Effect Of "Preservation From Both Physique And Spirit" On Cortical Neuronal Apoptosis And Astrocytes Proliferation In Rats After Cerebral Ischemia/Reperfusion Injury

Objectives To construct a set of enriched environments based on the theory of"preservation from both the physique and spirit".To investigate the effects of different enriched environments on cortical neuronal apoptosis and astrocytes proliferation in rats after focal cerebral ischemia/reperfusion(I/R)injury.Methods(1)Healthy adult male rats weighing 200-240 g were divided into five groups:physical enriched environment(PE),social enriched environment(SE),physical and social enriched environment(PSE),ischemia + standard(IS)and sham-operated + standard(SS).The rat model of focal cerebral I/R injury was established by suture method.Twenty-four hours later,the groups were sent to the corresponding cages for 14 days environmental intervention.The modified neurological severity score(mNSS)and limb-placing test were conducted at day 3,7 and 14.On day 14,the infarct volume was assessed by 2,3,5-triphenyl tetrazolium chloride(TTC)staining and the neuronal survival rate was evaluated by Nissl staining.Transmission electron microscopy was used to observe the morphology and ultrastructure of ischemic neurons.Double labeling of TdT-mediated dUTP-biotin nick end labeling(TUNEL)and NeuN was applied to assess neuronal apoptosis.The expressions of apoptosis-related proteins were tested by immunohistochemical labeling and western blotting.(2)The grouping methods were the same as experiment 1.Foot-fault test and cylinder test were conducted at day 3,7 and 14.The infarct volume was evaluated by magnetic resonance imaging(MRI).Double immunofluorescent labeling of glial fibrillary acidic protein(GFAP)and Ki67 was used to assess the astrocytes proliferation.The expression of protein brain-derived neurotropic factor(BDNF)was tested by western blotting.Results 1.(1)Compared with the SS group,the ischemic rats(PE,SE,PSE and IS)exhibited worse behavioral performances on the two behavioral tests at various time points.The PSE rats had significantly lower mNSS scores and significant improved limb placement compared with PE and SE rats,and PE rats outweighed SE rats.(2)Compared with the PE and SE groups,treatment with PSE significantly reduced infarct volume,increased neuronal viable rate and alleviated the morphological and ultrastructural damage of neurons after cerebral I/R injury.(3)The number of double-labeled cells was lower in the enriched rats(PSE,PE,SE)compared with the IS rats.PSE group showed the lowest number of TUNEL-positive neurons,followed by PE and SE groups.(4)PE,SE and PSE rats had higher Bcl-2 protein levels and lower Bax,and caspase-3 protein levels compared with IS rats.PSE rats exhibited the highest levels of Bcl-2 and lowest levels of Bax and caspase-3 compared with PE and SE groups,while PE was superior to SE.(5)Protein levels of Bcl-2 expression in the enriched groups(PE,SE and PSE)were higher while Bax,cytochrome c,caspase-3 and p53 protein levels were lower when compared with IS group.PSE rats exhibited the highest levels of Bcl-2 and lowest levels of Bax,cytochrome c,caspase-3 and p53.2.(1)PSE rats showed less left forefoot faults and lower asymmetry scores compared with PE and SE rats.(2)The infarct volume in PE and PSE rats was significantly reduced compared with IS rats.PSE was superior to PE.(3)The number of Ki67-positive astrocytes was higher in enriched rats(PSE,PE,SE)compared with the IS rats.PSE rats showed the highest number of Ki67-positive astrocytes and PE rats was superior to SE rats.(4)BDNF expression was significantly increased in the enriched groups compared with the IS group.PSE rats exhibited the highest levels of BDNF compared with PE and SE rats,while PE group was superior to the SE group.(5)There were significantly negative correlations between the mNSS and astrocytes proliferation and BDNF expression levels,and positive correlation was found between the astrocytes proliferation and BDNF protein levels.Conclusions1.The enriched environment of "preservation from both the physique and spirit"promotes functional recovery of stroke rats after focal cerebral I/R injury.2.The enriched environment of "preservation from both the physique and spirit"inhibits neuronal apoptosis in the periinfarct cortex after focal cerebral I/R injury,which may be one of the possible mechanisms underlying the neuroprotective effects.3.The "preservation from both the physique and spirit" promotes astrocytes proliferation and BDNF expression in the periinfarct cortex after focal cerebral I/R injury,which may contribute to the improved neurological function of stroke animals.