这项工作得到韩国政府 (NRF-2014R1A1A3049456) 资助的大韩民国国家研究基金会 (NRF) 的支持, 并通过韩国卫生产业发展研究所 (KHIDI) 提供了韩国卫生技术研发 & 开发项目的赠款,由大韩民国卫生部 & 福利部资助 (HI15C2854)。

所有实验程序均经韩国天主教大学道德委员会批准, 并按照国家卫生研究院的《实验室动物护理和使用指南》 (NIH 出版物80-23 号) 进行。
1. SE 感应
<ol><li>购买8周大的雄性 C57BL/6NHsd 小鼠, 并权衡每只老鼠。然后, 使用标记笔标记所有小鼠的尾巴, 以便在 SE 感应过程中容易识别。</li>
	<li>计算东莨菪碱的数量 (东莨菪碱; 2 毫克/千克), 特普他林硫酸盐 (特普他林; 2 毫克/千克), 和匹凡盐酸盐 (匹, 280 毫克/千克) 根据鼠标重量和添加生理盐水 (0.9% 氯化钠, 10 毫升/千克) 来解决。 注意:例如, 如果鼠标的重量是25克, 应用以下数额: 东莨菪碱和特普他林: 2 毫克/千克 * (25 g/1,000 克) = 0.05 毫克, 生理盐水:10 毫升/千克 * (25 g/1,000 g) = 0.25 毫升, 匹: 280 毫克/千克 * (25 g/1,000 g) = 7 毫克, 生理盐水:10 毫升/千克 * (25 g/1,000 g) = 0.25 毫升。</li>
	<li>用30克针将东莨菪碱和特普他林混合物装入1毫升注射器中。将解决方案腹腔到每个鼠标中, 然后将鼠标返回到它们的笼子中。30分钟后, 东莨菪碱和特普他林管理, 向每只老鼠 (ip; 1 毫升注射器; 30 克针) 注射在匹28-30°C 注射后, 立即将小鼠放在孵化器中进行观察。</li>
	<li>仔细监测小鼠的行为, 直到诱发硒。如果检测到3级或更高的边缘运动癫痫发作, 根据拉辛的比例, 记录时间和监测老鼠, 以确定癫痫发作是否更频繁。一旦连续运动癫痫持续超过2分钟, 将鼠标放置在一个新的笼子里, 在室温下, 保持监测3小时, 以确定他们抽搐发作继续和 SE 是否诱发。弄死在2小时后没有进入 SE 的老鼠。 注意:拉辛的规模;1阶段, 口腔和面部运动;阶段 2, 头点头;阶段 3, 前肢阵挛;4期, 前肢阵挛饲养;阶段 5, 饲养和下降与前肢阵挛 16.如果在 SE 感应后, 鼠标没有立即转移到笼子里, 老鼠就会因热疗而死亡。</li>
	<li>通过注射安定液 (ip; 10 毫克/千克; 1 毫升注射器; 30 克针), 在 SE 发病后3小时终止行为急性发作。加盐水 (ip; 10 毫升/千克; 1 毫升注射器; 30 克针), 在 10% polyoxyl 35 氢化蓖麻油中稀释5毫克/毫升的安定溶液。 注: 10% polyoxyl 35 氢化蓖麻油溶液: 1 毫升 polyoxyl 35 氢化蓖麻油溶液 + 9 毫升盐水。在室温下存储解决方案。例如, 如果鼠标体重是25克, 注入250µL 的安定溶液:50 µL 5 毫克/毫升安定 + 200 µL 10% polyoxyl 35 氢化蓖麻油溶液 = 250 µL 1 毫克/毫升安定。</li>
	<li>对于假鼠, 执行对东莨菪碱和特普他林硫酸盐混合物的 ip 注射 (ip; 2 毫克/千克, 10 毫升/千克; 1 毫升注射器; 30 克针)。30分钟后, 注入生理盐水腹腔 (ip; 10 毫升/千克; 1 毫升注射器; 30 克针)。 注意:如果鼠标体重是25克, 注射250µL 生理盐水而不是匹比。</li>
	<li>西泮注射液后 (ip; 1 毫升注射器; 30 克针), 每单个鼠标管理1毫升5% 葡萄糖 (ip; 1 毫升注射器; 26 克针)。 注意: 1 毫升5% 葡萄糖注射液提供能提高存活率的能量源和水化。</li>
	<li>在后护理在孵化器 (28-30 °c), 清除过量分泌物, 如唾液, 眼泪和粪便。</li>
	<li>1天后, 硒诱导, 体重的老鼠, 并保持他们在孵化器 (28-30 °c) 一个额外的一天。在2天, 在 SE 感应之后, 称老鼠体重并将它们返回他们的笼子。提供湿润的食物, 方便他们的康复。 注意:在这个实验中, C57BL/6NHsd 后的死亡率平均为 8.57% (3 的35小鼠被测试)。</li>
	<li>测量动物的每日体重, 直到7天后的匹克, 并注射5% 葡萄糖 (ip; 1 毫升注射器; 26 克针) 时, 体重没有增加的小鼠。停止体重监测时, 小鼠开始重拾体重, 并在2天后服用无困难, 在匹比注射后的湿润周。 注意:如果一只老鼠的体重在7天后没有增加, 那么从实验中排除老鼠, 弄死二氧化碳。根据老鼠的背景, 死亡有时会发生;然而, 在 C57BL/6NHsd 的情况下, 在这些实验中死亡的老鼠少于1%。随着硒诱导后7天的存活, 小鼠在潜伏期很少死亡。</li>
