Withanolide A prevents neurodegeneration by modulating hippocampal glutathione biosynthesis during hypoxia.

Withania somnifera root extract has been used traditionally in ayurvedic system of medicine as a memory enhancer. Present study explores the ameliorative effect of withanolide A, a major component of withania root extract and its molecular mechanism against hypoxia induced memory impairment. Withanolide A was administered to male Sprague Dawley rats before a period of 21 days pre-exposure and during 07 days of exposure to a simulated altitude of 25,000 ft. Glutathione level and glutathione dependent free radicals scavenging enzyme system, ATP, NADPH level, γ-glutamylcysteinyl ligase (GCLC) activity and oxidative stress markers were assessed in the hippocampus. Expression of apoptotic marker caspase 3 in hippocampus was investigated by immunohistochemistry. Transcriptional alteration and expression of GCLC and Nuclear factor (erythroid-derived 2)-related factor 2 (Nrf2) were investigated by real time PCR and immunoblotting respectively. Exposure to hypobaric hypoxia decreased reduced glutathione (GSH) level and impaired reduced gluatathione dependent free radical scavenging system in hippocampus resulting in elevated oxidative stress. Supplementation of withanolide A during hypoxic exposure increased GSH level, augmented GSH dependent free radicals scavenging system and decreased the number of caspase and hoescht positive cells in hippocampus. While withanolide A reversed hypoxia mediated neurodegeneration, administration of buthionine sulfoximine along with withanolide A blunted its neuroprotective effects. Exogenous administration of corticosterone suppressed Nrf2 and GCLC expression whereas inhibition of corticosterone synthesis upregulated Nrf2 as well as GCLC. Thus present study infers that withanolide A reduces neurodegeneration by restoring hypoxia induced glutathione depletion in hippocampus. Further, Withanolide A increases glutathione biosynthesis in neuronal cells by upregulating GCLC level through Nrf2 pathway in a corticosterone dependenet manner.

Enriched environment prevents hypobaric hypoxia induced memory impairment and neurodegeneration: role of BDNF/PI3K/GSK3ß pathway coupled with CREB activation.

Adverse environmental conditions such as hypobaric hypoxia (HH) cause memory impairment by affecting cellular machinery leading to neurodegeneration. Providing enriched environment (EE) is found to be beneficial for curing several neurodegenerative disorders. The protective role of EE in preventing HH induced neuronal death has been reported previously but the involved mechanism is still not clearly understood. The present study is an attempt to verify the impact of EE on spatial memory during HH and also to explore the possible role of neurotrophin in EE mediated neuroprotection. Signaling mechanism involved in neuroprotection was also explored. Male Sprague Dawley rats were simulated to HH condition in an Animal Decompression Chamber at an altitude of 25000 feet in standard and enriched cages for 7 days. Spatial memory was assessed through Morris Water Maze. Role of different neurotrophins was explored by gene silencing and inhibitors for their respective receptors. Further, using different blockers signaling pathway was also explored. Finding of the present study suggested that EE prevents HH mediated memory impairment and neurodegeneration. Also brain-derived neurotrophic factor (BDNF) plays a major role in EE mediated neuroprotection and it effectively prevented neurodegeneration by activating PI3K/AKT pathway resulting in GSK3ß inactivation which further inhibits apoptosis. Moreover GSK3ß phosphorylation and hence its inactivation upregulates CREB phosphorylation which may also accounts for activation of survival machinery in cells and provides neuroprotection. From these observations it can be postulated that EE has a therapeutic potential in amelioration of HH induced memory impairment and neurodegeneration. Hence it may be used as a non invasive and non pharmacological intervention against various neurological disorders.

