Photobiomodulation induces antinociception,recovers structural aspects and regulates mitochondrial homeostasis in peripheral nerve of diabetic mice

Diabetic peripheral neuropathy (DPN) is a nervous disorder caused by diabetes mellitus, affecting about 50% of patients in clinical medicine. Chronic pain is one of the major and most unpleasant symptoms developed by those patients, and conventional available treatments for the neuropathy, including the associated pain, are still unsatisfactory and benefit only a small number of patients. Photobiomodulation (PBM) has been gaining clinical acceptance once it is able to promote early nerve regeneration resulting in significant improvement in peripheral nerves disabilities. In this work, the effects of PBM (660 nm, 30 mW, 1.6 J/cm², 0.28 cm², 15 s in a continuous frequency) on treating DPN‐induced pain and nerve damage were evaluated in an experimental model of diabetic‐neuropathy induced by streptozotocin in mice. PBM‐induced antinociception in neuropathic‐pain mice was dependent on central opioids release. After 21 consecutive applications, PBM increased nerve growth factor levels and induced structural recovery increasing mitochondrial content and regulating Parkin in the sciatic nerve of DPN‐mice. Taking together, these data provide new insights into the mechanisms involved in the effects of PBM‐therapy emphasizing its therapeutic potential in the treatment of DPN.      

Penehyclidine hydrochloride protects against anoxia/reoxygenation injury in cardiomyocytes through ATP‐sensitive potassium channels,and the Akt/GSK‐3β and Akt/mTOR signaling pathways

Penehyclidine hydrochloride (PHC) can protect against myocardial ischemia/reperfusion (I/R) injury. However, the possible mechanisms of PHC in anoxia/reoxygenation (A/R)‐induced injury in H9c2 cells remain unclear. In the present study, H9c2 cells were pretreated with PI3K/Akt inhibitor LY294002, ATP‐sensitive K⁺ (KATP) channel blocker 5‐hydroxydecanoate (5‐HD), PHC, or KATP channel opener diazoxide (DZ) before subjecting to A/R injury. Cell viability and cell apoptosis were determined by cell counting kit‐8 assay and annexin V/PI assay, respectively. Myocardial injury was evaluated by measuring creatine kinase (CK) and lactate dehydrogenase (LDH) activities. Intracellular Ca²⁺ levels, reactive oxygen species (ROS) generation, mitochondrial membrane potential (ΔΨ_m), and mitochondrial permeability transition pore (mPTP) were measured. The levels of cytoplasmic/mitochondrial cytochrome c (Cyt‐C), Bax, Bcl‐2, cleaved caspase‐3, KATP channel subunits (Kir6.2 and SUR2A), and the members of the Akt/GSK‐3β and Akt/mTOR signaling pathways were determined by western blotting. We found that PHC preconditioning alleviated A/R‐induced cell injury by increasing cell viability, reducing CK and LDH activities, and inhibiting cell apoptosis. In addition, PHC preconditioning ameliorated intracellular Ca²⁺ overload and ROS production, accompanied by inhibition of both mPTP opening and Cyt‐C release into cytoplasm, and maintenance of ΔΨ_m. Moreover, PHC preconditioning activated mitochondrial KATP channels, and modulated the Akt/GSK‐3β and Akt/mTOR signaling pathways. Similar effects were observed upon treatment with DZ. Pretreatment with LY294002 or 5‐HD blocked the beneficial effects of PHC. These results suggest that the protective effects of PHC preconditioning on A/R injury may be related to mitochondrial KATP channels, as well as the Akt/GSK‐3β and Akt/mTOR signaling pathways.     

Cellular transformations in near‐infrared light‐induced apoptosis in cancer cells revealed by label‐free CARS imaging

