Name: pentylenetetrazole-induced kindling mouse model
Uploaded: 2018-06-12T12:30:00.000+00:00
Duration: 7 min 6 s
Description: Watch this Scientific Journal Video about Pentylenetetrazole-Induced Kindling Mouse Model at JoVE.com

这项工作得到部分支持的 jsp KAKENHI 赠款号 24700349, 24659093, 25293239, JP18H02536 和 17K07086, 下个 KAKENHI 赠款号码25110737和 23110525, 阿曼德赠款号码 JP18ek0109311, SENSHIN 医学研究基金会和日本癫痫研究基金会。

所有实验程序均经东京都医学科学院动物护理和使用委员会批准。建议产后 8-16 周大鼠。任何近交菌株都是可以接受的实验。C57BL/6 小鼠对 ptz 更有抵抗力, 而 BALB/c 和瑞士白化小鼠对 ptz 更敏感。C57BL/6 在这项研究中被使用。对 PTZ 的脆弱性也取决于鼠标的年龄。与小鼠相比, 大鼠更难在 PTZ35。这种方法使用的动物数量可能会有所不同, 但每种情况下至少需要 6-10 只动物。
1. 云台的研制
<ol><li>溶解2毫克/毫升 PTZ 在无菌 0.9% (瓦特/v) 氯化钠。在使用日准备云台。</li>
</ol>2. 云台的注入
<ol><li>在上午9:00 至下午12:00 (中午) 之间进行所有实验。</li>
	<li>测量动物的体重。</li>
	<li>将动物放在观察室中以适应习惯。</li>
	<li>在适应期 (3 分钟) 内, 根据动物的体重和先前确定的注射剂量, 计算出该注射液的云台溶液体积。 注: 例如, 当使用35毫克/千克 ptz 时, 0.875 毫克的 ptz 是需要的鼠标重25克 (35 毫克/千克 x 0.025 公斤 = 0.875 毫克)。因此, 437.5 ul 的2毫克/毫升 PTZ 解决方案应注射 (0.875 毫克/2 毫克/毫升 = 0.4375 毫升)。云台的注射剂量取决于小鼠的基因型和应变。对于野生型 C57BL/6 小鼠, 建议第一次试验的剂量为30-35 毫克 PTZ/千克体重。对 PTZ 的灵敏度取决于使用的鼠标应变36,37。注射剂量根据实验目的而异。</li>
	<li>注射 PTZ 腹腔与一个1毫升注射器连接到一个27口径的针, 到左或右象限的动物腹部。避免在中线注射。<ol>
<li>避免重复注射在同一位置。</li>
	</ol>
</li>
	<li>观察云台管理后30分钟的动物行为 (图 1A), 并对异常行为进行分类和评分, 如下所示。如果可能的话, 保持观察动物24小时, 或至少增加 6-10 h 后注射, 特别是一旦癫痫严重评分达到3或以上。<ol>
<li>注意在30分钟的观察期以外的动物轻微的癫痫发作或行为改变。这一长期观察期可以解决一个特别重要和完全的影响, subconvulsive 剂量 PTZ 在产生慢性无端癫痫发作。</li>
		<li>此外, 测量每项观察到的癫痫发作持续时间, 因为扣押期间的变化与缉获严重程度有关。此外, 云台注射后第一次癫痫发作的潜伏期是另一项重要的收集措施。监测癫痫发作的频率、持续时间和潜伏期对于任何癫痫后的分子研究都是至关重要的。</li>
	</ol>
</li>
	<li>每隔一天注入 PTZ (图 1A)。注射的数量取决于实验的目的。有代表性的例子如下。<ol>
<li>要生成完全被点燃的动物, 一旦动物经历了癫痫发作, 分数为 5 (补挛癫痫), 在额外的三个行政部门内完成注射。在这种情况下, 增加注射剂量, 如果癫痫评分不增加三个连续的管理。每种动物可以接受不同数量和剂量的 PTZ 注射。</li>
		<li>确定每种情况下 ptz 注射的数量和剂量, 以评估对 ptz 的脆弱性, 包括对抗癫痫药物的评估和转基因小鼠表型的研究。给所有动物相同数量的 PTZ 注射;建议共注射8至12针。如果动物死亡, 癫痫评分应记为6。</li>
		<li>确定注射剂量, 以维持高癫痫评分 (4 或 5) 10 或更多的注射, 以研究发生癫痫发作的病理变化。在这种情况下, 总注入数应介于25到30之间。如果癫痫评分达到 5, 则减少注射剂量。如果癫痫评分降低到3或更低, 增加注射剂量。</li>
