生命早期炎症对惊厥易感性及相关脑损伤的影响
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摘要
研究背景
癫痫(epilepsy)是一种大脑神经元发作性异常放电引起的,以反复痫样发作(seizure)为特征的临床症候群,是常见的神经系统慢性疾病。惊厥以强直或阵挛等骨骼肌运动性发作为主要表现,常伴意识障碍。由于惊厥是痫性发作的常见形式,常常被当做痫性发作的同义词。儿童及成人癫痫发病率高,长期反复痫性发作会导致进一步的脑损伤甚至持久性精神行为障碍,给患者本人、家庭及社会造成很大的困扰。癫痫的发病机制非常复杂,大量研究表明,癫痫发病与神经递质失衡、离子通道、遗传及神经胶质细胞异常有着密切关系。近年来,关于癫痫的病因、发病机制及治疗等的研究工作取得了很大进展,但是癫痫发生发展的确切机制尚未完全阐明。
星形胶质细胞和小胶质细胞是中枢神经系统的重要组成成分,与大脑的发育、正常生理活动、神经病理过程、以及损伤与修复有着密切的联系。神经炎症主要以胶质细胞(特别是小胶质细胞和星形胶质细胞)活化、增生并分泌促炎症细胞因子如白介素-1β (IL-1β)、肿瘤坏死因子α(TNFa)、白介素-6(IL-16)及抗炎细胞因子白介素-10(IL-10)等为特点。近年来,越来越多的研究数据表明大脑炎症反应参与癫痫发生及发展过程。许多研究显示癫痫发病过程中伴有小胶质细胞及星形胶质细胞过度活化增生与大量促炎症细胞因子生成。适度的神经炎症是大脑应对损伤的保护性反应。然而,过度持续胶质细胞活化及其产生的大量促炎细胞因子不仅可使血脑屏障完整性被破坏,通透性增加,促进炎症细胞进一步浸润,也可使神经元兴奋性增高,升高惊厥易感性并能加剧癫痫打击导致的脑损伤。因此,过度持续大脑炎症反应与癫痫发作互为因果,促进癫痫的发生发展。
近年来,大量研究表明,生命早期炎症不仅可导致急性脑损伤更可对大脑、心理及行为发育产生长远影响。胎儿发育关键时期子宫微环境的紊乱可导致大脑结构、功能和发育异常,增加生后罹患神经、精神及行为障碍的风险。临床上,母亲孕期病毒或细菌感染均与后代孤独症、精神分裂症及脑瘫的发生有关。例如,前瞻性流行病学研究显示母亲孕期弓形虫、生殖系感染及感冒均可使得后代精神分裂症发病危险性增高。此外,新生儿期炎症刺激也可导致代谢、免疫及神经内分泌系统发生长期功能性改变,增加青少年期甚至成年后神经精神疾病及行为障碍易感性。许多动物实验通过制作母体免疫激活(maternal immune activation, MIA)啮齿类动物模型来测定孕期感染对后代的生理及行为效应。在这些研究中,常通过给孕鼠注射脂多糖(LPS)或人工合成的双链RNA聚肌胞苷酸(polyI:C),以刺激孕鼠产生免疫应答,来分别模拟母体细菌或病毒感染。LPS是革兰氏阴性细菌细胞壁中的主要组成成分,可通过与某些细胞表面的Toll样受体4(toll-like receptor-4, TLR4)及TLR2结合,激活核因子-kB (nuclear factor-κB, NF-κB),从而诱导促炎细胞因子的表达。在MIA模型中,孕期LPS刺激可使得孕鼠血清、胎盘及羊水中促炎细胞因子mRNA及蛋白表达上调。大剂量LPS甚至可导致胎鼠大脑炎性细胞因子表达升高。另外,有研究显示大鼠孕期及新生儿期暴露于LPS可导致大脑海马区小胶质细胞及星形胶质细胞活化一直持续到成年期。由于大脑炎症反应可促进癫痫发生发展,我们推测孕期及新生儿期炎症刺激可能会增加儿童期及成年期惊厥易感性。
与我们的推测相符,近年来,流行病学调查资料证实母亲妊娠期巨细胞病毒感染、阴道酵母菌感染、膀胱炎、持续性咳嗽大于7天及持续性腹泻大于4天均可增加后代儿童期癫痫发病危险性。母亲产时感染也可升高新生儿惊厥易感性。最近国外动物实验结果显示大鼠新生儿期炎症刺激可增加成年期惊厥易感性。然而,孕期感染能否增加后代儿童期及成年期惊厥易感性,生命早期炎症能否加剧癫痫打击造成的脑损伤,能否导致长期脑发育及行为异常及其可能的机制,至今,未有动物实验报道。对上述问题的深入研究,有助于进一步阐明癫痫及其相关脑损伤的发病机制,为其临床防治提供新的思路。
第一章孕晚期免疫激活对子代青少年大鼠惊厥易感性的影响
研究目的：
研究母体孕晚期免疫激活对子代青少年大鼠惊厥易感性及相关脑损伤的影响及其可能的机制。研究方法：
1.孕晚期免疫激活模型的建立
给予雌性Sprague-Dawley (SD)大鼠在孕龄19天及20天时,连续两天腹腔注射脂多糖(LPS),每次300ug/kg,以构建孕晚期免疫激活大鼠模型；对照组孕鼠腹腔注射相同容积的生理盐水(NS)。观察两组孕鼠的分娩时间、产仔量及新生仔鼠出生体重。
2.子代青少年大鼠癫痫持续状态模型的建立
选用生后21天(postnatal day21, P21)的雄性子代青少年大鼠并随机分组。将海人酸(kainic acid, KA)按照7.5mg/kg给予癫痫持续状态(status epilepticus,SE)组大鼠腹腔注射,观察大鼠行为变化。痫性发作行为学评价根据Racine分级标准进行。具体分级标准如下：0级：正常行为状态；Ⅰ级：凝视、咀嚼、动须或头面部轻微颤动；Ⅱ级：点头、甩尾、搔抓,湿狗样抖动；Ⅲ级：一侧前肢局限性阵挛；Ⅳ级：伴后肢站立的全身强直性阵挛发作；Ⅴ级：出现站立并摔倒的全身强直-阵挛性发作。持续观察大鼠行为,记录发作的等级及出现时间。持续全身发作或连续多次全身发作之间不能恢复正常状态超过30min者为SEC(Ⅳ-Ⅴ级)。在SE开始后2小时给予水合氯醛(400mg/kg)腹腔注射终止发作。仅发作达Ⅳ或Ⅴ级的大鼠纳入后续试验。未达标准的子代大鼠予以剔除。对照组青少年大鼠给予腹腔注射相同容积的NS。子代大鼠腹腔注射观察行为变化后送回至原母鼠笼。
这样,腹腔注射KA或NS的青少年大鼠已经被随机分为4组,正常对照组(NS-NS),生前炎症组(LPS-NS),海人酸致痫组(NS-KA)和“二次打击”组(LPS-KA)。
3.惊厥易感性测定
海人酸注射后,首次癫痫发作开始时间(seizure onset time, SOT)作为癫痫发作的潜伏期。我们将大鼠前肢局限性阵挛、后肢站立或身体失衡作为首次痫性行为。本研究中,我们用SOT来衡量青少年大鼠对海人酸的惊厥易感性。
4.SE后6h,随机选择各组大鼠,采用实时定量多聚酶链反应(real-time polymerase chain reaction, RT-PCR)技术,检测炎性因子IL-1β、TNFα、IL-6及IL-10表达情况。
5.SE后24h及3d时,随机选择各组大鼠,分别采用Western blot方法及免疫组织化学染色技术检测海马区小胶质细胞标记物,离子钙接头蛋白分子1(Iba1)和星形胶质细胞标记物,胶原纤维酸性蛋白(GFAP)表达变化。
6.SE后3d,随机选择各组大鼠,采用NeuN免疫组织化学染色、尼氏染色和FJB染色观察海马CA1区神经元形态及损伤情况。
7.在P70时,采用Morris水迷宫实验,测定各组大鼠空间学习及记忆能力。
8. Morris水迷宫测试后,采用旷场试验测定各组大鼠自发活动水平及探索能力。结果：
1.孕期脂多糖刺激对孕鼠分娩时间、产仔量及子代鼠体重的影响。
暴露于LPS组孕鼠与NS注射组孕鼠在分娩时间及产仔量上无明显统计学差异。LPS暴露组新生鼠平均出生体重较NS注射组显著减低。此外,LPS暴露组子代鼠P9体重也较NS注射组子代鼠显著减低。LPS刺激组及NS注射组子代鼠P21体重无统计学差异。
2.孕期暴露于LPS导致子代鼠青少年期KA所致惊厥易感性增加。
腹腔注射KA后,LPS暴露组子代鼠(LPS-KA) SOT较NS注射组子代鼠(NS-KA) SOT缩短,表明孕期LPS刺激可导致子代鼠青少年期KA所致惊厥易感性增加。
3.KA诱导P21大鼠SE后6小时,各组大鼠海马炎症因子的表达情况。
RT-PCR定量分析结果显示：
(1)生前炎症组(LPS-NS)IL-1βmRNA、TNFamRNA、IL-6mRNA及IL-10mRNA的表达与正常对照组(NS-NS)无显著差异；
(2)KA致痫组(NS-KA)IL-1βmRNA、TNFamRNA、IL-6mRNA及IL-10mRNA的表达较正常对照组(NS-NS)均增高;
(3)与NS-KA组相比,LPS-KA组IL-1βmRNA及IL-6mRNA表达增高；然而,IL-10mRNA与TNFamRNA的表达在LPS-KA组与NS-KA组间无差异。
4.KA诱导P21大鼠SE后,各组大鼠海马小胶质细胞活化情况
Western blot与免疫组化染色均显示,与NS-NS组相比,LPS-NS组、NS-KA组大鼠海马Ibal蛋白表达水平升高；与NS-KA组相比,LPS-KA组大鼠海马Ibal蛋白表达水平升高。Ibal蛋白表达结果显示：生前炎症刺激不但可导致生后海马小胶质细胞长期活化,并能加剧KA所致海马小胶质细胞活化程度。
5.KA诱导P21大鼠SE后,各组大鼠海马星形胶质细胞活化情况
Western blot与免疫组化染色均显示,与NS-NS组相比,LPS-NS组、NS-KA组大鼠海马GFAP蛋白表达水平升高；与NS-KA组相比,LPS-KA组大鼠海马GFAP蛋白表达水平升高。GFAP蛋白表达结果显示：生前炎症刺激不但可导致生后海马星形胶质细胞长期活化,并能加剧KA所致海马星形胶质细胞活化程度。
6.KA诱导P21大鼠SE后3天,各组大鼠海马CA1区神经元损伤情况
尼氏染色显示NS-NS组大鼠海马CA1区无神经元损伤,形态正常。LPS-NS组、NS-KA组及LPS-KA组大鼠均出现神经元损伤,表现为尼氏染色阳性细胞数减少,形态异常。与NS-KA组相比,LPS-KA组大鼠海马CA1区神经元损伤加重。尼氏染色结果表明生前炎症刺激可产生长期效应：生前炎症刺激不但可导致生后海马神经元损伤,并能加剧KA所致海马神经元损伤。
NeuN与FJB染色结果同尼氏染色结果相似。7.P70时,Morris水迷宫实验中,各组大鼠空间学习及记忆情况
在Morris水迷宫寻找隐藏平台的学习实验中,随着训练进展,各组大鼠逃避潜伏期逐渐缩短。与NS-NS组相比,LPS-NS组及NS-KA组大鼠寻找到平台的潜伏期明显增长。与NS-KA组相比,LPS-KA组大鼠寻找到平台的潜伏期明显增长。
在Morris水迷宫探索试验中,与NS-NS组相比,LPS-NS组与NS-KA组大鼠目标象限活动时间百分比明显缩短。与NS-KA组相比,LPS-KA组大鼠目标象限活动时间百分比明显缩短。
8.旷场实验中,各组大鼠自发活动水平及探索能力表现情况
旷场实验自发活动结果显示,各组大鼠水平穿越方格数及竖直站立次数无明显不同。
探索行为结果显示,LPS-NS组大鼠与NS-NS组大鼠间点头次数无明显差别。与NS-NS组大鼠相比,NS-KA组大鼠点头次数减少。LPS-KA组大鼠比NS-KA组大鼠点头次数减少,表明LPS-KA组大鼠比NS-KA组大鼠探索能力降低。
结论：
1.孕期炎症刺激可导致子代鼠青少年期海人酸所致惊厥易感性增加；
2.孕期炎症刺激可增加惊厥所致青少年子代鼠海马神经元损伤、恶化神经炎症应答并能加剧惊厥所致空间学习记忆能力及探索能力损害；
3.孕期炎症刺激可导致青少年子代鼠海马神经元损伤、小胶质细胞及星形胶质细胞长期活化,但对海马区炎症因子表达无影响；
4.孕期炎症刺激可导致子代鼠成年期空间学习及记忆能力损害；
综上所述,生前炎症刺激可能通过启动小胶质细胞及星形胶质细胞长期活化以致惊厥所致脑损伤加剧。
第二章孕期感染增加氯化锂-匹罗卡品所致子代成年大鼠惊厥易感性研究目的：
研究孕期感染对氯化锂-匹罗卡品所致子代成年大鼠惊厥易感性及相关脑损伤的影响。研究方法：
1.孕期免疫激活模型的建立
雌性wistar大鼠在孕龄15及16天时,连续两天腹腔注射脂多糖(LPS),每次200ug/kg,构建孕期免疫激活大鼠模型；对照组孕鼠腹腔注射相同容积的生理盐水(NS)。
2.子代年轻成年大鼠癫痫持续状态模型(SE)的建立
选用生后45天(postnatal day45, P45)的雄性子代年轻成年大鼠并随机分组。腹腔注射氯化锂(LiCl,127mg/kg)后,将匹罗卡品(pilocarpine, Pilo)按照36mg/kg给予SE组大鼠腹腔注射,观察大鼠行为变化。痫性发作行为学评价根据Racine分级标准进行(具体分级标准见本研究第一章摘要部分)。持续观察大鼠行为,记录发作的等级及出现时间。在SE开始后1小时给予水合氯醛(400mg/kg)腹腔注射终止发作。仅发作达IV或V级的大鼠纳入后续试验。未达标准的子代大鼠予以剔除。对照组成年大鼠腹腔注射相同容积的NS。
