新生神经元异常融入齿状回神经环路介导卒中后癫痫发生

发布时间：2018-05-19 10:11

本文选题：卒中后癫痫 + 神经发生　；参考：《华中科技大学》2014年博士论文

【摘要】：[背景] 癫痫是脑卒中后常见的并发症。流行病学调查发现脑卒中后癫痫的发病率超过10%,占新确诊的成年癫痫患者的三分之一；60岁以上新诊断的癫痫患者中,近一半与脑血管病有关。随着生活质量和医疗水平的提高,脑卒中患者存活期延长,脑卒中后癫痫的患病率也随之增加。然而,目前仍然缺乏脑卒中后癫痫有效防治办法,对其发生机制也不甚了解。成年哺乳动物体内神经祖细胞主要位于海马齿状回颗粒下层和侧脑室的室下区,它们能够增殖分化成为神经元。在新生神经元的发育过程中,它们逐渐伸出轴突和树突,与周围的细胞形成功能性突触联系并整合到原有的神经环路中,发挥相应的作用。神经发生过程受多种因素影响。有研究表明脑卒中的新生大鼠成年后认知功能障碍、呈现高度兴奋状态并伴有癫痫发生。还有报道指出,脑卒中促进海马神经祖细胞活化增殖,新生细胞向损伤部位迁移,参与神经功能修复；脑卒中或癫痫后,海马新生神经元形态和分布异常。卒中后新生神经元的异常是否与癫痫的发生发展有关,至今仍是一个谜。 [目的] 观察缺血性脑卒中小鼠齿状回新生神经元的形态和功能以及与癫痫发生的关系。 [方法] 通过大脑中动脉栓塞(MCAO)构建4月龄小鼠脑缺血模型或相应的假手术对照,本实验分别采用了雄性C57BL/6J小鼠、Nestin-Cre小鼠、ChR2小鼠、TeTX小鼠为研究对象。通过磁共振(MRI)和Fluoro-Jade C染色检测小鼠卒中后脑部受损情况；采用逆转录病毒(RV-EGFP)、腺相关病毒(AAV-EGFP)和狂犬病毒(△Rabies-mCherry)感染以及荧光染料显微注射标记新生神经元；使用BrdU染色标记新生细胞；利用RT-PCR方法检测中间神经元、胶质细胞和颗粒细胞的GAD67、GFAP和Calbindin-D28K的mRNA表达水平；通过全细胞膜片钳电生理技术记录新生颗粒神经元与原有颗粒神经元的电生理特征；通过红藻酸氨(KA)诱发急性癫痫发作；通过视频脑电图监测小鼠的癫痫发作情况。 [结果] MCAO小鼠反复自发癫痫发作。4月龄雄性C57BL/6小鼠行MCAO (C57-MCAO小鼠)或假手术(C57-sham小鼠)处理,术后连续3个月记录脑电图和癫痫相关行为,结果显示C57-MCAO小鼠术后1月左右开始出现反复自发癫痫发作现象并伴有脑电图异常。 MCAO小鼠海马新生神经元形态异常。4月龄雄性C57BL/6小鼠行MCAO (C57-MCAO小鼠)或假手术(C57-sham小鼠)处理,海马齿状回注射逆转录病毒(RV-EGFP)标记新生神经元。2周及4周后,观察新生神经元数量和形态,结果显示C57-MCAO小鼠术后GFP+细胞数量显著增加,双极细胞所占比例增加,并出现多个基树突。 MCAO小鼠海马新生神经元突触连接异常。方法一：4月龄雄性C57BL/6小鼠行MCAO (C57-MCAO小鼠)或假手术(C57-sham小鼠)处理,海马齿状回注射逆转录病毒(RV-EGFP)标记新生神经元,术后4周取动物脑片,分别对GFP+细胞和GFP-细胞显微注射绿色和红色生物素染料,发现C57-MCAO小鼠GFP+细胞轴突纤维深入到分子层。方法二：4月龄雄性C57BL/6小鼠行MCAO (C57-MCAO小鼠)或假手术(C57-sham小鼠)处理,腹腔注射BrdU标记新生细胞,海马齿状回注射AAV-EGFP标记所有神经元,3周后同样的部位注射△Rabies-mCherry标记已被AAV感染的神经元及其上级神经元,此后1周取脑片观察齿状回EGFP+/mCherry+、EGFP+/mCherry"和EGFP-/mCherry+细胞的数量、形态和分布,与BrdU是否共定位,结果显示大部分EGFP-/mCherry+细胞是BrdU+细胞。 MCAO小鼠海马新生神经元异常融入齿状回神经环路。方法一：4月龄雄性Nes-Cre小鼠行MCAO手术(Nes-Cre-MCAO小鼠)或假手术(Nes-Cre-sham小鼠),术后1周海马齿状回注射腺相关病毒(AAV-ChR2-EGFP),同时连续5天腹腔注射BrdU;术后4周,进行NeuN和BrdU染色,观察两组动物海马GFP+/NeuN+细胞和GFP+/NeuN+/BrdU+细胞的数量、形态、分布以及NeuN+/BrdU+细胞占总GFP+细胞的比例,结果显示Nes-Cre-MCAO小鼠较Nes-Cre-sham小鼠新生神经元数量增加,两组动物NeuN+/BrdU+细胞数量没有明显变化。方法二：4月龄雄性ChR2小鼠行MCAO手术(ChR2-MCAO小鼠)或假手术(ChR2-sham小鼠),并给予他莫昔芬(TAM)诱导表达ChR2和EGFP,术后4周取脑片记录DG区ChR2+/EGFP+细胞与CA3区以及DG区的ChR2-/EGFP细胞之间的突触联系,结果显示ChR2-MCAO小鼠与ChR2-sham小鼠CA3区ChR2-/EGFP-细胞均可以记录到EPSCs, ChR2-MCAO组小鼠DG区ChR2-/EGFP细胞可以记录到EPSCs,而在ChR2-sham组小鼠则记录不到DG区ChR2-/EGFP细胞的EPSCs。 