这项工作由中国国家自然科学基金 (81671685) 和上海卫生与计划生育委员会 (201440538 号) 资助。

十五只雄性猎犬 (12 至18月大, 体重约 10.0-12.0 公斤) 被购买并接受实验。所有程序均遵照美国国立卫生研究院出版的《护理和使用实验动物指南》 (85-23 号修订版 1996), 并经复旦大学中山医院动物护理委员会批准。大学.图 1显示了所有协议步骤的示意图工作流。
1. 手术前准备和基线数据收集
<ol><li>剃掉一个后肢的头发, 以便静脉穿刺。用静脉导管 (22 克, 0.9 毫米 x 25 毫米), 通过实验性比格犬小隐静脉的侧支建立静脉通路。通过静脉导管缓慢注射戊巴比妥钠 (30 毫克/千克), 引起麻醉, 这是由于睫毛反射丧失所证实的。在手术中麻醉恢复的情况下, 给予戊巴比妥钠10毫克/千克的剂量。</li>
	<li>用粗绳将四肢固定在操作桌上, 使动物处于仰卧位。</li>
	<li>夹住四肢和胸部的毛发。将肢体铅电极贴在胸壁上六指定位置的肢体四肢和胸铅电极上。记录基线心电图 (ECG)。</li>
	<li>
超声心动图评估
	<ol>
<li>将超声心动图的铅电极粘贴到动物的四肢上。</li>
		<li>进行标准的超声心动图检查。从常规的顶端四室 (A4C) 和顶端两室 (A2C) 视图, 获得 lv 端舒张容积 (左), lv 端收缩容积 (LVESV), 和 lv 弹射分数 (LVEF) 计算使用双辛普森的方法。</li>
		<li>用二维散斑跟踪成像法评价低压纵应变。对 A4C、A2C 和顶端长轴视图 (APLAX) 执行散斑跟踪。</li>
		<li>从上述三顶端的角度, 获得每面墙的基底、中心室和顶端水平的纵应变曲线 (A4C: 隔膜和侧壁;A2C: 前壁和下壁;APLAX: 前隔膜和后壁)。该软件将自动集成这些数据, 以产生一个公牛的眼睛图17段, 包括6段在基底水平 (隔膜, 侧壁, 前壁, 下壁, 前隔膜, 后壁), 6 段在中心室水平 (隔膜, 侧壁, 前壁, 下壁, 前隔膜, 后壁), 4 段在顶端水平 (隔膜, 侧壁, 前壁, 下壁), 和一个顶盖。</li>
		<li>峰值应变时间由 QRS 波开始至应变曲线的最低点的时间间隔, 表明最大纵应变。计算17段的标准差 (PSD), 以评估 LV 机械同步。</li>
		<li>记录 transaortic 多普勒流速在顶端五室视图。测量和平均 aVTI 在3-4 连续节拍。</li>
	</ol>
</li>
	<li>
气管插管与机械通气
	<ol>
<li>轻轻拔出舌头, 保持舌头的延伸, 准备气管插管。将动物定位在 "嗅探" 的位置。</li>
		<li>慢慢地推进喉镜的弯曲的刀片, 直到刀片尖端位于舌头的底部和会厌之间。抬起喉镜向上露出声带。将气管插管插入口腔, 并将导管越过声带。用胶带把管子固定在动物头上。</li>
		<li>听诊两肺, 以确认适当的气管插管放置, 证明了双侧和对称呼吸声音在正压通气期间。</li>
		<li>将气管导管的外端连接至容积式循环呼吸机。使用室内空气启动和维护辅助机械通气。将呼吸频率设置为每分钟8-20 次, 潮汐量为8-15 毫升/千克。根据脉冲氧测量的2设置参数。</li>
	</ol>
</li>
</ol>2. 心外膜 LV 起搏电极植入术
<ol><li>将心脏除颤器/显示器引线连接到与四肢相连的皮肤电极上。用0.3 克左氧氟沙星静脉注射 guttae 对动物进行预用药。</li>
	<li>剃后颈部和胸部的头发, 消毒前胸区和左颈部地区与碘伏和铺消毒片。</li>
	<li>
切口
	<ol>
<li>在右侧压位位置执行肌肉备用开胸手术。经静脉连续输液 (0.01 毫克/千克/小时) 管理芬太尼后, 在第四肋间空间从左胸骨旁线横向切割皮肤。</li>
		<li>在钝性解剖3层胸肌 (胸大, 胸小, 肋), 打开左胸腔在第四肋间空间 (4th和 5th肋骨) 通过尖锐的解剖。将肋骨牵引器放入肋间空间。包不育纱布浸泡在0.9% 氯化钠周围的肺裂片, 以保护肺部和保持一个明确的视野。</li>
		<li>用烙小心切开侧心包。打开心包充分暴露 LV 侧壁与停留缝合 (0 缝合)。</li>
	</ol>
</li>
	<li>
LV 起搏电极植入术
	<ol>
<li>将单极 lv 起搏电极缝合到 lv 侧壁上的心肌上, 用一针4-0 缝合。在缝合上做一个温和的结, 以防止划破心肌组织。</li>
		<li>将起搏导线的端子金属销连接到桥接电缆以测试引线参数。在达到满意的引线参数后, 通过起搏阈值 < 2.0 V 在 0.48 ms 和引线阻抗 < 2000 Ω, 轻微拉电极引线保证牢固的固定。</li>
	</ol>
</li>
	<li>取出留缝线, 仔细检查手术区域, 消除活动出血。</li>
	<li>用 2-0/T 缝线缝合两针的心包。取出填充纱布和肋骨牵引器。</li>
	<li>使用两个 pericostal 缝合 (0 缝合) 近似 4th和 5th肋骨。用 2-0/T 缝线缝合几针肋间筋膜。在最后缝合前通过气管插管, 充分利用辅助气球充气肺部。通过肋确认肺正常扩张。</li>
	<li>将肌肉层重新定位在没有缝合的地方。起搏铅穿透心包、肋间筋膜和肌肉层, 通过手术结之间的间隙先后。</li>
	<li>切开左颈部的皮肤, 解剖皮下的组织, 直到用弯曲钳到达深筋膜。用直钳在胸区的深招牌上建立一个皮下隧道。</li>
	<li>使用直钳将引线的端子销通过隧道拉到左颈部区域。用 2-0/T 缝线将端子销盖上绝缘套筒。将套管周围的铅缝合到筋膜上, 并在颈部左侧嵌入铅。</li>
	<li>用0缝线闭合胸部和颈部切口的皮下组织和皮肤。</li>
	<li>停止麻醉诱导, 当动物正在进行自发性呼吸时, 将气管插管与呼吸机断开。当动物从麻醉中恢复后, 取出气管插管和静脉导管。保持观察动物直到完全恢复。</li>
	<li>术后2周, 每12小时注射肌肉 80万 U 青霉素。</li>
</ol>3. RA 和 RV 起搏电极植入术
<ol><li>植入 RA 和 RV 起搏电极2周后, LV 电极植入, 当动物从开胸恢复。在心脏导管手术室进行手术, 配备透视仪。</li>
	<li>在步骤1.1 诱导麻醉。将四肢固定在手术台上, 并保持仰卧位的动物。用0.3 克左氧氟沙星静脉注射 guttae 对动物进行预用药。</li>
