癫痫发作大鼠海马神经元凋亡与caspase-3 mRNA表达的研究

目的 :研究癫大鼠海马神经元凋亡与caspase 3mRNA表达的关系。方法 :采用大鼠红藻氨酸 (KA)致模型 ,以原位末端标记 (TUNEL)检测癫后不同时间海马神经元凋亡 ;RT PCR检测caspase 3mRNA的表达。结果 :KA致后 1d ,海马CA1、CA3及CA4区开始出现凋亡细胞 ,3d时明显增多 ,7d时最多。KA致后 6h ,海马组织caspase 3mRNA表达显著增高 ,1、3、7d仍持续高水平表达。结论 :癫大鼠海马神经元凋亡与caspase 3mRNA的表达密切相关 ,caspase 3在神经元凋亡过程中起着重要的作用     

Differential Regulation of Cytokine Expression Following Pilocarpine-Induced Seizure

While the pathological changes that occur in the brain following seizure have been well characterized, the molecular mechanisms underlying these events are poorly understood. Cell death, reactive gliosis, and axonal sprouting are among the best studied alterations in the epileptic brain. Previous work in both the peripheral and the central nervous systems suggests that cytokines are capable of affecting each of these processes. To better understand the role of cytokines in seizures and their sequelae, we have characterized cytokine expression in an animal model of epilepsy. Using pilocarpine to chemically induce seizures, and RNase protection assays to assess mRNA levels, we have quantified changes in expression of several members of the neuropoietic cytokine family following a single, prolonged seizure. Levels of oncostatin M (OSM), leukemia inhibitory factor (LIF), cardiotrophin-1, and ciliary neurotrophic factor were all increased in the hippocampus after seizure, though to differing extents and with markedly different time courses. Cells expressing the most dramatically up-regulated cytokines, LIF and OSM, were identified by combined in situ hybridization and immunohistochemistry. The majority of LIF(+) cells in the hippocampus were glial fibrillary acidic protein(+) astrocytes, while the majority of OSM(+) cells had the morphology of interneurons and were occasionally colabeled with neurofilament markers. Both the time course and the localization of cytokine up-regulation following seizure suggest possible roles for these intercellular signaling molecules in epilepsy.     

NDRG2通过Hes1参与癫痫发作后海马齿状回的神经发生

目的 探讨N-Myc下游调节基因2(N-Myc downstream regulated gene 2,NDRG2)与癫痫发作后海马齿状回神经发生的关系。方法 C57BL/6小鼠20只,随机分为癫痫组和对照组,每组又分为癫痫造模后1和7 d两个时间点,每个时间点5只,通过蛋白免疫印迹检测癫痫后海马齿状回NDRG2蛋白相对表达水平和mRNA相对表达水平变化; 使用双皮质素(DCX)染色标记未成熟神经元,神经巢蛋白(Nestin)标记神经干细胞,神经核蛋白(NeuN)标记成熟神经元,观察NDRG2对海马齿状回神经干细胞增殖影响; 采用RT-PCR检测发状分裂相关增强子1(hairy and enhancer of split 1,Hes 1)、NDRG2 mRNA相对表达表达水平,并分析两者之间的相关性; 观察NDRG2参与癫痫发作后神经发生的可能机制。结果 癫痫组与对照组比较,DCX、Nestin、NeuN、Hes1、NDRG2蛋白相对表达水平在1和7 d这2个时间点有显著性增高,并随时间逐渐递增。结论 癫痫发作后海马NDRG2蛋白相对表达水平增高,与癫痫发作后海马齿状回的神经细胞增值时间具有一致性和相关性,NDRG2可能参与癫痫发作后海马齿状回的神经发生过程; 同时发现海马NDRG2表达增加和Hes1分子表达增加具有相关性,故推测NDRG2可能通过Hes1参与癫痫发作后海马齿状回的神经发生。     

