OBJECTIVE Stroke has become the top ten leading cause of death in China.Isch⁃emic stroke accounts for 85%of stroke cases,and insufficiency of cerebral blood supply caused by atherosclerosis is one of the important cau...OBJECTIVE Stroke has become the top ten leading cause of death in China.Isch⁃emic stroke accounts for 85%of stroke cases,and insufficiency of cerebral blood supply caused by atherosclerosis is one of the important causes of ischemic stroke.Therefore,it is of posi⁃tive significance to study the molecular mecha⁃nism of stroke injury caused by hypoperfusion in the search for drug targets.Voltage-dependent potassium channels are a family of potassium channels widely expressed in the central ner⁃vous system.However,their roles in neurogene⁃sis after stroke insults have not been clearly illus⁃trated.The purpose of this experiment is to explore the expression changes of different sub⁃families of voltage-dependent potassium chan⁃nels after the occurrence of ischemic stroke and their influence on neuroregeneration,to study the molecular mechanism of stroke injury caused by hypoperfusion,and to find potential targets for drug therapy of ischemic stroke.METHODS C57BL/6 mice aged 7-8 weeks and C17.2 cells were used in vivo and in vitro in the experiment.The mice in the experimental group were suf⁃fered from bilateral common carotid artery occlu⁃sion(BCCO)for 1 h and reperfusion for 7 d.In the control group,bilateral carotid artery was dis⁃sected without occlusion.Behavioral assay of suspension test were performed to assess the motor deficits of the mice.In this assay,the time of the first drop(latency),the number of drops within one minute(frequency),and the final scores were recorded as the results of athletic ability.A lower score indicated more severe motor damage of the mice.TTC staining was used to observe the cerebral infarction areas caused by ligation of bilateral common carotid arteries.After seven days,mice were sacrificed and brain tissue protein samples were collected for real-time quantitative PCR(RT-PCR)and Western blotting test to detect the changes of potassium channel subfamily expression levels in different brain regions.Neuronal injuries in all brain regions were detected using Nissl staining methods 7 d following model establishment.To detect the effects and the underlying mechanism of the related potassium channel on neurogene⁃sis,recombinant plasmids of the potassium chan⁃nels were transfected in cultured C17.2 neural stem cells.Afterwards,oxygen glucose depriva⁃tion experiments were performed.RESULTS Behavioral tests showed that BCCO can cause impaired motor performance.TTC staining showed that cerebral infarction existed in the stri⁃atum region,and the motor function decline caused by the injury in this region was consistent with the behavioral experiment results which veri⁃fied the effectiveness of our surgical operation.Nissl staining revealed a large amount of neuronal cell necrosis in the cortex and striatum regions,and dense neuronal cells in the lateral ventricular limbic region,suggesting that neurogenesis may have occurred in this region.The results of real-time quantitative RT-PCR showed that among the detected potassium channels distributed in the measured nervous system,the expression of voltage-dependent potassium channel Kv4.2 decreased significantly in all brain regions after stroke,suggesting that it may be involved in the pathological process of stroke.Immunohisto⁃chemical staining showed that there was neuro⁃genesis in the subgranular zone(SGZ)and sub⁃ventricular zone(SVZ)of the mice,and Kv4.2 expression was significantly changed in the regions,suggesting that it may be involved in the regulation of neuro regeneration after stroke.The transfected Kv4.2 plasmid enhanced the dif⁃ferentiation of the C17.2 neural stem cells to neu⁃rons and astrocytes under normoxia and the oxy⁃gen-glucose deprivation,suggesting that Kv4.2 may induce the differentiation of neural stem cells after stroke.Kv4.2 could induce the neural stem cells to differentiate into neurons in vitro and in vivo,and Western blotting assay showed that Kv4.2 could up-regulate the expression level of ERK1/2,p-ERK1/2,p-STAT3,NGF,p-TtkA,and BDNF.Moreover,the calcium ions and CAMKⅡwas also increased by Kv4.2 in vitro.CONCLUSION BCCO insults can induce the expressions of the potassium channels in the brains,among which the expression of Kv4.2 is down-regulated in the cerebral cortex,hippocam⁃pus and striatum.In vitro experiments confirmed that Kv4.2 can induce the differentiation of C17.2 neural stem cells into neurons and astrocytes under the condition of normoxia and oxygen-glucose deprivation.We concluded that Kv4.2 possibly promoted neurogenesis through ERK1/2/STAT3,NGF/TrkA,and Ca2+/CAMKⅡsignal pathways after stroke.Regulating the physiologi⁃cal functions of Kv4.2 channel might contribute to the rehabilitation of neuronal damage after stroke.展开更多
