Enriched Environment Regulates Act A/NMDAR-Wnt Signal Transduction And Improves Vascular Cognitive Impairment Cognitive Dysfunction

Chronic cerebral hypoperfusion(CCH)is a crucial factor in the occurrence and development of vascular cognitive impairment(VCI).Glutamate binds to the N-methyl-D-aspartate receptor(NMDAR),which is widely distributed in the postsynaptic membrane of the central nervous system,induces excitotoxicity,leads to neuronal apoptosis,reduces synaptic plasticity,and impairs cognitive function.Activin A(Act A)can promote the phosphorylation of NMDAR.The previous research results of our group show that exogenous Act A may reduce neuronal damage during ischemia and hypoxia through an anti-apoptotic effect.The classical Wnt signaling,also known as Wnt/?-catenin signaling,can be activated by the upstream NMDAR and participate in the regulation of synaptic plasticity.Enriched environment(EE)is a classical positive environmental effect model,previous studies have confirmed that EE can improve cognitive impairment caused by a variety of diseases and affect the expression of NMDAR and the upregulation of synaptic plasticity.However,the mechanism of EE improving VCI is unclear.Act A,NMDAR,and Wnt/?-catenin signaling may involve.Therefore,this study clarifies the impact of enriched environment on VCI and the mechanism of Act A and NMDAR-Wnt signal transduction in this process to provide a new idea for improving the prognosis of patients with VCI.1.Enriched environment improves vascular cognitive impairment cognitive dysfunctionObjective:To explore the effect of enriched environment on vascular cognitive impairment.Method:SPF SD rats were randomly divided into chronic cerebral hypoperfusion+standard environment group(CCH group),sham+standard environment group(sham group),chronic cerebral hypoperfusion+enriched environment group(EE group).The CCH model was established by 2-vessel occlusion(2-VO)to simulate the occurrence of VCI.The 2-VO method was used in the CCH group and EE group.In the sham group,bilateral common carotid arteries were only separated without ligation.EE group was fed in an enriched environment,CCH group and the sham group were fed in a standard environment.At 4 and 8 weeks,Morris water maze was used to detect rats'learning and memory ability in each group.HE evaluation of histopathological damage,TUNEL detection of neuronal apoptosis,and Western blot were used to detect the expression of rats'apoptosis-related proteins Bcl-2 and Bax.Result:After four weeks,the escape latency of the CCH group was significantly longer than that of the EE group,and the difference was more significant after eight weeks.The result indicates that the CCH rats'cognitive function decreased,and an enriched environment can improve cognitive function.Compared with the CCH group,the neuron damage in the EE group was significantly reduced,the cell morphology of some neurons was restored,the degree of degeneration and necrosis of neurons was improved,and the neuron apoptosis was reduced.The neuron apoptosis rate after 8W was higher than that in the 4W group;Compared with the CCH group,the expression level of Bcl-2/Bax in the EE group was up-regulated,and the above changes were more significant after eight weeks.Conclusion:Enriched environment can affect the expression of apoptosis-related protein Bax/Bcl-2,reduce neuronal cell apoptosis and VCI rats'brain injury.2.Enriched environment improves vascular cognitive impairment cognitive function by inducing Act AObjective:To explore the effects of Act A on calcium influx,NMDAR,and Wnt/?-catenin signaling pathway in enriched environment.Method:A calcium ion probe was used as the primary antibody to label intracellular Ca2+,and a fluorescently labeled secondary antibody was added to detect the changes of calcium ion content in neurons.Immunofluorescence staining and western blot were used to detect Act A.Changes in the expression level of NMDAR related subunits(NMDAR,NR2A,and NR2B),synapse related proteins(GAP43,SYN,PSD-95,and MAP-2),and Wnt/?-catenin signaling(Wnt3a,?-catenin,VEGF,Cyclin D1,GSK-3?,and p-GSK-3)in hippocampal and prefrontal cortical neurons were detected by western blot.The changes in the dendritic structure of neurons were observed by Golgi staining.On this basis,the enriched environment+CCH+Act A inhibitor FSH group(EEF group)was established,and then the expression changes of Ca²⁺and Act A were detected by immunofluorescence staining.Act A,NMDAR,NR2A,NR2B,GAP43,SYN,PSD-95,MAP-2,Wnt3a,?-catenin,VEGF,Cyclin D1,GSK-3?,and p-GSK-3 expression changes were detected by western blot.Result:Compared with the CCH group,the concentration of Act A increased in the frontal cortex and hippocampal neurons in the EE group,and the concentration of Ca²⁺changed.The expression levels of NMDAR related subunits and synapse-related proteins increased in the EE group.The density of dendritic spines in the frontal cortex and hippocampus increased significantly in the EE group.Compared with the rats in the EE group,the expression of Act A was downregulated in the EEF group.The expression levels of NMDAR,NR2A,and NR2B were decreased,and the content of Ca²⁺was increased in the EEF group.The expression of GAP43,SYN,PSD-95,MAP-2,Wnt3a,?-catenin,VEGF,and cyclin D1 was downregulated,p-GSK-3?was up-regulated in the EEF group.The head volume of neuronal dendritic spines decreased,the neck was elongated,and the dendritic density decreased in EEF group.Conclusion:Enriched environment promotes the expression of NMDAR,and Wnt/?-catenin signaling pathways by activation of Act A,inducing up regulates synaptic plasticity of neurons in VCI rats.3.Enriched environment reduces VCI damage through the positive feedback loop of NMDAR-Wnt signal transduction and Act AObjective:To explore NMDAR and Wnt/?-catenin signal pathway on synaptic plasticity and further clarify the regulatory mechanism of Act A and NMDAR-Wnt signal transduction in the EE improving VCI rats'cognitive function.Method:The level of Ca²⁺in neurons was detected by immunofluorescence staining.The expression of Act A and synapse-associated proteins(GAP43,SYN,PSD-95,and MAP-2)in hippocampal and prefrontal cortical neurons were detected by Western blot.The changes of the dendritic structure were observed by Golgi staining.On this basis,the enriched environment+CCH+NMDAR blocker MK801group(EEM group)and the enriched environment+CCH+Wnt/?-catenin signaling pathway blocker Dickkopf-1 group(EED group)were established.Morris water maze was used to determine the effect of NMDAR on rats'learning and memory ability.The changes of Ca²⁺were detected by immunofluorescence staining.The expressions of Act A,GAP43,SYN,PSD-95,and MAP-2 were detected by Western blot.Result:Compared with the rats in the EE group,the escape latency of the EEM group was prolonged,the number of crossing the platform was reduced,and the learning and memory functions of rats were still impaired;The expression of Act A decreased,and the content of Ca²⁺increased;The expression of GAP43,SYN,PSD-95,and MAP-2 decreased,and the density of neuronal dendrites decreased in the EEM group.Compared with the EE group,the expression of GAP43,SYN,PSD-95,and MAP-2 decreased,and synaptic plasticity decreased in the EED group.Conclusion:A positive feedback loop exist between Act A,and NMDAR-Wnt signal transduction during enriched environment reduces VCI injury.