氯碘羟喹抑制癫痫小鼠海马神经元凋亡的实验研究

目的研究氯碘羟喹对癫痫持续状态模型小鼠海马神经元凋亡的影响。方法 18只CD1小鼠随机分为3组:对照组(Con)给予腹腔注射生理盐水;癫痫持续状态组(SE)给予腹腔注射匹罗卡品(300mg/kg);氯碘羟喹治疗组(Cli)在腹腔注射匹罗卡品(300mg/kg)后30min给予腹腔注射氯碘羟喹(15mg/kg·d)。每天观察动物的行为学变化,于7d后取海马组织。应用尼氏染色和TUNEL染色检测氯碘羟喹对癫痫持续状态小鼠海马神经元凋亡的影响。结果癫痫持续状态组动物萎靡,自发性痫性发作频繁;氯碘羟喹治疗组动物萎靡状态明显改善,自发性痫性发作次数明显减少。尼氏染色和TUNEL染色发现,与癫痫持续状态组相比,氯碘羟喹治疗组能改善海马神经元的细胞损伤,降低凋亡阳性细胞百分比(<0.01)。结论氯碘羟喹能抑制癫痫持续状态小鼠海马神经元凋亡。     

左乙拉西坦对红藻氨酸致发育期癫痫幼鼠海马主穹窿蛋白表达的影响

目的:建立发育期慢性癫痫大鼠模型,观察海马主穹窿蛋白(MVP)的表达及左乙拉西坦(LEV)对其表达的影响。方法:腹腔注射红藻氨酸(KA)1 mg/kg(浓度0.5 mg/ml),建立大鼠慢性癫痫模型,注射后连续观察8 h,癫痫发作达5级以上并出现癫痫持续状态的大鼠,且两周后出现自发性反复惊厥发作者视为模型成功。将造模成功后的40只大鼠随机分为未治疗组(KA组)20只,左乙拉西坦治疗组(KA+LEV组)20只,另取40只大鼠腹腔注射生理盐水,并分为NS组20只、NS+LEV组20只。用药组均于癫痫自发性反复发作后开始用药,疗程为6周,然后将所有大鼠断头取脑,用免疫组化及RT-PCR法测定大鼠海马内MVP及其mRNA的表达。结果:(1)大鼠海马MVP的阳性细胞计数与MVP的mRNA表达趋势相一致。(2)NS组、NS+LEV组大鼠海马有少量MVP阳性细胞及mRNA表达,NS+LEV组MVP阳性细胞数及mRNA的含量与NS组相比差异无统计学意义(P>0.05);KA组MVP阳性细胞计数及mRNA的含量与NS组相比显著增高(P<0.05);KA+LEV组与KA组相比,MVP的阳性细胞及mRNA含量减少(P<0.05)。结论:MVP可能参与慢性癫痫耐药的发生,LEV可以控制大鼠痫性发作,并下调MVP的表达。     

锂-匹罗卡品致颞叶癫痫小鼠模型的建立及苔藓纤维出芽的实验研究

目的探讨腹腔注射锂-匹罗卡品建立小鼠颞叶癫痫(EP)模型方法及其行为学改变与苔藓纤维出芽的关系。方法小鼠腹腔注射锂-匹罗卡品建立颞叶EP模型,应用ZnSe金属自显影技术(AMG)检测3d、7d、15d、30d及60d的苔藓纤维出芽情况。结果锂-匹罗卡品注射后,约40%的小鼠呈现癫痫持续状态,并出现慢性自发发作。形态学检查发现海马齿状回分子层苔藓纤维出芽,并且随着时间的延长,苔藓纤维出芽逐渐增多。结论腹腔注射锂-匹罗卡品小鼠模型是一种理想的颞叶EP动物模型,苔藓纤维出芽可作为判断SE模型是否成功的形态学标准。     