	</ol>
</li>
</ol>3. 扣押评分
<ol><li>观察动物行为, 记录分数。<ol>
<li>分类和评分的癫痫行为如下38,39 (图 1B): 0: 正常行为, 无异常。 1: 固定, 躺在腹部。 2: 头点头, 面部, 前肢, 或后肢挛。 3: 连续的全身挛, 挛的抽搐, 尾巴僵硬地举行了。 4: 养育, 滋补发作, 跌倒在它的边。 5: 补药-挛发作, 跌倒在它的后面, 狂放的冲和跳跃。 6: 死亡。</li>
	</ol>
</li>
	<li>根据实验条件, 更改基于拉辛评分40的行为准则。</li>
</ol>4. 扣押后分析
<ol><li>Immunohistopathological 分析<ol>
<li>灌注固定<ol>
<li>制备<ol>
<li>溶解 4% (w/v) 多聚甲醛 (粉煤灰) 在0.1 米磷酸盐缓冲 (pH 7.4)。加热解答到大约70°c 以加法 1 m 氢氧化钠 (大约1µL 1 m 氢氧化钠为50毫升煤灰解答)。</li>
				<li>准备冰冷4% 粉煤灰和磷酸盐缓冲盐水 (PBS)。</li>
				<li>用管子和针头设置蠕动泵。通过管子运行大约20毫升的水来清除残留物。然后, 把管子的开端放在冰冷的 PBS 里。</li>
			</ol>
</li>
			<li>Transcardial 灌注与固定脑的制备<ol>
<li>用 50% isoflurane/50%ethanol 在一个小罐子里深深地麻醉老鼠。</li>
				<li>将老鼠的前肢和后肢的尖端固定在仰卧位。</li>
				<li>切开腹部腹中线, 露出隔膜。</li>
				<li>沿肋边切开隔膜。然后, 切开两侧的肋骨和身体。用锁钳捏剑突过程, 埃弗特肋骨笼。保持剑突过程的位置, 用针钉住它或用镊子捏紧它。暴露心脏。</li>
				<li>将针头插入左心室, 用剪刀在右心房切开。注意不要把针推到心脏太远, 因为它可以刺穿内壁。</li>
				<li>通过针开始一个稳定的冰的 PBS 流, 以洗涤血液 (大约 15-20 毫升/分钟)。</li>
				<li>当血液从体内清除后, 将管子移至4% 多聚甲醛溶液 (大约60毫升), 而不添加气泡。然后, 停止灌注。</li>
				<li>切开后颈部和脊柱, 剥去头部皮肤和头骨的背部部分用解剖剪刀。</li>
				<li>在眼球轨道之间切开前上颌骨骨, 并完全移除头骨的背部。</li>
				<li>在大脑和泪骨之间建立一个间隙, 并在大脑下方切开三叉神经和视神经交叉。小心地取出大脑, 放在含有4% 只粉煤灰的瓶子里。后固定的大脑在 4°c 12-16 小时。</li>
			</ol>
</li>
		</ol>
</li>
		<li>脑切片制备<ol>
<li>将固定的大脑转移到20% 的蔗糖/PBS, 直到大脑吸收到溶液中, 以允许抗冻。</li>
			<li>根据所需的切片方向和大脑需要的部分来切割固定的大脑。</li>
			<li>设置 cryomicrotome。设置刀片位置, 并保持在-20 摄氏度以下的温度切片阶段。</li>
			<li>将预切的大脑放在舞台上, 用20% 的蔗糖/PBS 将大脑涂上。蔗糖/PBS 溶液将逐渐冻结, 覆盖大脑的舞台上, 直到大脑完全嵌入冷冻20% 蔗糖/pbs。</li>
			<li>通过将刀片滑入组织, 切片大脑 (30 µm 厚度)。将切片转移到 PBS 并保持在4摄氏度。</li>
		</ol>
</li>
		<li>荧光免疫组化<ol>
<li>清洗必要的切片在0.1% 海卫 X-100/PBS (PBST) 10 分钟, 3 倍室温 (RT)。</li>
			<li>在 PBST 中用2% 山羊血清或5% 牛血清白蛋白 (BSA) 在 RT 中进行 1-2 小时的孵化, 阻断切片。</li>
			<li>以2% 山羊血清或 5% BSA 为1至7过夜, 在4摄氏度的 PBST 中, 以适当浓度的原抗体在抗体溶液中孵化切片。</li>
			<li>将 PBST 中的切片洗净10分钟, RT 3 次。</li>
			<li>在二次抗体溶液中孵育切片, 适当浓度为 1-2 小时的 PBST 中的二级抗体, 光保护。</li>
