【作者】 树海峰；

【导师】 杨辉；

【作者基本信息】 第三军医大学 ， 外科学， 2010， 博士

【摘要】 皮质发育障碍(malformations of cortical development, MCDs)是小儿难治性癫痫的主要病因之一。随着临床诊断技术的不断发展,尤其是高分辨率核磁共振成像技术的出现,MCDs的诊断率逐年上升,最新资料显示大约有40%难治性癫痫是由MCDs引起的。因此,关于MCDs致病机制的研究成为人们关注的重点。MCDs的临床分型较为复杂,根据最新分类标准,局灶性皮质发育不良(focal cortical dysplasia,FCD)是MCDs中的常见类型之一。大量的研究证实,FCD属于神经细胞迁移异常类疾病(neuronal migration disorders, NMDs)。在皮层发育的后期阶段,多种外源性刺激因素引起新生神经元的异常迁移可能是引起FCD的原因之一。另外,临床研究也证实,在FCD的致痫病灶中存在一些与癫痫发作程度正相关的“异构神经元”,如:气球样细胞(balloon cells,BCs)。最近的研究发现,BCs是由室管膜下区(subventricular zone,SVZ)的神经前体细胞(Neural precursor cells,NPCs)异常迁移分化而来。由此可见,SVZ区NPCs的异常迁移与FCD的致病机制或者致痫过程密切相关。因此,针对性地研究SVZ区NPCs向FCD病灶中的迁移、分化过程以及FCD病灶中SVZ来源细胞的分子生物学特性,将有助于人们进一步认识和阐明MCDs的致痫机制。在本研究中,我们利用建立的FCD动物模型,结合使用荧光分子探针CM-DiI标记NPCs在体(in vivo)示踪技术,系统观察了SVZ区NPCs向FCD皮层病灶迁移、分化的过程。并且,利用膜片钳记录等神经电生理技术手段,研究了不同发育阶段FCD皮层损伤灶中SVZ区NPCs源性神经元的电生理特性。在动物模型的研究基础上,我们进一步以MCDs临床手术切除的标本为研究对象,初步研究了FCD和TSC皮层病灶中SVZ区NPCs来源的“异构神经元”的分子表型。主要结果如下:一、SVZ区NPCs参与皮质发育障碍致痫机制的动物实验研究1.联合利用局部皮层冰冻损伤和CM-DiI侧脑室注射的方法,我们建立了“DiI标记SVZ区NPCs—局灶性皮质发育不良”动物模型(DiI-FCD模型)。结果显示,DiI-FCD模型冰冻损伤灶内可见DiI(+)细胞形成的细胞迁移流(SFMS)。而且,在P10到P90各个阶段,DiI(+)细胞形成的SFMS一直存在。2.利用免疫荧光双标技术,我们对DiI-FCD模型SFMS中的DiI(+)细胞进行了进一步鉴定。结果显示,P10模型SFMS中DiI(+)不同程度地表现为Nestin、DCX以及OTX1/2阳性。这提示,局部皮层冰冻损伤激活了内源性SVZ区NPCs的迁移,DiI-FCD模型皮质损伤灶—微脑回畸形结构中的DiI(+)细胞主要来源于SVZ区NPCs的迁移分化。进一步的研究显示,在微脑回畸形中,大部分DiI(+)细胞分化为胶质细胞,参与局部胶质增生的形成。但仍有一部分DiI(+)细胞分化为神经元,并与周围的细胞形成了突触联系,整合入局部的神经环路。3.电生理的研究结果显示:①从SVZ迁移至微脑回畸形结构中的DiI(+)神经元表现为幼稚神经元的电生理特性,提示其比正常皮层神经元发育较迟缓。然而, DiI(+)神经元比正常皮层神经元兴奋性更高,成熟的DiI(+)神经元(P90)兴奋性增高更显著;②对单个动作电位的分析显示,DiI(+)神经元动作电位的去极化和复极化过程均较正常神经元快,而且,大多数DiI(+)神经元的AHP幅值绝对值显著增大;③DiI(+)神经元的sEPSC的幅值除在P10阶段高于正常神经元外,其他阶段(P30-P90)与正常皮层神经元差异不大,但sEPSC的频率在幼年至成年的各个时期均较正常皮层神经元增高;④DiI(+)神经元上记录的eEPSC主要是NMDA受体介导的突触后电流。与正常皮层神经元相比,DiI(+)神经元上eEPSC峰值略高,但没有达到统计学差异。而且,其10-90% rise time和decay time均较正常神经元有所增高;⑤DiI(+)神经元对突触前细胞的反馈抑制效应较正常皮层神经元降低。二、MCDs致痫灶内SVZ区域NPCs来源的异构神经元分子表型的研究1.利用Western blot的方法,我们进一步研究了P10-P90不同发育阶段DiI-FCD模型微脑回畸形皮层中NMDA/AMPA受体亚单位的表达变化情况,结果显示:在DiI-FCD模型的微脑回畸形中,GluR2,GluR 3蛋白含量在P10-P90各个阶段都呈不同程度的表达上调;NR1和NR2A在P30以后呈表达上调趋势;NR2B蛋白含量除了在P60时期DiI-FCD模型的微脑回畸形中NR2B蛋白含量略低于对照组外,其他时间点都表达上调。此外,我们发现IL-6、IL-6R以及gp130蛋白水平在DiI-FCD模型微脑回畸形皮层中显著表达上调,细胞外给予高浓度IL-6刺激可以引起DiI(+)神经元动作电位发放频率显著增加。这提示:①IL-6可能促进了SVZ区来源的DiI(+)神经元的存活和分化;②微脑回畸形结构中的IL-6,可能参与了FCD病灶高致痫性的形成过程。2.根据在MCDs动物模型上观察到的IL-6及其受体表达上调的现象,我们利用临床MCDs的手术切除标本,进一步研究了IL-6及其受体在两种常见的MCDs疾病类型(TSC和FCD)中的表达和细胞定位。RT-PCR和Western blot的结果分别从mRNA水平和蛋白水平证实,IL-6、IL-6R及gp130在TSC皮质结节和FCDIIb的皮质致痫灶中表达上调。进一步的免疫组化和免疫荧光双标的研究发现,IL-6和IL-6R在TSC皮质结节和FCDIIb的皮质致痫灶中的表达具有明显的细胞特异性,即:IL-6和IL-6R在来源于SVZ区NPCs的“异构神经元”上表达显著增高。这些IL-6/IL-6R阳性的异构神经元,除了FCDIIb中的一些BCs表现为胶质细胞谱系外,大多表现为神经元谱系。这些结果说明,SVZ区NPCs源性的异构神经元可能只MCDs致痫灶中IL-6的细胞来源之一。进一步的研究发现,IL-6信号转导通路gp130/JAK2/STAT3在TSC皮质结节和FCDIIb的皮质致痫灶中普遍被激活。这说明TSC皮质结节和FCDIIb的皮质致痫灶中表达上调的IL-6可能通过gp130/JAK2/STAT3通路发挥其生物学功能。结论如下:1.利用DiI-FCD模型,我们成功地观察到了SVZ区NPCs向微脑回畸形结构内部及其周围皮层迁移的过程,这进一步验证了SVZ区NPCs参与FCD病灶形成的推断。而且,迁移至微脑回畸形局部的NPCs分化为胶质细胞和神经元,并且能和周围细胞形成突触联系,这提示:在MCDs皮层病灶中,SVZ区NPCs来源的细胞具有影响局部神经环路兴奋性的形态学基础;2. SVZ区NPCs来源的神经元具有高兴奋性、接收更多的兴奋性传入以及输出较小的反馈抑制等电生理特性。这提示:SVZ区NPCs来源的神经元兴奋性增高,进而引起整个神经环路兴奋性上调,这些细胞可能是MCDs致痫灶“痫样放电”的“点燃”因素;3. DiI-FCD模型微脑回畸形局部皮层中NMDA/AMPA受体亚单位表达水平的改变,这在一定程度上反应了微脑回畸形局部皮层兴奋性受体分布的失衡状态,进一步支持了上述电生理研究的推断;4.动物实验和临床标本的研究同时证实:MCDs中SVZ区NPCs源性的“异构神经元”不但可以通过自身兴奋性的改变影响局部神经环路的兴奋性,还可能通过释放一些可溶性的蛋白因子(如:IL-6),以旁分泌或者自分泌的方式作用于自身或者其他细胞,进而影响局部皮层的兴奋性。综上所述,多方面的证据都显示:在MCDs致痫病灶中,SVZ区NPCs来源的异位神经元是MCDs癫痫发作的重要原因之一。更多还原