这样,腹腔注射Pilo或NS的子代成年大鼠已经被随机分为4组,正常对照组(NS-NS),生前炎症组(LPS-NS),氯化锂-匹罗卡品(LiPC)致痫组(NS-LiPC)和“二次打击”组(LPS-LiPC)。
3.惊厥易感性测定
LiPC注射后,首次痫性行为发作的潜伏期作为痫性发作开始时间(seizure onset time, SOT)。我们将大鼠前肢局限性阵挛、后肢站立或身体失衡作为首次痫性行为。本研究中,我们用SOT来衡量年轻成年大鼠对LiPC的惊厥易感性。
4.SE后3天,随机选择各组大鼠,采用尼氏染色观察大鼠海马CA1及CA3区神经元形态及损伤情况。
5.P61时,采用旷场试验测定各组大鼠自发活动水平及探索能力。
6.P68时,采用高架迷宫实验测定各组大鼠焦虑水平。
7.P75时,采用Morris水迷宫实验,测定各组大鼠空间学习及记忆能力。结果：
1.孕期暴露于LPS导致子代鼠成年期LiPC所致惊厥易感性增加。
腹腔注射LiPC后,LPS暴露组子代鼠(LPS-LiPC) SOT较NS注射组子代鼠(NS-LiPC) SOT缩短,表明孕期LPS刺激可导致子代鼠成年期LiPC所致惊厥易感性增加。
2. LiPC诱导P45大鼠SE后3天,各组大鼠海马CA1及CA3区神经元损伤情况。
尼氏染色显示NS-NS组大鼠海马CA1及CA3区神经元形态正常,无损伤。LPS-NS组、NS-LiPC组及LPS-LiPC组大鼠均出现神经元损伤,表现为CA1及CA3区尼氏染色阳性细胞数减少,形态异常。与NS-LiPC组相比,LPS-LiPC组大鼠海马CA1及CA3区神经元损伤加重。尼氏染色结果表明生前炎症刺激可产生长期效应：生前炎症刺激不但可导致生后成年期大脑海马神经元损伤,并能加剧惊厥所致海马神经元损伤。
3.旷场实验中,各组大鼠自发活动水平及探索能力表现情况。
旷场实验自发活动结果显示,各组大鼠间水平穿越方格数及竖直站立次数无明显不同。
探索行为结果显示,LPS-NS组与NS-NS组之间及NS-LiPC组与NS-NS组之间点头次数均无明显差别。LPS-LiPC组比NS-LiPC组点头次数减少,表明LPS-LiPC组大鼠比NS-LiPC组大鼠探索能力降低。
4.各组大鼠在高架迷宫实验中的表现情况。
高架迷宫实验中,用焦虑分数来反映焦虑程度。结果显示,LPS-NS组焦虑分数低于NS-NS正常对照组,表明生前炎症刺激导致大鼠生后成年期焦虑水平升高。此外,NS-LiPC组焦虑分数高于NS-NS组。LPS-LiPC组与NS-LiPC组大鼠间焦虑分数无明显差异。
5. Morris水迷宫实验中,各组大鼠空间学习及记忆情况。
在Morris水迷宫寻找隐藏平台的学习实验中,随着训练进展,各组实验动物逃避潜伏期逐渐缩短。与NS-NS组相比,LPS-NS组、NS-LiPC组及LPS-LiPC组大鼠寻找到平台的潜伏期明显延长。与NS-LiPC组相比,LPS-LiPC组大鼠寻找到平台的潜伏期明显延长。
此外,在Morris水迷宫探索试验中,与NS-NS组相比,LPS-NS组、NS-LiPC组及LPS-LiPC组大鼠目标象限活动时间百分比明显缩短。NS-LiPC组与LPS-LiPC组目标象限活动时间百分比无明显不同。结论：
1.孕期炎症刺激可导致子代鼠成年期LiPC所致惊厥易感性增高；
2.孕期炎症刺激可增加惊厥所致成年子代鼠海马神经元损伤；
3.孕期炎症刺激可加剧惊厥所致空间学习及探索能力损害。
4.孕期炎症刺激可升高子代鼠成年期焦虑水平并导致空间学习及记忆能力损害；
综上所述,生前炎症刺激可增加生后成年期惊厥易感性并加剧惊厥所致脑损伤。
第三章新生儿期炎症增加惊厥所致成年大鼠海马依赖性记忆损伤
研究目的：
研究新生儿期免疫应激能否导致小胶质细胞发生长期改变并探索这种变化对成年期癫痫发作所致神经行为结果的影响。研究方法：
1.新生儿期免疫激活模型的建立
给予雄性SD大鼠在生后第3天(postnatal day3,P3)及P5,腹腔注射脂多糖(LPS),每次50ug/kg,构建新生儿期免疫激活大鼠模型；对照组新生鼠腹腔注射相同容积的生理盐水(NS)。
2.免疫荧光法测定新生儿期LPS刺激对海马区小胶质细胞的影响
分别在P5注射LPS或NS后4天(P9)、16天(P21)及40天(P45)时处死大鼠,取海马组织。运用离子钙接头蛋白分子1(Ibal,小胶质细胞标记物)免疫荧光染色研究LPS刺激所致小胶质细胞反应的时程变化。
3.在P3注射LPS或NS后,连续6天给予新生鼠腹腔注射米诺环素(minocycline)或磷酸盐缓冲溶液(PBS)。新生鼠分为3组：SS组(正常对照组),LS组(新生儿期LPS刺激并PBS处理组)和LM组(新生儿期LPS刺激并米诺环素处理组)。分别在P9及P21时处死大鼠,运用Ibal免疫荧光染色研究米诺环素对LPS所致小胶质细胞变化的抑制效应。
4.以下设计以研究新生儿期LPS暴露能否对成年期海人酸(KA)所致神经行为结果产生影响,并探究米诺环素在其中的作用。新生儿期注射NS或LPS的大鼠PBS或米诺环素处理后,在P45时腹腔注射KA,以诱导癫痫发作。此研究共分4组：正常对照组(SSS),成年致痫组(SSK),“二次打击”组(LSK),及“二次打击”米诺环素治疗组(LMK)。具体方法如下：
(1)子代年轻成年大鼠癫痫持续状态(SE)模型的建立
P45时,将KA按照15mg/kg给予SE组大鼠腹腔注射,观察大鼠行为变化。痫性发作行为学评价根据Racine分级标准进行,具体分级标准见第一部分。本研究中,我们仍用痫性发作开始时间(seizure onset time,SOT)来衡量年轻成年大鼠对KA所致癫痫发作易感性。在SE开始后2小时给予大鼠水合氯醛(400mg/kg)腹腔注射终止发作；
(2)SE后6h,随机选择各组大鼠,采用实时定量多聚酶链反应(Real-time polymerase chain reaction, RT-PCR)技术,检测炎性因子IL-1p及TNFα表达情况;
(3)SE后3天,随机选择各组大鼠,采用1bal染色观察海马区小胶质细胞活化情况；
(4)从P46至P55,随机选择各组大鼠,运用Y-迷宫实验检测各组大鼠海马依赖性空间学习情况；
(5)Y-迷宫实验后,从P60至P65,运用水迷宫实验检测各组大鼠海马依赖性空间学习及记忆情况；
(6)水迷宫实验后,在P70,随机选择各组大鼠,运用抑制回避实验检测各组大鼠海马依赖性非空间记忆情况。结果
1.新生儿期暴露于LPS对成年期KA所致惊厥易感性无影响。
腹腔注射KA后,SSK、LSK及LMK大鼠间SOT无统计学差异,表明新生儿期LPS刺激对成年期KA所致惊厥易感性无影响。
2.新生儿期LPS免疫应激导致海马区小胶质细胞发生较长时间活化。
Iba1免疫荧光染色显示,新生儿期LPS暴露组大鼠在P9及P21,海马区Iba1阳性染色光密度(OD)值高于新生儿期NS注射组大鼠；但是在P45时,两组大鼠Iba1阳性染色OD值无差异。该结果显示,新生儿期LPS免疫应激导致海马区小胶质细胞发生较长时间活化至青少年时期。
3.米诺环素抑制LPS所致小胶质细胞活化。
Iba1免疫荧光染色显示,在P9及P21,LS组海马区Ibal阳性染色OD值高于LM与SS组；在P9及P21,SS组与LM组Iba1阳性染色OD值无差异。
4.SE后6h,各组大鼠炎性因子IL-1βmRNA及TNFαmRNA表达情况。
RT-PCR定量分析结果显示：
(1)SSK、LSK及LMK组IL-1β mRNA较SSS组升高；LSK组IL-1βmRNA表达较SSK组及LMK组升高；SSK与LMK间,IL-1β mRNA表达水平无差异；
(2) SSK、LSK及LMK组TNFα mRNA较SSS组升高；LSK组TNFα mRNA表达较SSK及LMK组升高；SSK与LMK间,NFa mRNA表达水平无差异。
5.SE后3天,各组大鼠海马区小胶质细胞活化情况。
Ibal免疫荧光染色显示：SSK、LSK及LMK组Ibal阳性染色OD值较SSS组升高；LSK组Ibal阳性染色OD值较SSK及LMK组升高；SSK与LMK间,Ibal蛋白表达水平无差异；
6.从P46至P55,各组大鼠Y-迷宫实验表现情况。
Y-迷宫实验中,自发交替反应率越高表示大鼠学习能力越强。结果显示SSK、LSK及LMK组自发交替反应率低于SSS组；LSK组自发交替反应率较SSK及LMK组降低,SSK与LMK间自发交替反应率无差别。
7.从P60至P65,各组大鼠水迷宫实验表现情况。
结果显示：在Morris水迷宫寻找隐藏平台的学习实验中,各组实验动物间学习能力无明显差异。
在探索试验中,SSK、LSK及LMK组目标象限活动时间百分比低于SSS组,LSK组目标象限活动时间百分比较SSK及LMK组降低,SSK与LMK间目标象限活动时间百分比无差别。
8.P70时,各组大鼠抑制回避实验表现情况。
抑制回避实验中,暗室逃避潜伏期越长反映大鼠海马依赖性非空间记忆力越好。结果显示：训练后1小时,LSK组暗室逃避潜伏期较SSS组明显缩短；SSS、LMK及SSK组之间,暗室逃避潜伏期无明显差异。训练后24小时,LSK组暗室逃避潜伏期较SSS、SSK及LMK组明显缩短；与SSS组相比,SSK及LMK组暗室逃避潜伏期缩短；SSK与LMK之间暗室逃避潜伏期无明显差异。结论：
1.新生儿期炎症刺激可导致海马小胶质细胞发生较长时间活化；
2.米诺环素可抑制LPS所致新生鼠海马小胶质细胞活化；
3.新生儿期炎症刺激可能通过长期启动小胶质细胞而加剧成年期癫痫发作所致神经炎症应答反应及海马依赖性行为损伤。
Background
Epilepsy, a common chronic neurologic disorder characterized by recurrent seizures, is caused by abnormal, paroxysmal changes in the electrical activity of the brain. It has high morbidity in children and adults. Convulsion, characterized by an abnormal, involuntary contraction of the muscles, is most typically seen with seizure disorders and is always accompanied by conscious disturbance. Because a convulsion is often a symptom of an epileptic seizure, the term convulsion is sometimes used as a synonym for seizure. Frequent and serious seizures can lead to further brain injury as well as persistent mental and behavioral disorders, bringing troubles to the patients, their families and society. Epileptogenesis is defined as the process of developing epilepsy. The mechanisms of epileptogenesis are complicated, which involve neurotransmitter imbalance, ion channel and genetic abnormalities, and glial dysfunction. Although great progress has been made in the etiology, pathogenesis and treatment of epilepsy, the exact mechanisms of epileptogenesis still remain unclear.