			<li>将切片安装到玻璃滑梯上, 并盖上安装介质和盖玻璃。</li>
			<li>用荧光显微镜观察切片。</li>
		</ol>
</li>
	</ol></li>
	<li>三室试验<ol>
<li>制备<ol>
<li>准备至少 2 2-6 月大的 129/Sv 小鼠相同的性别作为主题小鼠是 "陌生人老鼠"。习惯所有老鼠在笼子前进行分析。对于每个训练会话, 将鼠标放在笼子中, 并将鼠标留在15分钟。</li>
			<li>在每个适应阶段与主题老鼠, 清洗整个仪器和两个笼子通过擦拭表面与70% 乙醇。</li>
			<li>把空笼子放在3室器具的两个侧室里, 打开房间的门。</li>
			<li>要习惯主题鼠标, 请在中心腔内放置一个主题鼠标, 并允许鼠标在整个设备上自由移动, 直到鼠标对两个笼子进行了调查, 再加上5分钟. 可能是害怕笼子的主题动物不调查笼20分钟, 不应使用此分析。这样的动物避开笼子, 很少靠近笼子。</li>
			<li>动物进入中心腔后, 关闭车门, 让鼠标在中心房间内自由移动5分钟。在此期间, 把 129/Sv 老鼠的一个笼子里习惯到笼子里去。</li>
		</ol>
</li>
		<li>社会性分析<ol>
<li>在主题鼠标的适应期后立即进行分析。</li>
			<li>放置包含 129/Sv 鼠标的笼子, 称为 "陌生人1笼", 在其中一个侧面室和放置空笼, 称为 "对象笼", 在另一侧的房间。</li>
			<li>打开门并监视主题鼠标的行为。</li>
			<li>允许主题鼠标自由移动10分钟, 并记录以下行为参数。如果主题鼠标害怕在陌生人1笼中的鼠标, 如鼠标所示避免陌生人1笼和留在一个房间的角落里, 动物不应该用于分析。 a) 在每个会议厅、陌生人1室、对象室和中心室度过的时间。 b) 调查每个笼子、陌生人1笼和物体笼所花费的时间。(如果鼠标嗅探或增韧鼠标或笼子, 则将鼠标归类为调查笼子) c) 每个房间的入口数量 (可选) d) 旅行总距离 (可选)</li>
			<li>鼠标移动到中心房间后, 把门关上。</li>
		</ol>
</li>
		<li>社会新奇分析<ol>
<li>在社会性分析后立即进行分析。</li>
			<li>用70% 乙醇擦拭两个笼子。</li>
			<li>将另一只 129/Sv 鼠标放在其中一个笼子里, 称为 "陌生人2笼", 并将陌生人2笼放在其中一个房间里。将陌生人1只老鼠放在另一个笼子里, 称为 "陌生人1笼", 并将陌生人1笼放在另一个房间里。</li>
			<li>打开门并监视主题鼠标的行为。</li>
			<li>允许主题鼠标自由移动10分钟, 并测量相同的行为参数描述的社会性分析。 注: 陌生人1和陌生人2鼠标应随机选择。陌生人1房间和对象房间并且陌生人1个房间和陌生人2个房间应该被随机地确定。</li>
		</ol>
</li>
	</ol></li>
	<li>语境恐惧歧视<ol>
<li>调节<ol>
<li>每次调节试验前, 用70% 乙醇擦拭表面, 清洁实验装置。</li>
			<li>将鼠标放在具有特定条件的空调设备 (设备形状、墙壁颜色、地板材质、气味、照明和背景噪音音量应预先确定)。例如, 这里使用的空调设备是一个方形的设备, 具有清晰的有机玻璃墙, 金属网格地板, 乙醇气味, 100 勒克斯亮度和 65 dB 背景白噪声。</li>
			<li>在5分钟的时间内, 用 0.1 mA 电击的伪随机定时对小鼠进行足部冲击, 2 x 3 次。</li>
			<li>调节后将鼠标返回到主笼子。</li>
		</ol>
</li>
		<li>内存评估<ol>
<li>每次评估前, 用70% 乙醇擦拭表面, 清洁实验器具。</li>
			<li>第二天调理后, 将鼠标放到同一个装置中作为休克状态, 并在5分钟的过程中测量凝固时间。</li>
			<li>同一天晚些时候, 将鼠标放在一个新的仪器中, 在5分钟的评估中测量冰冻时间。在这里使用了一个三角形的设备, 白色有机玻璃墙, 平板地板, 没有气味, 30 莱克丝亮度和70分贝白噪音。</li>
			<li>比较了相同条件和新条件下的冻结时间。具有正常记忆能力的小鼠在相同的条件下会比在新的条件下冷冻更多。冰冻被定义为超过2秒的动物的静止。</li>
		</ol>
</li>
	</ol></li>
</ol>

