The Effect And Mechanism Of Excitatory Neuron Activity Inhibition In Ischemic Brain Injury

Background:Stroke is the second leading cause of death and the primary cause of adult disability in the world.However,the current treatment for stroke is very limited.Currently,fibrinogen activator?r-tPA?and mechanical thrombolysis are the only two effective treatment for ischemic stroke.Because of the short time window of treatment and secondary bleeding risk,only less than 5%of the patients have benefited from the treatment.Therefore,it is very important to further study the pathophysiological mechanism of ischemic brain injury and explore effective targets for drug therapy.In the decades of research on ischemic stroke,excitotoxicity has been considered to be one of the core theories of ischemic brain damage.Recent findings demonstrate that excitotoxicity is closely related to excessive release of glutamate,overactivation of NMDA receptor and cortical spreading depolarization,which are implicated in the depolarization state of cells.Whether cells are easy to depolarize is often related to their activity.The lower the cell activity is,the harder it is to depolarize.Therefore,we hypothesized that reducing neuronal activity,especially the activity of excitability neurons,may reduce the excitotoxic effect in ischemic brain injury.As every coin has two sides,at present,glutamic signaling pathways are also involved in the repair of ischemic brain injury.In some stages after stroke,there is an excitatory-inhibitory imbalance in peri-infarct,increasing in baseline glutamatergic excitability in peri-infarct tissue parallel recovery.Therefore,we deem that the excitability of neurons after stroke plays various roles in different stages of ischemia.But at present,we are still unclear the specific roles and mechanisms of different activated state of neurons,especially excitatory neurons,in diverse stages of ischemic brain damage.Objective:1?The transgenic mice,overexpression of inward rectifier potassium channels?Kir2.1?in excitatory neurons,were used to investigate the specific role of excitatory neuron activity inhibition in different stages of ischemic brain injury.2?The mechanism of activity inhibition of excitatory neurons in different stages of ischemic brain injury was investigated by means of RNA-seq and bioinformatics.Methods:CamK2a-Cre^ERT2 transgenic mice were crossed with the Kir2.1^loxp/loxp transgenic mice.After several generations,we obtained CamK2?-CreERT2/Kir 2.1^loxp/loxp and CamK2?-CreERT2/Kir 2.1^-/-transgenic mice.The excitatory neurons of the CamK2?-CreERT2/Kir 2.1^loxp/loxpoxp/loxp mice would overexpress the Kir2.1 protein after tamoxifen induction.Then we confirmed whether a mouse model of excitatory neuron activity inhibition would be successfully constructed using qPCR,Western blotting,immunofluorescence,3D reconstruction and electrophysiology.Subsequently,we randomly divided CamK2?-CreERT2/Kir 2.1^loxp/loxp?Kir 2.1+?and CamK2?-CreERT2/Kir 2.1^-/-?Kir 2.1-?transgenic mice after tamoxifen treatment into four group:Sham_Kir2.1?-?,MCAO_Kir2.1?-?,Sham_Kir2.1?+?and MCAO_Kir2.1?+?group.The model of cerebral ischemia in mice was constructed by middle cerebral artery occlusion?MCAO?.MRI were used to measure ischemic area after MCAO 1h reperfusion 1D and 3D.The number of neural degenerative cells were detected by Fluoro-Jade?FJ?staining after MCAO 1h reperfusion 7D and 14D.In the process of experiment,we also recorded the mortality of mice and the weight-gain rate of mice after operation.The neurology score of 1D,3D and 7D in mice after ischemia was evaluated with a modified National Institutes of Health Stroke rating method.The recovery of motor function of mice after stroke was checked by rotated test.RNA-seq technology was exploited to comprehensively detect the transcriptome of cortical tissue from Sham_Kir2.1?-?,MCAO_Kir2.1?-?,Sham_Kir2.1?+?and MCAO_Kir2.1?+?group at 1 day,3 day,7 day,14day and 28 day after operation?resampling three times at each time point in each group?.We applied Tophat-Cufflink-Cuffdiff procedure in hppRNA to screen for differentially expressed genes between MCAO_Kir2.1?-?and MCAO_Kir2.1?+?group mice at the time point on the 1st,3rd,7th,14th,28th postoperative day,and then respectively conducted GO?Gene Ontology?functional enrichment analysis and KEGG signal pathway enrichment analysis on the differentially expressed genes.Besides,we used the R package of MaSigPro to screen the time-series differentially expressed genes.By observing the genetic expression pattern of MC_MS group?Sham_Kir2.1?+?versus MCAO_Kir2.1?+??and WC_WS group?Sham_Kir2.1?-?versus MCAO_Kir2.1?