【作者】 杨璐；

【导师】 孙若鹏；

【作者基本信息】 山东大学 ， 儿科学， 2010， 博士

【摘要】 研究背景癫痫持续状态(SE)是指一次癫痫发作持续30分钟以上,或连续多次发作、发作间期意识和神经功能未恢复至通常水平,是神经科常见急症之一,若处理稍有不当,就会给患者带来终身残疾,甚至危及生命。从年龄分布看,SE在婴幼儿期发病率高：37%的SE发生于1岁以内,73%发生于3岁以内,83%发生于5岁以内。幼年时期发生的SE是否会损伤患儿的脑功能、影响患儿以后的智力行为发育是儿科医生和患儿家属关注的问题。早年的临床研究及动物实验表明：与成年人(鼠)相比,SE对幼儿(鼠)的未成熟脑造成的损伤较小。这具体表现在：与成年人相比,发生SE的幼儿死亡率低、神经系统后遗症发生率低、较少遗留神经行为缺陷；对SE模型的研究表明幼鼠SE后几乎无神经元损伤、苔藓纤维出芽等神经病理改变。然而,对癫痫二次打击模型的研究说明未成熟脑并非完全不受SE的影响。新生期有惊厥史的大鼠在青春期SE后,其脑损伤较新生期无惊厥大鼠更严重。同样,生后15天(P15)和生后45天(P45)各经历一次SE的大鼠,神经元损伤和空间学习记忆能力损伤比只在P45经历SE的大鼠严重。二次打击模型的研究结果说明幼年期的SE导致大脑对成年期SE的易损性增强。Somera-Molina发现大鼠在P15经历SE后海马区小胶质细胞短暂激活,星形胶质细胞可持续激活,且胶质细胞激活是导致大鼠对P45第二次SE的易损性增加的原因。然而激活的胶质细胞导致易损性增高的机制目前尚不明确。小胶质细胞和星形胶质细胞的激活表现为细胞形态的改变和一些特定的标志物的表达(小胶质细胞标志物为Iba-1,星形胶质细胞标志物为GFAP、S100β)。小胶质细胞激活是一个复杂的过程,包括药理学和电生理特性的改变、细胞迁移和增殖的改变以及释放一系列的炎症介质。激活的星形胶质细胞也有多种功能改变,包括谷氨酸的释放功能、代谢功能和转运功能、以及对细胞外水和钾离子的调节功能的变化；此外星形胶质细胞和小胶质细胞一样,也是脑组织内产生炎症因子的主要来源之一。越来越多的证据表明炎症反应参与了许多急性和慢性脑部疾病,其中包括与惊厥相关的神经系统疾病。在颞叶癫痫手术切除组织中可检测到炎症物质的表达增高。在颞叶癫痫的动物模型中也观察到海马区炎性细胞因子和固有免疫的标志物升高(白介素1β、白介素6、肿瘤坏死因子α、核因子κB、环氧合酶2、前列腺素、Toll样受体),这些物质在SE后1小时即可表达增高且可以持续数天。但对于惊厥后细胞因子表达的研究多集中于成年人或成年动物,对未成熟大脑的研究较少。对幼年期大鼠在SE之后海马细胞因子的表达改变尚未有系统研究,且海马细胞因子与海马易损性增强之间的关系尚有待于明确。幼年儿童经历一次癫痫持续状态后应该应用抗癫痫药物进行治疗。本研究将从脑保护角度探索,幼年期大鼠经历一次SE后给予不同抗癫痫药物,对大鼠海马部位的胶质细胞激活情况及细胞因子表达情况是否有所影响,以及应用抗癫痫药物后海马对第二次惊厥打击的易损性是否有所改变。纳洛酮是一种在儿科应用较广泛的药物,用于新生儿窒息的复苏及促醒。近几年的研究发现纳洛酮具有抑制炎症反应和保护神经元的作用。介于P15大鼠在SE后短期内小胶质细胞激活、而星形胶质细胞长期激活,且胶质细胞可产生细胞因子,我们猜想应用纳洛酮也可抑制幼年期SE后海马内的炎症反应,并对海马的易损性产生一定的影响。目的1研究幼年期(P15)大鼠经历SE后,在急性期和慢性期,海马区各种细胞因子在翻译和蛋白表达水平上的变化。2应用相应的细胞因子抗体研究细胞因子与海马易损性增强之间的关系。3在P15大鼠经历SE后,连续给予苯巴比妥、丙戊酸钠、托吡酯、拉莫三嗪、左乙拉西坦进行单药治疗,观察慢性期星形胶质细胞激活情况和细胞因子的变化及应用这些药物后大鼠对二次惊厥打击易损性的变化。4在P15大鼠经历SE后,给予纳洛酮治疗,观察急、慢性期胶质细胞激活情况和细胞因子的变化及大鼠对二次惊厥打击易损性的变化。方法1幼年期(P15)大鼠经历SE后海马细胞因子表达的改变：将P15大鼠随机分为2组,SE组(60只),对照组(60只)；SE组和对照组又各自分为6小组(每小组10只),在不同的时间点处死,取出海马组织进行细胞因子的检测。SE组给予海人藻酸(KA) 5mg/kg腹腔注射致SE,对照组给予同样体积的生理盐水腹腔注射。在模型建立后12小时、24小时、3天、10天、20天、30天6个时间点,分别取SE组和对照组中的10只大鼠,提取海马中的蛋白和1mRNA,应用酶联免疫吸附实验(Elisa)和荧光实时定量聚合酶链反应(RT-PCR)检测白介素1β(IL-1β)、白介素2(IL-2)、白介素4(IL-4)、白介素6(IL-6)、白介素10(IL-10)、白介素12(IL-12)、肿瘤坏死因子α(TNFα)、干扰素γ(IFNγ)的表达。2表达增高的细胞因子与大鼠海马易损性增强之间的关系：将P15大鼠分为SE组(10只)、干预组(40只)、对照组(10只)。其中干预组分为4小组(每小组10只)。SE组给予KA 5mg/kg腹腔注射致SE；干预组分别在KA致SE后12小时给予IL-1β抗体侧脑室内注射,20天时给予IL-1β抗体、IL-10抗体、TNFα抗体侧脑室内注射；对照组在P15时给予同等体积生理盐水腹腔注射。各组大鼠在P45时给予KA 15mg/kg腹腔注射致SE,P50时行Morris水迷宫测试,P55时原位末端标记(TUNEL)法标记海马凋亡细胞。3抗癫痫药物对幼年期SE后慢性期星形胶质细胞激活、细胞因子表达及易损性的影响：将P15大鼠分为6组,SE组、苯巴比妥治疗组、丙戊酸钠治疗组、托吡酯治疗组、拉莫三嗪治疗组、左乙拉西坦治疗组(每组20只)。SE组大鼠给予KA腹腔注射致SE。五个药物治疗组分别在P15经历SE后,连续给药7天(苯巴比妥30mg/kg/天、丙戊酸钠200mg/kg/天、托吡酯40mg/kg/天、拉莫三嗪50mg/kg/天、左乙拉西坦200mg/kg/天)。在P45每组取10只大鼠对星形胶质细胞标志物(GFAP)和细胞因子进行检测,每组剩余10只给予KA 15mg/kg腹腔注射后致第二次SE,P55时检测海马凋亡细胞。4纳洛酮对幼年期经历SE大鼠的保护作用：P15大鼠分为SE组、纳洛酮治疗组、对照组。SE组大鼠P15时给予KA 5mg/kg致SE,纳洛酮治疗组大鼠在P15时致SE后皮下植入微泵12小时内泵入不同剂量纳洛酮,生理盐水对照组大鼠腹腔注射与KA同等体积生理盐水。