【作者】 易芳；

【导师】 肖波；

【作者基本信息】 中南大学 ， 神经病学， 2010， 博士

【摘要】 第一部分：匹罗卡品致痫大鼠海马GABA能中间神经元的动态变化及轴突出芽背景与目的：颞叶癫痫是主要的难治性癫痫类型之一,发病人数日益增多,但其病因未明、发病机制不清、诊断困难、治疗效果差,已逐渐成为危害我国人群公共健康的一种主要疾病。但迄今为止其发病机制仍不明确。目前国内外学者较为公认的是颞叶癫痫发作可能由于海马区和齿状回区兴奋性和抑制性神经环路重组,导致对神经元的兴奋抑制不平衡。苔藓纤维出芽导致的异常兴奋性突触环路研究较多,但它并不足以解释慢性自发性痫性发作的原因；而构成抑制性神经环路的GABA能中间神经元逐渐成为近年来的研究热点。已有的研究发现：痫性发作早期出现部分GABA能中间神经元脱失,后期在海马和齿状回区则存在轴突出芽。但总体而言,国外已有的对GABA能中间神经元的研究范围较片面,结论亦存在较大争议。因此对GABA能中间神经元在颞叶癫痫模型中的早期缺失和后期的轴突出芽还需进一步深入研究。本课题组前期工作中利用免疫组化探讨了SS、NPY两种抑制性中间神经元的变化,本研究在此基础上继续对不同中间神经元的亚类进行深入探讨。方法：1.模型制作与分组：健康雄性SD大鼠(6-8周龄)180只随机分为实验组(A组,n=105)和对照组(B组,n=75),每组又随机分为5个亚组(A1-5亚组,n=21；B1-5亚组,n=15)。A组颞叶癫痫模型制作采用氯化锂加匹罗卡品腹腔注射法；B组大鼠腹腔注射等量无菌生理盐水。根据Racine标准判定癫痫发作的程度。选取痫性发作后1d、7d、15d、30d、60d为研究时间点,海马CA1区、CA3区、齿状回为研究部位。2.不同时间点行尼氏染色、NeuN免疫组化观察海马病理改变。3.免疫组化方法检测各时间点不同区域钙结合蛋白CB、PV阳性中间神经元数目变化及轴突出芽。4.免疫组化方法检测各时间点不同区域神经元NeuN的数量变化,免疫荧光双标记法检测SS/NPY与NeuN的共表达,进一步观察SS、NPY中间神经元及其轴突的动态变化。结果：1.实验组大鼠腹腔注射氯化锂-匹罗卡品后,SE诱发成功率为80%,死亡率为10.7%,模型成功率为71.4%。2.尼氏染色结果显示：实验组海马神经元缺失以SE后7d和60d最为明显。除齿状回颗粒细胞缺失相对较轻外(P<0.05),CA区域锥体细胞和门区神经元均显著减少(P<0.01)。3.CB免疫组化结果显示：阳性神经元CA1区锥体细胞亦部分表达CB,但相对于中间神经元染色很浅。SE后CA1区CB阳性神经元持续下降(P<0.05)。CA3区CB阳性神经元无明显变化。门区CB阳性神经元在1d时降至最低(P<0.05),然后有所恢复。SE15d后,CA1区辐状层出现CB染色的纤维。4.实验组大鼠海马门区PV阳性神经元在SE后早期无明显变化(P>0.05),慢性期时下降(P<0.01)。海马CA1区PV阳性神经元在SE后15d时开始明显增多(P<0.01),可见相应增多的PV阳性纤维；海马CA3区PV阳性神经元在SE后即持续增多,至60d时最多(P<0.01),并可见相应增多的PV阳性纤维。5. NeuN免疫组化显示：SE后门区和CA3区锥体细胞层、CA1区锥体细胞层显著减少(P<0.01)。齿状回外分子层细胞数目无显著变化,齿状回颗粒细胞、CA3区辐状层及腔隙分子层缺失相对较轻(P<0.05)。SE后60d变化最为明显,CA1区锥体细胞基本消失(P<0.01),但在CA1区的起始部位的各层NeuN免疫阳性神经元相对保留,始层和辐状层数目甚至超过正常,神经元形态各异,排列不齐。6.免疫荧光双标结果显示：对照组和实验组各时期均可见双标记的SS中间神经元,在门区7d时减至最低CA1区始层在SE后15d逐渐增多,30d、60d时均超过正常水平,60d时CA1全层可见增多的双标记SS中间神经元；CA3区7d时减至最低,后始层双标记的SS神经元逐渐恢复。NPY变化情况与SS类似,不同之处在于各时段CA3区双标记的NPY阳性神经元数目无明显变化。结论：1.同一类不同中间神经元在海马同一部位存在选择性易损性；同种中间神经元在海马不同区域易损性亦不同。中间神经元在海马的动态变化大致有如下几个特点：1)CA1区致痫后中间神经元数量下降,慢性期有所恢复,甚至超过正常；2)CA3区改变相对不明显；3)门区则下降。2.各亚型GABA能中间神经元均存在不同程度的轴突出芽,尤其是SE后60d海马CA1区全层出现大量增加的SS染色的纤维,可能部分来自于CA1区始层和辐状层增多的神经元,并在海马CA1区临近区域内构成病理性抑制性突触环路,从而在癫痫的发生和慢性期自发发作中发挥重要作用。第二部分：荧光金逆行示踪观察匹罗卡品致痫大鼠慢性期海马神经元的突触重建背景与目的：正常海马结构存在着主细胞(锥体细胞和颗粒细胞)介导的兴奋性回路和非主细胞(GABA能中间神经元)介导的抑制性回路。兴奋性回路被研究得较多,而有关抑制性回路的研究相对较少,以上两种回路的异常可能与颞叶癫痫的发生和自我修复密切相关,具体机制目前仍不清楚。虽然目前已有大量关于兴奋性回路重组的报道,但颞叶癫痫中兴奋性主细胞的突触联系变化仍不太清楚；而有关GABA能抑制性回路重组尤其是颞叶癫痫慢性自发发作期抑制性中间神经元的突触重建的研究则非常有限。神经定位示踪技术可以帮助我们从整体水平系统地观察神经元的突触联系变化,对于中间神经元的定位示踪研究可以深入了解颞叶癫痫中抑制性回路的异常及突触改变。迄今国内外有关颞叶癫痫海马GABA能中间神经元突触联系的神经示踪研究极少。本课题组前期研究发现海马CA1区SS阳性中间神经元之间可能存在异常的抑制性突触联系,本研究即在此基础上,深入探讨海马不同区域及不同亚类中间神经元的突触重建状况,以期明确颞叶癫痫大鼠海马抑制性回路的构成。方法：1.模型制作与分组：健康雄性SD大鼠(6-8周龄)80只随机分为实验组(A组,n=40)和对照组(B组,n=40)。A组颞叶癫痫模型制作采用氯化锂加匹罗卡品腹腔注射法；B组大鼠腹腔注射等量无菌生理盐水。根据Racine标准判定癫痫发作的程度。2.SE后60d左右,利用立体定位仪在活体内注射逆行性示踪剂荧光金(Fluoro Gold, FG)至海马CA1、CA3区,对照组大鼠亦于同一时期注射等量FG至海马同一区域,术后常规喂养7-10d后灌注取材。3.FG免疫组化方法观察致痫后大鼠海马异常兴奋性突触联系。4.激光扫描共聚焦显微镜下观察FG的分布,免疫荧光双标记法检测SS/NPY与FG的共表达情况,观察致痫大鼠海马异常抑制性突触联系。结果：1.实验组40只大鼠经腹腔注射匹罗卡品后,5只致痫失败,4只因抽搐致死,模型成功率77.5%。2.FG免疫组化结果显示：注射区域内可见大量FG免疫阳性神经元,注射区呈内外两带；FG注入大鼠海马CA1区,实验组远离注射部位的CA1区、CA3区锥体细胞被大量标记,海马下托亦发现有FG标记的锥体细胞,对照组未见；始层可见少量FG标记的非主细胞,门区亦可见FG标记的非主细胞,而对照组均未见。FG注入大鼠海马CA3区,对照组及实验组CA3区全层、门区锥体细胞均可见大量FG标记,实验组中CA1区部分锥体细胞被FG标记,对照组未见；对照组齿状回颗粒细胞层部分被标记,而实验组中未见；对照组及实验组门区、CA1区远离注射部位的始层均存在FG标记的非主细胞,对照组1m层偶见非主细胞呈FG阳性。3. SS/NPY与FG免疫荧光双标记染色显示：SS与NPY的荧光双标染色结果类似。FG注入CA1区后,实验组大鼠CA1区远离注射部位处、CA3区、门区均有FG标记的SS/NPY中间神经元,而对照组未见；FG注入海马CA3区,实验组大鼠门区有双标记的中间神经元,对照组未见。结论：1.颞叶癫痫大鼠海马慢性期CA1区锥体细胞之间、下托-CA1区、CA1-CA3区存在异常的兴奋性联系,可能构成兴奋性回路重组,导致海马持久的超兴奋性。2.颞叶癫痫慢性期CA1区中间神经元之间、CA3-CA1区、门区-CA1区、门区-CA3区存在异常的抑制性神经网络。这种与轴突出芽密切相关的回路重组,可能在颞叶癫痫慢性期的自发发作中起重要作用。更多还原

