High-Resolution Respirometry for Mitochondrial Function in Rodent Brain.

High-resolution mitochondrial respirometry is a modern technique that enables to measure mitochondrial respiration in various cell types. It contains chambers with oxygen sensors that measure oxygen concentration via polarography and calculate its consumption. The chamber contains plastic stoppers with injection ports that allow the injection of samples and different substrates, inhibitors, and uncoupler substances to measure mitochondrial respiration with high efficiency. These substances act on the mitochondrial electron transport chain (ETC) and help to assess the mitochondrial ATP production capacity and oxidative phosphorylation. The respirograph obtained with the help of software represents the oxygen consumption in each stage after adding different reagents.

Hypoxia and its effect on the cellular system.

Eukaryotic cells utilize oxygen for different functions of cell organelles owing to cellular survival. A balanced oxygen homeostasis is an essential requirement to maintain the regulation of normal cellular systems. Any changes in the oxygen level are stressful and can alter the expression of different homeostasis regulatory genes and proteins. Lack of oxygen or hypoxia results in oxidative stress and formation of hypoxia inducible factors (HIF) and reactive oxygen species (ROS). Substantial cellular damages due to hypoxia have been reported to play a major role in various pathological conditions. There are different studies which demonstrated that the functions of cellular system are disrupted by hypoxia. Currently, study on cellular effects following hypoxia is an important field of research as it not only helps to decipher different signaling pathway modulation, but also helps to explore novel therapeutic strategies. On the basis of the beneficial effect of hypoxia preconditioning of cellular organelles, many therapeutic investigations are ongoing as a promising disease management strategy in near future. Hence, the present review discusses about the effects of hypoxia on different cellular organelles, mechanisms and their involvement in the progression of different diseases.

Inosine attenuates post-stroke neuroinflammation by modulating inflammasome mediated microglial activation and polarization.

To date, various agents and molecules have been developed to treat post-stroke neuroinflammation; however, none of them are clinically successful. Post-stroke neuroinflammation is primarily attributed to microglial polarization as the generation of inflammasome complexes shifts microglia to their M1 phenotype and regulates the downstream cascade. Inosine, an adenosine derivative reported to maintain cellular energy homeostasis in stressed conditions. Although the exact mechanism is still unexplored, various studies have reported that it can stimulate axonal sprouting in different neurodegenerative diseases. Hence, our present study aims to decipher the molecular mechanism of inosine mediated neuroprotection by modulating inflammasome signaling towards altered microglial polarization in ischemic stroke. Inosine was administered intraperitoneally to male Sprague Dawley rats at 1 h post-ischemic stroke and was further evaluated for neurodeficit score, motor coordination and long-term neuroprotection. Brains were harvested for infarct size estimation, biochemical assays and molecular studies. Inosine administration at 1 h post ischemic stroke decreased infarct size, neurodeficit score, and improved motor co-ordination. Normalization of biochemical parameters were achieved in the treatment groups. Microglial polarization towards its anti-inflammatory phenotype and modulation of inflammation were evident by relevant gene and protein expression studies. The outcome provides preliminary evidence of inosine mediated alleviation of post-stroke neuroinflammation via modulation of microglial polarization towards its anti-inflammatory form through regulating the inflammasome activation.

Neuroendocrine regulation in stroke.

The neuroendocrine system, a crosstalk between the central nervous system and endocrine glands, balances and controls hormone secretion and their functions. Neuroendocrine pathways and mechanisms often get dysregulated following stroke, leading to altered hormone secretion and aberrant receptor expression. Dysregulation of the hypothalamus-pituitary-thyroid (HPT) axis and hypothalamus-pituitary-adrenal (HPA) axis often led to severe stroke outcomes. Post-stroke complications such as cognitive impairment, depression, infection etc. are directly or indirectly influenced by the altered neuroendocrine activity that plays a crucial role in stroke vulnerability and susceptibility. Therefore, it is imperative to explore various neurohormonal inter-relationships in regulating stroke, its outcome, and prognosis. Here, we review the biology of different hormones associated with stroke and explore their regulation with a view towards prospective therapeutics.

Stem cells alleviate OGD/R mediated stress response in PC12 cells following a co-culture: modulation of the apoptotic cascade through BDNF-TrkB signaling.

