戊四氮点燃模型大鼠GSK-3β活性的动态变化及其与苔藓纤维出芽的关系

目的 探讨戊四氮点燃模型大鼠GSK-3β活性变化与苔藓纤维出芽的关系.方法 取戊四氮点燃模型大鼠给药后3 d、1、2、4和6周的脑片,通过Timm染色观察苔藓纤维出芽的动态变化;取各时间点的大鼠海马组织,通过酶活性测定方法 检测GSK-3β活性变化.结果 戊四氮组各时间点GA3区苔藓纤维出芽评分均有显著性差异(P＜0.05);从3 d起评分逐渐增加,2周时达高峰并维持至6周.在点燃过程中海马GSK-3β蛋白活性逐渐增高,2周达高峰,4、6周表达逐渐下调到生理盐水对照组水平,除6周组外各时间点与相应时间点生理盐水对照组比较差异均有统计学意义.结论 GSK-3β通过活性上调参与了苔藓纤维出芽过程,促进了癫(癎)的发生.     

戊四氮致痫大鼠海马GSK-3β表达与苔藓纤维出芽关系研究

目的观察戊四氮(PTZ)致痫大鼠海马各区糖原合成酶激酶-3β(GSK-3β)蛋白及其mRNA表达和苔藓纤维出芽(MFS)情况,探讨GSK-3β在癫痫发病机制中的作用。方法SD雄性成年大鼠120只,随机分为实验组和对照组;实验组分为PTZ第1次注射后3d、1w、2w、4w、6w共5个亚组,每亚组12只。对照组同样随机分为5个亚组,每亚组12只,与实验组各时间点对应。以上各亚组再分2个小组,每小组6只大鼠,分别进行(1)GSK-3β的免疫组化和原位杂交染色并测定其相应的光密度值;(2)Timm染色并评分。结果实验组大鼠海马各区GSK-3β蛋白及其mRNA表达在点燃过程中逐渐增多,点燃后表达逐渐下调到正常对照组水平,在点燃前后除6w组外GSK-3β表达与对照组相应时间点比较差异有统计学意义(P<0.05);实验组CA3区在点燃前可见1~4级MFS,点燃后可见4~5级MFS;癫痫点燃过程中CA3区GSK-3β表达与MFS评分有线性正相关关系。结论GSK-3β在海马表达变化可能在苔藓纤维出芽的过程中起促进作用,从而促进癫痫的发生。     

TRPC6在匹罗卡品致痫大鼠海马苔藓纤维出芽中的作用

目的 观察传统型瞬时受体电位通道6(TRPC6)蛋白在匹罗卡品致痫大鼠海马中的表达变化,探讨其在海马苔藓纤维出芽中的作用.方法 72只SD大鼠随机分为实验组(n=60)和对照组(n=12).实验组采用氯化锂-匹罗卡品腹腔注射法建立颞叶癫痫模型;对照组腹腔注射等量无菌生理盐水.实验组按癫痫持续状态(SE)后1d、7d、15d、30 d和60 d分为5个亚组,每亚组12只大鼠.以上各亚组及对照组再分为2个小组,分别进行Western blot检测TRPC6及突触重建标志蛋白Synaptophysin在海马中的表达和Timm染色观察海马苔藓纤维出芽并评分.结果 实验组TRPC6蛋白表达量在SE后1d达高峰(P＜0.01),其他时间点均显著高于对照组(P＜0.01).Synaptophysin蛋白表达量在SE后7d、15d、30 d和60 d显著增加(7 d:P＜0.05;15 d、30 d、60 d:P＜0.01),30 d达峰值(P＜0.01).实验组大鼠齿状回内分子层在SE后7d出现Timm颗粒,并呈进行性增加.结论 TRPC6可能参与了苔藓纤维出芽这一过程.     

