这项工作得到了公民联合研究癫痫 (赠款编号 35489);国家卫生研究院 (赠款编号 R01NS100954, R01NS099188);和路易斯安那州立大学健康科学中心马尔科姆费斯特博士后奖学金。

所有的实验程序都应按照国家卫生研究院 (NIH) 的指导方针进行, 由机构的动物保育和使用委员会 (IACUC) 批准。此协议所需的主要手术工具如图 1所示。
1. 植入电极的制备
<ol><li>安置10插座女性 nanoconnector (i. e, 电极;图 2A)到一个桌面老虎钳与10线面对和黑色的电线在前面。使用细钳, 将第一条 (黑线) 线折叠到右侧, 第二条 (棕) 线向左折。接下来将红色、橙色、蓝色和紫色的线交替向下折叠 (图 2B)。切断在他们的附件底部的黄色, 绿色, 白色和灰色的电线。</li>
	<li>要准备心电图线, 使用永久性的标记在紫色导线上做标记在 ~ 3.2 cm 和 ~ 3.5 cm 从电极的基地和在蓝色导线在 ~ 2.2 cm 和 ~ 2.5 cm (图 2C)。将电极从虎钳上取出, 并通过剥开手术刀刀片 (图 2D), 将导线一侧的绝缘层剥离, 从而将银丝暴露在标记区域之间。 注: 刮线应在显微镜下进行。应谨慎使用, 以确保银丝没有损坏, 因为绝缘被刮掉。</li>
	<li>将电极放回虎钳中。用一层薄薄的强力, 在电极的长度和宽度上贴一条双面安装胶带, 预制到导线的顶端。 注: 在粘胶带之前, 请确保电线是平的, 笔直地贴在两侧, 而不是相互缠绕。</li>
	<li>将用于脑电图的导线修剪成一个略微 V 形的角, 长度约为7-9 毫米, 棕褐色和黑色的线切割最短。不要切断用于心电图的导线 (图 2E)。</li>
	<li>包装和灭菌电极, 以备日后使用。</li>
</ol>2. 为手术准备鼠标
<ol><li>称量鼠标。注射5毫克/千克剂量的卡洛芬皮下 (南卡罗来纳州)。麻醉用腹腔 (ip) 注射含有氯胺酮 (80 毫克/千克)、嗪 (10 毫克/千克) 和乙酰 (1 毫克/千克) 的小鼠麻醉鸡尾酒的动物。</li>
	<li>一旦鼠标变成麻醉, 应用一条细线的兽医眼软膏每只眼睛。使用电动微调器, 剃须两个小区域 (〜2厘米2) 在鼠标的躯干两侧, 对应的心电图线将被植入 (图 3A)。 注意: 右边的剃光区域应该位于动物右侧 "腋窝" 的侧位置。在左侧, 剃须的区域应位于动物一侧的更腹的方向, 但比右侧的剃光区域 (图 3A) 的后侧要多1厘米。</li>
	<li>取下修剪过的头发, 用洗必泰溶液清洗两个剃光区域。</li>
</ol>3. 将电极连接到头骨
<ol><li>在解剖显微镜的舞台上, 将小鼠置于俯卧位置, 并在没有脚趾夹紧反射的情况下确认麻醉深度。 注: 步骤3.2 至5.6 应借助显微镜来完成。</li>
	<li>在拇指和食指之间保持头部稳定, 将皮毛从耳朵中间从耳部到眼睛后面, 再用棉签浸泡在酒精中 (图 3B)。 注意: 虽然这项手术应该做无菌技术, 它不是一个无菌的程序, 因为头皮不能剃光和鼠标必须在手术过程中操作。</li>
	<li>使用手术刀, 使一个〜1厘米中线切口通过头皮之间的分离皮毛之间的只是在前面的耳朵之间的眼睛 (图 3C, D)。<ol>
<li>使用手术刀的一侧或棉签, 轻轻地刮在头骨上的粘液膜, 直到骨头出现干燥。</li>
		<li>在切口周围拔毛, 形成一个薄薄的秃皮边缘。小心地除去任何可能掉进手术场的毛皮, 用一副钳子。用不育的棉头涂抹器将颅骨表面干燥, 如果需要的话, 施加几秒钟的轻柔压力。</li>
	</ol>
</li>
	<li>在头骨上有一个无菌的永久性标记, 在钻孔将钻 (图 3E) 的位置上做四标记。放置两个标记, 一个在矢状缝合的两侧在 bregma 前, 大约4毫米前和5毫米侧向 bregma (在额叶皮层以上), 为参考和地线。另两个标记, 一个在两侧的矢状缝合后 bregma, 大约2毫米后部和7毫米侧向 bregma (高于颞皮层), 为两个脑电图记录线。 注意: 这不是一个立体定向手术, 所给的距离是近似的, 根据鼠标的大小会有所不同。确保将孔放置在足够的侧面, 以方便地容纳电极植入物的底座, 该基座将沿着矢状缝合线 (图 3F) 固定在中线上。</li>
	<li>使用无菌微钻头, 用0.8 毫米直径钻头在每个标记处制作小毛刺孔。<ol>
<li>在钻孔时施加轻柔的压力, 在每个标记点处创建小凹槽。钻过头骨通过脉冲钻头的钻孔接近完成, 确保不施加太大的压力, 这可能会导致穿透和损害的基础脑组织。</li>
		<li>钻完所有孔后, 用棉签将该区域擦拭干净。</li>
	</ol>
</li>
	<li>要将电极固定在头骨的顶部, 请从电极上的双面安装胶带上取下纸背。在磁带上应用一层薄薄的强力。使用一对镊子, 从钳子中取出电极。定位于此, 当沿矢状缝合时, 较短的 EEG 线是侧的, 而长的心电图导线是尾鳍。<ol>
<li>将电极粘在头骨上, 在孔之间的矢状缝合 (图 3F)。 注意: 头骨必须是完全干燥的胶水在电极上坚持。一定不要用电极或胶水咬合头骨上的毛刺孔。</li>
		<li>简要地, 保持电极到位, 以确保与头骨的粘附, 然后允许胶水干燥5-10 分钟。</li>
	</ol>
</li>
</ol>4. 植入心电图的导线
<ol><li>在保持头部直立的同时, 将鼠标稍稍旋转到右侧。将长的心电图线放在左侧, 并将其向下延伸到鼠标左侧的剃光区域。想象一下, 一旦被暴露的电线将被放置在皮肤下面的隧道。 注: 为参考, 一个小的标记可以在皮肤上有一个永久性的标志。</li>
	<li>使用手术刀, 做一个〜1厘米切口在皮肤上的位置, 裸露的电线将被定位。在用 Adson 钳切开切口的同时, 用一根镊钳将切口周围的皮肤从底层结缔组织中松开, 形成一个口袋。从切口部位开始在动物的一侧, 隧道皮下与一块无菌聚乙烯油管 (已准备通过切割到〜6厘米长度与领先的边缘斜面), 直到斜面边缘退出切口上作出的头部 (c0>Figure 4A, B)。</li>
	<li>使用 (图 4C) 将心电图线通过油管送进导管。取出油管时, 用 Adson 钳抓住电极丝, 退出侧向切口。拉紧线 (图 4D)。</li>
	<li>通过将6-0 尼龙 (图 4E) 缝合到皮肤下的组织, 将心电导线固定到位。使用镊子和奥尔森 Hegar 针持有人, 在暴露的部分之前或之后, 在裸露的花丝和另一缝合线上应用一缝合。</li>
	<li>切割的电极线约2-3 毫米过去的最后缝合和塔克结束到口袋的皮肤之前形成。将切口的两侧拉在一起, 并使用克瑞理-木针夹 (图 4F) 应用的伤口夹子缝合。</li>
	<li>转动鼠标, 使鼻子指向相反的方向。头部仍处于直立俯卧位置, 将鼠标稍稍旋转到其左侧。</li>
	<li>重复上述步骤, 将对侧心电导线。 注意: 要近似于导联心电图记录的配置, 右心电线应放置在比左心线稍多的背侧和前部, 应稍多一些腹侧和后方。</li>
</ol>5. 植入脑电图线
<ol><li>为脑电图植入导线, 将小鼠置于俯卧位置, 并用非显性手的拇指和食指将头皮切口打开。</li>
	<li>用钳子, 去除任何可能已被油管拉下皮肤的毛皮。如有必要, 再用棉签擦干颅骨。使用 "钻钳", 小心铲出并清除任何可能在毛刺孔中收集的碎片或血块。</li>
	<li>从一侧最前面的孔开始, 弯曲最靠近该孔的导线, 使其直接位于孔上, 但尚未插入。抓住导线的下端, 并尽可能地将其喂入到孔中, 直到2-3 毫米的导线在头骨下 (图 5A)。 注意: 导线应该水平地位于头骨和大脑的表面之间。电线不应该刺穿大脑。</li>
	<li>随着导线的一端固定在孔中, 轻轻地将导线的剩余部分折叠起来, 使其对着头骨平躺。</li>
	<li>继续以同样的方式与后方的电线在同一侧。对另一侧的前后线重复 (图 5B)。 注意: 线配置在图 5C中进行了总结。</li>
</ol>6. 用牙科用水泥封头切口
<ol><li>将两勺羧酸粉与5滴羧酸液混合。用牙签搅拌混合物, 使糊状物具有所需的粘度。 注: 后续步骤6.2 至6.4 必须快速执行, 因为牙科水泥在搅拌后1分钟内干燥。</li>