Withania somnifera root extract ameliorates hypobaric hypoxia induced memory impairment in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Withania somnifera (WS) root extract has been used traditionally in ayurvedic system of medicine as a memory enhancer and anti-stress agent. AIM OF THE STUDY: To evaluate the neuroprotective and prophylactic potential of WS root extract in ameliorating hypobaric hypoxia (HH) induced memory impairment and to explore the underlying molecular mechanism. MATERIALS AND METHODS: WS root extract was administered to male Sprague Dawley rats during a period of 21 days pre-exposure and 07 days exposure to a simulated altitude of 25,000 ft. Spatial memory was assessed by Morris Water Maze. Neurodegeneration, corticosterone, acetylcholine (Ach) levels, acetylcholine esterase (AchE) activity, oxidative stress markers and nitric oxide (NO) concentration were assessed in the hippocampus. Synaptic and apoptotic markers were also investigated by immunoblotting. To study the role of NO in regulating corticosterone mediated signaling, the neuronal nitric oxide synthase (n-NOS) inhibitor, L-Nitro-arginine methyl ester (L-Name) and NO agonist sodium nitroprusside (SNP) were administered from 3rd to 7th day of hypoxic exposure. RESULTS: Administration of WS root extract prevented HH induced memory impairment and neurodegeneration along with decreased NO, corticosterone, oxidative stress and AchE activity in hippocampal region. Inhibition of NO synthesis by administration of L-Name reduced corticosterone levels in hippocampus during hypoxic exposure while co-administration of corticosterone increased neurodegeneration. Administration of sodium nitroprusside (SNP) along with WS root extract supplementation during hypoxic exposure increased corticosterone levels and increased the number of pyknotic cells. CONCLUSION: WS root extract ameliorated HH induced memory impairment and neurodegeneration in hippocampus through NO mediated modulation of corticosterone levels.

Corticosterone synthesis inhibitor metyrapone ameliorates chronic hypobaric hypoxia induced memory impairment in rat.

Chronic exposure to hypobaric hypoxia causes oxidative stress and neurodegeneration leading to memory impairment. The present study aimed at investigating the role of corticosterone in hypoxia induced neurodegeneration and effect of metyrapone, a corticosterone synthesis inhibitor that reduces the stress induced elevation of corticosterone without affecting the basal level, in ameliorating chronic hypobaric hypoxia induced cognitive decline. Rats were exposed to simulated altitude of 25,000 ft for 0, 3, 7, 14 and 21 days to determine the temporal alterations in corticosterone and its receptors following exposure to hypobaric hypoxia. Our results showed an elevation of corticosterone in plasma and hippocampal tissue following 7 days of exposure, which declined on prolonged hypoxic exposure for 21 days. A concomitant increase in ROS and lipid peroxidation was observed along with depletion of intracellular antioxidants. Glucocorticoid and mineralocorticoid receptors were upregulated on 3 and 7 days of hypoxic exposure. Though expression of Glut1 and Glut3 were upregulated on 3 days of hypoxic exposure, sharp decline in Glut1 expression following 7 days of hypoxic exposure leads to reduced neuronal glucose uptake. Administration of metyrapone from 3rd to 7th day of hypoxic exposure to suppress hypoxia induced increase in corticosterone levels resulted in reduced oxidative damage, neurodegeneration and improvement of intracellular energy status. The metyrapone treated hypoxic animals performed better in the Morris Water Maze. Further, administration of exogenous corticosterone along with metyrapone during hypoxic exposure blunted the neuroprotective effect of metyrapone indicating a role for corticosterone in mediating hypobaric hypoxia induced neurodegeneration and memory impairment.

Role of cholinergic markers on memory function of rats exposed to hypobaric hypoxia.

