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通讯作者:

胡安康,E-mail:300112110963@stu.xzhmu.cn

中图分类号:R742.1

文献标识码:A

文章编号:1007-4368(2023)08-1094-08

DOI:10.7655/NYDXBNS20230808

参考文献 1
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参考文献 4
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参考文献 5
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参考文献 6
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参考文献 7
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参考文献 8
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参考文献 9
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参考文献 10
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参考文献 11
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参考文献 12
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参考文献 13
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参考文献 14
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参考文献 15
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参考文献 16
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参考文献 17
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参考文献 18
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参考文献 19
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参考文献 20
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参考文献 21
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参考文献 22
DONG X,HAO X,XU P,et al.RNA sequencing analysis of cortex and hippocampus in a kainic acid rat model of temporal lobe epilepsy to identify mechanisms and thera⁃ peutic targets related to inflammation,immunity and cog⁃ nition[J].Int Immunopharmacol,2020,87:106825
参考文献 23
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参考文献 24
JOSHI S,BAYAT A,JONES A,et al.The effects of ammo⁃ nia stimulation on kainate⁃induced status epilepticus and anterior piriform cortex electrophysiology[J].Epilepsy Behav,2020,104(Pt A):106885
目录contents

    摘要

    目的:建立和评价梨状皮层微量注射红藻氨酸(kainic acid,KA)激发的癫痫小鼠模型。方法:利用即刻早期基因(c- fos)免疫荧光染色技术、原位末端转移酶标记染色(TdT-mediated dUTP nick-end labeling,TUNEL)技术在急性癫痫小鼠模型中确定癫痫相关脑区梨状皮层。在梨状皮层脑立体定位注射KA后,观察小鼠癫痫发作程度(Racine评分)和癫痫发作潜伏期,行为学实验包括水迷宫检测小鼠学习记忆能力和新物体识别实验检测小鼠探索能力,癫痫发作结果和行为学实验结果均与海马立体定位注射KA的癫痫模型作对比。利用电生理技术检测新模型小鼠神经元电活动。结果:c-fos免疫荧光和TUNEL染色结果显示,在急性癫痫小鼠模型中除海马外,梨状皮层脑区神经元也被大量激活,且发生凋亡;在梨状皮层注射KA后成功激发癫痫,与海马注射KA的癫痫模型相比,癫痫发作评分没有显著性差异且发作潜伏期更短;行为学结果显示,梨状皮层注射KA 的癫痫模型小鼠空间记忆能力降低,新物体探索能力降低,该结果与海马注射KA的癫痫模型相比无显著性差异;膜片钳实验显示梨状皮层注射KA的癫痫模型神经元放电频率增加、幅值降低、静息膜电位上升。结论:在梨状皮层微量注射KA后成功激发癫痫,这为癫痫发病机制的研究提供了一种新的动物模型。

    Abstract

    Objective:The current study aims to establish and evaluatee the kindling model of epilepsy induced by kainic acid(KA) microinjection into piriform cortex. Methods:Using c -fos immunofluorescence staining and TdT -mediated dUTP nick - end labeling (TUNEL)staining techniques to find the piriform cortex associated with epilepsy in a acute epilepsy mouse model. Racine score and seizure latency were observed after injection of KA in piriform cortex. Behavioral experiments were performed including testing the learning and memory abilities of mice in a water maze and the exploration ability of mice in a new object. The results of seizures and behavioral experiments were compared with the epileptic model of stereotactic injection of KA into the hippocampus. The electrical activities of new model mouse neurons were measured by electrophysiological techniques. Results:The results of c - fos immunofluorescence and TUNEL staining showed that in addition to the hippocampus,neurons in the piriform cortex and hippocampus were also significantly activated,and apoptosis occurred in the acute epilepsy mouse model. After injecting KA into the piriform cortex, the epilepsy was successfully ignited. Compared with the epilepsy model injected KA into the hippocampus,there was no significant difference in the epileptic seizure score and the seizurelatency was shorter. The behavioral results showed that the epileptic model mice induced by KA injection into the piriform cortex had a reduced spatial memory ability and a reduced ability to explore new objects. These results showed no significant difference compared to the epileptic model induced by KA injection into the hippocampus. The patch clamp recording test showed an increase in firing frequency and the resting membrane potential,and a decrease in amplitude of neuronal discharge. Conclusion:The microinjection of KA into the piriform cortex has successfully induced epilepsy,which may provide a new animal model for studying the pathogenesis of epilepsy.