	<li>拿起一大滴水泥膏与牙签和应用它周围的底部电极开始尾 (图 6A)。在电极周围继续进行, 使水泥在植入物周围形成一个盖子的导线上滴水 (图 6B)。</li>
	<li>使用平钳, 拉在切口的边缘上的毛皮, 在水泥帽上, 并按在一起, 小心不要扰乱植入的电线下面。将毛皮压入水泥以帮助闭合。</li>
	<li>通过将毛皮与牙科水泥粘合 (图 6C), 在眼睛之间进行切口密封。</li>
</ol>7. 协助术后康复
<ol><li>将鼠标放在循环热垫上的空笼中。监测鼠标, 直到它恢复知觉, 并能保持胸骨卧床。</li>
	<li>手术后, 把老鼠单独放在笼子里, 放在笼子里的食物颗粒和保湿凝胶中。用微隔离器盖上的笼子。</li>
	<li>在 24 h 后手术, 注射 (南卡罗来纳州) 的鼠标与5毫克/千克卡洛芬。</li>
	<li>在记录前允许≥48小时的手术后恢复。</li>
</ol>8. 从栓鼠中记录 EEG-心电信号
<ol><li>恢复后, 将植入的鼠标转移到具有透明壁的记录室, 以方便视频监控。系绳 (即 "插入") 鼠标 (图 7A), 轻轻但牢牢地握住鼠标, 同时使用另一只手插入的10针 (男性) nanoconnector 与引导后, 到插座的脑电图-心电图电极植入 (女性) 在鼠标的头部。</li>
	<li>使用支撑杆固定在房间上方的布线, 确保在导线上有足够的松弛, 允许鼠标自由移动, 但不太多, 电线拖地板的房间。</li>
	<li>将10针 nanoconnector 的布线连接到计算机连接的信号采集接口单元, 并在图 7B中描述同步视频录制。</li>
	<li>将记录的采样率设置为≥2赫用于心电图和≥500赫兹的脑电图 (即, 至少两倍的频率, 你有兴趣学习)。</li>
	<li>为获得最佳的信号跟踪效果, 请按照先前的18: 对所有数据使用60赫兹陷波器、75赫兹低和0.3 赫兹的 EEG 高通频带滤波器, 以及用于心电图的 3 hz 高通滤波器。</li>
	<li>记录同步视频和脑电图-心电图 (图 7C), 并将数字化数据保存到信号处理软件进行离线分析。</li>
	<li>一旦录音完成, 仔细解开鼠标, 并返回到它的家庭笼。</li>
</ol>9. 分析脑电图记录
<ol><li>进行检出量化分析。<ol>
<li>目视检查整个脑电图记录, 以手动识别发作发作, 在这个模型中定义为高振幅 (至少两次基线), 有节律的图放电持续大于5秒 (图 8A)。检查与图癫痫发作相对应的视频, 以确定与癫痫相关的行为。</li>
		<li>要计算癫痫发作频率 (缉获量/小时), 将缉获次数除以记录小时数。</li>
		<li>要计算发作持续时间, 请测量从图发作开始直到停止峰值 (图 8A) 的经过时间。</li>
		<li>计算扣押负担, 定义为每小时占用的时间, 对扣押期间进行求和, 并除以总记录时间。</li>
	</ol>
</li>
	<li>对前、后发作脑电图进行频谱功率分析。<ol>
<li>选择一个30分钟 (或任何其他所需的时间段) 围发作脑电图数据中心周围的癫痫发作将被检查。将原始数据 (已删除过滤器设置) 导出为 ASCII 数据文件或与电源频谱软件兼容的某些其他文件类型。</li>
		<li>使用简单的文本编辑器应用程序将 ASCII 文件转换为文本文件。</li>
		<li>打开功率谱软件中 EEG 段的结果文本文件, 并指定以下设置: "忽略非数字行";"逗号作为数据分隔符";和1000赫兹的缺省采样率。</li>
		<li>一旦 EEG 信号出现在功率谱软件在其各自的通道, 点击频道下拉菜单, 并选择 "数字滤波"。应用与所需频率范围相对应的数字带通滤波器进行分析。</li>
		<li>打开 "频谱视图" 从菜单面板, 选择适当的脑电图显示频道进行分析, 然后单击 "设置"。在 "设置" 下, 为频谱图指定下列参数, 然后单击 "关闭" 以生成频谱谱 (图形 8C): FFT 大小: 8192, 数据窗口: 韦尔奇, 窗口重叠: 93.75%, 显示模式: 功率密度, 频谱图颜色: 彩虹,笑颜色:64、PSD 平均: 1、去除零频率分量: 检查为 "on"。</li>
		<li>根据需要调整色度刻度以实现频谱图的最佳可视化。</li>
		<li>从菜单面板打开 "分析管理器"。点击 "+ 新分析" 创建两个分析 (分析1和分析 2), 这将对应于前和后发作脑电片段进行分析。在频谱图上指定所需的前和后发作段, 并分别与分析1和分析2相关联。 注意: 只有在没有噪声和伪影的情况下, 才应该考虑脑电图数据, 并从分析中删除 eeg 记录与重要工件的周期。</li>
		<li>创建分析段后, 从 "菜单" 面板中打开 "数据键盘视图"。点击适当的脑电图频道, 打开该频道的 "数据键盘设置" 菜单。</li>
		<li>在 "数据垫列设置" 中, 选择 "频谱" 选项, 然后选择 "总功率百分比"。</li>
		<li>在 "数据键盘列设置" 中, 单击 "选项", 并指定要检查的频率范围。在 "频谱数据键盘选项" 和 "数据键盘列设置" 中单击 "确定", 并且指定频带的百分比 (%) 电源将显示在所选分析段 (即分析1或分析 2) 的 "数据键盘" 视图中, 如 "分析经理。 注: 每个波段的功率或相对功率表示为在指定频率范围内的总光谱功率的百分比。</li>
		<li>对每个要分析的频带重复上述步骤。 注: 五主要 EEG 频带的常用范围包括18: δ波段 = 0.5-3 赫兹, 带 = 3.5-7 hz, α波段 = 8-12 hz, β波段 = 13-20 hz, γ波段 = 21-50 hz。</li>
	</ol>
</li>
</ol>10. 分析心电图记录
<ol><li>量化跳过心脏跳动。<ol>
<li>目测检查整个心电图记录, 以手动识别跳过的心跳, 定义为 RR 间隔的延长等于≥1.5 倍以前 r r 间隔, 这通常与非传导性 P 波指示房室传导块 (图 9A)。</li>
		<li>要计算每小时跳过心跳的频率, 请将录制会话期间的跳过节拍总数除以录制时间的总持续时间。</li>
	</ol>
</li>
	<li>执行心率变异性 (HRV) 分析。<ol>
<li>在数据采集软件中, 将记录设置更改为心电图通道的1新纪元。为 ecg 记录生成分析器段: 在12小时的光相位期间每3小时一个5分钟的心电图段, 总共4段。 注: 为分析选择的心电图记录应该是在动物静止和数据没有运动伪影的时候。</li>
		<li>通过单击 "保存已分析的派生数据", 从选定的已分析的心电图段生成 r r 间隔值的电子表格。检查电子表格中是否有丢失的数据或错误数据, 并删除除 r r 间隔数据以外的所有其他数值。将此修改后的电子表格保存为文本文件, 选择 "制表符分隔" 选项。</li>
		<li>在 HRV 软件中打开文本文件作为自定义 ASCII 文件, 指定以下选项: 标题行数: 0, 列分隔符: 制表符/空格, 数据类型: RR, 数据列: 1, 数据单位: 毫秒和时间索引列: 无。</li>
		<li>在菜单的 "首选项" 部分中, 设置下面详细的选项。<ol>
<li>设置分析选项, 如所述。r r 区间 detrending, detrending 方法: smoothn 先验, 平滑参数: 500, 心率变异性频带19, 极低频: 0-0. 15 赫兹, 低频: 0.15-1. 5 hz 和高频: 1.5-5 hz</li>
			<li>设置如上所述的高级设置。频谱估计选项, RR 序列的插值: 20Hz, 频率域中的点: 500 点/Hz, 使用 Welchs 周期方法的 FFT 频谱, 窗口宽度: 32s, 窗口重叠: 50%</li>
		</ol>
</li>
		<li>运行心率变异性分析, 产生时域分析值的平均 rr, STD rr (即, SDNN), RMSSD 和频域分析值的 HF 功率, lf 功率, 和比率的低频/高频功率。如果需要, 请将结果保存为 PDF 文件。</li>
	</ol>
</li>
</ol>