作者声明没有利益冲突。

在这里, 我们提出了一个广泛的可访问的协议, 建立一个药理动物模型的癫痫。云台介导的化学点火具有悠久的历史, 是一种普遍接受的模型, 研究的病理学和细胞病理学的癫痫41。Suzdak 和詹森对癫痫的化学点燃模型进行了回顾, 199542。药物癫痫诱导, 特别是与 PTZ, 是一种简单和简单的方法来唤起严重癫痫发作。注射剂量的变化与癫痫严重程度相关。因此, 通过改?...

癫痫是一种慢性神经系统疾病, 其特点是反复发作, 影响大约1% 的人。在临床研究中不能充分阐明癫痫患者负反馈和癫痫产生的机制。因此, 对癫痫1的研究需要一个适宜的动物模型。
各种动物模型的癫痫已被用来调查癫痫的生理和识别抗癫痫药物2,3。在这些模型中, 药物癫痫诱导是一种常见的方法, 用于生成动物模型, 以调查4的癫痫病理。这种方法既便宜又简单。电极介导的点燃也是一种常用的方法, 但这一过程的成本较高, 该方法需要手术和电气技能, 以诱发重复性发作5。
药理诱导也有利, 因为发作的时间和数量容易控制。在癫痫的研究中也使用了表现为自发癫痫的基因小鼠模型。然而, 预测在这些基因模型中癫痫发作的时间和频率可能是不可能的6。为了观察基因修饰小鼠6的癫痫行为, 需要一个监测系统。
海仁酸、匹戊四氮 (PTZ) 被广泛用作癫痫诱导药物7。海仁酸是谷氨酸受体的促激动剂, 而匹属激活胆碱受体。PTZ 是一种伽玛丁酸 (GABA)-受体拮抗剂8。PTZ 抑制抑制性突触的功能, 导致神经元活动增加。这个章程导致普遍的缉获在动物9。单一注射海仁酸和匹特能诱发急性癫痫发作, 特别是癫痫持续状态 (SE)10,11和海仁酸-或匹特式介导硒促进慢性自发性和复发性癫痫12,13. 脑电图 (EEG) 录音和行为分析表明, 在一次注射12、13后一个月内, 自然复发性癫痫发作。单一注射的抽搐剂量的 PTZ 也诱发急性发作。然而, 单一注射 PTZ 后的慢性自发性癫痫很难推广。需要对 PTZ 进行慢性管理, 以诱发重复性癫痫14。在这两种方法中, 重复性癫痫的产生都能诱发一种与人类癫痫相似的病理, 而非急性癫痫的发生。在 PTZ 的情况下, 每次注射都能引起癫痫发作, 每次注射时, 检出的严重性都会逐步加重。最后, 单低剂量 PTZ 注射诱发严重的挛癫痫发作。在这个阶段, 每次注射都能唤起严重的癫痫发作。此外, 在注射过程中, 癫痫潜伏期和持续时间也会发生变化。在点燃15的后阶段, 补剂发作的潜伏期往往会缩短。此外, 癫痫发作加重还伴有长期发作期16。研究控制癫痫发作严重性、潜伏期和持续时间的分子机制对于筛查抗癫痫药物17、18、19有很重要的意义。
癫痫发作通常是由一个单一的系统管理的 PTZ, 恢复速度非常快, 在30分钟4,5。因此, 在云台点火模型中, 癫痫发作的次数更能控制。然而, 脑电图监测表明, 在云台介导的癫痫发作20后, 一般峰值可达12小时。因此, 动物最好保持观察24小时后, 挛或补药发作21 , 以更精确的分析点燃机制。
抗癫痫药物, 如 ethosuximide, 丙戊酸, 苯巴比妥, 氨乙烯酸, 和 retigabine3, 在 PTZ 注射之前或之后, 降低发作严重性3,22的恶化, 23。同样, 没有基因发作加重的击倒小鼠, 如基质 metalloproteinase-924, FGF-2225和 neuritin26, 已被证明在多台 PTZ 注射后, 检出严重程度降低。此外, 通过这种方法可以观察癫痫发作后的组织病理改变。颞叶癫痫患者有典型的组织学改变, 如苔藓纤维发芽27,28, 异常颗粒神经元迁移29, astrogliosis30, 神经细胞死亡在海马31,32和海马硬化症33。癫痫模型动物也观察到类似的变化。在现有的方法中, 云台介导的化学点火是一种很好的, 重现性和廉价的方法来产生一种动物模型的癫痫。在一个由匹凡达介导的 se 模型中, 癫痫控制是困难的, 许多小鼠死亡或无法发展 se34。相比之下, 在 PTZ 模型中, 死亡率和癫痫严重程度更可控制。此外, PTZ 比海仁酸便宜, 而且在小鼠脑外科的技能是不需要药物管理。