Photobiomodulation (PBM) involves light to activate cellular signaling pathways leading to cell proliferation or death. In this work, fluorescence and Coherent anti‐Stokes Raman Scattering (CARS) imaging techniques were applied to assess apoptosis in human cervical cancer cells (HeLa) induced by near infrared (NIR) laser light (808 nm). Using the Caspase 3/7 fluorescent probe to identify apoptotic cells, we found that the pro‐apoptotic effect is significantly dependent of irradiation dose. The highest apoptosis rate was noted for the lower irradiation doses, that is, 0.3 J/cm² (~58%) and 3 J/cm² (~28%). The impact of light doses on proteins/lipids intracellular metabolism and distribution was evaluated using CARS imaging, which revealed apoptosis‐associated reorganization of nuclear proteins and cytoplasmic lipids after irradiation with 0.3 J/cm². Doses of NIR light causing apoptosis (0.3, 3 and 30 J/cm²) induced a gradual increase in the nuclear protein level over time, in contrast to proteins in cells non‐irradiated and irradiated with 10 J/cm². Furthermore, irradiation of the cells with the 0.3 J/cm² dose resulted in lipid droplets (LDs) accumulation, which was apparently caused by an increase in reactive oxygen species (ROS) generation. We suggest that PBM induced apoptosis could be caused by the ability of NIR light to trigger excessive LDs formation which, in turn, induces cellular cytotoxicity.      

1064 nm Nd:YAG laser light affects transmembrane mitochondria respiratory chain complexes

Photobiomodulation (PBM) is a non‐plant‐cell manipulation through a transfer of energy by means of light sources at the non‐ablative or thermal intensity. Authors showed that cytochrome‐c‐oxidase (complex IV) is the specific chromophore's target of PBM at the red (600‐700 nm) and NIR (760‐900 nm) wavelength regions. Recently, it was suggested that the infrared region of the spectrum could influence other chromospheres, despite the interaction by wavelengths higher than 900 nm with mitochondrial chromophores was not clearly demonstrated. We characterized the interaction between mitochondria respiratory chain, malate dehydrogenase, a key enzyme of Krebs cycle, and 3‐hydroxyacyl‐CoA dehydrogenase, an enzyme involved in the β‐oxidation (two mitochondrial matrix enzymes) with the 1064 nm Nd:YAG (100mps and 10 Hz frequency mode) irradiated at the average power density of 0.50, 0.75, 1.00, 1.25 and 1.50 W/cm² to generate the respective fluences of 30, 45, 60, 75 and 90 J/cm². Our results show the effect of laser light on the transmembrane mitochondrial complexes I, III, IV and V (adenosine triphosphate synthase) (window effects), but not on the extrinsic mitochondrial membrane complex II and mitochondria matrix enzymes. The effect is not due to macroscopical thermal change. An interaction of this wavelength with the Fe‐S proteins and Cu‐centers of respiratory complexes and with the water molecules could be supposed.      

Photobiomodulation therapy in knee osteoarthritis reduces oxidative stress and inflammatory cytokines in rats

Knee osteoarthritis (OA) is a chronic disease that causes pain and gradual degeneration of the articular cartilage. In this study, MIA‐induced OA knee model was used in rats to test the effects of the photobiomodulation therapy (PBM). We analyzed the inflammatory process (pain and cytokine levels), and its influence on the oxidative stress and antioxidant capacity. Knee OA was induced by monosodium iodoacetate (MIA) intra‐articular injection (1.5 mg/50 μL) and the rats were treated with eight sessions of PBM 3 days/week (904 nm, 6 or 18 J/cm²). For each animal, mechanical and cold hyperalgesia and spontaneous pain were evaluated; biological analyses were performed in blood serum, intra‐articular lavage, knee structures, spinal cord and brainstem. Cytokine assays were performed in knee, spinal cord and brainstem samples. The effects of the 18 J/cm² dose of PBM were promising in reducing pain and neutrophil activity in knee samples, together with reducing oxidative stress damage in blood serum and spinal cord samples. PBM improved the antioxidant capacity in blood serum and brainstem, and decreased the knee pro‐inflammatory cytokine levels. Our study demonstrated that PBM decreased oxidative damage, inflammation and pain. Therefore, this therapy could be an important tool in the treatment of knee OA.     

Remifentanil preconditioning alleviating brain damage of cerebral ischemia reperfusion rats by regulating the JNK signal pathway and TNF-α/TNFR1 signal pathway