<ol>
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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+for+Seizures+in+Rodents.">Monitoring for Seizures in Rodents.</a> Models of Seizures and Epilepsy. , 97-109 (2017).</li><li>Mishra, V., 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=Scn2a+deletion+improves+survival+and+brain-heart+dynamics+in+the+Kcna1-null+mouse+model+of+sudden+unexpected+death+in+epilepsy+(SUDEP).">Scn2a deletion improves survival and brain-heart dynamics in the Kcna1-null mouse model of sudden unexpected death in epilepsy (SUDEP).</a> Hum. Mol. Genet. 26 (11), 2091-2103 (2017).</li><li>Thireau, J., Zhang, B. L., Poisson, D., Babuty, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+rate+variability+in+mice:+a+theoretical+and+practical+guide.">Heart rate variability in mice: a theoretical and practical guide.</a> Exp. Physiol. 93 (1), 83-94 (2008).</li><li>Smart, S. L., 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=Deletion+of+the+K(V)1.1+potassium+channel+causes+epilepsy+in+mice.">Deletion of the K(V)1.1 potassium channel causes epilepsy in mice.</a> Neuron. 20 (4), 809-819 (1998).</li><li>Glasscock, E., Yoo, J. W., Chen, T. T., Klassen, T. L., Noebels, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kv1.1+potassium+channel+deficiency+reveals+brain-driven+cardiac+dysfunction+as+a+candidate+mechanism+for+sudden+unexplained+death+in+epilepsy.">Kv1.1 potassium channel deficiency reveals brain-driven cardiac dysfunction as a candidate mechanism for sudden unexplained death in epilepsy.</a> J. Neurosci. 30 (15), 5167-5175 (2010).</li><li>Moore, B. M., Jerry Jou, ., Tatalovic, C., Kaufman, M., S, E., Kline, D. D., Kunze, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Kv1.1+null+mouse,+a+model+of+sudden+unexpected+death+in+epilepsy+(SUDEP).">The Kv1.1 null mouse, a model of sudden unexpected death in epilepsy (SUDEP).</a> Epilepsia. 55 (11), 1808-1816 (2014).</li><li>Ryvlin, P., 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=Incidence+and+mechanisms+of+cardiorespiratory+arrests+in+epilepsy+monitoring+units+(MORTEMUS):+a+retrospective+study.">Incidence and mechanisms of cardiorespiratory arrests in epilepsy monitoring units (MORTEMUS): a retrospective study.</a> Lancet Neurol. 12 (10), 966-977 (2013).</li><li>Stables, C. L., Auerbach, D. S., Whitesall, S. E., D'Alecy, L. G., Feldman, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+impact+of+type-1+and+type-2+diabetes+on+control+of+heart+rate+in+mice.">Differential impact of type-1 and type-2 diabetes on control of heart rate in mice.</a> Auton. Neurosci. 194, 17-25 (2016).</li><li>Gehrmann, J., Hammer, P. E., Maguire, C. T., Wakimoto, H., Triedman, J. K., Berul, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+screening+for+heart+rate+variability+in+the+mouse.">Phenotypic screening for heart rate variability in the mouse.</a> Am. J. Physiol. Heart Circ. Physiol. 279 (2), H733-H740 (2000).</li><li>Goldman, A. M., Glasscock, E., Yoo, J., Chen, T. T., Klassen, T. L., Noebels, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arrhythmia+in+heart+and+brain:+KCNQ1+mutations+link+epilepsy+and+sudden+unexplained+death.">Arrhythmia in heart and brain: KCNQ1 mutations link epilepsy and sudden unexplained death.</a> Sci. Transl. Med. 1 (2), 2ra6 (2009).</li></ol>

作者没有什么可透露的。

为了获得高质量的脑电图-心电记录, 没有任何文物, 应采取一切预防措施, 以防止退化或松动的植入电极和电线。当 EEG 头植入物变得松散时, 与大脑的导线接触会降低, 导致信号振幅降低。松散的植入物或不良的导线接触也会导致电信号失真, 将运动工件和背景噪音引入录音。为防止头部植入物可能松动, 在闭合头皮切口的基础上, 在种植体周围应用大量的牙科水泥, 以确保最大的强度和附着力。还应注意确保从颅骨中完全去除毛皮, 因为毛皮残留物可引起术后炎症, 导致种植体周围肿胀和过早植入剥离。随着时间的推移, 头部植入物有可能松动, 由于与反复堵塞和拔掉动物的压力。因此, 如果可能的话, 尝试通过执行单个长时间记录而不是多个短时间记录来最小化动物的插入/拔出次数。另一个潜在的来源后种植损伤和随后的动物损伤是身体接触之间的植入物和 wiretop 在动物的家庭笼。为了消除对 wiretops 的需要, 可以将食物颗粒和保湿凝胶放在笼底上。最后, 为了保持心电图导联的完整性, 应尽量减少对动物的处理, 特别是在心电导线运行的两侧。
除了对植入物或导线接触的降解外, 栓系记录配置的另一个潜在的并发症是, 在导致信号丢失的实验中, 动物被分离 (即, 拔出或脱钩) 的可能性。分离可能是特别麻烦的小鼠, 经历严重抽搐惊厥的运行和弹跳。为了最小化鼠标成为分离的可能性, 优化线缆的松弛量。最好的导线长度通常是一个平衡之间提供足够的松弛动物, 以探索所有角落的笼子, 但没有那么少, 有不必要的张力在电线, 可以促进支队。在确定最佳导线长度时, 确保没有太多的松弛, 鼠标可以很容易地咀嚼电线, 这可能导致信号丢失, 如果电线被打破。使用电极 nanoconnector 植入物的至少10线 (即10针/插座对) 对提供额外的稳定性的拴连接, 也很重要, 因为 nanoconnectors 与少于10线往往更频繁地解开。为了进一步减少动物成为分离的可能性, 这个协议可以很容易地通过连接从鼠标的头部到一个低扭矩换向器悬浮在记录室上方的电线来修改。换向器的工作方式是通过旋转鼠标移动, 以减轻电线中扭转应变的积聚, 从而防止鼠标拔出。
这种连线视频-EEG-心电图协议的主要优点是能够修改其他应用程序的方法。正如这里所描述的, 只有六的可用十电极导线被利用。然而, 其余的四根导线也可以被植入, 作为一个额外的四脑电图导致提供更好的空间分辨率的大脑活动。另外, 两个未使用的导线可以缝合到颈部肌肉, 以记录肌电图 (肌电信号), 这提供了一个衡量肌肉活动, 结合脑电图是重要的确定睡眠/唤醒状态。另一种可能的修改是将动物记录在整个身体的描记室中, 并被修改以适应钢丝绳。在描记中, 与灵感和失效相关的小压力变化转化为呼吸波形。因此, 通过结合描记, 在技术上可以同时记录视频、脑电图、心电图、肌电图和呼吸, 这将代表行为和大脑、心脏、肌肉和肺部活动的读数。这种全面的体内生理记录实际上是不可能的, 在今天的遥测系统中, 使栓的方法在这里描述了一个特别强大的工具, 同时审讯多生理在小鼠。

