这项工作得到了法国政府 (国家研究机构, 全国情报局) 的支持, 通过 "未来投资" LABEX SIGNALIFE 计划 (参考 ANR-11-LABX-0028-01) 和赠款的 k.d.w. 从协会倒 la 研究癌,法兰西基金会, 计划癌症 Inserm。库珀和影音从研究 Médicale 和尼斯城分别获得了奖学金。心动和换能器由飞利浦公司提供。我们感谢 a. Borderie、s Destree、m. Cutajar-Bossert、Landouar、Martres、Biancardini 和克里希纳瓦格纳的熟练技术援助。

此处所述的实验是按照相关的机构和法国动物福利法、指南和政策进行的。他们已获得法国伦理委员会 (Comit 和 #233; Institutionnel 和 #39; Ethique #39; 动物 de Laboratoire; 数字 NCE/2012-106). 
 1. 超声心动图 <ol> <li> 使用标准的实验室平衡来确定鼠标的体重, 同时将其轻轻地放在尾部以确保正确定位。
	<li> 麻醉通过腹腔 (ip) 注射50毫克/千克戊巴比妥 25 , 26 。
	注意: 如果在整个研究中使用相同的协议, 任何其他类型的麻醉都可以使用。下面将讨论优缺点. </li>
	<li> 将鼠标放回自己的笼子, 等到它没有反应, 它显示稳定的呼吸, 后足反射是缺席。为了测试这一点, 稍微挤压一只脚, 观察腿是否仍然缩回. </li>
	<li> 使用商用的啮齿动物剃须刀将胸腔和左腋窝的左侧剃须. 
	注意: 使用专用的啮齿动物剃须刀可以完全去除细小的小鼠毛, 以避免干涉超声心动图测量。商业脱毛膏或解决方案应避免, 因为它们通常是芳香, 这将扰乱动物后, 它唤醒。避免过度剃须, 因为它会增加热量损失. </li>
	<li> 将熟睡的动物放在一个暖垫上, 设置为 40-42 和 #176; C 在浅的左侧位置, 头部在 12 o 和 #39; 时钟和尾部在 6 o 和 #39; 时钟。用胶带固定左臂、左腿和尾部. </li>
	<li> 应用5ml 超声心动图凝胶到剃须的胸部和传感器的头部. </li>
	<li> 将传感器胸骨放在左侧, 将其定向到颈部的右侧, 以获得 two-dimensional (2D) 胸骨轴在乳突肌水平上的视面。转动传感器90和 #176; 顺时针方向以获得 papilary 肌肉水平的短视图。使用最小深度设置和缩放来最大化图像质量和帧速率。将扫描速度设置为最大值. 
	<ol> <li> 若要获取这些设置, 可以使用不同的缩放和深度设置选项, 具体取决于计算机和软件。在短视图 27 中记录 2 d 引导的 M 模式图像。请参阅 图 1A 和 B 27 . 
		注意: 小心避免对胸部施加过度压力, 因为这可能会导致心动过缓. </li>
	</ol> </li> <li> 记录每种动物至少3系列3心脏跳动的电影循环. 
	注: 对于本研究中使用的心动软件, 按 #34; 获取/保存/#34; 按钮一次。此方法特定于与此特定软件的心动。其他软件包可用于不同的计算机. </li>
	<li> 成功录音后, 从鼠标胸腔、发热垫和换能器中去除超声心动图凝胶。从四肢和尾部取下胶带. </li>
	<li> 将鼠标置于发热垫上, 用纸巾覆盖, 避免不必要的光照和热损耗, 直到它苏醒. </li>
	<li> 将动物放回笼子. </li>
	<li> 分析记录的 M 模式图像从胸骨短视图, 以确定左心室 (LV) 的尺寸和功能。在收缩期和舒张期 (LVAWs 和 LVAWd)、lv 内端收缩和舒张末期直径 (LVIDs 和 LVIDd) 和左心室后壁厚度 (LVPW) 的收缩和舒张期 (LVPWs 和 LVPWd) 中测量 lv 前壁的厚度。在存储的图像上标识组织-血液界面. </li>
	<li> 在 lv 后壁最前收缩期时, 测量表观最大左心室舒张维度和左心室终收缩维度时的舒张维度。点击触摸屏上的 #34; 分析和 #34; 图标, 然后在和 #34; LVAWd 和 #34; 图标。将电子卡尺定位于右心室腔与左前壁的舒张功能. </li>
	<li> 将电子卡尺放置在 lv 前壁和 lv 腔之间的接口上, 以获得 lv 舒张前壁厚度; 该软件将直接切换到 lv 内部舒张末期测量. </li>
	<li> 将卡钳放置在 lv 腔和 lv 后壁的接口上, 以获得 lv 内端舒张直径; 软件将切换到 lv 后壁厚度测量. </li>
	<li> 在 lv 后壁与心包的交界面上放置卡尺, 以获得左心室舒张后壁厚度。对于左心室收缩尺寸, 点击触摸屏上的和 #34; LVAWs 和 #34; 图标和位置在收缩期的右室腔和 lv 前壁的界面上的电子卡尺. </li>
	<li> 将电子卡尺放置在 lv 前壁和 lv 腔之间的接口上, 以获得 lv 收缩前壁厚度。重复上述过程, 为 lv 内端收缩直径和左心室收缩后壁厚度。使用美国超声心动图学会采用的前沿公约跟踪心内膜和心外膜边界 13 , 27 。