应用免疫印迹检测大鼠海马区急慢性期胶质细胞标志物(Iba-1、GFAP、S100β)表达情况,应用酶联免疫吸附试验、荧光实时定量PCR技术检测细胞因子表达情况。各组大鼠在P45时均再次给予KA 15mg/kg腹腔注射致SE,P50时行Morris水迷宫测试,P55时观察比较海马区凋亡细胞。结果1幼年期(P15)大鼠SE后细胞因子表达的改变：幼年期SE可导致海马区细胞因子在不同时段的改变。IL-1β在幼年期SE后早期(P<0.001)及慢性期(P<0.001)均表达显著增高,而IL-10(P<0.01)和TNFα(P<0.01)在幼年期SE后慢性期较对照组大鼠表达显著增高。其余细胞因子在各检测时间点无明显表达改变(P>0.05)。2表达增高的细胞因子与大鼠海马易损性增强之间的关系：SE组大鼠(在P15和P45均经历SE)水迷宫测试表现差于对照组大鼠(只在P45经历SE)(P<0.001)、SE组大鼠海马区凋亡细胞数显著高于对照组大鼠(P<0.01)；大鼠在P15经历SE后12小时和20天侧脑室内注射IL-1β抗体均可改善大鼠在水迷宫测试中的表现(P<0.01)、显著减少其在P45第二次SE后海马区凋亡细胞数(P<0.05)；在P15大鼠经历SE后20天海马内注射IL-10抗体可加剧大鼠在水迷宫测试中空间记忆能力减退(P<0.05)、明显增加其在P45第二次SE后海马凋亡细胞(P<0.05)；而SE后20天侧脑室内注射TNFα抗体可改善大鼠在水迷宫测试中的表现(P<0.05)、显著减少大鼠在P45第二次SE后海马凋亡细胞(P<0.01)。3抗癫痫药物对幼年期SE后慢性期星形胶质细胞激活、细胞因子表达及易损性的影响：苯巴比妥和拉莫三嗪治疗对P15大鼠经历SE后慢性期GFAP表达、细胞因子表达及易损性均无显著影响(P>0.05)。丙戊酸钠治疗对慢性期GFAP表达无明显影响(P>0.05),但可减少慢性期海马内IL-1β、IL-10、TNFα的表达(P<0.05),并减少其在P45时SE后凋亡细胞数(P<0.01)。托吡酯治疗组大鼠与SE组大鼠相比,GFAP和细胞因子表达无明显差异(P>0.05),但托吡酯治疗组大鼠在P45经历SE后海马区凋亡细胞数显著少于SE组大鼠(P<0.05)。左乙拉西坦治疗组大鼠慢性期海马内GFAP含量较SE组明显减少(P<0.01),且IL-1β、TNFα表达显著减少(P<0.01),在经历P 45时SE后海马内凋亡细胞数少于SE组大鼠(P<0.01)。4纳洛酮对幼年期经历SE大鼠的保护作用：P15大鼠经历SE后12小时内皮下微泵注射纳洛酮可显著降低急性期海马内Iba-1、GFAP、S100β表达水平(P<0.01),降低慢性期GFAP、S100β表达水平(P<0.001)。纳洛酮治疗可使急性期海马内IL-1β和慢性期海马内IL-1β、TNFα、IL-10表达减少(P<0.01)。纳洛酮治疗可改善大鼠在P 45第二次SE后在Morris水迷宫测试中的表现(P<0.01),使海马区凋亡细胞数显著减少(P<0.01)。结论1大鼠在幼年期(P15)经历SE可导致海马区急性期IL-1β表达增高,慢性期海马区IL-1β、IL-10、TNFα表达增高。2大鼠在P15经历SE后急性期及慢性期海马区增高的IL-1β增加了海马对P45时第二次SE的易损性；慢性期增高的IL-10对海马起保护作用,可以减轻海马对第二次SE打击的易损性；慢性期增高的TNFα可增加海马对成年期二次惊厥的易损性。3幼年期(P15)大鼠经历SE后给予苯巴比妥、拉莫三嗪治疗对于其海马易损性增强无改善作用；丙戊酸钠可通过抑制慢性期海马内细胞因子表达减轻其易损性；托吡酯虽然对星形胶质细胞激活和细胞因子表达无影响,仍可减轻海马易损性；左乙拉西坦可抑制慢性期海马内星形胶质细胞激活和细胞因子高表达并减轻海马易损性。4幼年期SE后急性期内给予纳洛酮治疗可以抑制急慢性期胶质细胞激活,并抑制急慢性期细胞因子的高表达,减轻大鼠对成年期第二次SE打击的易损性。更多还原

【Abstract】 BackgroundStatus epilepticus(SE) is defined as one continuous unremitting seizure lasting longer than 30 minutes, or recurrent seizures without regaining consciousness between seizures for greater than 30 minutes. It is one of the common emergencies in neurology department. The mortality rate and disability rate of SE is very high especially if treatment is not intiated quickly and properly. A high proportion of SE occur in younger people:37% occur in infants,73% in children younger than 3 years old,83% in children younger than 5 years old. Whether SE in childhood would change brain architecture and function and further influence intelligence and behavior development is a concern by parents and pediatricians. Previous clinical and animal studies indicated the immature brain is relatively resistant to seizure-induced brain damage. The mortality and incidence of sequelae following SE are low in children in the absence of an acute neurologic insult or progressive neurologic disorder. Similarly, long-term behavioral deficits or neuropathologic changes have not been found in immature