	<li>夹住四肢的毛发。将心电图监测导线与皮肤电极连接, 将皮肤电极粘贴到肢体四肢。打开心电图监控器, 并为程序内监控选择第二主角。</li>
	<li>剃掉颈部的毛发后, 用碘伏消毒左颈部区域, 并将不育的床单下垂。在全过程中静脉注射 (0.01 毫克/千克/小时) 连续输液 (芬太尼)。</li>
	<li>
静脉入路
	<ol>
<li>在颈部左侧的伤口处做一个小的垂直切口。采用钝夹层, 将筋膜分开, 露出左颈外静脉。小心地用蚊子钳把静脉从结缔组织中分离出来。</li>
		<li>用弯曲的钳轻轻地拉起静脉, 在静脉下面通过两 2-0/T 缝线。把远端缝合线系上。</li>
		<li>轻轻抬起远端缝线, 用虹膜剪刀在两条缝线中间切开一个小孔。使用静脉摘出, 插入被动 J 型 RA 铅和主动 RV 铅进入左外颈静脉。</li>
	</ol>
</li>
	<li>
RV 铅植入术
	<ol>
<li>一旦 rv 铅被推进到低右心房或下腔静脉下透视, 退出直接鞘从 RV 铅。在鞘的远端形成 J 形, 再通过 RV 引线插入。</li>
		<li>在弯曲的鞘的帮助下, 引入贯穿三尖瓣和流出道的铅。慢慢地撤回铅和鞘, 允许铅尖下垂向 RV 顶端。</li>
		<li>用直线替换曲线鞘。提前向顶端的铅。</li>
		<li>测试引线参数与鞘撤回约一半。满意的参数包括起搏阈值 < 1.0 V 在0.48 毫秒, R 波振幅 > 5.0 mV, 和铅阻抗 < 2000 Ω。一旦获得可接受的电气参数, 扩展活动螺旋线, 移除鞘, 并重新测量参数。</li>
	</ol>
</li>
	<li>
RA 铅植入术
	<ol>
<li>保持 RA 铅定向向高前心房, 慢慢地撤回直鞘, 允许收缩的预制 J 型铅与其尖端进入附属物。可以观察到具有心房活动的电极的特性来来回运动。</li>
		<li>满意的参数包括起搏阈值 < 1.0V 在0.48 毫秒, P 波振幅 > 2.0 mV, 和铅阻抗 < 2000 Ω。同样, 当获得可接受的参数时, 调整引线松弛并移除鞘。</li>
	</ol>
</li>
	<li>检查两个引线的稳定性后, 拧紧近 venotomy 的缝合线。把两个或三 2-0/T 缝合线围绕缝合袖子的基础深筋膜。在缝合后重新检查两条引线在透视下的电参数和位置。</li>
	<li>使一个脉冲发生器口袋附近的静脉进入和在一个平面上的筋膜层和下方的皮下脂肪。使用弯曲钳, 用钝夹层创建口袋。它应该只是足够大, 以适应发电机和冗余的线索。</li>
	<li>清洁并烘干引线针脚。用绝缘套筒盖住心房引线的端子销, 并将引线缝合到口袋的底部。将心室引线插入起搏器脉冲发生器中, 并将其与远端连接器引脚拧紧, 通过发电机的固定螺钉。</li>
	<li>将发电机放入口袋中, 并将多余的引线卷入设备下。用 2-0/T 缝线将发电机绑在发板上, 通过在发电机头上的栓孔缝合。对整个系统进行透视检查。</li>
	<li>检查止血后, 用 2-0/T 缝线关闭口袋和浅层筋膜。最后用0缝线近似皮肤边缘, 用遥测棒将起搏器编程到蛋模式。</li>
	<li>对于假组的动物, 植入 LV, RV 和 RA 的线索类似的方式, 但没有发电机插入。</li>
</ol>4. LBB 消融术
<ol><li>在 RV 和 RA 铅植入术后立即在透视指导下进行导管消融术。剃去胸部、背部和右腹股沟区域的毛发。使动物处于仰卧位。</li>
	<li>准备一个多通道的电生理记录仪, 用于同时进行表面和心内心电图记录, 过滤器设置为30-400 赫 (双极) 或0.05-500 赫 (单极), 信号放大5000倍。将无绳返回电极连接到背部, 并将标准的12引线电极固定在四肢和胸部。将所有引线连接到电生理记录器, 并以100毫米/秒的扫描速度记录心电图。</li>
	<li>
静脉和动脉方法
	<ol>
<li>在右腹股沟区域进行例行消毒和悬垂后, 通过皮肤垂直切开小切口。采用钝夹层分离筋膜, 识别右股静脉和股动脉。</li>
		<li>轻轻拉上股静脉和放置两个留缝线 (2-0/T 缝合) 的静脉。在远端系上静脉。在股动脉上做同样的动作。</li>
		<li>稍微拾起股静脉, 并在两条缝线之间引入微穿刺针。保持针稳定, 并插入一个灵活的尖端 (软盘 J 形) 导丝通过针。</li>
		<li>当足够的导丝进入静脉时, 取出针并将扩张和鞘组合 (6 法郎) 在导丝到股静脉内。取出导丝和扩张与鞘剩余的导管介绍。用静脉鞘在 venotomy 附近的静脉上系上松散的缝线。</li>
		<li>同样, 插入一个7法郎鞘到股动脉。将100的 U/千克生理盐水稀释肝素送到动脉鞘中, 以防止凝血。</li>
	</ol>
</li>
	<li>
右侧映射他的捆绑潜力
	<ol>
<li>通过静脉鞘推进6法郎导向 quadripolar 导管进入股静脉。通过电缆通过导管输入模块将导管末端连接到多通道电生理记录仪。</li>
		<li>通过导管进入右心房, 并通过三尖瓣, 直到它清楚地在右心室。在右前斜 (饶) 的30°透视视图, 撤回导管横跨三尖瓣孔, 直到心房电位出现并变得更大。轻微的顺时针扭矩有助于保持电极与隔膜接触。当心房和心室电位大小近似相等时, 两者之间出现双相或三相偏转, 代表右侧的束电位。</li>
	</ol>
</li>
	<li>
左束分支电位 (LBP) 映射和烧蚀
	<ol>
<li>通过动脉鞘引入7法郎4毫米的导向消融导管进入股动脉。将消融导管的末端连接到射频发生器和多通道电生理记录仪。</li>
		<li>通过动脉导管 retrogradely 横跨主动脉瓣膜和前进到 LV 在饶的看法。转移导管尖向室隔膜。保持电极与隔膜紧密接触。</li>
		<li>在左前斜 (老挝) 45°透视视图, 慢慢地撤回导管沿隔膜, 直到左侧他的捆绑电位被记录在心房和心室心电图之间, 就在主动脉瓣膜下方。然后慢慢推进导管沿隔膜和操作提示, 以确定一个离散的 LBP, 这是记录在主动脉瓣下, 通常 1-1. 5 厘米低于左侧他的捆绑记录网站。</li>
		<li>当电位-心室心电图间隔比高压间隔短约10毫秒时, 观察 < 1:10 的 A/V 心电图比, 确定了 LBP。通常的 lbp 到最早的心室心电图间隔 (lbp V) 短于20毫秒, 这可能会降低完全 A V 阻滞的风险。</li>