	注: LV 收缩功能参数将使用以前的测量自动计算。LV 分数缩短 (fs) 定义为 fs (%) = [(LVIDd-LVIDs)/LVIDd] x 100。LV 弹出分数 (ef) 是用修改后的 Teicholz 公式计算的, 其中 ef (%) = [(LVIDd 3 -LVIDs 3 )/LVIDd 3 ) x 100 12 。请参阅 图 1A 和 B 。
	<li> 将数据存储在光盘或 USB 内存棒上, 并制作备份副本. </li>
	<li> 使用适当的软件导入、分析和导出数据. 
	注意: 在这些基线测量之后, 可以暂停实验。如果对击倒动物和野生型窝进行直接比较, 请进行组织学分析 6 。Cre-ERT2;液氧/液氧鼠标线, 继续第二天与他莫昔芬诱导的 ip 注入, 如描述的 5 , 24 。如果通过结扎左冠状动脉来诱发实验性心肌梗死 5 , 28 , 手术可以直接在超声心动图测量后进行, 当小鼠都在麻醉之下否则, 应维持两轮麻醉之间的最短延迟一周, 以限制术后致死率. </li>
</ol> 2。组织学评价用心脏标本的制备 <ol> <li> 用颈椎脱位处死动物。测量他们的身体重量。用70% 的酒精拭子消毒 #160; 胸部和腹部. </li>
	<li> 在胸骨远端1厘米的皮肤上做一个横切口。使用钝钳, 从胸腔中取出皮肤, 向头部方向移动。用细钳轻轻握住胸骨, 用细剪刀的钝端打开隔膜. </li>
	<li> 将两侧的肋骨固定在胸骨上。向头部方向移动胸骨。在胸腔找到心脏用细钳夹住心脏的血管主干, 然后用细剪刀切割. </li>
	<li> 打开胸腔并切除整个心脏, 测量心脏重量, 并建立一个 heart-to-body 重量比 4 , 5 , 6 , 23 , 29 , 30/我的 </li>
	<li> 将2毫升10% 中性缓冲福尔马林溶液中的心脏固定在15毫升的管子中过夜, 在4和 #176; C. 
	小心: 危险!使用福尔马林溶液的工作必须在化学油烟罩中进行;戴上手套和安全眼镜. 
	注: 由于福尔马林溶液的缓冲成分因不同的供应商而异, 因此在整个研究中使用相同的供应商. </li>
	<li> 第二天, 在横切面上切开心脏, 在中间, 然后将它们转换成石蜡包埋盒, 这是在病理实验室使用自动嵌入装置进行的. </li>
	<li> 执行切片. 
	<ol> <li> 节石蜡块的厚度为3和 #181; m 使用切片, 并将其浮在40和 #176 中; C 水浴含蒸馏水. </li>
		<li> 将稿件转移到幻灯片上。允许幻灯片在37和 #176 中过夜; c 孵化器, 并将它们存储在4和 #176; c 直到准备就绪. 
		注意: 可以在此处暂停协议, 直到用户准备好染色 (步骤 3). </li>
	</ol> </li> <li> Deparaffinize 和水化组织幻灯片. 
	<ol> <li> 将幻灯片放入着色罐中, 并在55和 #176 中放置玻璃插入物; C 烤箱10分钟融化石蜡. </li>
		<li> Deparaffinize 将200毫升二甲苯或二甲苯的两个变化中的幻灯片替换为5分钟. 
		警告: 高度易燃和有毒!在化学油烟罩中工作;戴上手套和安全眼镜. </li>
		<li> 将幻灯片传送到200毫升的100% 酒精。做两个变化3分钟, 并转移一次通过200毫升95% 酒精3分钟. 
		警告: 高度易燃!远离点火源;禁止吸烟. </li>
		<li> 在200毫升磷酸盐缓冲盐水溶液 (PBS) 中冲洗两次, 每次5分钟, 然后继续步骤3、4或 5. </li>
	</ol> </li> </ol> 3。苏木精和曙红染色 <ol> <li> 在蒸馏水中用它们的剖面冲洗幻灯片. </li>
	<li> 用苏木精溶液将细胞核染色8分钟 </li>
	<li> 在自来水中冲洗10分钟. </li>
	<li> 用曙红溶液染色2分钟 </li>
	<li> 脱水三次, 2 分钟, 100% 乙醇 (乙醇)。二甲苯或二甲苯代用品2分钟清除三次。安装在 xylene-based 安装介质中. </li>
	<li> 照片的幻灯片和测量的心肌细胞直径在室隔膜的纵向部分的核心水平。每节至少测量100细胞, 每心脏三节. </li>
</ol> 4。WGA 染色 <ol> <li> 用甲基 (或其他荧光染料) 孵育从步骤2.7 获得的幻灯片--共轭 WGA (PBS 中的 1:100), 在潮湿的室内温度为60分钟. </li>
	<li> 用 PBS 洗涤三次, 每次5分钟. </li>
	<li> 装有带有 DAPI 的荧光安装介质的安装。存储在4和 #176; C 在黑暗中在分析之前. 
	注意: 佩戴防护眼罩和兼容的耐化学手套. </li>
	<li> 拍摄幻灯片. 