    关键词

    梨状皮层海马癫痫红藻氨酸c-fos

  • 癫痫是一种慢性神经系统疾病,其病理特征是神经元过度或超同步放电[1]。全球 1%~2% 的人患有癫痫并影响着全球 10% 人口的正常生活[2]。其反复无端发作使患者备受煎熬,给患者及其家人造成了很大困扰。目前癫痫的发病机制暂不明确,但普遍认为是神经元异常放电引起的大脑暂时性生理功能障碍[3]。内侧颞叶癫痫是成人中最常见的癫痫类型[4],据报道,颞叶癫痫患者会发生许多病变,包括海马硬化、细胞死亡[5]、颗粒细胞分散[6] 和神经胶质增生等。为了模拟颞叶癫痫,研究学者们已经开发了几种癫痫实验模型,包括毛果芸香碱模型、电点燃模型和红藻氨酸(kainic ac⁃ id,KA)模型等,其中KA癫痫模型被广泛应用于科学研究。

  • KA 是一种高亲和力激动剂,主要由红藻氨酸受体的激活发挥作用。KA 受体与 AMPA 受体(α⁃ amino⁃3⁃hydroxy⁃5⁃methyl⁃4⁃isoxazole⁃propionic acid receptor,α⁃氨基⁃3⁃羟基⁃5⁃甲基⁃4⁃异恶唑丙酸受体)和 NMDA 受体(N⁃methyl⁃D⁃aspartic acid recep⁃ tor,N⁃甲基⁃D⁃天冬氨酸受体)一起构成谷氨酸受体的离子型受体家族。天然红藻氨酸受体广泛分布在大脑中[7],且在海马区域较为密集。1970 年以来,海马就被认为是与癫痫发作相关的区域[8]。研究人员将 KA 注射到海马中,发现胶质增生[9] 和细胞凋亡等病理变化,其发病特征也与在人类颞叶癫痫患者中观察到的相似[10]。因为它能模拟在单侧海马发生硬化的颞叶癫痫病例且死亡率低[11],所以海马内给予 KA 诱导的自发性癫痫小鼠模型被科研界广泛应用,成为较成熟的癫痫模型[12]

  • 梨状皮层与海马结构相似,具有保守发育的三层结构,与边缘和皮层区域有广泛的联系,且拥有与海马体中类似的微电路,提供兴奋、前馈抑制和反馈抑制[13-14]。容易受到兴奋性毒性损伤,并且与癫痫发作有关[15]。早在 1993 年研究显示,在癫痫模型中,海马、杏仁核、嗅球、梨状皮质和内嗅皮质中检测到 c ⁃fos 信使 RNA(messenger RNA,mRNA)表达显著增加[16]。后续研究表明,电刺激梨状皮层也可以产生抗癫痫作用[17]。但是在人类癫痫研究中,人们大多关注内侧颞叶结构,尤其是海马[18],而梨状皮层在癫痫研究中未得到重视。

  • 本研究通过与海马注射 KA 癫痫模型对比,探讨梨状皮层注射KA癫痫模型的可行性及优势。

  • 1 材料和方法

  • 1.1 材料

  • 8 周龄C57BL/6雄鼠,体重20~25 g,由徐州医科大学实验动物中心提供。随机分为腹腔注射生理盐水对照组(4只)、腹腔注射15 mg/kg KA组(KA组, 4只)、海马注射KA模型组(Hi⁃KA组,5只)、海马注射生理盐水对照组(Hi⁃Con组5只)、梨状皮层注射 KA 模型组(Pir⁃KA 组,30 只)和梨状皮层注射生理盐水对照组(Pir⁃Con组,10只)。本研究经徐州医科大学伦理批准,实验伦理号:202207S128。