<table><tbody><tr><td>Pentylenetetrazole</td><td>Sigma-Aldrich</td><td>P6500</td><td></td></tr><tr><td>Sodium chloride</td><td>MANAC</td><td>7647-14-5</td><td></td></tr><tr><td>Mouse</td><td>CLEA Japan</td><td></td><td>C57Bl/6NJcl, postnatal 8 week, male</td></tr><tr><td>Syringe (1mL)</td><td>Terumo</td><td>SS-01T</td><td></td></tr><tr><td>Needle(27G x 3/4&quot;) (0.40 x 19 mm)</td><td>Terumo</td><td>NN-2719S</td><td></td></tr><tr><td>Weighing scale</td><td>Mettler</td><td>PE2000</td><td>This item is a discontinued product. Almost equivalent to FX-2000i with FXi-12-JA from A&amp;amp;D company.</td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td></td></tr><tr><td>Sodium hydroxide</td><td>nacalai tesque</td><td>31511-05</td><td></td></tr><tr><td>Peristatic pump</td><td>ATTO</td><td>SJ1211</td><td></td></tr><tr><td>Sucrose</td><td>nacalai tesque</td><td>30404-45</td><td></td></tr><tr><td>Microtome</td><td>Yamato</td><td>REM-700</td><td>This item is a discontinued product. Almost equivalent to REM-710</td></tr><tr><td>Microtome blade</td><td>Feather</td><td>S35</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X-100</td><td></td></tr><tr><td>anti-synaptoporin antibody</td><td>Synaptic systems</td><td>102 002</td><td></td></tr><tr><td>anti-ZnT3 antibody</td><td>Synaptic systems</td><td>197 002</td><td></td></tr><tr><td>anti-doublecortin</td><td>Santa Cruz</td><td>sc-8066</td><td>This item is a discontinued product. We did not test equivalent product (sc-271390).</td></tr><tr><td>Contextual fear discrimination test apparatus</td><td>O&#039;hara</td><td></td><td></td></tr><tr><td>Three chamber test apparatus</td><td>Muromachi</td><td></td><td></td></tr></tbody></table>

pentylenetetrazole-induced kindling mouse model

戊四氮 (PTZ) 是 GABA-受体拮抗剂。腹腔内注射 PTZ 进入动物诱导急性, 严重癫痫的高剂量, 而顺序注射 subconvulsive 剂量已用于发展化学点燃, 癫痫模型。单次低剂量的 PTZ 诱导轻度发作, 不抽搐。然而, 反复低剂量的 PTZ 注射降低了诱发痉挛性癫痫发作的门槛。最后, 云台连续低剂量管理诱发严重的挛癫痫发作。这种方法简单, 广泛适用于研究癫痫的病理生理学, 它被定义为一种涉及重复性癫痫的慢性疾病。这种化学点燃协议导致动物反复发作。利用这种方法, 估计易受云台介导的癫痫发作或癫痫发作加重的程度。这些优势导致了使用这种方法筛选抗癫痫药物和癫痫相关的基因。此外, 该方法已被用来调查癫痫发作后的神经细胞损伤, 因为在癫痫患者的大脑中观察到的组织学改变也出现在化学点燃动物的大脑中。因此, 该协议对于方便地制作癫痫动物模型是有益的。

反复注射 PTZ 诱发癫痫发作的严重性增加。对六 C57BL/6 小鼠进行了 PTZ 治疗, 另有6只小鼠以生理盐水作为对照组治疗。PTZ 剂量为35毫克/千克, 10 次注射。随着 PTZ 注射, 癫痫评分逐渐增加, 而生理盐水注射没有诱发癫痫发作或异常行为 (图 2)。方差分析后的 Bonferroni 试验显示, 云台治疗组与生理盐水治疗组有显著性差异。
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Watch this Scientific Journal Video about Pentylenetetrazole-Induced Kindling Mouse Model at JoVE.com

Pentylenetetrazole-Induced Kindling Mouse Model

本协议描述了一种戊四氮化学点燃方法, 并提供了一种小鼠癫痫模型。该协议还可用于调查小鼠癫痫发作后癫痫诱导和发病机制的脆弱性。

戊四氮诱导点燃小鼠模型

Pentylenetetrazole (PTZ) is a GABA-A receptor antagonist. An intraperitoneal injection of PTZ into an animal induces an acute, severe seizure at a high ...