		<li>一旦获得满意的 LBP 位置, 开始导管消融与射频发生器, 提供500赫 unmodulated 正弦波能量 (功率范围 30-40 W)。在电极-组织界面上调整功率以达到60摄氏度的目标温度。如果温度在十五年代内不超过摄氏50摄氏度, 停止能量传递, 调整导管尖端, 然后重新开始。</li>
		<li>在能量应用过程中不断地监测阻抗。在能量传递过程中, 阻抗大于6-8 Ω的下降被认为是良好的组织接触和足够的加热的标志。</li>
		<li>典型 LBBB 的定义是: QRS 持续时间的延长;QRS 阳性在主角 I, II, V5, V6 具缺口 R 波浪和阴性在主角 aVR, V1;和心电图的损失。如果十年代后 QRS 形态学没有变化, 停止能量传递, 调整导管以找到新的 LBP 靶。当一个典型的 LBBB QRS 形态学出现在表面心电图, 继续能量应用 60-90 s 或直到突然上升的阻抗。</li>
		<li>在完全 (3rd度) 房室阻滞或心室颤动 (VF) 的情况下立即停止能量传递。当 VF 发生时, 迅速实施电除颤。</li>
		<li>当 LBBB QRS 形态学达到时, 观察表面心电图稳定期为30分钟。如果正常 QRS 形态学再现, 重复上述消融过程。</li>
	</ol>
</li>
	<li>导管消融术完成后, 取出两个导管。切除静脉和动脉鞘, 并在近端缝合上迅速打结以防止出血。</li>
	<li>经过仔细检查, 以排除主动出血, 关闭筋膜层使用 2-0/T 缝合。最后用0缝线关闭皮肤。</li>
	<li>切断动物的所有电极, 并经常监测动物, 直到完全恢复麻醉。术后1周, 每12小时注射肌肉 80万 U 青霉素。</li>
	<li>LBB 消融不是为假组进行。</li>
</ol>5. 高频感应快速起搏
<ol><li>当动物从手术中恢复后, 再次记录表面心电图, 确认1周后永久 LBBB 存在。然后用遥测棒将起搏器编程到 VVI 模式, 每分钟260次 (bpm)。</li>
	<li>诱导麻醉 (如步骤 1.1) 和程序起搏器到蛋模式后4周快速起搏。</li>
	<li>执行超声心动图评估 LVEF (参照步骤 1.4)。如果 LVEF 低于 35%, 为 CRT 性能准备动物。如果 LVEF 仍然在35% 以上, 提交动物快速 RV 起搏再次通过重新编程起搏器到 VVI 模式在 260 bpm。</li>
	<li>每2周进行一次超声心动图, 直到 LVEF 低于35%。一旦达到 LVEF 目标, 终止快速 RV 起搏, 并准备 CRT 策略。</li>
	<li>假组动物不提交快速起搏。</li>
</ol>6. 心脏再同步治疗的性能
<ol><li>随机将与 HF 的动物分为对照组和 CRT 组。对于 HF 控制组, 让动物再活8周, 无需干预。对于 crt 组, 通过心室起搏启动 crt 性能。</li>
	<li>麻醉诱导后 (如步骤 1.1), 通过将四肢固定在手术台上, 使动物处于仰卧位。用0.3 克左氧氟沙星静脉注射 guttae 对动物进行预用药。剃掉颈部的毛发, 消毒左颈部区域, 并将不育的床单下垂。</li>
	<li>经静脉连续输液 (0.01 毫克/千克/小时) 治疗芬太尼后, 在颈部左侧的前伤口旁做一个小的垂直切口。使用钝解剖, 分开的筋膜, 以隔离脉冲发生器和起搏线索 (包括 RV, LV 和 RA 线索) 没有伤害。</li>
	<li>松开螺钉, 使 RV 引线的端子销脱离发电机头。小心地切开紧固在 LV 和 RA 主角和 uncap 终端别针。在乙醇中浸泡并连续使用干纱布, 清洁所有引线的针脚。</li>
	<li>将 RA、RV 和 LV 插入到 CRT 脉冲发生器的标头中, 并拧紧螺钉。用钝夹层放大口袋, 适合新发电机。把发电机放到口袋里, 用 2-0/T 缝合线把发电机绑在口袋的地板上。</li>
	<li>检查口袋里是否有止血。用 2-0/T 缝线关闭口袋和浅层筋膜。然后用0缝线关闭皮肤。</li>
	<li>将起搏器设置为与房室 (AV) 延迟的 DDD 模式, 其值为70毫秒和室 (), 延迟0毫秒. 检查动物经常, 直到恢复麻醉。注射 80万 U 青霉素肌肉每12小时1周。</li>
	<li>经过8周的 CRT 性能, 再次执行经胸超声心动图对所有组的动物 (如步骤 1.4)。</li>
</ol>7. 祭祀动物和组织学分析
<ol><li>在全身麻醉下, 将动物的四肢固定在手术台上。注射100毫克/千克静脉注射戊巴比妥进行动物牺牲。确保动物死亡的心脏跳动和呼吸运动没有。</li>
	<li>切开左侧宫颈区的皮肤。用尖锐和钝的解剖组合, 释放发电机和引线。松开螺钉, 从发电机上拔下所有引线。</li>
	<li>从皮下组织中逐渐释放出线索, 直到静脉进入点。识别缝合袖子并切开所有的固定缝线。收回房车的主动固定螺旋, 以方便去除。</li>
	<li>从胸骨线到左锁骨中线线, 在第四肋间空间进行横切口。释放 LV 铅从胸筋膜, 沿皮下隧道, 直到左颈部地区, 用一个直钳。</li>
	<li>胸部肌肉钝解剖后, 打开左胸腔。将肋骨牵引器放入肋间空间。完全打开心包。</li>
	<li>切断在心外膜 LV 铅电极上的缝合线。通过切断电极周围的一小块心肌组织, 将电极与心脏分开。</li>
	<li>切开与隔膜一起打开右室。用尖锐的和钝的解剖方法从心肌中分离 RA 和 RV 电极。通常电极提示是由纤维和心肌组织封装。如果需要, 从心脏切开周围的组织。</li>
	<li>通过终端引脚通过一个直鞘到每个引线的尖端。从静脉入口提取 RV 和 RA 引线, 从皮下隧道中取出 LV 铅。如果由于纤维粘连而无法摆脱引线, 则用钝夹层去除导线上的纤维粘连。</li>
	<li>轻轻拉起心脏, 通过靠近主动脉的心脏组织夹住心脏。把心脏放在一个不育的碗里, 用生理盐水冲洗几次。将壁心肌从侧 LV 壁切片进行组织学分析。</li>
	<li>用缓冲的福尔马林固定心肌组织, 然后脱水, 并嵌入石蜡。在切割成5µm 厚的切片后, deparaffinize 的样品和染色与苏木精和伊红 (他) 和马尾三色。</li>
	<li>测量与他染色的纵断面的细胞直径。表达 CVF 的百分比的胶原染色面积除以总组织面积的马尾三色染色部分。为每个节随机选择并计数五个高功率字段 (400x)。拍摄数字照片, 并使用高分辨率的数字图像分析系统进行分析。</li>
</ol>