Apoptosis mediated by endoplasmic reticulum (ER) stress plays a crucial role in several neurovascular disorders, including ischemia/reperfusion injury (I/R injury). Previous in vitro and in vivo studies have suggested that following I/R injury, ER stress is vital for mediating CCAT-enhancer-binding protein homologous protein (CHOP) and caspase-12-dependent apoptosis. However, its modulation in the presence of stem cells and the underlying mechanism of cytoprotection remains elusive. In vivo studies from our lab have reported that post-stroke endovascular administration of stem cells renders neuroprotection and regulates apoptosis mediated by ER stress. In the current study, a more robust in vitro validation has been undertaken to decipher the mechanism of stem cell-mediated cytoprotection. Results from our study have shown that oxygen-glucose deprivation/reoxygenation (OGD/R) potentiated ER stress and apoptosis in the pheochromocytoma 12 (PC12) cell line as evident by the increase of protein kinase R (PKR)-like ER kinase (p-PERK), p-Eukaryotic initiation factor 2α subunit (EIF2α), activation transcription factor 4 (ATF4), CHOP, and caspase 12 expressions. Following the co-culture of PC12 cells with MSCs, ER stress was significantly reduced, possibly via modulating the brain-derived neurotrophic factor (BDNF) signaling. Furthermore, inhibition of BDNF by inhibitor K252a abolished the protective effects of BDNF secreted by MSCs following OGD/R. Our study suggests that inhibition of ER stress-associated apoptotic pathway with MSCs co-culture following OGD/R may help to alleviate cellular injury and further substantiate the use of stem cells as a therapeutic modality toward neuroprotection following hypoxic injury or stroke in clinical settings.

Cardiolipin-Mediated Alleviation of Mitochondrial Dysfunction Is a Neuroprotective Effect of Statin in Animal Model of Ischemic Stroke.

In clinical settings, the benefit of statin for stroke is debatable as regular statin users may suffer from myalgia, statin-associated myopathy (SAM), and rarely rhabdomyolysis. Studies suggest that patients on statin therapy show lesser vulnerability toward ischemic stroke and post-stroke frailty. Both pre- and post-treatment benefits of statin have been reported as evident by its neuroprotective effects in both cases. As mitochondrial dysfunction following stroke is the fulcrum for neuronal death, we hereby explore the role of statin in alleviating mitochondrial dysfunction by regulating the mitochondrial dynamics. In the present study, we intend to evaluate the role of statin in modulating cardiolipin-mediated mitochondrial functionality and further providing a therapeutic rationale for repurposing statins either as preventive or an adjunctive therapy for stroke.

Glial Cells Response in Stroke.

As the second-leading cause of death, stroke faces several challenges in terms of treatment because of the limited therapeutic interventions available. Previous studies primarily focused on metabolic and blood flow properties as a target for treating stroke, including recombinant tissue plasminogen activator and mechanical thrombectomy, which are the only USFDA approved therapies. These interventions have the limitation of a narrow therapeutic time window, the possibility of hemorrhagic complications, and the expertise required for performing these interventions. Thus, it is important to identify the contributing factors that exacerbate the ischemic outcome and to develop therapies targeting them for regulating cellular homeostasis, mainly neuronal survival and regeneration. Glial cells, primarily microglia, astrocytes, and oligodendrocytes, have been shown to have a crucial role in the prognosis of ischemic brain injury, contributing to inflammatory responses. They play a dual role in both the onset as well as resolution of the inflammatory responses. Understanding the different mechanisms driving these effects can aid in the development of therapeutic targets and further mitigate the damage caused. In this review, we summarize the functions of various glial cells and their contribution to stroke pathology. The review highlights the therapeutic options currently being explored and developed that primarily target glial cells and can be used as neuroprotective agents for the treatment of ischemic stroke.

Advancement in CRISPR/Cas9 Technology to Better Understand and Treat Neurological Disorders.

Neurological disorders have complicated pathophysiology that may involve several genetic mutations. Conventional treatment has limitations as they only treat apparent symptoms. Although, personalized medicine is emerging as a promising neuro-intervention, lack of precision is the major pitfall. Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system is evolving as a technological platform that may overcome the therapeutic limitations towards precision medicine. In the future, targeting genes in neurological disorders may be the mainstay of modern therapy. The present review on CRISPR/Cas9 and its application in various neurological disorders may provide a platform for its future clinical relevance towards developing precise and personalized medicine.

Endovascular Stem Cell Therapy Promotes Neuronal Remodeling to Enhance Post Stroke Recovery by Alleviating Endoplasmic Reticulum Stress Modulated by BDNF Signaling.