Tumor necrosis factor (TNF) and the TNF receptor (TNFR) superfamily play very important roles for cell death as well as normal immune regulation. Previous studies have strongly suggested that c-Jun N-terminal kinase (JNK) signaling pathway plays a critical role in ischemic brain injury. The purpose of this investigation was to examine the protective effect of remifentanil preconditioning in cerebral ischemia/reperfusion injury (CIR) and its possible molecular mechanism. Results showed that Remifentanil pretreatment significantly decreased the CD4⁺ and increased the CD8⁺ in cerebral tissues. Additionally, CD4⁺/CD8⁺ in CIR + Remifentanil group was markedly lower than that in CIR group. TNF-α and TNFR1 in CIR + Remifentanil group rats was found to be significant lower than that in CIR group rats. The expression levels of Cyt-c, caspase-3, caspase-9 and pJNK proteins in brain of CIR + Remifentanil group rats were found to significantly decreased compared to CIR group rats. In addition, decreased ROS level indirectly inhibit JNK activation and cell death in CIR rat receiving Remifentanil preconditioning. From current experiment results, at least two signal pathways involve into the process of Remifentanil preconditioning inhibiting cerebral damage induced by ischemia reperfusion. The inhibitory effects of Remifentanil preconditioning on the brain damage are achieved probably through blocking the activation of TNF-α/TNFR1, JNK signal transduction pathways, which implies that Remifentanil preconditioning may be a potential and effective way for prevention of the ischemic/reperfusion injury through the suppression extrinsic apoptotic signal pathway induced by TNF-α/TNFR1, JNK signal pathways. Taken together, this study indicated that regulation of the TNF-α/TNFR1 and JNK signal pathways may provide a new therapy for cerebral damage induced by ischemia and reperfusion.     

Irradiation to the immature brain attenuates neurogenesis and exacerbates subsequent hypoxic‐ischemic brain injury in the adult

Cranial radiotherapy is common in pediatric oncology. Our purpose was to investigate if irradiation (IR) to the immature brain would increase the susceptibility to hypoxic‐ischemic injury in adulthood. The left hemisphere of postnatal day 10 (P10) mice was irradiated with 8 Gy and subjected to hypoxia‐ischemia (HI) on P60. Brain injury, neurogenesis and inflammation were evaluated 30 days after HI. IR alone caused significant hemispheric tissue loss, or lack of growth (2.8 ± 0.42 mm³, p < 0.001). Tissue loss after HI (18.2 ± 5.8 mm³, p < 0.05) was synergistically increased if preceded by IR (32.0 ± 3.5 mm³, p < 0.05). Infarct volume (5.1 ± 1.6 mm³) nearly doubled if HI was preceded by IR (9.8 ± 1.2 mm³, p < 0.05). Pathological scoring revealed that IR aggravated hippocampal, cortical and striatal, but not thalamic, injury. Hippocampal neurogenesis decreased > 50% after IR but was unchanged by HI alone. The number of newly formed microglia was three times higher after IR + HI than after HI alone. In summary, IR to the immature brain produced long‐lasting changes, including decreased hippocampal neurogenesis, subsequently rendering the adult brain more susceptible to HI, resulting in larger infarcts, increased hemispheric tissue loss and more inflammation than in non‐irradiated brains.     

Vagal nerve stimulation improves mitochondrial dynamics via an M3 receptor/CaMKKβ/AMPK pathway in isoproterenol‐induced myocardial ischaemia

Mitochondrial dynamics—fission and fusion—are associated with ischaemic heart disease (IHD). This study explored the protective effect of vagal nerve stimulation (VNS) against isoproterenol (ISO)‐induced myocardial ischaemia in a rat model and tested whether VNS plays a role in preventing disorders of mitochondrial dynamics and function. Isoproterenol not only caused cardiac injury but also increased the expression of mitochondrial fission proteins [dynamin‐related peptide1 (Drp1) and mitochondrial fission protein1 (Fis‐1)) and decreased the expression of fusion proteins (optic atrophy‐1 (OPA1) and mitofusins1/2 (Mfn1/2)], thereby disrupting mitochondrial dynamics and leading to increase in mitochondrial fragments. Interestingly, VNS restored mitochondrial dynamics through regulation of Drp1, Fis‐1, OPA1 and Mfn1/2; enhanced ATP content and mitochondrial membrane potential; reduced mitochondrial permeability transition pore (MPTP) opening; and improved mitochondrial ultrastructure and size. Furthermore, VNS reduced the size of the myocardial infarction and ameliorated cardiomyocyte apoptosis and cardiac dysfunction induced by ISO. Moreover, VNS activated AMP‐activated protein kinase (AMPK), which was accompanied by phosphorylation of Ca²⁺/calmodulin‐dependent protein kinase kinase β (CaMKKβ) during myocardial ischaemia. Treatment with subtype‐3 of muscarinic acetylcholine receptor (M₃R) antagonist 4‐diphenylacetoxy‐N‐methylpiperidine methiodide or AMPK inhibitor Compound C abolished the protective effects of VNS on mitochondrial dynamics and function, suggesting that M₃R/CaMKKβ/AMPK signalling are involved in mediating beneficial effects of VNS. This study demonstrates that VNS modulates mitochondrial dynamics and improves mitochondrial function, possibly through the M₃R/CaMKKβ/AMPK pathway, to attenuate ISO‐induced cardiac damage in rats. Targeting mitochondrial dynamics may provide a novel therapeutic strategy in IHD.     