【Abstract】 Malformations of cortical development (MCDs) are increasingly recognized as important causes of medically intractable epilepsy in pediatric patients. With the continuous development of clinical diagnostic techniques, particularly the emergence of high-resolution magnetic resonance imaging, the diagnosis rate of MCDs is increasing, the latest data showed that about 40% of refractory epilepsy is caused by the MCDs. Therefore, the pathogenic mechanism of MCDs become a major concern.Clinical classification of MCDs is more complex, according to the latest classification, focal cortical dysplasia (FCD) are the common types of MCDs. A large number of studies confirmed that FCD is abnormal neuronal migration diseases (NMDs). During the late stage of cortical development, a variety of exogenous stimuli cause the abnormal migration of newborn neurons resulted in FCD sequently. In addition, clinical studies have also indicated that the misshapen cells, such as balloon cells (BCs), within the cortical lesions of FCD are in some degree of positive correlation with the severity of seizure. Recent studies have demonstrated that BCs are derived in part from the neural precursor cells (NPCs) in subventricular zone (SVZ). Accordingly, we speculate that the abnormal migration of SVZ-derived NPCs is closely related to the epileptogenicity of MCDs. Therefore, to observe the the migration and differentiation of SVZ-derived cells in FCD lesions and to study the roles and neurochemical features of misshapen cells within the cortical dysplastic lesions of MCDs will help people better understand and clarify the mechanisms of epileptogenicity of MCDs.In this study, we have established a rat model of a microgyral malformation, and continuously observed the process of the migration and differentiation of SVZ-derived NPCs labelled with CM-DiI (named as“DiI-FCD”model). In addition, by using the patch-clamp recording techniques in the brain slice, we investigated the electrophysiological characteristics of the SVZ-NPCs- derived pyramidal neurons in the microgyral and paramicrogyral regions. Furthermore, the alterations of neurochemical features in the microgyral/ paramicrogyral of the DiI-FCD model and the cortical lesions of MCDs have also been studied in the present study. The main results are as follows:1. Experimental study of the the involvement of SVZ-derived neural precursor cells in the epileptogenicity of malformations of cortical development1.1 Combined use of neonatally cortical freez-lesion and CM-DiI intracerebroventricular injection methods, we successfully established a“DiI labeled SVZ NPCs- focal cortical dysplasia”animal model (DiI-FCD model). The results showed that DiI-positive (DiI+) cells, which form a migratory stream from SVZ to FCD cortical lesion (SFMS), could be continuously observed from P10 to P90.1.2 By using double immunofluorescence techniques, the molecular phenotype of the DiI(+) were further identified. The results showed that the DiI(+) cells