锌螯合剂对匹罗卡品致痫小鼠行为学及海马神经元的影响

目的:研究锌螯合剂氯碘羟喹对匹罗卡品致痫小鼠行为学及海马神经元的影响。方法:将CD1小鼠随机分成3组,对照组小鼠腹腔注射等量的生理盐水;癫痫组小鼠腹腔注射匹罗卡品300 mg/kg;氯碘羟喹治疗组(治疗组)小鼠腹腔注射匹罗卡品300 mg/kg,30 min之后腹腔注射氯碘羟喹15 mg/kg。观察各组小鼠的癫痫发作次数。Morris水迷宫实验测定逃避潜伏期和目标象限停滞时间。应用尼氏染色计数海马神经元数量;利用免疫组织化学方法、免疫印迹结合图像分析技术检测凋亡基因caspase-3在各组小鼠海马的表达变化。结果:与对照组相比,其他2组小鼠的癫痫发作次数明显增多,发作级别明显增高;其中,治疗组小鼠的发作次数和发作级别均低于癫痫组。水迷宫实验结果显示,与对照组相比,其他2组小鼠的逃避潜伏期明显延长,目标象限停滞时间明显缩短;其中,治疗组的逃避潜伏期比癫痫组短,目标象限停滞时间比癫痫组长。其他2组小鼠海马神经元数量明显少于对照组;治疗组的海马神经元数量比癫痫组多。其他2组小鼠海马caspase-3表达明显高于对照组,阳性细胞数量增多,光密度增加;治疗组海马caspase-3的表达低于癫痫组,阳性细胞数量减少,光密度降低。结论:锌螯合剂氯碘羟喹能抑制癫痫发作,保护海马神经元,提高癫痫小鼠的学习记忆能力。     

头孢曲松对脑损伤后海马谷氨酸及谷氨酸转运体-1的影响

目的:观察β-内酰胺抗生素头孢曲松对创伤性脑损伤大鼠海马谷氨酸(Glutamate,Glu)及谷氨酸转运体-1(Glutamate transporter subtype-1,GLT-1)的影响。方法:将60只健康成年SD大鼠随机分三组:假手术组(Sham组)、脑创伤模型组(TBI组)、头孢曲松组(CTX组),采用改良Feeney法制备大鼠创伤性脑损伤模型,致伤后立即腹腔注射头孢曲松(200mg/kg)。伤后6 h、12 h、24 h及48 h取材,采用干湿比重法测定脑组织含水量;高效液相色谱法检测大鼠海马兴奋性氨基酸Glu水平;免疫组织化学法及Western blot法检测大鼠海马组织GLT-1的分布及表达情况。结果:模型组大鼠较Sham组相比,脑组织含水量明显增加(P0.05),海马区Glu水平显著升高(P0.05),大鼠海马GLT-1的蛋白表达量明显下调(P0.05)。与模型组大鼠比较,头孢曲松治疗组大鼠脑水肿减轻(P0.05),大鼠海马Glu水平明显下降(P0.05),GLT-1的蛋白表达量上调(P0.05)。结论:β-内酰胺抗生素头孢曲松可以阻断兴奋性神经毒性,减轻脑水肿程度。     

卡马西平增加癫痫模型大鼠脑内P-糖蛋白的表达

目的探讨癫痫发作与抗癫痫药物卡马西平(CBZ)对多药耐药基因编码的P-糖蛋白(P-gp)表达的影响。方法给雌性SD大鼠侧脑室注射海人酸(KA)或PBS,成模后再口服CBZ。成模或给药后3、5、7、14、21和28d灌杀大鼠取脑,用免疫组化和双标免疫染色对脑中P-gp进行半定量及定位分析。尼氏染色观察海马神经元的变化。结果模型大鼠脑电图及行为变化与人类癫痫相似,CBZ对癫痫发作没有作用。癫痫发作后第5天脑P-gp表达水平增加,7d时最高,21d后恢复到正常水平。阳性产物主要位于海马的毛细血管内皮细胞,也见于皮层的毛细血管及神经元。CBZ对正常大鼠P-gp的表达没有影响。CBZ KA组动物海马P-gp的表达与KA组动物相似,但7d时其表达明显增强。结论癫痫发作可诱导P-gp的表达;卡马西平在癫痫发作中也参与了P-gp的高表达。     