<ol>
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G., 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=The+effect+of+cardiac+resynchronization+on+morbidity+and+mortality+in+heart+failure.">The effect of cardiac resynchronization on morbidity and mortality in heart failure.</a> N Engl J Med. 352 (15), 1539-1549 (2005).</li><li>Rickard, J., 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=Predictors+of+response+to+cardiac+resynchronization+therapy:+A+systematic+review.">Predictors of response to cardiac resynchronization therapy: A systematic review.</a> Int J Cardiol. 225, 345-352 (2016).</li><li>Ponikowski, 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=2016+ESC+Guidelines+for+the+diagnosis+and+treatment+of+acute+and+chronic+heart+failure:+The+Task+Force+for+the+diagnosis+and+treatment+of+acute+and+chronic+heart+failure+of+the+European+Society+of+Cardiology+(ESC).+Developed+with+the+special+contribution+of+the+Heart+Failure+Association+(HFA)+of+the+ESC.">2016 ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure: The Task Force for the diagnosis and treatment of acute and chronic heart failure of the European Society of Cardiology (ESC). 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作者声明他们没有竞争的财政利益。

扩张型心肌病是充血性心力衰竭的主要病因, 其特点为心室扩张、收缩功能不全、LVEF 减少、舒张期异常 (11)。由于慢性心动过速介导 HF 是公认的临床条件, 快速起搏的心房或心室至少3至4周作为一种常用的动物模型诱发 CHF11。快速起搏后, 血流动力学的变化发生在24小时后, 心脏功能持续恶化长达3至5周。然而, 从起搏诱发的 HF 的恢复是这个模型的戏剧性和独特的...

晚期慢性 HF 是导致各种心血管疾病死亡的主要原因。充血性心力衰竭患者 (CHF) 的一个子集也发展心室传导 discoordination, 加重症状和预后。CRT, 也称为心室起搏, 已被介绍为这些病人的替代治疗20年以上1,2。不幸的是, 大约20-40% 的患者对 CRT 反应不佳。此后, 为了最大限度地提高 CRT 响应3, 已经进行了许多研究。现在人们已经认识到, LBBB 患者比非 LBBB4更能受益于 CRT, 因为 LBBB 模式会导致较大的心脏不同步, 因为间隔和侧壁之间的壁运动自由不对称。.同时, 最近的研究已经开始探索与 CRT5相关的基因表达和分子重塑的变化。伴随着 CRT 诱导的结构反向重构, 细胞和分子恢复到正常水平是非常感兴趣的6。因此, 有必要建立一个独立 LBBB 的 CHF 的最佳模型, 以研究 CRT 的优点。
慢性, 快速心室起搏曾经用来产生 CHF 的犬模型。RV 起搏无疑会产生延迟的 LV 收缩作为 LBBB 收缩模式的模型。然而, 这种类型的功能异步与完整的传导系统可能不会模拟解剖 LBBB, 并没有被认为是一个适当的模型, 研究 CRT 性能, 其本质是协调受损的电激活和心肌收缩。在停止起搏后, 快速恢复 lv 收缩力和部分恢复 lv 尺寸也报告了7。
实验研究通过射频消融诱导慢性 LBBB 建立异步心室收缩8。全球泵功能减少和区域无效机械工作的结合, 可能会导致心力衰竭, 产生心脏低效和心脏重塑在组织, 细胞和分子水平。在 LBBB 的心脏, 工作负荷是最低的隔膜和最高的 LV 侧壁。因此, 心脏重塑最明显的侧壁9。本研究的目的是: (i) 采用快速 RV 起搏与 LBB 消融相结合的方法, 在室和脑室机械异步的情况下, 推进一种稳定、慢性 HF 模型;(ii) 确认 dyssynchronous HF 在我们的模型和 CRT 的好处, 协调收缩的二维散斑跟踪超声心动图和 aVTI;(iii) 初步探讨 CRT 诱发的细胞反向重塑。