脑电图 (eeg) 和心电图 (ECG) 是强有力的和广泛使用的技术, 以评估在体内脑和心脏功能, 分别。EEG 是通过将电极连接到头皮1来记录电脑活动。用非侵入性脑电图记录的信号是由皮层锥体神经元产生的发货兴奋和抑制突触后电位引起的电压波动1,2。脑电图是评价和管理癫痫患者的最常见的 neurodiagnostic 测试3,4。这是特别有用的, 当癫痫发作发生时, 没有明显的抽搐行为表现, 如缺勤发作或无抽搐状态持续癫痫症5,6。反之, 非癫痫相关的情况, 导致抽搐发作或意识丧失可能被误诊为癫痫发作没有视频脑电图监测7。除了在癫痫领域的用处之外, EEG 还广泛用于检测与睡眠障碍、脑和记忆障碍有关的异常脑活动, 以及在手术中补充全身麻醉2,8,9。
与脑电图相反, 心电图 (有时简称心电图) 是心脏电活动的记录10。心电图通常是通过在四肢和胸壁上贴上电极来进行的, 这样可以在收缩和松弛的每个心脏周期中检测到心肌产生的电压变化10,11。正常心脏周期的主要心电图波形成分包括 P 波、QRS 复合体和 T 波, 分别对应于心房去极化、心室去极化和心室复极,10, 11. 心电图监测通常用于确定心脏传导系统的心律失常和缺陷12。在癫痫患者中, 使用心电图识别潜在的危及生命的心律失常的重要性被放大了, 因为他们是在明显增加心脏骤停的风险, 以及突然意外死亡的癫痫13, 14,15。
除了临床应用, 脑电图和心电图记录已经成为识别小鼠疾病模型中大脑和心脏功能障碍的不可或缺的工具。尽管传统上这些录音是单独进行的, 我们在这里描述了一种在小鼠中同时记录视频、脑电图和心电图的技术。在这里详细介绍的同步视频脑电图-心电图方法采用栓式记录配置, 在鼠标头上植入的电极硬连接到记录设备。历史上, 这种拴, 或有线, 配置已成为标准和最广泛使用的方法, 脑电图记录小鼠;然而, 无线脑电波遥测系统也在最近得到了发展, 并且越来越受欢迎16。
与无线脑电图系统相比, 栓系安排具有若干技术优势, 可根据所需的应用而使其更可取。这些优势包括更多的频道记录脑电图或其他 biopotentials;降低电极成本;电极一次性;对信号损耗的敏感性较小;和更大的频率带宽 (i. e, 采样率) 的录音17。正确地做, 这里描述的栓录音方法能提供高质量, 无工件的 EEG 和心电图数据同时, 与相应的录影为行为监视。这种脑电图和心电图数据, 然后可以挖掘, 以确定神经, 心脏, 或 neurocardiac 异常, 如癫痫发作, 改变脑电图功率谱, 心脏传导阻滞 (i. e, 跳过心脏跳动), 和心率变异性的变化。为了演示这些 EEG-心电图定量方法的应用, 我们提出了一个使用Kcna1挖空 (-) 鼠标的示例实验。Kcna1--小鼠缺乏电压门控的 Kv1.1 α亚基, 结果显示自发性癫痫发作、心功能不全和过早死亡, 使它们成为同时脑电图-心电图评价有害癫痫相关性的理想模型neurocardiac 功能障碍。