癫痫部分性发作的基本机制

癫痫部分性发作源于中枢神经系统的某限定区域。许多病因都能导致痫灶的产生。新皮质区的致痫灶通常是因发育改变、肿瘤及其它畸变、脑外伤引起，少数情况下为基因异常引起，而成年后开始的癫痫发作也可能是中风后大脑皮质改变的结果。癫痫部分性发作最常见的形式是始自颞叶内侧结构如杏仁、内嗅皮质和海马，通常以不同程度的海马神经元丢失为特征。当海马的门区，CA1和CA3区细胞丢失少于50％时，颞叶内侧癫痫（mesial temopral lobe epilepsy，MTLE）归类于非海马硬化；而当这些区域细胞丢失大于50％时则是颞叶内侧癫痫综合征伴海马硬化的特征之一。海马硬化经常是由于以前的癫痫持续状态、复杂性热性惊厥、脑炎或者脑缺血损伤引起。在非海马硬化病人，通常在海马门区，     

腺苷A1受体激动剂减轻糖尿病并发抑郁症大鼠的认知功能障碍

目的观察腺苷A1受体(A1R)激动剂对糖尿病并发抑郁症大鼠学习记忆功能的影响。方法将大鼠分为对照组、模型组、二甲双胍(Met)组[Met组:灌胃给予二甲双胍(0.18 g/kg) 28 d]和二甲双胍联合腺苷A1受体激动剂(Met+CPA)干预组[Met+CPA组:灌胃给予二甲双胍28 d,于第22天开始腹腔注射CPA(1 mg/kg),注射给药7 d]。观察大鼠血糖水平和学习记忆功能;ELISA检测大鼠海马谷氨酸的含量;用免疫组化法和Western blot检测海马谷氨酸转运体GLT-1和脑源性神经营养因子(BDNF)的表达。结果与正常组比较,模型组大鼠血糖含量显著升高(P0.01);学习和记忆功能明显下降(P0.01);海马中谷氨酸含量明显升高(P0.01);而GLT-1和BDNF表达显著下降(P0.01或P0.05)。与模型组比较,Met组大鼠仅血糖含量显著下降(P0.01),其余指标均未见显著变化;而Met+CPA组大鼠血糖含量显著下降(P0.01),认知功能得到明显的改善(P0.01),海马中谷氨酸含量显著减少(P0.01),且GLT-1和BDNF表达显著增加(P0.01或P0.05)。结论激活腺苷A1受体可有效改善糖尿病并发抑郁症大鼠的认知功能,这可能与其通过减少谷氨酸释放和增加神经营养作用双途径共同减轻神经元兴奋性毒性有关。     

食欲素A促进短暂性全脑缺血大鼠的海马CA1区谷氨酸摄取并抑制迟发性神经元死亡

目的探讨食欲素A(OX-A)对短暂性全脑缺血(tGCI)大鼠的海马CA1区胶质细胞谷氨酸转运体-1(GLT-1)的表达与功能以及迟发性神经元死亡(DND)的影响及机制。方法将大鼠分为假手术(sham)组、模型(model)组、OX-A组和OX-A+LY294002组。用[~3H]-谷氨酸标记法检测海马CA1区GLT-1的结合能力和对谷氨酸的摄取能力;用谷氨酸定量试剂盒检测海马CA1区组织中谷氨酸含量;用Nissol、NeuN和Fluro-Jade C染色法观察海马CA1区神经元病理改变;用Western blot检测海马CA1区组织中GLT-1、p-PI3K和p-AKT的表达。结果 OX-A能促进tGCI大鼠海马CA1区的GLT-1的结合能力和GLT-1对谷氨酸摄取能力、增加谷氨酸含量、GLT-1表达和PI3K/AKT信号活性并抑制DND(P0.05);而LY294002能减弱OX-A的上述治疗效应(P0.05)。结论 OX-A能通过增强PI3K/AKT信号活性来增加tGCI大鼠海马CA1区GLT-1的表达与功能并减轻DND。     

Acupuncture suppresses kainic acid-induced neuronal death and inflammatory events in mouse hippocampus

