A Study On Mechanism Of Cerebral Ischemia-reperfusion Tolerance Induced By Hyperbaric Oxygen Preconditioning In Rats

Ischemic stoke is a major source of increased morbidity,mortality and disability rate, which is a threat to people's health and results in heavy pressure of spirits and economy on the society and families.Nowdays there aren't good strateges about the secondary prevention of ischemic stroke.The emphases of ischemic cerebrovascular disease are transferred form therapy to prevetion gradually and the primary prevention has attracted extensive attention.Interventions means are taken before the episode of stoke or the existence of risk factors to lower the stroke risk and reinforce the resistance to harmful stress.The aim is to avoid the happening of stoke or reduce the severity.It has been demonstrated that many pretreatment means can induce tolerance of tissues and cells to ischemia-reperfusion injury,including ischemia,hypoxia,hyperbaric oxygen,chemical agents,cortical spreading depression,sleep deprivation,dietary restriction,and both hyperthermia and hypothermia.However,the possibility of clinical application of these substances is yet to be elucidated because of their toxicity or side effects.At present HBO has been applied in clinic as a treatment way to be proved safe and feasible,for example,CO poisoning,air embolism and decompression sickness or other adjunctive therapy related to insufficient oxygen supply.HBO is more feasible in clinic. Yet the mechanisms on ischemic tolerance induced HBO pretreatment remained poorly defined which has restricted its practical application.We used an established middle cerebral artery occlusion(MCAO) model described initially by Zea-Longa with some improvement.This model is typical for stoke research because vascular construction of Sprague-Dawley rats is similar with that of human being, it can mimic the process of cerebral ischemia-reperfusion in human.We investigated the effect of HBO preconditioning on ischemia-reperfusion injury reflected by the means of symptomatology,pathology and morphology to estimate the effect of HBO preconditioning on cortical ischemic penumbra,core or hippocampus.There were two aspects of mechanisms that have been studied;one is the change of ROS and related antioxidase after repeated HBO intervention.The other is the change of mitochondrial apoptosis after HBO. We hope to provide experimental evidence for the clinical application of HBO preconditioning.PartⅠThe protective effect of HBO preconditioning on transient focal ischemia-reperfusion injury in ratsObjective:To investigate whether the short-term HBO protocol is of protective effect on ischemia-reperfusion injury and the degree of reponse on HBO pretreatment in cortical ischemic penumbra,core or hippocampus,respectively.Methods:HBO preconditioning was administered by using 100%oxygen at 2.5 ATA for 1 hour at 12 hours interval for four times in 2 days.The last HBO preconditioning was performed at 24 hours before MCAO produced by the filament model initially reported by Zea-Longa et al with some modifications.Rats were randomly assigned to one of the following three groups:sham group,MCAO group,and HBO plus MCAO group.At 24 hours after the models were made,we observed the general state of spirit and behavior,the change of weight,survival rate,assessed the neurological functional decect by the scales descriped by Johansson and Garcia,determined the brain water content and TTC staining. After perfused and sliced,the sections were performed by HE,NISSL,Spoerrl staining and counted under the microscope.Results:During 24h after surgery we observed the rats in MCAO group were depressed and less active,whose coat was in mess.They seemed to lose their interest in food and water.In contrast,the rats in HBO preconditioning group were more vigorous. HBO preconditioning improved survival rate from 78%in MCAO group to 92%(P＜0.05) and reduced body weight reduction from 33.60±9.53g in the MCAO group to 22.07±9.03 g (P＜0.01).Animals subjected to sham surgery showed no neurobehavioral functional deficit. HBO-PC animals showed better neurological functional recovery at 24 hours when compared with MCAO alone animals(9.5±1.1 vs.6.8±1.5;P＜0.05).The water content of the MCAO group was significantly higher than the control(P＜0.01) and HBO-PC groups (0.78±0.01vs.0.86±0.02;P＜0.01).The infarct ratio were 0.21±0.04 in MCAO group and 0.14±0.03 in HBO-PC group(P＜0.05).There are more intact neurons in the penumbra than in the core at 24 hrs in MCAO and HBO-PC+MCAO groups.The intact neuron numbers per mm2 in the