法舒地尔对戊四氮点燃大鼠海马丝切蛋白及苔藓纤维出芽的影响

目的 探讨法舒地尔对戊四氮(PTZ)点燃大鼠海马组织中丝切蛋白(cofilin,非磷酸化形式)表达与苔藓纤维出芽程度关系的影响.方法 210只SD雄性大鼠分成戊四氮组、法舒地尔干预组和生理盐水对照组,采用PTZ慢性点燃癫癎模型,应用SABC法检测cofilin表达,用Timm染色检测苔鲜纤维出芽情况.结果 PTZ组大鼠点燃率、病死率与法舒地尔组比较差异无统计学意义.PTZ组和法舒地尔组CA3区苔藓纤维出芽评分差异无统计学意义,与对照组相比差异均有统计学意义(P＜0.05).PTZ组和法舒地尔组海马非磷酸化cofilin表达差异无统计学意义.结论 丝切蛋白可能通过苔藓纤维出芽与癫癎的发生相关.     

Semaphorin-3A与苔藓纤维出芽在毛果芸香碱致痫大鼠中的改变及意义

目的探讨海马Semaphorin-3A(Sema3A)与苔藓纤维出芽(MFS)在癫痫发病机制中的作用。方法通过小剂量多次腹腔注射氯化锂-毛果芸香碱建立癫痫大鼠模型,随机将大鼠分为生理盐水对照组和痫性发作组。痫性发作组分别于药物注射后1、5、7d及3、4周时间点,应用Western blotting法检测大鼠海马Sema3A的表达,同时采用neo-Timm银染观察海马MFS情况。结果生理盐水对照组Sema3A仅有少量表达,痫性发作组1、5d无表达,7d表达明显,3周后亦无表达;痫性发作组1、5d未见MFS,7d可见MFS至齿状回内分子层,3周后明显可见MFS至齿状回分子层。痫性发作组Timm评分与生理盐水对照组比较差异有统计学意义(P<0.05)。结论海马区Sema3A表达变化伴有MFS可能是癫痫发病机制的重要因素之一。     

Tau蛋白在点燃大鼠海马苔藓纤维出芽中的作用探讨

目的观察总tau蛋白及其p-ptauser202在戊四氮点燃癫模型海马中的表达变化,探讨其在苔藓纤维出芽中的作用。方法 180只雄性SD大鼠随机分为对照组(腹腔注射生理盐水)和模型组(腹腔注射戊四氮),均n=90。于不同的时间点应用Timm染色观察苔藓纤维出芽,免疫组化和Western blot检测各时间点海马总tau蛋白及p-ptauser202蛋白表达情况。结果模型组各时间点CA1、CA3、DG区苔藓纤维出芽较对照组明显增多,差异有统计学意义(P<0.05);模型组第1周CA1区、CA3区,第2周DG区的p-ptauser202蛋白表达与对照组比较明显增多,差异均有统计学意义(分别为CA1区P<0.05,CA3区P<0.01,DG区P<0.01);门区(H区)tau蛋白及p-ptauser202蛋白在各时间点的表达明显增多,差异有显著统计学意义(P<0.01);对照组无动态变化。结论 tau蛋白可能通过其磷酸化水平的增高参与癫点燃大鼠的苔藓纤维出芽,在癫的发生和发展中起重要作用。     

杏仁核点燃癫痫大鼠P-糖蛋白及苔藓纤维出芽变化的研究

目的 观察杏仁核点燃癫痫大鼠海马区P-糖蛋白(P-gp)表达及苔藓纤维出芽(MFS)的动态变化. 方法 90只大鼠采用随机数字表法分为假手术对照组(10只)、癫痫组(40只)和治疗组(40只),假手术对照组只安装电极,不予刺激;癫痫组和治疗组制作杏仁核点燃模型,治疗组加用左乙拉西坦灌胃治疗[100 mg/(kg·d),2次/d)].采用Timm银染组织化学方法观察海马区MFS,免疫组化法检测P-gp的表达. 结果 (1)成功制造癫痫模型后,在海马CA3区透明层出现异常MFS,其中S1亚组大鼠MFS评分最低,与假手术对照组比较差异无统计学意义(P＞0.05);S2亚组大鼠评分开始增高,S4亚组大鼠明显增高,S8亚组大鼠达到高峰,与假手术对照组比较差异均有统计学意义(P＜0.05).而治疗组大鼠MFS评分各时间点与假手术对照组比较差异均无统计学意义(P＞0.05).(2)癫痫发作后癫痫组大鼠P-gp表达量呈现出逐渐降低的趋势,S1、S2、S4亚组与假手术对照组差异有统计学意义(P＜0.05);S8亚组接近正常水平,与假手术对照组比较差异无统计学意义(P＞0.05).治疗组大鼠除Y1亚组外,余各亚组P-gp表达量与假手术对照组比较差异均无统计学意义(P＞0.0S). 结论 MFS是慢性癫痫形成的重要机制,P-gp是癫痫发生的产物,是癫痫药物耐药的主要原因.     