Astrocyte and microglia, the important component cells of the central nervous system (CNS), play an essential role in maintaining brain homeostasis, development and pathologies and participate actively in the process of neuronal injury and repair. Neuroinflammation is the term used to describe CNS inflammatory responses characterized by activation and proliferation of glia (especially astrocytes and microglia) and the associated up-regulation of proinflammatory cytokines such as interleukin (IL)-1β, tumor necrosis factor (TNF)a, and IL-6, and anti-inflammatory cytokines such as IL-10. In recent years, accumulated data have indicated that neuroinflammation participates in the pathogenesis of epilepsy. A recognized response to seizures and a potential contributor to mechanisms of epileptogenesis is excessive or prolonged glial activation and the associated increase in proinflammatory cytokine production. Appropriate neuroinflammation occurs as protective responses to CNS insults. However, excessive neuroinflammation can cause damage to the blood-brain-barrier (BBB), leading to increased BBB permeability and exaggerated leukocyte infiltration and enhance neuronal excitability and exacerbate seizure-induced brain injury. Thus, the relationship between neuroinflammation and epileptogenesis is represented as an amplifying feedback loop.
There is an increasing body of evidence suggesting that infection occurring within the intrauterine or perinatal environment can not only produce acute brain injury but affect brain as well as psychological and behavioral development in the long term. Insults to the fetus during critical times of development may alter fetal brain structure and function thereby acting as risk factors for mental, psychiatric and behavioral disorders later in life. Maternal infections of both viral and bacterial origin have been linked with later development in the offspring of autism, schizophrenia and cerebral palsy. For instance, prospective epidemiological studies indicate that maternal influenza, toxoplasmosis, and genital/reproductive infection are associated with schizophrenia. Moreover, inflammation in the neonatal period can also have long-term functional effects on metabolic, immune, behavioral and neuroendocrine system that persist into adulthood, leading to increased susceptibility to neuropsychiatric and behavioral disorders. Rodent models have been developed to determine the behavioral and biological effects of maternal immune activation (MIA) on offspring. In these studies, lipopolysaccharide (LPS) or the synthetic, double-stranded RNA polyriboinosinic-polyribocytidilic acid (polyI：C) are administered to pregnant dams to mimic the immune-stimulating actions of live bacterial or viral infections, respectively. LPS is a large lipid-polysaccharide complex released from the outer cell wall of Gram-negative bacteria that binds to the toll-like receptor4(TLR4) and TLR2expressed on the surface of certain cell types resulting in the activation of nuclear factor-KB (NF-kB) which induces proinflammatory cytokine expression. In the MIA model, maternal LPS increases proinflammatory cytokine mRNA expression and/or proteins in the maternal serum, amniotic fluid, and placenta. Increased cytokine gene expression in fetal brain has been reported in some studies that used a much higher dose of LPS. Recently, accumulating data from animal studies have indicated that prenatal or neonatal LPS can induce long-term hippocampal microglial and astrocytic activation that persisted into adulthood. Since excessive neuroinflammation can cause increased seizure susceptibility, we hypothesize that early life immune challenge may predispose the maturing brain more vulnerable to later-life seizures.
In accordance with our hypothesis, recent epidemiological studies have indicated that maternal infection during pregnancy, such as cystitis, vaginal yeast infection, consistent diarrhea lasting4days and coughs lasting1week was associated with an increased risk for childhood epilepsy. Moreover, intrapartum infection is associated with a higher risk for newborn seizures. A recent animal study has shown that neonatal immune challenge can increase seizure susceptibility in adult rats. However, whether early life infection causes long-lasting brain injury and heightened susceptibility to later seizures and the possible mechanisms underlying this association remain largely unexplored experimentally. To make clealy these questions will help to clarify the mechanisms of epileptogenesis, and will be possible to find new methods to treat this disease.
Chapter I Effect of maternal immune activation during late gestation on seizure susceptibility in juvenile rat offspring
Objectives
The study was designed to investigate whether maternal immune challenge during late gestation could influence juvenile seizure susceptibility and the associated brain injury as well as the possible mechanisms.
Methods
1. The construction of maternal immune activation model
Lipopolysaccharide (LPS) diluted in saline was injected intraperitoneally into pregnant Sprague-Dawley (SD) rats on gestational day (GD)19and20at a dose of300ug/kg. Control dams were injected with a corresponding volume of normal saline (NS) at the same GDs. Pregnant females were monitored for the parturition date. In addition, litter size and birth weight of pups were recorded.
2. Kainic acid (KA)-induced status epilepticus (SE) model in juvenile rat offspring
Juvenile offspring whose mothers received NS or LPS during late pregnancy received7.5mg/kg KA intraperitoneally at postnatal day (P)21. Instead of KA administration, control rats received comparable volume of NS as given to KA-injected rats. A seizure severity grade was assigned according to a modified Racine scale as follows:0—no response; Ⅰ—motionless staring, and slight facial and mouth movements; Ⅱ—pawing, head nobbing, and wet-dog shake; Ⅲ—unilaternal forelimb clonus; Ⅳ—bilaternal forelimb clonus with rearing; Ⅴ—rearing, generalized tonic-clonic seizures and transient loss of postural control. Animals were closely observed and seizure severity and latency to the first sign of seizure were recorded. Seizures were terminated with chloral hydrate (400mg/kg, intraperitoneally)2h after onset of SE. Only animals with grade IV or V seizures were included in this study. Animals were returned to their dams after behavioral monitoring.
Thus, four experimental groups were studied, including normal control (NS-NS), prenatal inflammation (LPS-NS), juvenile seizure (NS-KA), and "two-hit"(LPS-KA) groups.
3. KA seizure susceptibility testing
The latency to the first behavioral seizure after KA administration, referred to as seizure onset time (SOT), was defined by the occurrence of forelimb clonus, rearing and loss of balance, and was recorded to the nearest second for each animal by an individual blind to the prenatal treatment of the animal. SOT is a commonly used measure to describe seizure susceptibility to convulsant compounds in rats.
4. At acute stage, rats were killed6h after SE, and examined for production of cytokines, including IL-1β, TNFa, IL-6and IL-10using real-time polymerase chain reaction (RT-PCR).
5. Glial activation state was analyzed by western blotting of glial markers, glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule1(Ibal)24h after SE.
6. Three days after SE, rats were sacrificed for further examination of glial activation by immunohistological methods.
7. Three days after SE, rats were sacrificed for examination of neuronal injury by NeuN, Nissl and Fluoro-Jade B (FJB) staining.
8. At P70, water maze task was used to evaluate the long-term impact of maternal inflammation on spatial learning and memory ability of adult rat offspring.
9. After water maze test, locomotor activity and exploratory ability were tested using an open-field apparatus.
Results:
1. The effect of maternal LPS exposure on gestational length, litter size and birth weight of pups
We did not observe any significant difference in gestation length or litter size between LPS-exposed dams and controls, though some LPS-exposed pregnant rats died after LPS application, suffered from miscarriage or had stillborn pups. The offspring of LPS-treated dams displayed significantly reduced birth weight and weight gain at P9compared to controls. Pup body weight did not differ between groups at P21.
2. Prenatal LPS increases juvenile seizure susceptibility to KA
Rats exposed to LPS prenatally showed faster mean SOT compared with controls, indicating that maternal LPS exposure increased juvenile seizure susceptibility.
3. Cytokine expression in the hippocampus of P21rats after KA-induced SE
There was no difference in the mRNA expression of the four cytokines, including IL-1β, TNF-a, IL-6and IL-10between LPS-NS and NS-NS groups. The expression of the four cytokines was higher in NS-KA group compared with NS-NS group. Compared with NS-KA group, the expression of IL-1β mRNA and IL-6mRNA was higher in LPS-KA group. However, no difference in the expression of IL-10mRNA and TNFa mRNA was observed between LPS-KA and NS-KA rats.
4. Hippocampal microgilal activation after KA-induced SE
Western blot analysis of Iba1expression and immunohistochemical staining for Ibal showed similar results. Compared with NS-NS group, LPS-NS and NS-KA rats had higher hippocampal Ibal expression. Compared with NS-KA group, LPS-KA group had higher hippocampal Ibal expression. These results demonstrated that prenatal inflammation not only caused prolonged microglial response but also enhanced KA-induced microglial activation.
5. Hippocampal astrocyte activation after KA-induced SE
Western blot analysis of GFAP expression and immunohistochemical staining for GFAP showed similar results. Compared with NS-NS group, LPS-NS and NS-KA rats had higher hippocampal GFAP expression. Compared with NS-KA group, LPS-KA group had higher hippocampal GFAP expression. These results demonstrated that prenatal inflammation not only caused prolonged astrocytic response but also enhanced KA-induced astrocytic activation.
6. Neuronal damage in the hippocampus3days after KA-induced SE
Nissl staining showed that there was no neuronal damage in NS-NS rats. Compared to NS-NS group, Nissl-positive cell counts were significantly reduced in LPS-NS, NS-KA and LPS-KA groups. Fewer Nissl-staining cells were detected in the LPS-KA group compared with NS-KA group.
The results for NeuN and FJB staining were consistent with those of Nissl staining.
7. The performance of adult rat offspring in the Morris water maze after KA-induced SE
Performance improved in all groups over the four training days in the place navigation test. Compared to NS-NS group, escape latency was significantly increased in LPS-NS, NS-KA and LPS-KA groups. Escape latency in LPS-KA rats was greater than that in NS-KA rats.
On the day of probe testing, NS-NS rats spent significantly more time in the (target quadrant) TQ than animals from the other three groups."Two-hit" rats spent significantly less time in the TQ than animals in the NS-KA group.