【Abstract】 Background and Objective:Temporal lobe epilepsy(TLE) is one of the most common refractory epilepsy in clinical, but so far the epileptogenesis is still not clear. The enhancement of excitatory circuit and decrease of inhibitory circuit in the hippocampus play an important role in temporal lobe epilepsy. Researches revealed although the foundation of excitatory circuit such as mossy fiber sprouting participated in the generation of temporal lobe epilepsy, it still couldn’t fully explain the reasons of spontaneous seizures. So currently GABAergic interneurons which are deeply associated with hippocampal inhibitory circuit have gradually been the research hotspot. Most studies have showed loss of GABAergic inhibitory interneurons in early period of SE, while some axonal sprouting exist in hippocampus and dentate gyrus in chronic period. But totally the range of research on GABAergic interneurons in temporal lobe epilepsy is incomplete and there are still much great arguments on it, so further studies are needed to be done. We have already observed the dynamic changes and axonal sprouting of SS&NPY by immunohistochemistry technology in our early study, and the aim of this research is to find out transmissions of other subtypes of GABAergic interneurons in temporal lobe epilepsy.Methods:1.180 healthy male SD rats were divided randomly into epilepsy groups(n=105) and control groups(n=75). Two groups were divided randomly into 5 subsets at 1,7,15,30,60d after pilocarpine or normal sodium(NS) intraperitoneal injection. The models of epilepsy were established by intraperitoneal injection of pilocarpine and lithium, while the controls were injected with NS. The degree of seizure was judged according to Racine standard.2. Nissl stain and NeuN immunohistochemistry was used to observe the pathological changes of hippocampus.3. Immunohistochemistry method was used to detect number changes and axonal sprouting of CB\PV positive interneurons in different domains of the hippocampus at different time points.4. Immunohistochemistry method was used to detect number changes of NeuN positive neurons in different domains of the hippocampus at different time points, and coexpression of SS/NPY positive interneurons combined with NeuN was detected by the technique of double immunofluorescence.Results:1. After lithium-chloride and pilocarpine administration,80% rats were induced SE successfully, the mortality rate was 10.7%, and the success rate of the model was 71.4%.2. Nissl stain revealed that in the experimental group, loss of hippocampal neurons was most evident on 7d and 60d after SE. Significant loss of hilar neurons and pyramidal neurons was present in CA area(P<0.01), while loss of granular cells in the dentate gyrus was relatively slight(P<0.05).3. CB expression could also be seen in CA1 pyramidal neurons, but weaker than that of CB interneurons. The number of CB positive neurons in CA1 area decreased after SE(P<0.05), but no obvious change in CA3 area. CB neurons in hilus decreased to minimum on 1d(P<0.05), and recoverd gragrully in chronic phase. On 15d after SE, plenty of CB positive neurophils could be seen in stratum radiatum of CA1 area.4. There was no evident