酸敏感离子通道2a对缺血预处理诱导大鼠海马缺血耐受的作用

目的探讨脑缺血时的组织病理学变化及酸敏感离子通道2a（ASIC2a）在缺血预处理诱导的脑缺血模型耐受中的作用。方法取成年雄性SD大鼠160只,随机分为假手术组、预缺血组、缺血组、缺血预处理组共4组。行焦油紫染色观察各组大鼠海马CAI区存活神经元密度,TUNEL染色观察大鼠海马CA1区神经元凋亡情况,RT—PCR和Western blotting检测ASIC2a在大鼠海马CAl区mRNA和蛋白表达情况。结果缺血预处理能够显著减少大鼠海马CA1区锥体神经元的死亡和凋亡,PC＋Isch组和Isch组相比具有显著性差异（P〈0．01）。全脑缺血能够上调ASIC2a在大鼠海马CA1区mRNA和蛋白表达,在24h达到高峰,而缺血预处理进一步上调ASIC2a表达,呈进行性上升,在24h和72h时相点,PC＋Isch组和Isch组相比具有显著性差异（P〈0．01）。结论在脑缺血耐受中,缺血预处理对第二次致死性缺血表现出保护作用。在这个过程中,大鼠海马CA1区ASIC2a基因和蛋白表达上调发挥了重要的保护作用。     

红藻氨酸致痫大鼠海马神经元凋亡及其与caspase-3关系的研究

目的　研究大鼠癫痫发作后海马神经元凋亡及其与天冬氨酸特异性半胱氨酸蛋白酶 -3 (cysteinylasparate-specific proteinase,caspase-3 )表达的关系。方法　采用红藻氨酸 (kainic acid,KA)诱导大鼠癫痫模型 ,以原位末端标记 (TUNEL)及透射电镜检测癫痫发作后 6h及 1、3、7d海马神经元凋亡 ;半定量 RT-PCR及免疫组化法检测 caspase-3 m RNA及 caspase-3阳性表达。结果　KA致痫后 1 d,海马 CA1、CA3及 CA4区开始出现凋亡细胞 ,3 d时明显增多 ,7d时最多。 3个时间组相应区域间凋亡神经元数比较差异均有显著性 (P<0 .0 0 1 )。透射电镜观察可见典型的凋亡细胞形态学改变。 RT-PCR结果显示 ,KA致痫后 6h,海马组织 caspase-3 m RNA表达较对照组显著增高 (P <0 .0 5 ) ,1、3、7d caspase-3 m RNA仍持续高水平表达 (P <0 .0 5 )。免疫组化结果显示 ,KA致痫后 1 d,海马 CA1、CA3、CA4区开始出现 caspase-3阳性表达 ,3 d时阳性表达进一步增强 ,7d时表达最强。结论　凋亡参与 KA致痫大鼠癫痫发作后海马神经元迟发性死亡过程 ,caspase-3可能在癫痫后神经元凋亡过程中具重要的作用。     

Decreased epileptic susceptibility correlates with neuropeptide Y overexpression in a model of tolerance to excitotoxicity

Prior epileptic episodes have been shown to decrease markedly the neuronal damage induced by a second epileptic episode, similar to the tolerance following an episode of mild ischemia. Endogenous neuroprotective effects mediated by various mechanisms have been put forward. This study investigated whether neuroprotection against the excitotoxic damage induced by re-exposure to an epileptic challenge can reflect a change in epileptic susceptibility. Tolerance was elicited in rats by a preconditioning session using intrahippocampal kainic acid (KA) administration followed at 1, 7 and 15-day intervals by a subsequent intraventricular KA injection. The degree of pyramidal cell loss in the vulnerable CA3 subfield contralateral to the KA-injected hippocampus was extensively reduced in animals experiencing KA ventricular administration. This neuroprotection was highly significant 1 and 7 days after injection, but not 15 days after injection. In preconditioned animals, the after-discharge threshold was assessed as an index of epileptic susceptibility. It increased significantly from 1 to 15 days after intrahippocampal KA administration. Finally, an enhancement of neuropeptide Y expression in both non-principal cells and mossy fibers was detected, occurring at the same time as the decrease in epileptic susceptibility. These results provide further evidence of an 'epileptic tolerance' as shown by the substantial neuroprotective effect of a prior episode of epileptic activity upon subsequent epileptic insult and suggest that the prevention of excitotoxic damage after preconditioning results from an endogenous neuroprotective mechanism against hyperexcitability and seizures.     