<table>
	<tbody>
		<tr>
			<td>Closed iv catheter system (0.9mm&#215;25mm)</td>
			<td>Becton Dickinson Medical</td>
			<td>5264442</td>
			<td>Used as venous retention needle</td>
		</tr>
		<tr>
			<td>Sodium pentobarbital</td>
			<td>Sigma-Aldrich Company</td>
			<td>130205</td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>Pet clipper</td>
			<td>Wuhan Shernbao pet supplies Co., Ltd.</td>
			<td>PGC-660</td>
			<td>For hair shaving</td>
		</tr>
		<tr>
			<td>Electrocardiograph</td>
			<td>Shanghai photoelectric medical electronic instrument Co., Ltd.</td>
			<td>ECG-6511</td>
			<td>For electrocardiogram recording</td>
		</tr>
		<tr>
			<td>Echocardiograph</td>
			<td>GE-Vingmed Ultrasound Company</td>
			<td>VIVID E9</td>
			<td>For echocardiographic assessment</td>
		</tr>
		<tr>
			<td>EchoPAC software</td>
			<td>GE healthcare</td>
			<td>Version201</td>
			<td>Offline analysis</td>
		</tr>
		<tr>
			<td>Laryngoscope</td>
			<td>Shanghai Medical Instrument Co., Ltd</td>
			<td></td>
			<td>Orotracheal intubation</td>
		</tr>
		<tr>
			<td>Endotracheal tube</td>
			<td>SIMS Portex Inc, UK</td>
			<td>274093</td>
			<td>Orotracheal intubation</td>
		</tr>
		<tr>
			<td>Volume cycled respirator</td>
			<td>Newport Corporation</td>
			<td>C100</td>
			<td>Artificial ventilation</td>
		</tr>
		<tr>
			<td>HeartStart XL Defibrillator/Monitor</td>
			<td>Philips Medical Systems</td>
			<td>M4735A</td>
			<td>Electrocardiogram monitor during operation</td>
		</tr>
		<tr>
			<td>Benzalkonium Bromide Tincture</td>
			<td>Shanghai Yunjia Pharmaceutical Co., Ltd.</td>
			<td>H31022694</td>
			<td>Used for skin disinfection</td>
		</tr>
		<tr>
			<td>Rib retractor</td>
			<td>Shanghai Medical Instrument Co., Ltd.</td>
			<td></td>
			<td>For thoracotomy</td>
		</tr>
		<tr>
			<td>4-0 suture</td>
			<td>Shanghai Pudong Jinhuan Medical Products Co., LTD.</td>
			<td>24L1005</td>
			<td>Suture of LV epicardial electrode</td>
		</tr>
		<tr>
			<td>2-0/T suture</td>
			<td>Shanghai Pudong Jinhuan Medical Products Co., LTD.</td>
			<td>11M0505</td>
			<td>Suture of pacing leads, fascia, vessels, etc.</td>
		</tr>
		<tr>
			<td>0-suture</td>
			<td>Shanghai Pudong Jinhuan Medical Products Co., LTD.</td>
			<td>11P0501</td>
			<td>Skin suture</td>
		</tr>
		<tr>
			<td>penicillin powder</td>
			<td>North China Pharmaceutical Co., Ltd.</td>
			<td>F6034105</td>
			<td></td>
		</tr>
		<tr>
			<td>DSA X-ray machine</td>
			<td>Philips</td>
			<td>Allura Xper FD10</td>
			<td>X-ray for fluoroscopy</td>
		</tr>
		<tr>
			<td>LV pacing electrode</td>
			<td>Medtronic, Inc.</td>
			<td>LBT 4965</td>
			<td></td>
		</tr>
		<tr>
			<td>RV pacing electrode</td>
			<td>St. Jude Medical</td>
			<td>Tendril 1888</td>
			<td></td>
		</tr>
		<tr>
			<td>RA pacing electrode</td>
			<td>St. Jude Medical</td>
			<td>IsoFlex 1642T</td>
			<td></td>
		</tr>
		<tr>
			<td>Pacemaker pulse generator</td>
			<td>Medtronic, Inc.</td>
			<td>Enpulse E2DR01</td>
			<td>For rapid RV pacing</td>
		</tr>
		<tr>
			<td>CRT pulse generator</td>
			<td>St. Jude Medical</td>
			<td>Anthem PM 3212</td>
			<td>For CRT performance</td>
		</tr>
		<tr>
			<td>Multi-channel electrophysiologic recorder</td>
			<td>GE Medical Systems</td>
			<td>2003232-004</td>
			<td>For surface and intracardiac electrogram</td>
		</tr>
		<tr>
			<td>Catheter input module</td>
			<td>GE Medical Systems</td>
			<td>301-00202-08</td>
			<td>Multiple pole switches for stimulation or recording</td>
		</tr>
		<tr>
			<td>Radiofrequency generator</td>
			<td>Johnson-Johnson Company</td>
			<td>ST-4460</td>
			<td>For RF current delivery</td>
		</tr>
		<tr>
			<td>Cordless return electrode</td>
			<td>Covidien</td>
			<td>E7509</td>
			<td>For current circuit formation</td>
		</tr>
		<tr>
			<td>Cordis 6-Fr sheath</td>
			<td>Johnson-Johnson Company</td>
			<td>504-606X</td>
			<td>Access for mapping catheter</td>
		</tr>
		<tr>
			<td>Cordis 7-Fr sheath</td>
			<td>Johnson-Johnson Company</td>
			<td>504-607X</td>
			<td>Access for mapping and ablation catheter</td>
		</tr>
		<tr>
			<td>6-Fr quadripolar catheter</td>
			<td>Johnson-Johnson Company</td>
			<td>F6QRA005RT</td>
			<td>Mapping catheter</td>
		</tr>
		<tr>
			<td>7-Fr 4mm-tip steerable ablation catheter</td>
			<td>St. Jude Medical</td>
			<td>402823</td>
			<td>Mapping and ablation catheter</td>
		</tr>
		<tr>
			<td>Prucka Cardio-Lab&#174;2000</td>
			<td>GE Medical Systems</td>
			<td>6.9.00.000</td>
			<td>Software package for electrogram recording</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Haitong Pharmaceutical Co., Ltd</td>
			<td>160505</td>
			<td>Anticoagulant during catheter ablation</td>
		</tr>
		<tr>
			<td>Digital image analysis system</td>
			<td>Leica Microsystems</td>
			<td>Qwin V3</td>
			<td>For histologic analysis</td>
		</tr>
	</tbody>
</table>

benefits of cardiac resynchronization therapy in an asynchronous heart failure model induced by left bundle branch ablation and rapid pacing