Hypobaric hypoxia is encountered at high altitude. It has a deleterious effect on cognitive functions. An important cause of memory impairment at high altitude is the impairment of neurotransmission. The present study investigates the role of cholinergic markers in hypobaric hypoxia-induced memory impairment. Rats were exposed to hypobaric hypoxia at 6,100 m for 7 days in a simulated-decompression chamber. Memory performance was assessed using the Morris water maze task. Cholinergic markers such as acetylcholine, acetylcholinesterase, choline acetyltransferase, α-7-nicotinic acetylcholine receptor and M(1) muscarinic acetylcholine receptor were also evaluated along with neuronal morphology and DNA fragmentation. We found impairment in memory function along with a decrease in acetylcholine levels, increase in acetylcholinesterase activity, down regulation of choline acetyltransferase, α-7-nicotinic acetylcholine receptor and M(1) muscarinic acetylcholine receptor. We also found that cellular damage is associated with a significant increase in DNA fragmentation. However, administration of acetylcholinesterase inhibitors, such as physostigmine and galantamine, resulted in amelioration of the hypobaric hypoxia induced deleterious effects. It improved acetylcholine level, decreased acetylcholinesterase activity and increased the synthesis of acetylcholine by increasing choline acetyltransferase activity. Also, the acetylcholinesterase inhibitors improved neuronal morphology, perhaps by increasing the expression of α-7-nicotinic acetylcholine receptor and by reducing the acetylcholinesterase level in the cortex and the hippocampus. Therefore, our results suggest cholinergic dysfunction is one of the mechanisms involved in hypobaric hypoxia-induced memory impairment and that acetylcholinesterase inhibitors were able to restore cholinergic function and thus improve memory function.

Cordyceps sinensis promotes exercise endurance capacity of rats by activating skeletal muscle metabolic regulators.

ETHNOPHARMACOLOGICAL RELEVANCE: Cordyceps sinensis is a traditional Chinese medicine used for promotion of health, longevity and athletic power. However, the molecular mechanism for anti-fatigue activity and physical fitness has not yet been reported. AIM OF THE STUDY: The present study was conducted to evaluate the exercise endurance promoting activities of fungal traditional Chinese medicine (FTCM) Cordyceps sinensis cultured whole mycelium (CS) and the underlying mechanisms. MATERIALS AND METHODS: CS was orally supplemented (200mg/kg body weight/day) to rats for 15days with or without swimming exercise along with exercise and placebo groups. RESULTS: Both CS supplementation and supplementation concurrent with exercise improved exercise endurance by 1.79- (P<0.05) and 2.9-fold (P<0.01) respectively as compared to placebo rats. CS supplementation concurrent with exercise also increased the swimming endurance by 1.32-fold (P<0.05) over the exercise group. To study the molecular mechanism of the observed effect, we measured the expression levels of endurance responsive skeletal muscle metabolic regulators AMPK, PGC-1α and PPAR-δ as well as endurance promoting and antioxidant genes like MCT1, MCT4, GLUT4, VEGF, NRF-2, SOD1 and TRX in red gastrocnemius muscle. Our results indicate that CS supplementation significantly upregulates the skeletal muscle metabolic regulators, angiogenesis, better glucose and lactate uptake both in exercised and non-exercised rats. We have also observed increased expression of oxidative stress responsive transcription factor NRF-2 and its downstream targets SOD1 and TRX by CS supplementation. CONCLUSION: CS supplementation with or without exercise improves exercise endurance capacity by activating the skeletal muscle metabolic regulators and a coordinated antioxidant response. Consequently, CS can be used as a potent natural exercise mimetic.

Possible role of cholinesterase inhibitors on memory consolidation following hypobaric hypoxia of rats.

High altitude (HA) generates a deleterious effect known as hypobaric hypoxia (HBH). This causes severe physiological and psychological changes such as acute mountain sickness (AMS) and cognitive functions in terms of learning and memory. The present study has evaluated the effect of cholinesterase inhibitors on memory consolidation following HBH. Adult male Sprague Dawley rats (80-90 days old) with an average body weight of 250 ± 25 g were used. Rats were assessed memory consolidation by using Morris water maze (MWM) for 8 days. After assessment of memory consolidation, rats were then exposed to HBH in stimulated chamber for 7 days at 6,100 m. After exposure to HBH, the memory consolidation of rats has been assessed in MWM. The results showed that there was memory consolidation impairment in HBH-exposed rats as compared to normoxic rats in terms of time spent in quaradents, rings, and counters. The rats which have been treated with physostigmine (PHY) and galantamine (GAL) showed better time spent in quaradents, rings, and counters as compared with hypoxic rats. In conclusion, the cholinesterase inhibitors could ameliorate the impairment of memory consolidation following HBH.