	<ol> <li> 使用带有荧光 epi 照明的显微镜和 DAPI 和甲基的滤镜集 (请参阅 材料表 )。将光圈设置为最大值和亮度为曝光. </li>
		<li> 获取蓝色和红色通道的400x 放大倍数的单独图像。打开 ImageJ 中的图像。根据需要调整亮度和对比度 (图像和 #62; 调整和 #62; 亮度/对比度). </li>
		<li> 将每个图像设置为8位 (图像和 #62; 类型和 #62; 8 位)。覆盖 DAPI 和 WGA 图像。使用蓝色通道为 DAPI 和红色渠道为 WGA;将绿色和灰色通道设置为 none (图像和 #62; 颜色和 #62; 合并通道) 31 . </li>
	</ol> </li> <li> 确定室隔膜横截面上的细胞核水平的心肌细胞直径。
	<ol> <li> 定义 ImageJ 中图像的比例。为此, 拍摄已知大小的对象 ( 例如, 与心脏部分相同的放大倍数的例室; 步骤 4.4). </li>
		<li> 使用 straight-line 工具从已知结构的开始到结束处绘制直线 (分析和 #62; 设置刻度). 
		注意: 以像素为单位的距离将自动显示;必须输入已知的距离和长度单位。在细胞核的水平上进行心肌细胞的测量. </li>
		<li> 通过 DAPI 阳性核向 WGA 阳性细胞膜 (分析和 #62; 测量) 的相反位置绘制一条从 WGA 阳性膜的直线。在 "结果" 窗口中, 确保长度值有一个有意义的小数位 (分析和 #62; 设置度量和 #62; 小数位置). </li>
		<li> 每节至少测量100单元格, 每个心脏三节。将结果导出到 Excel (单击结果和 #62; 文件和 #62; 保存为和 #62;. csv) 6 , 31 . </li>
	</ol> </li> </ol> 5。Pecam-1 免疫 <ol> <li> 执行抗原揭露. 
	<ol> <li> 将1600毫升柠檬酸钠缓冲液 (10 毫米柠檬酸钠, 0.05% 吐温 20, pH 6.0) 添加到压力锅中。将压力锅放在板上, 并将其完全通电。在这一点上, 不要保证压力锅的盖子;简单地在上面休息. 
		注意: 热! </li>
		<li> 一旦柠檬酸钠缓冲液沸腾, 将幻灯片从步骤2.5 转移到压力锅。保护压力锅盖。一旦炊具已达到全部压力, 等待7分钟。当7分钟已经过去了, 关闭板和放置高压锅在一个空水槽。启动压力释放阀, 在炊具上运行冷水。一旦它有 de-pressurized, 打开盖子和运行冷水到炊具5分钟, 放置在200毫升 PBS 的幻灯片. 
		注: 另外, 可使用微波抗原揭露, 虽然过热的风险增加. </li>
	</ol> </li> <li> 在甲醇中使用0.3% 过氧化氢来阻止内源性过氧化物酶活性为5分钟. 在200毫升的 PBS 中冲洗三次, 每张2分钟. 
	警告: 易燃和有毒! </li>
	<li> 在稀释的正常阻断血清 (PBS 中5% 正常山羊血清) 中孵育15分钟的幻灯片, 其中也含有素块 (4 滴1毫升). </li>
	<li> 小心地从切片中挖掘液体, 并用家兔的 Pecam-1 抗体孵育它们, 在 PBS 中稀释 1:50, 其中含有2.5% 正常山羊血清和4滴生物素块每毫升。在4和 #176 的潮湿的室内, 在一夜之间孵化幻灯片; C. </li>
	<li> 在 PBS 200 毫升中每5分钟清洗一次幻灯片三次。在室温下, 用化山羊兔 IgG 抗体稀释1:200 的 PBS 中含有2.5% 正常山羊血清1小时。在200毫升 PBS 中每三次洗涤5分钟的幻灯片. </li>
	<li> 孵化 t他的部分与素/biotin-based 过氧化物酶系统20分钟 (试剂 A 和试剂 B 需要结合30分钟之前使用)。在200毫升 PBS 中每三次洗涤5分钟的幻灯片. </li>
	<li> 溶解 1 33 和 #39;-diaminobenzidine (民建联) 和1尿素过氧化氢片剂5毫升的双水。用民建联的溶液孵育约3分钟, 仔细监测颜色的发展。通过在200毫升 PBS 中轻轻地冲洗幻灯片来停止颜色反应. 
	警告: 致癌;穿防化学手套! </li>
	<li> 用苏木精将细胞核 Counterstain 6 分钟。在自来水中冲洗2分钟脱水三次, 2 分钟, 每200毫升100% 酒精。在200毫升的二甲苯或二甲苯替代品中, 每2分钟清除三次。安装在 xylene-based 安装介质中. </li>
	<li> 在每颗心脏的室间隔40x 倍的情况下拍摄幻灯片 (至少十字段), 并使用自由可用的 ImageJ 软件 31 来测量 Pecam-1 区域的密度。使用颜色反褶积 32 插件, 为民建联和苏木精和调整图像对比度相同的水平. 
	注意: 如果褐色和紫色/蓝色颜色有明显的光谱重叠, 可能会导致颜色反褶积困难, 你可以尝试不同品牌的苏木精溶液, 以获得明确的, 蓝色核染色。或者, 可以对毛细血管进行免疫荧光或人工计数. </li>
</ol>

