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

Neuroprotective Role of L-N^G-Nitroarginine Methyl Ester (L-NAME) against Chronic Hypobaric Hypoxia with Crowding Stress (CHC) Induced Depression-Like Behaviour - Fig 2

<p>Changes in the A) time immobility in FST B) sucrose intake C) body weight D) food intake following exposure to Hypobaric Hypoxia, Crowded housing stress and CHC. E) Time of immobility, F) sucrose intake in separate groups of rats exposed to CHC compared to positive control for depression (corticosterone treatment) and following treatment with known antidepressant (Imipramine). *p < 0.05, **p < 0.01 when compare to Control+Veh; values expressed mean percentage of Control ± SEM (n = 10 in each group).</p

Details of antibodies used in the experiment.

<p>Details of antibodies used in the experiment.</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

Neurodegeneration in hippocampal sub-regions CA1 and CA3 following exposure to hypobaric hypoxia, crowding stress alone and CHC.

<p>Slides showing A) hoechst positive cells in the CA1 region and B) CA3 region of hippocampus following crowding, hypobaric hypoxia alone and CHC. C) Quantitative data showing changes in the number of hoescht positive cells in CA1 and CA3 region of hippocampus following exposure to crowding, hypobaric hypoxia alone and CHC. Slides showing D) Fluoro Jade B positive cells in the CA1 region and E) CA3 region of hippocampus following crowding, hypobaric hypoxia alone and CHC. F) Quantitative data showing changes in the number of Fluoro Jade B positive cells in Ca and CA3 region of hippocampus following exposure to crowding, hypobaric hypoxia alone and CHC.</p

Changes in number of active microglia.

<p>A) Representative slides of ED-1 stained hippocampus. Slides showing ED-1 expression in B) DG (C) CA1 and (D) CA3 region of hippocampus. E) ED-1 representative cells, F) Number of ED-1 positive cells in DG, CA1 and CA3 region of hippocampus. *p < 0.05; **p < 0.01; ***p < 0.001 when compare to CHC+Veh; values expressed mean percentage of Control ± SEM (n = 20 in each group).</p

Changes in NF-κB expression.

<p>Representative pictures of NF-κB expression in A) CA3, B) CA1 and C) DG region of hippocampus. Quantitative data showing number of NF-κB positive cell in D) CA3, E) CA1 and F) DG region of hippocampus. *p < 0.05; **p < 0.01; ***p < 0.001 when compare to CHC+Veh; values expressed mean percentage of Control ± SEM (n = 20 in each group).</p

Slides showing Microglial Phenotypes (Ramified and Active) in hippocampus during stress exposure and L-NAME administration.

<p>Representative slides showing Iba-1 expression in neurons of A) Entire hippocampus B) Dentate Gyrus C) CA1 and D) CA3 region of hippocampus. Changes in the number of Iba-1 positive cells in E) DG, F) CA1 and G) CA3 region of hippocampus. *p < 0.05; **p < 0.01; ***p < 0.001 when compare to CHC+Veh; values expressed mean percentage of Control ± SEM (n = 20 in each group).</p