Pain management using photobiomodulation: Mechanisms,location, and repeatability quantified by pain threshold and neural biomarkers in mice

Photobiomodulation (PBM) is a simple, efficient and cost‐effective treatment for both acute and chronic pain. We previously showed that PBM applied to the mouse head inhibited nociception in the foot. Nevertheless, the optimum parameters, location for irradiation, duration of the effect and the mechanisms of action remain unclear. In the present study, the pain threshold in the right hind paw of mice was studied, after PBM (810 nm CW laser, spot size 1 or 6 cm², 1.2–36 J/cm²) applied to various anatomical locations. The pain threshold, measured with von Frey filaments, was increased more than 3‐fold by PBM to the lower back (dorsal root ganglion, DRG), as well as to other neural structures along the pathway such as the head, neck and ipsilateral (right) paw. On the other hand, application of PBM to the contralateral (left) paw, abdomen and tail had no effect. The optimal effect occurred 2 to 3 hours post‐PBM and disappeared by 24 hours. Seven daily irradiations showed no development of tolerance. Type 1 metabotropic glutamate receptors decreased, and prostatic acid phosphatase and tubulin‐positive varicosities were increased as shown by immunofluorescence of DRG samples. These findings elucidate the mechanisms of PBM for pain and provide insights for clinical practice.      

Remote preconditioning reduces ischemic injury in the explanted heart by a KATP channel-dependent mechanism

Local and remote ischemic preconditioning (IPC) reduce ischemia-reperfusion (I/R) injury and preserve cardiac function. In this study, we tested the hypothesis that remote preconditioning is memorized by the explanted heart and yields protection from subsequent I/R injury and that the underlying mechanism involves sarcolemmal and mitochondrial ATP-sensitive K(+) (K(ATP)) channels. Male Wistar rats (300-350 g) were randomized to a control (n = 10), a remote IPC (n = 10), and a local IPC group (n = 10). Remote IPC was induced by four cycles of 5 min of limb ischemia, followed by 5 min of reperfusion. Local IPC was induced by four cycles of 2 min of regional myocardial ischemia, followed by 3 min of reperfusion. The heart was excised within 5 min after the final cycle of preconditioning, mounted in a perfused Langendorff preparation for 40 min of stabilization, and subjected to 45 min of sustained ischemia by occluding the left coronary artery and 120 min of reperfusion. I/R injury was assessed as infarct size by triphenyltetrazolium staining. The influence of sarcolemmal and mitochondrial K(ATP) channels on remote preconditioning was assessed by the addition of glibenclamide (10 microM, a nonselective K(ATP) blocker), 5-hydroxydecanoic acid (5-HD; 100 microM, a mitochondrial K(ATP) blocker), and HMR-1098 (30 microM, a sarcolemmal K(ATP) blocker) to the Langendorff preparation before I/R. The role of mitochondrial K(ATP) channels as an effector mechanism for memorizing remote preconditioning was further studied by the effect of the specific mitochondrial K(ATP) activator diaxozide (10 mg/kg) on myocardial infarct size. Remote preconditioning reduced I/R injury in the explanted heart (0.17 +/- 0.03 vs. 0.39 +/- 0.05, P < 0.05) and improved left ventricular function during reperfusion compared with control (P < 0.05). Similar effects were obtained with diazoxide. Remote preconditioning was abolished by the addition of 5-HD and glibenclamide but not by HMR-1098. In conclusion, the protective effect of remote preconditioning is memorized in the explanted heart by a mechanism that involves mitochondrial K(ATP) channels.     