The administration of kainic acid (KA) causes seizures and produces neurodegeneration in hippocampal CA3 pyramidal cells. The present study investigated a possible role of acupuncture in reducing hippocampal cell death and inflammatory events, using a mouse model of kainic acid-induced epilepsy. Male C57BL/6 mice received acupuncture treatments at acupoint HT8 or in the tail area bilaterally once a day for 2 days and again immediately after an intraperitoneal injection of KA (30 mg/kg). HT8 is located on the palmar surface of the forelimbs, between the fourth and fifth metacarpal bones. Twenty-four hours after the KA injection, neuronal cell survival, the activations of microglia and astrocytes, and mRNA expression of two proinflammatory cytokines, interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), were measured in the hippocampus. Acupuncture stimulation at HT8, but not in the tail area, significantly reduced the KA-induced seizure, neuron death, microglial and astrocyte activations, and IL-1β mRNA expression in the hippocampus. The acupuncture stimulation also decreased the mRNA expression of TNF-α, but it was not significant. These results indicate that acupuncture at HT8 can inhibit hippocampal cell death and suppress KA-induced inflammatory events, suggesting a possible role for acupuncture in the treatment of epilepsy.     

Acupuncture inhibits kainic Acid-induced hippocampal cell death in mice

We examined whether acupuncture can reduce both the incidence of seizures and hippocampal cell death using a mouse model of kainic acid (KA)-induced epilepsy. ICR mice were given acupuncture once a day at acupoint HT8 (sobu) bilaterally during 2 days before KA injection. After an intracerebroventricular injection of 0.1 microg of KA, acupuncture treatment was subsequently administered once more (total 3 times), and the degree of seizure was observed for 20 min. Three hours after injection, the survival of neuronal cells and the expressions of c-Fos, c-Jun, and glutamate decarboxylase (GAD)-67 in the CA1 and CA3 were determined using immunohistochemistry and Western blotting techniques. Acupuncture reduced the severity of the KA-induced epileptic seizure and the rate of neural cell death, and it also decreased the expressions of c-Fos and c-Jun induced by KA in the hippocampus. Furthermore, acupuncture increased GAD-67 expressions in the same areas. These results demonstrated that it could inhibit the KA-induced epileptic seizure and hippocampal cell death by increasing GAD-67 expressions.     

c-JUN expression and apoptotic cell death in kainate-induced temporal lobe epilepsy

Following kainate (KA)-induced epilepsy, rat hippocampal neurons strongly express immediate early gene (IEG) products, i.e., c-FOS and c-JUN, and neural stress protein, HSP72. Prolonged expression of c-JUN and c-FOS 48 hr after cerebral ischemia has been underwent delayed neuronal death. However, it is not yet clear whether IEGs actually assume the essential roles in the cell death process or simply as a by-product due to external stimuli because of the prolonged expression of c-FOS, more than one week, on intact CA2 neurons of the hippocampus in a KA-induced epilepsy model. This study investigated the relationships between prolonged expression of c-JUN and hippocampal neuronal apoptosis in a KA-induced epilepsy model. Epileptic seizure was induced in rats by a single microinjection of KA (1 microgram/microL) into the left amygdala. Characteristic seizures and hippocampal neuronal injury were developed. The expression of c-JUN was evaluated by immunohistochemistry, and neuronal apoptosis by in situ end labeling. The seizures were associated with c-JUN expression in the hippocampal neurons, of which the level showed a positive correlation with that of apoptosis. Losses of hippocampal neurons, especially in the CA3 region, were partly caused by apoptotic cell death via a c-JUN-mediated signaling pathway. This is thought to be an important component in the pathogenesis of hippocampal neuronal injury via KA-induced epilepsy.     