8. The performance of adult rat offspring in the open field after KA-induced SE
No difference in the number of squares crossed and rearing frequency was observed among NS-NS, LPS-NS, NS-KA and LPS-KA groups.
No difference in the number of head dippings was observed between NS-NS and LPS-NS groups. NS-KA and LPS-KA groups demonstrated smaller number of head dippings compared with that of NS-NS control."Two-hit" rats showed smaller number of head dippings compared with NS-KA group.
Conclusins
1. Maternal infection increases seizure susceptibility to KA in juvinile rat offspring;
2. Maternal infection, without perturbation of cytokine expression, causes long-lasting neuronal injury, glial alteration and cognitive deficit in juvenile offspring;
3. Maternal infection during late pregnancy enhances neuronal injury, exaggerates neuroinflammatory responses, and exacerbates long-term neurobehavioral impairment associated with a second adolescent KA insult.
In sum, primed glia by prenatal infection may underlie, at least in part, long-term "perinatal programming" of later-life vulnerability to seizure insult.
Chapter II Prenatal immune challenge in rats increases susceptibility to seizure-induced brain injury in adulthood
Objectives
The study was designed to investigate whether maternal immune challenge during gestation could influence adult seizure susceptibility and the associated brain injury.
Methods
1. The construction of maternal immune activation model
Lipopolysaccharide (LPS, Escherichia coli, serotype055:B5) diluted in saline was injected intraperitoneally into pregnant Wistar rats on gestational day (GD)15and16at a dose of200ug/kg. Control dams were injected with a corresponding volume of normal saline (NS) at the same GDs.
2. Lithium-pilocarpine (LiPC)-induced status epilepticus (SE) model in adult rat offspring
At P45, male offspring from LPS-or NS-treated mothers received intraperitoneal injections of lithium chloride (127mg/kg) followed16h later by pilocarpine (36mg/kg, i.p.)(LiPC). Instead of pilocarpine application, control rats received corresponding volumes of NS as given to the pilocarpine-injected rats. A seizure severity grade was assigned according to a modified Racine scale (detailed in part1). Animals were closely observed and seizure severity and latency to the first sign of seizure were recorded. Seizures were terminated with chloral hydrate (400mg/kg, intraperitoneally)1h after the onset of SE. Only animals with grade IV or V seizures were included in this study.
Four experimental groups were studied, including normal controls (NS-NS), prenatal inflammations (LPS-NS), adult seizures (NS-LiPC), and "two-hit"(LPS-LiPC) animals.
3. LiPC seizure susceptibility testing
SOT was used to assess seizure susceptibility (detailed in part1).
4. Three days after SE, rats were sacrificed for examination of neuronal injury by Nissl staining.
5. At P61, locomotor activity and exploratory ability were tested using an open-field apparatus.
6. At P68, anxiety was examined using the elevated plus maze.
7. Begining at P75, water maze task was used to evaluate long-term impact of maternal inflammation on spatial learning and memory ability of adult rat offspring.
Results:
1. Prenatal LPS increases adult seizure susceptibility to LiPC
Rats exposed to LPS prenatally showed faster mean SOT compared with controls, indicating that maternal LPS exposure increased adult seizure susceptibility.
2. Neuronal damage in the hippocampus3days after KA-induced SE
Nissl staining showed that there was no hippocampal neuronal damage in NS-NS rats. Compared to NS-NS group, Nissl-positive cell counts were significantly reduced in LPS-NS and NS-LiPC groups. Fewer Nissl-staining cells were detected in the LPS-LiPC group compared with NS-LiPC group. These results demonstrated that prenatal inflammation not only caused neuronal injury but also enhanced LiPC-induced neuronal damage.
3. The performance of adult rat offspring in the open field after LiPC-induced SE
No difference in the number of squares crossed and rearing frequency was observed among NS-NS, LPS-NS, NS-LiPC and LPS-LiPC groups.
No difference in the number of head dippings was observed between NS-NS and LPS-NS groups. In addition, no difference in the number of head dippings was observed between NS-NS and NS-LiPC groups."Two-hit" rats showed smaller number of head dippings compared with NS-LiPC group.
4. The performance of adult rat offspring in the elevated plus maze after LiPC-induced SE
The offspring of LPS-treated mothers (LPS-NS) had significantly lower anxiety score than NS-NS controls, indicating that maternal immune challenge caused increased anxiety in offspring. No significant difference in anxiety scores was found in LPS-LiPC rats compared with NS-LiPC rats. The "single hit" NS-LiPC animals had higher anxiety scores than NS-NS controls.
5. The performance of adult rat offspring in the Morris water maze after KA-induced SE
Performance improved in all groups over the four training days in the place navigation test. Compared to NS-NS group, escape latency was significantly increased in LPS-NS and NS-LiPC groups. Escape latency in LPS-LiPC rats was greater than that in NS-LiPC rats.
On the day of probe testing, NS-NS rats spent significantly more time in the TQ than animals from the other three groups. No significant difference existed between NS-LiPC and LPS-LiPC groups. Conclusins
1. Maternal infection increases seizure susceptibility to LiPC in the adult rat offspring;
2. Maternal infection alone causes long-lasting neuronal injury, increased anxiety level and cognitive deficit in the offspring rats;
3. Maternal infection during late pregnancy enhances neuronal injury and exacerbates long-term neurobehavioral impairment associated with a second adult LiPC insult.
In sum, prenatal infection can increase adult seizure susceptibility and exacerbates the associated brain injury.
Chapter Ⅲ Neonatal inflammation exacerbates seizure-induced hippocampus-dependent memory impairment in adult rats
Objectives
The study was designed to examine whether neonatal lipopolysaccharide (LPS) exposure is associated with changes in microglia and whether these alternations could influence later seizure-induced neurobehavioral outcomes.
Methods
1. The construction of neonatal immune activation model
LPS diluted in saline was injected intraperitoneally (i.p.) into male Sprague-Dawley pups at postnatal (P) day3and P5at a dose of50ug/kg. Control pups were injected with a corresponding volume of normal saline (NS).
2. To study the effect of neonatal LPS on hippocampal microglia using immunofluorescence staining, male pups were sacrificed4,16, or40days after the last administration of NS or LPS on P5. Immunofluorescence staining of Ibal, a marker of microglia, was used to study the time course of LPS-induced microglia alternation.
3. To study the inhibitory effect of minocycline on neonatal LPS-induced microglia alternation, pups administered saline or LPS neonatally were treated with phosphate buffered saline (PBS) or minocycline for6consecutive days. Three groups of pups were studied, including normal controls treated with PBS (SS), neonatally LPS-exposed pups treated with PBS (LS), and neonatally LPS-exposed pups treated with minocycline (LM). On P9(4days after administration of NS or LPS on P5) and P21, rats were sacrificed and evaluated for hippocampal microglial activation using immunofluorescence staining of Ibal.
4. To investigate the effect of early-life LPS exposure on seizure-induced neurobehavioral impairment and to further explore whether minocycline would prevent changes in susceptibility to later seizure-induced brain injury, a separate study was conducted. Rat pups injected with saline or LPS neonatally were administered kainic acid (KA) at P45. Four groups were studied, including SSS (normal controls), SSK.(adult seizures treated with PBS neonatally), LSK ("two-hit" animals treated with PBS at the time of neonatal LPS exposure), and LMK ("two-hit" animals treated with minocycline at the time of neonatal LPS exposure).
(1) KA-induced status epilepticus (SE) model in adult rats
At P45, KA dissolved in saline (15mg/kg) was administered i.p. to rats that were injected with saline or LPS neonatally. Controls received equal volumes of NS. A seizure severity grade was assigned according to a modified Racine scale (detailed in part1). Animals were closely observed and seizure severity and latency to the first sign of seizure were recorded. Seizure onset time (SOT) was used to assess seizure susceptibility. Seizures were terminated with chloral hydrate (400mg/kg, i.p.)2h after the onset of SE. Only animals with grade IV or V seizures were included in this study;
(2) At acute stage (6h after SE), animals were killed for quantification of hippocampal IL-1β and TNFa production using real-time polymerase chain reaction (RT-PCR);
(3) Three days after SE, rats were sacrificed for further examination of microglial activation by immunofluorescence staining of Ibal;
(4) From P46to P55, Y maze task was used to evaluate hippocampus-dependent spatial learning ability of adult rats;
(5) From P60to P65, water maze task was used to evaluate hippocampus-dependent spatial learning and memory ability of adult rats;
(6) At P70, inhibitory avoidance task was used to evaluate hippocampus-dependent nonspatial memory ability of adult rats.
Results:
1. Neonatal LPS has no effect on KA-induced seizure susceptibility in adult rats.
There were no significant difference in SOTs among SSK, LSK, and LMK groups, indicating that neonatal LPS has no effect on KA-induced seizure susceptibility in adulthood.
2. Transient microglia activation after neonatal LPS exposure.
The analysis of immunostaining of Ibal revealed that LPS-treated pups exhibited significantly greater hippocampal expression of Ibal than NS-treated pups at P9and P21. However, we observed no significant difference in the expression of Ibal at P45. In short, dual exposure to LPS on P3and P5caused persistent activation of hippocampal microglial cells. This activation, however, was not permanent and subsided within40days.
3. Minocyclne inhibits neonatal LPS-induced microglial activation
Compared with SS, LS showed higher expression of Ibal at P9and P21. Minocycline administration significantly reduced LPS-induced microglia activation in LM pups compared with LS pups at both P9and P21. There was no difference in the expression of Ibal between SS controls and LM pups at both P9and P21.
4. Cytokine expression in the hippocampus of adult rats after KA-induced SE
Compared with SSS group, SSK, LSK and LMK rats showed higher expression of IL-1βmRNA. Compared with SSK group, the expression of IL-1β mRNA was higher in LSK group. There was no difference in the expression of IL-1(3mRNA between SSK and LMK groups.
Compared with SSS group, SSK, LSK and LMK rats showed higher expression of TNFa mRNA. Compared with SSK group, the expression of TNFa mRNA was higher in LSK group. There was no difference in the expression of TNFamRNA between SSK and LMK groups.
5. Hippocampal microgilal activation after KA-induced SE
Compared with SSS group, SSK, LSK and LMK rats had higher hippocampal Ibal expression. Compared with SSK and LMK groups, LSK group had higher hippocampal Ibal expression. There was no difference in the expression of Ibal between SSK and LMK groups.
6. The performance of adult rats in the Y maze after KA-induced SE
Animals in SSK group, LSK group, and LMK group all demonstrated lower spontaneous alternation scores when compared with SSS controls. Compared with SSK and LMK rats, the percent of alternation in "two-hit" rats (LSK) was significantly reduced. There was no difference in the percent alternation between SSK and LMK groups.
7. The performance of adult rats in the water maze after KA-induced SE
All groups of rats have comparable rate of acquisition. On the day of probe testing, SSS rats spent significantly more time in the TQ than animals from the other three groups. LSK rats spent significantly less time in the TQ than animals in SSK and LMK groups. SSK and LMK rats spent comparable time in the TQ.
8. The performance of adult rats in the inhibitory avoidance task after KA-induced SE
SSS, SSK, and LMK rats demonstrated comparable retention time at1h after training. However, the latency to step into the dark compartment was significantly decreased in LSK rats compared with SSS group.
At24h, the latency to step into the dark compartment was significantly increased in SSS rats compared with SSK, LSK and LMK groups. Compared with LSK group, SSK and LMK groups spent longer time to step into the dark compartment. No difference in escape latency was observed between SSK and LMK groups.
Conclusins
1. Neonatal inflammation caused persistent activation of hippocampal microglial cells;
2. Minocycline inhibited neonatal LPS-induced microglial activation;
3. Neonatal inflammation predisposed the immatured brain to exacerbated neuroinflammatory response and worse hippocampus-dependent behavioral deficit following seizures in adulthood, possibly by priming microglia.