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作者没有什么可透露的。

对小鼠心脏结构和功能进行了不同的评价, 包括超声心动图、造影增强 MRI、微 CT 和 PET 扫描。由于其 cost-effectiveness 和简单, 超声心动图是最广泛使用的技术, 功能分析小鼠11。一般来说, 由于心脏的小尺寸和心率的高频率在小鼠, 传感器以频率和 #62; 10 兆赫应该使用, 虽然成功的测量被报告了与8或9兆赫传感器4,7.由于心功能与体温和心脏...

有大量的转基因小鼠模型可供使用, 而在老鼠体内药理学研究的数量是高1,2。超声心动图和 MRI 是常用的方法的表型表征心脏功能和形态学在这些小鼠模型3。该方案的目的是分析成年小鼠的心功能和形态学。它结合了超声心动图、组织学和免疫组化测量。超声心动图检测在小鼠中广泛应用4,56,78910 12。Pachon et al.11已确定在循环中发布的205项研究,循环研究,美国生理学杂志-心脏和循环系统生理学, 和心血管研究之间的2012和2015用超声心动图检查动物。
超声心动图用于鉴别基因修饰小鼠心脏表型5,6,13,1415,1617,18,19,20,21,22, 以及分析小鼠慢性超负荷诱发肥大、心肌缺血和心肌病模型的心功能 (在12中进行了回顾)。改进的超声心动图设备允许对左心室收缩和舒张维度、组织多普勒成像、心肌对比声像的标准测量, 以及对 lv 区域功能和冠脉储备的评价12. 理想情况下, 应在有意识的小鼠中进行超声心动图检查, 以避免麻醉对收缩功能、自主反射控制和心率的负面影响11。然而, 这种方法受训练动物的要求的限制;保持体温稳定的困难;运动工件;应力;非常高的心脏频率;并且要求至少两名调查员进行实验, 特别是在大量动物被调查的情况下。有趣的是, 最近的一项研究报告在经过训练和未经训练的动物中, 超声心动图参数没有差异19。我们在麻醉小鼠身上进行超声心动图测量。下面将讨论不同的麻醉方案。
虽然标准分辨率超声心动图 (和 #62; 10 兆赫) 是足够的测量左心室收缩和舒张功能的成年小鼠, 该方法是有限的, 其基本结构现象的描述。因此, 我们结合的在体内测量与组织学和免疫的分析, 以测量, 例如, 心肌细胞直径和血管密度。其他组织学和免疫的研究, 如测定增殖, 细胞凋亡的检测, 梗死面积的测量, 纤维化的测定, 和特定的标记表达, 也可以进行相同类型的加工过的组织, 但不是本议定书的主题。体内超声心动图与组织学分析相结合, 为基础结构改变提供了更多的见解。另外一步, 我们可以通过分子和微的调查来完成这些测量。组织学分析不仅完成了超声心动图检查, 而且在超声心动描记术的分辨率不足时也成为必不可少的。这是特别的情况下, 在模型的转基因小鼠是胚胎致命的23,24。