戊四氮点燃癫(癎)大鼠海马巢蛋白和骨形成蛋白-4的表达研究

目的观察巢蛋白(nestin)和骨形成蛋白4(BMP4)基因在戊四氮(PTZ)点燃癫大鼠海马中的表达,并探讨两者与癫发病机制的关系。方法将81只成年雄性SD大鼠随机分为实验组(n=54)和对照组(n=27)。实验组采用PTZ点燃癫大鼠,按点燃中的不同时相点,又随机分为9组。用免疫组化技术、地高辛标记特异性寡核苷酸探针原位杂交组织化学技术,观察海马nestin和BMP4表达的变化。结果nestin阳性细胞在PTZ注射后3d开始出现在齿状回、CA3区和CA1区,到7d达到高峰,以后逐渐减少。BMP4在PTZ注射后7d开始增多,在点燃后1d达到高峰,以后逐渐减少,主要分布在齿状回、CA3区和CA1区。结论PTZ点燃可引起海马内星形胶质细胞增生、活化和神经发生,这可能是癫海马组织胶质化、神经元可塑性的病理基础;BMP4可能在PTZ癫形成过程中起重要作用。     

戊四氮点燃癫癎大鼠海马5-羟色胺能神经递质的动态研究

目的：观察戊四氮（PTZ）点燃癫癎形成过程中大鼠海马5-羟色胺（5-HT）能神经递质的变化。方法：用PTZ制作癫癎大鼠模型,将造模成功大鼠分为戊四氮急性发作组（PTZ 1组）和戊四氮慢性点燃组（PTZ 2组）,同时设立对照组（腹腔注射生理盐水）。在体微透析取样,观察大鼠行为、脑电图（EEG）和海马5-HT能神经递质的变化。结果：PTZ 1组癫癎发作时EEG自发放电逐级加重;癫癎发作时海马5-HT水平与对照组、发作前和发作后比较显著升高（P〈0.05）;海马5-羟吲哚乙酸（5-HIAA）水平差异无统计学意义;5-HT转化率（5-HIAA/5-HT）降低,差异有统计学意义（P〈0.05）。PTZ 2组点燃后大鼠出现自发癫癎发作,EEG在发作间期出现自发放电;5-HT和5-HIAA水平在点燃期、维持点燃期、对照组间比较差异有统计学意义（P〈0.05）。结论：大鼠癫癎发作时海马5-HT水平显著升高,发作后恢复正常;在癫癎形成过程中,早期5-HT水平一过性升高、PTZ点燃后和发作间期海马5-HT水平逐渐降低。     

PTZ诱导孕鼠痫性发作对胎鼠海马中bax、bcl-2、capase-3表达的影响

目的探讨母鼠孕期痫性发作对胎鼠海马中bax、bcl-2、capase-3表达的影响。方法将雌性SD大鼠随机分为正常对照组(NC组)、盐水组(NS组)和癫痫组(PTZ组),采用戊四氮(pentylenetetrazol,PTZ)致痫模型,PTZ组大鼠每天给予腹腔下注射PTZ 35 mg/kg,点燃成功后将雌性SD大鼠与雄性SD大鼠合笼;发现阴栓记为孕0d,孕鼠继续孕前处理至孕20d。NS组腹腔注射相应剂量的生理盐水,NC组不做任何处理。Western Blot方法测定胎鼠海马组织中bax、bcl-2及caspase-3蛋白表达变化。结果 PTZ组胎鼠海马caspase-3蛋白(2.57±0.08)较NC组(0.53±0.13)明显升高(P<0.05);PTZ组胎鼠海马bax蛋白(3.24±0.32)较NC组(1.55±0.11)明显升高(P<0.05);PTZ组子鼠海马bcl-2表达水平(1.42±0.074)较NC组(2.45±0.07)明显降低(P<0.05)。结论孕期痫性发作使胎鼠海马促凋亡蛋白bax表达增加,抑凋亡蛋白bcl-2表达降低,凋亡执行蛋白caspase-3表达增加,推测孕期痫性发作可能使胚胎海马神经元凋亡增加。     

