| Objective A raft of studies shows that an enriched environment(EE)can promote the recovery of cognitive function after ischemic stroke,but its specific mechanism remains unclear.In recent years,the effect of white matter on brain function has been gradually paid attention,among which white matter is closely related to cognitive function.Studies have found that stroke can lead to severe white matter lesions,the main pathological change of white matter is demyelination.Previous studies have shown that enriched environment can promote myelin regeneration,and microglia and astrocytes have been found to participate in the repair process after stroke and have an impact on myelin regeneration.This study will verify the mechanism of action of microglia and astrocytes in post-stroke remyelination,provide an experimental basis for enriched environment to promote remyelination in stroke,and provide further in-depth research on enriched environment and remyelination.The molecular mechanism of regulation between them,as well as the combined application of enriched environment and repair drugs of myelin-damaged in clinical rehabilitation after stroke provide experimental evidence.MethodsA total of 120 male SD rats(8 weeks old)were selected in this experiment.Middle cerebral artery occlusion and reperfusion(MCAO/R)was used in the experimental group to make ischemic stroke disease models.A sham operation is performed,that is,the surgical procedure is the same,but no monofilament is inserted.During MCAO/R surgery,Laser Doppler Flowmeter was used to monitor blood flow,and a 70%reduction in blood flow was considered successful.72 hours after the operation,3 rats were taken for TTC staining to determine the formation of infarcts.Rats that survived one week after the operation were randomly divided into four groups according to the operation and intervention methods: modeling group(MCAO/R)+ enriched environment group(EE);modeling group(MCAO/R)+standard environment group(SE);sham operation group(Sham)+EE;Sham +SE.Each group was intervened for28 days,and Longa score was used to detect the neurological deficit of the model rats on the 7th,14 th,and 28 th days;the cognitive and memory function of the rats was measured by the Morris water maze on the 7th and 14 th days.The hippocampus was sacrificed on the 28 th day,and the degree of myelin loss was evaluated by LFB staining.Western blot and immunofluorescence methods were used to detect remyelination-related proteins,microglia,astrocytes,brain-derived neurotrophic factor(BDNF),and vascular endothelial growth factor(VEGF).The expression and colocalization of GFAP/BDNF and GFAP/VEGF detected by immunofluorescence double staining,and the levels of inflammatory factors were evaluated by ELISA.Results1.Enriched environment can promote the recovery of neurological and cognitive functions in ischemic strokeAfter ischemic stroke,Longa scores were performed on the 7th,14 th,and 28 th days of intervention.The results presented that the scores of the MCAO+EE group were lower than those of the MACA+SE group,and the scores of the MCAO+EE group in the three stages were significant in turn.decreased(P<0.05).It shows that the enriched environment can lessen the degree of neurological impairment caused by cerebral ischemia injury.Morris water maze was used to test the learning and memory function of rats.The results displayed that on the 14 th and 28 th day of intervention,the escape latency(response learning ability)of the rats in the MCAO+EE group was shorter than that in the MCAO+SE group.The crossed platform times of MCAO+EE rats were more than those in the MCAO+EE group,and there was a significant difference(P<0.05).2.Enriched environment can reduce demyelination damage caused by ischemic strokeAfter 28 days of enriched environmental intervention,the rats were anesthetized and sacrificed.Luxol fast blue tissue staining showed that the blue area of the sections in the MCAO+EE group was significantly larger than that in the MCAO+SE group,indicating that the degree of demyelination was reduced.This suggests that enriching the environment can reduce the demyelination damage caused by cerebral ischemia,which is beneficial to the recovery of white matter after stroke.The results of western blot and immunofluorescence showed that in the model group rats,the myelin marker MBP in the EE group was significantly higher than that in the SE group(P<0.05),but the oligodendrocyte precursor markers(PDGFR-α)had no difference in expression between groups.It is suggested that EE can promote the differentiation and maturation of oligodendrocytes into myelin sheath but cannot promote the proliferation of oligodendrocyte precursor cells.3.Enriched environment can reduce the expression of microglial protein markers and pro-inflammatory factors after ischemic strokeTo explore whether EE affects post-stroke remyelination by regulating the number of microglia and the expression levels of inflammatory factors,we detected the expression of microglia protein markers by western blot and immunofluorescence.Comparing with the SE group,the EE group significantly reduced the number of microglia.In addition,we also observed the expressions of pro-inflammatory factors IL-6 and IL-1β in serum by ELISA,and we found that the expression contents of pro-inflammatory factors in the MCAO+EE group were significantly lower than those in the MACO+SE group.From the results,we can conclude that EE can reduce the inflammatory damage produced by microglia and pro-inflammatory factors after stroke,thereby providing a good physiological microenvironment for remyelination.4.Enriched environment can increase the expression of astrocyte protein markers and their secreted factors BDNF and VEGF after ischemic stroke We detected the protein expressions of astrocytes,BDNF and VEGF in the hippocampus of the rat affected side by western blotting.The sstatistical analysis indicates that in the MCAO model group,the expressions of the three markers in the EE group were more than those in the SE group,and they had significant difference(P<0.05).Then,we detected the co-localization of BDNF,VEGF and GFAP(astroglia marker)by immunofluorescence co-localization.The results showed that the co-localization area of BDNF/GFAP and VEGF/GFAP was higher in the MCAO+EE group.This suggests that EE may promote remyelination after stroke by stimulating astroglia proliferation and secreting BDNF and VEGF.Conclusion1.Enriched environment can improve cognitive function and promote white matter recovery after stroke2.Enriched environment can reduce the inflammatory damage caused by microglia and pro-inflammatory factors after stroke3.Enriched environment can increase the expression levels of astrocytes and their secretions BDNF and VEGF,which may be one of the important mechanisms for promoting remyelination... |
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