Temporal analysis of P15 hippocampus in kainate-induced seizures. Koh-2K08NS002068-04

Early childhood convulsions have been correlated with hippocampal neuron loss in patients with intractable temporal lobe epilepsy. Using a "two-hit" rat seizure model, we have shown that animals subjected to kainate (KA)- or hypoxia-induced seizures during early postnatal period showed no cell death, yet sustained more extensive neuronal death after second seizures in adulthood. An early life seizure, without causing overt cellular injury, predisposes the brain to the damaging effect of seizures in later life. Cellular and molecular changes that accompany early seizures and that lead to subsequent epileptogenesis and increased susceptibility to seizure-induced neuronal injury, however, remain poorly understood. We propose to investigate age-specific, time-dependent changes in gene expression that may underlie this priming effect of early-life seizures. We will determine the sequence of gene expression pattern in the hippocampus at various times following KA induced seizures at postnatal day (P) 15. Previous studies have shown that AMPA receptor subtype of glutamate receptors play a crucial role in the age-specific vulnerability and in the long-term epileptogenic effects of perinatal hypoxia seizures. We found that AMPA receptor antagonists block the increased susceptibility caused by early life seizures to later seizures and seizure-induced brain damage. We hypothesize that an alteration of AMPA receptor composition is one of many changes caused by early-life seizures that leads to an increase in Ca2+ permeability, which then results in cascade of downstream events and modifies array of gene expression that promote epileptogenesis and susceptibility to neuronal death in later life. We will examine three time points: 1hr, 72 hr, and 15 days following systemic KA-induced seizures at P15 as we have previously observed structural changes within the hippocampus at these time points. Within an hour of KA seizures, a marked swelling of dendrites, disassembly of dendritic microtubules and glycogen depletion are observed by electron microscopy. Within 5 days, basal dendrites of CA3 hippocampal pyramidal neurons show abnormal spine morphology and decreased branching pattern. 15 days after the seizures, aberrant growth of mossy fibers in the CA3 stratum oriens is observed in animals exposed to KA. Ten hippocampi will be pooled from five animals treated with KA (3mg/kg i.p.) and from five littermate controls injected with PBS. Animals will be decapitated and hippocampi will be rapidly dissected from the brain, flash frozen in liquid nitrogen, and stored at -80C until extraction of total RNA, which will be sent to the center. We will provide 4 tissue samples-2 controls and 2 KA, each a pool of five animals - for each time points. Mixing tissues from multiple rats will normalize single nucleotide polymorphisms and tissue heterogeneity. Keywords: time-course

儿童早期惊厥与难治性颞叶癫痫患者的海马神经元丢失存在相关性。本研究采用「二次打击」大鼠癫痫模型（two-hit rat seizure model），已证实：出生后早期经红藻氨酸（kainate, KA）或缺氧诱导惊厥的大鼠，初始惊厥未引发细胞死亡，但在成年期再次发作惊厥后，会出现更广泛的神经元死亡。可见，早期惊厥虽未造成明显细胞损伤，却会使大脑在后续生命阶段更易受到惊厥的损伤作用。然而，伴随早期惊厥发生、并最终导致后续癫痫发生及惊厥诱导神经元损伤易感性升高的细胞与分子机制，目前仍未被充分阐明。
本研究拟探究可能介导早期惊厥致敏效应的年龄特异性、时间依赖性基因表达变化。我们将明确出生后第15天（postnatal day, P15）经KA诱导惊厥后，不同时间点海马组织的基因表达模式时序。既往研究表明，谷氨酸受体的AMPA受体亚型（AMPA receptor subtype of glutamate receptors）在围产期缺氧惊厥的年龄特异性易感性及远期致痫效应中发挥关键作用。本团队已证实，AMPA受体拮抗剂可阻断早期惊厥所引发的后期惊厥易感性升高及惊厥诱导的脑损伤。
我们提出如下假说：AMPA受体组成改变是早期惊厥引发的诸多变化之一，该改变会提升钙离子（Ca²+）通透性，进而触发下游级联反应，并调控一系列促进癫痫发生及后期神经元死亡易感性的基因表达谱。
本研究将选取P15大鼠经全身性KA诱导惊厥后的3个时间点：1小时、72小时及15天——我们前期已观察到海马组织在这些时间点存在结构变化：惊厥后1小时内，电子显微镜可观察到树突显著肿胀、树突微管解聚及糖原耗竭；惊厥后5天内，海马CA3区锥体神经元的基底树突出现异常棘突形态及分支减少；惊厥后15天，KA暴露大鼠可见海马CA3区或层（CA3 stratum oriens）内苔藓纤维异常增生。
本研究将分别从5只KA处理组（3mg/kg 腹腔注射（intraperitoneal, i.p.））及5只同窝对照（注射磷酸盐缓冲液（phosphate-buffered saline, PBS））大鼠中收集海马组织并混合。大鼠将被断头处死，快速剥离海马组织，经液氮速冻后保存于-80℃，直至进行总RNA提取，随后送至测序中心。每个时间点我们将提供4份组织样本：2份对照样本与2份KA处理样本，每份样本均为5只大鼠的混合组织。混合多只大鼠的组织可归一化单核苷酸多态性（single nucleotide polymorphisms）及组织异质性。
关键词：时间进程（time-course）