</ol>2. 视频脑电图监测植入术
<ol><li>准备12周大的小鼠 (SE 诱导后4周) 进行脑电图监测的遥测植入。 注意:根据实验目的和小鼠菌株, 可以对植入时间进行修改。</li>
	<li>手术前, 麻醉小鼠的混合物 (4:0, 5) 氯胺酮 (50 毫克/毫升) 和甲苯噻嗪 (23.3 毫克/毫升) 溶液溶于生理盐水, 剂量2.5 µL/克体重 (ip; 1 毫升注射器; 26 克针)。定期监测小鼠的呼吸速率和呼吸努力 (15 分钟间隔最大值), 通过踏板退出反射评估麻醉深度。</li>
	<li>将鼠标放在一个有耳棒和咬板的立体定向的框架内, 并在两只眼睛上应用兽医软膏, 以避免失明。</li>
	<li>为了在手术期间保持不育的条件, 用剃刀刀片剃掉手术部位。小心防止毛皮污染的手术领域。剃须后, 用70% 乙醇和碘溶液对皮肤进行消毒。 注意:手术应以无菌的方式进行, 佩戴无菌手套和口罩, 使用灭菌设备和无菌技术。</li>
	<li>确认麻醉深度后, 对皮肤进行中线切开, 使颅骨暴露。在前后轴 (AP) 的 Bregma 上的坐标处, 使两个毛刺孔与所述立体定向装置的钻头连接: +0.1 毫米, mediolateral 轴 (ML): +0.1 毫米 (参考) 和 AP: −0.2 毫米, ML: +0.22 毫米 (左顶叶皮质)13。<ol>
<li>用 PBS 浸泡的棉花掉期擦拭皮肤, 然后70% 乙醇和碘溶液。在头部中间用剪刀做纵切口, 以暴露 Lambda (矢状和 lambdoid 缝合的点), Bregma (冠状缝合的交点) 和靶位置。</li>
		<li>识别解剖学地标, 如 Lambda 和 Bregma。在 Bregma 上定位钻头, 并记下此点的 X、Y 坐标。</li>
		<li>使用大脑图谱或发表的参考资料, 以确定大脑区域的适当坐标的脑电图记录。然后, 使用步骤2.5.2 测量的 Bregma 坐标计算螺钉 (参考和记录) 的坐标。</li>
		<li>慢慢地降低钻头, 使一个毛刺孔小心, 以避免插入钻头太远的地步, 头骨变得更薄。</li>
	</ol>
</li>
	<li>将单通道无线脑电图发射机的正文插入颈部后部, 并在头骨附近放置灵活的引线。</li>
	<li>用镊子将不锈钢螺钉 (2 毫米长) 放入每个毛刺孔中, 并通过顺时针旋转拧紧螺钉驱动。为了防止硬脑膜或脑组织受损, 请确保螺钉的插入深度不能太深。</li>
	<li>将绝缘层从引线尖端剥离2毫米, 并将导线拉伸足够长的时间以圆螺钉轴, 以使导线与螺钉之间有良好的连接。将参考导线连接到前螺钉, 记录导致顶叶皮层中的螺钉。然后, 在不暴露金属部件的情况下, 应用牙科水泥将整个装配固定在原处。</li>
	<li>经检验, 牙科水泥经目视检查完全干燥后, 缝合皮肤, 应用外用莫匹罗星软膏。将鼠标放在一个30摄氏度的孵化器中, 直到它从手术中恢复 (大约30分钟)。然后, 将鼠标返回到主笼, 直到连续视频-EEG 监视开始。 注意:手术后, 每只老鼠注射 (ip; 1 毫升注射器; 30 克针), 5 毫克/千克庆大霉素 (抗生素) 和5毫克/千克酮酮 (止痛药)。动物应该被监视, 直到它们完全 ambulant。发射机植入的小鼠有大约1周的恢复期。</li>
</ol>3. 视频和脑电图监测和分析
<ol><li>将鼠标放在单独的笼子中, 然后将笼子放在装有法拉第笼的无线接收板的顶部, 以避免从任何来源 (包括计算机、交流线路和相邻接收器) 中断电信号。 注意:请参阅制造商提供的手册。必须注意避免盲点。</li>
	<li>使用无线视频脑电图遥测系统记录电气活动。<ol>
<li>打开视频脑电图记录软件。单击 "硬件" 并选择 "编辑配置"。匹配接收器和发射机植入到每个鼠标。单击 "通道配置" 并确认所有通道都处于活动状态。单击 "视频配置" 将视频与 EEG 数据同步 (分辨率为 40 x 480 和帧速率 30.00)。 注意:安装一个高灵敏度的 IP 相机, 如果它是关键的收集良好的图像质量, 可以识别细微的行为变化的老鼠在夜间。</li>
		<li>单击 "设置" 和 "P3 设置" 以输入单个动物信息、匹配的照相机和图形设置。</li>
		<li>单击 "获取" 和 "a/d 采样率" 以设置采样率。单击 "数据集名称" 以指定每个实验。 注意:脑电图的采样率需要高于500赫兹. 由于视频和脑电图数据量大, 建议每个会话只处理24小时的数据, 而不是使用连续的2周记录作为一个文件。数据可以单独保存在8小时或 12 h 间隔, 以防 RAM 内存不足。</li>
		<li>用磁性棒激活发射机, 点击 "开始采集" 收集视频脑电图记录。通过视频脑电图系统连续监测鼠标至少2周。 注意:对于 C57BL/6 小鼠, SRS 倾向于发生在持续约5.2 天的簇群中, 而癫痫释放期的持续时间约为6.7 天17。</li>