changes of PV positive neurons in early phase in hilus(P>0.05), the number of PV neurons decreased in chronic phase(P<0.01); PV positive neurons in CA1 area increased significantly after 15d after SE(P<0.01), increased neurophils also could be seen; PV neurons in CA3 area increased as early as the phase after SE, and increased to maximum on 60d(P<0.01), correspondingly increased neurophils also could be seen.5. NeuN immunohistochemistry revealed that in the experimental group, significant loss of hilar neurons, pyramidal neurons in CA area was present (P<0.01), while loss of granular cells in the dentate gyrus, neurons in stratum radiatum and lacunosum-moleculare of CA3 was relatively slight(P<0.05). Changes in outer molecular layer of dentate were not notable. On 60d after SE, neurons of pyramidal layer in CA1 almost disappeared(P<0.01), but in initiation site of CA1 area, part of NeuN positive neurons reserved, even beyond normal in stratum oriens and stratum radiatum.6. Double immunofluorescence revealed that both in experimental and control group, double labeled SS interneurons could be seen at each time point. The number of it was least on 7d in hilus; partially recovered on 15d in stratum oriens of CA1,even exceeded the controls in all layers of CA1 on 60d; decreased to minimum on 7d in CA3 and also recovered gragrully in chronic phase. Similar changes could be seen in double labeled NPY interneurons, and differences were that little change happened in CA3 area.Conclusions:1. Different subtypes of interneurons have different sensitivities to injuries induced by seizures in different time points and different domains. On the whole, there are some features of interneurons after SE:a) the number in CA1 decreases in early phase and recovers in chronic phase, even beyond normal; b) not obvious in CA3; c) decreases in hilus.2. Level of axonal sprouting vary from different subtype of interneurons, especially numerous increase of SS positive neurophils within area CA1 in chronic phase may come from increased interneurons in stratum oriens and stratum radiatum of CA1,and form a pathological inhibitory loop to take in part in generation and compensation of temporal lobe epilepsy. Background and Objective:There are trisynaptic loop in normal hippocampal stuctrue, involving excitatory circuits mediated by pyramidal neurons and granule cells and inhibitory circuits by interneurons. Rearrangements of hippocampal excitatory and inhibitory circuits are deeply related to the generation of temporal lobe epilepsy. Nowadays there have been a lot of reports about excitatory circuit rearrangement, but the elucidation of synaptic connections among principal cells in temporal lobe epilepsy remain topics of intensive incestigation. Meanwhile researches about GABAergic inhibitory rearrangement are few, especially studies of synaptic reconstruction among inhibitory interneurons during chronic phase of temporal lobe epilepsy are much more less. Neuroanatomical tracing can help us to observe the synaptic connections through a total level, and aberrant inhibitory circuits will be revealed through interneurons tracing technique. Results of our previous study showed numerous SS positive neurophils present within CA1 area in chronic phase of temporal lobe epilepsy, and in this study, we hoped to use