大鼠骨髓源性神经干细胞移植治疗颞叶癫痫实验研究

目的 探讨骨髓源性神经干细胞移植至KA大鼠海马后与宿主细胞的整合及其对损伤宿主的修复作用,从而为干细胞移植治疗颞叶癫痫提供理论依据.方法 首先分离大鼠骨髓基质细胞,并在特定的条件下培养使其诱导分化为神经干细胞,且使用Feridex对干细胞进行标记.然后建立大鼠颞叶癫痫模型,将Feridex标记的神经干细胞自体移植至KA大鼠的海马内,观察移植后1周、2周、4周、8周和16周大鼠海马的脑电图、病理学和MRI改变情况.结果 与KA未移植组相比,移植组大鼠海马脑电图的波幅明显降低,最高降低达40%以上;KA移植组海马CA3区锥体细胞数与未移植组相比有极显著性差异（P＜0.01）;KA移植组海马损伤侧的Timm染色与未移植组相比也有极显著性差异（P＜0.01）;MRI检查发现在神经干细胞移植后1周和2周时低信号改变区比较局限,但移植4周、8周和16周后低信号改变明显增大,且随着时间的推移低信号改变区逐渐增大.结论 骨髓源性神经干细胞移植至KA大鼠后能与宿主细胞进行整合,且对宿主海马具有显著的修复作用,但其具体的作用机制尚待进一步研究.     

海马干细胞移植对癫痫鼠苔状纤维发芽抑制作用的研究

Turner等证实大鼠海马胚胎细胞移植对于海人酸（KA）所致癫痫鼠神经元的损害具有一定的修复作用，但胚胎细胞移植受细胞来源和伦理学等多方面限制。神经干细胞的发现为细胞移植治疗颞叶癫痫等神经系统疾病提供了广阔前景。本研究将分离培养的大鼠海马干细胞移植至KA所致癫痫模型鼠CA3区，     

神经干细胞移植于慢性癫痫鼠海马CA3区对其癫痫发作影响的研究

目的 研究神经干细胞(neural stem cells,NSCs)移植到慢性海人酸(kainic acid,KA)癫痫鼠海马CA3区后对大鼠癫痫发作的影响.方法 用KA脑审注射制作慢性癫痫模型.将原代培养的、EGFP标记的NSCs移植到慢件癫痫鼠的海马CA3区.分别在移植后第2周、第4周、第8周和第12周连续进行7天观察大鼠癫痫发作频率和程度,在移植后第10周进行发作间期右侧海屿深部脑电监测.然后取脑冰冻切片,在倒置荧光显微镜下直接观察移植细胞的存活和迁移,用免疫荧光染色观察移植细胞分化情况,Timm's染色观察海马齿状回异常苔状纤维发芽.结果 移植后12周仍有大量移植细胞存活(65,045.00±881.72).NSCs在移植区以胶质细胞分化为主,在齿状回和海马各区以神经元分化为主,γ-氨基丁酸(GABA)能神经元在齿状回门区和海马CA3区分化比率较高.NSCs移植后第4周移植组癫痫鼠的发作次数与对照组相比开始减少,Timm's染色计分和发作程度也有明显改善,两组发作问期脑电图尖、棘波在每个观察期的发放次数分别是3.83±4.96和27.16±21.08,,结论 将NSCs移植到慢性KA癫痫鼠的海马CA3区,移植细胞不仪能够长期存活、迁移到海马齿状回的各区,而且能够分化为神经元和神经胶质细胞,特别是GABA能神经元,同时还能够抑制齿状回颗粒细胞的苔状纤维发芽,从而减少癫痫发作次数,减轻癫痫发作程度.     

骨髓源性神经干细胞自体移植海藻酸致痫大鼠海马后脑电图的改变

目的探讨骨髓源性神经干细胞移植至海藻酸（KA）致痫大鼠海马后对宿主海马脑电图的影响，从而为神经干细胞移植治疗颞叶癫痫提供理论依据。方法无菌条件下分离大鼠骨髓基质细胞，在特定的条件下培养使其诱导分化为神经干细胞。建立大鼠颞叶癫痫模型，将诱导分化的神经干细胞自体移植至KA大鼠的右侧海马内，观察移植后1周、2周、4周、8周和16周大鼠海马的MRI和脑电图改变情况。结果MRI检查发现在神经干细胞移植后1周和2周时低信号改变区比较局限，但移植4周、8周和16周后低信号改变明显增大，且随着时间的推移低信号改变区逐渐增大。与未移植组相比，移植组大鼠海马脑电图的波幅明显降低，且随着时间的推移降低幅度也明显增加，最高至移植8周后脑电图波幅降低达40％以上。结论骨髓源性神经干细胞自体移植至KA大鼠后对宿主海马的癫痫样放电有一定的抑制作用。     