pentylenetetrazole-induced-kindling-mouse-model

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Education

JoVE Core

JoVE Journal

Pharmacology

Neuroscience

Unit 1

Pharmacotherapy of the Epilepsies

SCIENTISTS IN ACTION

32.7K 次观看.  Tokyo Metropolitan Institute of Medical Science. 本协议描述了一种戊四氮化学点燃方法, 并提供了一种小鼠癫痫模型。该协议还可用于调查小鼠癫痫发作后癫痫诱导和发病机制的脆弱性。

视频: Pentylenetetrazole-Induced Kindling Mouse Model

Long-term Potentiation of Perforant Pathway-dentate Gyrus Synapse in Freely Behaving Mice

Studies of long-term potentiation of synaptic efficacy, an activity-dependent synaptic phenomenon having properties that make it attractive as a potential cellular mechanism underlying learning and information storage, have long been used to elucidate the physiology of various neuronal circuits in the hippocampus, amygdala, and other limbic and cortical structures. With this in mind, transgenic mouse models of neurological diseases represent useful platforms to conduct long-term&#160;potentiation (LTP) studies to develop a greater understanding of the role of genes in normal and abnormal synaptic communication in neuronal networks involved in learning, emotion and information processing. This article describes methodologies for reliably inducing LTP in the freely behaving mouse. These methodologies can be used in studies of transgenic and knockout freely&#160;behaving mouse models of neurodegenerative diseases.

Transgenic and knockout mouse models of neurological diseases are useful for studying the role of genes in normal and abnormal neurophysiology. This article describes methodologies which can be used to study long-term potentiation, a cellular mechanism which may underlie learning and memory, in transgenic and knockout freely&#160;behaving mouse models of neuropathology.

穿通通路 - 齿状回突触在自由活动的小鼠长时程增强

Studies of long-term potentiation of synaptic efficacy, an activity-dependent synaptic phenomenon having properties that make it attractive as a potential ...

科研

行为学

Manipulation of Epileptiform Electrocorticograms (ECoGs) and Sleep in Rats and Mice by Acupuncture

Ancient Chinese literature has documented that acupuncture possesses efficient therapeutic effects on epilepsy and insomnia. There is, however, little research to reveal the possible mechanisms behind these effects. To investigate the effect of acupuncture on epilepsy and sleep, several issues need to be addressed. The first is to identify the acupoints, which correspond between humans, rats, and mice. Furthermore, the depth of insertion of the acupuncture needle, the degree of needle twist in manual needle acupuncture, and the stimulation parameters for electroacupuncture (EA) need to be determined. To evaluate the effects of acupuncture on epilepsy and sleep, a feasible model of epilepsy in rodents is required. We administer pilocarpine into the left central nucleus of the amygdala (CeA) to simulate focal temporal lobe epilepsy (TLE) in rats. Intraperitoneal (IP) injection of pilocarpine induces generalized epilepsy and status epilepticus (SE) in rats. Five IP injections of pentylenetetrazol (PTZ) with a one-day interval between each injection successfully induces spontaneous generalized epilepsy in mice. Recordings of electrocorticograms (ECoGs), electromyograms (EMGs), brain temperature, and locomotor activity are used for sleep analysis in rats, while ECoGs, EMGs, and locomotor activity are employed for sleep analysis in mice. ECoG electrodes are implanted into the frontal, parietal, and contralateral occipital cortices, and a thermistor is implanted above the cerebral cortex by stereotactic surgery. EMG electrodes are implanted into the neck muscles, and an infrared detector determines locomotor activity. The criteria for categorizing vigilance stages, including wakefulness, rapid eye movement (REM) sleep, and non-REM (NREM) sleep are based on information from ECoGs, EMGs, brain temperature, and locomotor activity. Detailed classification criteria are stated in the text.

Manipulation of Epileptiform Electrocorticograms ECoGs and Sleep in Rats and Mice by Acupuncture

This paper demonstrates the performance of acupuncture, epilepsy models, and the analysis of sleep in rodents. The acupuncture procedure and the identification of acupoints are described. Pilocarpine or pentylenetetrazol (PTZ) is used to induce epilepsy. Electrocorticogram (ECoG), electromyogram (EMG), brain temperature, and locomotor activity recordings are employed for sleep analysis.

癫痫Electrocorticograms（ECoGs）的操作，并睡在针刺大鼠和小鼠

Ancient Chinese literature has documented that acupuncture possesses efficient therapeutic effects on epilepsy and insomnia. There is, however, little ...