Neuroprotective potential of ceftriaxone in in vitro models of stroke

Astrocytic glutamate transporters are considered an important target for neuroprotective therapies as the function of these transporters is abnormal in stroke and other neurological disorders associated with excitotoxicity. Recently, Rothstein et al., [Rothstein JD, Patel S, Regan MR, Haenggeli C, Huang YH, Bergles DE, Jin L, Dykes Hoberg M, Vidensky S, Chung DS, Toan SV, Bruijn LI, Su ZZ, Gupta P, Fisher PB (2005) Beta-lactam antibiotics offer neuroprotection by increasing glutamate transporter expression. Nature 433:73-77] reported that beta-lactam antibiotics (including ceftriaxone, which easily crosses the blood-brain barrier) increase glutamate transporter 1 (GLT-1) expression and reduce cell death resulting from oxygen-glucose deprivation (OGD) in dissociated embryonic cortical cultures. To determine whether a similar neuroprotective mechanism operates in more mature neurons, which show a different pattern of response to ischemia than primary cultures, we exposed acute hippocampal slices obtained from rats treated with ceftriaxone for 5 days (200 mg/kg; i.p.) to OGD. Whole-cell patch clamp recording of glutamate-induced N-methyl-d-aspartate (NMDA) currents from CA1 pyramidal neurons showed a larger potentiation of these currents after application of 15 microM dl-threo-beta-benzyloxyaspartic acid (TBOA; a potent blocker of glutamate transporters) in ceftriaxone-injected animals than in untreated animals, indicating increased glutamate transporter activity. Western blot analysis did not reveal GLT-1 upregulation in the hippocampus. Delay to OGD-induced hypoxic spreading depression (HSD) recorded in slices obtained from ceftriaxone-treated rats was longer (6.3+/-0.2 vs. 5.2+/-0.2 min; P<0.001) than that in the control group, demonstrating a neuroprotective action of the antibiotic in this model. The effect of ceftriaxone was also tested in organotypic hippocampal slices obtained from P7-9 rats (>14 days in vitro). OGD or glutamate (3.5-5.0 mM) damaged CA1 pyramidal neurons as assessed by propidium iodide (PI) fluorescence. Similar damage was observed after pre-treatment with ceftriaxone (10-200 microM; 5 days) and ceftriaxone exposure did not result in GLT-1 upregulation as assayed by Western blot. Treatment of slice cultures with dibutyryl cAMP (100-250 microM; 5 days) increased GLT-1 expression but did not reduce cell damage induced by OGD or glutamate. Thus we confirm the neuroprotective effect of antibiotic exposure on OGD-induced injury, but suggest that this action is related to independent modulation of transporter activity rather than to the level of GLT-1 protein expression. In addition, our results indicate that the protective effects of beta-lactam antibiotics are highly dependent on the experimental model.     

The immunosuppressant cyclosporin A inhibits recurrent seizures in an experimental model of temporal lobe epilepsy

The immunosuppressant, cyclosporin A (CsA), is neuroprotective following brain injury. Previous studies suggest that CsA treatment ameliorates seizure severity during status epilepticus (SE) or cell death following SE. The antiepileptic effects of CsA on recurrent seizures, however, have not been investigated. In the present study, the effects of CsA on spontaneous recurrent seizures (SRSs) in a kainate (KA)-induced mouse model of mesial temporal lobe epilepsy (TLE) were examined. Moreover, the effects of CsA on epileptiform activity in a 4-aminopyridine (4-AP)-induced in vitro seizure model were investigated. A mesial TLE mouse model was generated with a unilateral intrahippocampal injection of KA. SRSs were determined in the ipsilateral hippocampal CA1 region with a long-term video-EEG. CsA was systemically administrated to the epileptic mice exhibiting a stable occurrence of SRSs. A 1-mg/kg dose of CsA did not have any effect on SRSs in the epileptic mice. However, a 5-mg/kg dose of CsA significantly reduced the number of SRSs and decreased the severity of the seizures in the epileptic mice. Additionally, CsA treatment inhibited spontaneous burst discharges in 4-AP-treated hippocampal slices. The results of the present study demonstrate that CsA inhibits recurrent seizures in a mouse model of mesial TLE and suggest that CsA may afford both neuroprotection against SE and antiepileptic effects during the chronic period of epilepsy.     

The beta-lactam antibiotic, ceftriaxone, inhibits the development of opioid-induced hyperalgesia in mice

The glutamate transporter GLT-1 is primarily responsible for glutamate clearance in the spinal cord. beta-Lactam antibiotics have been shown to attenuate neuropathic pain behaviors by promoting GLT-1 expression and function in the CNS. The present study tested the hypothesis that ceftriaxone, a prototype beta-lactam antibiotic, can prevent the development of opioid-induced hyperalgesia (OIH) in mice. Repeated morphine administration produced mechanical allodynia and thermal hyperalgesia, signs of OIH, and reduced spinal GLT-1 expression in mice. Ceftriaxone (200 mg/kg/d, i.p., for 7 d) inhibited OIH. Correlating with the behavioral effects, ceftriaxone reversed downregulation of GLT-1 expression that was induced by OIH. These results suggest that ceftriaxone inhibited the development of OIH by up-regulating spinal GLT-1 expression.     