<table><tbody><tr><td>Wheat germ agglutinin (WGA) conjugated tetramethylrhodamine</td><td>Life Technologies, Molecular Probes</td><td>W849</td><td></td></tr><tr><td>Biotinylated Goat Anti-Rabbit IgG Antibody</td><td>Vectorlabs</td><td>BA-1000</td><td></td></tr><tr><td>Avidin/Biotin Blocking Kit</td><td>Vectorlabs</td><td>SP-2001</td><td></td></tr><tr><td>VECTASTAIN Elite ABC HRP Kit (Peroxidase, Standard)</td><td>Vectorlabs</td><td>PK-6100</td><td></td></tr><tr><td>VECTASHIELD Antifade Mounting Medium with DAPI</td><td>Vectorlabs</td><td>H-1200</td><td></td></tr><tr><td>SIGMAFAST 3,3&#039;-Diaminobenzidine tablets</td><td>Sigma</td><td>D4168</td><td></td></tr><tr><td>Hydrogen peroxide solution</td><td>Sigma</td><td>H1009</td><td></td></tr><tr><td>Anti-Pecam-1 (CD31) antibody</td><td>Abcam</td><td>ab28364</td><td></td></tr><tr><td>Ultrasound transmission gel, Gel Aquasonic 100</td><td>Parker</td><td></td><td></td></tr><tr><td>Linear ultrasound probe, L15-7io</td><td>Philips Healthcare</td><td></td><td></td></tr><tr><td>Echocardiograph, IE33 xMATRIX</td><td>Philips Healthcare</td><td></td><td></td></tr><tr><td>Microscope, Leica DMi8</td><td>Leica</td><td></td><td></td></tr><tr><td>Fluorescence Filterset DAPI</td><td>Leica</td><td>11525304</td><td></td></tr><tr><td>Filterset TxR</td><td>Leica</td><td>11525310</td><td></td></tr><tr><td>Digital Camera, SPOT RT3 Color Slider</td><td>Spot Imaging</td><td></td><td></td></tr><tr><td>Imaging Software, SPOT 5.2 Advanced and Basic Software</td><td>Spot Imaging</td><td></td><td></td></tr><tr><td>Imaging Computer</td><td>Dell</td><td></td><td></td></tr><tr><td>Fine Scissors</td><td>Fine Science Tools</td><td>14028-10</td><td></td></tr><tr><td>Large Scissors</td><td>Fine Science Tools</td><td>14501-14</td><td></td></tr><tr><td>Scalpel blades</td><td>Fine Science Tools</td><td>10023-00</td><td></td></tr><tr><td>Graefe Forceps</td><td>Fine Science Tools</td><td>11650-10</td><td></td></tr><tr><td>Rodent shaver</td><td>Harvard Apparatus</td><td>34-0243</td><td></td></tr><tr><td>cassettes for paraffin embedding</td><td>Sakura</td><td>4155F</td><td></td></tr><tr><td>neutral buffered Formalin</td><td>Sakura</td><td>8727</td><td></td></tr><tr><td>Xylene</td><td>Sakura</td><td>8733</td><td></td></tr><tr><td>Paraffine TEK III</td><td>Sakura</td><td>4511</td><td></td></tr><tr><td>automated embedding apparatus, Tissue-Tek VIP</td><td>Sakura</td><td>6032</td><td></td></tr><tr><td>paraffin-embedding station Tissue-Tek TEC 5</td><td>Sakura</td><td>5229</td><td></td></tr><tr><td>microtome blades,Accu-Edge S35</td><td>Sakura</td><td>4685</td><td></td></tr><tr><td>microscopy slides, Tissue-Tek</td><td>Sakura</td><td>9533</td><td></td></tr><tr><td>cover slips, Tissue-Tek</td><td>Sakura</td><td>9582</td><td></td></tr><tr><td>Mounting medium Tissue-Tek</td><td>Sakura</td><td>1408</td><td></td></tr><tr><td>slide boxes</td><td>Sakura</td><td>3958</td><td></td></tr><tr><td>eosine solution</td><td>Sakura</td><td>8703</td><td></td></tr><tr><td>hematoxyline solution</td><td>Sakura</td><td>8711</td><td></td></tr><tr><td>microtome, RM2125RT</td><td>Leica</td><td>720-1880 (VWR)</td><td></td></tr><tr><td>water bath, Leica HI1210</td><td>Leica</td><td>720-0113(VWR)</td><td></td></tr><tr><td>Ethanol</td><td>VWR</td><td>ACRO444220050</td><td></td></tr><tr><td>15 ml tubes</td><td>VWR</td><td>734-0451</td><td></td></tr><tr><td>staining glass dish</td><td>VWR</td><td>MARI4220004</td><td></td></tr><tr><td>staining jars</td><td>VWR</td><td>MARI4200005</td><td></td></tr><tr><td>Incubator</td><td>Binder</td><td>9010-0012</td><td></td></tr><tr><td>DAB and urea hydrogen peroxide tablets, SIGMAFAST 3,3&amp;prime;-Diaminobenzidine tablets</td><td>Sigma</td><td>D4293</td><td></td></tr><tr><td>PBS (10X)</td><td>Thermo Fisher Scientific</td><td>70011044</td><td></td></tr></tbody></table>

echocardiographic and histological examination of cardiac morphology in the mouse