rats in several seizure models. However, several two-hit seizure model studies demonstrate that the immature brain does not appear to be immune to injury following prolonged seizures. Schmid et al. reported that SE in adolescent rats with a history of neonatal seizures caused substantially more damage than in animals without a history of neonatal seizures. Likewise, Koh et al. found that although SE on postnatal day (P) 15 did not result in any detectable cell death or impairment of spatial learning, it predisposed rats to more extensive neuronal injury and worse performance in Morris water maze after the second seizure on P45. Recently, Somera-Molina explored the molecular events after kainic acid (KA)-induced early life seizures and found that transient microglial activation and persistent astrocyte activation may result in increased vulnerability to seizures in adulthood. However, the definite mechanism by which activated glia lead to increased vulnerability is unclear.Microglia and astrocyte activation are demonstrated as cell morphology change and expression of specific markers(Iba-1 for microglia and GFAP for astrocytes). Microglia activation is a complex process, including changes in pharmacological and electrophysiological properties, changes in migration and proliferation. Activated astrocytes also show many functional alterations, including glutamate release function, metabolism of glutamate, glutamate transport, water and potassium balance regulation. Apart from all above function changes, microglia and astrocytes are the two main sources of inflammatory factors in brain. More and more studies reveal that inflammation is involved in many acute and chronic brain diseases, including the diseases related to seizure. The expression of inflamamtory factors are elevated in resected tissue from temporal lope epilepsy patients. Likewise, inflammatory cytokines and markers for innate immunity(IL-1β, IL-6, TNFα, NF-κB system, COX-2, prostaglandins, Toll-like receptors) are upregulated in 1 hour after SE and persist for several days in hippocampus in temporal lope epilepsy animal models. The information about cytokine expression after seizure are mostly from studies in human adults and adult rats. There are few studies on immature brain. The cytokine expression after SE in immature brain have not been widely explored. The relationship between cytokine expression and increased vulnerability deserves further studies.Antiepileptic drugs are necessary after for children after SE in clinical practice. In this study, we will deliver several different antiepileptic drugs after SE in immature rats and observe whether these drugs would change cytokine expression and alleviate the increased vulnerability to second SE.Naloxone is a traditional