  • KA(K2389,Sigma 公司,美国),c ⁃ fos 抗体 (2250,Cell Signaling Technology 公司,美国),DAPI 染色液(C1005,上海碧云天),TUNEL 试剂盒 (11684795910,罗氏公司,美国),荧光显微镜 (BX53/DP73,Olympus公司,日本),脑立体定位仪、台式动物手术显微镜(深圳瑞沃德公司),冰冻切片机(Leica 公司,德国),微量移液器(Gilson 公司,法国),膜片钳放大器(MultiClamp700B,Axon公司,美国),数据采集器(Digidata1400A,Molecular Devices 公司,美国),微操纵器(MPC⁃200,Scientifica 公司,英国),硼硅酸玻璃电极(1.2 mm 外径,0.69 mm 内径)、P97微电极拉制仪拉制(Sutter Instrument公司,美国)

  • 1.2 方法

  • 1.2.1 c⁃fos免疫荧光染色

  • 小鼠腹腔注射15 mg/kg KA约2 h后,经4%多聚甲醛灌注固定,取脑,固定过夜,经30%蔗糖脱水沉糖。冰冻切片,切片厚25 μm,10%山羊血清封闭于 37℃烘箱 40 min,滴加 c⁃fos 抗体(1∶500),4℃孵育过夜。PBS 清洗 3 次,10 min/次,滴加荧光二抗 (1∶500)孵育 1.5 h;PBS 清洗 3 次,10 min/次,DAPI 染色10 min;PBS清洗3次,10 min/次,加防荧光淬灭剂封片并于荧光显微镜下观察拍照。

  • 1.2.2 TUNEL染色

  • 冰冻切片25 μm 充分晾干,PBS漂洗2次;玻片干后,加 50 μL TUNEL 反应混合液(阴性对照组仅加50 μL 2号液)于标本上,加盖玻片或封口膜在暗湿盒中反应 37℃ 1 h。PBS 漂洗 3 次后加 1 滴 PBS 在荧光显微镜下拍摄。

  • 1.2.3 模型建立方法

  • 小鼠麻醉后固定于立体定位仪上,剪开头皮、暴露颅骨,利用前、后囟调平颅骨,利用玻璃电极连接微量注射器将 200 nL KA(1 mg/mL)注射于海马(AP:-2.0,ML:-1.5,DV:-1.5)或梨状皮层(AP:2.2, ML:-2.0,DV:-4.5),注射完成后留针10 min。缓慢拔针并完成缝合。

  • 梨状皮层使用剂量为 0.1 μg、0.2 μg 和 0.4 μg,分别立体定位注射 200 nL KA(0.5 mg/mL)、200 nL KA(1 mg/mL)和200 nL KA(2 mg/mL)。

  • 1.2.4 行为学方法

  • 1.2.4.1 水迷宫

  • 前4 d为训练期,分别从水迷宫4个象限将小鼠依次放入。每次训练时间为1 min,1 min内找到平台,使其在平台上停留15 s。若1 min内未找到平台则引导小鼠到达平台并停留 15 s。第 5 天为测试期,撤掉平台,将小鼠从第二象限放入,记录第一次到达平台的时间和到达总次数等信息。

  • 1.2.4.2 新物体识别

  • 第一阶段,在实验装置里放入两个相同物品,并让小鼠自由探索10 min。第二阶段,与第一阶段间隔1 h以上,将其中一个物品换成新物品,再次放入测试小鼠,让其自由探索10 min,完成测试。更换测试小鼠之前,用75%酒精擦拭实验装置。

  • 1.2.5 电生理记录

  • 对照组和处理组小鼠分别使用异氟烷麻醉后,使用 20 mL 冰冻记录用人工脑脊液(ASCF)灌注小鼠。使用大剪刀迅速断头,取出鼠脑,剥离至预充氧(95%O2+5%CO2)的高糖 ASCF 中。2 min 后取出鼠脑修块,并转移固定到震荡切片机的载脑台上并一起置于切片机的切片槽中,调节刀片角度,迅速将鼠脑切成250 μm厚脑片。用吸管将切好的脑片转移至预充氧的32℃ ASCF中,孵育30 min,然后再转移至室温,稳定至少1 h后开始记录。