	</ol>
</li>
	<li>使用分析软件确定检出频率和持续时间。<ol>
<li>标记显示 SRS 的区域并导出数据进行统计分析。小心排除可以通过抓头或咀嚼产生的脑电图工件;可以通过检查视频数据来删除这些信息。 注意:SRS 被定义为重复性癫痫发作 (≥3赫兹) 的突然出现, 持续了超过十年代. 抽搐行为可以经常伴有突发的扣球活动。无抽搐发作也可以显示短束的峰值活动, 不抽搐行为。当触发峰值返回基线活动时, 检出终止被定义为发生。</li>
		<li>对于扣押频率的分析, 将2周内检测到的 SRS 病例总数除以每只动物的记录天数。</li>
		<li>对于癫痫发作时间的分析, 测量癫痫发作的发病时间。</li>
	</ol>
</li>
	<li>视频脑电图记录完成后, 用 transcardial 灌注4% 多聚甲醛修复脑部。要麻醉鼠标, 请注射一溶液 (4:0, 5) 氯胺酮 (50 毫克/毫升) 和甲苯噻嗪 (23.3 毫克/毫升) 溶于生理盐水, 剂量为 10 ul/克体重 (ip; 1 毫升注射器; 26 克针)。一旦老鼠被证实被深深麻醉, 执行 transcardial 灌注。</li>
	<li>在隔离鼠标的大脑之前, 从鼠标检索发射机, 以便在清洗后再使用。<ol>
<li>使用镊子去除引线周围的牙科水泥。</li>
		<li>将引线的尖端与牙科水泥浸泡在100% 丙酮中, 直到牙科水泥溶解为止。</li>
		<li>用蒸馏水冲洗发射机周围的组织污染物。</li>
		<li>用70% 乙醇冲洗发射器3小时, 空气干燥以贮存。在下次使用之前, 立即用70% 乙醇冲洗螺钉和发射机, 然后用蒸馏水将其放入盐水中。 注意:斩首时, 必须小心避免切割挠性导线, 因为如果挠性引线太短, 下一个植入和视频脑电图记录中的噪声可能会增加。</li>
	</ol>
</li>
</ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accumulation+of+abnormal+adult-generated+hippocampal+granule+cells+predicts+seizure+frequency+and+severity.">Accumulation of abnormal adult-generated hippocampal granule cells predicts seizure frequency and severity.</a> J Neurosci. 33 (21), 8926-8936 (2013).</li><li>Shibley, H., Smith, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pilocarpine-induced+status+epilepticus+results+in+mossy+fiber+sprouting+and+spontaneous+seizures+in+C57BL/6+and+CD-1+mice.">Pilocarpine-induced status epilepticus results in mossy fiber sprouting and spontaneous seizures in C57BL/6 and CD-1 mice.</a> Epilepsy Research. 49 (2), 109-120 (2002).</li><li>Borges, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+and+glial+pathological+changes+during+epileptogenesis+in+the+mouse+pilocarpine+model.">Neuronal and glial pathological changes during epileptogenesis in the mouse pilocarpine model.</a> Exp Neurol. 182 (1), 21-34 (2003).</li><li>Pavlova, M. K., Shea, S. A., Bromfield, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Day/night+patterns+of+focal+seizures.">Day/night patterns of focal seizures.</a> Epilepsy Behav. 5 (1), 44-49 (2004).</li></ol>