in the SFMS of P10 rats expressed Nestin, DCX and OTX1/2, which confirmed that these cells are from SVZ-derived NPCs. Further studies revealed that most of the DiI(+) cells in SFMS differentiate into glial cells, which probably in the formation of local gliosis. However, part of the DiI (+) cells differentiate into neurons, and with the surrounding cells to form a synapse.1.3 Electrophysiological studies showed that: (1) The intrinsic membrane properties of DiI(+) neurons in the microgyral and paramicrogyral regions suggest that these cells are more immature, but higher excitability, than the control neurons from age-matched animals. (2) The depolarization and repolarization process of the DiI(+) neurons shows faster than those of normal neurons, and the absolute value of the AHP amplitude increased significantly in DiI(+) cells. (3) During the P10 phase, the amplitude of spontaneous EPSC (sEPSC) in the DiI(+) neurons are higher than normal neurons; however, during the other stages (P30-P90), there is no significant difference between each group. In addition, the sEPSC frequency in the early years to adulthood are significant higher than normal cortical neurons. (4) Compared with normal cortical neurons, the amplitude of evoked-EPSC (eEPSC) in DiI (+) neurons increased slightly, but did not achieve statistical significance. Moreover, the 10-90% rise time and decay time has increased compared with normal neurons. Interestingly, by analysising the paired-plus eEPSC, the data shows an impairment of feedback inhibition in DiI(+) neurons.2. The molecular phenotype of SVZ-derived neural precursor cells in the malformations of cortical development2.1 Using western blot method, we further studied the expression of NMDA/ AMPA receptor subunits in the microgyral and paramicrogyral regions compared with the control cortex from sham-operated group. The results show that the GluR2 and GluR3 proteins increased in DiI-FCD group in each stage of P10-P90, and NR1 and NR2A were upregulated from P30 to P90. Except the P60 stage, NR2B protein levels are upregulated in the DiI-FCD group. In addition, we also found that IL-6, IL-6R and gp130 proteins in DiI-FCD model was significantly upregulated, extracellular high concentration of IL-6 stimulation can cause DiI (+) neuron spike frequency increased significantly.2.2 According to the data about the upregulation of the IL-6 and its receptors in the microgyral and paramicrogyral regions of DiI-FCD model, we futher studied the expression of IL-6 system in the cortical lesions of TSC and FCDIIb. The results demonstrated that upregulation of mRNA and increased protein levels of IL-6 and its receptors, including the IL-6 receptor (IL-6R) and gp130, were observed in both TSC and FCDIIb lesions. Immunohistochemical analyses indicated that IL-6 and IL-6R were strongly expressed in misshapen cells such as dysmorphic neurons, TS cells and balloon cells (BCs), while gp130 was expressed in almost all cells following a diffuse distribution pattern. Co-localization assays further revealed that most of the IL-6/IL-6R-positive misshapen cells were co-labeled with neuronal markers rather than astrocytic markers. This