Spreading depression and selective neuronal death

我们的研究结果分为三个部分：1）扩散性抑郁后海马区MAP 2的缺失2）反复诱导扩散性抑郁后SNAP-25的表达3）扩散性抑郁时谷氨酸的释放1）扩散性抑郁后海马区MAP 2的缺失创伤性脑损伤导致海马CA 3区神经元细胞丢失。然而，尚未确定为什么皮层创伤会导致偏远区域的神经损伤。扩散性抑制（SD）可能是这种病理生理学的一个潜在机制。本研究评估了皮质上的SD是否引起海马的病理变化。将42只Fisher大鼠随机分为4组：I组：假手术组（n=7）; II组：右侧颈动脉闭塞（UO）7 d。（n=7），组III：连续7天（n=7），连续3小时（n =7），连续7天（n = 14），连续7天（n=14），连续3小时（n=14），连续7天（n=14） ...更多信息 我们的（n=7）在第III组和第IV组中的7只动物中的5只，在SD期间用激光多普勒血流仪监测脑血流3 h，并对脑组织进行微管相关蛋白2（MAP 2）的免疫组织化学分析。SD诱导的反应性充血未被右侧颈动脉闭塞显著抑制（III组和IV组分别为194（]SY+-[））25%和181（]SY+-[））42% UO）。在组IV的7天模型中的7只动物中的6只和组III的7天模型中的3只动物中，观察到海马CA 3区（部分包括CA 2）中的MAP 2耗尽，尽管在其他组中没有观察到海马的变化。总之，SD联合UO在CA 3中产生了可重现的病变。脑外伤后海马神经元损伤可能与SD和血流限制有关。2）反复诱导扩散性抑制后SNAP-25的表达反复诱导皮质扩散性抑制（SD）可增加单侧颈动脉阻断血流限制后海马CA 3区神经元细胞的丢失。该模型可能能够外推到由于创伤性脑损伤引起的远程神经元损伤。在这项研究中，我们评估是否皮质SD增加海马谷氨酸释放SNAP-25，胞吐相关蛋白的免疫组化分析。将15只大鼠分为三组，每组5只：I ;对照; II ; SD诱导和单侧颈动脉闭塞（UO）3小时; III ; SD和UO 7天。处理大脑用于SNAP-25和MAP 2的免疫组织化学分析。组II中的5只动物中有2只显示海马CA 3区的MAP 2耗竭在组II中对应于MAP 2耗尽区域的区域中存在相当强的SNAP-25免疫反应性。五只组III动物中的四只显示海马CA-3中的MAP 2耗尽，但是，没有观察到明显的SNAP-25免疫染色，甚至在MAP 2耗竭区也没有。总之，CA-3中的谷氨酸释放可通过皮质诱导的SD在皮质上持续3小时来增强。皮层诱发SD引起的海马远端神经元损伤可能受到谷氨酸释放的调节。3）扩散性抑郁时谷氨酸的释放扩散性抑郁（SD）似乎在扩大半影区神经元损伤中起关键作用。单独的SD不会引起神经元损伤，因此检查了可能将SD转化为有害刺激的补充因素。将11只雄性Fischer大鼠麻醉并在立体定位框架中机械通气。激光多普勒流量计，将一个用于测量谷氨酸的电酶电极和一个用于测量DC电位的甘汞电极通过位于第二个窗口3 mm处的颅骨窗口放置，在第二个窗口处放置KCl浸泡的棉花以引发SD。结扎左侧颈动脉以抑制SD的反应性充血。电酶电极连续监测被L-氧化的细胞外谷氨酸。谷氨酸氧化酶通过电极灌注。谷氨酸水平在SD开始时立即从34（SY+-[）24 mM/L增加到颈动脉阻断前的48（SY+-[）47 mM/L（p<0.05），从41（SY+-[）25 mM/L增加到颈动脉结扎后的53（SY+-[）41 mM/L（p<0.05），当DC电位恢复时恢复到对照值。SD持续时间与反应性充血抑制率无明显正相关，但与谷氨酸浓度呈正相关（p<0.05）。总之，高细胞外谷氨酸水平伴随SD延长。缺血半暗带SD延长可能是由高细胞外谷氨酸引起的。少

Our research results are divided into three parts ; 1) MAP2 depletion in the hippocampus following spreading depression 2) SNAP-25 expression after repeated induction of spreading depression 3) glutamate release during spreading depression1) MAP2 depletion in the hippocampus following spreading depressionTraumatic brain injury induces neuronal cell loss in area CA3 of the hippocampus. However, it has not yet been established why trauma of the cortex induces neuroral damage in a remote area. Spreading depression (SD) may be one potential mechanism for this pathophysiology. The present study evaluated whether SD on the cortex evokes a pathological change in the hippocampus. Forty-two Fisher rats were assigned to four groups : Group I : sham operation (n=7), Group II : right carotid occlusion (UO) for 7days. (n=7), GroupIII : repeated induction of SD by KCl application on dura for 7 days (n=7), GroupIII'for 3hours (n=7), Group IV : SD induction and UO for seven days (n=14) GroupIV' for 3h … More ours (n=7) In five out of 7 animals in GroupsIII' and IV', cerebral blood flow was monitored using laser Doppler flowmetry for 3h during the passage of SD.The brains were pocessed for immunohistochemical analysis of microtubule-associated protein 2 (MAP2). Reactive hyperemia induced by SD was not significantly suppressed by right carotid occlusion (194(]SY+-[))25% and 181(]SY+-[))42% UO in Groups III and IV,respectively). In six out of seven animals in 7 days model of Group IV,and three animals in 7 days model of Group III,MAP2 depletion in the CA3 area of the hippocampus (partly including CA2) was observed, although no change in the