近年来, 越来越多的基因改良的小鼠模型已成为可用。此外, 在小鼠体内进行药理学研究的数量也很高。这些小鼠模型的表型特征也需要检查心脏功能和形态学。超声心动图和磁共振成像 (MRI) 是常用的方法来表征心脏功能和形态学的小鼠。专门用于小型啮齿动物的超声心动图和 MRI 设备昂贵, 需要专用空间。该协议描述了使用15兆赫人体血管探针的临床超声心动图系统对小鼠的心脏测量。对麻醉成年小鼠进行了测量。在胸骨短视图中, 每种动物在 M 模式下记录和分析至少三图像序列。术后进行心脏组织学检查, 在苏木素-曙红-或麦胚凝集素 (WGA) 染色石蜡切片上测定心肌细胞直径。Pecam-1 免疫后, morphometrically 测定血管密度。该协议已成功应用于基线条件下的药理学研究和不同的遗传动物模型, 以及经永久性结扎左前降冠状动脉后的实验性心肌梗死后 (小伙子)。根据我们的经验, 超声心动图的研究仅限于麻醉动物, 在体重至少25克的成年小鼠中是可行的。

在图 1中, 有代表性的超声心动图显示了超声心动描记对转基因小鼠心脏表型的鉴别作用。有正常心功能的小鼠 (图 1A) 和左心室扩张的动物和减少的 LV 功能 (图 1B) 之间的区别可以很容易地被识别出来。图 2显示了在没有 (图 2A) 的动物中进行心肌细胞直径测量的...

Watch this Scientific Journal Video about Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse at JoVE.com

Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse

超声心动图检查常用于小鼠。为此, 开发了昂贵的高分辨率超声波装置。该协议描述了一个负担得起的超声心动图方法结合组织学形态学分析, 以确定心脏形态学。

超声心动图与小鼠心脏形态学的组织学检查

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed ...

echocardiographic-histological-examination-cardiac-morphology

Research

JoVE Journal

Medicine

21.4K 次观看.  CNRS, INSERM, iBV. 超声心动图检查常用于小鼠。为此, 开发了昂贵的高分辨率超声波装置。该协议描述了一个负担得起的超声心动图方法结合组织学形态学分析, 以确定心脏形态学。

视频: Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse

Transthoracic Echocardiography in Mice

In recent years, murine models have become the primary avenue for studying the molecular mechanisms of cardiac dysfunction resulting from changes in gene expression. Transgenic and gene targeting methods can be used to generate mice with altered cardiac size and function,1-3 and as a result, in vivo techniques are needed to evaluate their cardiac phenotype. Transthoracic echocardiography, pulse wave Doppler (PWD), and tissue Doppler imaging (TDI) can be used to provide dimensional measurements of the mouse heart and to quantify the degree of cardiac systolic and diastolic performance. Two-dimensional imaging is used to detect abnormal anatomy or movements of the left ventricle, whereas M-mode echo is used for quantification of cardiac dimensions and contractility.4,5 In addition, PWD is used to quantify localized velocity of turbulent flow,6 whereas TDI is used to measure the velocity of myocardial motion.7 Thus, transthoracic echocardiography offers a comprehensive method for the noninvasive evaluation of cardiac function in mice.

Transthoracic echocardiography offers a noninvasive method for the evaluation of cardiac function in mice. A combination of ultrasound and Doppler imaging modalities can be used to obtain dimensional measurements of the heart and intracardiac blood flow, which together provide an assessment of cardiac systolic and diastolic performance.

经胸超声心动图在小鼠

In recent years, murine models have become the primary avenue for studying the molecular mechanisms of cardiac dysfunction resulting from changes in gene ...

科研

医学

Murine Echocardiography and Ultrasound Imaging

Rodent models of cardiac pathophysiology represent a valuable research tool to investigate mechanism of disease as well as test new therapeutics.1 Echocardiography provides a powerful, non-invasive tool to serially assess cardiac morphometry and function in a living animal.2 However, using this technique on mice poses unique challenges owing to the small size and rapid heart rate of these animals.3 Until recently, few ultrasound systems were capable of performing quality echocardiography on mice, and those generally lacked the image resolution and frame rate necessary to obtain truly quantitative measurements. Newly released systems such as the VisualSonics Vevo2100 provide new tools for researchers to carefully and non-invasively investigate cardiac function in mice. This system generates high resolution images and provides analysis capabilities similar to those used with human patients. Although color Doppler has been available for over 30 years in humans, this valuable technology has only recently been possible in rodent ultrasound.4,5 Color Doppler has broad applications for echocardiography, including the ability to quickly assess flow directionality in vessels and through valves, and to rapidly identify valve regurgitation. Strain analysis is a critical advance that is utilized to quantitatively measure regional myocardial function.6 This technique has the potential to detect changes in pathology, or resolution of pathology, earlier than conventional techniques. Coupled with the addition of three-dimensional image reconstruction, volumetric assessment of whole-organs is possible, including visualization and assessment of cardiac and vascular structures. Murine-compatible contrast imaging can also allow for volumetric measurements and tissue perfusion assessment.

This video demonstrates use of a rail-mounted high-frequency ultrasound probe to perform echocardiography on an anesthetized mouse. The methods describe both conventional two-dimensional and M-mode measurements of cardiac function in addition to newer, more powerful tools such as color Doppler, strain analysis, as well as general and targeted contrast imaging.

小鼠超声心动图和超声成像

Rodent models of cardiac pathophysiology represent a valuable research tool to investigate mechanism of disease as well as test new therapeutics.1  ...