癫痫(epilepsy)是一种大脑神经元发作性异常放电引起的,以反复痫样发作(seizure)为特征的临床症候群,是常见的神经系统慢性疾病。惊厥以强直或阵挛等骨骼肌运动性发作为主要表现,常伴意识障碍。由于惊厥是痫性发作的常见形式,常常被当做痫性发作的同义词。儿童及成人癫痫发病率高,长期反复痫性发作会导致进一步的脑损伤甚至持久性精神行为障碍,给患者本人、家庭及社会造成很大的困扰。癫痫的发病机制非常复杂,大量研究表明,癫痫发病与神经递质失衡、离子通道、遗传及神经胶质细胞异常有着密切关系。近年来,关于癫痫的病因、发病机制及治疗等的研究工作取得了很大进展,但是癫痫发生发展的确切机制尚未完全阐明。
星形胶质细胞和小胶质细胞是中枢神经系统的重要组成成分,与大脑的发育、正常生理活动、神经病理过程、以及损伤与修复有着密切的联系。神经炎症主要以胶质细胞(特别是小胶质细胞和星形胶质细胞)活化、增生并分泌促炎症细胞因子如白介素-1β (IL-1β)、肿瘤坏死因子α(TNFa)、白介素-6(IL-16)及抗炎细胞因子白介素-10(IL-10)等为特点。近年来,越来越多的研究数据表明大脑炎症反应参与癫痫发生及发展过程。许多研究显示癫痫发病过程中伴有小胶质细胞及星形胶质细胞过度活化增生与大量促炎症细胞因子生成。适度的神经炎症是大脑应对损伤的保护性反应。然而,过度持续胶质细胞活化及其产生的大量促炎细胞因子不仅可使血脑屏障完整性被破坏,通透性增加,促进炎症细胞进一步浸润,也可使神经元兴奋性增高,升高惊厥易感性并能加剧癫痫打击导致的脑损伤。因此,过度持续大脑炎症反应与癫痫发作互为因果,促进癫痫的发生发展。
近年来,大量研究表明,生命早期炎症不仅可导致急性脑损伤更可对大脑、心理及行为发育产生长远影响。胎儿发育关键时期子宫微环境的紊乱可导致大脑结构、功能和发育异常,增加生后罹患神经、精神及行为障碍的风险。临床上,母亲孕期病毒或细菌感染均与后代孤独症、精神分裂症及脑瘫的发生有关。例如,前瞻性流行病学研究显示母亲孕期弓形虫、生殖系感染及感冒均可使得后代精神分裂症发病危险性增高。此外,新生儿期炎症刺激也可导致代谢、免疫及神经内分泌系统发生长期功能性改变,增加青少年期甚至成年后神经精神疾病及行为障碍易感性。许多动物实验通过制作母体免疫激活(maternal immune activation, MIA)啮齿类动物模型来测定孕期感染对后代的生理及行为效应。在这些研究中,常通过给孕鼠注射脂多糖(LPS)或人工合成的双链RNA聚肌胞苷酸(polyI:C),以刺激孕鼠产生免疫应答,来分别模拟母体细菌或病毒感染。LPS是革兰氏阴性细菌细胞壁中的主要组成成分,可通过与某些细胞表面的Toll样受体4(toll-like receptor-4, TLR4)及TLR2结合,激活核因子-kB (nuclear factor-κB, NF-κB),从而诱导促炎细胞因子的表达。在MIA模型中,孕期LPS刺激可使得孕鼠血清、胎盘及羊水中促炎细胞因子mRNA及蛋白表达上调。大剂量LPS甚至可导致胎鼠大脑炎性细胞因子表达升高。另外,有研究显示大鼠孕期及新生儿期暴露于LPS可导致大脑海马区小胶质细胞及星形胶质细胞活化一直持续到成年期。由于大脑炎症反应可促进癫痫发生发展,我们推测孕期及新生儿期炎症刺激可能会增加儿童期及成年期惊厥易感性。
与我们的推测相符,近年来,流行病学调查资料证实母亲妊娠期巨细胞病毒感染、阴道酵母菌感染、膀胱炎、持续性咳嗽大于7天及持续性腹泻大于4天均可增加后代儿童期癫痫发病危险性。母亲产时感染也可升高新生儿惊厥易感性。最近国外动物实验结果显示大鼠新生儿期炎症刺激可增加成年期惊厥易感性。然而,孕期感染能否增加后代儿童期及成年期惊厥易感性,生命早期炎症能否加剧癫痫打击造成的脑损伤,能否导致长期脑发育及行为异常及其可能的机制,至今,未有动物实验报道。对上述问题的深入研究,有助于进一步阐明癫痫及其相关脑损伤的发病机制,为其临床防治提供新的思路。
第一章孕晚期免疫激活对子代青少年大鼠惊厥易感性的影响
研究目的：
研究母体孕晚期免疫激活对子代青少年大鼠惊厥易感性及相关脑损伤的影响及其可能的机制。研究方法：
1.孕晚期免疫激活模型的建立
给予雌性Sprague-Dawley (SD)大鼠在孕龄19天及20天时,连续两天腹腔注射脂多糖(LPS),每次300ug/kg,以构建孕晚期免疫激活大鼠模型；对照组孕鼠腹腔注射相同容积的生理盐水(NS)。观察两组孕鼠的分娩时间、产仔量及新生仔鼠出生体重。
2.子代青少年大鼠癫痫持续状态模型的建立
选用生后21天(postnatal day21, P21)的雄性子代青少年大鼠并随机分组。将海人酸(kainic acid, KA)按照7.5mg/kg给予癫痫持续状态(status epilepticus,SE)组大鼠腹腔注射,观察大鼠行为变化。痫性发作行为学评价根据Racine分级标准进行。具体分级标准如下：0级：正常行为状态；Ⅰ级：凝视、咀嚼、动须或头面部轻微颤动；Ⅱ级：点头、甩尾、搔抓,湿狗样抖动；Ⅲ级：一侧前肢局限性阵挛；Ⅳ级：伴后肢站立的全身强直性阵挛发作；Ⅴ级：出现站立并摔倒的全身强直-阵挛性发作。持续观察大鼠行为,记录发作的等级及出现时间。持续全身发作或连续多次全身发作之间不能恢复正常状态超过30min者为SEC(Ⅳ-Ⅴ级)。在SE开始后2小时给予水合氯醛(400mg/kg)腹腔注射终止发作。仅发作达Ⅳ或Ⅴ级的大鼠纳入后续试验。未达标准的子代大鼠予以剔除。对照组青少年大鼠给予腹腔注射相同容积的NS。子代大鼠腹腔注射观察行为变化后送回至原母鼠笼。
这样,腹腔注射KA或NS的青少年大鼠已经被随机分为4组,正常对照组(NS-NS),生前炎症组(LPS-NS),海人酸致痫组(NS-KA)和“二次打击”组(LPS-KA)。
3.惊厥易感性测定
海人酸注射后,首次癫痫发作开始时间(seizure onset time, SOT)作为癫痫发作的潜伏期。我们将大鼠前肢局限性阵挛、后肢站立或身体失衡作为首次痫性行为。本研究中,我们用SOT来衡量青少年大鼠对海人酸的惊厥易感性。
4.SE后6h,随机选择各组大鼠,采用实时定量多聚酶链反应(real-time polymerase chain reaction, RT-PCR)技术,检测炎性因子IL-1β、TNFα、IL-6及IL-10表达情况。
5.SE后24h及3d时,随机选择各组大鼠,分别采用Western blot方法及免疫组织化学染色技术检测海马区小胶质细胞标记物,离子钙接头蛋白分子1(Iba1)和星形胶质细胞标记物,胶原纤维酸性蛋白(GFAP)表达变化。
6.SE后3d,随机选择各组大鼠,采用NeuN免疫组织化学染色、尼氏染色和FJB染色观察海马CA1区神经元形态及损伤情况。
7.在P70时,采用Morris水迷宫实验,测定各组大鼠空间学习及记忆能力。
8. Morris水迷宫测试后,采用旷场试验测定各组大鼠自发活动水平及探索能力。结果：
1.孕期脂多糖刺激对孕鼠分娩时间、产仔量及子代鼠体重的影响。
暴露于LPS组孕鼠与NS注射组孕鼠在分娩时间及产仔量上无明显统计学差异。LPS暴露组新生鼠平均出生体重较NS注射组显著减低。此外,LPS暴露组子代鼠P9体重也较NS注射组子代鼠显著减低。LPS刺激组及NS注射组子代鼠P21体重无统计学差异。
2.孕期暴露于LPS导致子代鼠青少年期KA所致惊厥易感性增加。
腹腔注射KA后,LPS暴露组子代鼠(LPS-KA) SOT较NS注射组子代鼠(NS-KA) SOT缩短,表明孕期LPS刺激可导致子代鼠青少年期KA所致惊厥易感性增加。
3.KA诱导P21大鼠SE后6小时,各组大鼠海马炎症因子的表达情况。
RT-PCR定量分析结果显示：
(1)生前炎症组(LPS-NS)IL-1βmRNA、TNFamRNA、IL-6mRNA及IL-10mRNA的表达与正常对照组(NS-NS)无显著差异；
(2)KA致痫组(NS-KA)IL-1βmRNA、TNFamRNA、IL-6mRNA及IL-10mRNA的表达较正常对照组(NS-NS)均增高;
(3)与NS-KA组相比,LPS-KA组IL-1βmRNA及IL-6mRNA表达增高；然而,IL-10mRNA与TNFamRNA的表达在LPS-KA组与NS-KA组间无差异。
4.KA诱导P21大鼠SE后,各组大鼠海马小胶质细胞活化情况
Western blot与免疫组化染色均显示,与NS-NS组相比,LPS-NS组、NS-KA组大鼠海马Ibal蛋白表达水平升高；与NS-KA组相比,LPS-KA组大鼠海马Ibal蛋白表达水平升高。Ibal蛋白表达结果显示：生前炎症刺激不但可导致生后海马小胶质细胞长期活化,并能加剧KA所致海马小胶质细胞活化程度。
5.KA诱导P21大鼠SE后,各组大鼠海马星形胶质细胞活化情况
Western blot与免疫组化染色均显示,与NS-NS组相比,LPS-NS组、NS-KA组大鼠海马GFAP蛋白表达水平升高；与NS-KA组相比,LPS-KA组大鼠海马GFAP蛋白表达水平升高。GFAP蛋白表达结果显示：生前炎症刺激不但可导致生后海马星形胶质细胞长期活化,并能加剧KA所致海马星形胶质细胞活化程度。
6.KA诱导P21大鼠SE后3天,各组大鼠海马CA1区神经元损伤情况
尼氏染色显示NS-NS组大鼠海马CA1区无神经元损伤,形态正常。LPS-NS组、NS-KA组及LPS-KA组大鼠均出现神经元损伤,表现为尼氏染色阳性细胞数减少,形态异常。与NS-KA组相比,LPS-KA组大鼠海马CA1区神经元损伤加重。尼氏染色结果表明生前炎症刺激可产生长期效应：生前炎症刺激不但可导致生后海马神经元损伤,并能加剧KA所致海马神经元损伤。
NeuN与FJB染色结果同尼氏染色结果相似。7.P70时,Morris水迷宫实验中,各组大鼠空间学习及记忆情况
在Morris水迷宫寻找隐藏平台的学习实验中,随着训练进展,各组大鼠逃避潜伏期逐渐缩短。与NS-NS组相比,LPS-NS组及NS-KA组大鼠寻找到平台的潜伏期明显增长。与NS-KA组相比,LPS-KA组大鼠寻找到平台的潜伏期明显增长。
在Morris水迷宫探索试验中,与NS-NS组相比,LPS-NS组与NS-KA组大鼠目标象限活动时间百分比明显缩短。与NS-KA组相比,LPS-KA组大鼠目标象限活动时间百分比明显缩短。
8.旷场实验中,各组大鼠自发活动水平及探索能力表现情况
旷场实验自发活动结果显示,各组大鼠水平穿越方格数及竖直站立次数无明显不同。
探索行为结果显示,LPS-NS组大鼠与NS-NS组大鼠间点头次数无明显差别。与NS-NS组大鼠相比,NS-KA组大鼠点头次数减少。LPS-KA组大鼠比NS-KA组大鼠点头次数减少,表明LPS-KA组大鼠比NS-KA组大鼠探索能力降低。
结论：
1.孕期炎症刺激可导致子代鼠青少年期海人酸所致惊厥易感性增加；
2.孕期炎症刺激可增加惊厥所致青少年子代鼠海马神经元损伤、恶化神经炎症应答并能加剧惊厥所致空间学习记忆能力及探索能力损害；
3.孕期炎症刺激可导致青少年子代鼠海马神经元损伤、小胶质细胞及星形胶质细胞长期活化,但对海马区炎症因子表达无影响；
4.孕期炎症刺激可导致子代鼠成年期空间学习及记忆能力损害；
综上所述,生前炎症刺激可能通过启动小胶质细胞及星形胶质细胞长期活化以致惊厥所致脑损伤加剧。
第二章孕期感染增加氯化锂-匹罗卡品所致子代成年大鼠惊厥易感性研究目的：
研究孕期感染对氯化锂-匹罗卡品所致子代成年大鼠惊厥易感性及相关脑损伤的影响。研究方法：
1.孕期免疫激活模型的建立
雌性wistar大鼠在孕龄15及16天时,连续两天腹腔注射脂多糖(LPS),每次200ug/kg,构建孕期免疫激活大鼠模型；对照组孕鼠腹腔注射相同容积的生理盐水(NS)。
2.子代年轻成年大鼠癫痫持续状态模型(SE)的建立
选用生后45天(postnatal day45, P45)的雄性子代年轻成年大鼠并随机分组。腹腔注射氯化锂(LiCl,127mg/kg)后,将匹罗卡品(pilocarpine, Pilo)按照36mg/kg给予SE组大鼠腹腔注射,观察大鼠行为变化。痫性发作行为学评价根据Racine分级标准进行(具体分级标准见本研究第一章摘要部分)。持续观察大鼠行为,记录发作的等级及出现时间。在SE开始后1小时给予水合氯醛(400mg/kg)腹腔注射终止发作。仅发作达IV或V级的大鼠纳入后续试验。未达标准的子代大鼠予以剔除。对照组成年大鼠腹腔注射相同容积的NS。