作者没有什么可透露的。

这项工作描述了 SE 诱导和评估慢性癫痫的实验程序。
有几个因素会影响成功的 SE 诱导。根据拉辛尺度进行精确的行为监测对 SRS 的发展至关重要。头点头, 前肢阵挛, 饲养和下降是急性发作的行为特征发展到 SE 阶段 4, 16.一旦检测到第一次癫痫发作, 在进入 SE 之前, 后痉挛性癫痫发作间隔的时间长度会减少。此外, SE 应保持至少6小时...

癫痫是最常见的获取的1之一。患有癫痫的人由于大脑中异常的神经元活动而经常发作, 如2,3。鉴于这一问题往往难以解决, 因此了解癫痫发展的基本机制至关重要。
可以概括人类的关键特征的动物模型可以更好地了解人体病理生理学, 使我们能够很容易地监测和操作负反馈中的关键因素。其中, chemoconvulsants 诱导 SE 已被广泛使用4,5。不像其他癫痫模型, 如电刺激显示没有海马硬化和强健的 SRS6,7,8, 系统注射 chemoconvulsants 可以模仿人类的临床发病机制,即、初始脑损伤、潜伏期和慢性癫痫阶段, 表现为 SRS5、9、10。因此, 这项技术可以用于解释急性脑损伤、负反馈或癫痫发作机制的各种研究。此外, chemoconvulsants 引起的组织病理学改变类似于人类所看到的, 提供了一个额外的理由使用的啮齿动物模型10,11,12。值得注意的是, 包括海马的结构损伤在海仁酸和匹属的硒模型中一直被重现。然而, 与海仁酸注射液相比, 该模型能在小鼠体内产生更强的 SRS, 在考虑到转基因小鼠线的广泛可用性时, 可以为研究慢性癫痫提供相当大的优势5,13,14,15. 此外, 在使用海仁酸模型后, 在匹比注射后的检出进展通常更快, 这为有效利用匹属的癫痫模型提供了更多的证据。
在这里, 我们演示了一种诱导硒的方法, 通过 ip 注射的匹比和视频和脑电图监测的慢性癫痫。

