jun and HSP27 were present in, respectively, ~60 and ~20% of the axotomized respiratory neurons, whereas the apoptotic factor caspase 3 was not detected in these cells. NOS appeared belatedly, and it was detected in ~20% of the axotomized respiratory neurons at 30DPL. At 30DPL, these different CBR markers were strongly colocalized in a sub-population of axotomized respiratory neurons and also in a sub-population of injured neurons within the gigantocellularis nucleus. Such CBR was also accompanied by a sustained alteration of the neural mature phenotype, as indicated by a loss of NeuN immunoreactivity selectively in HSP27+ bulbospinal neurons at 7DPL and 30DPL. Altogether, this study shows that a subset of axotomized medullary respiratory neurons remains in a growth-competent state after a chronic injury, suggesting that they may play a preferential role in long-lasting respiratory neuroplasticity processes.

Spinal cord injury (SCI) causes an interruption of descending motor and autonomic nervous tracts. However, a partial injury, and particularly a unilateral section, is generally followed by spontaneous locomotor and respiratory recovery. Although locomotor functional recovery has been correlated to spontaneous anatomical plasticity of the corticospinal tract, the remodeling of the bulbospinal tract that sustains respiratory improvement is unknown and has therefore been investigated here after chronic lateral cervical injury in rats (90 days post-lesion by comparison to 7 days post-lesion). We show that chronic lateral C2 SCI leads both to a decreased thickness of the ipsilateral ventrolateral funiculus at sus and sub-lesional levels and to an opposite effect on the contralateral side. At C1 level, the number of ventrolateral bulbospinal fibers, stained with anterograde tracer was reduced within the ipsilateral ventrolateral funiculi while collateral arborization toward the gray matter and growth associated protein-43 levels was increased. At C2 lesional level, fibers rerouting toward the gray matter were also identified for 5% of the axotomized axon terminals. Despite these chronic sprouting processes respiratory bulbospinal projections to ipsilateral phrenic nucleus remained poor (less than 10% compared to non-injured conditions). Retrograde labeling of projections onto the phrenic nucleus revealed, after chronic injury, an increased recruitment of C1 propriospinal interneurons which moreover received more contacts from bulbospinal collaterals. This chronic remodeling was correlated with chronic diaphragm recovery under conditions of respiratory stress. Thus, despite extensive axonal loss and absence of direct phrenic reinnervation by bulbospinal respiratory neurons, sprouting processes toward cervical propriospinal neurons may contribute to the observed partial respiratory recovery.

High cervical spinal cord injury (SCI) interrupts bulbospinal respiratory pathways innervating phrenic motoneurons, and induces an inactivation of phrenic nerves (PN) and diaphragm. We have previously shown that the ipsilateral (ipsi) PN was inactivated following a lateral C2 SCI, but was spontaneously partially reactivated 7 days post-SCI. This phrenic reactivation depended on contralateral (contra) descending pathways, located laterally, that cross the spinal midline. We analysed here whether long-term post-lesional changes may occur in the respiratory network. We showed that ipsi PN reactivation was greater at 3 months compared with 7 days post-SCI, and that it was enhanced after acute contra phrenicotomy (Phx), which also induced a substantial reactivation of the ipsi diaphragm (not detected at 7 days post-SCI). At 3 months post-SCI (compared with 7 days post-SCI), ipsi PN activity was only moderately affected by ipsi Phx or by gallamine treatment, a nicotinic neuromuscular blocking agent, indicating that it was less dependent on ipsi sensory phrenic afferents. After an additional acute contra SCI (C1) performed laterally, ipsi PN activity was abolished in rats 7 days post-SCI, but persisted in rats 3 months post-SCI. This activity thus depended on new functional descending pathways located medially rather than laterally. These may not involve newly recruited neurons as retrograde labelling showed that ipsi phrenic motoneurons were innervated by a similar number of medullary respiratory neurons after a short and long post-lesional time. These results show that after a long post-lesional time, phrenic reactivation is reinforced by an anatomo-functional reorganization of spinal respiratory pathways.