Effects of photobiomodulation combined with MSCs transplantation on the repair of spinal cord injury in rat

Stem cell transplantation has shown promising regenerative effects against neural injury, and photobiomodulation (PBM) can aid tissue recovery. This study aims to evaluate the therapeutic effect of human umbilical cord mesenchymal stem cells (hUCMSCs) and laser alone or combined on spinal cord injury (SCI). The animals were divided into SCI, hUCMSCs, laser treatment (LASER) and combination treatment (hUCMSCs + LASER) groups. Cell‐enriched grafts of hUCMSCs (1 × 10⁶ cells/ml) were injected at the site of antecedent trauma in SCI model rats. A 2 cm² damaged area was irradiated with 630 nm laser at 100 mW/cm² power for 20 min. Locomotion was evaluated using Basso–Beattie–Bresnahan (BBB) scores, and neurofilament repair were monitored by histological staining and diffusion tensor imaging (DTI). First, after SCI, the motor function of each group was restored with different degrees, the combination treatment significantly increased the BBB scores compared to either monotherapy. In addition, Nissl bodies were more numerous, and the nerve fibers were longer and thicker in the combination treatment group. Consistent with this, the in situ expression of NF‐200 and glial fibrillary acidic protein in the damaged area was the highest in the combination treatment group. Finally, DTI showed that the combination therapy optimally improved neurofilament structure and arrangement. These results may show that the combination of PBM and hUCMSCs transplantation is a feasible strategy for reducing secondary damage and promoting functional recovery following SCI.     

Therapeutic Benefits of Delayed Lithium Administration in the Neonatal Rat after Cerebral Hypoxia-Ischemia

Aim

We have previously shown that lithium treatment immediately after hypoxia-ischemia (HI) in neonatal rats affords both short- and long-term neuroprotection. The aim of this study was to evaluate possible therapeutic benefits when lithium treatment was delayed 5 days, a time point when most cell death is over.

Methods

Eight-day-old male rats were subjected to unilateral HI and 2 mmol/kg lithium chloride was injected intraperitoneally 5 days after the insult. Additional lithium injections of 1 mmol/kg were administered at 24 h intervals for the next 14 days. Brain injury was evaluated 12 weeks after HI. Serum cytokine measurements and behavioral analysis were performed before sacrificing the animals.

Results

Brain injury, as indicated by tissue loss, was reduced by 38.7%, from 276.5±27.4 mm³ in the vehicle-treated group to 169.3±25.9 mm³ in the lithium-treated group 12 weeks after HI (p<0.01). Motor hyperactivity and anxiety-like behavior after HI were normalized by lithium treatment. Lithium treatment increased neurogenesis in the dentate gyrus as indicated by doublecortin labeling. Serum cytokine levels, including IL-1α, IL-1β, and IL-6, were still elevated as late as 5 weeks after HI, but lithium treatment normalized these cytokine levels.

Conclusions

Delayed lithium treatment conferred long-term neuroprotection in neonatal rats after HI, and this opens a new avenue for future development of treatment strategies for neonatal brain injury that can be administered after the acute injury phase.     

Mitochondrial dynamics in the neonatal brain – a potential target following injury?

The impact of birth asphyxia and its sequelae, hypoxic–ischaemic (HI) brain injury, is long-lasting and significant, both for the infant and for their family. Treatment options are limited to therapeutic hypothermia, which is not universally successful and is unavailable in low resource settings. The energy deficits that accompany neuronal death following interruption of blood flow to the brain implicate mitochondrial dysfunction. Such HI insults trigger mitochondrial outer membrane permeabilisation leading to release of pro-apoptotic proteins into the cytosol and cell death. More recently, key players in mitochondrial fission and fusion have been identified as targets following HI brain injury. This review aims to provide an introduction to the molecular players and pathways driving mitochondrial dynamics, the regulation of these pathways and how they are altered following HI insult. Finally, we review progress on repurposing or repositioning drugs already approved for other indications, which may target mitochondrial dynamics and provide promising avenues for intervention following brain injury. Such repurposing may provide a mechanism to fast-track, low-cost treatment options to the clinic.     

Recovering the osteoblastic differentiation potential of mesenchymal stem cells derived from diabetic rats by photobiomodulation therapy

Autologous cell-based therapy for bone regeneration might be impaired by diabetes mellitus (DM) due to the negative effects on mesenchymal stem cells (MSCs) differentiation. Strategies to recover their osteogenic potential could optimize the results. We aimed to evaluate the effect of photobiomodulation (PBM) therapy on osteoblast differentiation of rats with induced DM. Bone marrow MSCs of healthy and diabetic rats were isolated and differentiated into osteoblasts (OB and dOB, respectively). dOB were treated with PBM therapy every 72 hour (660 nm; 0.14 J; 20 mW; 0.714 W/cm², and 5 J/cm²). Cell morphology, viability, gene and protein expression of osteoblastic markers, alkaline phosphatase (ALP) activity, and the mineralized matrix production of dOB-PBM were compared to dOB. PBM therapy improved viability of dOB, increased the gene and protein expression of bone markers, the ALP activity and the mineralized matrix production. PBM therapy represents an innovative therapeutic approach to optimize the treatment of bone defects in diabetic patients.     