core of HBO-PC+MCAO group were slightly higher than MCAO group, but not significant(34.57±8.62 vs.25.32±9.53,P＞0.05).The intact neuron numbers of the penumbra and the hippocampus were higher in HBO-PC+MCAO group than those of MCAO group(99.87±19.57 vs.54.52+17.63,p＜0.05 for penumbra; 98.95±20.32vs.35.85±15.47,P＜0.01 for hippocampus,respectively).HE and Spoerrl also showed the similar results.Conclusion:1 the making method of MCAO models is simple and repeatable.They are suitable for comparing the effect of HBO preconditioning and provide clear ischemic penumbra and core.2 short-term HBO preconditioning protocol is pretective for cerebral ischemia-reperfusion injury.3 the protective effect of HBO preconditioning is better in ischemic penumbra than core.PartⅡHyperbaric oxygen preconditioning induces tolerance against brain ischemiareperfusion injury by upregulation of antioxidant enzymes in ratsObjective:To investigate whether the ischemic tolerance induced by HBO preconditioning is related to the activity of antioxidase stimulated by ROS after ischemia-reperfusion injury.Methods:The animal models were made and the HBO was perfomed according to the protocol introduced in PartⅠ.The ischemic core and penumbra were dissected according to well-established protocols in rodent models of unilateral proximal MCAO and the samples were homogenized.The activity of anti-negative oxygen ions and GSH-px were assayed by the means of chromatometry,SOD was assayed by the means of hydroxylamine, and CAT was assayed by the means of ultraviolet chromatometry according to the instruments contained in the kits.The content of MDA was measured by the means of TBA.Results:HBO preconditioining improved the activity of anti-negative oxygen ions in the cortical ischemic penumbra(369.22±25.94 vs.315.49±24.16,P＜0.05) and hippocampus (381.19±27.54 vs.321.25±30.11,P＜0.05),decreased the content of MDA(6.94±2.21 vs.11.43±1.18,P＜0.05 for penumbra;8.28±1.89 vs.12.25±1.65,P＜0.05 for hippocampus), enhanced the activity of SOD(162.55±14.44 vs.124.47±13.86,P＜0.01 for penumbra; 146.75±11.48 vs.105.29±10.55,P＜0.01 for hippocampus),CAT(22.47±2.89 vs. 9.69±3.27 for penumbra;19.66±3.98 vs.7.62±3.77 for hippocampus,P＜0.01 for both; 14.62±4.51vs.6.28±3.88,P＜0.05 for ischemic core),but had no significant effect on the activity of GSH-px.Conclusion:1 HBO preconditioning may be related to enhanced antioxidant activity of brain tissues triggered by ROS.2 There were differences in the reactive degree of oxidative stress after HBO preconditioning followed ischemia-reperfusion injury between ischemic penumbra and core.PartⅢHyperbaric oxygen preconditioning reduces ischemic-reperfusion injury by inhibition of apoptosis in a middle cerebral artery occlusion rat modelObjective:To investigate the influence of HBO preconditioning on mitochondrial apoptosis.Methods:The animal models were made and the HBO was perfomed according to the protocol introduced in PartⅠ.The terminal deoxynucleotidyl transferase-mediated Dutp nick end labeling(TUNEL) assay was performed on paraffin-embedded sections according to the manufacture's instructions.The activity of caspase-3 and -9 were measured with caspase-3 and caspase-9/CPP32 Fluorometric Assay Kit.The expression levels of cytochourome c,Bcl-2 and Bax proteins were detected by Western Blot.Results:There were more apoptotic neurons in the core of HBO-preconditioning rats than MCAO rats(80.43±15.47vs.59.23±14.35,P＞0.05).HBO-preconditioning redeuced the number of TUNEL positive cells in the penumbra(84.57±18.75 vs.145.82±17.02, P＜0.01) and CA1 sector(67.58±18.30 vs.163.68±20.35,P＜0.01) when compared with MCAO group.At 24 hour after MCAO the relative activity of caspase-3 and -9 in the ischemic core were similar for MCAO and HBO-preconditioning,respectively(P＞0.05). HBO-preconditioning reduced caspase-3 and -9 activity in the penumbra(P＜0.05) and in the hippocampus(P＜0.05).Western blot analysis of brain samples at 24 hour after MCAO showed the similar levels of cytoplasmic cytochourome c in the ischemic core in MCAO and HBO-preconditioning groups(P＞0.05).HBO-PC reduced cytochourome c in the penumbra and in the hippocampus(P＜0.05).HBO-PC enhanced Bcl-2 levels in the ischemic core,penumbra and hippocampus.The level of Bax remains constant in all groups after MCAO.Conclusion:1 the inhibition of mitochondrial apoptosis may be one of the mechanisms of ischemic tolerance induced by HBO preconditioning.2 there are different dead mechanisms after ischemia-reperfusion between ischemic penumbra and core.HBO demonstrated different influence on both two.