To examine whether near-infrared light (NIr) treatment reduces clinical signs and/or offers neuroprotection in a subacute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) monkey model of Parkinson disease.We implanted an optical fiber device that delivered NIr (670 nm) to the midbrain of macaque monkeys, close to the substantia nigra of both sides. MPTP injections (1.5-2.1mg/kg) were made over a 5- to 7-day period, during which time the NIr device was turned on. This was then followed by a 3-week survival period. Monkeys were evaluated clinically (eg, posture, bradykinesia) and behaviorally (open field test), and their brains were processed for immunohistochemistry and stereology.All monkeys in the MPTP group developed severe clinical and behavioral impairment (mean clinical scores = 21-34; n = 11). By contrast, the MPTP-NIr group developed much less clinical and behavioral impairment (n = 9); some monkeys developed moderate clinical signs (mean scores = 11-15; n = 3), whereas the majority--quite remarkably--developed few clinical signs (mean scores = 1-6; n = 6). The monkeys that developed moderate clinical signs had hematic fluid in their optical fibers at postmortem, presumably limiting NIr exposure and overall clinical improvement. NIr was not toxic to brain tissue and offered neuroprotection to dopaminergic cells and their terminations against MPTP insult, particularly in animals that developed few clinical signs.Our findings indicate NIr to be an effective therapeutic agent in a primate model of the disease and create the template for translation into clinical trials.

La maladie de Parkinson (MP) est une maladie neurodégénérative chronique affectant le système dopaminergique et se traduisant par une perte des neurones dopaminergiques (DA) de la substance noire compacta (SNc) et des faisceaux nigro-striés. Le diagnostic précoce de la MP est difficile car 70 % des neurones dopaminergiques de la substance noire sont détruits, avec une dysfonction mitochondriale, quand les premiers signes moteurs apparaissent. Les traitements actuels sont tous des traitements symptomatiques, à ce jour il n’existe pas de traitement curatif de la MP. Des études antérieures ont montré que des molécules, dont CoQ10 et la mélatonine, ont un effet neuroprotecteur sur les cellules DA de la SNc contre la dégénérescence induite dans des modèles animaux de la MP qui induisent un dysfonctionnement mitochondrial. Des résultats récents ont rapporté des propriétés neuroprotectrices de la thérapie par illumination de faible intensité (ou photobiomodulation par lumière proche infrarouge (NIR)) par l'activation de la cytochrome C oxydase et la synthèse d'ATP dans les cellules endommagées. Des expériences préliminaires ont démontré « in vivo » une protection par NIR des cellules DA dans la SNc, de souris MPTP et rats 6-OHDA aigus et chroniques, associée à une amélioration de l'activité locomotrice. L’objectif de notre étude est de montrer l’effet de NIR chez des primates (PNH) MPTP. Nous utilisons un prototype d'implant intracérébral chronique lumineux (longueur d'onde 670 nm) permettant de délivrer le traitement voulu à proximité de la zone d’intérêt. La puissance de l'implant est de 10 mW avec un cycle d’illumination 5 sec. ON/ 60 sec. OFF. L’effet thermique de l'appareil a déjà été testé pour se limiter à + 1 °C maximum. Les groupes contrôles aigu et chronique (5 PNH chacun) ont reçu du MPTP (0,3 mg/kg/jour) pendant 5 jours pour le groupe aigu et avec 2 injections supplémentaires (1 fois/semaine) pour le groupe chronique. Les groupes traités avec NIR (5PNH chacun, avec implantation du dispositif) sont également soumis au protocole MPTP en aigu ou chronique et reçoivent le traitement NIR : aigu (24 h autour de l’injection de MPTP) ou chronique (en continu pendant les 3 semaines du protocole). Le score clinique et l'activité motrice ont été évalués tous les jours pendant 1 semaine avant et 3 semaines après le début de l'injection de MPTP. Les animaux sont sacrifiés 3 semaines après le début des injections, et les cerveaux traités pour une histologie (coloration de Nissl et TH, alphasynucléine), pour la quantification de l'effet neuroprotecteur du traitement NIR. Les résultats préliminaires montrent un effet comportemental du traitement NIR sur ce modèle de MP, effet qui semble corrélé au niveau histologique. D'autres expériences sont nécessaires pour étudier l’effet-dose. Cette étude est la première étape vers un essai clinique humain visant à évaluer le potentiel neuroprotecteur de NIR chez des patients parkinsoniens au stade précoce.

We explored whether 810 nm near-infrared light (NIr) offered neuroprotection and/or improvement in locomotor activity in an acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mouse model of Parkinson's disease. Mice received MPTP and 810 nm NIr treatments, or not, and were tested for locomotive activity in an open-field test. Thereafter, brains were aldehyde-fixed and processed for tyrosine hydroxylase immunohistochemistry. Our results showed that MPTP-treated mice that were irradiated with 810 nm NIr had both greater locomotor activity (∼40%) and number of dopaminergic cells (∼20%) than those that were not. In summary, 810 nm (as with 670 nm) NIr offered neuroprotection and improved locomotor activity in MPTP-treated mice.