丙戊酸钠对戊四氮致痫大鼠海马神经元凋亡的影响

目的 探讨丙戊酸钠(VAP)对戊四氮(PTZ)致痫大鼠海马神经元凋亡的影响.方法 将48只成年Wistar大鼠随机平均分为正常对照(NC)组、PTZ组和VAP组,PTZ组和VAP组大鼠腹腔注射阈下剂量的PTZ 35mg/(kg·d),直至达到点燃标准.点燃后,VAP组大鼠经腹腔注入VAP15mg/(kg·d),PTZ组大鼠经腹腔注入生理盐水,30min后,再腹腔注射PTZ诱发癫痫发作.应用免疫组化法检测大鼠海马神经元Fas、Caspase-3和Survivin的表达.结果 PTZ组大鼠海马神经元Fas、Caspase-3阳性细胞数和光密度明显高于VAP组和NC组(均P<0.01),Survivin阳性细胞数和光密度明显低于VAP组(P<0.01),但高于NC组(P<0.05);VAP组大鼠海马神经元Fas、Caspase-3阳性细胞数和光密度明显高于NC组(均P<0.05),Survivin阳性细胞数和光密度明显高于NC组(P<0.01).结论 癫痫发作可以导致大鼠海马神经元的凋亡,而丙戊酸钠有对抗癫痫发作导致细胞凋亡的作用,主要通过促进Survivin的表达和抑制Fas和Caspase-3的表达发挥作用.     

Prolonged infusion of tetrodotoxin does not block mossy fiber sprouting in pilocarpine-treated rats

PURPOSE: Mossy fiber sprouting is a common abnormality found in patients and models of temporal lobe epilepsy. The role of mossy fiber sprouting in epileptogenesis is unclear, and its blockade would be useful experimentally and perhaps therapeutically. Results from previous attempts to block mossy fiber sprouting have been disappointing or controversial. In some brain regions, prolonged application of the sodium channel blocker tetrodotoxin prevents axon sprouting and posttrauma epileptogenesis. The present study tested the hypothesis that prolonged, focal infusion of tetrodotoxin would block mossy fiber sprouting after an epileptogenic treatment. METHODS: Adult rats were treated with pilocarpine to induce status epilepticus. Several hours to 3 days after pilocarpine treatment, a pump with a cannula directed toward the dentate gyrus was implanted to deliver 10 microM tetrodotoxin or vehicle alone at 0.25 microl/h. This method blocks local EEG activity in the hippocampus (Galvan et al. J Neurosci 2000; 20:2904-16). After 28 days of continuous infusion, rats were perfused with fixative, and their hippocampi analyzed anatomically with stereologic techniques. RESULTS: Tetrodotoxin infusion was verified immunocytochemically in tetrodotoxin-treated but not vehicle-treated hippocampi. Tetrodotoxin-infused and vehicle-infused hippocampi displayed similar levels of hilar neuron loss. The Timm stain revealed mossy fiber sprouting regardless of whether hippocampi were treated with tetrodotoxin infusion, vehicle infusion, or neither. CONCLUSIONS: Prolonged infusion of tetrodotoxin did not block mossy fiber sprouting. This finding suggests that sodium channel-mediated neuronal activity is not necessary for mossy fiber sprouting after an epileptogenic treatment.     

Physiologic and Morphologic Characteristics of Granule Cell Circuitry in Human Epileptic Hippocampus