海马干细胞移植对癫癎鼠脑电影响的初步研究

目的研究神经干细胞移植对癫癎鼠脑电的影响,为神经干细胞移植治疗癫癎提供理论依据.方法分离、培养新生鼠海马干细胞,将其移植至海人酸(KA)所致癫癎模型鼠的右侧海马,比较移植组及未移植组大鼠在移植前和移植后1周、4周、8周及24周海马及杏仁核的脑电变化.结果海马干细胞移植可减少癫癎动物脑电的痫性发放,并降低癫癎波幅约50%.结论神经干细胞移植对于KA诱发癫癎鼠的癫癎具有一定的抑制作用,但其具体作用机制还有待于进一步的研究.     

慢性癫鼠海马内移植神经干细胞的存活、迁移、分化及与宿主整合的研究

目的研究神经干细胞(NSC)移植到慢性海人酸癫疒间鼠海马CA3区后细胞的存活、迁移、分化及与宿主细胞的整合情况。方法将增强型绿色荧光(EGFP)标记的原代培养NSC移植到慢性癫疒间鼠的海马CA3区。分别在移植后第2、4、8、12周4个时间点取脑冷冻切片,在倒置荧光显微镜下观察移植细胞的存活和迁移。采用免疫荧光染色观察移植细胞的分化和整合情况。结果NSC在移植后2周未见迁移;移植后4周,可见移植细胞迁移到距移植区最近的齿状回;8周和12周,移植细胞可迁移到齿状回和海马各区。12周仍有大量移植细胞(65 045.00±881.72)存活。NSC在移植区以分化为胶质细胞为主,在齿状回和海马各区以分化为神经元为主;-γ氨基丁酸(GABA)能神经元在齿状回门区和海马CA3区分化比例较高。突触素免疫荧光染色示移植细胞与宿主细胞间产生了有机的整合。结论NSC移植于慢性癫疒间鼠的海马CA3区后,能够迁移到齿状回和海马各区并长期存活,且可分化为神经元,特别是GABA能神经元,并能与宿主细胞有机整合。     

慢性癫(癎)鼠海马内移植神经干细胞的存活、迁移、分化及与宿主整合的研究

目的 研究神经干细胞(NSC)移植到慢性海人酸癫(癎)鼠海马CA3区后细胞的存活、迁移、分化及与宿主细胞的整合情况.方法 将增强型绿色荧光(EGFP)标记的原代培养NSC移植到慢性癫(癎)鼠的海马CA3区.分别在移植后第2、4、8、12周4个时间点取脑冷冻切片,在倒置荧光显微镜下观察移植细胞的存活和迁移.采用免疫荧光染色观察移植细胞的分化和整合情况.结果 NSC在移植后2周未见迁移;移植后4周,可见移植细胞迁移到距移植区最近的齿状回;8周和12周,移植细胞可迁移到齿状回和海马各区.12周仍有大量移植细胞(65 045.00±881.72)存活.NSC在移植区以分化为胶质细胞为主,在齿状回和海马各区以分化为神经元为主;γ-氨基丁酸(GABA)能神经元在齿状回门区和海马CA3区分化比例较高.突触素免疫荧光染色示移植细胞与宿主细胞间产生了有机的整合.结论 NSC移植于慢性癫(癎)鼠的海马CA3区后,能够迁移到齿状回和海马各区并长期存活,且可分化为神经元,特别是GABA能神经元,并能与宿主细胞有机整合.     

神经干细胞移植在颞叶损伤后癫痫治疗中的应用

颞叶脑损伤后海马神经元的破坏和异常修复的结果形成了后期癫痫发作的基础。哺乳动物中枢神经系统损伤后自身修复能力有限,临床现行治疗多着重于改善脑代谢以加强细胞保护和防止存活神经元继发性损伤。随着干细胞研究的深入,其在特定环境和因子的诱导下能定向分化成不同的神经细胞类型,体内移植并使它们存活、分化、整合到宿主正常神经环路成为新的治疗方向。本文结合神经组织移植治疗颞叶皮层和海马损伤后癫痫的实验结果,探讨神经干细胞移植治疗的可行性及其与移植脑环境的关系。     