<table>
	<tbody>
		<tr>
			<td>VistaVision stereozoom dissecting microscope</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dolan-Jenner MI-150 microscopy illuminator, with ring light</td>
			<td>VWR</td>
			<td>MI-150RL</td>
			<td></td>
		</tr>
		<tr>
			<td>CS Series scale</td>
			<td>Ohaus</td>
			<td>CS200</td>
			<td>for weighing animal</td>
		</tr>
		<tr>
			<td>T/Pump professional</td>
			<td>Stryker</td>
			<td></td>
			<td>recirculating water heat pad system</td>
		</tr>
		<tr>
			<td>Ideal Micro Drill</td>
			<td>Roboz Surgical Instruments</td>
			<td>RS-6300</td>
			<td></td>
		</tr>
		<tr>
			<td>Ideal Micro Drill Burr Set</td>
			<td>Cell Point Scientific</td>
			<td>60-1000</td>
			<td>only need the 0.8-mm size</td>
		</tr>
		<tr>
			<td>electric trimmer</td>
			<td>Wahl</td>
			<td>9962</td>
			<td>mini clipper</td>
		</tr>
		<tr>
			<td>tabletop vise</td>
			<td>Eclipse Tools</td>
			<td>PD-372</td>
			<td>PD-372 Mini-tabletop suction vise</td>
		</tr>
		<tr>
			<td>fine scissors</td>
			<td>Fine Science Tools</td>
			<td>14058-11</td>
			<td>ToughCut, Straight, Sharp/Sharp, 11.5 cm</td>
		</tr>
		<tr>
			<td>Crile-Wood needle holder</td>
			<td>Fine Science Tools</td>
			<td>12003-15</td>
			<td>Straight, Serrated, 15 cm, with lock - For applying wound clips</td>
		</tr>
		<tr>
			<td>Dumont #7 forceps</td>
			<td>Fine Science Tools</td>
			<td>11297-00</td>
			<td>Standard Tips, Curved, Dumostar, 11.5 cm</td>
		</tr>
		<tr>
			<td>Adson forceps</td>
			<td>Fine Science Tools</td>
			<td>11006-12</td>
			<td>Serrated, Straight, 12 cm</td>
		</tr>
		<tr>
			<td>Olsen-Hegar needle holder with suture cutter</td>
			<td>Fine Science Tools</td>
			<td>12002-12</td>
			<td>Straight, Serrated, 12 cm, with lock</td>
		</tr>
		<tr>
			<td>scalpel handle #3</td>
			<td>Fine Science Tools</td>
			<td>10003-12</td>
			<td></td>
		</tr>
		<tr>
			<td>surgical blades #15</td>
			<td>Havel&#39;s</td>
			<td>FHS15</td>
			<td></td>
		</tr>
		<tr>
			<td>6-0 surgical suture</td>
			<td>Unify</td>
			<td>S-N618R13</td>
			<td>non-absorbable, monofilament, black</td>
		</tr>
		<tr>
			<td>gauze sponges</td>
			<td>Coviden</td>
			<td>2346</td>
			<td>12 ply, 7.6 cm x 7.6 cm</td>
		</tr>
		<tr>
			<td>cotton-tipped swabs</td>
			<td>Constix</td>
			<td>SC-9</td>
			<td>15.2-cm total length</td>
		</tr>
		<tr>
			<td>super glue&#160;</td>
			<td>Loctite</td>
			<td>LOC1364076</td>
			<td>gel control</td>
		</tr>
		<tr>
			<td>Michel wound clips, 7.5mm</td>
			<td>Kent Scientific</td>
			<td>INS700750</td>
			<td></td>
		</tr>
		<tr>
			<td>polycarboxylate dental cement kit</td>
			<td>Prime-dent</td>
			<td>010-036</td>
			<td>Type 1 fine grain</td>
		</tr>
		<tr>
			<td>tuberculin syringe</td>
			<td>BD</td>
			<td>309623</td>
			<td></td>
		</tr>
		<tr>
			<td>polyethylene tubing</td>
			<td>Intramedic</td>
			<td>427431</td>
			<td>PE160, 1.143 mm (ID) x 1.575 mm (OD)</td>
		</tr>
		<tr>
			<td>chlorhexidine&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C9394</td>
			<td></td>
		</tr>
		<tr>
			<td>ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>E7023-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Puralube vet ointment</td>
			<td>Dechra Veterinary Products</td>
			<td></td>
			<td>opthalamic eye ointment</td>
		</tr>
		<tr>
			<td>mouse anesthetic cocktail</td>
			<td></td>
			<td></td>
			<td>Ketamine (80 mg/kg), Xylazine (10 mg/kg), and Acepromazine (1 mg/kg)</td>
		</tr>
		<tr>
			<td>carprofen</td>
			<td></td>
			<td></td>
			<td>Rimadyl (trade name)</td>
		</tr>
		<tr>
			<td>HydroGel</td>
			<td>ClearH20</td>
			<td>70-01-5022</td>
			<td>hydrating gel; 56-g cups</td>
		</tr>
		<tr>
			<td>Ponemah&#160; software</td>
			<td>Data Sciences International</td>
			<td></td>
			<td>data acquisition and analysis software; version 5.2 or greater with Electrocardiogram Module</td>
		</tr>
		<tr>
			<td>7700 Digital Signal conditioner</td>
			<td>Data Sciences International</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>12 Channel Isolated Bio-potential Pod</td>
			<td>Data Sciences International</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>fish tank</td>
			<td>Topfin</td>
			<td></td>
			<td>for use as recording chamber; 20.8 gallon aquarium; 40.8 cm (L) X 21.3 cm (W) X 25.5 cm (H)</td>
		</tr>
		<tr>
			<td>Digital Communication Module (DCOM)</td>
			<td>Data Sciences International</td>
			<td>13-7715-70</td>
			<td></td>
		</tr>
		<tr>
			<td>12 Channel Isolated Bio-potential Pod</td>
			<td>Data Sciences International</td>
			<td>12-7770-BIO12</td>
			<td></td>
		</tr>
		<tr>
			<td>serial link cable</td>
			<td>Data Sciences International</td>
			<td>J03557-20</td>
			<td>connects DCOM to bio-potential pod</td>
		</tr>
		<tr>
			<td>Acquisition Interface (ACQ-7700USB)</td>
			<td>Data Sciences International</td>
			<td>PNM-P3P-7002</td>
			<td></td>
		</tr>
		<tr>
			<td>network video camera</td>
			<td>Axis Communications</td>
			<td></td>
			<td>P1343, day/night capability</td>
		</tr>
		<tr>
			<td>8-Port Gigabit Smart Switch</td>
			<td>Cisco</td>
			<td>SG200-08</td>
			<td>8-port gigabit ethernet swith with 4 power over ethernet supported ports (Cisco Small Business 200 Series)</td>
		</tr>
		<tr>
			<td>10-pin male nanoconnector with guide post hole</td>
			<td>Omnetics</td>
			<td>NPS-10-WD-30.0-C-G</td>
			<td>electrode for implantation on the mouse head</td>
		</tr>
		<tr>
			<td>10-socket female nanoconnector with guide post</td>
			<td>Omnetics</td>
			<td>NSS-10-WD-2.0-C-G</td>
			<td>connector for electrode implant</td>
		</tr>
		<tr>
			<td>1.5-mm female touchproof connector cables</td>
			<td>PlasticsOne</td>
			<td>441</td>
			<td>1 signal, gold-plated; for connecting the wiring from the head-mount implant to the bio-potential pod</td>
		</tr>
		<tr>
			<td>soldering iron</td>
			<td>Weller</td>
			<td>WESD51 BUNDLE</td>
			<td>digital soldering station</td>
		</tr>
		<tr>
			<td>solder</td>
			<td>Bernzomatic</td>
			<td>327797</td>
			<td>lead free, silver bearing, acid flux core solder</td>
		</tr>
		<tr>
			<td>heat shrink tubing</td>
			<td>URBEST</td>
			<td></td>
			<td>collection of tubing with 1.5- to 10-mm internal diameters</td>
		</tr>
		<tr>
			<td>heat gun</td>
			<td>Dewalt</td>
			<td>D26960</td>
			<td></td>
		</tr>
		<tr>
			<td>mounting tape (double-sided)</td>
			<td>3M Scotch</td>
			<td>MMM114</td>
			<td>114/DC Heavy Duty Mounting Tape, 2.54 cm x 1.27 m&#160;</td>
		</tr>
		<tr>
			<td>desktop computer</td>
			<td>Dell</td>
			<td></td>
			<td>recommended minimum requirements: 3rd Gen Intel Core i7-3770 processor with HD4000 graphics; 4 GB RAM, 1 GB AMD Radeon HD 7570 video card; 1 TB hard drive; Windows 7 OS&#160;</td>
		</tr>
		<tr>
			<td>permanent marker</td>
			<td>Sharpie</td>
			<td>37001</td>
			<td>black color, ultra fine point</td>
		</tr>
		<tr>
			<td>toothpicks</td>
			<td></td>
			<td></td>
			<td>for mixing and applying the polycarboxylate dental cement</td>
		</tr>
		<tr>
			<td>LabChart Pro software</td>
			<td>ADInstruments</td>
			<td></td>
			<td>power spectrum software; version 8.1.3 or greater</td>
		</tr>
		<tr>
			<td>Kubios HRV software</td>
			<td>Univ. of Eastern Finland</td>
			<td></td>
			<td>HRV analysis software; version 2.2</td>
		</tr>
		<tr>
			<td>Notepad</td>
			<td>Microsoft</td>
			<td></td>
			<td>simple text editor software</td>
		</tr>
	</tbody>
</table>

simultaneous video-eeg-ecg monitoring to identify neurocardiac dysfunction in mouse models of epilepsy

癫痫发作可以引起心律紊乱, 如心率变化、传导阻滞、asystoles 和心律失常, 这可能增加癫痫猝死的风险 (SUDEP)。脑电图 (eeg) 和心电图 (ECG) 是广泛使用的临床诊断工具, 以监测异常的大脑和心律失常患者。在这里, 一项技术, 同时记录视频, 脑电图和心电图的小鼠, 以测量行为, 大脑和心脏活动, 分别描述。本文所描述的技术采用了栓 (即, 有线) 记录配置, 其中植入的电极头上的鼠标是硬连接到录音设备。与无线遥测记录系统相比, 栓系布置具有多种技术优势, 如可能有更多的记录脑电图或其它 biopotentials 的通道数;降低电极成本;和更大的频率带宽 (即, 采样率) 的录音。这项技术的基础, 也可以很容易地修改, 以适应记录其他生理, 如肌电图 (肌电信号) 或描记评估的肌肉和呼吸活动, 分别。除了描述如何执行脑电图-心电记录, 我们还详细的方法, 以量化的结果数据的癫痫发作, 脑电图频谱功率, 心脏功能和心率变异性, 我们在一个例子实验演示使用鼠标癫痫由于Kcna1基因缺失。在小鼠癫痫或其他神经系统疾病模型中, 视频脑电图-心电图监测提供了一个强有力的工具来识别大脑、心脏或大脑-心脏相互作用的功能障碍。