现在人们已经认识到, 心力衰竭 (HF) 患者左束支阻滞 (LBBB) 从心脏再同步治疗 (crt) 获得了大量的临床好处, LBBB 已成为 CRT 反应的重要预测因素之一。传统的 tachypacing 诱发 HF 模型有几个主要的局限性, 包括没有稳定的 LBBB 和快速逆转左心室 (LV) 功能障碍停止后起搏。因此, 有必要建立一个具有孤立 LBBB 的慢性 HF 的最佳模型来研究 CRT 的优点。在本研究中, 建立了由左束支 (LBB) 消融和4周快速右心室 (RV) 起搏引起的异步 HF 犬模型。RV 和右心房 (RA) 起搏电极通过颈静脉的方法, 连同心外膜 LV 起搏电极, 被植入的 CRT 性能。本文介绍了射频导管消融、起搏引导植入和快速起搏策略的详细协议。术中还提供心内和表面电图, 以更好地了解 LBB 消融。采用二维散斑跟踪成像和主动脉速度时间积分 (aVTI) 验证了具有 LV 异步和 CRT 优点的慢性稳定 HF 模型。通过协调心室活化和收缩, CRT 制服 lv 机械工作和恢复 lv 泵功能, 其次是逆转的 lv 扩张。此外, 病理学研究显示, 显着恢复的心肌细胞直径和胶原体积分数 (CVF) 后 crt 的表现, 表明组织学和细胞反向重塑诱发的 crt。在本报告中, 我们描述了一种可行的和有效的方法来发展一个慢性异步 HF 模型, 这是适合研究结构和生物逆向重塑后, CRT。

成功的 LBB 消融:
图 2表示导管消融过程中的典型表面和心内心电图。测量的平均 LBP 为18.8 ±2.8 ms, 比基线 h-v 间隔短10毫秒 (28.8 ±2.6 ms, p < 0.01)。在 LBB 消融后, QRS 持续时间从59.2 ±6.8 ms 延长到94.2 ±8.6 ms (p < 0.01)。心电图的丢失证实了成功的 LBB 消融。
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Watch this Scientific Journal Video about Benefits of Cardiac Resynchronization Therapy in an Asynchronous Heart Failure Model Induced by Left Bundle Branch Ablation and Rapid Pacing at JoVE.com

Benefits of Cardiac Resynchronization Therapy in an Asynchronous Heart Failure Model Induced by Left Bundle Branch Ablation and Rapid Pacing

心脏再同步治疗在左束分支消融和快速起搏诱发的异步心力衰竭模型中的益处

采用快速起搏联合左束分支消融法建立慢性心力衰竭 (HF) 模型。应用二维散斑跟踪成像和主动脉速度时间积分验证了该模型的左心室异步性和心脏再同步治疗的益处。

It is now well recognized that heart failure (HF) patients with left bundle branch block (LBBB) derive substantial clinical benefits from cardiac ...

benefits-cardiac-resynchronization-therapy-an-asynchronous-heart

Research

JoVE Journal

Biology

10.4K 次观看.  Zhongshan Hospital, Fudan University. 采用快速起搏联合左束分支消融法建立慢性心力衰竭 (HF) 模型。应用二维散斑跟踪成像和主动脉速度时间积分验证了该模型的左心室异步性和心脏再同步治疗的益处。

视频: Benefits of Cardiac Resynchronization Therapy in an Asynchronous Heart Failure Model Induced by Left Bundle Branch Ablation and Rapid Pacing

Imaging In-Stent Restenosis: An Inexpensive, Reliable, and Rapid Preclinical Model

Preclinical models of restenosis are essential to unravel the pathophysiological processes that lead to in-stent restenosis and to optimize existing and future drug-eluting stents.
A variety of antibodies and transgenic and knockout strains are available in rats. Consequently, a model for in-stent restenosis in the rat would be convenient for pathobiological and pathophysiological studies. 
In this video, we present the full procedure and pit-falls of a rat stent model suitable for high throughput stent research. We will show the surgical procedure of stent deployment, and the assessment of in-stent restenosis using the most elegant technique of OCT (Optical Coherence Tomography). This technique provides high accuracy in assessing plaque CSAs (cross section areas) and correlates well with histological sections, which require special and time consuming embedding and sectioning techniques. OCT imaging further allows longitudinal monitoring of the development of in-stent restenosis within the same animal compared to one-time snapshots using histology.

This video demonstrates how to use a preclinical inexpensive and reliable model to study pathobiological and pathophysiological processes of in-stent restenosis development. Longitudinal in vivo monitoring using OCT (Optical Coherence Tomography) and analysis of OCT images are also demonstrated.

支架内再狭窄的影像：一种廉价的，可靠，快速的临床前模型

Preclinical models of restenosis are essential to unravel the pathophysiological processes that lead to in-stent restenosis and to optimize existing and ...

