| Objective:There are 80.97% stroke patients accompanied by post-stroke cognitive impairment(PSCI),which leads to a significant prolongation of patients’ rehabilitation progress and a significant decline in their quality of life.Enriched environment(EE)is a safe and convenient rehabilitation intervention.Growing evidence suggests that EE significantly improves PSCI.However,the underlying mechanism has not to be clarified.α7n ACh R is widely existed in the central nervous system and immune system.Recent studies proved that α7n ACh R plays an important role in neuronal synaptic plasticity and serves as an important target for improving cognitive function.α7n ACh R is also a necessary "switch" to activate the cholinergic anti-inflammatory pathway(CAP).Whether α7n ACh R mediated CAP is involved in EE improving PSCI remains to be confirmed.This study is expected to perfect the mechanism theory of EE improving PSCI,and also to provide new therapeutic targets for the clinical treatment of PSCI.Methods:8 weeks aged male SD rats were selected as the animal subjects.The cerebral ischemia injury model was established through middle cerebral artery occlusion and reperfusion(MCAO/R).Rats were divided into three groups according to the operation method and intervention method,which includes the control group: Rats were reared in a standard environment(SE)housing condition for 28 days after sham operation;EE group: MCAO/R rats were reared in an EE housing condition for 28 days;SE group:MCAO/R rats were reared in a SE housing condition for 28 days.The neurological function scores at 7,14,and 28 days after MCAO/R were evaluated by the modified neurological severity score(m NSS).The Morris water maze test was used to evaluate the learning and memory ability of rats from 21 to 28 days after MCAO/R surgery.Then the rats were sacrificed by general anesthesia and the tissue was removed.The cerebral infarct volume of rats was detected by TTC staining.The effect of EE intervention on hippocampal synaptic plasticity was assessed by electrophysiology.Moreover,nicotine(α7n ACh R agonist)or α-BGT(α7n ACh R inhibitor)was added to confirm the role ofα7n ACh R in EE enhancing synaptic plasticity.Furthermore,immunohistochemistry was applied to detect the expression of α7n ACh R protein in hippocampal CA1 region.ELISA was applied to detect the content of serum cytokines at 7,14,28 days after MCAO/R and cholinergic proteins at 28 days after MCAO/R.Results:1.EE significantly improves the neurological and cognitive function of ischemic stroke rats.Compared with the control group undergoing sham operation,m NSS scores at 7,14,28 days after MCAO/R were significantly increased(P<0.001),while the m NSS scores of rats in the EE group was significantly lower than that of the SE group(P<0.01),suggesting that cerebral ischemia injury leads to obvious neurological deficit,and EE intervention significantly promotes the recovery of neurological function.In the first five days of the Morris water maze test,the rat’s spatial learning ability was tested by the escape latency that rats reached the platform.Then the platform was removed and the number of times the rat crossed the original platform was observed to evaluate the rat’s spatial memory ability.It was found that MCAO/R induced significant learning and memory deficits,which is especially obvious from the 2nd to the 4th day of the test.Furthermore,EE intervention promotes the recovery of the spatial learning and memory ability of cerebral ischemic rats.2.EE intervention has no obvious effect on the cerebral infarct volume of rats.The infarct volume of MCAO/R rats at 28 day was 40.48%±2.69% in SE group,the infarcted volume of EE group was 38.13%±1.44%.There was no significant difference in the infarcted volume(P>0.05).3.EE intervention enhances hippocampal synaptic plasticity of ischemic stroke ratsAt 28 days after MCAO/R,the neuroplasticity of the hippocampus was detected by electrophysiology.The results showed that compared with the control group,the long-term potentiation(LTP)of the CA3-CA1 pathway in the hippocampus of the SE group was significantly reduced(P<0.001),suggesting that MCAO/R surgery severely damages the hippocampal synaptic plasticity,while EE intervention partially reversed the decrease in LTP induced by MCAO/R(P<0.001),and this effect is enhanced by nicotine(α7n ACh R agonist)(P<0.001),and Weakened by α-BGT(α7n ACh R inhibitor)(P<0.01).4.EE intervention promotes the recovery of α7n ACh R expression in hippocampal CA1 region.At 28 day after MCAO/R,the rats were anesthetized and sacrificed,and the number of positive cells of α7n ACh R in the hippocampal CA1 region on the ipsilateral side of the ischemic injury was detected by immunohistochemical analysis.The results showed that the α7n ACh R in the control group were highly expressed in the hippocampal CA1 region,but the number of α7n ACh R positive cells in the hippocampal CA1 region in the SE group was significantly reduced,suggesting that MCAO/R surgery induces decreased expression of α7n ACh R in the hippocampus CA1region(P<0.001),and EE intervention promotes the recovery of α7n ACh R expression(P<0.001).5.EE intervention inhibits the expression of serum pro-inflammatory factors.The expression of pro-inflammatory factors IL-1β and IL-6 in serum was detected by ELISA on 7,14,and 28 days after MCAO/R.Results showed that serum IL-1β and IL-6 were significantly up-regulated at 7 days after MCAO/R.As time went by,the expression of serum cytokines gradually decreased but always higher than the control group(P<0.01).Compared with the SE group,the serum pro-inflammatory factors IL-1βand IL-6 of the EE group at each time point significantly decreased but were always higher than those of the control group(P<0.05).It is suggested that MCAO/R induces obvious peripheral inflammation,while EE inhibits the expression of pro-inflammatory factors to reduce inflammation.6.EE intervention significantly increases the expression of cholinergic-related proteins in the hippocampus.28 days after MCAO/R,the expression of cholinergic-related proteins in the hippocampus was detected by ELISA.The results showed that compared with the control group,the levels of acetylcholine(ACh)and acetylcholine acetyltransferase(Ch AT)in the SE group decreased significantly(P<0.001),and the expression of acetylcholinesterase(ACh E)increased significantly(P<0.001).Compared with the SE group,EE intervention increased the expression of ACh and Ch AT in the hippocampus,and reduced the expression of ACh E(P<0.001).Conclusions:1.EE significantly reduces neurological deficit and cognitive impairment induced by MCAO/R2.α7nAChR is a necessary regulatory factor for EE enhancing synaptic plasticity,and is also expected to become an important clinical treatment target for PSCI.3.The CAP mediated by α7nAChR is one of the possible mechanisms for EE improving PSCI... |
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