Lithium reduces apoptosis and autophagy after neonatal hypoxia�Cischemia

Lithium is used in the treatment of bipolar mood disorder. Reportedly, lithium can be neuroprotective in models of adult brain ischemia. The purpose of this study was to evaluate the effects of lithium in a model of neonatal hypoxic–ischemic brain injury. Nine-day-old male rats were subjected to unilateral hypoxia–ischemia (HI) and 2 mmol/kg lithium chloride was injected i.p. immediately after the insult. Additional lithium injections, 1 mmol/kg, were administered at 24-h intervals. Pups were killed 6, 24 or 72 h after HI. Lithium reduced the infarct volume from 24.7±2.9 to 13.8±3.3 mm³ (44.1%) and total tissue loss (degeneration + lack of growth) from 67.4±4.4 to 38.4±5.9 mm³ (43.1%) compared with vehicle at 72 h after HI. Injury was reduced in the cortex, hippocampus, thalamus and striatum. Lithium reduced the ischemia-induced dephosphorylation of glycogen synthase kinase-3β and extracellular signal-regulated kinase, the activation of calpain and caspase-3, the mitochondrial release of cytochrome c and apoptosis-inducing factor, as well as autophagy. We conclude that lithium could mitigate the brain injury after HI by inhibiting neuronal apoptosis. The lithium doses used were in the same range as those used in bipolar patients, suggesting that lithium might be safely used for the avoidance of neonatal brain injury.     

In vivo inhibition of the mitochondrial H+‐ATP synthase in neurons promotes metabolic preconditioning

A key transducer in energy conservation and signaling cell death is the mitochondrial H⁺‐ATP synthase. The expression of the ATPase inhibitory factor 1 (IF1) is a strategy used by cancer cells to inhibit the activity of the H⁺‐ATP synthase to generate a ROS signal that switches on cellular programs of survival. We have generated a mouse model expressing a mutant of human IF1 in brain neurons to assess the role of the H⁺‐ATP synthase in cell death in vivo. The expression of hIF1 inhibits the activity of oxidative phosphorylation and mediates the shift of neurons to an enhanced aerobic glycolysis. Metabolic reprogramming induces brain preconditioning affording protection against quinolinic acid‐induced excitotoxicity. Mechanistically, preconditioning involves the activation of the Akt/p70S6K and PARP repair pathways and Bcl‐xL protection from cell death. Overall, our findings provide the first in vivo evidence highlighting the H⁺‐ATP synthase as a target to prevent neuronal cell death.     

AMP‐activated protein kinase (AMPK)–induced preconditioning in primary cortical neurons involves activation of MCL‐1

Neuronal preconditioning is a phenomenon where a previous exposure to a sub‐lethal stress stimulus increases the resistance of neurons towards a second, normally lethal stress stimulus. Activation of the energy stress sensor, AMP‐activated protein kinase (AMPK) has been shown to contribute to the protective effects of ischaemic and mitochondrial uncoupling‐induced preconditioning in neurons, however, the molecular basis of AMPK‐mediated preconditioning has been less well characterized. We investigated the effect of AMPK preconditioning using 5‐aminoimidazole‐4‐carboxamide riboside (AICAR) in a model of NMDA‐mediated excitotoxic injury in primary mouse cortical neurons. Activation of AMPK with low concentrations of AICAR (0.1 mM for 2 h) induced a transient increase in AMPK phosphorylation, protecting neurons against NMDA‐induced excitotoxicity. Analysing potential targets of AMPK activation, demonstrated a marked increase in mRNA expression and protein levels of the anti‐apoptotic BCL‐2 family protein myeloid cell leukaemia sequence 1 (MCL‐1) in AICAR‐preconditioned neurons. Interestingly, over‐expression of MCL‐1 protected neurons against NMDA‐induced excitotoxicity while MCL‐1 gene silencing abolished the effect of AICAR preconditioning. Monitored intracellular Ca²⁺ levels during NMDA excitation revealed that MCL‐1 over‐expressing neurons exhibited improved bioenergetics and markedly reduced Ca²⁺ elevations, suggesting a potential mechanism through which MCL‐1 confers neuroprotection. This study identifies MCL‐1 as a key effector of AMPK‐induced preconditioning in neurons.     

