Oxygen Enrichment Reduces Reductive Stress In Rat Hippocampus Under Acute Hypobaric Hypoxia

The reduced atmospheric pressure is one of the most distinct features of the altitude environment.Low oxygen partial pressure causes insufficient oxygen supply to the body,resulting in symptoms such as headache,insomnia,weakness,cognitive impairment,and gastrointestinal reactions.Among them,cognition impairment is a common occurrence.More than 30 million people ascend to high altitudes every year in China.The intellectual ability of plain troops and construction laborers was significantly diminished after rapid ascent to 4000 m.Therefore,it is of great importance to explore the progression of brainpower decline at altitude.Learning and memory deficits are the main factors of brainpower decline,which has been reported to be closely related to oxidative stress.Since the hippocampus may be the iconic brain region associated with learning and memory,it’s crucial to clarify the effects of hypobaric hypoxia on redox signal in rat hippocampus.It has been suggested that hippocampal impairment under acute hypobaric hypoxia(HH)is caused by oxidative stress,which results from both excess reactive oxygen species and reduced endogenous antioxidant capacity,but the endogenous antioxidant system could be activated by oxidative stress.Therefore,it remains controversial whether acute exposure to high altitude activates the endogenous antioxidant system.Considering excessive activation of Nrf2(the key regulator of the endogenous antioxidant system)may lead to reductive stress and enhance oxidative stress,it has not been reported whether reductive stress mediates the development of hippocampal impairment under acute HH and whether oxygen enrichment(an effective intervention at altitude)could alleviate hippocampal injury by regulating redox signal.In conclusion,this study observed the redox status in rats hippocampus under HH with and without oxygen enrichment intervention to answer the question refined previously.This study was mainly divided into three parts:Part Ⅰ: Time domain analysis of differentially expressed genes(DEGs)in rat hippocampus under HH Objective: To clarify whether antioxidant capacity can be regulated in the hippocampus of rats rapidly exposed to HH.Methods: After preprocessing and analysis of published microarray profiles of GSE66287 by R software,DEGs were screened out.Then,the genes were clustered and their functions were analyzed by short-time sequence expression analysis software.Results: The consistently up-regulated DEGs are mainly involved in redox-related biological processes such as cellular detoxification,antioxidant,and transition metal ion homeostasis.Conclusion: Redox-related biological processes could be regulated in rat hippocampus under acute HH exposure,which implied a possible activation of Nrf2.Part Ⅱ: Analysis of the redox status in rat hippocampus under different HH circumstances Objective: To clarify whether Nrf2 can be activated/hyper-activated in rat hippocampus under acute HH.Methods: We set 8 HH groups with equivalent altitudes of 5000,6000,7000,and 7600 m for 1 and 3 d exposure.For each duration,we set a control group in Xi’an(400 m).The rats were euthanized with an anesthetic overdose after the model were built.Except H&E staining,TUNEL,4-HNE,and Nrf2 fluorescence staining which were co-labeled with Neu N fluorescence were performed on the brain tissues to detect neuronal damage,percentage of apoptotic-like neurons,lipid peroxidation damage,and Nrf2 activation in the CA1 region of rat hippocampus;quantification of Klf9 and Gpx4 in the hippocampus were performed to determine the activation and antioxidant capacity of Nrf2.Results: H&E staining showed decreasing neurons with increasing altitude when exposed for 1 d;HH exposure for 3 d resulted in a reduced number of neurons in the rat hippocampus,which was obvious at 7600 m.TUNEL staining revealed an elevated percentage of apoptotic-like neurons at 1 d exposure;acute exposure to 7600 m for 3 d increased the percentage of apoptotic-like neurons.4-HNE staining showed that the lipid peroxidation of neurons increased with altitude at 1 d exposure;3 d exposure led to an increase in the level of lipid peroxidation,which was obvious at 6000 m and above altitude.Nrf2 staining and the quantification of Klf9 and Gpx4 showed a tendency of hyper-activation of Nrf2 at altitudes above 6000 m without statistical significance,and Nrf2 was sustainably up-regulated when exposed to 7000 m for 1 and 3 d,in which the corresponding mean expressions of Klf9 and Gpx4 were higher than those of the control group.Conclusion: Neuronal damage increased with altitude when exposed to HH for 1 d.There was a hyper-activation of Nrf2 in rat hippocampus under acute HH,which implies that antioxidant supplementation may not be always effective in alleviating hippocampus injury after HH and that enhancing the ability to maintain redox homeostasis or lessening the effects of environmental stimuli may be a more reliable protective way.In addition,hyper-activation of Nrf2 may occur after rapid ascent to 7000 m.This part of the study provides new insights into the protection against learning and memory impairment under acute HH and provides experimental parameters for the next part of the oxygen enrichment intervention study.Part III: The mechanism of oxygen enrichment intervention based on redox regulation Objective: To clarify whether reductive stress mediates the hippocampal impairment and whether oxygen enrichment can protect the hippocampus under HH.Methods: H&E and TUNEL staining were performed to determine the effects of oxygen enrichment intervention;the detection of glutathione(GSH)and the ratio of glutathione to oxidized glutathione(GSH/ GSSG)was conducted to determine the redox status of the hippocampus;fluorescent co-labeling staining with 4-HNE and Neu N was performed to observe lipid peroxidation in neurons;the expression of Nrf2,Klf9,and Gpx4 were detected to evaluate the level of reductive agents in the hippocampus.Results: The concentration of oxygen enrichment was stabilized at(28.0±0.2)%.GSH and GSH/GSSG were significantly elevated(1.89 times higher than the control group under 1 d exposure and about 8.25 times higher than the control group under 3 d exposure)in rat hippocampus after acute HH exposure,while GSH/GSSG was significantly decreased by oxygen enrichment intervention.The results of 4-HNE staining showed that the oxygen enrichment intervention effectively reduced the extent of lipid peroxidation in hippocampal neurons under acute HH.Assays of Nrf2,Klf9,and Gpx4 showed that the oxygen enrichment intervention inhibited the expression of Nrf2,Klf9,and Gpx4 under acute HH.Conclusion: Oxygen enrichment intervention can alleviate hippocampal impairment by inhibiting the hyper-activation of Nrf2 and secondary reductive stress under acute HH.