为了演示如何分析从 eeg-心电图记录的数据, 以确定 neurocardiac 异常, 结果显示为一个24小时脑电图-心电图记录的Kcna1-/-鼠标 (2 月老)。这些突变的动物, 被设计为缺乏电压门控的 Kv1.1 α亚基编码的Kcna1基因, 是一个经常使用的遗传模型的癫痫, 因为他们表现出可靠和频繁的广义补药挛发作活动开始在大约2-3 周的年龄20。除了自发的癫痫发作, Kcna1-/小鼠也表现出与癫痫发作同时出现的过早死亡, 以及发作和癫痫相关的心脏功能障碍21, 22. 因此, Kcna1-/-小鼠也经常被用来研究潜在的病理生理过程中潜藏的突发性意外死亡 (SUDEP), 导致癫痫相关的主要原因死亡率, 据信, 这涉及与癫痫有关的心肺骤停, 截至目前为止, 不太了解的机制23。
在本实验中, 来自Kcna1-/-鼠标的记录的 EEG 组件显示频繁的自发发作, 通常被观察为在癫痫发作时的初始大穗, 紧接着是短暂的电压抑郁症, 转换成高振幅的扣球和终止在突发抑制模式 (图 8A)。使用同时录制的视频, 发现这些图发作与癫痫样行为相吻合, 其特点是饲养和前肢挛, 随后发展为全身强直挛惊厥。值得注意的是, 脑电图的一个主要优点是能够识别 "无声" 的图癫痫发作, 而这并不与明显的行为有关, 这意味着他们将会被一个仅凭行为的观察者评分的癫痫所忽略。在此特定的Kcna1-/-鼠标中的检出率的量化显示在24小时记录期间 (图 8B) 中有15的癫痫发作。这些癫痫发作的持续时间平均为六十年代, 从大约15-105 秒 (图 8B) 不等。为了证明发作期前后的相对谱功率密度分析, 选择了80秒的检出时间, 使用功率谱软件和生成的发作频谱图 (图形 8C) 进行评估。与前发作基线 (图 8D) 相比, 增量频率波段的后发作相对光谱功率增加了50%。另外, 相对于前发作周期 (图 8D), 其他较高频率的 EEG 波段的后发作相关功率呈现相应的下降。后发作三角洲功率的增加和其他波段后发作功率的下降表明, EEG 减慢, 这一模式的长期, 严重的癫痫发作的特点, 在这个模型18。
从Kcna1-/-鼠标分析录制的心电图组件, 发作跳过的心跳次数按上述方式手动计数。跳过心跳的频率在这个Kcna1-/鼠标是 5.84/小时 (表 1), 这是一个 > 5 倍的增长相比, 在我们以前的研究的小鼠 18,21。在Kcna1-/-小鼠的心电图中, 跳过的心跳经常会出现一个 P 波, 它没有跟随 QRS 复合体, 如图 9A所示, 表示房室 (AV) 传导阻滞21。其次, 利用心率变异性软件对心率变异性进行了分析, 为该动物的心功能提供了一项影响自主神经系统的措施。在Kcna1-/-鼠标上计算 HRV 的以下时间域度量值: 节拍节拍间隔 (SDNN) 的标准偏差, 这是一个总自主性变异性的指标;和根平均平方的连续跳动的差异 (RMSSD), 这是一个指数的副交感神经基调。24使用信号采集软件生成的 Kcna1/鼠标 ( 图 9B) 的 r r 间隔值, 心率变异性软件计算出的心率为737次/分钟 (表 1), 这与我们以前的研究18中的小鼠相似。SDNN 和 RMSSD 的值分别计算为2.4 毫秒和3.2 毫秒 (表 1), 其中大约2到3倍, 比正常的鼠标18高。升高的时域心率变异性在这个Kcna1-/-鼠标中显示了增加的副交感神经音, 提示心脏的异常自主控制。接下来, 我们用 hrv 软件计算频率域中 hrv 的下列值, 并在表 1中进行了总结: 低频功率百分比 (LF);高频功率百分比 (高频);和 LF/HF 比。HF 组分被认为反射副交感神经调制, 而 LF 组分被认为反射共鸣和副交感神经的组合影响25。低频/高频比值是用来捕获副交感神经和交感神经活动的相对平衡。
最后, 除了对神经和心功能障碍的定量测量, 还可以定性地分析脑电图和心电图异常之间的时间关系, 以确定潜在的 neurocardiac 功能障碍。, 如前所述21,26。例如, 当癫痫发作或发作样放电被确定在 EEG, 相应的心电图可以检查心脏异常, 如传导阻滞或心律失常, 可能诱发癫痫脑活动。在Kcna1-/-小鼠中, 癫痫发作有时会引起心动过缓或停止, 从而使其致死21,22。在另一种癫痫模型中, Kcnq1突变鼠、传导阻滞和 asystoles 与发作脑电图放电同时发生, 提示它们是病理 neurocardiac 相互作用的结果26。因此, 同时记录脑电图和心电图提供了一个更完整的图像的互动大脑和心脏, 这是特别重要的癫痫, 因为癫痫发作可能引起致命的心脏功能障碍。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57300/57300fig1.jpg"> 图1。手术所需的操作工具.(1)手术刀片 #15;(2)手术刀柄 #3;(3) Adson 钳;(4) Hegar 针夹;(5)精细剪刀;(6) #7 钳;(7)米歇尔伤口夹;(8)克瑞理木针架;(9)微钻头, 0.8 毫米钻头;(10)电动微调。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57300/57300fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57300/57300fig2.jpg"> 图2。准备植入电极.(a)一个10插槽的女性 nanoconnector (即电极) 的例子。(B)在桌面虎钳上的电极, 将植入的导线用于脑电图和心电图折叠。线颜色被表明。其余的电线, 这是指向上, 将被切断。插图显示了从电极上出来的线的放大视图。(C)标记蓝色心电图线, 以指示从何处剥离保温材料。(D)使用手术刀刀片剥去暴露银丝内部的电线绝缘。(E)所准备的电极的最终配置, 显示已修剪的脑电图线和带贴在顶部的已剥离的 ECG 导线。该插页显示了一个放大的看法, 安装磁带和电线出来的电极。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57300/57300fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57300/57300fig3.jpg"> 图3。电极的外科连接到头骨.(a)用于心电图线植入的侧边 (箭头表示) 的鼠标的示例。(B)眼睛和耳朵之间的毛皮分开, 使切口的路径。(C)使用手术刀进行头皮切口。(D)头皮切口。(E)用于指示钻孔位置的头骨上的四标记的示例。(F)钻孔后在颅骨上放置电极。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57300/57300fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57300/57300fig4.jpg"> 图4。心电图导线的隧道和植入.(a)一个聚乙烯管的例子, 它已被切割为约6厘米, 一端为斜面, 便于皮下隧道。(B)隧道与聚乙烯管从侧面切口处开始。(C)通过导管将电极上的心电导线送到头部。(D)卸下管道后拉紧钢丝。(E)对心电导线的绝缘外露部分进行缝合, 使其在所述的组织上保持到位。(F)侧切口用伤口夹闭。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57300/57300fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57300/57300fig5.jpg"> 图5。植入脑电图线(A)抓住红色的脑电图线, 并将其水平地喂入颅骨的毛刺孔, 然后放置黑色的地线。(B)植入后的 nanoconnector 和导线的最终配置。(C)示意图, 显示双侧脑电图和心电图线的位置, 以及参考 (REF) 和地线 (接地) 线。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57300/57300fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57300/57300fig6.jpg"> 图6。关闭头部切口(A)在电极开始尾和进行 rostrally 的基础上使用牙科水泥。(B)在切口最后闭合之前, 在整个 nanoconnector 和导线周围的牙科水泥帽的示例。(C)最后的密封切口的示例。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57300/57300fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/57300/57300fig7.jpg"> 图7。录像脑电图-心电图信号。(a)录制过程中连线鼠标的示例。(B)示意图显示了在体内连线视频-EEG-心电图记录系统的设备配置。从10针男性 nanoconnector 的布线, 插入到女性 nanoconnector 植入头骨, 是焊接到1.5 毫米的女性电缆连接到一个12通道隔离生物电位 pod 接口。然后, 该 pod 由串行链路电缆连接到数字通信模块 (DCOM), 将数字化数据传输到与台式计算机相连的信号采集接口单元 (ACQ), 并使用数据采集软件。视频也可以同时获取使用的网络摄像机定位在笼子的外面和毗邻。摄像头通过以太网电源智能交换机链接到计算机。(C)典型 eeg 和 ECG 信号数据的代表性痕迹, 适用于以下滤镜:60 赫兹缺口、75赫兹低和0.3 赫兹高通频带滤波器用于脑电图;和一个3赫兹高通滤波器的心电图。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57300/57300fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/57300/57300fig8.jpg"> 图8。分析脑电图信号.(a)脑电图示踪, 显示在Kcna1-/ 鼠标中有代表性的自发性癫痫发作。(B)在Kcna1- -鼠标中的24小时录制会话期间观察到的每次扣押的时间段的图。条形对应于平均±标准偏差。(C)周发作频谱图, 显示在代表癫痫发作之前、期间和之后的频率和功率密度。(D)在前和后发作期间, 每个 EEG 频带中相对功率的比较显示相对三角洲功率的增加, 而θ、α、β和伽马功率的下降。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57300/57300fig8large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/57300/57300fig9.jpg"> 图9。心电图信号的分析.(a)从Kcna1-/ 鼠标上显示正常窦性心律的样本心电图示踪在房室传导阻滞前表现为 P 波, 不随 QRS 复合体。P 波, QRS 复合体, r r 间隔标记为参考。(B)一个 r r 间隔序列的有代表性的图, 它由Kcna1/ 鼠标的 ECG 记录获得, 显示节拍之间时间的波动。红线显示了从 r r 区间序列中删除的低频趋势分量 (detrending)。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57300/57300fig9large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td rowspan="4">跳过心脏跳动/小时</td>
			<td colspan="6">心率变异性 (HRV)</td>
		</tr>
<tr>
<td colspan="3">时间域</td>
			<td colspan="3">频域</td>
		</tr>
<tr>
<td>人力资源</td>
			<td>SDNN</td>
			<td>RMSSD</td>
			<td>如果</td>
			<td>高频</td>
			<td rowspan="2">LF/高频比</td>
		</tr>
<tr>
<td>(节拍/分钟)</td>
			<td>女士)</td>
			<td>女士)</td>
			<td>(%)</td>
			<td>(%)</td>
		</tr>
<tr>
<td>5.84</td>
			<td>736。8</td>
			<td>2。4</td>
			<td>3。2</td>
			<td>52.27</td>
			<td>46.38</td>
			<td>1.127</td>
		</tr>
</tbody></table>表 1.量化心跳跳动、心率 (HR) 和心率变异性 (HRV)在 Kcna1-/- 鼠标给出了心率变异性的以下时域测度: 节拍间隔 (SDNN) 的标准差和连续节拍差 (RMSSD) 的根均方根。在频域中, 以下 HRV 测量显示: 低频功率百分比 (lf%); 高频功率百分比 (hf%); 低频功率与高频功率的比值 (低频/高频比)。