Low‐level laser therapy (810 nm) protects primary cortical neurons against excitotoxicity in vitro

Excitotoxicity describes a pathogenic process whereby death of neurons releases large amounts of the excitatory neurotransmitter glutamate, which then proceeds to activate a set of glutamatergic receptors on neighboring neurons (glutamate, N‐methyl‐D‐aspartate (NMDA), and kainate), opening ion channels leading to an influx of calcium ions producing mitochondrial dysfunction and cell death. Excitotoxicity contributes to brain damage after stroke, traumatic brain injury, and neurodegenerative diseases, and is also involved in spinal cord injury. We tested whether low level laser (light) therapy (LLLT) at 810 nm could protect primary murine cultured cortical neurons against excitotoxicity in vitro produced by addition of glutamate, NMDA or kainate. Although the prevention of cell death was modest but significant, LLLT (3 J/cm² delivered at 25 mW/cm² over 2 min) gave highly significant benefits in increasing ATP, raising mitochondrial membrane potential, reducing intracellular calcium concentrations, reducing oxidative stress and reducing nitric oxide. The action of LLLT in abrogating excitotoxicity may play a role in explaining its beneficial effects in diverse central nervous system pathologies. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Photobiomodulation suppresses JNK3 by activation of ERK/MKP7 to attenuate AMPA receptor endocytosis in Alzheimer's disease

Alzheimer's disease (AD), a severe age‐related neurodegenerative disorder, lacks effective therapeutic methods at present. Physical approaches such as gamma frequency light flicker that can effectively reduce amyloid load have been reported recently. Our previous research showed that a physical method named photobiomodulation (PBM) therapy rescues Aβ‐induced dendritic atrophy in vitro. However, it remains to be further investigated the mechanism by which PBM affects AD‐related multiple pathological features to improve learning and memory deficits. Here, we found that PBM attenuated Aβ‐induced synaptic dysfunction and neuronal death through MKP7‐dependent suppression of JNK3, a brain‐specific JNK isoform related to neurodegeneration. The results showed PBM‐attenuated amyloid load, AMPA receptor endocytosis, dendrite injury, and inflammatory responses, thereby rescuing memory deficits in APP/PS1 mice. We noted JNK3 phosphorylation was dramatically decreased after PBM treatment in vivo and in vitro. Mechanistically, PBM activated ERK, which subsequently phosphorylated and stabilized MKP7, resulting in JNK3 inactivation. Furthermore, activation of ERK/MKP7 signaling by PBM increased the level of AMPA receptor subunit GluR 1 phosphorylation and attenuated AMPA receptor endocytosis in an AD pathological model. Collectively, these data demonstrated that PBM has potential therapeutic value in reducing multiple pathological features associated with AD, which is achieved by regulating JNK3, thus providing a noninvasive, and drug‐free therapeutic strategy to impede AD progression.     

Photobiomodulation and estrogen stabilize mitochondrial membrane potential in angiotensin–II challenged porcine aortic smooth muscle cells

Rupture of Abdominal aortic aneurysm (AAA) is among the 15 leading causes of death after age 65. Using high frequency ultrasound, we showed that photobiomodulation (PBM) prevents formation and progression of AAA in the angiotensin-II (Ang-II)-infused, apolipoprotein-e-deficient mouse model. In the current study we report that while challenge of porcine aortic Smooth Muscle Cells (SMCs) with Ang-II (1 μM) resulted in a marked decay in mitochondrial membrane potential (MitMP) vs non-challenged cells, treatment with PBM (continuous diode laser, 780 nm, 6.7 mW/cm², 5 minutes, 2 J/cm²) or pre-incubation with estrogen (50 nM, 1 hour) significantly attenuated this deterioration in MitMP. We also report that PBM and estrogen markedly affected porcine aortic SMC contraction and modified mitochondrial dispersion reflecting important influence on SMC function. These studies provide strong evidence of the important underlying role of mitochondria in the preventive effect of PBM on formation and progression of AAA and its reduced incidence and delayed onset in women.