氯化锂-匹罗卡品致痫大鼠海马电压门控性Ⅰ型钠通道的变化

Change in hippocampal voltage-gated Ⅰ sodium channel of Lithium chloride-Pilocarpine epileptic rat model

目的研究氯化锂-匹罗卡品癫痫模型大鼠海马Ⅰ型电压门控性钠通道α亚基蛋白(Nav1.1)及其钠通道功能的变化。方法建立氯化锂-匹罗卡品癫痫大鼠模型。采用免疫组织化学染色法(IHC)检测实验大鼠海马Nav1.1的表达;采用全细胞膜片钳技术检测钠通道功能(电流-电压曲线、激活曲线、失活曲线及失活后恢复曲线)的变化。结果1.成功复制氯化锂-匹罗卡品大鼠模型。观察到实验大鼠行为学3期(急性期、潜伏期和慢性期)的表现,而空白组未见发作。2.IHC结果:致痫大鼠海马CA1区和DG区神经元结构基本正常,且Nav1.1的表达变化不明显。在CA3区,神经元变性、坏死明显,Nav1.1在神经元变性、坏死部位染色变浅,甚至消失;在变性、坏死神经元周围的正常组织中染色增强,与空白组比较,模型组大鼠Nav1.1的表达增高(0.235±0.008比0.210±0.002),差异有统计学意义(t′=-7.426,P<0.05)。3.全细胞膜片钳技术记录钠电流结果:与空白组比较,模型组的钠电流密度明显增加[(-319.70±28.24)pA/pF比(-229.06±26.01)pA/pF,t=8.178,P<0.05]、激活曲线阈值下降(4.15±0.80比4.50±0.85,t=11.020,P<0.05)、失活曲线阈值上升(7.47±0.53比6.24±0.31,t=6.940,P<0.05)、失活后恢复时间缩短[(1.36±0.15)ms比(1.86±0.21)ms,t=6.712,P<0.05],差异均有统计学意义。结论反复癫痫发作可以导致Nav1.1代偿性表达增多,钠通道电流密度明显增高,而激活曲线阈值下降、失活曲线阈值上升、失活后恢复时间缩短,最终引起大鼠神经元兴奋性增高,更易引起癫痫发作。

Objective To research the changes in hippocampal voltage-gated sodium channel of Lithium chloride-Pilocarpine epileptic rat models, includingⅠsodium channel α subunit protein (Nav1.1), mRNA of Ⅰso-dium channel alpha subunit protein gene and function of sodium channel. Methods Epileptic rat models of Lithium chloride-Pilocarpine were established.Nav1.1 expression in the hippocampus of experimental rats was detected by immunohistochemical staining method, and the changes in voltage-gated sodium channel function (the current-voltage curves, activation and inactivation curves and the recovery curve) of hippocampus nerve cells were detected by whole-cell patch-clamp technique. Results (1)The Lithium chloride-Pilocarpine rat models were successfully reproduced.Three stages of behavior (acute, latent and chronic) of rat models were observed.The blank control group was free of seizure.(2)Immunohistochemistry results: neurons in CA1 and DG regions of hippocampal of epileptic rats were normal, and there was no obvious change in the expression of Nav1.1.In CA3 area, the degeneration and necrosis of neurons were obvious.Staining of Nav1.1 became superficial and even disappeared in these areas, but the normal tissues were enhanced around degenerative and necrotic neurons.Compared with the blank control group, the expression of Nav1.1 in the model group was higher(0.235±0.008 vs.0.210±0.002), and there was statistically significant diffe-rence (t′=-7.426, P<0.05). (3) The whole-cell patch-clamp technique showed that the sodium current density of the model group increased significantly compared with that of the blank group [(-319.70±28.24) pA/pF vs. (-229.06±26.01) pA/pF, t=8.178, P<0.05], the threshold value of activation curve decreased (4.15±0.80 vs. 4.50±0.85, t=11.020, P<0.05), the threshold value of inactivation curve increased (7.47±0.53 vs. 6.24±0.31, t=6.940, P<0.05), and the recovery time after inactivation shortened [(1.36±0.15) ms vs. (1.86±0.21) ms, t=6.712, P<0.05], and there were all statistically significant differences. Conclusion Repeated seizures can lead to increase Nav1.1 compensatory expression of, and significantly increase sodium channel current density, while the threshold value of activation curve decreases, the threshold value of inactivation curve rises, and the recovery time after inactivation is shortened, which eventually leads to increased neuron excitability and is more likely to cause seizures.