MCAO小鼠海马新生神经元与原有成熟颗粒细胞形成功能性突触连接。ChR2小鼠持续给予TAM,行MCAO(ChR2+-MCAO小鼠)或假手术(ChR2+-sham小鼠),术后4周,取脑片记录DG区ChR2+/EGFP+细胞与CA3区以及DG区的ChR2-/EGFP细胞之间的突触联系。结果显示ChR2+-MCAO组小鼠和ChR2+-sham小鼠CA3区ChR2-/EGFP-细胞以及ChR2-MCAO组小鼠DG区ChR2-/EGFP细胞可以记录到EPSCs;该EPSCs与蓝光光强呈线性正相关,对AMPA受体拮抗剂敏感。小鼠海马新生神经元中特异性表达TeTX可特异性阻断其异常突触传递,并不影响MCAO小鼠新生神经元存活和电生理特征。4月龄雄性TeTX小鼠行MCAO处理(TeTX-MCAO小鼠),一组动物给予TAM诱导新生神经元中表达TeTX (TeTX+-MCAO小鼠),另外一组动物给予正常对照溶剂(TeTX-MCAO小鼠),4周后,使用Fluoro-Jade C染色了解TeTX对细胞存活的影响,结果表明两组动物F-J+细胞数量没有明显差异；进一步使用电生理记录两组动物GFP+和GFP-细胞的EPSCs和IPSCs,结果显示两组动物的GFP+和GFP细胞的EPSCs之间以及GFP+和GFP-细胞的IPSCs之间没有明显差异。TeTX小鼠行MCAO (TeTX--MCAO小鼠),术后1周海马齿状回注射腺相关病毒(AAV-ChR2),4周后,两组动物分别给予多西环素(DOX)(TeTX+-ChR2+-MCAO-mice)和正常对照溶剂(vehicle)(TeTX--ChR2+-MCAO-mice)1周,然后第一次记录光刺激新生神经元和电刺激苔藓纤维时CA3区和DG区的EPSCsNMDA,然后两组全部更换为正常饮水1周,进行第二次记录(同第一次记录)。结果显示,TeTX--ChR2+-MCAO小鼠可以在CA3区和DG区记录到光刺激和电刺激后的EPSCsNMDA,TeTX+-ChR2+-MCAO小鼠只能在CA3区记录到电刺激后的EPSCsNMDA。特异性阻断海马新生神经元突触传递能降低MCAO小鼠癫痫发作的频率和强度。4月龄雄性TeTX小鼠行MCAO (TeTX-MCAO小鼠)或假手术(TeTX-sham小鼠)处理,3周后开始持续给予多西环素(TeTX+-MCAO/sham小鼠)或对照溶剂(TeTX"-MCAO/sham小鼠),1周后,开始连续观察脑电图和行为学变化。结果显示TeTX-sham小鼠无癫痫发生；TeTX--MCAO小鼠癫痫有发作,并且随着脑缺血恢复时间的延长其发作次数增加；TeTX+-MCAO小鼠癫痫发作次数和单次发作持续时间较TeTX--MCAO小鼠低。我们也将4月龄雄性TeTX小鼠持续给予多西环素(TeTX+小鼠)或对照溶剂(TeTX小鼠),再行MCAO (TeTX+/--MCAO小鼠)或假手术(TeTX+/--sham小鼠)处理,给予10-30mg/kg的红藻氨酸(KA)腹腔注射诱发急性癫痫发作,观察注射KA后各组动物脑电图、癫痫发作情况以及死亡率。结果发现随着KA剂量的增加,各组动物死亡率增加,以TeTX--MCAO小鼠居多；TeTX+/--sham小鼠和TeTX+-MCAO小鼠脑电图基本正常；TeTX--MCAO小鼠脑电图波形出现明显异常,癫痫指数高于其他组小鼠。进一步使用TeTX小鼠行MCAO (TeTX-MCAO小鼠)或假手术(TeTX-sham小鼠),术后4周开始持续记录两组动物(TeTX--MCAO/sham小鼠)癫痫发作情况4周(第一次记录,R1),然后两组动物持续5周给予多西环素,记录后四周两组动物(TeTX+-MCAO/sham小鼠)癫痫发作情况(第二次记录,R2),然后两组动物持续5周给予正常饮水,记录后四周两组动物(TeTX--MCAO/sham小鼠)癫痫发作情况(第三次记录,R3)。结果发现,整个实验期间,TeTX+/--sham小鼠几乎没有癫痫发作；对于TeTX-MCAO小鼠,R1时,其癫痫发作强度频率均明显高于假手术组小鼠,R2时,癫痫发作强度频率较R1有明显下降,R3时,癫痫发作强度频率恢复到R1时的水平。 [结论] 新生神经元异常整合到原有的神经环路是卒中后癫痫反复发作的细胞基础,阻断该异常突触连接可以降低MCAO小鼠癫痫发生频率和强度。该研究结果为卒中后癫痫的干预和治疗提供了新的思路。
[Abstract]:[background]