Expression, interaction, and proteolysis of death-associated protein kinase and p53 within vulnerable and resistant hippocampal subfields following seizures

Death-associated protein (DAP) kinase is a novel regulator of cell death whose in vivo target(s) and role in neuronal cell death remain uncertain. Since DAP kinase has been implicated in p53-mediated apoptosis, a pathway activated following epileptic brain injury, we examined the relationship between DAP kinase and p53 following seizures. Rats underwent brief (40-min) seizures evoked by intraamygdala kainic acid, which caused the death of ipsilateral CA3 neurons while preserving the contralateral CA3 subfield. Seizures caused a small decline in levels of the approximately 160-kD DAP kinase within injured ipsilateral hippocampus, commensurate with the appearance of an approximately 60-kD fragment, and proteolysis of the p53 inhibitor, murine double minute gene 2 (MDM2). Expression of p53 increased within the ipsilateral hippocampus, and DAP kinase was detected within p53 immunoprecipitates. In contrast, DAP kinase and MDM2 were not proteolyzed within the seizure damage-resistant contralateral hippocampus. Furthermore, DAP kinase and p53 did not interact within the contralateral hippocampus, and p53 cellular localization redistributed from the nucleus to cytoplasm commensurate with p53 proteolysis. These data suggest that DAP kinase may be involved in the p53 pathway during seizure-induced neuronal death.     

Involvement of extracellular regulated kinase and p38 kinase in hippocampal seizure tolerance

The mechanisms underlying brain seizure tolerance, a phenomenon in which brief periods of seizures protect brain against the lethal effects of subsequent sustained seizures, are poorly understood. Because brain seizure tolerance and brain ischemia tolerance likely share certain common mechanisms, the recent evidence that activation of extracellular regulated kinase (ERK) and p38 kinase pathways plays a critical role in ischemic preconditioning suggests that a similar mechanism may underlie brain seizure tolerance. We investigated the hypothesis in a rat kainic acid preparation of seizure preconditioning and tolerance, which was established by induction of one episode of priming epileptic status lasting for 20 min on the first day and another episode of sustained epileptic status lasting for 2 hr on the second day. We observed that acute seizures lead to a rapid activation of ERK and p38 in the hippocampal CA3 area, the brain region most susceptible to the lethal effects of epileptic status. Pretreatment with the ERK inhibitor PD98059 and the p38 inhibitor SB203580 selectively reduces seizure-elicited activation of ERK and p38, respectively, and significantly reduces priming seizure-induced protection of CA3 neurons. These findings indicate that, similar to brain ischemia tolerance, brain seizure tolerance also involves the ERK and p38 signaling pathways.     

红藻氨酸致惊大鼠海马神经元Caspase—3表达与神经元凋亡

目的：研究Caspase-3在红藻氨酸（Kainate,KA)致惊大鼠海马中的变化及其在海马神经元凋亡中的作用。方法：在KA所致大鼠惊厥模型中，用免疫组织化学方法检测惊厥后不同时间点大鼠海马中Caspase-3的表达，用电子显微镜和原位末端标记法（TUNEL）检测惊厥后不同时间点大鼠海马神经元凋亡。结果：惊厥后1d,大鼠海马内Caspase-3的表达就明显升高，一直持续到惊厥3d；大鼠海马内凋亡细胞从惊厥后3d即明显增多，一直持续到惊厥后7d。结论：KA所致惊厥后，大鼠海马内Caspase-3表达明显升高，神经元凋亡明显增多，而且Caspase-3的变化发生在神经元亡增多之前，提示Caspase-3可能参与了KA致惊厥大鼠海马神经元凋亡的发生。     