这样,腹腔注射Pilo或NS的子代成年大鼠已经被随机分为4组,正常对照组(NS-NS),生前炎症组(LPS-NS),氯化锂-匹罗卡品(LiPC)致痫组(NS-LiPC)和“二次打击”组(LPS-LiPC)。
3.惊厥易感性测定
LiPC注射后,首次痫性行为发作的潜伏期作为痫性发作开始时间(seizure onset time, SOT)。我们将大鼠前肢局限性阵挛、后肢站立或身体失衡作为首次痫性行为。本研究中,我们用SOT来衡量年轻成年大鼠对LiPC的惊厥易感性。
4.SE后3天,随机选择各组大鼠,采用尼氏染色观察大鼠海马CA1及CA3区神经元形态及损伤情况。
5.P61时,采用旷场试验测定各组大鼠自发活动水平及探索能力。
6.P68时,采用高架迷宫实验测定各组大鼠焦虑水平。
7.P75时,采用Morris水迷宫实验,测定各组大鼠空间学习及记忆能力。结果：
1.孕期暴露于LPS导致子代鼠成年期LiPC所致惊厥易感性增加。
腹腔注射LiPC后,LPS暴露组子代鼠(LPS-LiPC) SOT较NS注射组子代鼠(NS-LiPC) SOT缩短,表明孕期LPS刺激可导致子代鼠成年期LiPC所致惊厥易感性增加。
2. LiPC诱导P45大鼠SE后3天,各组大鼠海马CA1及CA3区神经元损伤情况。
尼氏染色显示NS-NS组大鼠海马CA1及CA3区神经元形态正常,无损伤。LPS-NS组、NS-LiPC组及LPS-LiPC组大鼠均出现神经元损伤,表现为CA1及CA3区尼氏染色阳性细胞数减少,形态异常。与NS-LiPC组相比,LPS-LiPC组大鼠海马CA1及CA3区神经元损伤加重。尼氏染色结果表明生前炎症刺激可产生长期效应：生前炎症刺激不但可导致生后成年期大脑海马神经元损伤,并能加剧惊厥所致海马神经元损伤。
3.旷场实验中,各组大鼠自发活动水平及探索能力表现情况。
旷场实验自发活动结果显示,各组大鼠间水平穿越方格数及竖直站立次数无明显不同。
探索行为结果显示,LPS-NS组与NS-NS组之间及NS-LiPC组与NS-NS组之间点头次数均无明显差别。LPS-LiPC组比NS-LiPC组点头次数减少,表明LPS-LiPC组大鼠比NS-LiPC组大鼠探索能力降低。
4.各组大鼠在高架迷宫实验中的表现情况。
高架迷宫实验中,用焦虑分数来反映焦虑程度。结果显示,LPS-NS组焦虑分数低于NS-NS正常对照组,表明生前炎症刺激导致大鼠生后成年期焦虑水平升高。此外,NS-LiPC组焦虑分数高于NS-NS组。LPS-LiPC组与NS-LiPC组大鼠间焦虑分数无明显差异。
5. Morris水迷宫实验中,各组大鼠空间学习及记忆情况。
在Morris水迷宫寻找隐藏平台的学习实验中,随着训练进展,各组实验动物逃避潜伏期逐渐缩短。与NS-NS组相比,LPS-NS组、NS-LiPC组及LPS-LiPC组大鼠寻找到平台的潜伏期明显延长。与NS-LiPC组相比,LPS-LiPC组大鼠寻找到平台的潜伏期明显延长。
此外,在Morris水迷宫探索试验中,与NS-NS组相比,LPS-NS组、NS-LiPC组及LPS-LiPC组大鼠目标象限活动时间百分比明显缩短。NS-LiPC组与LPS-LiPC组目标象限活动时间百分比无明显不同。结论：
1.孕期炎症刺激可导致子代鼠成年期LiPC所致惊厥易感性增高；
2.孕期炎症刺激可增加惊厥所致成年子代鼠海马神经元损伤；
3.孕期炎症刺激可加剧惊厥所致空间学习及探索能力损害。
4.孕期炎症刺激可升高子代鼠成年期焦虑水平并导致空间学习及记忆能力损害；
综上所述,生前炎症刺激可增加生后成年期惊厥易感性并加剧惊厥所致脑损伤。
第三章新生儿期炎症增加惊厥所致成年大鼠海马依赖性记忆损伤
研究目的：
研究新生儿期免疫应激能否导致小胶质细胞发生长期改变并探索这种变化对成年期癫痫发作所致神经行为结果的影响。研究方法：
1.新生儿期免疫激活模型的建立
给予雄性SD大鼠在生后第3天(postnatal day3,P3)及P5,腹腔注射脂多糖(LPS),每次50ug/kg,构建新生儿期免疫激活大鼠模型；对照组新生鼠腹腔注射相同容积的生理盐水(NS)。
2.免疫荧光法测定新生儿期LPS刺激对海马区小胶质细胞的影响
分别在P5注射LPS或NS后4天(P9)、16天(P21)及40天(P45)时处死大鼠,取海马组织。运用离子钙接头蛋白分子1(Ibal,小胶质细胞标记物)免疫荧光染色研究LPS刺激所致小胶质细胞反应的时程变化。
3.在P3注射LPS或NS后,连续6天给予新生鼠腹腔注射米诺环素(minocycline)或磷酸盐缓冲溶液(PBS)。新生鼠分为3组：SS组(正常对照组),LS组(新生儿期LPS刺激并PBS处理组)和LM组(新生儿期LPS刺激并米诺环素处理组)。分别在P9及P21时处死大鼠,运用Ibal免疫荧光染色研究米诺环素对LPS所致小胶质细胞变化的抑制效应。
4.以下设计以研究新生儿期LPS暴露能否对成年期海人酸(KA)所致神经行为结果产生影响,并探究米诺环素在其中的作用。新生儿期注射NS或LPS的大鼠PBS或米诺环素处理后,在P45时腹腔注射KA,以诱导癫痫发作。此研究共分4组：正常对照组(SSS),成年致痫组(SSK),“二次打击”组(LSK),及“二次打击”米诺环素治疗组(LMK)。具体方法如下：
(1)子代年轻成年大鼠癫痫持续状态(SE)模型的建立
P45时,将KA按照15mg/kg给予SE组大鼠腹腔注射,观察大鼠行为变化。痫性发作行为学评价根据Racine分级标准进行,具体分级标准见第一部分。本研究中,我们仍用痫性发作开始时间(seizure onset time,SOT)来衡量年轻成年大鼠对KA所致癫痫发作易感性。在SE开始后2小时给予大鼠水合氯醛(400mg/kg)腹腔注射终止发作；
(2)SE后6h,随机选择各组大鼠,采用实时定量多聚酶链反应(Real-time polymerase chain reaction, RT-PCR)技术,检测炎性因子IL-1p及TNFα表达情况;
(3)SE后3天,随机选择各组大鼠,采用1bal染色观察海马区小胶质细胞活化情况；
(4)从P46至P55,随机选择各组大鼠,运用Y-迷宫实验检测各组大鼠海马依赖性空间学习情况；
(5)Y-迷宫实验后,从P60至P65,运用水迷宫实验检测各组大鼠海马依赖性空间学习及记忆情况；
(6)水迷宫实验后,在P70,随机选择各组大鼠,运用抑制回避实验检测各组大鼠海马依赖性非空间记忆情况。结果
1.新生儿期暴露于LPS对成年期KA所致惊厥易感性无影响。
腹腔注射KA后,SSK、LSK及LMK大鼠间SOT无统计学差异,表明新生儿期LPS刺激对成年期KA所致惊厥易感性无影响。
2.新生儿期LPS免疫应激导致海马区小胶质细胞发生较长时间活化。
Iba1免疫荧光染色显示,新生儿期LPS暴露组大鼠在P9及P21,海马区Iba1阳性染色光密度(OD)值高于新生儿期NS注射组大鼠；但是在P45时,两组大鼠Iba1阳性染色OD值无差异。该结果显示,新生儿期LPS免疫应激导致海马区小胶质细胞发生较长时间活化至青少年时期。
3.米诺环素抑制LPS所致小胶质细胞活化。
Iba1免疫荧光染色显示,在P9及P21,LS组海马区Ibal阳性染色OD值高于LM与SS组；在P9及P21,SS组与LM组Iba1阳性染色OD值无差异。
4.SE后6h,各组大鼠炎性因子IL-1βmRNA及TNFαmRNA表达情况。
RT-PCR定量分析结果显示：
(1)SSK、LSK及LMK组IL-1β mRNA较SSS组升高；LSK组IL-1βmRNA表达较SSK组及LMK组升高；SSK与LMK间,IL-1β mRNA表达水平无差异；
(2) SSK、LSK及LMK组TNFα mRNA较SSS组升高；LSK组TNFα mRNA表达较SSK及LMK组升高；SSK与LMK间,NFa mRNA表达水平无差异。
5.SE后3天,各组大鼠海马区小胶质细胞活化情况。
Ibal免疫荧光染色显示：SSK、LSK及LMK组Ibal阳性染色OD值较SSS组升高；LSK组Ibal阳性染色OD值较SSK及LMK组升高；SSK与LMK间,Ibal蛋白表达水平无差异；
6.从P46至P55,各组大鼠Y-迷宫实验表现情况。
Y-迷宫实验中,自发交替反应率越高表示大鼠学习能力越强。结果显示SSK、LSK及LMK组自发交替反应率低于SSS组；LSK组自发交替反应率较SSK及LMK组降低,SSK与LMK间自发交替反应率无差别。
7.从P60至P65,各组大鼠水迷宫实验表现情况。
结果显示：在Morris水迷宫寻找隐藏平台的学习实验中,各组实验动物间学习能力无明显差异。
在探索试验中,SSK、LSK及LMK组目标象限活动时间百分比低于SSS组,LSK组目标象限活动时间百分比较SSK及LMK组降低,SSK与LMK间目标象限活动时间百分比无差别。
8.P70时,各组大鼠抑制回避实验表现情况。
抑制回避实验中,暗室逃避潜伏期越长反映大鼠海马依赖性非空间记忆力越好。结果显示：训练后1小时,LSK组暗室逃避潜伏期较SSS组明显缩短；SSS、LMK及SSK组之间,暗室逃避潜伏期无明显差异。训练后24小时,LSK组暗室逃避潜伏期较SSS、SSK及LMK组明显缩短；与SSS组相比,SSK及LMK组暗室逃避潜伏期缩短；SSK与LMK之间暗室逃避潜伏期无明显差异。结论：
1.新生儿期炎症刺激可导致海马小胶质细胞发生较长时间活化；
2.米诺环素可抑制LPS所致新生鼠海马小胶质细胞活化；
3.新生儿期炎症刺激可能通过长期启动小胶质细胞而加剧成年期癫痫发作所致神经炎症应答反应及海马依赖性行为损伤。
Background
Epilepsy, a common chronic neurologic disorder characterized by recurrent seizures, is caused by abnormal, paroxysmal changes in the electrical activity of the brain. It has high morbidity in children and adults. Convulsion, characterized by an abnormal, involuntary contraction of the muscles, is most typically seen with seizure disorders and is always accompanied by conscious disturbance. Because a convulsion is often a symptom of an epileptic seizure, the term convulsion is sometimes used as a synonym for seizure. Frequent and serious seizures can lead to further brain injury as well as persistent mental and behavioral disorders, bringing troubles to the patients, their families and society. Epileptogenesis is defined as the process of developing epilepsy. The mechanisms of epileptogenesis are complicated, which involve neurotransmitter imbalance, ion channel and genetic abnormalities, and glial dysfunction. Although great progress has been made in the etiology, pathogenesis and treatment of epilepsy, the exact mechanisms of epileptogenesis still remain unclear.