Watch this Scientific Journal Video about Simultaneous Video-EEG-ECG Monitoring to Identify Neurocardiac Dysfunction in Mouse Models of Epilepsy at JoVE.com

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

在这里, 我们提出了一个协议来记录大脑和心脏的生物信号, 在小鼠使用同步视频, 脑电图 (eeg) 和心电图 (ECG)。我们还描述的方法来分析产生的脑电图-心电图记录癫痫发作, 脑电图谱功率, 心功能, 心率变异性。

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 ...

simultaneous-video-eeg-ecg-monitoring-to-identify-neurocardiac

Nanyang Technological University

Research

JoVE Journal

Genetics

25.2K 次观看.  Louisiana State University Health Sciences Center. 在这里, 我们提出了一个协议来记录大脑和心脏的生物信号, 在小鼠使用同步视频, 脑电图 (eeg) 和心电图 (ECG)。我们还描述的方法来分析产生的脑电图-心电图记录癫痫发作, 脑电图谱功率, 心功能, 心率变异性。

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

Monitoring Spatial Segregation in Surface Colonizing Microbial Populations

Microbes provide an intriguing system to study social interaction among individuals within a population. The short generation times and relatively simple genetic modification procedures of microbes facilitate the development of the sociomicrobiology field. To assess the fitness of certain microbial species, selected strains or their genetically modified derivatives within one population, can be fluorescently labelled and tracked using microscopy adapted with appropriate fluorescence filters. Expanding colonies of diverse microbial species on agar media can be used to monitor the spatial distribution of cells producing distinctive fluorescent proteins.
Here, we present a detailed protocol for the use of green- and red-fluorescent protein producing bacterial strains to follow spatial arrangement during surface colonization, including flagellum-driven community movement (swarming), exopolysaccharide- and hydrophobin-dependent growth mediated spreading (sliding), and complex colony biofilm formation. Non-domesticated isolates of the Gram-positive bacterium, Bacillus subtilis can be utilized to scrutinize certain surface spreading traits and their effect on two-dimensional distribution on the agar-solidified medium. By altering the number of cells used to initiate colony biofilms, the assortment levels can be varied on a continuous scale. Time-lapse fluorescent microscopy can be used to witness the interaction between different phenotypes and genotypes at a certain assortment level and to determine the relative success of either.

The role of assortment (spatial segregation) in evolutionary scenarios can be examined using simple microbial systems in the laboratory that allow controlled adjustment of spatial distribution. By modifying the founder cell density, various assortment levels can be visualized using fluorescently labelled bacterial strains in the colony biofilms of Bacillus subtilis.

在表面定植微生物种群监测空间分异

Microbes provide an intriguing system to study social interaction among individuals within a population. The short generation times and relatively simple ...

科研

遗传学

Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation of gene functions. Their therapeutic potential in the heart can also be determined. There are limited approaches for in vivo molecular intervention in the mouse heart. Recombinant adeno-associated virus (rAAV)-based genome engineering has been utilized as an essential tool for in vivo cardiac gene manipulation. The specific advantages of this technology include high efficiency, high specificity, low genomic integration rate, minimal immunogenicity, and minimal pathogenicity. Here, a detailed procedure to construct, package, and purify the rAAV9 vectors is described. Subcutaneous injection of rAAV9 into neonatal pups results in robust expression or efficient knockdown of the gene(s) of interest in the mouse heart, but not in the liver and other tissues. Using the cardiac-specific TnnT2 promoter, high expression of GFP gene in the heart was obtained. Additionally, target mRNA was inhibited in the heart when a rAAV9-U6-shRNA was utilized. Working knowledge of rAAV9 technology may be useful for cardiovascular investigations.

In this manuscript, a method to prepare recombinant adeno-associated virus 9 (rAAV9) vectors to manipulate gene expression in the mouse heart is described.

在小鼠心脏rAAV9的制备过表达或敲除基因

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation ...

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the identification and verification of a drug resistance gene in the Apicomplexan parasite Toxoplasma gondii, the method of Quantitative Trait Locus (QTL) mapping using a Whole Genome Sequence (WGS) -based genetic map and the method of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 -based gene editing. The approach of QTL mapping allows one to test if there is a correlation between a genomic region(s) and a phenotype. Two datasets are required to run a QTL scan, a genetic map based on the progeny of a recombinant cross and a quantifiable phenotype assessed in each of the progeny of that cross. These datasets are then formatted to be compatible with R/qtl software that generates a QTL scan to identify significant loci correlated with the phenotype. Although this can greatly narrow the search window of possible candidates, QTLs span regions containing a number of genes from which the causal gene needs to be identified. Having WGS of the progeny was critical to identify the causal drug resistance mutation at the gene level. Once identified, the candidate mutation can be verified by genetic manipulation of drug sensitive parasites. The most facile and efficient method to genetically modify T. gondii is the CRISPR/Cas9 system. This system comprised of just 2 components both encoded on a single plasmid, a single guide RNA (gRNA) containing a 20 bp sequence complementary to the genomic target and the Cas9 endonuclease that generates a double-strand DNA break (DSB) at the target, repair of which allows for insertion or deletion of sequences around the break site. This article provides detailed protocols to use CRISPR/Cas9 based genome editing tools to verify the gene responsible for sinefungin resistance and to construct transgenic parasites.

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

Details are presented on how QTL mapping with a whole genome sequence based genetic map can be used to identify a drug resistance gene in Toxoplasma gondii and how this can be verified with the CRISPR/Cas9 system that efficiently edits a genomic target, in this case the drug resistance gene.

QTL定位和CRISPR / Cas9编辑识别抗药性基因<em&gt;弓形虫</em

Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the ...

RNA Pull-down Procedure to Identify RNA Targets of a Long Non-coding RNA

Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding RNA&#39;s family. Although their biological functions remain largely unknown, the number of these lncRNAs has steadily increased and it is now estimated that humans may have more than 10,000 such transcripts. Some of these are known to be involved in important regulatory pathways of gene expression which take place at the transcriptional level, but also at different steps of RNA co- and post-transcriptional maturation. In the latter cases, RNAs that are targeted by the lncRNA have to be identified. That&#39;s the reason why it is useful to develop a method enabling the identification of RNAs associated directly or indirectly with a lncRNA of interest.
This protocol, which was inspired by previously published protocols allowing the isolation of a lncRNA together with its associated chromatin sequences, was adapted to permit the isolation of associated RNAs. We determined that two steps are critical for the efficiency of this protocol. The first is the design of specific anti-sense DNA oligonucleotide probes able to hybridize to the lncRNA of interest. To this end, the lncRNA secondary structure was predicted by bioinformatics and anti-sense oligonucleotide probes were designed with a strong affinity for regions that display a low probability of internal base pairing. The second crucial step of the procedure relies on the fixative conditions of the tissue or cultured cells that have to preserve the network between all molecular partners. Coupled with high throughput RNA sequencing, this RNA pull-down protocol can provide the whole RNA interactome of a lncRNA of interest.