<table><tbody><tr><td>C57BL/6</td><td>Envigo</td><td>C57BL/6NHsd</td><td></td></tr><tr><td>Scopolamine methyl nitrate</td><td>Sigma</td><td>S2250</td><td>Make 10X stock</td></tr><tr><td>Terbutaline hemisulfate salt</td><td>Sigma</td><td>T2528</td><td>Make 10X stock</td></tr><tr><td>Pilocarpine hydrochloride</td><td>Sigma</td><td>P6503</td><td></td></tr><tr><td>Ketamine hydrochloride</td><td>Yuhan corporation</td><td></td><td></td></tr><tr><td>Xylazine hydrochloride</td><td>Bayer Korea</td><td></td><td></td></tr><tr><td>Diazepam</td><td>SAMJIN</td><td></td><td></td></tr><tr><td>Castor oil (Kolliphor EL)</td><td>Sigma</td><td>C5135</td><td>Polyoxyl 35 hydrogenated castor oil</td></tr><tr><td>Saline</td><td>Daihan pharm. Co.</td><td></td><td></td></tr><tr><td>5% Dextrose</td><td>Daihan pharm. Co.</td><td></td><td></td></tr><tr><td>Iodine solution (Povidin)</td><td>Firson</td><td></td><td></td></tr><tr><td>vet ointment (Terramycin)</td><td>Pfizer</td><td></td><td></td></tr><tr><td>Blue Nylon</td><td>AILEE</td><td>NB617</td><td></td></tr><tr><td>Mupirocin (Bearoban)</td><td>Daewoong Pharmaceutical Co., Ltd</td><td></td><td></td></tr><tr><td>Ketoprofen</td><td>Samchundang Pharm. Co., Ltd</td><td></td><td>5 mg/kg</td></tr><tr><td>Gentamicin</td><td>Huons, Ltd.</td><td></td><td>5 mg/kg</td></tr><tr><td>1 mL syringe</td><td>Sung shim medial Co., Ltd.</td><td></td><td></td></tr><tr><td>26 guage needle</td><td>Sung shim medial Co., Ltd.</td><td></td><td>26 G * 13 mm (1/2&quot;)</td></tr><tr><td>30 guage needle</td><td>Sung shim medial Co., Ltd.</td><td></td><td>30 G * 13 mm (1/2&quot;)</td></tr><tr><td>Razor blade</td><td>Dorco</td><td></td><td></td></tr><tr><td>Drill</td><td>Saeshin precision Co., Ltd.</td><td></td><td>207A, 35K (speed)</td></tr><tr><td>Telemetry video/EEG system</td><td>Data sciences International. Inc.</td><td>Version 5.20-SP6</td><td></td></tr><tr><td>Implantable transmitter</td><td>Data sciences International. Inc.</td><td>ETA-F10</td><td></td></tr><tr><td>Screw</td><td>Sungho Steel</td><td></td><td>M1.4, 2 mm length stainless steel</td></tr><tr><td>Vertex dental material&amp;nbsp;</td><td>Dentimex</td><td></td><td></td></tr><tr><td>Acetone</td><td>Duksan pure chemicals Co., Ltd.</td><td></td><td>CAS 67-64-1</td></tr><tr><td>Paraformaldehyde (PFA)</td><td>millipore</td><td>1.04005.1000</td><td>4 %&amp;nbsp;</td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S9378</td><td>30 % solution in 0.01 M PBS</td></tr><tr><td>Cresyl violet acetate</td><td>Sigma</td><td>C5042</td><td></td></tr><tr><td>Ethanol</td><td>EMD Millipore Co.</td><td>UN1170</td><td></td></tr><tr><td>xylene</td><td>Duksan pure chemicals Co., Ltd.</td><td>UN1307</td><td></td></tr><tr><td>Acetic acid glacial</td><td>Junsei chemical</td><td>31010-0350</td><td></td></tr><tr><td>FSC33 Clear&amp;nbsp;</td><td>Leica biosystems</td><td></td><td>OCT compound for tissue freezing</td></tr><tr><td>DPX Mounting for histology</td><td>Sigma</td><td>6522</td><td></td></tr><tr><td>Forceps</td><td>Fine science tools</td><td>11002-12</td><td></td></tr><tr><td>Scissors</td><td>Solco biomedical</td><td>02-2445</td><td></td></tr><tr><td>Stereotaxic frame</td><td>David Kopf Instruments</td><td>E51070012</td><td></td></tr></tbody></table>

the pilocarpine model of temporal lobe epilepsy and eeg monitoring using radiotelemetry system in mice