  • 吸管将孵育好的小鼠脑片转移到充满ASCF的脑片槽中,用缠有尼龙丝的铂金圏压住脑片。采用全细胞膜片钳的方法来记录膜电位和电流诱发的动作电位。玻璃电极灌入电极内液后给予正压,接近细胞后释放正压并用嘴短促地吸气,给予细胞负压,同时用Multi Clamp700B迅速将细胞钳制到70 mV,等待电极与细胞间形成高阻封接(>1 GΩ)。使用 Zap 方式进行破膜,观察破膜后串联电阻 Ra (<30 MΩ)和漏电流(<-100 pA)的值。对于良好破膜的细胞进行全细胞膜片钳记录。在电流钳模式下,I= 0时给予细胞不同电流刺激(步阶,-200 pA+Δ50 pA) 以诱发动作电位,刺激时间为500 ms,2次trial间隔为5 s。全细胞记录过程中,观察和记录Ra值,变动范围超过25%的细胞去除。刺激电流为0 pA时,记录的膜电位为细胞静息电位(RMP)。

  • 1.3 统计学方法

  • 应用GraphPad Prism 9.0进行统计学分析,符合正态分布的计量资料,两组比较采用t检验;多组比较采用单因素方差分析或者双因素方差分析。P< 0.05 为差异有统计学意义。

  • 2 结果

  • 2.1 腹腔注射KA的急性癫痫模型中梨状皮层和海马脑区被激活

  • 利用经典急性癫痫小鼠模型寻找癫痫相关脑区。在小鼠腹腔注射15 mg/kg KA 2 h后灌注取脑,冰冻切片后进行c⁃fos免疫荧光染色。结果显示,小鼠大脑梨状皮层和海马区域内神经元被大量激活 (图1A),c⁃fos阳性细胞数量显著增加(P <0.001,图1B),与海马相比无显著性差异。结果表明,在腹腔注射 KA 的急性癫痫模型中,梨状皮层和海马脑区一样,神经元会被大量激活。

  • 2.2 腹腔注射KA的急性癫痫模型中梨状皮层和海马脑区神经元发生凋亡

  • 已有研究显示,在 KA 的刺激下神经元过度激活会对大脑有进一步损伤,造成细胞凋亡[19]。利用 TUNEL检测技术,对急性癫痫小鼠模型的梨状皮层和海马脑区进行凋亡检测(图2A),结果显示,在急性癫痫模型小鼠中梨状皮层和海马脑区的神经元凋亡数量与对照组相比显著增多(P <0.001,图2B)。与海马区域相比,梨状皮层神经元凋亡数量也显著增多(图2B)。这表明梨状皮层和海马脑区的神经元在急性癫痫小鼠模型中都发生凋亡,且梨状皮层神经元凋亡数量比海马显著增多。

  • 2.3 梨状皮层脑立体定位注射KA的最佳剂量

  • 上述结果表明除海马外,梨状皮层神经元在急性癫痫模型中也被大量激活,海马立体定位注射 KA的癫痫模型已被广泛应用,于是将KA分别定位注射到小鼠梨状皮层。参考海马立体定位注射KA 的癫痫模型使用1 nmol(约0.213 μg)KA剂量[20],在梨状皮层探索了0.1 μg、0.2 μg和0.4 μg3种使用剂量,立体定位注射后观察小鼠行为。对小鼠痫性发作强度进行惊厥分级,分级标准采用Racine分级方法:0级,行为正常;Ⅰ级,动须、眼,湿狗样抖动,节律性咀嚼;Ⅱ级,点头,甩尾;Ⅲ级,一侧前肢阵挛; Ⅳ级,双侧前肢阵挛,站立;Ⅴ级,全身阵挛,跌倒。其中 Racine Ⅰ~Ⅲ级行为代表了部分点燃,Racine Ⅳ、Ⅴ级行为代表完全点燃。结果显示KA 0.1 μg组小鼠完全点燃率为20%,KA 0.2 μg组小鼠完全点燃率为90%,KA 0.4 μg组小鼠完全点燃率为90%,但 20%因过度癫痫造成死亡。结果表明,梨状皮层脑立体定位注射KA能完全点燃癫痫,且KA的最佳剂量为0.2 μg /只,该剂量也与海马模型使用的剂量最为接近(表1)。

  • 图1 急性癫痫模型小鼠中梨状皮层和海马神经元被激活

  • Figure1 Neurons in the piriform cortex and hippocampus were activated in acute epileptic mice