科研

生物学

An Alternant Method to the Traditional NASA Hindlimb Unloading Model in Mice

The Morey-Holton hindlimb unloading (HU) method is a widely accepted National Aeronautics and Space Administration (NASA) ground-based model for studying disuse-atrophy in rodents 4-6. Our study evaluated an alternant method to the gold-standard Morey-Holton HU tail-traction technique in mice. Fifty-four female mice (4-8 mo.) were HU for 14 days (n=34) or 28 days (n=20). Recovery from HU was assessed after 3 days of normal cage ambulation following HU (n=22). Aged matched mice (n=76) served as weight-bearing controls.
Prior to HU a tail ring was formed with a 2-0 sterile surgical steel wire that was passed through the 5th, 6th, or 7th inter-vertebral disc space and shaped into a ring from which the mice were suspended. Vertebral location for the tail-ring was selected to appropriately balance animal body weight without interfering with defecation.
We determined the success of this novel HU technique by assessing body weight before and after HU, degree of soleus atrophy, and adrenal mass following HU. Body weight of the mice prior to HU (24.3 &plusmn; 2.9g) did not significantly decline immediately after 14d of HU (22.7 &plusmn; 1.9g), 28d of HU (21.3 + 2.1g) or after 3 days recovery (24.0 &plusmn; 1.8g). Soleus muscle mass significantly declined (-39.1%, and -46.6%) following HU for 14 days and 28 days respectively (p&lt;0.001). Following 3 days of recovery soleus mass significantly increased to 74% of control values. Adrenal weights of HU mice were not different compared to control mice.
The success of our novel HU method is evidenced by the maintenance of animal body weight, comparable adrenal gland weights, and soleus atrophy following HU, corresponding to expected literature values 2, 7, 8. The primary advantages of this HU method include: 1) ease of tail examination during suspension; 2) decreased likelihood of cyanotic, inflamed, and/or necrotic tails frequently observed with tail-taping and HU; 3) no possibility of mice chewing the traction tape and coming out of the suspension apparatus; and 4) rapid recovery and normal cage activity immediately after HU.

We developed an alternant hindlimb unloading model in mice. The primary advantage of our hindlimb unloading tail-ring method over the conventional Morey-Holton tail-traction technique is a simple straightforward procedure that minimizes stress upon the animal.

传统美国宇航局后肢卸小鼠模型的一个交替的方法

The Morey-Holton hindlimb unloading (HU) method is a widely accepted National Aeronautics and Space Administration (NASA) ground-based model for studying ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

在鼠标提睾肌为外周干细胞迁移分析的微循环活体显微镜

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

在造血干细胞和祖细胞利用共聚焦和多光子显微镜五荧光蛋白标记的活体克隆跟踪

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and develops most often in the setting of immunosuppression. Despite decades of research, optimal treatment of KS remains poorly defined and clinical outcomes are especially unfavorable in resource-limited settings. KS lesions are driven by pathological angiogenesis, chronic inflammation, and oncogenesis, and various in vitro cell culture models have been developed to study these processes. KS arises from KSHV-infected cells of endothelial origin, so EC-lineage cells provide the most appropriate in vitro surrogates of the spindle cell precursor. However, because EC have a limited in vitro lifespan, and as the oncogenic mechanisms employed by KSHV are less efficient than those of other tumorigenic viruses, it has been difficult to assess the processes of transformation in primary or telomerase-immortalized EC. Therefore, a novel EC-based culture model was developed that readily supports transformation following infection with KSHV. Ectopic expression of the E6 and E7 genes of human papillomavirus type 16 allows for extended culture of age- and passage-matched mock- and KSHV-infected EC and supports the development of a truly transformed (i.e., tumorigenic) phenotype in infected cell cultures. This tractable and highly reproducible model of KS has facilitated the discovery of several essential signaling pathways with high potential for translation into clinical settings.

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is a tumor induced by infection with the oncogenic virus human herpesvirus-8/KS herpesvirus (HHV-8/KSHV). The endothelial cell culture model described here is uniquely suited for studying the mechanisms by which KSHV transforms host cells.

一种用于研究卡波西肉瘤细胞转化的体外模型

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and ...

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Here, we describe the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) for the analysis of the gap junction-dependent shuttling of miRNA. In contrast to commonly applied methods, 3D-FRAP allows for the quantification of the intercellular transfer of small RNAs in real time, with high spatio-temporal resolution.

用3D-FRAP显微镜分析miRNA的间隙连接依赖性转移

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

Induction of Right Ventricular Failure by Pulmonary Artery Constriction and Evaluation of Right Ventricular Function in Mice

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of RVF. Previous rat models imitating RVF progression have been described. Compared with rats, mice are more accessible, economical, and widely used in animal experiments. We developed a pulmonary artery constriction (PAC) approach which is comprised of banding the pulmonary trunk in mice to induce right ventricular (RV) hypertrophy. A special surgical latch needle was designed that allows for easier separation of the aorta and the pulmonary trunk. In our experiments, the use of this fabricated latch needle reduced the risk of arteriorrhexis and improved the surgical success rate to 90%. We used different padding needle diameters to precisely create quantitative constriction, which can induce different degrees of RV hypertrophy. We quantified the degree of constriction by evaluating the blood flow velocity of the PA, which was measured by noninvasive transthoracic echocardiography. RV function was precisely evaluated by right heart catheterization at 8 weeks after surgery. The surgical instruments made inhouse were composed of common materials using a simple process that is easy to master. Therefore, the PAC approach described here is easy to imitate using instruments made in the lab and can be widely used in other labs. This study presents a modified PAC approach that has a higher success rate than other models and an 8-week postsurgery survival rate of 97.8%. This PAC approach provides a useful technique for studying the mechanism of RVF and will enable an increased understanding of RVF.

Here, we provide a useful approach for studying the mechanism of right ventricular failure. A more convenient and efficient approach to pulmonary artery constriction is established using surgical instruments made inhouse. In addition, methods to evaluate the quality of this approach by echocardiography and catheterization are provided.

小鼠肺动脉收缩引起的右心室衰竭及右心室功能评价

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of ...

Analyzing the &alpha;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. Despite the development of multiple differentiation protocols, the generation of iPSC cardiomyocytes resembling an adult-like phenotype remains challenging. One major aspect of cardiomyocytes maturation involves the formation of a well-organized sarcomere network to ensure high contraction capacity. Here, we present a super resolution-based approach for semi-quantitative analysis of the &#945;-actinin network in cardiomyocytes. Using photoactivated localization microscopy a comparison of sarcomere length and z-disc thickness of iPSC-derived cardiomyocytes and cardiac cells isolated from neonatal tissue was performed. At the same time, we demonstrate the importance of proper imaging conditions to obtain reliable data. Our results show that this method is suitable to quantitatively monitor the structural maturity of cardiac cells with high spatial resolution, enabling the detection of even subtle changes of sarcomere organization.