Epilepsy is a common complication after stroke. Epidemiological investigation has found that the incidence of epilepsy after stroke is more than 10%, accounting for 1/3 of the newly diagnosed adult epileptic patients; nearly half of the newly diagnosed epilepsy patients over 60 years old are related to cerebrovascular disease. With the improvement of quality of life and medical treatment, the survival period of stroke patients is prolonged, The incidence of epilepsy after stroke is also increasing. However, there is still a lack of effective prevention and control of epilepsy after stroke, and it is not well understood. The neural progenitor cells in adult mammals are mainly located in the subgranular layer of the dentate gyrus and the subventricular zone in the lateral ventricle, and they can proliferate and differentiate into neurons. During the development of the cells, they gradually extend the axons and dendrites to form functional synapses with the surrounding cells and integrate them into the original nerve loop and play a corresponding role. The process of neurogenesis is influenced by many factors. It is also reported that cerebral apoplexy promotes the activation and proliferation of hippocampal neural progenitor cells, the migration of new cells to the injured part and the repair of neural function. After stroke or epilepsy, the morphology and distribution of newborn neurons in the hippocampus are abnormal. A mystery.
[Objective]
To observe the morphology and function of newborn neurons in dentate gyrus of ischemic stroke and the relationship with the occurrence of epilepsy.
[method]
4 month old mice model of cerebral ischemia or corresponding sham control was constructed by middle cerebral artery embolism (MCAO). Male C57BL/6J mice, Nestin-Cre mice, ChR2 mice and TeTX mice were used to test the brain damage in mice after stroke by magnetic resonance (MRI) and Fluoro-Jade C, and retrovirus was used. (RV-EGFP) infection of adeno-associated virus (AAV-EGFP) and rabies virus (delta Rabies-mCherry) and microinjection of fluorescent dyes for newborn neurons; BrdU staining was used to mark new cells; GAD67, GFAP and Calbindin-D28K in the middle neurons, glia and granulosa cells were detected by RT-PCR. Electrophysiological characteristics of neonatal granular neurons and original granular neurons were recorded by cell patch clamp electrophysiology; acute epileptic seizures were induced by KA, and the epileptic seizures of mice were monitored by video electroencephalogram.