骨髓源性神经干细胞自体移植对癫(癎)大鼠海马的修复作用

目的 探讨骨髓源性神经干细胞自体移植对癫(癎)大鼠海马的修复作用.方法 雄性SD大鼠随机分为正常对照组、移植组和非移植组.无菌条件下分离大鼠骨髓基质细胞,在特定条件下培养、诱导其分化为神经干细胞;对移植组和非移植组大鼠建立颞叶癫(癎)模型,将诱导分化的神经干细胞自体移植至移植组大鼠右侧海马内,观察移植后1周、2周、4周、8周和16周模型鼠海马的形态学变化.结果 移植组与非移植组海马CA3区锥体细胞数各时间点显著少于正常对照组(均P＜0.01);与非移植组比较,移植组海马CA3区锥体细胞数于移植后第2～16周明显增多(均P＜0.01);移植组各时间段之间差异有统计学意义(均P＜0.01).与正常对照组相比,移植组和非移植组海马损伤侧的Timm染色评分显著增高(均P＜0.01);但移植组移植2周后各时间点评分显著低于非移植组(均P＜0.01);非移植组随制模时间延长评分持续升高.MRI检查显示在神经干细胞移植后1周和2周时低信号改变区比较局限,此后低信号影随着时间的推移逐渐增大.结论 骨髓源性神经干细胞自体移植至癫(癎)大鼠后能够在海马中生存并迁移,具有减轻海马CA3区锥体细胞缺失、抑制海人酸引起的苔状纤维发芽的修复作用.     

神经干细胞和GABA能神经元治疗大鼠颞叶癫痫实验研究

目的探讨并比较神经干细胞(NSCs)和γ-氨基丁酸(GABA)能神经元移植治疗大鼠颞叶癫痫的疗效。方法取孕12 d SD大鼠胎鼠脑组织,分离培养NSCs并鉴定,取第3代NSCs定向诱导分化为GABA能神经元。48只SD大鼠随机分为4组,空白对照组、未移植组、NSCs移植组和GABA能神经元移植组,移植细胞用5-溴脱氧尿苷(BrdU)标记,在模型建立后的第4 d将上述两种细胞移植到癫痫大鼠右侧海马。分别在细胞移植后的4 w、8 w、12 w处死大鼠留取脑标本。常规HE染色和Nissl染色观察大鼠右侧海马的损伤与治疗情况并进行评价。结果 NSCs移植组和GABA能神经元移植组均于移植后第8 w时海马CA3(CA3)区神经元计数最多,组内比较时,与另外两个时间点之间的差异具有统计学意义(P<0.05)。进而取第8 w时间点进行组间比较,结果各组海马区神经元计数之间的差异均具有统计学意义(P<0.05)。结论两治疗组在移植后第8 w时海马区神经元计数最多,且NSC移植组的疗效优于GABA能神经元移植组。     

GFP转基因骨髓基质干细胞移植对癫痫鼠脑电的影响

目的　观察绿色荧光蛋白（GFP）转基因骨髓基质干细胞（BMSCs）移植至致痫鼠后的存活、迁移及其对癫痫鼠脑电的影响。方法　分离、培养GFP转基因小鼠BMSCs，移植至青霉素致痫鼠的右侧海马内，比较移植后1w、2w、4wBMSCs在脑内的存活和迁移情况及大鼠脑电改变。结果　BMSCs可以在致痫鼠脑内存活和迁移，随移植时间延长，细胞存活数逐渐减少（P〈0．01）；BMSCs移植可减少癫痫大鼠脑电的痫性放电，降低癫痫波波幅。结论　BMSCs移植于青霉素诱发的癫痫鼠脑内后能够存活、迁移，并能够改善癫痫鼠的脑电生理功能，提示干细胞移植可能成为一种有效的癫痫     

Increased excitatory synaptic input to granule cells from hilar and CA3 regions in a rat model of temporal lobe epilepsy

One potential mechanism of temporal lobe epilepsy is recurrent excitation of dentate granule cells through aberrant sprouting of their axons (mossy fibers), which is found in many patients and animal models. However, correlations between the extent of mossy fiber sprouting and seizure frequency are weak. Additional potential sources of granule cell recurrent excitation that would not have been detected by markers of mossy fiber sprouting in previous studies include surviving mossy cells and proximal CA3 pyramidal cells. To test those possibilities in hippocampal slices from epileptic pilocarpine-treated rats, laser-scanning glutamate uncaging was used to randomly and focally activate neurons in the granule cell layer, hilus, and proximal CA3 pyramidal cell layer while measuring evoked EPSCs in normotopic granule cells. Consistent with mossy fiber sprouting, a higher proportion of glutamate-uncaging spots in the granule cell layer evoked EPSCs in epileptic rats compared with controls. In addition, stimulation spots in the hilus and proximal CA3 pyramidal cell layer were more likely to evoke EPSCs in epileptic rats, despite significant neuron loss in those regions. Furthermore, synaptic strength of recurrent excitatory inputs to granule cells from CA3 pyramidal cells and other granule cells was increased in epileptic rats. These findings reveal substantial levels of excessive, recurrent, excitatory synaptic input to granule cells from neurons in the hilus and proximal CA3 field. The aberrant development of these additional positive-feedback circuits might contribute to epileptogenesis in temporal lobe epilepsy.     

