Effect Of "Preservation From Both Physique And Spirit" On Angiogenesis In Rats After Cerebral Ischemia/reperfusion Injury

Objectives To build three different kinds of enriched environments based on the theory of "preservation from both physique and spirit" which originated from traditional Chinese medical book "Huang Di Nei Jing".To investigate the effects of different enriched environments on angiogenesis in rats after cerebral ischemia/reperfusion injury.Methods(1)Rats were randomly divided into five groups:physical enriched environment(PE),social enriched environment(SE),physical and social enrichment group(PSE),ischemia + standard group(IS)and sham-operated + standard group(SS).The PE,SE,PSE and IS rats were cerebral ischemia/reperfusion(I/R)model built by occlusion the right middle cerebral artery.Twenty-four hours after surgery,the rats were sent for environmental intervention in corresponding cages which lasted for 14 days.The modified neurological severity score(mNSS)and rota-rod test were conducted at day3,7 and 14.HE staining was used for observation of the brain cortex pathological changes.The infarct volume was assessed by Nissl staining.Laser scanning confocal microscopy(LSCM)and 3D reconstruction was conducted for quatitative analysis of cerebral microvessels.Immunoglobulin(IgG)extraction assay was used to detect the blood brain barrier(BBB)leakage in the ischemic boundary cortex.CD31 and Ki67 double immunofluorescent staining was applied to quantify the proliferation of brain endothelial cells,CD31 immunofluorescent staining was also analyzed for microvessel density(MVD).The erythropoietin(EPO)protein and gene expression were detected using immunohistochemical assessment and real time quantitative PCR.The expressions of VEGF/Flk-1,Ang-1/Tie-2 proteins were detected by western blotting and immunohistochemical labeling.Results(1)No significant differences in mNSS results or rota-rod test were seen among ischemic groups(PE,SE,PSE,and IS)on day 3(P>0.05).The PE,SE and PSE rats exhibited better functional recovery compared to IS rats by day 7(P<0.05),and the advantages were more obvious by day 14.Among enrivhed conditioning groups,PSE rats performed the best,while PE rats performed better than SE rats(P<0.05).(2)The pathological damage of ischemia boundary cortex was more severe in IS group than the enriched conditioning groups.(3)PSE and PE rats had smaller infarct volume than IS rats(P<0.05).(4)Rats in enriched conditioning groups had less IgG leakage than IS rats(P<0.05),PSE conditioning preserved blood brain barrier(BBB)to the greatest extent(P<0.05),while PE outweighed SE(P<0.05)in this result.(5)The quatitative analysis of LSCM results showed rats in IS group had smaller capillary,less total vessel surface area and less branch points compared to rats in SS group,while enriched conditioning rats had larger capillary,more total vessel surface area and more branch points compared to IS rats(P<0.05),PSE conditioning promoted angiogenesis to the greatest extent(P<0.05),while PE outweighed SE(P<0.05)in this result.(6)The enriched groups(PSE,PE,SE)had more CD31/Ki67 double-labeled cells than IS group(P<0.05),PSE rats exceeded PE and SE rats(P<0.05)while PE group outweighed SE group(P<0.05).The same trend was seen in MVD results.(7)EPO protein and gene level in PE,SE and PSE rats were higher than IS group(P<0.05),PSE conditioning promoted its expression to the largest extent(P<0.05),while PE conditioning outweighed SE conditioning(P<0.05).(8)The protein levels of VEGF/Flk-1 and Ang-l/Tie-2 in the enriched groups(PE,SE and PSE)were higher than IS rats(P<0.05),PSE rats exhibited the highest levels of VEGF/Flk-1 and Ang-1/Tie-2(P<0.05),while PE outweighed SE group(P<0.05)in these results.Conclusions1.The enriched enviroments derived from "preservation from both the physique and spirit" promoted fuctional recovery of rats after cerebral I/R injury.2.The enriched enviroments derived from "preservation from both the physique and spirit" promoted angiogenesis in the periinfarct cortex after focal cerebral I/R injury,which might be one of the possible mechanisms underlying their neuroprotective effects.3.The enriched enviroments derived from "preservation from both the physique and spirit" up-regulated VEGF/Flk-1 and Ang-1/Tie-2 expression in the periinfarct cortex,which might be the potential pathway through which the different enriched enviroments promoted angiogenesis.