Preconditioning With Intermittent Hypobaric Hypoxia Attenuates Stroke Damage And Its Possible Mechanism

Objective: Human life and health are threatened by cerebral vascular diseases,including stroke.Preconditioning has become one of the primary research hotspots for increasing brain ischemia tolerance.However,the neuroprotective effect of hypoxic preconditioning on ischemic stroke has always been controversial.This study aims to investigate the impact of hypobaric hypoxic stimulation at an altitude of5000 meters on normal rats over 10 days.It also keens on the issue of whether hypobaric hypoxic preconditioning has a neuroprotective effect on a stroke animal model.Whether intermittent and persistent preconditioning have the same effect on MCAO rats,and which condition is more effective.We explored the possible mechanism of the neuroprotective effect of hypobaric hypoxic preconditioning through proteomics.Furthermore,different experiments were conducted to develop basic theories and research ideas for the prevention and treatment of ischemic brain injury.Methods: In this study,148 SPF male SD rats(220～240g)were randomly divided into nine groups.Non-surgical groups included: the control group(C,n=6),2hours/day hypoxic stimulation group(H2,n=6),6 hours/day hypoxic stimulation group(H6,n=6),and persistent hypoxic stimulation group(Hp,n=6).The surgical groups were: sham operation group(S,n=20),MCAO group(M,n=28),MCAO group with 2 hours/day intermittent preconditioning(H2M,n=20),MCAO group with6 hours/day preconditioning(H6M,n=28),and cerebral infarction group with persistent hypoxic preconditioning(Hp M,n = 28).The modified neurological function score(m NSS)and Zea-Longa score were used to determine the extent of neurological injury 24 hours after the operation.Transmission electron microscopy was used to examine the ultrastructure of the cortex and hippocampus.Observation of the condition of neurons was made by Nissl staining.Immunofluorescence staining was performed to analyze the injury in the cortex and the CA1 area of the hippocampus.The number of Neu N-positive cells in peri-infarct and CA1 areas was calculated.Western blots were used to detect the level of caspase3,cleaved-caspase3,and interleukin-6(IL-6)in the cortex and hippocampus.Label-free proteomics was used to detect the differentially expressed proteins between the H6 M group and the M group,and the corresponding differentially expressed proteins were analyzed to screen out the meaningful signal pathways.Western blotting and immunofluorescence staining were used to verify the results.Results: As a result,the percentage of infarct volume and neurological score in the H6 M group were significantly lower than those in the M group;the morphology and arrangement of neurons in the cortex and hippocampal CA1 area of the H6 M group were significantly better than those of the other 3 MCAO model groups.According to the Nissl staining results,neurons,tissue integrity,and distribution of Nissl bodies in both the cortex and hippocampal areas in the S group were similar to those in the control group.There were significantly Neu N positive cells in the core area and peri-infarct area in the H6 M group than in the M group.In the hippocampal CA1 area,there was no significant difference in the number of Neu N positive cells between the four groups.Neurons and endothelial cells were apoptotic in the H2 and H2 M groups,autophagy bodies appeared in brain tissue in the Hp group,and mitochondria appeared enlarged.Transmission electron microscopy showed that the ultrastructure of the cortex and hippocampus in the hypoxic preconditioning cerebral infarction group was significantly better than that in the simple cerebral infarction group,with the H6 M group performing the best,followed by the Hp M group.Compared with group M,the expression of cleaved-caspase3 in the cortex of the H2 M group,H6 M group,and Hp M group decreased significantly.Proteomics screened out28 proteins with a significant difference between the H6 M group and the M group.KEGG analysis identified the top ten main signal pathways that hypoxia pretreatment affected protein expression in the cortex of MCAO rats.In the cortex of the injured side,protein expression changes were consistent with the observations of proteomics indicating that chmp1 a,rabep1,arpc5,and Hspa2 are closely associated with the nervous system.The expression of clathrin protein and EEA1,which were the main markers of clathrin-dependent endocytosis,was down-regulated compared with that in group M.Conclusion: Hypobaric hypoxic preconditioning at an altitude of 5000 meters,6 h/day and 10 days can effectively reduce the cerebral infarction volume and improve the behavioral score of MCAO rats.At an altitude of 5000 meters,short-term hypobaric hypoxia stimulation does not have a serious impact on the protein synthesis ability of neurons in normal rat brain tissue and the ultrastructure of neurons and the blood-brain barrier.6 h/day preconditioning effectively improves the number and ultrastructure of residual neurons in cortex and hippocampus,and reduces expression of IL-6,caspase 3 and cleaved-caspase 3 in the cortex.The neuroprotective effect of intermittent hypobaric hypoxic preconditioning is better than persistent preconditioning,and the neuroprotective effect of 6 h/day preconditioning is better than 2 h/day preconditioning.The neuroprotective effect of intermittent hypobaric hypoxic preconditioning on MCAO rats may be associated with the regulation of a variety of signal pathways including endocytosis,longevity regulation pathway,RNA transport,etc.Neurons in the core necrotic area and peri-infarct tissue are regulated by clathrin dependent endocytosis,which may participate in the protective process of hypobaric hypoxic preconditioning on MCAO rats.