颞叶癫痫是成人常见的神经系统疾病。对于慢性癫痫的翻译研究, 匹特诱发的癫痫持续状态 (SE) 经常被选择来重述自发性复发性癫痫 (SRS)。在这里, 我们提出了一个通过腹腔 (ip) 注射的匹属和监测在活体动物慢性复发性惊厥使用无线遥测视频和脑电图系统。我们表现出显著的行为变化, 需要注意后, 匹凡和与海马神经元丢失的关系, 在7天和6周后的匹。我们还描述了视频和脑电图记录电极植入的实验过程, 分析了慢性复发性癫痫的频率和持续时间。最后, 我们讨论了在每种情况下未能实现预期结果的可能原因。这为小鼠慢性癫痫模型的建模和诊断指南提供了基本的概述。我们认为, 该议定书可以作为一个基线, 为适当的模型慢性癫痫和负反馈。

成功的 SE 可以诱发海马细胞死亡和 SRS (图 1和图 2)。在 SE 发病后3小时, 我们终止了西泮注射液的行为性急性发作, 并在7天或6周后对小鼠进行了牺牲。
对于视频脑电图监测, 小鼠接受植入术后4周 se, 和 SRS 病例评估2周后5-7 周的 se 发病。
...

Watch this Scientific Journal Video about The Pilocarpine Model of Temporal Lobe Epilepsy and EEG Monitoring Using Radiotelemetry System in Mice at JoVE.com

The Pilocarpine Model of Temporal Lobe Epilepsy and EEG Monitoring Using Radiotelemetry System in Mice

这篇手稿描述了一种方法, 以诱发癫痫持续状态的系统的匹里注射和监测自发复发性癫痫活动物使用无线遥测视频和脑电图系统。本协议可用于研究慢性癫痫、负反馈和急性发作的病理生理机制。

小鼠颞叶癫痫的 Radiotelemetry 模型及脑电图监测

Temporal lobe epilepsy (TLE) is a common neurological disorder in adulthood. For translational studies of chronic epilepsy, pilocarpine-induced status ...

the-pilocarpine-model-temporal-lobe-epilepsy-eeg-monitoring-using

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

19.5K 次观看.  The Catholic University of Korea. 这篇手稿描述了一种方法, 以诱发癫痫持续状态的系统的匹里注射和监测自发复发性癫痫活动物使用无线遥测视频和脑电图系统。本协议可用于研究慢性癫痫、负反馈和急性发作的病理生理机制。

视频: The Pilocarpine Model of Temporal Lobe Epilepsy and EEG Monitoring Using Radiotelemetry System in Mice

Simultaneous Video-EEG-ECG Monitoring to Identify Neurocardiac Dysfunction in Mouse Models of Epilepsy

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can potentially increase risk of sudden unexpected death in epilepsy (SUDEP). Electroencephalography (EEG) and electrocardiography (ECG) are widely used clinical diagnostic tools to monitor for abnormal brain and cardiac rhythms in patients. Here, a technique to simultaneously record video, EEG, and ECG in mice to measure behavior, brain, and cardiac activities, respectively, is described. The technique described herein utilizes a tethered (i.e., wired) recording configuration in which the implanted electrode on the head of the mouse is hard-wired to the recording equipment. Compared to wireless telemetry recording systems, the tethered arrangement possesses several technical advantages such as a greater possible number of channels for recording EEG or other biopotentials; lower electrode costs; and greater frequency bandwidth (i.e., sampling rate) of recordings. The basics of this technique can also be easily modified to accommodate recording other biosignals, such as electromyography (EMG) or plethysmography for assessment of muscle and respiratory activity, respectively. In addition to describing how to perform the EEG-ECG recordings, we also detail methods to quantify the resulting data for seizures, EEG spectral power, cardiac function, and heart rate variability, which we demonstrate in an example experiment using a mouse with epilepsy due to Kcna1 gene deletion. Video-EEG-ECG monitoring in mouse models of epilepsy or other neurological disease provides a powerful tool to identify dysfunction at the level of the brain, heart, or brain-heart interactions.