drug in pediatrics, mainly used in neonatal resuscitation and wake promoting. Recent studies find that naloxone possesses inflammation-inhibitory and neuron protective property. Because transient microglial activation and persistent astrocyte activation appear after SE in P15 rats, we suppose that naloxone could inhibit inflammatory reaction in hippocampus after P15 SE and further change the increased vulnerability to second SE hit in adulthood.Objectives1. To investigate cytokine transcription and expression at acute and chronic stage after P15 SE.2. To explore the relationship of upregulated cytokines and increased vulnerability after P15 SE by application of cytokine antibodies.3. To observe after P15 SE, the effects of phenobarbital, depakine, topiramate, lamotrigine, levetiracetam monotherapy on chronic cytokine expression and vulnerability to second SE hit.4. To test the influence of naloxone on glia activation, cytokine expression and increased vulnerability after P15 SE.Methods1. Cytokine expression after P15 SE:P15 rats were randomly assigned to two groups:SE group(n=60) and Control group(n=60). Rats in SE group and Control group were further randomly divided into 6 groups(n=10 in each group) and sacrificed at 6 different time points for cytokine detection in hippocampus. Rats in SE group were intraperitoneally injected with kainic acid(KA) 5mg/kg to induce SE. Rats in control group were intraperitoneally injected with the identical volume of saline. Ten rats from each groups were sacrificed at 12h,24h,3d, 10d,20d,30d after administration of KA or saline. Hippocampal protein and mRNA were extracted. The cytokine(IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, TNFα, IFNγ)transcription level were determined by real-time PCR and the cytokine protein expression were determined by enzyme linked immunoabsorbent assay(Elisa).2. The relationship between increased cytokines(IL-1β, IL-10, TNFα) and increased vulnerability:P15 rats were randomly divided into SE group(n=10), Antibody intervention group(n=40) and Control group(n=10). Antibody intervention group were further divided into 4 intervention groups(n=10 in each group). Rats in SE group were given KA(5mg/kg injected i.p.) to induce SE. Rats in control group were injected intraperitoneally with the same volume of saline. Rats in four Antibody intervention groups were intracerebroventricularly injected with IL-1βantibody at 12h after SE, IL-1βantibody at 20d after SE, IL-10 antibody at 20d after SE, TNFa antibody at 20d after KA-induced SE at P15, respectively. Rats from all groups were injected with KA 15mg/kg intraperitoneally to induce SE at P45. Rats were tested for spatial learning and memory by Morris water maze on P50 and sacrificed on P55 for apoptosis