Summary: Morphological and electrophysiological techiques were used to examine granule cells and their mossy fiber axons in nine surgically resected hippocampal specimens from temporal lobe epilepsy (TLE) patients. Timm histochemistry showed mossy fiber sprouting into the inner molecular layer (IML) of the dentate in a subset of tissue samples. In slices from five tissue samples, stimulus-induced bursting activity could be induced with a low concentration (2.5 μM) of bicuculline; bursts were sensitive to the N -methyl- d -aspartate (NMDA) blocker, APV. There was a general correlation between such sprouting and experimentally induced yperexcit ability. Fourteen granule cells from five tissue samples were intracellularly stained [with lucifer yellow (LY) or neurobiotin]. Axons from a subset of these neurons showed axon collaterals reaching into the IML, but this axon projection pattern for single cells was not directly correlated with degree of mossy fiber sprouting shown grossly by Timm staining. Electron microscopic examination of intracellularly stained elements showed mossy fiber axon terminals making asymmetric synaptic contacts (including autapses on the granule cell dendrite) with dendritic shafts and spines in both apical and basal domains. These data are consistent with the hypothesis that mossy fiber sprouting provides a structural basis for recurrent excitation of granule cells, but does not provide direct support of the hypothesis that mossy fiber sprouting causes hyperexcitability. The data suggest that granule cell bursting activity is at least in part a function of compromised synaptic inhibition, since levels of γ-aminobutyric acid (GABA) blockade that are generally subthreshold for burst induction were epileptogenic in some tissue samples from human epileptic hippocampus.     

托吡酯对戊四氮致癫癎大鼠海马AQP4表达水平的影响

目的探讨托吡酯对戊四氮致癫癎大鼠海马AQP4表达水平的影响。方法将30只Wistar大鼠随机分为戊四氮致癫癎组、托吡酯干预组和正常对照组，每组各10只；癫癎模型点燃后在不同时相点灌注取材，通过HE染色观察大鼠海马神经元的变化，并应用免疫组化法检测大鼠海马AQP4表达水平。结果HE染色显示托吡酯干预组神经元变性和坏死较戊四氮致癫癎组明显减轻；免疫组化显示戊四氮致癫癎组在致癫癎后12hAQP4的表达显著增强，致癫癎后24h达高峰，托吡酯干预组在致癫癎后12h～36h各时相点AQP4表达水平均分别低于戊四氮致癫癎组相应时间点（P〈0．05）。结论托吡酯通过下调大鼠海马AQP4的表达可能参与了对大鼠海马神经元的保护过程。     

Activation of the dentate gyrus by pentylenetetrazol evoked seizures induces mossy fiber synaptic reorganization

Kindled seizures evoked by electrical stimulation of limbic pathways in the rat induce sprouting and synaptic reorganization of the mossy fiber pathway in the dentate gyrus (DG). To investigate whether seizures evoked by different methods also induce reorganization of this pathway, the distribution of mossy fiber terminals in the DG was examined with Timm histochemistry after systemic administration of pentylenetetrazol, a chemoconvulsant that reduces Cl- mediated GABAergic inhibition. Myoclonic seizures evoked by subconvulsant doses of pentylenetetrazol (24 mg/kg i.p.) were not accompanied by electrographic seizures in the DG, and did not induce mossy fiber sprouting. Generalized tonic-clonic seizures evoked by repeated administration of PTZ (24 mg/kg i.p.) were consistently accompanied by electrographic seizure activity in the DG, and induced sprouting and synaptic reorganization of the mossy fiber pathway. The results demonstrated that repeated generalized tonic-clonic seizures evoked by pentylenetetrazol induced mossy fiber synaptic reorganization when ictal electrographic discharges activated the circuitry of the DG.     

Association between mossy fiber sprouting and expression of semaphorin-3f protein in dentate gyrus of hippocampus in lithium-pilocarpine-induced status epilepticus mouse model

Objectives: Mossy fiber sprouting is involved in the pathogenesis of mesial temporal lobe epilepsy. But the exact mechanism of formation of mossy fiber sprouting is still unclear. Semaphorin-3f protein could inhibit the growth of neuron axons. The aim of this research is to evaluate the association between semaphorin-3f expression and mossy fiber sprouting.

Methods: We established pilocarpine-induced status epilepticus (PISE) models firstly. Then, mossy fiber sprouting in the hippocampus of PISE models was examined by Timm staining. Expression of semaphorin-3f was evaluated by western blot analysis and immunohistochemical examination. Expression of semaphorin-3f protein in different subregions of hippocampus and its relationship with mossy fiber sprouting were studied.