Here, we present a protocol to record brain and heart bio signals in mice using simultaneous video, electroencephalography (EEG), and electrocardiography (ECG). We also describe methods to analyze the resulting EEG-ECG recordings for seizures, EEG spectral power, cardiac function, and heart rate variability.

同步视频脑电图-心电图监测对小鼠癫痫模型 Neurocardiac 功能障碍的鉴别

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can ...

科研

遗传学

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 ...

行为学

Use of a Wireless Video-EEG System to Monitor Epileptiform Discharges Following Lateral Fluid-Percussion Induced Traumatic Brain Injury

The lateral fluid percussion injury (FPI) model is well established and has been used to study TBI and post-traumatic epilepsy (PTE). However, considerable variability has been reported for the specific parameters used in different studies that have employed this model, making it difficult to harmonize and interpret the results between laboratories. For example, variability has been reported regarding the size and location of the craniectomy, how the Luer lock hub is placed relative to the craniectomy, the atmospheric pressure applied to the dura and the duration of the pressure pulse. Each of these parameters can impact injury severity, which directly correlates with the incidence of PTE. This has been manifested as a wide range of mortality rates, righting reflex times and incidence of convulsive seizures reported. Here we provide a detailed protocol for the method we have used to help facilitate harmonization between studies. We used FPI in combination with a wireless EEG telemetry system to continuously monitor for electrographic changes and detect seizure activity.&#160; FPI is induced by creating a 5 mm craniectomy over the left hemisphere, between the Bregma and Lambda and adjacent to the lateral ridge. A Luer lock hub is secured onto the skull over the craniectomy. This hub is connected to the FPI device, and a 20-millisecond pressure pulse is delivered directly to the intact dura through pressure tubing connected to the hub via a twist lock connector. Following recovery, rats are re-anesthetized to remove the hub. Five 0.5 mm, stainless steel EEG electrode screws are placed in contact with the dura through the skull and serve as four recording electrodes and one reference electrode. The electrode wires are collected into a pedestal connector which is secured into place with bone cement. Continuous video/EEG recordings are collected for up to 4 weeks post TBI.

Here we present a protocol to induce severe TBI with the lateral fluid percussion injury (FPI) model in adult, male Wistar rats. We also demonstrate the use of a wireless telemetry system to collect continuous video-EEG recordings and monitor for epileptiform discharges consistent with post-traumatic epileptogenesis.&#160;

使用无线视频-EEG系统监测侧流冲击后癫痫的放电诱发创伤性脑损伤

The lateral fluid percussion injury (FPI) model is well established and has been used to study TBI and post-traumatic epilepsy (PTE). However, ...

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 ...

Long-term Continuous EEG Monitoring in Small Rodent Models of Human Disease Using the Epoch Wireless Transmitter System

Many progressive neurologic diseases in humans, such as epilepsy, require pre-clinical animal models that slowly develop the disease in order to test interventions at various stages of the disease process. These animal models are particularly difficult to implement in immature rodents, a classic model organism for laboratory study of these disorders. Recording continuous EEG in young animal models of seizures and other neurological disorders presents a technical challenge due to the small physical size of young rodents and their dependence on the dam prior to weaning. Therefore, there is not only a clear need for improving pre-clinical research that will better identify those therapies suitable for translation to the clinic but also a need for new devices capable of recording continuous EEG in immature rodents. Here, we describe the technology behind and demonstrate the use of a novel miniature telemetry system, specifically engineered for use in immature rats or mice, which is also effective for use in adult animals.

Here we demonstrate the use of a wireless enabling technology for electroencephalogram (EEG) in neonatal rodent models of human disease. With telemetry, there are no encumbering connections, thus allowing natural behaviors.

在人类疾病中使用大纪元无线传输系统的小型啮齿动物模型的长期连续监测脑电图

Many progressive neurologic diseases in humans, such as epilepsy, require pre-clinical animal models that slowly develop the disease in order to test ...

神经科学

Non-restraining EEG Radiotelemetry: Epidural and Deep Intracerebral Stereotaxic EEG Electrode Placement

Implantable EEG radiotelemetry is of central relevance in the neurological characterization of transgenic mouse models of neuropsychiatric and neurodegenerative diseases as well as epilepsies. This powerful technique does not only provide valuable insights into the underlying pathophysiological mechanisms, i.e., the etiopathogenesis of CNS related diseases, it also facilitates the development of new translational, i.e., therapeutic approaches. Whereas competing techniques that make use of recorder systems used in jackets or tethered systems suffer from their unphysiological restraining to semi-restraining character, radiotelemetric EEG recordings overcome these disadvantages. Technically, implantable EEG radiotelemetry allows for precise and highly sensitive measurement of epidural and deep, intracerebral EEGs under various physiological and pathophysiological conditions. First, we present a detailed protocol of a straight forward, successful, quick and efficient technique for epidural (surface) EEG recordings resulting in high-quality electrocorticograms. Second, we demonstrate how to implant deep, intracerebral EEG electrodes, e.g., in the hippocampus (electrohippocampogram). For both approaches, a computerized 3D stereotaxic electrode implantation system is used. The radiofrequency transmitter itself is implanted into a subcutaneous pouch in both mice and rats. Special attention also has to be paid to pre-, peri- and postoperative treatment of the experimental animals. Preoperative preparation of mice and rats, suitable anesthesia as well as postoperative treatment and pain management are described in detail.

Non-restraining EEG radiotelemetry is a valuable methodological approach to record in vivo long-term electroencephalograms from freely moving rodents. This detailed protocol describes stereotaxic epidural and deep intracerebral electrode placement in different brain regions in order to obtain reliable recordings of CNS rhythmicity and CNS related behavioral stages.

非约束脑电遥测:硬膜外和脑深部脑电立体定位放置电极

Implantable EEG radiotelemetry is of central relevance in the neurological characterization of transgenic mouse models of neuropsychiatric and ...

Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain interstitial space with the behavior and/or with the specific outcome of a pathology (e.g., seizures for epilepsy). When studying epilepsy, the microdialysis technique is often combined with short-term or even long-term video-electroencephalography (EEG) monitoring to assess spontaneous seizure frequency, severity, progression and clustering. The combined microdialysis-EEG is based on the use of several methods and instruments. Here, we performed in vivo microdialysis and continuous video-EEG recording to monitor glutamate and aspartate outflow over time, in different phases of the natural history of epilepsy in a rat model. This combined approach allows the pairing of changes in the neurotransmitter release with specific stages of the disease development and progression. The amino acid concentration in the dialysate was determined by liquid chromatography. Here, we describe the methods and outline the principal precautionary measures one should take during in vivo microdialysis-EEG, with particular attention to the stereotaxic surgery, basal and high potassium stimulation during microdialysis, depth electrode EEG recording and high-performance liquid chromatography analysis of aspartate and glutamate in the dialysate. This approach may be adapted to test a variety of drug or disease induced changes of the physiological concentrations of aspartate and glutamate in the brain. Depending on the availability of an appropriate analytical assay, it may be further used to test different soluble molecules when employing EEG recording at the same time.

Here, we describe a method for in vivo microdialysis to analyze aspartate and glutamate release in the ventral hippocampus of epileptic and non-epileptic rats, in combination with EEG recordings. Extracellular concentrations of aspartate and glutamate may be correlated with the different phases of the disease.

自由运动大鼠脑电图记录过程中兴奋氨基酸的微透析

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain ...

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 ...

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 ...

Systemic Pilocarpine Treatment: Mouse Model of Temporal Lobe Epilepsy

Source: Kim, J. E. and Cho, K. O. <a href="https://jove.unicartagenaproxy.elogim.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. ...