文摘OBJECTIVE Stroke has become the top ten leading cause of death in China.Isch⁃emic stroke accounts for 85%of stroke cases,and insufficiency of cerebral blood supply caused by atherosclerosis is one of the important causes of ischemic stroke.Therefore,it is of posi⁃tive significance to study the molecular mecha⁃nism of stroke injury caused by hypoperfusion in the search for drug targets.Voltage-dependent potassium channels are a family of potassium channels widely expressed in the central ner⁃vous system.However,their roles in neurogene⁃sis after stroke insults have not been clearly illus⁃trated.The purpose of this experiment is to explore the expression changes of different sub⁃families of voltage-dependent potassium chan⁃nels after the occurrence of ischemic stroke and their influence on neuroregeneration,to study the molecular mechanism of stroke injury caused by hypoperfusion,and to find potential targets for drug therapy of ischemic stroke.METHODS C57BL/6 mice aged 7-8 weeks and C17.2 cells were used in vivo and in vitro in the experiment.The mice in the experimental group were suf⁃fered from bilateral common carotid artery occlu⁃sion(BCCO)for 1 h and reperfusion for 7 d.In the control group,bilateral carotid artery was dis⁃sected without occlusion.Behavioral assay of suspension test were performed to assess the motor deficits of the mice.In this assay,the time of the first drop(latency),the number of drops within one minute(frequency),and the final scores were recorded as the results of athletic ability.A lower score indicated more severe motor damage of the mice.TTC staining was used to observe the cerebral infarction areas caused by ligation of bilateral common carotid arteries.After seven days,mice were sacrificed and brain tissue protein samples were collected for real-time quantitative PCR(RT-PCR)and Western blotting test to detect the changes of potassium channel subfamily expression levels in different brain regions.Neuronal injuries in all brain regions were detected using Nissl staining methods 7 d following model establishment.To detect the effects and the underlying mechanism of the related potassium channel on neurogene⁃sis,recombinant plasmids of the potassium chan⁃nels were transfected in cultured C17.2 neural stem cells.Afterwards,oxygen glucose depriva⁃tion experiments were performed.RESULTS Behavioral tests showed that BCCO can cause impaired motor performance.TTC staining showed that cerebral infarction existed in the stri⁃atum region,and the motor function decline caused by the injury in this region was consistent with the behavioral experiment results which veri⁃fied the effectiveness of our surgical operation.Nissl staining revealed a large amount of neuronal cell necrosis in the cortex and striatum regions,and dense neuronal cells in the lateral ventricular limbic region,suggesting that neurogenesis may have occurred in this region.The results of real-time quantitative RT-PCR showed that among the detected potassium channels distributed in the measured nervous system,the expression of voltage-dependent potassium channel Kv4.2 decreased significantly in all brain regions after stroke,suggesting that it may be involved in the pathological process of stroke.Immunohisto⁃chemical staining showed that there was neuro⁃genesis in the subgranular zone(SGZ)and sub⁃ventricular zone(SVZ)of the mice,and Kv4.2 expression was significantly changed in the regions,suggesting that it may be involved in the regulation of neuro regeneration after stroke.The transfected Kv4.2 plasmid enhanced the dif⁃ferentiation of the C17.2 neural stem cells to neu⁃rons and astrocytes under normoxia and the oxy⁃gen-glucose deprivation,suggesting that Kv4.2 may induce the differentiation of neural stem cells after stroke.Kv4.2 could induce the neural stem cells to differentiate into neurons in vitro and in vivo,and Western blotting assay showed that Kv4.2 could up-regulate the expression level of ERK1/2,p-ERK1/2,p-STAT3,NGF,p-TtkA,and BDNF.Moreover,the calcium ions and CAMKⅡwas also increased by Kv4.2 in vitro.CONCLUSION BCCO insults can induce the expressions of the potassium channels in the brains,among which the expression of Kv4.2 is down-regulated in the cerebral cortex,hippocam⁃pus and striatum.In vitro experiments confirmed that Kv4.2 can induce the differentiation of C17.2 neural stem cells into neurons and astrocytes under the condition of normoxia and oxygen-glucose deprivation.We concluded that Kv4.2 possibly promoted neurogenesis through ERK1/2/STAT3,NGF/TrkA,and Ca2+/CAMKⅡsignal pathways after stroke.Regulating the physiologi⁃cal functions of Kv4.2 channel might contribute to the rehabilitation of neuronal damage after stroke.