Regional brain glucose metabolism in chemically-induced seizures in the rat

Measurement of regional brain glucose metabolism may give information concerning the mechanism of neuronal cell death developing after prolonged periods of epileptic activity. Regional brain glucose utilization was measured in paralyzed ventilated rats during seizures induced by L-allylglycine, kainic acid and bicuculline using the [14C]deoxyglucose method. Regional brain glucose concentration was measured in another series of rats, after similar periods of seizure activity, to permit a more accurate calculation of the lumped constant. In L-allylglycine-induced seizures regional brain glucose concentration did not vary from control values, so no correction of the lumped constant was necessary. Regional brain glucose utilization increased throughout the brain, the largest increase being in the hippocampus (control 36 +/- 6 mumol 100 g-1 min-1; seizure 120 +/- 12 mumol 100 g-1 min-1). In kainic acid-induced seizures, brain glucose concentration fell in the hippocampus, involving some correction of the lumped constant. Increases in glucose utilization were limited primarily to the hippocampus, with some involvement of the inferior colliculus. The ventral hippocampus showed the largest increase in glucose utilization (control 34 +/- 5 mumol 100 g-1 min-1; seizure 167 +/- 10 mumol 100 g-1 min-1). In bicuculline-induced seizures, in starved rats, brain glucose concentration fell in all regions investigated and no increase in regional glucose utilization was recorded. In L-allylglycine and kainic acid-induced seizures, the hippocampus, a region vulnerable to neuronal damage, shows the largest increase in glucose utilization. Studies involving bicuculline need further investigation, due to severe perturbation of brain and plasma glucose concentration.     

癫痫患者血清和脑脊液神经元特异性烯醇化酶的测定

目的 探讨癫痫发作对脑神经元的损伤。方法 应用酶联免疫反应法动态测定癫痫发作后患者血清和脑脊液（CSF）中神经元特异性烯醇化酶（NSE）的含量。结果 癫痫组血清和CSF中NSE含量在发作后明显升高，血清NSE水平在发作后第1天最高，1周左右开始下降，2周左右降至正常；抽搐发作及频繁发作时，血清和CSF中NSE升高更明显。结论 癫痫发作引起血清和CSF中NSE水平的升高；癫痫发作导致神经元损伤，抽搐发作及频繁发作时神经元损伤更严重。     

Glutamate transporters alterations in the reorganizing dentate gyrus are associated with progressive seizure activity in chronic epileptic rats.

The expression of glial and neuronal glutamate transporter proteins was investigated in the hippocampal region at different time points after electrically induced status epilepticus (SE) in the rat. This experimental rat model for mesial temporal lobe epilepsy is characterized by cell loss, gliosis, synaptic reorganization, and chronic seizures after a latent period. Despite extensive gliosis, immunocytochemistry revealed only an up-regulation of both glial transporters localized at the outer aspect of the inner molecular layer (iml) in chronic epileptic rats. The neuronal EAAC1 transporter was increased in many somata of individual CA1-3 neurons and granule cells that had survived after SE; this up-regulation was still present in the chronic epileptic phase. In contrast, a permanent decrease of EAAC1 immunoreactivity was observed in the iml of the dentate gyrus. This permanent decrease in EAAC1 expression, which was only observed in rats that experienced progressive spontaneous seizure activity, could lead to abnormal glutamate levels in the iml once new abnormal glutamatergic synaptic contacts are formed by means of sprouted mossy fibers. Considering the steady growth of reorganizing mossy fibers in the iml, the absence of a glutamate reuptake mechanism in this region could contribute to progression of spontaneous seizure activity, which occurs with a similar time course.     