BACKGROUND AND PURPOSE: The impact of increased BDNF expression in brain by endovascular delivered mesenchymal stem cells (MSCs) post stroke towards modulating endoplasmic reticulum (ER) stress mediated neuronal remodeling has not been directly studied. Therefore, the present study investigates ER stress mediated neuronal remodeling following IA MSCs infusion in rodent model of ischemic stroke. METHODS: Ovariectomized Sprague Dawley rats were subjected to MCAO followed by 1 × 105 IA MSCs administration at 6 h. Infarct and functional outcomes at different time points post-stroke were evaluated. Further, various genes and protein expression studies were performed to determine the underlying mechanisms of the effect of IA MSCs towards ER stress mediated neuronal remodeling. RESULTS: Post-stroke IA MSCs administration significantly increased BDNF expression and decreased ER stress markers expression at day 1 post-stroke. A gradual rise in the expression of growth associate protein-43 (GAP 43) and spinophilin were observed at 7, 14- and 28-days post-stroke indicating an increase in neuronal remodeling towards functional restoration. CONCLUSIONS: The results suggest that IA MSCs post-stroke can modulate neuronal remodeling by BDNF-mediated reduction in ER stress that contribute towards functional recovery.

Pharmacological Strategies for Stroke Intervention: Assessment of Pathophysiological Relevance and Clinical Trials.

OBJECTIVES: The present review describes stroke pathophysiology in brief and discusses the spectrum of available treatments with different promising interventions that are in clinical settings or are in clinical trials. METHODS: Relevant articles were searched using Google Scholar, Cochrane Library, and PubMed. Keywords for the search included ischemic stroke, mechanisms, stroke interventions, clinical trials, and stem cell therapy. RESULTS AND CONCLUSION: Stroke accounts to a high burden of mortality and morbidity around the globe. Time is an important factor in treating stroke. Treatment options are limited; however, agents with considerable efficacy and tolerability are being continuously explored. With the advances in stroke interventions, new therapies are being formulated with a hope that these may aid the ongoing protective and reparative processes. Such therapies may have an extended therapeutic time window in hours, days, weeks, or longer and may have the advantage to be accessible by a majority of the patients.

Phyllanthus emblica L. Regulates BDNF/PI3K Pathway to Modulate Glutathione for Mitoprotection and Neuroprotection in a Rodent Model of Ischemic Stroke.

INTRODUCTION: Ischemic stroke remains the leading cause of death worldwide and is the primary cause of disability globally. Numerous studies have shown that plant-origin medicines are promising and can influence the treatment of neurological disorders. Phyllanthus embilica L. (P. emblica or Amla) is one of the herbal plants whose medicinal properties are widely studied. The objective of the present study is to determine the neuroprotective effects of an aqueous extract of the fruit of P. emblica (hereinafter referred to as just P. emblica) on cerebral ischemia-reperfusion injury and explore if it can regulate BDNF/PI3K pathway to modulate glutathione for mitoprotection and neuroprotection. METHODS: In vivo studies were conducted on male Sprague Dawley rats, where rats were prophylactically administered 100 mg/kg P. emblica for 30 days. In the treatment group, rats were given 100 mg/kg P. emblica, 1 h post middle cerebral artery occlusion (MCAo). Rats were evaluated for neuro deficit and motor function tests. Brains were further harvested for infarct size evaluation, biochemical analysis, protein expression studies, and mitochondrial studies. RESULTS: Prophylaxis and treatment with P. emblica demonstrated significant improvement in functional outcome with a reduction in infarct size. Normalization of glutathione, nitrite, and malondialdehyde levels was also observed. Improvement in mitochondrial complex I and IV activities was also reported. Expressions of BDNF, PI3K, SDF1 and VEGF increased while that of ROCK2 decreased following P. emblica administration. CONCLUSION: P. emblica regulates BDNF/PI3K pathway to modulate glutathione in ischemic stroke to confer mitoprotection and neuroprotection.

Drug repurposing for stroke intervention.

Despite the availability of advanced interventions, stroke remains one of the most significant causes of mortality and morbidity worldwide. US Food and Drug Administration (FDA)-approved treatment options for stroke include tissue plasminogen activators (tPAs) and mechanical thrombectomy (MT). However, these are limited by a narrow therapeutic time window. Additionally, poststroke rehabilitation therapies can provide functional recovery but take a long time to show benefits. Drug repurposing could be a novel approach to broaden treatment options in this scenario. In this review, we summarize marketed drugs that could be repurposed based on their safety and efficacy data. We also briefly discuss their mechanisms of action and provide a list of repurposed drugs under trials for ischemic stroke therapy.