-??,We conducted GO functional enrichment analysis and KEGG signal pathway enrichment analysis on the differential genes with characteristic time point.Combining these two analytical methods,we can find out the mechanism mediated by excitatory neuron activity inhibition in different stages of ischemic brain injury.According to the results of the bioinformatics analysis,we used colorimetry to detect the content of Evans blue in the surgical lateral brain tissue,utilized Iba immunostain to assess the number of activated microglia at 7 day after MCAO,and made use of ELASA assay to detect the content of inflammatory cytokines such as IL-6,IL-1?and TNF-?in the brain tissue 1 D,3 D,7 D and 14 D after operation of the above four groups of mice.Results:We successfully constructed a mouse model of excitatory neuron activity inhibition.At the time of MCAO 1h and 1D reperfusion,the result of MRI staining showed that there was no difference in the ischemic area between MCAO_Kir2.1?-?group and MCAO_Kir2.1?+?group?P>0.05?.But,at the time of MACO 1h and 3D reperfusion,the mice showed a marked increasement of infraction area in the MCAO_Kir2.1?+?group.FJ staining also showed much more neuronal death at the 7D and 14D after MCAO in the MCAO_Kir2.1?+?group.We also observed that the mortality of MCAO_Kir2.1?-?group mice was nil,but that of MCAO_Kir2.1?+?group was up to57%in the 7D after MCAO.Moreover,the weight recovery of the survival of MCAO_Kir2.1?+?mice was slower than that of MCAO_Kir2.1?-?group mice.In addition,compared with the MCAO_Kir2.1?-?mice,neurological score was higher in the MCAO_Kir2.1?+?group mice and the duration of MCAO_Kir2.1?+?mice on the rotating rod also decreased significantly.These results suggested that excitatory neuron activity inhibition gradually aggravated ischemic brain damage and delayed the recovery of neural function in the mice after MCAO 1D.According to the RNA-seq data,there were 30,92,350,275 and 51 differential genes by applying the Tophat-Cufflink-Cuffdiff procedure in hppRNA to screen differential genes at each time point on the 1st,3rd,7th,14th,28th postoperative day in MCAO_Kir2.1?-?and MCAO_Kir2.1?+?mice,respectively.We also found that the inflammatory and immune response of MCAO_Kir2.1?+?mice was stronger than MCAO_Kir2.1?-?mice at the time of 3rd to 14th day after MCAO by GO biological functional clustering and KEGG pathway enrichment analysis.Moreover,on the 7th day after MCAO operation,we found that the genetic pathways for neural functional recovery were down-regulated in the mice of MCAO_Kir2.1?+?group.Screening by R package of maSigPro,there were 1938 time-series differential genes in MC_MS group while 1731 in WC_WS group,and these two groups shared1402 common genes.By classifying the two groups of differential genes into 7 clusters,we found that the genetic expression patterns between the two groups of differentially expressed genes were significantly different.The main difference was that 54.5%genes showed expression peak period on the 7th day after MCAO operation in WC_WS group,while 73.1%genes showed expression peak period on the 14th day after MCAO operation in MC_MS group,and 173 genes in WC_WS group showed expression peak period on the 1st day after MCAO operation,however,there was basically no differential gene showing expression peak period on the 3rd day,and the expression peak period of 246 genes in MC_MS group appeared on the 1st to 3rd day after MCAO.Next,we conducted GO?Gene Ontology?functional analysis and KEGG signal pathway enrichment analysis on the two groups of differential genes with characteristic time point.Then compared the WC_WS group with MC_MS group,we found that the duration of expression was longer and strogner in the differentially expressed genes of MC_MS group associated to injury and inflammatory response.Moreover,we also found that 58 of the 93 genes expressed in the MC_MS group at the peak of MCAO 7D were the specific differentially expressed genes in Kir2.1?+?mice after ischemic stroke.These differentially expressed genes are mainly related to cell cycle and DNA replication.In addition,we found that related genes for recovery of neural function were sustainably inhibited among the down-regulated time-series differentially expressed genes of MC_MS group.Furthermore,the content of Evans blue in ischemic brain tissue of Kir2.1?+?mice was higher than that of Kir2.1?-?mice 3 days after MCAO.7 days after MCAO,the number of activated microglia of Kir2.1?+?mice was also significantly higher than that of Kir2.1?-?mice.And the protein fold change of inflammatory cytokinesTNF-?of Kir2.1?+?mice was significantly higher than that of Kir2.1?-?mice at 3,7 and 14 day after cerebral ischemia,while the protein fold change of IL-6 and IL-1?were markedly higher than that of Kir2.1?-?mice at 7 and 14 day after cerebral ischemia.Conclusion:Overexpression of Kir2.1 channel protein on excitatory neurons lead to activity inhibition of themselves.Inhibition of excitatory neurons activity can aggravate the injury of ischemic stroke in subacute phase,promote neuronal death,and retard the recovery of neural function after ischemic stroke in mice.And which is concerned with the fact that the inhibition of excitatory neurons activity altered the genetic expression pattern after ischemic stroke,aggravated the post-ischemic inflammatory and immune response,promoted cell cycle derangement and sustainably inhibited the expression of related genes for the recovery of neural function.