Fujimoto S, Katsuki H, Ohnishi M, Takagi M, Kume T, Akaike A
Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.
Neuroscience. 2007 Jan 19;144(2):694-701. doi: 10.1016/j.neuroscience.2006.09.049. Epub 2006 Nov 2.
Intracerebral hemorrhage represents stroke characterized by formation and expansion of hematoma within brain parenchyma. Blood-derived factors released from hematoma are considered to be involved in poor prognosis of this disorder. We previously reported that thrombin, a blood-derived serine protease, induced cytotoxicity in the cerebral cortex and the striatum in organotypic slice cultures, which depended on mitogen-activated protein kinase (MAPK) pathways. Here we investigated the mechanisms of thrombin cytotoxicity in the striatum in vivo. Thrombin microinjected into the striatum of adult rats induced neuronal death and microglial activation around the injection site. Neuronal loss without any sign of nuclear fragmentation was observed as early as 4 h after thrombin injection, which was followed by gradual neuronal death exhibiting nuclear fragmentation. Thrombin-induced damage assessed at 72 h after injection was partially but significantly reduced by concomitant administration of inhibitors of MAPK pathways. Activation of extracellular signal-regulated kinase (ERK) and p38 MAPK in response to thrombin was verified by Western blot analysis. Moreover, phosphorylated ERK and p38 MAPK were localized prominently in reactive microglia, and inhibition of microglial activation by minocycline attenuated thrombin-induced damage, suggesting that reactive microglia were responsible for thrombin-induced neuronal death. Thus, MAPK pathways and microglial activation may serve as therapeutic targets of pathogenic conditions associated with hemorrhagic stroke.
脑出血是一种以脑实质内血肿形成和扩大为特征的中风类型。血肿释放的血液源性因子被认为与该疾病的不良预后有关。我们之前报道过，凝血酶作为一种血液源性丝氨酸蛋白酶，在器官型脑片培养中可诱导大脑皮层和纹状体产生细胞毒性，这一过程依赖于丝裂原活化蛋白激酶（MAPK）信号通路。在此，我们研究了凝血酶在体内纹状体中产生细胞毒性的机制。向成年大鼠纹状体内微量注射凝血酶可诱导注射部位周围的神经元死亡和小胶质细胞活化。凝血酶注射后4小时即可观察到神经元丢失，且无任何核碎裂迹象，随后逐渐出现表现为核碎裂的神经元死亡。注射72小时后评估发现，同时给予MAPK信号通路抑制剂可部分但显著减轻凝血酶诱导的损伤。通过蛋白质免疫印迹分析证实了凝血酶可激活细胞外信号调节激酶（ERK）和p38 MAPK。此外，磷酸化的ERK和p38 MAPK主要定位于反应性小胶质细胞中，而米诺环素抑制小胶质细胞活化可减轻凝血酶诱导的损伤，这表明反应性小胶质细胞是凝血酶诱导神经元死亡的原因。因此，MAPK信号通路和小胶质细胞活化可能是出血性中风相关致病条件的治疗靶点。