detection by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL).3. The effect of antiepileptic monotherapy on cytokine expression at chronic stage and increased vulnerability after P15 SE. P15 rats were randomly divided into six groups:SE group, phenobarbital group, sodium valproate group, topiramate group, lamotrigine group, levetiracetam group(n=20 in each group). Rats in SE group were injected with KA (5mg/kg) intraperitoneally to induce SE. Rats in four antiepileptic monotherapy groups were dosed with phenobarbital(30mg/kg/day), sodium valproate(200mg/kg/day), topiramate(40mg/kg/day), lamotrigine(50mg/kg/day), levetiracetam(200mg/kg/day) respectively for 7 days after P15 SE. At P 45, ten rats from each group were tested for astrocyte activation and cytokine expression. The residual ten rats from each group were injected with KA(15mg/kg) to induce second SE at P45 and went on TUNEL examination on P55.4. The protective effect of naloxone after P15 SE. P15 rats were divided into three groups:SE group, naloxone group and control group. Rats in SE group were injected with KA 5mg/kg to induce SE at P15. Rats from naloxone group were implanted subcutaneously with naloxone minipump of different doses after P15 SE. Rats from control group were intraperitoneally injected with saline. Glia activation states were examined by western blotting of Iba-1, GFAP and S100β. De novo synthesis of cytokines were assayed by real-time PCR and Elisa. Rats from each group were intraperitoneally injected with KA(15mg/kg) to induce SE at P 45 and compared for spatial learning ability in Morris water maze and neuron injury in TUNEL assay.Results1. Cytokine expression after P 15 SE. P 15 SE caused certain changes of cytokines in hippocampus at different time points. IL-1βwas significantly upregulated at both early(P<0.001) and chronic stage(P<0.001) after P15 SE. IL-10 and TNFαwere enhanced at chronic stage(P<0.01) after P15 SE compared with control group. However, the expression of IL-2, IL-4, IL-6, IL-12, IFNγwere not significantly changed after P15 SE at all time points examined(P>0.05).2. The relationship between increased cytokines(IL-1β, IL-10 and TNFα) and increased vulnerability to second SE. Rats in SE group performed worse in Morris water maze than rats in control group(P<0.001) and exhibited more apoptotic cells in hippocampus than rats in control group(P<0.01). Intracerebroventricular administration of IL-1βat both 12h and 20d after P 15 SE could improve performance in Morris water maze(P<0.01) and reduce the apoptotic cells in hippocampus after second SE at P45(P<0.05). Antagonism of IL-10 at 20 days after P15 SE could deteriorate learning ability(P<0.05) and aggravate apoptosis after second SE at P45(P<0.05). Administration of TNFαantibody