finding suggests that these cells have a neuronal lineage, with the exception of the IL-6/IL-6R-positive BCs in FCDIIb, the most of which have an astrocytic lineage. Additionally, the protein levels of JAK2 and phosphorylated STAT3 were strongly increased, suggesting the involvement of the gp130/JAK2/STAT3 pathway in IL-6 signal transduction in both TSC and FCDIIb. Soluble IL-6R, but not soluble gp130, was significantly increased in both TSC and FCDIIb lesions relative to CTX, indicating the activation of this trans-signaling pathway in TSC and FCDIIb lesions. These results suggest that overexpression within the IL-6 system and activation of IL-6 signal transduction pathway in TSC and FCDIIb cortical lesions may contribute to intrinsic and increased epileptogenicity.Conclusion:1. Using DiI-FCD model, we successfully observe the migration process of SVZ-derived NPCs to the microgyral/ paramicrogyral regions. The data further confirm the hypothesis that the SVZ-derived NPCs are involved in the formation of the epileptogenic foci of FCD.2. In the microgyral/paramicrogyral regions, the majority of the SVZ-derived NPCs differeniate into glial cells and neurons, and to format synaptic connections with the surround cells,suggesting the possibility of these cells to influence the local neural circuit.3. Electrophysiological studies indicated that the SVZ-NPCs-derived neurons in the the microgyral/paramicrogyral regions showed high excitability and recevied more excitatory input, while the feedback suppression to the presynaptic cells reduced. These data suggested that the high excitability of the SVZ-NPCs-derived neurons would result in the hyperexcitability of the local neural circuit, and consequently trigger the kindling of the epileptiform discharge.4. The alterations of the expression of the NMDA/AMPA receptor subunits suggested the imbalance of the excitatory receptors in the microgyral/paramicrogyral regions, which further supported our electrophysiological finding above.5. The clinical study demonstrated a strong association between overproduction and overexpression in the IL-6 system and MCDs. Our morphological study further indicated the cell-specific expression patterns of IL-6 and its receptors, which were mainly distributed in the DNs, GNs, BCs and TS cells. This finding suggests that these misshapen cells are the potential source of IL-6 in MCDs cortical lesions. Additionally, we showed that the gp130/JAK2/STAT3 signal-transduction pathway might be involved in the response of these cortical lesions to locally high concentrations of IL-6. Our data do not provide insight into the mechanism(s) of IL-6 that underlie the epileptogenic properties of TSC tubers and FCDIIb cortical lesions. However, it is likely that either the strong expression or cell-specific distribution of IL-6 and its receptors, or the activation of IL-6 signal transduction in these cortical lesions, contributes to the intrinsic and high epileptogenicity of MCDs.In summary, a wide range of evidence show that the SVZ–NPCs-derived ectopic neurons in the epileptogenic lesions are one of the possible epileptogenic factors for the epileptogenicity of MCDs.更多还原