hippocampus was observed in other groups. In conclution, SD in combination with UO yielded reproducible lesions in CA3. Neuronal injury in the hippocampus after brain trauma may be attributable to SD in combination with the blood flow restriction.2) SNAP-25 expression after repeated induction of spreading depressionRepeated induction of cortical spreading depression (SD) elicits neuronal cell loss in area CA3 of the hippocampus, when the blood flow is restricted by unilateral carotid occlusion. This model may be able to extrapolated to remote neuronal injury due to traumatic brain injury. In this study, we evaluated whether cortical SD augmented hippocampal glutamate release by immunohistochemical analysis of SNAP-25, an exocytosis-related protein. Fifteen rats were assigned to three groups of five : I ; control ; II ; SD induction and unilateral carotid artery occlusion (UO) for 3h ; III ; SD and UO for seven days. The brains were processed for immunohistochemical analysis of SNAP-25 and MAP2. Two of the five animals in Group II showed MAP2 depletion in the CA3 area of the hippocampus (including partly of CA2), was observed, whereas hippocampal changes were observed in Group I.Fairly strong SNAP-25 immunoreactivity present, in the area corresponding to the MAP2 depleted region in Group II.Four of the five Group III animals showed MAP2 depletion in CA-3 of the hippocampus, but, no distinct SNAP-25 immunostaining was observed, not even in the MAP2 depletion area. In conclusion, glutamate release in CA-3 may be augmented by cortically induced SD on the cortex for three hours. Remote neuronal injury in the hippocampus resulting from cortically induced SD may be modulated by glutamate release.3) glutamate release during spreading depressionSpreading depression (SD) seems to play a key role in extending neuronal damage in the penumbra. SD alone does not elicit neuronal damage, so supplemental factors which might turn SD into a harmful stimulus were examined. Eleven male Fischer rats were anesthetized and mechanically ventilated in a stereotaxic frame. A laser Doppler flowmeter, an electroenzymatic electrode for measuring glutamate and a calomel electrode for measuring DC potential were placed through a cranial window positioned 3 mm away from a second window where KCl soaked cotton was placed to initiate SD.The left carotid artery was ligated to suppress reactive hyperemia of SD.The electroenzymatic electrode continuously monitored extracellular glutamate which was oxidized by L-glutamate oxidase perfused through the electrode. Glutamate levels increased immediately on initiation of SD from 34(]SY+-[)24 mM/L to 48(]SY+-[)47 mM/L (p<0.05) before carotid occlusion, from 41(]SY+-[)25 mM/L to 53(]SY+-[)41 mM/L (p<0.05) after ligation of the carotid artery, returned to the control value when the DC potential recovered. There was no positive correlation between the duration of SD and suppression of reactive hyperemia, however, a positive correlation (p<0.05) was found between the duration of SD and the glutamate concentration. In conclusion, high extracellular glutamate levels accompany prolonged SD.Prolonged SD in ischemic penumbra may be caused by high extracelluar glutamate. Less