Neuroprotective effects of sea buckthorn leaf extract against hypobaric hypoxia and post-hypoxic reoxygenation induced hippocampal damage in rats.

Exposure to hypobaric hypoxia (HBH) and reoxygenation (H/R) causes hippocampal neuronal damage leading to memory dysfunction and mood state alteration. The present study aimed at investigating the potential neuroprotective effect of seabuckthorn leaf extract ((SBTLE)) against HBH and reoxygenation induced neuronal injury in brain. Six groups of male sprague dawley rats were taken and exposed to simulated HBH equivalent at an altitude of 7600m in an animal decompression chamber for 7 days followed by reoxygenation. Rats were supplemented with SBTLE (100mg/kgBW) 20 days prior and during hypoxic exposure. Recovery from injuries following HBH exposure and subsequent reoxygenation was carried out in separate group of animals. Our study revealed that exposure to reoxygenation stress following hypoxia enhanced hypoxia induced oxidative stress in hippocampal neurons which was reversed with SBTLE supplementation. SBTLE restored Hypoxia/Reoxygenation(H/R) induced downregulation of  γ-glutamylcysteine synthetase (GCLC) enzymes responsible for glutathione biosynthesis. Post hypoxic supplementation of SBTLE decreased the reoxygenation induced enhanced oxidative markers, however, administration of SBTLE in conjunction with the inhibitor of GCLC resulted in slow recovery from H/R injuries. These results provide the first evidence of SBTLE   induced increase in glutathione biosynthesis by upregulating GCLC enzyme expression and hence can be used as a promising drug to cure H/R induced neuronal damages

Neuroprotective effects of sea buckthorn leaf extract against hypobaric hypoxia and post-hypoxic reoxygenation induced hippocampal damage in rats.

Exposure to hypobaric hypoxia (HBH) and reoxygenation (H/R) causes hippocampal neuronal damage leading to memory dysfunction and mood state alteration. The present study aimed at investigating the potential neuroprotective effect of seabuckthorn leaf extract ((SBTLE)) against HBH and reoxygenation induced neuronal injury in brain. Six groups of male sprague dawley rats were taken and exposed to simulated HBH equivalent at an altitude of 7600m in an animal decompression chamber for 7 days followed by reoxygenation. Rats were supplemented with SBTLE (100mg/kgBW) 20 days prior and during hypoxic exposure. Recovery from injuries following HBH exposure and subsequent reoxygenation was carried out in separate group of animals. Our study revealed that exposure to reoxygenation stress following hypoxia enhanced hypoxia induced oxidative stress in hippocampal neurons which was reversed with SBTLE supplementation. SBTLE restored Hypoxia/Reoxygenation(H/R) induced downregulation of  γ-glutamylcysteine synthetase (GCLC) enzymes responsible for glutathione biosynthesis. Post hypoxic supplementation of SBTLE decreased the reoxygenation induced enhanced oxidative markers, however, administration of SBTLE in conjunction with the inhibitor of GCLC resulted in slow recovery from H/R injuries. These results provide the first evidence of SBTLE   induced increase in glutathione biosynthesis by upregulating GCLC enzyme expression and hence can be used as a promising drug to cure H/R induced neuronal damages

Enriched environment prevents hypobaric hypoxia induced memory impairment and neurodegeneration: role of BDNF/PI3K/GSK3β 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 GSK3β inactivation which further inhibits apoptosis. Moreover GSK3β 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

Effect of Trk antagonist on Behavior was studied using Morris Water Maze test and representative tack sheet showing memory test of Normoxia (I), Hypoxia (II), Enriched environment + Hypoxia (III) and Enriched environment + hypoxia + K252a (IV) exposure.

<p>Inhibition of TrkB with K252a nullifies the protective effect of Enriched environment on hypobaric hypoxia induced memory impairment as evident from increased pathlength (A) and latency (B) and decreased No. of platform crossing (C) and time spent in target quadrant (D). Data represents Mean ± SEM. “*” and “#” represents p<0.001 when compared to control and hypoxic group whereas “**” represents p<0.05 when compared to hypoxic group.</p

Schematic diagram of signaling events in enriched environment mediated neuroprotection during hypobaric hypoxia.

<p>Schematic diagram of signaling events in enriched environment mediated neuroprotection during hypobaric hypoxia.</p

Role of neurotrophins was assessed using gene silencing technique for TrkA and TrkB receptors.

<p>Results showed that knockdown of TrkB significantly increased caspase3 activity even in presence of enriched environment whereas blocking of TrkA has diminutive effect on caspase3 activity in comparison to TrkB inhibition. To further explore the signaling cascade, ERK and PI3K pathway was blocked with U0216 and Wortmannin respectively and it has been observed that inhibition of PI3K pathway significantly increases caspase3 activity (B) whereas inhibiting ERK pathway have no significant effect in group exposed to hypobaric hypoxia in enriched cage (C). Data represents Mean ± SEM. In “*” and “#” represents p<0.001 when compared to control and hypoxic group while “?”represents p<0.05 when compared to control group.</p

Shows experimental design describing groups, number of individuals, exposure duration and interventions.

<p>Shows experimental design describing groups, number of individuals, exposure duration and interventions.</p

Withanolide A Prevents Neurodegeneration by Modulating Hippocampal Glutathione Biosynthesis during Hypoxia

<div><p><i>Withania somnifera</i> 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.</p></div

Showing schedules and doses of drug administered during exposure to hypobaric hypoxia.

<p>* Indicate the administration of drug started from 3^rd day of hypoxic exposure.</p>#<p> denotes the administration of Withanolide A started 21 days prior to hypoxic exposure and was continued during hypoxic exposure.</p><p>Showing schedules and doses of drug administered during exposure to hypobaric hypoxia.</p

Schematic diagram showing Withanolide A mediated modulation of glutathione biosynthesis and neuroprotection during hypobaric hypoxia.

<p>Schematic diagram showing Withanolide A mediated modulation of glutathione biosynthesis and neuroprotection during hypobaric hypoxia.</p