【作者】 金融冰；

【导师】 隋鸿锦；

【作者基本信息】 大连医科大学 ， 人体解剖与组织胚胎学， 2004， 硕士

【摘要】 癫痫(Epilepsy)是最常见的神经系统疾病之一,是由于脑细胞的异常放电所引起的反复性惊厥发作。患者除临床发作症状外,常伴有学习记忆能力减退的表现。癫痫发作可对海马结构造成损伤,而海马结构被认为是调节行为和学习记忆的重要神经解剖基础。海马内神经元数量的改变和突触可塑性的变化是影响学习记忆的重要因素。Morris 水迷宫是用来测定大鼠的定位航行和空间搜索能力的测量精度较好的方法,已广泛用来检测大鼠的学习记忆(尤其是空间记忆)能力。GFAP是星形胶质细胞的特异性标记物,其表达的增加反映了星形胶质细胞的增生。p38是一种突触前囊泡膜上的膜整合糖蛋白,可以准确反映突触的分布和密度。本文用KA诱发大鼠急性癫痫发作;采用Morris水迷宫进行行为学试验;应用硫堇染色和免疫组织化学染色技术,结合HPIAS病理图文分析系统,对NS组和KA组大鼠的海马内锥体细胞、GFAP和p38免疫反应进行观察、分析,从而为探讨癫痫所致的学习记忆障碍的机制提供形态学依据。1)水迷宫试验结果显示:在水迷宫的定位航行试验的4天学习期间,KA组大鼠的逃避潜伏期的差异无显著性。而NS组大鼠逃避潜伏期依次减少,差异有显著性(P<0.01);在空间搜索试验中,KA组8只大鼠只有1只穿过平台区一次。而NS组8只大鼠中有6只共穿过平<WP=5>台区14次。平台象限和相对象限内游泳搜索时间的比较结果也表明,KA组大鼠与NS组大鼠之间差异有显著性(P<0.05)。2)组织切片的观察结果:①NS组海马结构内锥体细胞层染色清晰,细胞排列整齐,条带完整;KA组CA1锥体细胞层硫堇染色变浅,细胞减少,条带不完整;②NS组切片海马结构内,GFAP反应阳性的星形胶质细胞数量少、散在分布、胞体较小、突起呈细丝状;在KA组切片中,齿状回门区、CA3和CA1区都出现了大量的肥大的GFAP反应阳性的星形胶质细胞,CA1区尤其严重,其全层出现了大量胞体肿胀分支粗大浓染的星形胶质细胞。有的区域还形成了胶质瘢痕。③KA组透明层p38免疫组化染色明显深于NS组,粗大颗粒的密度和着色深度都有增加。经统计学分析,KA组COD值的增加有统计学意义(P<0.05)。实验结论:KA急性致痫4周后,大鼠的学习和记忆能力下降。这种学习和记忆能力的下降可能与KA诱发急性癫痫发作后大鼠海马内CA1锥体细胞大量缺失以及胶质细胞增生有关。KA后4周学习记忆障碍大鼠的CA3透明层p38免疫反应COD值增加,提示此区出现新生突触。更多还原

【Abstract】 Epilepsy is one of the commonest nervous system diseases. It is a group of temporarily mal-functional chronic disorders which results from repeated discharges of neurons in central nervous system, and which is characterized by spontaneously recurrent and refractory seizures. Clinically, the seizures of the patients are often accompanied with decline of learning and memory abilities. It has been proved by many researches that seizures destroyed the hippocampal formation, which is an important neuroanatomical basis for adjusting behavior and learning and memory. The changes of the number of the hippocampal neurons and the synaptic plasticity are important factors influencing learning and memory abilities.Morris water maze is a precise method for detecting the abilities of place navigation and spatial probe, which has been widely used for examining the abilities of learning and memory (special spatial memory ability). Glial fibrillary acidic protein (GFAP) is a specific marker for astrocyte. The increased expression of GFAP indirectly reflects the proliferation of the astrocytes. Synaptophysin (p38), an integral membrane glycoprotein of presynaptic vesicles, has been widely used to research synaptogenesis in both animal models and human patients and reflect the distribution and density of the synapses. <WP=7>In this study, the acute epileptic seizures of rats were induced by subcutaneous injection of a convulsive dose of KA. Morris water maze was used to detect learning and memory abilities of the rats. Thionine staining, GFAP and p38 immunohistochemistry staining and HPLAS pathology image analysis system were used to demonstrate the loss of pyramidal cells and the difference of the immunoreactive products of GFAP and p38 in the hippocampus between the KA group and NS group. The purpose of present study is to supply the morphological evidence for the mechanisms that the seizures cause the learning and memory deficits. 1)The MWM results showed: In place navigation test, there were no significant differences among the escape latencies of the 4 study days in the KA group. However, significant differences were found among the escape latencies of the 4 days in the NS group(P<0.01) and the escape latency reduced according to the increase of the study time. In spatial probe test, only one of the eight rats in the KA group crossed the flat once, while the six of the eight rats in the NS group rats crossed the flat 14 times in all. The probe time in the flat quadrant and the opposite quadrant were significantly different between the NS group and KA group(P<0.05). 2)The results of tissue slices showed: ① The thionine staining displayed that the hippocampal pyramidal stratum in the NS group was stained clearly and arranged orderly, moreover, the stratum was rather integrated. However, the staining of CA1 pyramidal cells in the KA group was weakened and the number of the cells decreased, in addition, the stratum was not integrated. ②The GFAP-immunohistochemistry stainting displayed that a few GFAP-immunoreactivity (IR) positive astrocytes scattered all over the slices which had small bodies and protruded many filamentous processes in the NS group, whereas, large numbers of proliferative GFAP-IR positive astrocytes appeared in dentate hilus, CA3 and CA1 in the KA group. The proliferation of the astrocytes in CA1 was the most distinct. There were so many proliferative astrocytes whose bodies and processes were tumid and <WP=8>strongly stained that some collagenous scars were formed in this area. ③ Compared with the NS group, the staining of p38-IR in the stratum lucidum of the CA3 in the KA group was deeper and the density of the big granules were increased. The COD of p38-IR in the stratum lucidum of the KA group was increased significantly than that of the NS group(P<0.05). The results suggest that: (1) the learning and memory abilities of the rats decline 4 weeks after KA-induced acute epileptic seizures. (2) This decline is perhaps associated with the loss of pyramidal cells and the proliferation of the astrocyte更多还原