Upregulation of transcription factor NRF2-mediated oxidative stress response pathway in rat brain under short-term chronic hypobaric hypoxia.

Exposure to high altitude (and thus hypobaric hypoxia) induces electrophysiological, metabolic, and morphological modifications in the brain leading to several neurological clinical syndromes. Despite the known fact that hypoxia episodes in brain are a common factor for many neuropathologies, limited information is available on the underlying cellular and molecular mechanisms. In this study, we investigated the temporal effect of short-term (0-12 h) chronic hypobaric hypoxia on global gene expression of rat brain followed by detailed canonical pathway analysis and regulatory network identification. Our analysis revealed significant alteration of 33, 17, 53, 81, and 296 genes (p < 0.05, <1.5-fold) after 0.5, 1, 3, 6, and 12 h of hypoxia, respectively. Biological processes like regulation, metabolic, and transport pathways are temporally activated along with anti- and proinflammatory signaling networks like PI3K/AKT, NF-κB, ERK/MAPK, IL-6 and IL-8 signaling. Irrespective of exposure durations, nuclear factor (erythroid-derived 2)-like 2 (NRF2)-mediated oxidative stress response pathway and genes were detected at all time points suggesting activation of NRF2-ARE antioxidant defense system. The results were further validated by assessing the expression levels of selected genes in temporal as well as brain regions with quantitative RT-PCR and western blot. In conclusion, our whole brain approach with temporal monitoring of gene expression patterns during hypobaric hypoxia has resulted in (1) deciphering sequence of pathways and signaling networks activated during onset of hypoxia, and (2) elucidation of NRF2-orchestrated antioxidant response as a major intrinsic defense mechanism. The results of this study will aid in better understanding and management of hypoxia-induced brain pathologies.

Ship-borne journey induces Th1 cytokines level in antarctic summer expeditioners.

It has become apparent that extreme environmental conditions of Antarctic continent alters many immune responses. The present study was conducted on 28th Indian Antarctic expeditioners. The investigations were carried out to explore the effect of multiple stresses like isolation, cold and UV exposure on human immunity. Thirty blood samples were collected between 6 and 7 AM, after an overnight fast at different stages of the expedition - viz. the pre-exposure sample was collected at Delhi on 25(th) October 2008. The expedition started its ship journey from Capetown, on 6(th) January, 2009 and on-board blood was collected on 31(st) January 2009. After 1 month stay at Maitri, blood was collected on 3(rd) March 2009. Different parameters studied included levels of cytokines, chemokines and cortisol. The ship-borne journey induced a dramatic increase in TNF-α, IFN-γ, and B cell activating factor (BAFF) levels and moderate decreases in TGF-ß and cortisol levels. However, after being off board for 1 month at Maitri station, levels of above cytokines, cortisol and BAFF were decreased but MIP-1α was significantly increased. These data for the first time suggest that ship-borne journey to the Antarctic continent results in tremendous stress to the body, which eventually resulted in increased TH1-biased immunity.

Cholinesterase inhibitors ameliorate spatial learning deficits in rats following hypobaric hypoxia.