  • 2.4 梨状皮层脑立体定位注射KA可以产生癫痫样行为

  • 在梨状皮层脑立体定位注射 KA 后,观察 Ra⁃ cine评分和癫痫发作潜伏期,并与既往海马立体定位注射KA的癫痫模型作对比。结果发现与海马注射 KA(Hi⁃KA)的癫痫模型相比,梨状皮层注射 KA (Pir⁃KA)的癫痫模型在癫痫发作评分没有差异(图3A),但发作潜伏期更短(图3B),更具优势。手术恢复1周后进行新物体识别实验和水迷宫实验,结果显示,与对照组相比,Pir⁃KA 组新物体探索时间和总探索时间都显著降低,与Hi⁃KA组相比无显著性差异(图3C~E),表明梨状皮层癫痫模型小鼠的探索能力降低。在水迷宫实验中,Pir⁃KA组与对照组相比逃避潜伏期显著提高(P <0.001,图3G),找到平台次数显著降低(P <0.001,图3H),总路程不变(图3I),结果与Hi⁃KA组相比均无显著性差异。结果表明,梨状皮层癫痫模型和海马癫痫模型一致,记忆能力和探索能力都显著降低。

  • 2.5 梨状皮层脑立体定位注射KA后神经元兴奋性增加

  • 癫痫模型的典型特征是神经元的异常放电,为了验证梨状皮层脑立体定位注射KA癫痫模型的可行性,本研究利用膜片钳技术在不同电流刺激下记录了小鼠梨状皮层的放电频率、放电幅值和静息膜电位的变化(图4A)。结果显示,与对照组相比Pir⁃ KA组小鼠梨状皮层神经元放电频率显著增加(P <0.05,图4B、C),放电幅值显著减小(P <0.05,图4D),膜电位显著上升(P <0.001,图4E)。结果表明,在梨状皮层局部注射KA的癫痫模型中,梨状皮层神经元的兴奋性增加。梨状皮层脑立体定位注射KA可以作为新的癫痫模型。

  • 图2 在急性癫痫小鼠中梨状皮层和海马区域发生凋亡

  • Figure2 Apoptosis in the piriform cortex and hippocampus of mice with acute epilepsy

  • 表1 各组小鼠癫痫发作程度Racine分级结果

  • Table1 Results of Racine grading of seizure severity in mice

  • 3 讨论

  • 近年来,对癫痫的病理学、组织学、分子生物学和发病机制的理论研究都直接或者间接地来自癫痫动物模型。现已建立的成熟的模型包括 KA 模型、戊四氮模型、匹罗卡品模型、电点燃模型、电休克模型等,其中KA 模型在潜伏期、行为症状以及脑电图特征等方面与人类颞叶癫痫非常相似,有助于更好地了解颞叶癫痫过程及相关重要共病,开发更有效的靶向治疗药物。但在腹腔注射KA的全身性癫痫小鼠模型中,当 KA 注射剂量过大时死亡率较高,剂量过低时不足以诱发癫痫发作以及形成明显的痫后脑损伤。全身给药不仅KA使用剂量大且无法应用于局灶性癫痫发病机制的研究,脑内局部给药相比全身给药可以避免血⁃脑脊液屏障的阻隔、减少药物使用量,还可以应用于局灶性癫痫。海马脑内局部给药已被广泛应用[21],而本研究结果显示,在腹腔注射KA的急性癫痫模型中,除海马外,梨状皮层神经元也被大量激活且存在凋亡。提示梨状皮层或许能和海马一样作为脑局部注射位点。参考海马注射KA的使用剂量,在梨状皮层使用了0.1 μg、0.2 μg 和 0.4 μg 3 种剂量,结果显示,梨状皮层注射KA能成功激发癫痫,且KA最佳剂量为0.2 μg / 只,该剂量也与海马模型使用剂量 1 nmol/只接近。本研究结果显示,与海马注射KA的癫痫模型相比,梨状皮层注射KA的癫痫模型的癫痫发作潜伏期更短,更具优势。

  • 图3 梨状皮层脑立体定位注射KA可以产生癫痫样行为

  • Figure3 Epileptic behavior can be induced by stereotactic injection of KA into piriform cortex

  • Morris水迷宫是一种常用测试学习记忆和认知功能的实验方法,通过水迷宫实验发现小鼠梨状皮层注射KA 0.2 μg时逃避潜伏期较对照组显著延长,穿越平台的次数明显减少,说明小鼠出现了记忆力恢复缓慢,符合注射KA后神经元损伤、认知功能障碍的效果[22]。新物体探索实验结果显示小鼠梨状皮层注射KA 0.2 μg时对新物体探索兴趣降低,这与 Batterman 等[23] 的研究一致。