Post-stroke Impairment of the Blood-Brain Barrier and Perifocal Vasogenic Edema Is Alleviated by Endovascular Mesenchymal Stem Cell Administration: Modulation of the PKCδ/MMP9/AQP4-Mediated Pathway.

Post-stroke edema and upregulation of aquaporin 4 (AQP4) water transport channels play a significant role in the progression of stroke pathology and deteriorating stroke outcomes. Prior studies from our lab have demonstrated the safety and efficacy of intra-arterial (IA) 1 × 105 mesenchymal stem cells (MSCs) administration post-stroke towards functional restoration and neuroprotection. Protein kinases have been reported to be involved in the signaling cascade of edema, with evidence supporting both its upregulation and downregulation at 24 h post-stroke. Among different protein kinase C (PKC) isoforms, the δ isoform is widely reported to play a pivotal role in the progression of ischemic reperfusion injury. Our present study aims to decipher the molecular mechanism of post-stroke IA MSCs mediated alleviation of perifocal vasogenic edema by PKCδ-mediated AQP4 regulation. Ovariectomized female SD rats were infused with 1 × 105 IA MSCs at 6 h post middle cerebral artery occlusion (MCAo). Animals were evaluated for behavioral and functional outcomes. Brains were harvested for evaluating infarct size and brain edema. Further, brain tissues were used for biochemical and molecular studies to decipher the possible molecular mechanism related to the regulation of PKCδ-mediated AQP4 expression. 1 × 105 IA MSCs at 6 h post-stroke confers neuroprotection as evident by the reduction in infarct size, edema, and improvement of functional outcome. An increase in GSH and catalase and a reduction in nitrite and MDA were observed along with a decrease in AQP4 and PKCδ expressions within the cortical brain regions of IA MSC-infused animals. The study gives preliminary evidence that IA MSCs administration post-stroke modulates PKCδ to regulate AQP4 expression which alleviates vasogenic edema towards neuroprotection. The study is novel and clinically relevant as no previous studies have looked into this aspect following IA delivery of stem cells in an animal model of ischemic stroke.

Sirtuin-1 - Mediated NF-κB Pathway Modulation to Mitigate Inflammasome Signaling and Cellular Apoptosis is One of the Neuroprotective Effects of Intra-arterial Mesenchymal Stem Cell Therapy Following Ischemic Stroke.

AIM: Stroke results in long term serious disability that affect millions across the globe. Several clinical and preclinical studies have reinforced the therapeutic use of stem cells in stroke patients to enhance their quality of life. Previous studies from our lab have demonstrated that 1*105 allogeneic bone marrow-derived mesenchymal stem cells (BM-MSCs) when given intraarterially (IA) render neuroprotection by modulating the expression of inflammasomes. Sirtuins are a class of important deacylases having a significant role in cellular functioning. Sirtuin-1 (SIRT-1) is an important enzyme essential for regulating cellular metabolism, which is reduced following an ischemic episode. The present study aims to unviel the role of MSCs in regulating the brain SIRT-1 levels following stroke and the involvement of SIRT-1 in regulating inflammasome signaling to reduce cellular apoptosis towards rendering neuroprotection. MATERIALS AND METHODS: 6 h post-reversible middle cerebral artery occlusion (MCAo), ovariectomized Sprague Dawley (SD) rats were infused intraarterially with 1*105 MSCs. 24 h after MCAo animals were examined for functional and behavioral outcomes. Brains were collected for assessing size of infarct and neuronal morphology. Molecular and immunofluroscence studies were also performed for assessing changes in gene and protein expressions. Extent of apoptosis was also determined in different groups. Inhibition study with SIRT-1 specific inhibitor EX-527 was also performed. RESULTS: A reduction in infarct size and improvement in motor functional and behavioral outcomes following infusion of MSCs IA at 6 h post-stroke was observed. Increase in average neuronal density and neuronal length was also seen. Increased expression of SIRT-1, BDNF and concomitant reduction in the expression of different inflammatory and apoptotic markers in the brain cortical regions were observed following MSCs treatment. CONCLUSION: Our study provides a preliminary evidence that post-stroke IA MSCs therapy regulates SIRT-1 to modulate NF-κB pathway to mitigate inflammasome signaling and cellular apoptosis. This study using IA approach for administering MSCs is highly relevant clinically. Our study is the first to report that neuroprotective effects of IA MSCs in rodent focal ischemia is mediated by SIRT-1 regulation of inflammasome signaling.