Results: We found that in PISE group, mossy fiber sprouting appeared in dentate gyrus (DG) region. It started to develop in the latent phase of PISE group and increased significantly in the chronic phase. Expression of semaphorin-3f protein in DG region started to decrease in the latent phase, and stayed at low level in the chronic phase. No such change was found in the other groups.

Conclusions: These results indicate that the decrease in semaphorin-3f expression in DG region was in parallel to the change of mossy fiber sprouting in PISE models, suggesting that mossy fiber sprouting is closely associated with reduced expression of semaphorin-3f in this model.     

Prolonged infusion of cycloheximide does not block mossy fiber sprouting in a model of temporal lobe epilepsy

PURPOSE: The role of protein synthesis in mossy fiber sprouting is unclear. Conflicting reports exist on whether a single dose of the protein synthesis-blocker cycloheximide administered around the time of an epileptogenic injury can block the eventual development of mossy fiber sprouting. METHODS: In rats, osmotic minipumps and cannulae were implanted to deliver 8 mg/ml cycloheximide to one dentate gyrus and vehicle to the other. This method has been used to block protein synthesis in the infused region for up to 5 days with minimal neurotoxic effects (Taha and Stryker, Neuron 2002;34:425-36). After 2 days of infusion, rats were treated with pilocarpine to induce status epilepticus. Pumps were removed 3 days later. Thirty days after pilocarpine treatment, rats were perfused, and hippocampal sections were processed for Timm staining. RESULTS: Timm staining revealed aberrant mossy fiber sprouting in the inner molecular layer regardless of whether hippocampi were treated with cycloheximide or vehicle. Cycloheximide-treated hippocampi displayed more aberrant Timm staining and more tissue damage around the infusion site than did vehicle-treated hippocampi. CONCLUSIONS: Prolonged infusion of cycloheximide, spanning the period of pilocarpine treatment, did not block mossy fiber sprouting. This finding suggests that protein-dependent mechanisms around the time of an epileptogenic injury are not necessary for the eventual development of synaptic reorganization.     

Mossy fiber synaptic reorganization in the epileptic human temporal lobe

The distribution of the mossy fiber synaptic terminals was examined using the Timm histochemical method in surgically excised hippocampus and dentate gyrus from patients who underwent lobectomy of the anterior part of the temporal lobe for refractory partial complex epilepsy. The dentate gyrus of epileptic patients demonstrated intense Timm granules and abundant mossy fiber synaptic terminals in the supragranular region and the inner molecular layer. In contrast, the dentate gyrus of presenescent nonepileptic primates demonstrated no Timm granules in the supragranular region. In nonepileptic senescent primates, occasional very sparse supragranular Timm granules were results are morphological evidence of mossy fiber synaptic reorganization in the temporal lobe of epileptic humans, and suggest the intriguing possibility that mossy fiber sprouting and synaptic reorganization induced by repeated partial complex seizures may play a role in human epilepsy.     

Amygdala kindling develops without mossy fiber sprouting and hippocampal neuronal degeneration in rats

Repeated electrical stimulation of limbic structures has been reported to produce the kindling effect together with morphological changes in the hippocampus such as mossy fiber sprouting and/or neuronal loss. However, to argue against a causal role of these neuropathological changes in the development of kindling-associated seizures, we examined mossy fiber sprouting in amygdala (AM)-kindled rats using Timm histochemical staining, and evaluated the hippocampal neuronal degeneration in AM-kindled rats by terminal deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick end labelling (TUNEL). Amygdala kindling was established by 10.3 +/- 0.7 electrical stimulations, and no increase in Timm granules (neuronal sprouting) was observed up to the time of acquisition of a fully kindled state. However, the density and distribution of Timm granules increased significantly in the dentate gyrus compared with unkindled rats after 29 after-discharges or more than 10 kindled convulsions. In addition, no significant increase in TUNEL-positive cells was found in the hilar polymorphic neurons or in CA3 pyramidal neurons of the kindled rats that had fewer than 29 after-discharges. However, a significant increase of TUNEL-positive cells was found in the granule cell layer in the dentate gyrus of the stimulated side after 18 after-discharges or 10 kindled convulsions. Our result show that AM kindling develops without evidence of mossy fiber sprouting, and that mossy fiber sprouting may appear after repeated kindled convulsions, following death of the granule cells in the dentate gyrus.