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

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

Details of antibodies used in the experiment.

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

Validation of CHC stress as a Depression stress model.

<p>Changes in A) immobility time in FST B) sucrose intake in SPT C) time spent in open arm D) number of entries in open arm in EPM and D) time spent in central zone of OFT.</p

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

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

Amelioration of spatial memory function following Withanolide A administration during hypoxic exposure.

<p>Exposure to hypobaric hypoxia increased (i) path length and (ii) latency during spatial memory test but decreased (iii) number of platform crossing and (iv) time spent in target quadrant during probe trial when was reversed following withanolide A supplementation before and during exposure to hypobaric hypoxia. Data expressed as percentage change taking normoxic value as 100% and represents Mean ± SEM. ‘a’ denotes p≤0.05 vs. when compared to normoxic group and ‘b’ denotes p≤0.05 vs. when compared to 7 days hypoxic group treated with vehicle only.</p