International audience; After incomplete spinal cord injury (SCI), patients and animals may ă exhibit some spontaneous functional recovery which can be partly ă attributed to remodeling of injured neural circuitry. This post-lesion ă plasticity implies spinal remodeling but increasing evidences suggest ă that supraspinal structures contribute also to the functional recovery. ă Here we tested the hypothesis that partial SCI may activate ă cell-signaling pathway(s) at the supraspinal level and that this ă molecular response may contribute to spontaneous recovery. With this ă aim, we used a rat model of partial cervical hemisection which injures ă the bulbospinal respiratory tract originating from the medulla oblongata ă of the brainstem but leads to a time-dependent spontaneous functional ă recovery of the paralyzed hemidiaphragm. We first demonstrate that after ă SCI the PI3K/Akt signaling pathway is activated in the medulla oblongata ă of the brainstem, resulting in an inactivation of its pro-apoptotic ă downstream target, forkhead transcription factor (FKHR/FOXO1A). ă Retrograde labeling of medullary premotoneurons including respiratory ă ones which project to phrenic motoneurons reveals an increased FKHR ă phosphorylation in their cell bodies together with an unchanged cell ă number. Medulla infusion of the PI3K inhibitor, LY294002, prevents the ă SCI-induced Akt and FKHR phosphorylations and activates one of its ă death-promoting downstream targets, Fas ligand. Quantitative EMG ă analyses of diaphragmatic contractility demonstrate that the inhibition ă of medulla PI3K/Akt signaling prevents spontaneous respiratory recovery ă normally observed after partial cervical SCI. Such inhibition does not ă however affect either baseline contractile frequency or the ventilatory ă reactivity under acute respiratory challenge. Together, these findings ă provide novel evidence of supraspinal cellular contribution to the ă spontaneous respiratory recovery after partial SCI. (C) 2014 Elsevier ă Inc All rights reserved.

In several populations of adult central nervous system neurons, axon damage can lead to an up-regulation of some transcription factors among which is c-Jun, known to be a key regulator of neuron cell body response to injury and of its intrinsic potential for axon regeneration. Thus, cervical spinal hemisection leads to c-Jun up-regulation in bulbospinal and rubrospinal axotomized neurons. The aims of the present study were to specify, after a unilateral cervical spinal cord injury, the expression of another marker of the neuronal stress response, heat shock protein 27 (HSP27) in axotomized neurons of the medulla (labeled by fluorogold retrograde tracer), and to compare it to that of c-Jun. In the medulla of injured rats, HSP27 and phospho-HSP27 were expressed in sub-populations of axotomized neurons, principally in the rostral ventral respiratory group (rVRG) (20%), the dorsal part of the gigantocellularis (Gi) (50%), and vestibular nucleus, but seldom in the ventral Gi and raphe nucleus, indicating a heterogeneous post-lesion cell body response between these different neuron populations. By contrast, phospho-c-Jun was up-regulated in axotomized neurons in all nuclei containing bulbospinal neurons, including the rVRG and Gi. In these areas, phospho-c-Jun was also up-regulated in uninjured bulbospinal neurons which project caudal to the spinal cord injury (labeled by fluorogold retrograde tracer). In contrast to phospho-c-Jun, HSP27 immunoreactivity was generally not present in neurons with spared axons. Our results show that various bulbospinal neuron populations react differentially to the injury and that spinal cord injury affects also bulbospinal neurons that are spared by the injury. However, the molecular cell body response of spared neurons is distinct from that of axotomized neurons since they can up-regulate c-Jun but not HSP27.

To evaluate the plasticity processes occurring in the spared and injured tissue after partial spinal cord injury, we have compared the level of axon growth markers after a C2 cervical hemisection in rats between the contralateral (spared) and ipsilateral (injured) cervical cord using western blotting and immunohistochemical techniques. In the ipsilateral spinal cord 7 days after injury, although GAP-43 levels were increased in the ventral horn caudal to the injury, they were globally decreased in the whole structure (C1-C6). By contrast, in the contralateral intact side 7 days and 1 month after injury, we have found an increase of GAP-43 and betaIII tubulin levels, suggesting that processes of axonal sprouting may occur in the spinal region contralateral to the injury. This increase of GAP-43 in the contralateral spinal cord after cervical hemisection may account, at least partially, to the spontaneous ipsilateral recovery observed after a cervical hemisection.