Analyzing the &#945;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The formation of a proper sarcomere network is important for the maturation of iPSC-derived cardiomyocytes. We present a super resolution-based approach that allows for the quantitative evaluation of the structural maturation of stem cell derived cardiomyocytes, to improve culture conditions promoting cardiac development.

利用单分子定位显微镜分析人类 iPSC 衍生肌细胞中的α-Actinin 网络

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. ...

Isolation of Human Primary Valve Cells for In vitro Disease Modeling

Calcific aortic valve disease (CAVD) is present in nearly a third of the elderly population. Thickening, stiffening, and calcification of the aortic valve causes aortic stenosis and contributes to heart failure and stroke. Disease pathogenesis is multifactorial, and stresses such as inflammation, extracellular matrix remodeling, turbulent flow, and mechanical stress and strain contribute to the osteogenic differentiation of valve endothelial and valve interstitial cells. However, the precise initiating factors that drive the osteogenic transition of a healthy cell into a calcifying cell are not fully defined. Further, the only current therapy for CAVD-induced aortic stenosis is aortic valve replacement, whereby the native valve is removed (surgical aortic valve replacement, SAVR) or a fully collapsible replacement valve is inserted via a catheter (transcatheter aortic valve replacement, TAVR). These surgical procedures come at a high cost and with serious risks; thus, identifying novel therapeutic targets for drug discovery is imperative. To that end, the present study develops a workflow where surgically removed tissues from patients and donor cadaver tissues are used to create patient-specific primary lines of valvular cells for in vitro disease modeling. This protocol introduces the utilization of a cold storage solution, commonly utilized in organ transplant, to reduce the damage caused by the often-lengthy procurement time between tissue excision and laboratory processing with the benefit of greatly stabilizing cells of the excised tissue. The results of the present study demonstrate that isolated valve cells retain their proliferative capacity and endothelial and interstitial phenotypes in culture upwards of several days after valve removal from the donor. Using these materials allows for the collection of control and CAVD cells, from which both control and disease cell lines are established.

This protocol describes the collection of human aortic valves extracted during surgical aortic valve replacement procedures or from cadaveric tissue, and the subsequent isolation, expansion, and characterization of patient specific primary valve endothelial and interstitial cells. Included are important details regarding the processes needed to ensure cell viability and phenotype specificity.

分离人原代瓣膜细胞进行体外疾病建模

Calcific aortic valve disease (CAVD) is present in nearly a third of the elderly population. Thickening, stiffening, and calcification of the aortic valve ...

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of the cells. These adjustments are critical to eukaryotic cell survivability and &#39;damage control&#39; during fluctuating nutrient and salinity levels, temperature, and various chemical and radiation stresses. Due to the highly dynamic nature of the RNA-level responses, and the instability of many of the RNA:RNA and RNA:protein intermediates, obtaining a meaningful snapshot of the cytoplasmic RNA state is only possible with a limited number of methods. Transcriptome-wide, RNA-seq-based ribosome profiling-type experiments are among the most informative sources of data for the control of translation. However, absence of a uniform RNA and RNA:protein intermediate stabilization can lead to different biases, particularly in the fast-paced cellular response pathways. In this article, we provide a detailed protocol of rapid fixation applicable to eukaryotic cells of different permeability, to aid in RNA and RNA:protein intermediate stabilization. We further provide examples of isolation of the stabilized RNA:protein complexes based on their co-sedimentation with ribosomal and poly(ribo)somal fractions. The separated stabilized material can be subsequently used as part of ribosome profiling-type experiments, such as in Translation Complex Profile sequencing (TCP-seq) approach and its derivatives. Versatility of the TCP-seq-style methods has now been demonstrated by the applications in a variety of organisms and cell types. The stabilized complexes can also be additionally affinity-purified and imaged using electron microscopy, separated into different poly(ribo)somal fractions and subjected to RNA sequencing, owing to the ease of the crosslink reversal. Therefore, methods based on snap-chilling and formaldehyde fixation, followed by the sedimentation-based or other type of RNA:protein complex enrichment, can be of particular interest in investigating finer details of rapid RNA:protein complex dynamics in live cells.

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

We present a technique to rapidly stabilize translational (protein biosynthesis) complexes with formaldehyde crosslinking in live yeast and mammalian cells. The approach enables dissecting transient intermediates and dynamic RNA:protein interactions. The crosslinked complexes can be used in multiple downstream applications such as in deep sequencing-based profiling methods, microscopy, and mass-spectrometry.

从真核细胞 中快速体内 固定和分离转化复合物

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of ...

心脏再同步治疗在左束分支消融和快速起搏诱发的异步心力衰竭模型中的益处

摘要

探索更多视频

此视频中的章节

支架内再狭窄的影像：一种廉价的，可靠，快速的临床前模型

传统美国宇航局后肢卸小鼠模型的一个交替的方法

在鼠标提睾肌为外周干细胞迁移分析的微循环活体显微镜

在造血干细胞和祖细胞利用共聚焦和多光子显微镜五荧光蛋白标记的活体克隆跟踪

一种用于研究卡波西肉瘤细胞转化的体外模型

用3D-FRAP显微镜分析miRNA的间隙连接依赖性转移

小鼠肺动脉收缩引起的右心室衰竭及右心室功能评价

利用单分子定位显微镜分析人类 iPSC 衍生肌细胞中的α-Actinin 网络

分离人原代瓣膜细胞进行体外疾病建模

从真核细胞中快速体内固定和分离转化复合物

心脏再同步治疗在左束分支消融和快速起搏诱发的异步心力衰竭模型中的益处

摘要

探索更多视频

此视频中的章节

支架内再狭窄的影像：一种廉价的，可靠，快速的临床前模型

传统美国宇航局后肢卸小鼠模型的一个交替的方法

在鼠标提睾肌为外周干细胞迁移分析的微循环活体显微镜

在造血干细胞和祖细胞利用共聚焦和多光子显微镜五荧光蛋白标记的活体克隆跟踪

一种用于研究卡波西肉瘤细胞转化的体外模型

用3D-FRAP显微镜分析miRNA的间隙连接依赖性转移

小鼠肺动脉收缩引起的右心室衰竭及右心室功能评价

利用单分子定位显微镜分析人类 iPSC 衍生肌细胞中的α-Actinin 网络

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