Echocardiographic Assessment of the Right Heart in Mice

Transgenic and toxic models of pulmonary arterial hypertension (PAH) are widely used to study the pathophysiology of PAH and to investigate potential therapies. Given the expense and time involved in creating animal models of disease, it is critical that researchers have tools to accurately assess phenotypic expression of disease. Right ventricular dysfunction is the major manifestation of pulmonary hypertension. Echocardiography is the mainstay of the noninvasive assessment of right ventricular function in rodent models and has the advantage of clear translation to humans in whom the same tool is used. Published echocardiography protocols in murine models of PAH are lacking.
In this article, we describe a protocol for assessing RV and pulmonary vascular function in a mouse model of PAH with a dominant negative BMPRII mutation; however, this protocol is applicable to any diseases affecting the pulmonary vasculature or right heart. We provide a detailed description of animal preparation, image acquisition and hemodynamic calculation of stroke volume, cardiac output and an estimate of pulmonary artery pressure.

This article provides a protocol for the echocardiographic assessment of right ventricular size and pulmonary hypertension in mice. Applications include phenotype determination and serial assessment in transgenic and toxin-induced mouse models of cardiomyopathy and pulmonary vascular disease.

右心小鼠的超声心动图评价

Transgenic and toxic models of pulmonary arterial hypertension (PAH) are widely used to study the pathophysiology of PAH and to investigate potential ...

Analysis of Congenital Heart Defects in Mouse Embryos Using Qualitative and Quantitative Histological Methods

Congenital heart defects (CHD) are the most common type of birth defect in humans, affecting up to 1% of all live births. However, the underlying causes for CHD are still poorly understood. The developing mouse constitutes a valuable model for the study of CHD, because cardiac developmental programs between mice and humans are highly conserved. The protocol describes in detail how to produce mouse embryos of the desired gestational stage, methods to isolate and preserve the heart for downstream processing, quantitative methods to identify common types of CHD by histology (e.g., ventricular septal defects, atrial septal defects, patent ductus arteriosus), and quantitative histomorphometry methods to measure common muscular compaction phenotypes. These methods articulate all the steps involved in sample preparation, collection, and analysis, allowing scientists to correctly and reproducibly measure CHD.

In this protocol, we describe procedures to qualitatively and quantitatively analyze developmental phenotypes in mice associated with congenital heart defects.

使用定性和定量组织方法对小鼠胚胎先天性心脏缺陷的分析

Congenital heart defects (CHD) are the most common type of birth defect in humans, affecting up to 1% of all live births. However, the underlying causes ...

发育生物学

A Pipeline to Characterize Structural Heart Defects in the Fetal Mouse

Congenital heart diseases (CHDs) are major causes of infant death in the United States. In the 1980s and earlier, most patients with moderate or severe CHD died before adulthood, with the maximum mortality during the first week of life. Remarkable advances in surgical techniques, diagnostic approaches, and medical management have led to marked improvements in outcomes. To address the critical research needs of understanding congenital heart defects, murine models have provided an ideal research platform, as they have very similar heart anatomy to humans and short gestation rates. The combination of genetic engineering with high-throughput phenotyping tools has allowed for the replication and diagnosis of structural heart defects to further elucidate the molecular pathways behind CHDs. The use of noninvasive fetal echocardiography to screen the cardiac phenotypes in mouse models coupled with the high fidelity of Episcopic fluorescence image capture (EFIC) using Episcopic confocal microscopy (ECM) histopathology with three-dimensional (3D) reconstructions enables a detailed view into the anatomy of various congenital heart defects. This protocol outlines a complete workflow of these methods to obtain an accurate diagnosis of murine congenital heart defects. Applying this phenotyping protocol to model organisms will allow for accurate CHD diagnosis, yielding insights into the mechanisms of CHD. Identifying the underlying mechanisms of CHD provide opportunities for potential therapies and interventions.

This article details murine congenital heart disease (CHD) diagnostic methods using fetal echocardiography, necropsy, and Episcopic fluorescence image capture (EFIC) using Episcopic confocal microscopy (ECM) followed by three-dimensional (3D) reconstruction.

表征胎儿小鼠结构性心脏缺陷的管道

Congenital heart diseases (CHDs) are major causes of infant death in the United States. In the 1980s and earlier, most patients with moderate or severe ...

Assessment of Cardiac Morphological and Functional Changes in Mouse Model of Transverse Aortic Constriction by Echocardiographic Imaging

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable noninvasive method is essential to assess real-time cardiac morphological and functional changes in animal models of heart disease. Transthoracic echocardiography represents an important tool for noninvasive assessment of cardiac structure and function2. Here we used a high-resolution ultrasound imaging system to monitor myocardial remodeling and heart failure progression over time in a mouse model of TAC. B-mode, M-mode, and Doppler imaging were used to precisely assess cardiac hypertrophy, ventricular dilatation, and functional deterioration in mice following TAC. Color and pulse wave (PW) Doppler imaging was used to noninvasively measure pressure gradient across the aortic constriction created by TAC and to assess transmitral blood flow in mice. Thus transthoracic echocardiographic imaging provides comprehensive noninvasive measurements of cardiac dimensions and function in mouse models of heart disease.