Endovascular Stem Cell Therapy Post Stroke Rescues Neurons from Endoplasmic Reticulum Stress-Induced Apoptosis by Modulating Brain-Derived Neurotrophic Factor/Tropomyosin Receptor Kinase B Signaling.

Ischemic stroke is devastating, with serious long-term disabilities affecting millions of people worldwide. Growing evidence has shown that mesenchymal stem cells (MSCs) administration after stroke provides neuroprotection and enhances the quality of life in stroke patients. Previous studies from our lab have shown that 1 × 105 MSCs administered intra-arterially (IA) at 6 h post stroke provide neuroprotection through the modulation of inflammasome and calcineurin signaling. Ischemic stroke induces endoplasmic reticulum (ER) stress, which exacerbates the pathology. The current study intends to understand the involvement of brain-derived neurotrophic factor/tropomyosin receptor kinase B (BDNF/TrkB) signaling in preventing apoptosis induced by ER stress post stroke following IA MSCs administration. Ischemic stroke was induced in ovariectomized female Sprague Dawley rats. The MSCs were administered IA, and animals were sacrificed at 24 h post stroke. Infarct area, neurological deficit score, motor coordination, and biochemical parameters were evaluated. The expression of various genes and proteins was assessed. An inhibition study was also carried out to confirm the involvement of BDNF/TrkB signaling in ER stress-induced apoptosis. IA-administered MSCs improved functional outcomes, reduced infarct area, increased neuronal survival, and normalized biochemical parameters. mRNA and protein expression of ER stress markers were reduced, while those of BDNF and TrkB were increased. Reduction in ER stress-mediated apoptosis was also observed. The present study shows that IA MSCs administration post stroke provides neuroprotection and can modulate ER stress-mediated apoptosis via the BDNF/TrkB signaling pathway.

Pyruvate kinase M2 in chronic inflammations: a potpourri of crucial protein-protein interactions.

Chronic inflammation (CI) is a primary contributing factor involved in multiple diseases like cancer, stroke, diabetes, Alzheimer's disease, allergy, asthma, autoimmune diseases, coeliac disease, glomerulonephritis, sepsis, hepatitis, inflammatory bowel disease, reperfusion injury, and transplant rejections. Despite several expansions in our understanding of inflammatory disorders and their mediators, it seems clear that numerous proteins participate in the onset of CI. One crucial protein pyruvate kinase M2 (PKM2) much studied in cancer is also found to be inextricably woven in the onset of several CI's. It has been found that PKM2 plays a significant role in several disorders using a network of proteins that interact in multiple ways. For instance, PKM2 forms a close association with epidermal growth factor receptors (EGFRs) for uncontrolled growth and proliferation of tumor cells. In neurodegeneration, PKM2 interacts with apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) to onset Alzheimer's disease pathogenesis. The cross-talk of protein tyrosine phosphatase 1B (PTP1B) and PKM2 acts as stepping stones for the commencement of diabetes. Perhaps PKM2 stores the potential to unlock the pathophysiology of several diseases. Here we provide an overview of the notoriously convoluted biology of CI's and PKM2. The cross-talk of PKM2 with several proteins involved in stroke, Alzheimer's, cancer, and other diseases has also been discussed. We believe that considering the importance of PKM2 in inflammation-related diseases, new options for treating various disorders with the development of more selective agents targeting PKM2 may appear.

Neuroimmune crosstalk and evolving pharmacotherapies in neurodegenerative diseases.

Neurodegeneration is characterized by gradual onset and limited availability of specific biomarkers. Apart from various aetiologies such as infection, trauma, genetic mutation, the interaction between the immune system and CNS is widely associated with neuronal damage in neurodegenerative diseases. The immune system plays a distinct role in disease progression and cellular homeostasis. It induces cellular and humoral responses, and enables tissue repair, cellular healing and clearance of cellular detritus. Aberrant and chronic activation of the immune system can damage healthy neurons. The pro-inflammatory mediators secreted by chief innate immune components, the complement system, microglia and inflammasome can augment cytotoxicity. Furthermore, these inflammatory mediators accelerate microglial activation resulting in progressive neuronal loss. Various animal studies have been carried out to unravel the complex pathology and ascertain biomarkers for these harmful diseases, but have had limited success. The present review will provide a thorough understanding of microglial activation, complement system and inflammasome generation, which lead the healthy brain towards neurodegeneration. In addition to this, possible targets of immune components to confer a strategic treatment regime for the alleviation of neuronal damage are also summarized.