Cognitive functions especially learning and memory are severely affected by high altitude (HA) exposure. Hypobaric hypoxia (HBH) encountered at HA is known to cause oxidative stress, alterations of neurotransmitters and cognitive impairment. We hypothesized that alteration in cholinergic system may be involved in HBH-induced learning impairment. The present study has investigated the cholinergic dysfunctions associated with simulated HBH-induced impairment of learning in rats and protective role of acetylcholine esterase inhibitors (AChEIs). Male Sprague-Dawley rats were exposed to HBH equivalent to 6,100 m for 7 days in a simulated decompression chamber. After stipulated period of exposure, learning ability was assessed using Morris water maze (MWM) task. Cholinergic markers like acetylcholine (ACh) and acetyl cholinesterase (AChE) were evaluated from cortex and hippocampus. Morphological changes were evaluated from cortex, CA1, and CA3 region of hippocampus by Nissle staining and by electron microscopy. We found that exposure to HBH led to impairment of learning ability in MWM task, and it was accompanied by decrease in ACh level, increase in AChE activity, and revealed critical cellular damage. Administration of AChEIs like physostigmine (PHY) and galantamine (GAL) resulted in amelioration of the deleterious effects induced by HBH. The AChEIs were also able to restore the neuronal morphology. Our data suggest that cholinergic system is affected by HBH, and AChEIs were able to improve HBH-induced learning impairment in rats.

Oxidative-stress-induced alterations in Sp factors mediate transcriptional regulation of the NR1 subunit in hippocampus during hypoxia.

Ascent to high altitude is associated with tissue hypoxia resulting from the decrease in partial pressure of atmospheric oxygen. The hippocampus, in particular, is highly vulnerable to hypoxic insult, which at least in part can be attributed to the occurrence of glutamate excitotoxicity. Although this excitotoxic damage is often related to increased NMDA receptor activation and subsequent calcium-mediated free radical generation, the mechanisms involving the transcriptional regulation of NMDA receptor subunit expression by hypoxic stress remains to be explored. Our study reveals a novel mechanism for the regulation of expression of the NR1 subunit of NMDA receptors by the Sp family of transcription factors through an oxidative-stress-mediated mechanism that also involves the molecular chaperone Hsp90. The findings not only show the occurrence of lipid peroxidation and DNA damage in hippocampal cells exposed to hypoxia but also reveal a calcium-independent mechanism of selective oxidation and degradation of Sp3 by the 20S proteasome. This along with increased DNA binding activity of Sp1 leads to NR1 upregulation in the hippocampus during hypoxic stress. The study therefore provides evidence for free radical-mediated regulation of gene expression in hypoxia and the scope of the use of antioxidants in preventing excitotoxic neuronal damage during hypoxia.

Acetylcholinesterase inhibitors enhance cognitive functions in rats following hypobaric hypoxia.

Hypobaric hypoxia (HBH) can produce neuropsychological disorders such as insomnia, dizziness, memory deficiencies, headache and nausea. It is well known that exposure to HBH cause alterations of neurotransmitters and cognitive impairment in terms of learning and memory. But the mechanisms are poorly understood. The present study aimed to investigate the cholinergic system alterations associated with simulated HBH induced cognitive impairment. Male Sprague-Dawley rats were exposed to HBH equivalent to 6100 m for 7 days in a simulation chamber. The cognitive performance was assessed using Morris Water Maze (MWM) task. Cholinergic markers like acetylcholine (ACh) and acetylcholinesterase (AChE) were evaluated in hippocampus and cortex of rats. Neuronal damage was also studied through morphological changes. Exposure to HBH led to impairment in relearning ability and memory retrieval and it was accompanied by decrease in ACh level and increase in AChE and led to morphological damage. Administration of AChE inhibitor (AChEI), physostigmine (PHY) and galantamine (GAL) to rats during HBH exposure resulted in amelioration of the deleterious effects induced by HBH. The AChEIs were able to improve the cholinergic activity by restoring the level of ACh by blocking the AChE activity. In addition, the AChEIs also prevented neurodegeneration by reducing the AChE level in cortical and hippocampal neurons.

Bacopa monniera leaf extract ameliorates hypobaric hypoxia induced spatial memory impairment.