Astrocyte and microglia, the important component cells of the central nervous system (CNS), play an essential role in maintaining brain homeostasis, development and pathologies and participate actively in the process of neuronal injury and repair. Neuroinflammation is the term used to describe CNS inflammatory responses characterized by activation and proliferation of glia (especially astrocytes and microglia) and the associated up-regulation of proinflammatory cytokines such as interleukin (IL)-1β, tumor necrosis factor (TNF)a, and IL-6, and anti-inflammatory cytokines such as IL-10. In recent years, accumulated data have indicated that neuroinflammation participates in the pathogenesis of epilepsy. A recognized response to seizures and a potential contributor to mechanisms of epileptogenesis is excessive or prolonged glial activation and the associated increase in proinflammatory cytokine production. Appropriate neuroinflammation occurs as protective responses to CNS insults. However, excessive neuroinflammation can cause damage to the blood-brain-barrier (BBB), leading to increased BBB permeability and exaggerated leukocyte infiltration and enhance neuronal excitability and exacerbate seizure-induced brain injury. Thus, the relationship between neuroinflammation and epileptogenesis is represented as an amplifying feedback loop.
There is an increasing body of evidence suggesting that infection occurring within the intrauterine or perinatal environment can not only produce acute brain injury but affect brain as well as psychological and behavioral development in the long term. Insults to the fetus during critical times of development may alter fetal brain structure and function thereby acting as risk factors for mental, psychiatric and behavioral disorders later in life. Maternal infections of both viral and bacterial origin have been linked with later development in the offspring of autism, schizophrenia and cerebral palsy. For instance, prospective epidemiological studies indicate that maternal influenza, toxoplasmosis, and genital/reproductive infection are associated with schizophrenia. Moreover, inflammation in the neonatal period can also have long-term functional effects on metabolic, immune, behavioral and neuroendocrine system that persist into adulthood, leading to increased susceptibility to neuropsychiatric and behavioral disorders. Rodent models have been developed to determine the behavioral and biological effects of maternal immune activation (MIA) on offspring. In these studies, lipopolysaccharide (LPS) or the synthetic, double-stranded RNA polyriboinosinic-polyribocytidilic acid (polyI：C) are administered to pregnant dams to mimic the immune-stimulating actions of live bacterial or viral infections, respectively. LPS is a large lipid-polysaccharide complex released from the outer cell wall of Gram-negative bacteria that binds to the toll-like receptor4(TLR4) and TLR2expressed on the surface of certain cell types resulting in the activation of nuclear factor-KB (NF-kB) which induces proinflammatory cytokine expression. In the MIA model, maternal LPS increases proinflammatory cytokine mRNA expression and/or proteins in the maternal serum, amniotic fluid, and placenta. Increased cytokine gene expression in fetal brain has been reported in some studies that used a much higher dose of LPS. Recently, accumulating data from animal studies have indicated that prenatal or neonatal LPS can induce long-term hippocampal microglial and astrocytic activation that persisted into adulthood. Since excessive neuroinflammation can cause increased seizure susceptibility, we hypothesize that early life immune challenge may predispose the maturing brain more vulnerable to later-life seizures.
In accordance with our hypothesis, recent epidemiological studies have indicated that maternal infection during pregnancy, such as cystitis, vaginal yeast infection, consistent diarrhea lasting4days and coughs lasting1week was associated with an increased risk for childhood epilepsy. Moreover, intrapartum infection is associated with a higher risk for newborn seizures. A recent animal study has shown that neonatal immune challenge can increase seizure susceptibility in adult rats. However, whether early life infection causes long-lasting brain injury and heightened susceptibility to later seizures and the possible mechanisms underlying this association remain largely unexplored experimentally. To make clealy these questions will help to clarify the mechanisms of epileptogenesis, and will be possible to find new methods to treat this disease.
Chapter I Effect of maternal immune activation during late gestation on seizure susceptibility in juvenile rat offspring
Objectives
The study was designed to investigate whether maternal immune challenge during late gestation could influence juvenile seizure susceptibility and the associated brain injury as well as the possible mechanisms.
Methods
1. The construction of maternal immune activation model
Lipopolysaccharide (LPS) diluted in saline was injected intraperitoneally into pregnant Sprague-Dawley (SD) rats on gestational day (GD)19and20at a dose of300ug/kg. Control dams were injected with a corresponding volume of normal saline (NS) at the same GDs. Pregnant females were monitored for the parturition date. In addition, litter size and birth weight of pups were recorded.
2. Kainic acid (KA)-induced status epilepticus (SE) model in juvenile rat offspring
Juvenile offspring whose mothers received NS or LPS during late pregnancy received7.5mg/kg KA intraperitoneally at postnatal day (P)21. Instead of KA administration, control rats received comparable volume of NS as given to KA-injected rats. A seizure severity grade was assigned according to a modified Racine scale as follows:0—no response; Ⅰ—motionless staring, and slight facial and mouth movements; Ⅱ—pawing, head nobbing, and wet-dog shake; Ⅲ—unilaternal forelimb clonus; Ⅳ—bilaternal forelimb clonus with rearing; Ⅴ—rearing, generalized tonic-clonic seizures and transient loss of postural control. Animals were closely observed and seizure severity and latency to the first sign of seizure were recorded. Seizures were terminated with chloral hydrate (400mg/kg, intraperitoneally)2h after onset of SE. Only animals with grade IV or V seizures were included in this study. Animals were returned to their dams after behavioral monitoring.
Thus, four experimental groups were studied, including normal control (NS-NS), prenatal inflammation (LPS-NS), juvenile seizure (NS-KA), and "two-hit"(LPS-KA) groups.
3. KA seizure susceptibility testing
The latency to the first behavioral seizure after KA administration, referred to as seizure onset time (SOT), was defined by the occurrence of forelimb clonus, rearing and loss of balance, and was recorded to the nearest second for each animal by an individual blind to the prenatal treatment of the animal. SOT is a commonly used measure to describe seizure susceptibility to convulsant compounds in rats.
4. At acute stage, rats were killed6h after SE, and examined for production of cytokines, including IL-1β, TNFa, IL-6and IL-10using real-time polymerase chain reaction (RT-PCR).
5. Glial activation state was analyzed by western blotting of glial markers, glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule1(Ibal)24h after SE.
6. Three days after SE, rats were sacrificed for further examination of glial activation by immunohistological methods.
7. Three days after SE, rats were sacrificed for examination of neuronal injury by NeuN, Nissl and Fluoro-Jade B (FJB) staining.
8. At P70, water maze task was used to evaluate the long-term impact of maternal inflammation on spatial learning and memory ability of adult rat offspring.
9. After water maze test, locomotor activity and exploratory ability were tested using an open-field apparatus.
Results:
1. The effect of maternal LPS exposure on gestational length, litter size and birth weight of pups
We did not observe any significant difference in gestation length or litter size between LPS-exposed dams and controls, though some LPS-exposed pregnant rats died after LPS application, suffered from miscarriage or had stillborn pups. The offspring of LPS-treated dams displayed significantly reduced birth weight and weight gain at P9compared to controls. Pup body weight did not differ between groups at P21.
2. Prenatal LPS increases juvenile seizure susceptibility to KA
Rats exposed to LPS prenatally showed faster mean SOT compared with controls, indicating that maternal LPS exposure increased juvenile seizure susceptibility.
3. Cytokine expression in the hippocampus of P21rats after KA-induced SE
There was no difference in the mRNA expression of the four cytokines, including IL-1β, TNF-a, IL-6and IL-10between LPS-NS and NS-NS groups. The expression of the four cytokines was higher in NS-KA group compared with NS-NS group. Compared with NS-KA group, the expression of IL-1β mRNA and IL-6mRNA was higher in LPS-KA group. However, no difference in the expression of IL-10mRNA and TNFa mRNA was observed between LPS-KA and NS-KA rats.
4. Hippocampal microgilal activation after KA-induced SE
Western blot analysis of Iba1expression and immunohistochemical staining for Ibal showed similar results. Compared with NS-NS group, LPS-NS and NS-KA rats had higher hippocampal Ibal expression. Compared with NS-KA group, LPS-KA group had higher hippocampal Ibal expression. These results demonstrated that prenatal inflammation not only caused prolonged microglial response but also enhanced KA-induced microglial activation.
5. Hippocampal astrocyte activation after KA-induced SE
Western blot analysis of GFAP expression and immunohistochemical staining for GFAP showed similar results. Compared with NS-NS group, LPS-NS and NS-KA rats had higher hippocampal GFAP expression. Compared with NS-KA group, LPS-KA group had higher hippocampal GFAP expression. These results demonstrated that prenatal inflammation not only caused prolonged astrocytic response but also enhanced KA-induced astrocytic activation.
6. Neuronal damage in the hippocampus3days after KA-induced SE
Nissl staining showed that there was no neuronal damage in NS-NS rats. Compared to NS-NS group, Nissl-positive cell counts were significantly reduced in LPS-NS, NS-KA and LPS-KA groups. Fewer Nissl-staining cells were detected in the LPS-KA group compared with NS-KA group.
The results for NeuN and FJB staining were consistent with those of Nissl staining.
7. The performance of adult rat offspring in the Morris water maze after KA-induced SE
Performance improved in all groups over the four training days in the place navigation test. Compared to NS-NS group, escape latency was significantly increased in LPS-NS, NS-KA and LPS-KA groups. Escape latency in LPS-KA rats was greater than that in NS-KA rats.
On the day of probe testing, NS-NS rats spent significantly more time in the (target quadrant) TQ than animals from the other three groups."Two-hit" rats spent significantly less time in the TQ than animals in the NS-KA group.