Region-specific differentiation of embryonic stem cell-derived neural progenitor transplants into the adult mouse hippocampus following seizures

Embryonic stem (ES) cells can generate neural progenitors and neurons in vitro and incorporate into the adult central nervous system (CNS) following transplantation, suggesting their therapeutic potential for treating neurological disorders. However, our understanding of the conditions that direct ES-derived neural progenitor (ESNP) migration and differentiation within different regions of the adult CNS is incomplete. Rodents treated with the chemoconvulsant kainic acid (KA) experience seizures and display hippocampal sclerosis, as well as enhanced hippocampal neurogenesis, similar to pathological findings in patients with temporal lobe epilepsy (TLE). To examine the potential for ESNPs to incorporate into the adult hippocampus and differentiate into hippocampal neurons or glia following seizure-induced damage, we compared the fates of ESNPs after they were transplanted into the CA3 region or fimbria 1 week following KA-induced seizures. After 4-8 weeks, ESNPs grafted into the CA3 region had migrated to the dentate gyrus (DG), where a small subset adopted neural stem cell fates and continued to proliferate, based on bromodeoxyuridine uptake. Others differentiated into neuroblasts or dentate granule neurons. In contrast, most ESNPs transplanted into the fimbria migrated extensively along existing fiber tracts and differentiated into oligodendrocytes or astrocytes. Hippocampal grafts in mice not subjected to seizures displayed a marked tendency to form tumors, and this effect was more pronounced in the DG than in the fimbria. Taken together, these data suggest that seizures induce molecular changes in the CA3 region and DG that promote region-specific neural differentiation and suppress tumor formation.     

荧光金逆行示踪观察匹罗卡品致痫大鼠CA1区锥体细胞突触联系变化的研究

目的通过荧光金(FG)逆行示踪观察氯化锂-匹罗卡品致痫模型大鼠慢性自发发作期海马CA1区锥体细胞之间的突触联系变化。方法 SD大鼠2 0只随机分为实验组和对照组。癫痫持续状态后6 0 d左右,利用立体定位仪在活体内注射逆行性示踪剂FG至海马CA1区,术后常规喂养5~7 d后灌注取材。激光扫描共聚焦显微镜下观察FG的分布。结果 7只实验组大鼠中有5只可见有FG标记的锥体细胞,对照组未见。实验组中有2只大鼠在海马下托亦可见有FG标记的锥体细胞,而对照组未见。结论颞叶癫痫大鼠海马CA1区锥体细胞之间和下托至CA1区有异常兴奋性突触联系,其可能是构成异常兴奋性回路的解剖学基础。     

Human neural stem cell transplantation reduces spontaneous recurrent seizures following pilocarpine-induced status epilepticus in adult rats

Transplantation of neural stem cells (NSCs) can replace lost neurons and improve the functional deficits. Cell transplantation strategies have been tried in the epileptic disorder, but the effect of exogenous NSCs is unknown. In this study, we attempted to test the anti-epileptogenic effect of NSCs in adult rats with status epilepticus. Experimental status epilepticus was induced by lithium-pilocarpine injection, and beta galactosidase-encoded human NSCs were transplanted intravenously on the next day of status epilepticus. Spontaneous recurrent seizures were monitored with Racine's seizure severity scale. Immunohistochemistry with anti-beta gal, Tuj-1, NeuN, GFAP, CNPase, GluR2, parvalbumin, and GABA were performed and extracellular field excitatory postsynaptic potentials (fEPSP) were recorded. Human NSCs suppressed spontaneous recurrent seizure formation and transplanted NSCs were differentiated into GABA-immunoreactive interneurons in the damaged hippocampus. Amplitude of fEPSP in the hippocampal CA1 was reduced, which was reversed by picrotoxin. These findings suggest that NSCs could be differentiated into inhibitory interneurons and decrease neuronal excitability, which could prevent spontaneous recurrent seizure formation in adult rats with pilocarpine-induced status epilepticus.     