Expression and differential processing of caspases 6 and 7 in relation to specific epileptiform EEG patterns following limbic seizures

The caspase family of cell death proteases has been implicated in the mechanism of neuronal death following seizures. We investigated the expression and processing of caspases 6 and 7, putative executioner caspases. Brief limbic seizures were evoked by intraamygdala kainic acid to elicit unilateral death of target hippocampal CA3 neurons in the rat. Seizures rapidly induced cleavage of constitutively expressed caspase-6, followed by elevated VEIDase activity and the proteolysis of lamin A. Neuronal caspase-6 immunoreactivity was markedly upregulated within cortex and hippocampus in relation to bursts of polyspike paroxysmal discharges. In contrast, while caspase-7 expression also increased within cortical and hippocampal neuronal populations in response to the same seizure patterns, caspase-7 was not proteolytically activated. These data highlight differences in expression and activation of caspases 6 and 7 in response to identifiable seizure patterns, focusing potential therapeutic targets for neuroprotection in epilepsy.     

Experimental complex partial seizure and its possible clinical application

Clinical applications of experimental models of complex partial seizure were studied using kainic acid-induced limbic seizures and amygdaloid kindling models. The following experiments were done aiming to study the basic approach for the treatment of the intractable complex partial seizures. 1) Degenerative focal lesions were made in bilateral substantia nigra and substantia innominata by a local microinjection of the ibotenic acid and influences upon limbic seizures were studied. Substantia innominata has a facilitatory effect upon secondary generalization of the limbic seizure while substantia nigra has an inhibitory influence. Degenerative lesions of the bilateral hippocampus inhibited development process as well as establishment of the kindling. 2) Resection of the primary epileptic focus in a limbic seizure status resulted in seizure control in cats with a single focus but not in another with multiple foci. 3) An autoradiography was done during limbic seizure status induced by kainic acid microinjection, and local cerebral glucose utilization (LCGU) and local cerebral blood flow were studied in order to study the relationship between cerebral metabolism and cerebral blood flow during limbic seizures. In the pyramidal cell of the hippocampus, an increased ratio of LCGU (x 4.1) is larger than that of LCBF (x 1.6). This uncoupling may be one reason of the neuronal cell damage during the limbic seizure status. 4) Autoradiography of the calcium suggested that one of the causes of hippocampal degeneration in intractable complex partial seizures should be a consequence of calcium influx into pyramidal cells during repeated limbic seizures.     

海人酸致大鼠海马结构GABA_B受体亚单位表达的研究

目的:探讨海人酸诱导大鼠颞叶癫(EP)发作后2种γ-氨基丁酸(GABA)受体亚单位GABABR亚单位1a(GBR1a)和GABABR亚单位2(GBR2)在EP发生、发展中的作用。方法:运用原位杂交及免疫组化法,检测EP发作后GABABR亚单位mRNA及蛋白在海马的表达。结果:致早期CA1和CA3区2种亚单位mRNA表达持续低下后逐渐增加,DG区则暂时性下降后很快回升;而免疫反应早期却未见明显改变,随后CA1和CA3区表达处于低水平,DG区和颞叶皮质表达下降后很快恢复。结论:致后2种GABAB受体亚单位基因和蛋白表达上调为颞叶EP的内源性自我保护机制。     

Expression time course and spatial distribution of activated caspase-3 after experimental status epilepticus: contribution of delayed neuronal cell death to seizure-induced neuronal injury

Pilocarpine-induced status epilepticus (PCSE) is a widely used model to study neurodegeneration in limbic structures after prolonged epileptic seizures. However, mechanisms mediating neuronal cell death in this model require further characterization. Examining the expression time course and spatial distribution of activated caspase-3, we sought to determine the role of apoptosis in PCSE-mediated neuronal cell death. Expression of activated caspase-3, predominantly located in neurons, was detected 24 h (amygdala; piriform and temporal cortex) and 7 days (hippocampus; amygdala; piriform, temporal and parietal cortex; thalamus) after PCSE with strongest induction being observed in the amygdala, the piriform cortex, and the hippocampus. Further analysis revealed TUNEL positivity (24 h and 7 days after SE) and a significant, progressive neuronal cell loss in all brain regions displaying caspase-3 activation. Corresponding to high levels of activated caspase-3 expression, neuronal cell loss was most pronounced in the amygdala, piriform cortex, and dorsal CA-1 hippocampus. These results demonstrate that apoptosis contributes significantly to PCSE-induced neuronal cell death.