Dentate neurogenesis and neural maturation in animal models of epilepsy

【目的】神经元再生被认为是由缺血或癫痫发作的刺激诱导的，并补偿这些损伤引起的神经元坏死或凋亡。也有人说神经元再生是海马癫痫发生的一个原因，尽管对新形成的神经元的周转机制或意义尚不清楚。采用免疫染色和电生理（膜片钳）技术观察匹罗卡平致癫痫发作动物模型中海马神经发生随时间的变化。观察海马神经发生在癫痫发生中的作用。【方法】C57/BL小鼠腹腔注射匹罗卡品诱导癫痫发作48 h后，用逆转录病毒向海马区注射绿色荧光蛋白（GFP）。因此，癫痫发作诱导的新神经被GFP标记。分别于癫痫持续状态后7天（1周）、14天（2周）、28天（4周）、6个月进行免疫染色，观察正常神经元的成熟程度（NeuN染色）、DCX、NeuroD、PSA、BrdU及凋亡比例（ssDNA染色）。利用膜片钳直接记录GFP标记细胞的放电模式，观察细胞的兴奋性。【观察结果】癫痫发作后在亚颗粒区产生了许多新细胞，但它们体积小且脆弱，未成熟的gfp阳性细胞几乎没有进入成熟过程。癫痫发作后1 ~ 4周消失。早期消失的gfp阳性细胞ssDNA和NeuN均为阴性。癫痫发作后1-2周，gfp阳性细胞的输入电阻（IRs）为1.2 GΩ-2.0 GΩ，静息膜电位（RMPs）为-55 mV至-70 mV。第4周gfp阳性细胞的IRs为300 MΩ-1000 MΩ， RMPs为-70 mV至-80 mV。在癫痫诱导的新神经元中，较不成熟细胞的膜阻力较高，放电率较低。少

【Purpose】Neuronal regeneration is thought to be induced by stimulation from ischemia or epileptic seizures, as well as to compensate for neuronal necrosis or apoptosis from such insults. It is also said that neuronal regeneration is a cause of hippocampal epileptogenesis, although much is unknown about the turnover mechanism or significance of newly formed neurons. Using an animal model in which epileptic seizures were induced by pilocarpine, changes in hippocampal neurogenesis with time brought about by epilepsy were investigated using immunostaining and electrophysiological (patch-clamp) techniques. The role of hippocampal neurogenesis in epileptogenesis was then examined.【Methods】Forty-eight hours after epileptic seizures were induced by intraperitoneal injection of pilocarpine in C57/BL mice, the hippocampus was injected with a green fluorescent protein (GFP) using a retrovirus. Thus, new nerves induced by the epileptic seizure were marked with GFP. Immunostaining was done 7 days ( … More 1 week), 14 days (2 weeks), and 28 days (4 weeks), 6 months after status epilepticus, and the degree of maturation of normal neurons (NeuN staining), DCX, NeuroD, PSA, BrdU and the proportion of apoptosis (ssDNA staining) were investigated. With the patch-clamp, the firing pattern in the cells marked with GFP was directly recorded to investigate cell excitability.【Observation results】Many new cells were produced in the subgranule zone after the epileptic seizures, but they were small and fragile, and almost none of the immature GFP-positive cells entered the maturation process. They disappeared by 1-4 weeks after the seizure. The GFP-positive cells that disappeared at an early stage were negative for both ssDNA and NeuN. The GFP-positive cells at 1-2 weeks after the seizure had input resistances (IRs) of 1.2 GΩ-2.0 GΩ and resting membrane potentials (RMPs) of -55 mV to -70 mV. The GFP-positive cells at 4 weeks had IRs of 300 MΩ-1000 MΩ and RMPs of -70 mV to -80 mV. The membrane resistance in the new neurons induced by epilepsy was higher in the less mature cells, and they had a low firing rate. Less