Inducing Post-Traumatic Epilepsy in a Mouse Model of Repetitive Diffuse Traumatic Brain Injury

Traumatic brain injury (TBI) is a leading cause of acquired epilepsy. TBI can result in a focal or diffuse brain injury. Focal injury is a result of direct mechanical forces, sometimes penetrating through the cranium, creating a direct lesion in the brain tissue. These are visible during brain imaging as areas with contusion, laceration, and hemorrhage. Focal lesions induce neuronal death and glial scar formation and are present in 20%&minus;25% of all people who incur a TBI. However, in the majority of TBI cases, injury is caused by acceleration-deceleration forces and subsequent tissue shearing, resulting in nonfocal, diffuse damage. A subpopulation of TBI patients continues to develop post-traumatic epilepsy (PTE) after a latency period of months or years. Currently, it is impossible to predict which patients will develop PTE, and seizures in PTE patients are challenging to control, necessitating further research. Until recently, the field was limited to only two animal/rodent models with validated spontaneous post-traumatic seizures, both presenting with large focal lesions with massive tissue loss in the cortex and sometimes subcortical structures. In contrast to these approaches, it was determined that diffuse TBI induced using a modified weight drop model is sufficient to initiate development of spontaneous convulsive and non-convulsive seizures, even in the absence of focal lesions or tissue loss. Similar to human patients with acquired post-traumatic epilepsy, this model presents with a latency period after injury before seizure onset. In this protocol, the community will be provided with a new model of post-traumatic epilepsy, detailing how to induce diffuse non-lesional TBI followed by continuous long-term video-electroencephalographic animal monitoring over the course of several months. This protocol will detail animal handling, the weight drop procedure, the electrode placement for two acquisition systems, and the frequent challenges encountered during each of the steps of surgery, postoperative monitoring, and data acquisition.

This systematic protocol describes a new animal model of post-traumatic epilepsy after repetitive mild traumatic brain injury. The first part details steps for traumatic brain injury induction using a modified weight drop model. The second part provides instructions on the surgical approach for single- and multi-channel electroencephalographic data acquisition systems.

在重复性扩散创伤性脑损伤小鼠模型中诱导创伤后癫痫

Traumatic brain injury (TBI) is a leading cause of acquired epilepsy. TBI can result in a focal or diffuse brain injury. Focal injury is a result of ...

Preparation and Implantation of Electrodes for Electrically Kindling VGAT-Cre Mice to Generate a Model for Temporal Lobe Epilepsy

It was discovered that electrical kindling of VGAT-Cre mice led to the spontaneous motor and electrographic seizures. A recent paper focused on how unique VGAT-Cre mice were used in developing spontaneous recurring seizures (SRS) after kindling and a likely mechanism - insertion of Cre into the VGAT gene - disrupted its expression and reduced GABAergic tone. The present study extends these observations to a larger cohort of mice, focusing on key issues such as how long the SRS continues after kindling and the effect of the animal's sex and age. This report describes the protocols for the following key steps: making headsets with hippocampal depth electrodes for electrical stimulation and for reading the electroencephalogram; surgery to affix the headset securely on the mouse's skull so that it does not fall off; and key details of the electrical kindling protocol such as duration of the pulse, frequency of train, duration of train, and amount of current injected. The kindling protocol is robust in that it reliably leads to epilepsy in most VGAT-Cre mice, providing a new model to test for novel antiepileptogenic drugs.

This report describes the methods to generate a model of temporal lobe epilepsy based on the electrical kindling of transgenic VGAT-Cre mice. Kindled VGAT-Cre mice may be useful in determining what causes epilepsy and for screening novel therapies.

电点燃VGAT-CRE小鼠电极的制备和植入，以生成颞叶癫痫模型

It was discovered that electrical kindling of VGAT-Cre mice led to the spontaneous motor and electrographic seizures. A recent paper focused on how unique ...

神经科学

Using a Bipolar Electrode to Create a Temporal Lobe Epilepsy Mouse Model by Electrical Kindling of the Amygdala

The amygdala is one of the most common origins of seizures, and the amygdala mouse model is essential for the illustration of epilepsy. However, few studies have described the experimental protocol in detail. This paper illustrates the whole process of amygdala electrical kindling epilepsy model making, with the introduction of a method of bipolar electrode fabrication. This electrode can both stimulate and record, reducing brain injury caused by implanting separate electrodes for stimulation and recording. For long-term electroencephalogram (EEG) recording purposes, slip rings were used to eliminate the record interruption caused by cable tangles and falling off.
After periodic stimulation (60 Hz, 1 s every 15 min) of the basolateral amygdala (AP: 1.67 mm, L: 2.7 mm, V: 4.9 mm) for 19.83 &plusmn; 5.742 times, full kindling was observed in six mice (defined as induction of three continuous grade V episodes classified by Racine's scale). An intracranial EEG was recorded throughout the entire kindling process, and an epileptic discharge in the amygdala lasting 20-70 s was observed after kindling. Therefore, this is a robust protocol for modeling epilepsy originating from the amygdala, and the method is suitable for revealing the role of the amygdala in temporal lobe epilepsy. This research contributes to future studies on the mechanisms of mesial temporal lobe epilepsy and novel antiepileptogenic drugs.