intracerebroventricularly at 20 days after P15 SE improved performance in Morris water maze(P<0.05) and reduced the number of apoptotic cells in hippocampus after second SE at P45(P<0.01).3. The effect of antiepileptic monotherapy on glial activation, cytokine expression increased vulnerability after P15 SE. Phenobarbital or lamotrigine monotherapy after PI5 SE did not change the glia activation states, cytokines(IL-1β, IL-10, TNFα) expression and hippocampal vulnerability to second SE(P>0.05). Although sodium valproate monotherapy had no influence on glia activation states(P>0.05), it reduced expression of IL-1β,IL-10 and TNFα(P<0.05) and mitigate apoptosis after second SE (P<0.01). Glia activation states and cytokine(IL-1β, IL-10, TNFα) expression were not different between topiramate group and SE group(P>0.05). The number of apoptotic cells was significantly reduced in topiramate group compared with SE group(P<0.05). Glia activation states and expression of cytokines(IL-1β, TNFα) were mitigated by levetiracetam monotherapy(P<0.01). Tunel-positive cells in hippocampus was reduced by levetiracetam treatment(P<0.01).4. The protective effect of naloxone after P15 SE. Subcutaneous minipump administration of naloxone could inhibit Iba-1, GFAP and S100βupregulation at acute stage(P<0.01) and reduce GFAP and and S100βexpression in hippocampus at chronic stage(P<0.001). IL-β, TNFαand IL-10 expression in hippocampus were reduced in naloxone-treated rats(P<0.01). Rats with naloxone treatment exhibited better performance in Morris water maze after P 45 SE(P<0.01). The apoptotic cells in hippocampus were greatly reduced after second SE in nalxone-treated rats(P<0.01).Conclusions1. P15 SE could induce IL-1βexpression at acute stage in hippocampus and induce IL-1β, TNF-αand IL-10 expression at chronic stage in hippocampus.2. IL-1βproduction at acute and chronic stages after P15 SE increased the hippocampal vulnerability to second SE at P45. Increased IL-10 at chornic stage is a protective cytokine in hippocampus, alleviating the vulnerability to second SE at P45. Upregulation of TNFαat chronic stage after P15 SE aggravates hippocampal vulnerability to second SE at P45.3. Phenobarbital and lamotrigine monotherapy has no influence on increased vulnerability after P15 SE. Sodium valproate could decrease hippocampal vulnerability by inhibiting cytokine expression. Topiramate mitigate vulnerability to second SE through unknown mechanisms. Levetiracetam could decrease the vulnerability to second SE by depressing astrocyte activation and inhibiting cytokine expression.4. Naloxone treatment could alleviate glia activation and cytokine expression after P15 SE. Naloxone could alleviate the increased vulnerability to P 45 SE after P 15 SE.更多还原