Different Pattern Of Cortical Neuronal Calcium Activity And Gene Expression In Response To Different Degree Of Ischemia

Stroke causes a dramatic reduction in cerebral blood supply,resulting in disruption of neuronal structure and function.As neurons are highly energy-consuming cells,they are susceptible to disruption of energy supply and to metabolic imbalance.Once decreased,neuronal population is barely regenerated after stroke.Previous studies have shown that neuronal structures are rapidly damaged after global cerebral ischemia,but if blood flow is reperfused promptly,the disrupted neuronal structures can be restored to some extent.However,it is not completely clear whether different degree of global cerebral ischemia can differentially affect neuronal calcium activity.Therefore,in this study,we investigated neuronal calcium activity after different degree of ischemia using in vivo two-photon imaging,combined with the use of transgenic mice and customed viral tool;and explored the effects of different degree of ischemia on gene expression in mouse cerebral cortex using transcriptome sequencing technology.In this study,Thy1-GCa MP6 s transgenic mice were used to assess the excitotoxic calcium overload in cortical neurons after ischemia.The results showed that neuronal calcium level increased after blood flow deprivation;it gradually restored within 1 h of reperfusion.The neuronal calcium level of mice in the 30 min ischemic group restored to a near pre-ischemia level,while the 60 min group restored relatively slower;after 2and 3 days of reperfusion,neuronal calcium levels were higher in the 60 min ischemic group than those in the 30 min ischemic group,and no significant difference was observed after 7 days of reperfusion.Customized AAV9-Ca MKII-GCa MP6 s was used to label cortical neurons in layer 2/3 and to image calcium activity in awake C57BL/6mice.No significant difference was shown between the two groups before ischemia;after 1 day of reperfusion,the intensity of neuronal calcium activity,firing time and firing rate were higher in the mice of 30 min BCAL group than those in 60 min BCAL group;after 2-3 days of reperfusion,the intensity of neuronal calcium activity was significantly higher in the 60 min BCAL group than that in the 30 min BCAL group,but the firing time and firing rate were both lower than those in the 30 min BCAL group;after 7 days of reperfusion,the firing rate was significantly higher in the 30 min BCAL group than that in the 60 min BCAL group,while the intensity and the firing rate showed no significant difference.After 1,2,3,5 and 7 days of reperfusion,Fluoro-Jade C staining showed that the density of degenerative neurons was significantly higher in the 60 min BCAL group than those in the 30 min BCAL group.Results of transcriptome sequencing showed that global ischemia led to the upregulation of genes related to immune cell activation,cell proliferation and inflammatory response.Importantly,the60 min BCAL group had a higher level of upregulation.In addition,60 min ischemia led to the upregulation of a series of genes related to neuronal calcium activity.Immunofluorescence staining was also used to investigate microglial polarization,and the number of pro-inflammatory microglia was significantly higher than that of antiinflammatory microglia,and the number of polarized microglia was higher in the 60 min ischemic group than that in the 30 min group.In conclusion,different degree of global ischemia differentially affected the neuronal calcium activity and the gene expression profile in cortical tissue in mice.Our study can provide some theoretical and experimental guidance for the clinical study of stroke.