fluorogold(FG) to observe synaptic reconstruction in different subtypes of interneurons in every area of hippocampus during chronic phase, in order to reveal aberrant formation of inhibitory circuit rearrangements in temporal lobe epilepsy.Methods:1.80 healthy male SD rats were divided randomly into epilepsy groups(n=40) and control groups(n=40). The models of epilepsy were established by intraperitoneal injection of pilocarpine and lithium, while the controls were injected with NS. The degree of seizure was judged according to Racine standard.2. On about 60d after SE, we injected retrograde tracer FG into CA1 and CA3 area of the hippocampus in vivo by using the stereotaxic apparatus, the same method was performed in control animals. After surgery, animals were allowed to survive for 7-10 days before perfusion-fixation.3. Immunohistochemistry about FG to observe aberrant excitatory circuit rearrangements.4. Confocal microscopy was used to observe the distribution of FG Double immunofluorescence combined with SS/NPY and FG was performed, to observe aberrant inhibitory circuit rearrangements.Results:1. After lithium-chloride and pilocarpine administration, five animals were failed to induce SE, four were dead because of severe seizures. The success rate of the model was 77.5%.2. FG immunohistochemitry showed abundant FG-labeled neurons at the zone of FG-injected site, and the injection site could be divided into two bands. After injected FG into CA1 area, FG-labeled pyramidal cells could be seen remote from the zone of dye spread in CA1 area, CA3 area, and subiculm, while FG-labeled non-principle neurons could be seen in stratum oriens of CA1 and hilus in experimental group. When injection was taken place in CA3 area, FG-labeled pyramidal cells could be seen in the whole CA3 area and hilus in two groups; additional part of pyramidal cells in CA1 in experimental group; additional part of granule cells in dentate in control group; also additional some FG-labeled non-principle cells could be seen in hilus and remote from the zone of dye spread in CA1 area.3. Double immunofluorescence about FG and SS/NPY revealed that, at the zone of FG-injected site, FG-labeled SS/NPY positive interneurons could be seen in two groups, and there were no significant differences about the ratio of double labeled neurons to SS/NPY total neurons between two groups(P>0.05). As FG injection into CA1, double labeled interneurons could be seen remote from the zone of dye spread in CA1 area, CA3 area and hilus in experimental rats. When injection was taken place in CA3 area, FG-labeled SS/NPY neurons could be seen in hilus in experimental rats.Conclusions:1. Aberrant synaptic connections among pyramidal cells in CA1 area, pyramidal cells between CA1 and subiculum and pyramidal cells between CA1 and CA3 in the hippocampus in chronic phase of temporal lobe epilepsy may form an aberrant excitatory circuit arrangement, which will eventually develop into hyperexcitability in hippocampus.2. Aberrant synaptic connections among dendritic interneurons exist in CA1 area, CA3 to CA1, hilus to CA1, and hilus to CA3 area of the hippocampus in chronic phase of temporal lobe epilepsy. The inhibitory circuit arrangement related to axonal sprouting may play an important role in the generation and compensation of epilepsy.更多还原