Hypobaric hypoxia induced memory impairment has been attributed to several factors including increased oxidative stress, depleted mitochondrial bioenergetics, altered neurotransmission and apoptosis. This multifactorial response of the brain to hypobaric hypoxia limits the use of therapeutic agents that target individual pathways for ameliorating hypobaric hypoxia induced memory impairment. The present study aimed at exploring the therapeutic potential of a bacoside rich leaf extract of Bacopa monniera in improving the memory functions in hypobaric conditions. The learning ability was evaluated in male Sprague Dawley rats along with memory retrieval following exposure to hypobaric conditions simulating an altitude of 25,000 ft for different durations. The effect of bacoside administration on apoptosis, cytochrome c oxidase activity, ATP levels, and oxidative stress markers and on plasma corticosterone levels was investigated. Expression of NR1 subunit of N-methyl-d-aspartate receptors, neuronal cell adhesion molecules and was also studied along with CREB phosphorylation to elucidate the molecular mechanisms of bacoside action. Bacoside administration was seen to enhance learning ability in rats along with augmentation in memory retrieval and prevention of dendritic atrophy following hypoxic exposure. In addition, it decreased oxidative stress, plasma corticosterone levels and neuronal degeneration. Bacoside administration also increased cytochrome c oxidase activity along with a concomitant increase in ATP levels. Hence, administration of bacosides could be a useful therapeutic strategy in ameliorating hypobaric hypoxia induced cognitive dysfunctions and other related neurological disorders.

NR1 and GluR2 expression mediates excitotoxicity in chronic hypobaric hypoxia.

Hypobaric hypoxia has been reported to cause memory dysfunction. The possible molecular mechanism involved, however, remains to be explored. The role that glutamate and its receptors play in causing excitotoxicity in ischemia and neurodegenerative diseases indicates the possible occurrence of a similar phenomenon in hypobaric hypoxia. The present study aimed to elucidate the molecular events occurring at glutamatergic synapses in hypobaric hypoxia using Sprague-Dawley rats as a model system. The animals were exposed to an altitude of 7,600 m for different durations. Hypobaric hypoxia was found to cause oxidative stress, chromatin condensation, and neurodegeneration. A temporal change in the expression of the ionotropic receptors of glutamate was also observed. Expression of the N-methyl-D-aspartate (NMDA) receptor increased, and expression of glutamate receptor subunit 2 of the alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate receptor decreased. We also observed increased activity of glutamate dehydrogenase, indicating greater synthesis and release of glutamate after 3 and 7 days of exposure. Administration of a selective NMDA antagonist during exposure was found to ameliorate neuronal degeneration, providing evidence for the occurrence of excitotoxicity in hypobaric hypoxia. Our study indicates that excitotoxicity occurs in hypobaric hypoxia. This study also indicates the appropriate period for drug administration during exposure to hypobaric hypoxia and establishes ionotropic receptors of glutamate as potential therapeutic targets for ameliorating high-altitude-induced cognitive dysfunction.

Immunomodulatory effects of Premna tomentosa (L. Verbenaceae) extract in J 779 macrophage cell cultures under chromate (VI)-induced immunosuppression.

OBJECTIVE: In the present study, the immunomodulatory effects of Premna tomentosa extract against chromate (VI)-induced toxicity was assessed in J 779 macrophage cell line. DESIGN: The cells were analyzed for cytotoxicity, phagocytosis, oxidant burst, antioxidant status, and cell proliferation. RESULT: Chromate treatment resulted in a significant increase in cytotoxicity and free radical production. Furthermore, there is a significant decrease in reduced glutathione (GSH) levels and glutathione peroxidase activity (GPx). There was an appreciable decrease in cell proliferation and phagocytosis by macrophages in the presence of chromate. However, pretreatment of the cells with P. tomentosa extract (500 microg concentration), 30 minutes prior to chromate (VI) treatment resulted in a significant inhibition of chromate-induced cytotoxicity and reactive oxygen species production. The extract also restored the antioxidant status, cell proliferation, and phagocytosis similar to that of control cells. CONCLUSION: The results confirm the cytoprotective and immunomodulatory effects of the leaves of P. tomentosa and its possible usage in immunosuppressed conditions.