The amygdala plays a key role in temporal lobe epilepsy, which originates in and propagates from this structure. This article provides a detailed description of the fabrication of deep brain electrodes with both recording and stimulating functions. It introduces a model of medial temporal lobe epilepsy originating from the amygdala.

使用双极电极通过电点燃杏仁核创建颞叶癫痫小鼠模型

The amygdala is one of the most common origins of seizures, and the amygdala mouse model is essential for the illustration of epilepsy. However, few ...

Handling-Induced Seizures in a Mouse Model

Source: Batot, G., et al. <a href="https://app.jove.com/v/63673">A Model for Epilepsy of Infectious Etiology using Theiler&#39;s Murine Encephalomyelitis Virus.</a>&#160;J. Vis. Exp. (2022).
This video demonstrates the induction of handling-induced seizures in a mouse infected with Theiler&#39;s Murine Encephalomyelitis Virus (TMEV). The infected mouse, which has hyperexcitable neurons, is exposed to external physical stimuli. This exposure causes stress, leading to abnormal electrical activity in the neurons, which results in seizures.

Source: Batot, G., et al. A Model for Epilepsy of Infectious Etiology using Theiler&#39;s Murine Encephalomyelitis Virus.&#160;J. Vis. Exp. (2022).
This ...

JoVE Encyclopedia of Experiments

Neuropathology

Epilepsy and Seizure Disorders

Triggering and Monitoring Heat-Induced Seizures in a Transgenic Mouse Model

Source: Das, A., et al. <a href="https://app.jove.com/v/62846">A Behavioral Screen for Heat-Induced Seizures in Mouse Models of Epilepsy.</a> J. Vis. Exp. (2021)
This video demonstrates the procedure to induce heat-induced seizures in a transgenic mouse with epilepsy-related gene mutations by gradually increasing body temperature in a controlled chamber. This protocol is used to study seizure mechanisms and potential therapies for epilepsy.

Source: Das, A., et al. A Behavioral Screen for Heat-Induced Seizures in Mouse Models of Epilepsy. J. Vis. Exp. (2021)
This video demonstrates the ...

Establishing a Pentylenetetrazole-Induced Epileptic Seizure Model in Mice

Source: Shimada, T., et al. <a href="https://app.jove.com/v/56573">Pentylenetetrazole-Induced Kindling Mouse Model.</a> J. Vis. Exp. (2018).
This video demonstrates the process of inducing epilepsy in mice using pentylenetetrazole (PTZ) injections, a GABA-A receptor antagonist. Repeated intraperitoneal administration of PTZ progressively lowers the seizure threshold by inhibiting chloride ion influx, disrupting inhibitory neurotransmission, and leading to hyperexcitability in neurons that trigger seizures. This process results in kindling and the development of spontaneous seizures, mimicking chronic epilepsy.

Source: Shimada, T., et al. Pentylenetetrazole-Induced Kindling Mouse Model. J. Vis. Exp. (2018).
This video demonstrates the process of inducing epilepsy ...

Systemic Pilocarpine Treatment: Mouse Model of Temporal Lobe Epilepsy

Source: Kim, J. E. and Cho, K. O. <a href="https://www.jove.com/video/56831/the-pilocarpine-model-temporal-lobe-epilepsy-eeg-monitoring-using">The Pilocarpine Model of Temporal Lobe Epilepsy and EEG Monitoring Using Radiotelemetry System in Mice</a>. J. Vis. Exp. (2018).
This video presents the procedure for inducing status epilepticus (SE) in mice by systemic administration of pilocarpine to model temporal lobe epilepsy.

全身性毛果芸香碱治疗:颞叶癫痫小鼠模型

Source: Kim, J. E. and Cho, K. O. The Pilocarpine Model of Temporal Lobe Epilepsy and EEG Monitoring Using Radiotelemetry System in Mice. J. Vis. Exp. ...

生物学

Rodent Models

Adult

Technique to Induce Post-Traumatic Seizures in a Mouse Model via a Weight Drop Traumatic Injury

Source: Shandra, O. et al., <a href="https://app.jove.com/v/60360">Inducing Post-Traumatic Epilepsy in a Mouse Model of Repetitive Diffuse Traumatic Brain Injury.</a> J. Vis. Exp. (2020)