[results]
The.4 month old male C57BL/6 mice of MCAO mice were treated with MCAO (C57-MCAO) or sham operation (C57-sham mice). Electroencephalogram and epilepsy related behavior were recorded for 3 months after operation. The results showed that the spontaneous epileptic seizures and abnormal EEG occurred in the C57-MCAO mice around January.
The morphological abnormalities of hippocampal neurons in MCAO mice were.4 month old male C57BL/6 mice treated with MCAO (C57-MCAO mice) or sham operation (C57-sham mice). Hippocampal dentate gyrus injection retrovirus (RV-EGFP) was used to mark newborn neurons after.2 weeks and 4 weeks. The number and morphology of newborn neurons were observed. The results showed that the number of GFP+ cells in the C57-MCAO mice after operation was significant. The proportion of bipolar cells increased and multiple basal dendrites appeared.
The synaptic connections in the hippocampal neurons of MCAO mice were abnormal. Method 1: 4 month old male C57BL/6 mice were treated with MCAO (C57-MCAO mice) or sham operation (C57-sham mice). The hippocampal dentate gyrus was injected with retrovirus (RV-EGFP) to mark the newborn neurons, and the animal brain slices were taken for 4 weeks after the operation, and the GFP+ cells and GFP- cells were microinjected into green and red respectively. Biotin dye, found that C57-MCAO mice GFP+ cell axon fiber penetrated into the molecular layer. Method two: 4 month old male C57BL/6 mice were treated with MCAO (C57-MCAO mice) or sham operation (C57-sham mice), intraperitoneal injection of BrdU labeled new cells, hippocampal dentate gyrus injected with AAV-EGFP labeled all neurons, 3 weeks after the same site injection of delta Rabies-m. Cherry was labeled with AAV infected neurons and their superior neurons. After 1 weeks, the brain slices were taken to observe the EGFP+/mCherry+ of the dentate gyrus, the number, morphology and distribution of EGFP+/mCherry "and EGFP-/mCherry+ cells, and whether the BrdU was Co located with BrdU, and the results showed that most of the EGFP-/mCherry+ cells were BrdU+ cells.
Method one: 4 month old male Nes-Cre mice were treated with MCAO surgery (Nes-Cre-MCAO mice) or sham operation (Nes-Cre-sham mice). 1 weeks postoperatively, the adenoassociated virus (AAV-ChR2-EGFP) was injected into the sea dentate gyrus, and BrdU was injected intraperitoneally for 5 days. NeuN and BrdU staining was performed at 4 weeks after the operation. The number, morphology and distribution of GFP+/NeuN+ cells and GFP+/NeuN+/BrdU+ cells in the hippocampus of two groups of animals and the proportion of NeuN+/BrdU+ cells to total GFP+ cells were observed. The results showed that the number of newborn neurons in the Nes-Cre-MCAO mice was higher than that of the Nes-Cre-sham mice, and the number of NeuN+/BrdU+ cells in the two groups was not significantly changed. Method two: 4 month old male ChR2. The mice were treated with MCAO operation (ChR2-MCAO mice) or sham operation (ChR2-sham mice), and tamoxifen (TAM) was given to induce the expression of ChR2 and EGFP. The synaptic connections between DG zone ChR2+/EGFP+ cells and CA3 region and ChR2-/EGFP cells in DG region were recorded at 4 weeks after the operation. EPSCs was recorded in DG region ChR2-/EGFP cells of group ChR2-MCAO, but EPSCs. in ChR2-/EGFP group of DG region was not recorded in group EPSCs.
The synaptic connections between the hippocampal neurons of MCAO mice and the original mature granulosa cells formed a functional synaptic connection between.ChR2 mice and TAM. MCAO (ChR2+-MCAO mice) or sham operation (ChR2+-sham mice) were performed. 4 weeks after the operation, the brain slices were taken to record the synaptic connections between the ChR2+/EGFP+ cells of the DG region and the ChR2-/EGFP cells in the CA3 region and DG region. The results showed ChR2+-MCAO The ChR2-/EGFP- cells in the CA3 region of the group of mice and ChR2+-sham mice and the ChR2-/EGFP cells in the DG region of group ChR2-MCAO mice could be recorded to EPSCs; the EPSCs was linearly and positively correlated with the light intensity of blue light, and was sensitive to the AMPA receptor antagonist.