This RNA pull-down method allows identifying the RNA targets of a long non-coding RNA (lncRNA). Based on the hybridization of home-made, designed anti-sense DNA oligonucleotide probes specific to this lncRNA in an appropriately fixed tissue or cell line, it efficiently allows the capture of all RNA targets of the lncRNA.

rna 下拉法识别长非编码 rna 的 rna 靶点

Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding ...

Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome editing, embryonic stem (ES) cell-based gene-targeting technology does not have these shortcomings and is widely used to modify animal/mouse genome ranging from large deletions/insertions to single nucleotide substitutions. Notably, identifying the relatively few homologous recombination (HR) events necessary to obtain desired ES clones is a key step, which demands accurate and reliable methods. Southern blotting and/or conventional PCR are often utilized for this purpose. Here, we describe the detailed procedures of using those two methods to identify HR events that occurred in mouse ES cells in which the endogenous Myh9 gene is intended to be disrupted and replaced by cDNAs encoding other nonmuscle myosin heavy chain IIs (NMHC IIs). The whole procedure of Southern blotting includes the construction of targeting vector(s), electroporation, drug selection, the expansion and storage of ES cells/clones, the preparation, digestion, and blotting of genomic DNA (gDNA), the hybridization and washing of probe(s), and a final step of autoradiography on the X-ray films. PCR can be performed directly with prepared and diluted gDNA. To obtain ideal results, the probes and restriction enzyme (RE) cutting sites for Southern blotting and the primers for PCR should be carefully planned. Though the execution of Southern blotting is time-consuming and labor-intensive and PCR results have false positives, the correct identification by Southern blotting and the rapid screening by PCR allow the sole or combined application of these methods described in this paper to be widely used and consulted by most labs in the identification of genotypes of ES cells and genetically modified animals.

Here, we present a detailed protocol for identifying homologous recombination events that occurred in mouse embryonic stem cells using Southern blotting and/or PCR. This method is exemplified by the generation of nonmuscle myosin II genetic replacement mouse models using traditional embryonic stem cell-based homologous recombination-mediated targeting technology.

利用南方印迹和聚合酶链反应鉴定小鼠胚胎干细胞的同源重组事件

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

酵母神经退行性蛋白病模型中组蛋白后转化修饰改变的表征

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

Enhanced Yeast One-hybrid Screens To Identify Transcription Factor Binding To Human DNA Sequences

Identifying the sets of transcription factors (TFs) that regulate each human gene is a daunting task that requires integrating numerous experimental and computational approaches. One such method is the yeast one-hybrid (Y1H) assay, in which interactions between TFs and DNA regions are tested in the milieu of the yeast nucleus using reporter genes. Y1H assays involve two components: a &#8216;DNA-bait&#8217; (e.g., promoters, enhancers, silencers, etc.) and a &#8216;TF-prey,&#8217; which can be screened for reporter gene activation. Most published protocols for performing Y1H screens are based on transforming TF-prey libraries or arrays into DNA-bait yeast strains. Here, we describe a pipeline, called enhanced Y1H (eY1H) assays, where TF-DNA interactions are interrogated by mating DNA-bait strains with an arrayed collection of TF-prey strains using a high density array (HDA) robotic platform that allows screening in a 1,536 colony format. This allows for a dramatic increase in throughput (60 DNA-bait sequences against &#62;1,000 TFs takes two weeks per researcher) and reproducibility. We illustrate the different types of expected results by testing human promoter sequences against an array of 1,086 human TFs, as well as examples of issues that can arise during screens and how to troubleshoot them.

Here, we present an enhanced yeast one-hybrid screening protocol to identify the transcription factors (TFs) that can bind to a human DNA region of interest. This method uses a high-throughput screening pipeline that can interrogate the binding of &#62;1,000 TFs in a single experiment.

强化酵母单杂交筛网识别与人 dna 序列结合的转录因子

Identifying the sets of transcription factors (TFs) that regulate each human gene is a daunting task that requires integrating numerous experimental and ...

Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins

Gene regulation plays an important role in all cells. Transcriptional, post-transcriptional (or RNA processing), translational, and post-translational steps are used to regulate specific genes. Sequence-specific nucleic acid-binding proteins target specific sequences to control spatial or temporal gene expression. The binding sites in nucleic acids are typically characterized by mutational analysis. However, numerous proteins of interest have no known binding site for such characterization. Here we describe an approach to identify previously unknown binding sites for RNA-binding proteins. It involves iterative selection and amplification of sequences starting with a randomized sequence pool. Following several rounds of these steps-transcription, binding, and amplification-the enriched sequences are sequenced to identify a preferred binding site(s). Success of this approach is monitored using in vitro binding assays. Subsequently, in vitro and in vivo functional assays can be used to assess the biological relevance of the selected sequences. This approach allows identification and characterization of a previously unknown binding site(s) for any RNA-binding protein for which an assay to separate protein-bound and unbound RNAs exists.

Sequence specificity is critical for gene regulation. Regulatory proteins that recognize specific sequences are important for gene regulation. Defining functional binding sites for such proteins is a challenging biological problem. An iterative approach for identification of a binding site for an RNA-binding protein is described here and is applicable to all RNA-binding proteins.

探索序列空间,识别调节RNA结合蛋白的结合位点

Gene regulation plays an important role in all cells. Transcriptional, post-transcriptional (or RNA processing), translational, and post-translational ...

Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) are a rigorous method for studying mammalian development and disease. Our laboratory has developed several GEMMs of prostate cancer (PCa) that lack expression of one or multiple tumor suppressor genes using the site-specific Cre-loxP recombinase system and a prostate-specific promoter. In this article, we describe our method for necropsy of these PCa GEMMs, primarily focusing on dissection of mouse prostate tumors. New methods developed over the last decade have facilitated the culture of epithelial-derived cells to model organ systems in vitro in three dimensions. We also detail a 3D cell culture method to generate tumor organoids from mouse PCa GEMMs. Pre-clinical cancer research has been dominated by 2D cell culture and cell line-derived or patient-derived xenograft models. These methods lack tumor microenvironment, a limitation of using these techniques in pre-clinical studies. GEMMs are more physiologically-relevant for understanding tumorigenesis and cancer progression. Tumor organoid culture is an in vitro model system that recapitulates tumor architecture and cell lineage characteristics. In addition, 3D cell culture methods allow for growth of normal cells for comparison to tumor cell cultures, rarely possible using 2D cell culture techniques. In combination, use of GEMMs and 3D cell culture in pre-clinical studies has the potential to improve our understanding of cancer biology.&#160;

We show a method for necropsy and dissection of mouse prostate cancer models, focusing on prostate tumor dissection. A step-by-step protocol for generation of mouse prostate tumor organoids is also presented.

从前列腺癌的遗传工程小鼠模型生成肿瘤有机体

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) ...

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic screening using chemical mutagens is a useful strategy for mapping mutant phenotypes to genes among Drosophila and other model organisms that share conserved cellular pathways with humans. If the mutated gene of interest is not lethal in early developmental stages of flies, a climbing assay can be conducted to screen for phenotypic indicators of decreased brain functioning, such as low climbing rates. Subsequently, secondary histological analysis of brain tissue can be performed in order to verify the neuroprotective function of the gene by scoring neurodegeneration phenotypes. Gene mapping strategies include meiotic and deficiency mapping that rely on these same assays can be followed by DNA sequencing to identify possible nucleotide changes in the gene of interest.

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

We present a protocol using a forward genetic approach to screen for mutants exhibiting neurodegeneration in Drosophila melanogaster. It incorporates a climbing assay, histology analysis, gene mapping and DNA sequencing to ultimately identify novel genes related to the process of neuroprotection.

在Vivo前向遗传屏幕识别新的神经保护基因在果蝇黑色素

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic ...