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。

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			<td height="40" style="height:40px;width:189px;">C57BL/6</td>
			<td style="width:233px;">Envigo</td>
			<td style="width:175px;">C57BL/6NHsd</td>
			<td style="width:232px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;">Scopolamine methyl nitrate</td>
			<td>Sigma</td>
			<td>S2250</td>
			<td>Make 10X stock</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Terbutaline hemisulfate salt</td>
			<td>Sigma</td>
			<td>T2528</td>
			<td>Make 10X stock</td>
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		<tr height="40">
			<td height="40" style="height:40px;">Pilocarpine hydrochloride</td>
			<td>Sigma</td>
			<td>P6503</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Intensive care unit</td>
			<td>Daejong instrument industry Co., Ltd.</td>
			<td></td>
			<td>28~30&#8451;&#160;</td>
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		<tr height="40">
			<td height="40" style="height:40px;">Ketamine hydrochloride</td>
			<td>Yuhan corporation</td>
			<td></td>
			<td></td>
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			<td height="40" style="height:40px;">Xylazine hydrochloride</td>
			<td>Bayer Korea</td>
			<td></td>
			<td></td>
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			<td height="40" style="height:40px;">Diazepam</td>
			<td>SAMJIN</td>
			<td></td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;">Castor oil (Kolliphor EL)</td>
			<td>Sigma</td>
			<td>C5135</td>
			<td>Polyoxyl 35 hydrogenated castor oil</td>
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			<td height="40" style="height:40px;">Saline</td>
			<td>Daihan pharm. Co.</td>
			<td></td>
			<td></td>
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			<td height="40" style="height:40px;">5% Dextrose</td>
			<td>Daihan pharm. Co.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Iodine solution (Povidin)</td>
			<td>Firson</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">vet ointment (Terramycin)</td>
			<td>Pfizer</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Blue Nylon</td>
			<td>AILEE</td>
			<td>NB617</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Mupirocin (Bearoban)</td>
			<td>Daewoong Pharmaceutical Co., Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ketoprofen</td>
			<td>Samchundang Pharm. Co., Ltd</td>
			<td></td>
			<td>5 mg/kg</td>
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		<tr height="40">
			<td height="40" style="height:40px;">Gentamicin</td>
			<td>Huons, Ltd.</td>
			<td></td>
			<td>5 mg/kg</td>
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		<tr height="40">
			<td height="40" style="height:40px;">1 mL syringe</td>
			<td>Sung shim medial Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">26 guage needle</td>
			<td>Sung shim medial Co., Ltd.</td>
			<td></td>
			<td>26 G * 13 mm (1/2&#34;)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">30 guage needle</td>
			<td>Sung shim medial Co., Ltd.</td>
			<td></td>
			<td>30 G * 13 mm (1/2&#34;)</td>
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		<tr height="40">
			<td height="40" style="height:40px;">Razor blade</td>
			<td>Dorco</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Drill</td>
			<td>Saeshin precision Co., Ltd.</td>
			<td></td>
			<td>207A, 35K (speed)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Telemetry video/EEG system</td>
			<td>Data sciences International. Inc.</td>
			<td>Version 5.20-SP6</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Implantable transmitter</td>
			<td>Data sciences International. Inc.</td>
			<td>ETA-F10</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;">Screw</td>
			<td>Sungho Steel</td>
			<td></td>
			<td>M1.4, 2 mm length stainless steel</td>
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			<td height="40" style="height:40px;">Vertex dental material&#160;</td>
			<td>Dentimex</td>
			<td></td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;">Acetone</td>
			<td>Duksan pure chemicals Co., Ltd.</td>
			<td></td>
			<td>CAS 67-64-1</td>
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			<td height="40" style="height:40px;">Paraformaldehyde (PFA)</td>
			<td>millipore</td>
			<td>1.04005.1000</td>
			<td>4 %&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sucrose</td>
			<td>Sigma</td>
			<td>S9378</td>
			<td>30 % solution in 0.01 M PBS</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cresyl violet acetate</td>
			<td>Sigma</td>
			<td>C5042</td>
			<td></td>
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			<td height="40" style="height:40px;">Ethanol</td>
			<td>EMD Millipore Co.</td>
			<td>UN1170</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">xylene</td>
			<td>Duksan pure chemicals Co., Ltd.</td>
			<td>UN1307</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Acetic acid glacial</td>
			<td>Junsei chemical</td>
			<td>31010-0350</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">FSC33 Clear&#160;</td>
			<td>Leica biosystems</td>
			<td></td>
			<td>OCT compound for tissue freezing</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DPX Mounting for histology</td>
			<td>Sigma</td>
			<td>6522</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Forceps</td>
			<td>Fine science tools</td>
			<td>11002-12</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;">Scissors</td>
			<td>Solco biomedical</td>
			<td>02-2445</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">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

Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal models of acute ischemic stroke allows to confirm successful arterial occlusion and exclude subarachnoid hemorrhage. Cerebral perfusion monitoring can also be used to study intracranial collateral circulation, which is emerging as a powerful determinant of stroke outcome and a possible therapeutic target. Despite a recognized role of Laser Doppler perfusion monitoring as part of the current guidelines for experimental cerebral ischemia, a number of technical difficulties exist that limit its widespread use. One of the major issues is obtaining a secure and prolonged attachment of a deep-penetration Laser Doppler probe to the animal skull. In this video, we show our optimized system for cerebral perfusion monitoring during transient middle cerebral artery occlusion by intraluminal filament in the rat. We developed in-house a simple method to obtain a custom made holder for twin-fibre (deep-penetration) Laser Doppler probes, which allow multi-site monitoring if needed. A continuous and prolonged monitoring of cerebral perfusion could easily be obtained over the intact skull.

Cerebral perfusion monitoring has been demonstrated to improve accuracy in ischemic stroke models. Technical difficulties often limit the use of this essential tool for cerebrovascular research. In this video, an optimized system is shown to obtain a single or multi-site hemodynamic monitoring during intraluminal middle cerebral artery occlusion in rats.

优化系统的脑灌注监测在中风模型大鼠的大脑中动脉闭塞腔内

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal ...

科研

医学

Endotracheal Intubation in Mice via Direct Laryngoscopy Using an Otoscope

Mice, both wildtype and transgenic, are the principal mammalian model in biomedical research currently. Intubation and mechanical ventilation are necessary for whole animal experiments that require surgery under deep anesthesia or measurements of lung function. Tracheostomy has been the standard for intubating the airway in these mice to allow mechanical ventilation. Orotracheal intubation has been reported but has not been successfully used in many studies because of the substantial technical difficulty or a requirement for highly specialized and expensive equipment. Here we report a technique of direct laryngoscopy using an otoscope fitted with a 2.0 mm speculum and using a 20 G&#160;intravenous catheter as an endotracheal tube. We have used this technique extensively and reliably to intubate and conduct accurate assessments of lung function in mice. This technique has proven safe, with essentially no animal loss in experienced hands. Moreover, this technique can be used for repeated studies of mice in chronic models.

Endotracheal Intubation in Mice via Direct Laryngoscopy Using an Otoscope

We have developed a simple, reliable, and relatively inexpensive method for endotracheal intubation in mice via direct laryngoscopy using an otoscope with a 2.0 mm speculum. This technique is atraumatic and can be used for repeated measurements in chronic experiments. We find it superior to tracheostomy or previously reported nonsurgical techniques.

气管插管小鼠<em&gt;通过</em&gt;直接喉镜使用耳镜

Mice, both wildtype and transgenic, are the principal mammalian model in biomedical research currently. Intubation and mechanical ventilation are ...

Network Analysis of the Default Mode Network Using Functional Connectivity MRI in Temporal Lobe Epilepsy

Functional connectivity MRI (fcMRI) is an fMRI method that examines the connectivity of different brain areas based on the correlation of BOLD signal fluctuations over time. Temporal Lobe Epilepsy (TLE) is the most common type of adult epilepsy and involves multiple brain networks. The default mode network (DMN) is involved in conscious, resting state cognition and is thought to be affected in TLE where seizures cause impairment of consciousness. The DMN in epilepsy was examined using seed based fcMRI. The anterior and posterior hubs of the DMN were used as seeds in this analysis. The results show a disconnection between the anterior and posterior hubs of the DMN in TLE during the basal state. In addition, increased DMN connectivity to other brain regions in left TLE along with decreased connectivity in right TLE is revealed. The analysis demonstrates how seed-based fcMRI can be used to probe cerebral networks in brain disorders such as TLE.

The Default Mode Network (DMN) in Temporal Lobe Epilepsy (TLE) is analyzed in the resting state of the brain using seed-based functional connectivity MRI (fcMRI).

使用功能性磁共振成像连接在颞叶癫痫默认模式网络的网络分析

Functional connectivity MRI (fcMRI) is an fMRI method that examines the connectivity of different brain areas based on the correlation of BOLD signal ...

Monitoring of Systemic and Hepatic Hemodynamic Parameters in Mice

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. However, due to the small size of the mouse, technical details of the intraoperative monitoring procedure suitable for the mouse were rarely described. Previously we have reported a monitoring procedure to obtain hemodynamic parameters for rats. Now, we adapted the procedure to acquire systemic and hepatic hemodynamic parameters in mice, a species ten-fold smaller than rats. This film demonstrates the instrumentation of the animals as well as the data acquisition process needed to assess systemic and hepatic hemodynamics in mice. Vital parameters, including body temperature, respiratory&#160;rate&#160;and heart&#160;rate were recorded throughout the whole procedure. Systemic hemodynamic parameters consist of carotid artery pressure (CAP) and central venous pressure (CVP). Hepatic perfusion parameters include portal vein pressure (PVP), portal flow rate as well as the flow rate of the common hepatic artery (table 1). Instrumentation and data acquisition to record the normal values was completed within 1.5 h. Systemic and hepatic hemodynamic parameters remained within normal ranges during this procedure.
This procedure is challenging but feasible. We have already applied this procedure to assess hepatic hemodynamics in normal mice as well as during 70% partial hepatectomy and in liver lobe clamping experiments. Mean PVP after resection (n= 20), was 11.41&#177;2.94 cmH2O which was significantly higher (P&#60;0.05) than before resection (6.87&#177;2.39 cmH2O). The results of liver lobe clamping experiment indicated that this monitoring procedure is sensitive and suitable for detecting small changes in portal pressure and portal flow rate. In conclusion, this procedure is reliable in the hands of an experienced micro-surgeon but should be limited to experiments where mice are absolutely needed.

This film demonstrates how to acquire systemic and hepatic hemodynamics in mice. The whole monitoring includes acquisition of vital parameters, systemic blood pressure, central venous pressure, common hepatic artery flow rate, and portal vein pressure as well as the portal flow rate in mice.

全身及肝脏血流动力学参数的监测小鼠

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. ...

Brain Source Imaging in Preclinical Rat Models of Focal Epilepsy using High-Resolution EEG Recordings

Electroencephalogram (EEG) has been traditionally used to determine which brain regions are the most likely candidates for resection in patients with focal epilepsy. This methodology relies on the assumption that seizures originate from the same regions of the brain from which interictal epileptiform discharges (IEDs) emerge. Preclinical models are very useful to find correlates between IED locations and the actual regions underlying seizure initiation in focal epilepsy. Rats have been commonly used in preclinical studies of epilepsy1; hence, there exist a large variety of models for focal epilepsy in this particular species. However, it is challenging to record multichannel EEG and to perform brain source imaging in such a small animal. To overcome this issue, we combine a patented-technology to obtain 32-channel EEG recordings from rodents2 and an MRI probabilistic atlas for brain anatomical structures in Wistar rats to perform brain source imaging. In this video, we introduce the procedures to acquire multichannel EEG from Wistar rats with focal cortical dysplasia, and describe the steps both to define the volume conductor model from the MRI atlas and to uniquely determine the IEDs. Finally, we validate the whole methodology by obtaining brain source images of IEDs and compare them with those obtained at different time frames during the seizure onset.

This video introduces the preparation, recording, and source analysis procedures of high-resolution EEG on sedated rats with a particular preclinical model of focal epilepsy under noninvasive conditions.

采用高分辨率脑电图记录脑源成像局灶性癫痫的临床前模型大鼠

Electroencephalogram (EEG) has been traditionally used to determine which brain regions are the most likely candidates for resection in patients with ...

Network Analysis of Foramen Ovale Electrode Recordings in Drug-resistant Temporal Lobe Epilepsy Patients

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control seizures. However, a significant minority of patients continues to exhibit post-operative seizures, even in those cases in which the suspected source of seizures has been correctly localized and resected. The protocol presented here combines a clinical procedure routinely employed during the pre-operative evaluation of temporal lobe epilepsy (TLE) patients with a novel technique for network analysis. The method allows for the evaluation of the temporal evolution of mesial network parameters. The bilateral insertion of foramen ovale electrodes (FOE) into the ambient cistern simultaneously records electrocortical activity at several mesial areas in the temporal lobe. Furthermore, network methodology applied to the recorded time series tracks the temporal evolution of the mesial networks both interictally and during the seizures. In this way, the presented protocol offers a unique way to visualize and quantify measures that considers the relationships between several mesial areas instead of a single area.

This protocol describes a procedure to track the evolution of mesial network measures in temporal lobe epilepsy (TLE) patients. It is based on the combination of intracranial recordings with a novel numerical technique for data analysis. Specifically, we present a protocol for network analyses of foramen ovale recordings.

在耐药性颞叶癫痫患者卵圆孔电极记录的网络分析

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Induction and Characterization of Pulmonary Hypertension in Mice using the Hypoxia/SU5416 Model

Pulmonary Hypertension (PH) is a pathophysiological condition, defined by a mean pulmonary arterial pressure exceeding 25 mm Hg at rest, as assessed by right heart&#160;catheterization. A broad spectrum of diseases can lead to PH, differing in their etiology, histopathology, clinical presentation, prognosis, and response to&#160;treatment. Despite significant progress in the last years, PH remains an uncured disease. Understanding the underlying mechanisms can pave the way for the development of new therapies. Animal models are important research tools to achieve this goal. Currently, there are several models available for recapitulating PH. This protocol describes a two-hit mouse PH model. The stimuli for PH development are hypoxia and the injection of SU5416, a vascular endothelial growth factor (VEGF) receptor antagonist. Three weeks after initiation of Hypoxia/SU5416, animals develop pulmonary vascular remodeling imitating the histopathological changes observed in human PH (predominantly Group 1). Vascular remodeling in the pulmonary circulation results in the remodeling of the right&#160;ventricle (RV). The procedures for measuring RV pressures (using the open chest method), the morphometrical analyses of the RV (by dissecting and weighing both cardiac ventricles) and the histological assessments of the remodeling (both pulmonary by assessing vascular remodeling and cardiac by assessing RV cardiomyocyte hypertrophy and fibrosis) are described in detail. The advantages of this protocol are the possibility of the application both in wild type and in genetically modified mice, the relatively easy and low-cost implementation, and the quick development of the disease of interest (3 weeks). Limitations of this method are that mice do not develop a severe phenotype and PH is reversible upon return to normoxia. Prevention, as well as therapy studies, can easily be implemented in this model, without the necessity of advanced skills (as opposed to surgical rodent models).

This protocol describes the induction of pulmonary hypertension (PH) in mice based on the exposure to hypoxia and the injection of a VEGF receptor antagonist. The animals develop PH and right ventricular (RV) hypertrophy 3 weeks after the initiation of the protocol. The functional and morphometrical characterization of the model is also presented.

使用缺氧/SU5416模型对小鼠的肺高血压进行诱导和特征

Pulmonary Hypertension (PH) is a pathophysiological condition, defined by a mean pulmonary arterial pressure exceeding 25 mm Hg at rest, as assessed by ...

Assessment of Static Graviceptive Perception in the Roll-Plane using the Subjective Visual Vertical Paradigm

Vestibular disorders are among the most common syndromes in medicine. In recent years, new vestibular diagnostic systems have been introduced that allow the examination of all semicircular canals in the clinical setting. Assessment methods of the otolithic system, which is responsible for the perception of linear acceleration and perception of gravity, are far less in clinical use. There are several experimental approaches for measuring the perception of gravity. The most frequently used method is the determination of the subjective visual vertical. This is usually measured with the head in an upright position. We present here an assessment method for testing otolith function in the roll plane. The subjective visual vertical is measured in the head upright position as well as with head inclination of &#177; 15&#176; and &#177; 30&#176; in the roll plane. This extended functional paradigm is an easy-to-perform clinical test of otolith function and ensures increased information content for the detection of impaired graviceptive perception.

The perception of gravity is commonly determined by the subjective visual vertical in the head upright position. The additional assessment at head tilts of &#177; 15&#176; and &#177; 30&#176; in the roll plane ensures increased information content for the detection of impaired graviceptive perception.

使用主观视觉垂直范式对卷平面中的静态视觉感知的评估

Vestibular disorders are among the most common syndromes in medicine. In recent years, new vestibular diagnostic systems have been introduced that allow ...

Multi-system Monitoring for Identification of Seizures, Arrhythmias and Apnea in Conscious Restrained Rabbits

Patients with ion channelopathies are at a high risk of developing seizures and fatal cardiac arrhythmias. There is a higher prevalence of heart disease and arrhythmias in people with epilepsy (i.e., epileptic heart.) Additionally, cardiac and autonomic disturbances have been reported surrounding seizures. 1:1,000 epilepsy patients/year die of sudden unexpected death in epilepsy (SUDEP). The mechanisms for SUDEP remain incompletely understood. Electroencephalograms (EEG) and electrocardiograms (ECG) are two techniques routinely used in the clinical setting to detect and study the substrates/triggers for seizures and arrhythmias. While many studies and descriptions of this methodology are in rodents, their cardiac electrical activity differs significantly from humans. This article provides a description of a non-invasive method for recording simultaneous video-EEG-ECG-oximetry-capnography in conscious rabbits. As cardiac electrical function is similar in rabbits and humans, rabbits provide an excellent model of translational diagnostic and therapeutic studies. In addition to outlining the methodology for data acquisition, we discuss the analytical approaches for examining neuro-cardiac electrical function and pathology in rabbits. This includes arrhythmia detection, spectral analysis of EEG and a seizure scale developed for restrained rabbits.

Using simultaneous video-EEG-ECG-oximetry-capnography, we developed a methodology to evaluate the susceptibility of rabbit models to develop provoked arrhythmias and seizures. This novel recording system establishes a platform to test the efficacy and safety of therapeutics and can capture the complex cascade of multi-system events that culminate in sudden death.

意识约束兔子中癫痫发作、心律失常和呼吸暂停的多系统监测

Patients with ion channelopathies are at a high risk of developing seizures and fatal cardiac arrhythmias. There is a higher prevalence of heart disease ...