  • 图4 梨状皮层脑立体定位注射KA后神经元兴奋性增加

  • Figure4 Increased neuronal excitability after stereotactic injection of KA into piriform cortex

  • 本研究显示在梨状皮层脑区注射KA可以产生癫痫样放电,放电频率增加,静息膜电位降低,这表示梨状皮层注射KA后神经元更容易兴奋。Joshi等[24] 研究也表明,KA腹腔注射诱导的急性癫痫模型中会引起梨状皮层明显的脑电改变[24],与本研究一致。

  • 本研究结果显示,小鼠梨状皮层注射KA 0.2 μg 后,结合癫痫评分、行为学和电生理结果,该模型均符合癫痫模型特征。相比海马注射KA的癫痫模型来说,癫痫发作潜伏期更短。综上,梨状皮层注射 KA 0.2 μg的小鼠模型可作为新癫痫模型。

  • 参考文献

    • [1] BRENNAN G P,HENSHALL D C.MicroRNAs as regula⁃ tors of brain function and targets for treatment of epilepsy [J].Nat Rev Neurol,2020,16(9):506-519

    • [2] FALCO ⁃WALTER J.Epilepsy ⁃ definition,classification,pathophysiology,and epidemiology[J].Semin Neurol,2020,40(6):617-623

    • [3] LIU A H,CHU M,WANG Y P.Up⁃regulation of trem2 in⁃ hibits hippocampal neuronal apoptosis and alleviates oxi⁃ dative stress in epilepsy via the PI3K/Akt pathway in mice [J].Neurosci Bull,2019,35(3):471-485

    • [4] BRUXEL E M,DO CANTO A M,BRUNO D C F,et al.Multi⁃omic strategies applied to the study of pharmacore⁃ sistance in mesial temporal lobe epilepsy[J].Epilepsia Open,2022,7(Suppl 1):S94-S120

    • [5] HAGHANI S,JAMALI ⁃RAEUFY N,ZEINIVAND M,et al.Hepatocyte growth factor attenuates the severity of sta⁃ tus epilepticus in kainic acid ⁃induced model of temporal lobe epilepsy by targeting apoptosis and astrogliosis[J].Basic Clin Neurosci,2021,12(6):805-816

    • [6] CUTIA C A,LEVERTON L K,GE X,et al.Phenotypic differences based on lateralization of intrahippocampal kainic acid injection in female mice[J].Exp Neurol,2022,355:114118

    • [7] BAHN S,VOLK B,WISDEN W.Kainate receptor gene ex⁃ pression in the developing rat brain[J].J Neurosci,1994,14(9):5525-5547

    • [8] BEN ⁃ARI Y,COSSART R.Kainate,a double agent that generates seizures:two decades of progress[J].Trends Neurosci,2000,23(11):580-587

    • [9] REYES ⁃MENDOZA J,MORALES T.Prolactin treatment reduces kainic acid ⁃ induced gliosis in the hippocampus of ovariectomized female rats[J].Brain Res,2020,1746:147014

    • [10] BABB T L,PEREIRA ⁃LEITE J,MATHERN G W,et al.Kainic acid induced hippocampal seizures in rats:com⁃ parisons of acute and chronic seizures using intrahippo⁃ campal versus systemic injections[J].Ital J Neurol Sci,1995,16(1⁃2):39-44

    • [11] RIBAN V,BOUILLERET V,PHAM⁃LÊ B T,et al.Evolu⁃ tion of hippocampal epileptic activity during the develop⁃ ment of hippocampal sclerosis in a mouse model of tempo⁃ ral lobe epilepsy[J].Neuroscience,2002,112(1):101-111

    • [12] CHEN B,XU C,WANG Y,et al.A disinhibitory nigra ⁃parafascicular pathway amplifies seizure in temporal lobe epilepsy[J].Nat Commun,2020,11(1):923

    • [13] CHENG H,WANG Y,CHEN J,et al.The piriform cortex in epilepsy:What we learn from the kindling model[J].Exp Neurol,2020,324:113137

