【作者】 李灏；

【导师】 徐安定；

【作者基本信息】 暨南大学 ， 神经病学， 2010， 硕士

【摘要】 目的通过观察经侧脑室注射内皮素受体(endothelin receptor, ETR)拮抗剂对匹罗卡品(pilocarpine, Pilo)致痫大鼠急性癫痫发作及海马硬化(hippocampal sclerosis, HS)的影响,进一步了解内源性内皮素(ET)以及ETR亚型在癫痫发生发展中的作用。方法氯化锂(lithum chloride, LiCl)联合匹罗卡品小剂量3次重复腹腔注射建立大鼠急性癫痫发作模型。在腹腔注射匹罗卡品前,通过立体定位在大鼠左侧脑室内分别注射容积均为1.5μL的生理盐水(NS)(空白对照组,9只；模型组,30只)、特异性内皮素A受体拮抗剂(ETAR)BQ123(15nmol,30只)特异性ETBR拮抗剂BQ788(15nmol,30只1、ETA&BR非特异性拮抗剂PD145065(15nmol,30只)。建立模型后,观察大鼠的急性期行为学、海马组织匀浆ET含量及海马的病理学改变。结果1.空白对照组大鼠未见痫性发作,模型组大鼠有17只(56.7%)出现惊厥性痫性发作(convulsive seizure, CS),其中15只(88.2%)出现癫痫持续状态(status epilepticus, SE)。BQ123及PD145065能显著减少匹罗卡品诱导大鼠CS成功率。BQ788组及PD145065组大鼠匹罗卡品诱导SE发生率显著低于模型组。3个ETR拮抗剂均不影响匹罗卡品诱导大鼠惊厥性痫性发作平均潜伏期。2.模型组CS大鼠7 d时间点海马组织匀浆ET含量显著高于对照组及3个ETR拮抗剂预处理组。3.对照组及各组NCS大鼠海马亚区各时间点未出现明显的神经元脱失(neuron loss)。胶质细胞增生(gliosis)与苔藓纤维发芽(mossy fiber sprouting, MFS)。模型组及3个ETR拮抗剂预处理的4组CS大鼠海马CA1、CA3及hilar区在2d、7 d及28 d时间点出现不同程度的神经元脱失与胶质细胞增生。7d时的神经元脱失及胶质细胞增生最严重。CS大鼠CA3区神经元脱失与胶质细胞增生的程度重于CA1,并且海马锥体细胞层胶质细胞增生与神经元脱失相关。28 d时,4组CS大鼠CA3区出现明显的苔藓纤维发芽。海马内ET免疫组化阳性的细胞主要分布在CA区的锥体细胞层,尤以CA3的锥体细胞含量丰富,广泛散在的胶质细胞未见明显的ET表达。结论1.内源性ET可能通过作用于中枢性ETAR诱导惊厥性行为的产生,并且很可能通过激活ETBR参与癫痫泛化发展过程,内源性ET至少部分参与了匹罗卡品诱导大鼠急性癫痫发作的发生及发展过程。CS是匹罗卡品诱导海马硬化的重要因素,ETR拮抗剂预处理不能直接阻断匹罗卡品致痫大鼠海马硬化的发生。2.匹罗卡品致痫大鼠海马组织匀浆中内源性ET在7d时明显升高。海马内ET免疫组化阳性的细胞主要分布在CA区的锥体细胞层,尤以CA3的锥体细胞含量丰富,广泛散在的胶质细胞未见明显的ET表达。更多还原

【Abstract】 ObjectiveTo better understand the role of endogenous endothelin (ET) and endothelin receptor (ETR) subtypes in the initiation and development of acute epileptic seizures, we observed the effects of intracerebroventricular (ICV) administration of endothelin receptor (ETR) antagonists on piloca-rpine induced acute epileptic seizures and hippocampus sclerosis in rats.MethodsRepeated low-dose treatment with lithium chloride combined with pilocarpine to produce acute epileptic seizures in rats. Normal saline (control group,9 rats; pilocarpine group, n= 30), a specific endothelin ETAR antagonist (BQ123,15 nmol, n= 30), a specific endothelin ETBR antagonist (BQ788,15 nmol, n= 30) or a non specific endothelin ETA&BR antagonist (PD145065, 15 nmol, n= 30) was ICV administration into the left lateral ventricle of Sprague-Dawley male rats in a total volume of 1.5μL respectively before intraperitoneal (i.p.) injection of pilocarpine. Rats in control group were treated with NS instead of pilocarpine. All pilocarpine induced behavioral alterations, the ET concentration in tissue homogenate of hippocampus and pathological changes were evaluated.Results1. No seizures were seen in control group. Pilocarpine produced convulsive seizures (CS) in 56.7% animals of pilocarpine group.88.2% of rats that experienced convulsive seizures developed into SE. BQ123 and significantly decreased the incidence of animals that produced convulsive seizures compared to pilocarpine group. The incidence of rats that showed convulsive seizures subsequently developed SE in BQ788 group and PD145065 group were significant lower than that in pilocarpine group. However, the average latency to onset of convulsive seizure remained no significant differences among all the 4 group of animals treated with pilocarpine.2. The ET concentration in hippocampal tissue homogenate of rats with convulsive seizu-res in pilocarpine group was significantly higher than control group and the ETR antagonists pretreated animals at 7 d.3. There is no significant neuron loss, gliosis and mossy fiber sprouting (MFS) in rats of control group and animals with non convulsive seizures (NCS) at each time point. In the CA1、CA3 and hilar subfield, varying degrees of neuron loss and gliosis were observed among 4 groups of animals treated with pilocarpine and developed CS at 7 d and 28 d. The most significant neuron loss and gliosis occur at 7 d time point. It is more severe that the extent of neuron loss and gliosis in CA3 subfield compared to CA1, and gliosis was related to the severity of pyramidal cell loss. The CA3 subfield of all animals developed CS presented significantly MFS at 28 d. The ET immunohistochemistry positive cells mostly located in the pyramidal cell layer, especially the pyramidal cell of CA3 which contained high level of ET. No significant expression of ET was observed in the widespread glial cells.Conclusions1. Endogenous ET may produce convulsive seizures by acting on central ETAR, and stimulation of central ETBR may be involved in the generalizing process of epileptic seizures. Endogenous ET, at least, is partly involved in the initiation and development of acute epileptic seizures. CS is an important factory of hippocampal sclerosis, Pretreatment with ETR antagonists can not directly stop the process of hippocampal sclerosis in the rats with lithum-pilocarpine induced CS.2. Endogenous ET in hippocampal tissue homogenate of rats with lithum-pilocarpine induced CS significantly increases at 7d time point. The ET immunohistochemistry positive cells mostly locate in the pyramidal cell layer of CA subfield, the pyramidal cell of CA3 contains high level of ET. No significant expression of ET is observed in the widespread glial cells.更多还原