Restoration of calbindin after fetal hippocampal CA3 cell grafting into the injured hippocampus in a rat model of temporal lobe epilepsy

Degeneration of the CA3 pyramidal and dentate hilar neurons in the adult rat hippocampus after an intracerebroventricular kainic acid (KA) administration, a model of temporal lobe epilepsy, leads to permanent loss of the calcium binding protein calbindin in major fractions of dentate granule cells and CA1 pyramidal neurons. We hypothesize that the enduring loss of calbindin in the dentate gyrus and the CA1 subfield after CA3-lesion is due to disruption of the hippocampal circuitry leading to hyperexcitability in these regions; therefore, specific cell grafts that are capable of both reconstructing the disrupted circuitry and suppressing hyperexcitability in the injured hippocampus can restore calbindin. We compared the effects of fetal CA3 or CA1 cell grafting into the injured CA3 region of adult rats at 45 days after KA-induced injury on the hippocampal calbindin. The calbindin immunoreactivity in the dentate granule cells and the CA1 pyramidal neurons of grafted animals was evaluated at 6 months after injury (i.e. at 4.5 months post-grafting). Compared with the intact hippocampus, the calbindin in "lesion-only" hippocampus was dramatically reduced at 6 months post-lesion. However, calbindin expression was restored in the lesioned hippocampus receiving CA3 cell grafts. In contrast, in the lesioned hippocampus receiving CA1 cell grafts, calbindin expression remained less than the intact hippocampus. Thus, specific cell grafting restores the injury-induced loss of calbindin in the adult hippocampus, likely via restitution of the disrupted circuitry. Since loss of calbindin after hippocampal injury is linked to hyperexcitability, re-expression of calbindin in both dentate gyrus and CA1 subfield following CA3 cell grafting may suggest that specific cell grafting is efficacious for ameliorating injury-induced hyperexcitability in the adult hippocampus. However, electrophysiological studies of KA-lesioned hippocampus receiving CA3 cell grafts are required in future to validate this possibility.     

红藻氨酸致痫大鼠海马S100B、CGRP蛋白表达及病理改变

目的 研究红藻氨酸(KA)致痫大鼠海马S100B、降钙素基因相关肽(CGRP)的表达及病理改变.方法 雄性SD大鼠按照完全随机数字表法分成对照组(8只)和模型组(40只),模型组再根据处死时间分为造模后6 h、12 h、24 h、72 h、1周5个亚组,每组8只.模型组采用KA建立颞叶癫痫动物模型,对照组用等体积生理盐水代替KA注射.模型组造模后6 h、12 h、24 h、72 h、1周,对照组注射后24 h取大鼠海马组织行Nissl染色、Timm染色和免疫组化染色,观察S100B、CGRP蛋白的表达情况以及海马神经元和胶质细胞的病理变化.结果 Nissl染色结果显示,模型组大鼠1周后CA3区出现大量固缩的坏死神经元,胞体萎缩,尼氏体消失.Timm染色结果显示,模型组大鼠1周后CA3区始层出现条带状分布的棕色颗粒,齿状回内分子层亦可见少量棕色颗粒.免疫组化染色结果显示,模型组大鼠海马CGRP蛋白大量表达,72 h时达到高峰,同时伴随大量神经元丧失及胶质细胞增生.结论 KA致痫大鼠出现S100B、CGRP蛋白高表达,尼氏体消失,苔藓纤维发芽等一系列病理学改变,推测S100B、CGRP蛋白参与了癫痫发生.

Abstract：

Objective To investigate the expressions of S100B and calcitonin gene related peptide (CGRP) and the pathologic alterations of the hippocampus in kainic acid (KA)-induced epileptic rats. Methods Male SD rats were randomly divided into control group (n=8) and model group (n=40).Animal models of temporal lobe epilepsy were established by intracerebroventricular injection of KA; the same volume of saline was injected into the rats in the control group. Hippocampal tissues within various phases after seizures (6, 12, 24 and 72 h, and 24 h after the success of model making) were performed Nissl staining, Timm staining and immunohistochemical staining. The expressions of S100B and CGRP were observed, and the pathologic alterations of the hippocampal neurons and glial cells were studied.Results All rat models were successfully induced with epileptic seizures. Nissl staining showed that pyknotic neuronal necrosis appeared in the CA3 area of the hippocampus in the model group with cell body atrophy and disappearance of Nissl bodies 1 week after the injection. Timm staining showed that brown particles showed stripped distribution in the CA3 area of the hippocampus and some brown particles in the molecular layer of fascia dentate. Immunohistochemical staining indicated that significant neurons lost and gliosis appeared after seizures with abundant expressions of S100B and CGRP.Conclusion KA-induced epileptic rats express abundant S100B and CGRP and appear such pathological changes as disappearance of Nissl bodies and mossy fiber sprouting, indicating that both S100B and CGRP participate in the onset of epilepsy.