8. The performance of adult rat offspring in the open field after KA-induced SE
No difference in the number of squares crossed and rearing frequency was observed among NS-NS, LPS-NS, NS-KA and LPS-KA groups.
No difference in the number of head dippings was observed between NS-NS and LPS-NS groups. NS-KA and LPS-KA groups demonstrated smaller number of head dippings compared with that of NS-NS control."Two-hit" rats showed smaller number of head dippings compared with NS-KA group.
Conclusins
1. Maternal infection increases seizure susceptibility to KA in juvinile rat offspring;
2. Maternal infection, without perturbation of cytokine expression, causes long-lasting neuronal injury, glial alteration and cognitive deficit in juvenile offspring;
3. Maternal infection during late pregnancy enhances neuronal injury, exaggerates neuroinflammatory responses, and exacerbates long-term neurobehavioral impairment associated with a second adolescent KA insult.
In sum, primed glia by prenatal infection may underlie, at least in part, long-term "perinatal programming" of later-life vulnerability to seizure insult.
Chapter II Prenatal immune challenge in rats increases susceptibility to seizure-induced brain injury in adulthood
Objectives
The study was designed to investigate whether maternal immune challenge during gestation could influence adult seizure susceptibility and the associated brain injury.
Methods
1. The construction of maternal immune activation model
Lipopolysaccharide (LPS, Escherichia coli, serotype055:B5) diluted in saline was injected intraperitoneally into pregnant Wistar rats on gestational day (GD)15and16at a dose of200ug/kg. Control dams were injected with a corresponding volume of normal saline (NS) at the same GDs.
2. Lithium-pilocarpine (LiPC)-induced status epilepticus (SE) model in adult rat offspring
At P45, male offspring from LPS-or NS-treated mothers received intraperitoneal injections of lithium chloride (127mg/kg) followed16h later by pilocarpine (36mg/kg, i.p.)(LiPC). Instead of pilocarpine application, control rats received corresponding volumes of NS as given to the pilocarpine-injected rats. A seizure severity grade was assigned according to a modified Racine scale (detailed in part1). Animals were closely observed and seizure severity and latency to the first sign of seizure were recorded. Seizures were terminated with chloral hydrate (400mg/kg, intraperitoneally)1h after the onset of SE. Only animals with grade IV or V seizures were included in this study.
Four experimental groups were studied, including normal controls (NS-NS), prenatal inflammations (LPS-NS), adult seizures (NS-LiPC), and "two-hit"(LPS-LiPC) animals.
3. LiPC seizure susceptibility testing
SOT was used to assess seizure susceptibility (detailed in part1).
4. Three days after SE, rats were sacrificed for examination of neuronal injury by Nissl staining.
5. At P61, locomotor activity and exploratory ability were tested using an open-field apparatus.
6. At P68, anxiety was examined using the elevated plus maze.
7. Begining at P75, water maze task was used to evaluate long-term impact of maternal inflammation on spatial learning and memory ability of adult rat offspring.
Results:
1. Prenatal LPS increases adult seizure susceptibility to LiPC
Rats exposed to LPS prenatally showed faster mean SOT compared with controls, indicating that maternal LPS exposure increased adult seizure susceptibility.
2. Neuronal damage in the hippocampus3days after KA-induced SE
Nissl staining showed that there was no hippocampal neuronal damage in NS-NS rats. Compared to NS-NS group, Nissl-positive cell counts were significantly reduced in LPS-NS and NS-LiPC groups. Fewer Nissl-staining cells were detected in the LPS-LiPC group compared with NS-LiPC group. These results demonstrated that prenatal inflammation not only caused neuronal injury but also enhanced LiPC-induced neuronal damage.
3. The performance of adult rat offspring in the open field after LiPC-induced SE
No difference in the number of squares crossed and rearing frequency was observed among NS-NS, LPS-NS, NS-LiPC and LPS-LiPC groups.
No difference in the number of head dippings was observed between NS-NS and LPS-NS groups. In addition, no difference in the number of head dippings was observed between NS-NS and NS-LiPC groups."Two-hit" rats showed smaller number of head dippings compared with NS-LiPC group.
4. The performance of adult rat offspring in the elevated plus maze after LiPC-induced SE
The offspring of LPS-treated mothers (LPS-NS) had significantly lower anxiety score than NS-NS controls, indicating that maternal immune challenge caused increased anxiety in offspring. No significant difference in anxiety scores was found in LPS-LiPC rats compared with NS-LiPC rats. The "single hit" NS-LiPC animals had higher anxiety scores than NS-NS controls.
5. The performance of adult rat offspring in the Morris water maze after KA-induced SE
Performance improved in all groups over the four training days in the place navigation test. Compared to NS-NS group, escape latency was significantly increased in LPS-NS and NS-LiPC groups. Escape latency in LPS-LiPC rats was greater than that in NS-LiPC rats.
On the day of probe testing, NS-NS rats spent significantly more time in the TQ than animals from the other three groups. No significant difference existed between NS-LiPC and LPS-LiPC groups. Conclusins
1. Maternal infection increases seizure susceptibility to LiPC in the adult rat offspring;
2. Maternal infection alone causes long-lasting neuronal injury, increased anxiety level and cognitive deficit in the offspring rats;
3. Maternal infection during late pregnancy enhances neuronal injury and exacerbates long-term neurobehavioral impairment associated with a second adult LiPC insult.
In sum, prenatal infection can increase adult seizure susceptibility and exacerbates the associated brain injury.
Chapter Ⅲ Neonatal inflammation exacerbates seizure-induced hippocampus-dependent memory impairment in adult rats
Objectives
The study was designed to examine whether neonatal lipopolysaccharide (LPS) exposure is associated with changes in microglia and whether these alternations could influence later seizure-induced neurobehavioral outcomes.
Methods
1. The construction of neonatal immune activation model
LPS diluted in saline was injected intraperitoneally (i.p.) into male Sprague-Dawley pups at postnatal (P) day3and P5at a dose of50ug/kg. Control pups were injected with a corresponding volume of normal saline (NS).
2. To study the effect of neonatal LPS on hippocampal microglia using immunofluorescence staining, male pups were sacrificed4,16, or40days after the last administration of NS or LPS on P5. Immunofluorescence staining of Ibal, a marker of microglia, was used to study the time course of LPS-induced microglia alternation.
3. To study the inhibitory effect of minocycline on neonatal LPS-induced microglia alternation, pups administered saline or LPS neonatally were treated with phosphate buffered saline (PBS) or minocycline for6consecutive days. Three groups of pups were studied, including normal controls treated with PBS (SS), neonatally LPS-exposed pups treated with PBS (LS), and neonatally LPS-exposed pups treated with minocycline (LM). On P9(4days after administration of NS or LPS on P5) and P21, rats were sacrificed and evaluated for hippocampal microglial activation using immunofluorescence staining of Ibal.
4. To investigate the effect of early-life LPS exposure on seizure-induced neurobehavioral impairment and to further explore whether minocycline would prevent changes in susceptibility to later seizure-induced brain injury, a separate study was conducted. Rat pups injected with saline or LPS neonatally were administered kainic acid (KA) at P45. Four groups were studied, including SSS (normal controls), SSK.(adult seizures treated with PBS neonatally), LSK ("two-hit" animals treated with PBS at the time of neonatal LPS exposure), and LMK ("two-hit" animals treated with minocycline at the time of neonatal LPS exposure).
(1) KA-induced status epilepticus (SE) model in adult rats
At P45, KA dissolved in saline (15mg/kg) was administered i.p. to rats that were injected with saline or LPS neonatally. Controls received equal volumes of NS. A seizure severity grade was assigned according to a modified Racine scale (detailed in part1). Animals were closely observed and seizure severity and latency to the first sign of seizure were recorded. Seizure onset time (SOT) was used to assess seizure susceptibility. Seizures were terminated with chloral hydrate (400mg/kg, i.p.)2h after the onset of SE. Only animals with grade IV or V seizures were included in this study;
(2) At acute stage (6h after SE), animals were killed for quantification of hippocampal IL-1β and TNFa production using real-time polymerase chain reaction (RT-PCR);
(3) Three days after SE, rats were sacrificed for further examination of microglial activation by immunofluorescence staining of Ibal;
(4) From P46to P55, Y maze task was used to evaluate hippocampus-dependent spatial learning ability of adult rats;
(5) From P60to P65, water maze task was used to evaluate hippocampus-dependent spatial learning and memory ability of adult rats;
(6) At P70, inhibitory avoidance task was used to evaluate hippocampus-dependent nonspatial memory ability of adult rats.
Results:
1. Neonatal LPS has no effect on KA-induced seizure susceptibility in adult rats.
There were no significant difference in SOTs among SSK, LSK, and LMK groups, indicating that neonatal LPS has no effect on KA-induced seizure susceptibility in adulthood.
2. Transient microglia activation after neonatal LPS exposure.
The analysis of immunostaining of Ibal revealed that LPS-treated pups exhibited significantly greater hippocampal expression of Ibal than NS-treated pups at P9and P21. However, we observed no significant difference in the expression of Ibal at P45. In short, dual exposure to LPS on P3and P5caused persistent activation of hippocampal microglial cells. This activation, however, was not permanent and subsided within40days.
3. Minocyclne inhibits neonatal LPS-induced microglial activation
Compared with SS, LS showed higher expression of Ibal at P9and P21. Minocycline administration significantly reduced LPS-induced microglia activation in LM pups compared with LS pups at both P9and P21. There was no difference in the expression of Ibal between SS controls and LM pups at both P9and P21.
4. Cytokine expression in the hippocampus of adult rats after KA-induced SE
Compared with SSS group, SSK, LSK and LMK rats showed higher expression of IL-1βmRNA. Compared with SSK group, the expression of IL-1β mRNA was higher in LSK group. There was no difference in the expression of IL-1(3mRNA between SSK and LMK groups.
Compared with SSS group, SSK, LSK and LMK rats showed higher expression of TNFa mRNA. Compared with SSK group, the expression of TNFa mRNA was higher in LSK group. There was no difference in the expression of TNFamRNA between SSK and LMK groups.
5. Hippocampal microgilal activation after KA-induced SE
Compared with SSS group, SSK, LSK and LMK rats had higher hippocampal Ibal expression. Compared with SSK and LMK groups, LSK group had higher hippocampal Ibal expression. There was no difference in the expression of Ibal between SSK and LMK groups.
6. The performance of adult rats in the Y maze after KA-induced SE
Animals in SSK group, LSK group, and LMK group all demonstrated lower spontaneous alternation scores when compared with SSS controls. Compared with SSK and LMK rats, the percent of alternation in "two-hit" rats (LSK) was significantly reduced. There was no difference in the percent alternation between SSK and LMK groups.
7. The performance of adult rats in the water maze after KA-induced SE
All groups of rats have comparable rate of acquisition. On the day of probe testing, SSS rats spent significantly more time in the TQ than animals from the other three groups. LSK rats spent significantly less time in the TQ than animals in SSK and LMK groups. SSK and LMK rats spent comparable time in the TQ.
8. The performance of adult rats in the inhibitory avoidance task after KA-induced SE
SSS, SSK, and LMK rats demonstrated comparable retention time at1h after training. However, the latency to step into the dark compartment was significantly decreased in LSK rats compared with SSS group.
At24h, the latency to step into the dark compartment was significantly increased in SSS rats compared with SSK, LSK and LMK groups. Compared with LSK group, SSK and LMK groups spent longer time to step into the dark compartment. No difference in escape latency was observed between SSK and LMK groups.
Conclusins
1. Neonatal inflammation caused persistent activation of hippocampal microglial cells;
2. Minocycline inhibited neonatal LPS-induced microglial activation;
3. Neonatal inflammation predisposed the immatured brain to exacerbated neuroinflammatory response and worse hippocampus-dependent behavioral deficit following seizures in adulthood, possibly by priming microglia.
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