| Objective:An animal model of high altitude cerebral edema(HACE)was established to detect the differential expression of genes in brain tissues of HACE mice and control mice,focusing on screening differential genes related to angiogenesis,and to investigate the regulation effect and mechanism of differential expression genes on blood-brain barrier permeability in HACE animal models.Materials and methods:In the first part of this study to establish and identify an animal model of high altitude cerebral edema(HACE)by using a decompression chamber to simulate a high altitude environment,and to lay a foundation for further research on the mechanism of HACE.Balbc mice(8 weeks)were randomly divided in to sham and experimental group.For experimental group,mice were placed into a chamber mimicking the oxygen level and atmosphere pressure of different altitude of 4000 m,5000 m and 6000 m,respectively.For sham group,the mice deal with normal pressure and normal oxygen.Brain tissures were collected at 6 h,12 h,24 h,48 h,72 h after treatment,respectively.HE staining was used to observe the pathological changes of brains.An appropriate altitude for HACE animal model was dertermined according to H&E results.Cerebral water content was estimated according to weight of wet brains.Evan blue(EB)was injected by tail vein,and EB concentration in brains were measured with spectrophotometer.The second part of this study to compare the gene expression profiles of mice brains between HACE and sham group,and to find out the differential genes asscociated to angiogenesis,and thus to lay a foundation for the subsequent mechanism study of HACE.Balbc mice(Eight-weeks)were divided into sham group and experimental group.The mice in the experimental group were placed in decompression chamber mimicking an altitude of 6000 m.For sham group,the mice deal with normal pressure and normal oxygen.At 24 h after the exposure,mice were sacrificed under isoflurane anesthesia and the brain tissues were removed.H&Estaining was used to observe the pathological changes of HACE.Gene microarray was performed to detect gene expression profiles.Real-time PCR was performed to verify data of gene microarray.The third part of this study to investigate the effect and underlying mechamisms of Ang2 on blood brain barrier in HACE animal model.Balbc male mice were randomly divided into sham and experimental group.For sham group,the mice deal with normal pressure and normal oxygen.For experimental group,the mice were placed in decompression chamber mimicking the oxygen level and atmosphere pressure at an altitude of 6000 m.Brain tissue was collected after animal anesthetizing at 6 h,12 h,24 h,48 h,72 h after treatment,respectively.Neutralizing antibody was injected intracerebroventricularly to block the effect of Ang2.Western blot was used to detect expression of Ang2,Occludin,Claudin5 and ZO-1,RhoA,ROCK,p-ROCK,p-Occludin,p-Claudin5 protein.Immunofluorescence was used to detect Ang2 expression.Cerebral water content was evaluated by comparing brain weight.Evan blue was used to detect brain-blood barrier permeability.Pathological changes of brain specimen was evaluated by HE staining.Result:1.The mice in group mimicking an altitude of 4000 m generally performed well in the chamber,with no significant changes in body weight,and no significant changes in brain tissue morphology and structure showed by H&E staining.The mice in group mimicking an altitude of 5000 m showed a slightly reduced activity,appetite and response to stimulation and the body weight was significantly decreased after 48 h of low pressure and low oxygen insult.H&E staining showed that there were few cells were swelled and edematous in the 48 h and 72 h groups,but no obvious abnormality in the other groups.The mice in group mimicking an altitude of6000 m showed significantly reduce in activity,appetite,response to stimulation,shortness of breath and body weight.After exposed to acute hypobaric hypoxia for 12 h,the body weight was significantly reduced,and the mortality rate was 12.2%.H&E staining showed that there were a little edema of brain cells was observed at 6 h,12 h,while at 24 h,48 h and 72 h,more edema of brain cells,disordered cell arrangement,enlarged intercellular space and nuclear pyknosis were observed,which was most obvious at 24 h,48 h and 72 h.Based on the abvove results,altitude of 6000 m was choosed as the appropriate altitude in further study.In the group of altitude of 6,000 m,the brain weight and EB permeability began to increase at 12 h after hypobaric-hypoxic exposure,H&E staining showed cellular swelling at 6 h and 12 h after hypobaric hypoxia insult,cellular swelling,disordered cell arrangement,increased space between cells,and nuclear pyknosis at 24 h,48 h and 72 h after hypobaric-hypoxic insult.There is no obvious apoptosis both in the experimental group of 6000 m altitude of6000 m and the sham group.2.Compared with the sham group,there were 293 differentially expressed genes.among which 130 genes were down-regulated and 163 genes were up-regulated.GO biological process analysis revealed that a total of 6 differential genes involved in angiogenesis,including Ang2,Col4a1,Col4a2 mRNA,HIF3 a,EGF,and KDR.Except for down-regulated EGF expression,the expression of other differential genes was up-regulated,among which mRNA of Ang2 had the largest Fold change of 8.29442.RT-PCR verification showed that compared with Sham,the expression of Ang2 mRNA was significantly up-regulated after ≥6 h of hypoxia treatment,and there was no significant difference in the expression among the experimental groups.Compared with sham group,mRNA expression of COL4α1 increased after ≥24 h hypoxia treatment.The mRNA expression level of COL4α2 increased after ≥6 h hypoxia treatment,and the most significant after ≥24h hypoxia treatment.The expression level of HIF3αmRNA was significantly increased after ≥6h hypoxia treatment.The expression level of KDR mRNA increased after ≥24h hypoxia treatment.The expression level of EGF mRNA decreased after 6 h,12 h and 24 h hypoxia treatment,but increased after 48 h and 72 h hypoxia treatment.3.Ang2 was significantly increased in the brains of mice beginning at12 h after acute hypobariaand hypoxia,and maintained at a high level until72 h after treatment.There was no significant difference among 12 h,24 h,48 h and 72 h groups in terms of Ang2 expression.The expression levels of Occludin and Claudin5 began to decrease at 12 h and maintained at a low level until 72 h after of hypobaric-hypoxic reatment.There was no significant difference among groups of 12 h,24 h,48 h and 72 h.Administration of Ang2 neutralization antibody significantly abated cerebral water content,permeability of evan blue,and alleviated pathological changes of brain in terms of cell morphology,and tissue space.Applying of Ang2 neutralization antibody also increased protein expressions of Occludin and Claudin5,and decreased the expression of p-Occludin,RhoA and p-ROCK,but did not change ZO-1 protein expression.Conclusion:1.Simulating an altitude of 6000 m with a low-pressure and hypoxic chamber can cause changes in brain water content,EB permeability and brain histology,and the longer the treatment time is,the more serious the above changes are,and the most obvious changes occur after 24 h of treatment.The HACE model can be effectively established by using a low-pressure hypoxic chamber to simulate an altitude of 6000 m(atmospheric pressure 47.19 k Pa,oxygen partial pressure 9.73 k Pa).2.HACE signigicantly changed the gene expression profile of mice brains.Both gene microarray and Realtime PCR results showed that 6 genes associated to angiogenesis were dramatically changed in HACE mice brain.3.Ang2 can regulate the expression of Occludin and Claudin5 through RhoA/ROCK signaling pathway,increase the phosphorylation of Occludin,increase the permeability of blood-brain barrier,and lead to the pathological damage of HACE.The RhoA/ROCK signaling pathway was also inhibited after Ang2 inhibition,and the pathological damage of HACE was significantly reduced. |
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