The specific expression of TeTX in the newborn hippocampal neurons of the mice can specifically block the abnormal synaptic transmission, which does not affect the survival and electrophysiological characteristics of the newborn neurons in MCAO mice, and the male TeTX mice of.4 months of age.4 (TeTX-MCAO mice). A group of animals give TAM to express TeTX (TeTX+-MCAO mice) in the newborn neurons, and another group of animals. Given normal control solvent (TeTX-MCAO mice), after 4 weeks, Fluoro-Jade C staining was used to understand the effect of TeTX on cell survival. The results showed that there was no significant difference in the number of F-J+ cells in the two groups. Further electrophysiology was used to record the EPSCs and IPSCs of GFP+ and GFP- cells in two groups of animals, and the results showed EPSCs GFP+ and GFP cells of the two groups of animals. There was no significant difference between the IPSCs and the IPSCs in the GFP+ and GFP- cells..TeTX mice were treated with MCAO (TeTX--MCAO mice), and 1 weeks after the operation, the sea dentate gyrus was injected with adeno-related virus (AAV-ChR2). After 4 weeks, the two groups were given 1 weeks, respectively, DOX (TeTX+-ChR2+-MCAO-mice) and normal control solvent (vehicle) (TeTX--ChR2+-MCAO-mice), and then first. The EPSCsNMDA in CA3 and DG regions was recorded by light stimulation of new neurons and electrical stimulation of moss fibers, and all of the two groups were changed to normal drinking water for 1 weeks, and second records were recorded (the same first record). The results showed that the TeTX--ChR2+-MCAO mice were able to record the EPSCsNMDA, TeTX+-ChR2+-MCAO mice after light stimulation and electrical stimulation in CA3 and DG areas. EPSCsNMDA. can only be recorded in the CA3 area after electrical stimulation
Specifically blocking the synaptic transmission of hippocampal neurons could reduce the frequency and intensity of epileptic seizures in MCAO mice..4 months old male TeTX mice were treated with MCAO (TeTX-MCAO mice) or sham operation (TeTX-sham mice), and 3 weeks later, they began to continue to give doxycycline (TeTX+-MCAO/sham mice) or control solvent (TeTX "-MCAO/sham mice"), and 1 weeks later, it began to connect. The results showed that the electroencephalogram and behavioral changes were observed. The results showed that there was no epilepsy in TeTX-sham mice; TeTX--MCAO mice had seizures and increased with the time of cerebral ischemia. The number of epileptic seizures and the duration of single attack in TeTX+-MCAO mice were lower than that of TeTX--MCAO mice. We also had 4 month old male TeTX mice. Continuous administration of TeTX+ (TeTX mice) or control solvent (TeTX mice), then MCAO (TeTX+/--MCAO mice) or sham operation (TeTX+/--sham mice) treatment, and injection of 10-30mg/kg's kainic acid (KA) intraperitoneally to induce acute epileptic seizures, and observe the electroencephalogram, epileptic seizures and mortality of each group of animals after KA injection. The results were found with K. The increase of A dose increased the mortality of animals in each group, mostly in TeTX--MCAO mice, and the electroencephalogram of TeTX+/--sham mice and TeTX+-MCAO mice was basically normal, and the EEG waveforms of TeTX--MCAO mice were obviously abnormal and the epilepsy index was higher than that of other groups. The TeTX mice were further used for MCAO (TeTX-MCAO mice) or sham operation (TeTX-sham mice). 4 weeks after the operation, the seizures of two groups of animals (TeTX--MCAO/sham mice) were recorded for 4 weeks (the first record, R1), and then the two groups were given doxycycline for 5 weeks. The seizures of two groups of animals (TeTX+-MCAO/sham mice) were recorded (second times, R2), and then the two groups of animals were given normal drinking water for 5 weeks, and the records were recorded. Two groups of animals (TeTX--MCAO/sham mice) were epileptic (third records, R3). The results showed that there were almost no epileptic seizures in TeTX+/--sham mice during the whole period of the experiment. For TeTX-MCAO mice, the frequency of epileptic seizures was significantly higher than that of the sham operation group, while R2, the frequency of epileptic seizures was significantly lower than that of R1, R3, R3. The frequency of seizure intensity returned to R1 level.
[Conclusion]
The abnormal integration of newborn neurons to the original nerve loop is the cell basis of recurrent seizures after stroke. Blocking the abnormal synaptic connection can reduce the frequency and intensity of epilepsy in MCAO mice. The results provide a new way of thinking for the intervention and treatment of epilepsy after stroke.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R743.3;R742.1

【共引文献】