    • [14] VISMER M S,FORCELLI P A,SKOPIN M D,et al.The piriform,perirhinal,and entorhinal cortex in seizure gen⁃ eration[J].Front Neural Circuits,2015,9:27

    • [15] YOUNG J C,VAUGHAN D N,NASSER H M,et al.Ana⁃ tomical imaging of the piriform cortex in epilepsy[J].Exp Neurol,2019,320:113013

    • [16] MAGGIO R,LANAUD P,GRAYSON D R,et al.Expres⁃ sion of c⁃fos mRNA following seizures evoked from an epi⁃ leptogenic site in the deep prepiriform cortex:regional dis⁃ tribution in brain as shown by in situ hybridization[J].Exp Neurol,1993,119(1):11-19

    • [17] KURADA L,BAYAT A,JOSHI S,et al.Antiepileptic ef⁃ fects of electrical stimulation of the piriform cortex[J].Exp Neurol,2020,325:113070

    • [18] LI X,YANG C,SHI Y,et al.Abnormal neuronal damage and inflammation in the hippocampus of kainic acid ⁃in⁃ duced epilepsy mice[J].Cell Biochem Funct,2021,39(6):791-801

    • [19] PARK J A,LEE C H.Effect of rufinamide on the kainic acid⁃induced excitotoxic neuronal death in the mouse hip⁃ pocampus[J].Arch Pharm Res,2018,41(7):776-783

    • [20] QIN Z,SONG J,LIN A,et al.GPR120 modulates epilep⁃ tic seizure and neuroinflammation mediated by NLRP3 in⁃ flammasome[J].J Neuroinflammation,2022,19(1):121

    • [21] VAN DEN HERREWEGEN Y,DENEWET L,BUCKINX A,et al.The barnes mazetask reveals specific impairment of spatial learning strategy in the intra hippocampal Kain⁃ ic acid model for temporal lobe epilepsy[J].Neurochem Res,2019,44(4):600608

    • [22] DONG X,HAO X,XU P,et al.RNA sequencing analysis of cortex and hippocampus in a kainic acid rat model of temporal lobe epilepsy to identify mechanisms and thera⁃ peutic targets related to inflammation,immunity and cog⁃ nition[J].Int Immunopharmacol,2020,87:106825

    • [23] BATTERMAN A I,DECHIARA J,ISLAM A,et al.Cogni⁃ tive and behavioral effects of brief seizures in mice[J].Epilepsy Behav,2019,98(Pt A):249-257

    • [24] JOSHI S,BAYAT A,JONES A,et al.The effects of ammo⁃ nia stimulation on kainate⁃induced status epilepticus and anterior piriform cortex electrophysiology[J].Epilepsy Behav,2020,104(Pt A):106885

  • 参考文献

    • [1] BRENNAN G P,HENSHALL D C.MicroRNAs as regula⁃ tors of brain function and targets for treatment of epilepsy [J].Nat Rev Neurol,2020,16(9):506-519

    • [2] FALCO ⁃WALTER J.Epilepsy ⁃ definition,classification,pathophysiology,and epidemiology[J].Semin Neurol,2020,40(6):617-623

    • [3] LIU A H,CHU M,WANG Y P.Up⁃regulation of trem2 in⁃ hibits hippocampal neuronal apoptosis and alleviates oxi⁃ dative stress in epilepsy via the PI3K/Akt pathway in mice [J].Neurosci Bull,2019,35(3):471-485

    • [4] BRUXEL E M,DO CANTO A M,BRUNO D C F,et al.Multi⁃omic strategies applied to the study of pharmacore⁃ sistance in mesial temporal lobe epilepsy[J].Epilepsia Open,2022,7(Suppl 1):S94-S120

    • [5] HAGHANI S,JAMALI ⁃RAEUFY N,ZEINIVAND M,et al.Hepatocyte growth factor attenuates the severity of sta⁃ tus epilepticus in kainic acid ⁃induced model of temporal lobe epilepsy by targeting apoptosis and astrogliosis[J].Basic Clin Neurosci,2021,12(6):805-816

    • [6] CUTIA C A,LEVERTON L K,GE X,et al.Phenotypic differences based on lateralization of intrahippocampal kainic acid injection in female mice[J].Exp Neurol,2022,355:114118