地塞米松对海人酸致（癇）大鼠脑P-糖蛋白表达的影响

目的：探讨地塞米松对海人酸致痫大鼠脑P-糖蛋白（P-gp）表达的影响.方法：将SD大鼠随机分为假手术组（Sham组,n=8）、癫痫组（EP组,n=12）、地塞米松干预癫痫组（DEX组,n=12）.后两组采用海马注射海人酸方法制作癫痫模型,DEX组癫痫造模前30 min给予腹腔注射地塞米松0.4 mg/kg.分别记录各组大鼠达到Ⅲ级和Ⅴ级发作时所需的时间（潜伏期）,初次至第6次≥Ⅳ级发作的间隔时间作为评价癫痫发作严重程度的指标;大鼠术后24 h处死,使用HE染色和免疫组织化学方法,比较各组海马CA3区、齿状回、杏仁核复合体区P-gp表达及脑损伤情况.结果：①Sham 组未见癫痫发作;DEX组与EP组达到Ⅲ级发作的潜伏期分别为（87.92±45.80）min和（67.50±22.91）min,达到Ⅴ级发作的潜伏期分别为（103.33±51.27 ）min和（75.60±22.10）min,差异均无统计学意义（P〉0.05）;与EP组相比,DEX组样发作严重程度降低（P=0.004）;②与EP组相比,DEX组于所观察的脑区损伤均减轻,以海马CA3区和杏仁核复合体区较为显著;③与Sham组比较,EP组各观察脑区P-gp表达均明显升高（P〈0.01）;与EP组相比,DEX组海马CA3区和杏仁核复合体区P-gp表达显著减少（P〈0.05）,而在齿状回表达量差异无统计学意义（P=0.078）.结论：地塞米松可降低海人酸致痫大鼠发作严重程度和脑损伤,抑制P-gp表达上调,其中以海马CA3区和杏仁核区较为显著.     

急性癫痫发作后海马齿状回颗粒细胞的树突改变

目的：为探讨癫痫发作敏感性的形成机制提供形态学依据。方法：海人酸(KA)或戊四氮(PTZ)诱导大鼠短暂癫痫发作后，用高尔基染色法对海马齿状回颗粒细胞的树突形态改变进行观察，并进行图像分析。结果：癫痫大鼠齿状回颗粒细胞树突的树突总长度、树突棘密度、树突分支点数和树突野最大伸展距离等均出现改变；除KA模型外，在PTZ模型中也得到了同样的证实。结论：一次癫痫发作后7d时，海马齿状回颗粒细胞(1)出现了明显增生改变，这可能是癫痫发作敏感性的原因之一；(2)出现的树突增生可能是各类癫痫模型的普遍性的变化。     

Increased afterdischarge threshold during kindling in epileptic rats

The effects of daily electrical kindling stimulation of the perforant pathway were investigated in an excitotoxic rat model of epilepsy with chronic seizures in order to learn whether the preexisting epileptic condition would facilitate or retard kindling. Sprague-Dawley rats with recurrent spontaneous seizures 4-8 months after unilateral intrahippocampal kainic acid (KA) injection were implanted with recording electrodes in the hippocampus and stimulating electrodes in the perforant path. Daily stimulation for 10 s at 5 Hz was given for 15 days. The afterdischarge (AD) threshold and the AD duration of kindled KA rats were compared before and during kindling with those of a kindled control group. In the control group, as expected, mean AD thresholds decreased ( P<0.01), while AD duration progressively increased. Although AD threshold was the same in KA and control groups at the start of kindling, in the KA group a significant increase in threshold occurred from the beginning to the end of kindling ( P<0.01). Behaviorally, KA rats showed stage 4 or 5 seizures on the first stimulation, and stage 3-5 seizures during the remainder of kindling. Paired pulse testing showed facilitation of late components of the dentate gyrus field potential at the beginning of kindling, and suppression of late components at the end, in the KA rats. A significant decrease in the rate of spontaneous seizures in KA rats was noted during the period of kindling ( P=0.04). These results suggest that electrical stimulation of the perforant path may strengthen homeostatic seizure suppressing mechanisms, and may provide insights into novel approaches to the treatment of clinical seizures in temporal lobe epilepsy.