    • [7] BAHN S,VOLK B,WISDEN W.Kainate receptor gene ex⁃ pression in the developing rat brain[J].J Neurosci,1994,14(9):5525-5547

    • [8] BEN ⁃ARI Y,COSSART R.Kainate,a double agent that generates seizures:two decades of progress[J].Trends Neurosci,2000,23(11):580-587

    • [9] REYES ⁃MENDOZA J,MORALES T.Prolactin treatment reduces kainic acid ⁃ induced gliosis in the hippocampus of ovariectomized female rats[J].Brain Res,2020,1746:147014

    • [10] BABB T L,PEREIRA ⁃LEITE J,MATHERN G W,et al.Kainic acid induced hippocampal seizures in rats:com⁃ parisons of acute and chronic seizures using intrahippo⁃ campal versus systemic injections[J].Ital J Neurol Sci,1995,16(1⁃2):39-44

    • [11] RIBAN V,BOUILLERET V,PHAM⁃LÊ B T,et al.Evolu⁃ tion of hippocampal epileptic activity during the develop⁃ ment of hippocampal sclerosis in a mouse model of tempo⁃ ral lobe epilepsy[J].Neuroscience,2002,112(1):101-111

    • [12] CHEN B,XU C,WANG Y,et al.A disinhibitory nigra ⁃parafascicular pathway amplifies seizure in temporal lobe epilepsy[J].Nat Commun,2020,11(1):923

    • [13] CHENG H,WANG Y,CHEN J,et al.The piriform cortex in epilepsy:What we learn from the kindling model[J].Exp Neurol,2020,324:113137

    • [14] VISMER M S,FORCELLI P A,SKOPIN M D,et al.The piriform,perirhinal,and entorhinal cortex in seizure gen⁃ eration[J].Front Neural Circuits,2015,9:27

    • [15] YOUNG J C,VAUGHAN D N,NASSER H M,et al.Ana⁃ tomical imaging of the piriform cortex in epilepsy[J].Exp Neurol,2019,320:113013

    • [16] MAGGIO R,LANAUD P,GRAYSON D R,et al.Expres⁃ sion of c⁃fos mRNA following seizures evoked from an epi⁃ leptogenic site in the deep prepiriform cortex:regional dis⁃ tribution in brain as shown by in situ hybridization[J].Exp Neurol,1993,119(1):11-19

    • [17] KURADA L,BAYAT A,JOSHI S,et al.Antiepileptic ef⁃ fects of electrical stimulation of the piriform cortex[J].Exp Neurol,2020,325:113070

    • [18] LI X,YANG C,SHI Y,et al.Abnormal neuronal damage and inflammation in the hippocampus of kainic acid ⁃in⁃ duced epilepsy mice[J].Cell Biochem Funct,2021,39(6):791-801

    • [19] PARK J A,LEE C H.Effect of rufinamide on the kainic acid⁃induced excitotoxic neuronal death in the mouse hip⁃ pocampus[J].Arch Pharm Res,2018,41(7):776-783

    • [20] QIN Z,SONG J,LIN A,et al.GPR120 modulates epilep⁃ tic seizure and neuroinflammation mediated by NLRP3 in⁃ flammasome[J].J Neuroinflammation,2022,19(1):121

    • [21] VAN DEN HERREWEGEN Y,DENEWET L,BUCKINX A,et al.The barnes mazetask reveals specific impairment of spatial learning strategy in the intra hippocampal Kain⁃ ic acid model for temporal lobe epilepsy[J].Neurochem Res,2019,44(4):600608

    • [22] DONG X,HAO X,XU P,et al.RNA sequencing analysis of cortex and hippocampus in a kainic acid rat model of temporal lobe epilepsy to identify mechanisms and thera⁃ peutic targets related to inflammation,immunity and cog⁃ nition[J].Int Immunopharmacol,2020,87:106825

    • [23] BATTERMAN A I,DECHIARA J,ISLAM A,et al.Cogni⁃ tive and behavioral effects of brief seizures in mice[J].Epilepsy Behav,2019,98(Pt A):249-257

    • [24] JOSHI S,BAYAT A,JONES A,et al.The effects of ammo⁃ nia stimulation on kainate⁃induced status epilepticus and anterior piriform cortex electrophysiology[J].Epilepsy Behav,2020,104(Pt A):106885

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