The goal of this protocol is to noninvasively assess cardiac structural and functional changes in a mouse model of heart disease created by transverse aortic constriction, using B- and M-mode echocardiography and color/pulse wave Doppler imaging.

在横向主动脉缩窄的小鼠模型和心脏形态学评估功能改变通过超声心动图成像

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable ...

Echocardiographic Approaches and Protocols for Comprehensive Phenotypic Characterization of Valvular Heart Disease in Mice

The aim of this manuscript and accompanying video is to provide an overview of the methods and approaches used for imaging heart valve function in rodents, with detailed descriptions of the appropriate methods for anesthesia, the echocardiographic windows used, the imaging planes and probe orientations for image acquisition, the methods for data analysis, and the limitations of emerging technologies for the evaluation of cardiac and valvular function. Importantly, we also highlight several future areas of research in cardiac and heart valve imaging that may be leveraged to gain insights into the pathogenesis of valve disease in preclinical animal models. We propose that using a systematic approach to evaluating cardiac and heart valve function in mice can result in more robust and reproducible data, as well as facilitate the discovery of previously underappreciated phenotypes in genetically-altered and/or physiologically-stressed mice.

This protocol provides a detailed description of the echocardiographic approach for comprehensive phenotyping of heart and heart valve function in mice.

超声心动图方法和协议心脏瓣膜疾病的综合表型特征小鼠

The aim of this manuscript and accompanying video is to provide an overview of the methods and approaches used for imaging heart valve function in ...

Murine Echocardiography of Left Atrium, Aorta, and Pulmonary Artery

The present methodology teaches the investigator how to measure and use the LAV as a surrogate of chronic elevations in Left Ventricular diastolic pressure through echocardiography, as well as to obtain measurements of the Aorta and PA diameter in mice.
Mice older than 10 d of age can be analyzed using the present technique. The technique is composed of 3 main steps: set-up, image acquisition, and image analysis. The set-up step consists of getting the mouse anesthetized with 1% isoflurane, shaving it, and taping it in a supine position to a heated EKG board where the image acquisition will take place. The image acquisition step consists of learning to identify the cardiac structures and obtaining all the required images with its correspondent probe and axis in order to be able to calculate volumes and diameters. The image analysis step consists of measuring the previously acquired images with the aid of computer software.
Advantages of the proposed technique include a fast (15 min) procedure that would allow the researcher to evaluate interventions in a non-invasive, non-terminal approach and therefore follow the same mouse over time; each mouse can be used as its own control. This fact plus having the same operator perform all the acquisition and analysis for the entire experiment minimizes the limitation of operator-dependency. The present methodology is useful for mouse researchers in cardiovascular and pulmonary medicine.

The following protocol describes the methodology for the acquisition and analysis of echocardiographic images used to obtain the Left Atrial Volume (LAV), Aorta (Ao) diameter, and Pulmonary Artery (PA) diameter in mice. This technique is a non-invasive, non-terminal procedure that allows assessment of the cardiopulmonary function.

左心房，主动脉和肺动脉的超声心动图小鼠

The present methodology teaches the investigator how to measure and use the LAV as a surrogate of chronic elevations in Left Ventricular diastolic ...

Echocardiographic Measurement of Right Ventricular Diastolic Parameters in Mouse

Diastolic dysfunction is a prominent feature of right ventricular (RV) remodeling associated with conditions of pressure overload. However, the RV diastolic function is rarely quantified in experimental studies. This might be due to technical difficulties in the visualization of the RV in the apical four-chamber view in rodents. Here we describe two positions facilitating the visualization of the apical four-chamber view in mice to assess the RV diastolic function.
The apical four-chamber view is enabled by tilting the mouse fixation platform to the left and caudally (LeCa) or to the right and cranially (RiCr). Both positions provide images of comparable quality. The results of the RV diastolic function obtained from two positions are not significantly different. Both positions are comparably easy to perform. This protocol can be incorporated into published protocols and enables detailed investigations of the RV function.

Here we describe and compare two positions for obtaining the apical four-chamber view in mice. These positions enable the quantification of the right ventricular function, provide comparable results, and can be used interchangeably.

小鼠右心室舒张参数的超声心电图测量

Diastolic dysfunction is a prominent feature of right ventricular (RV) remodeling associated with conditions of pressure overload. However, the RV ...

Echocardiographic Characterization of Left Ventricular Structure, Function, and Coronary Flow in Neonate Mice

Echocardiography is a non-invasive procedure that enables the evaluation of structural and functional parameters in animal models of cardiovascular disease and is used to assess the impact of potential treatments in preclinical studies. Echocardiographic studies are usually conducted in young adult mice (i.e., 4-6 weeks of age). The evaluation of early neonatal cardiovascular function is not usually performed because of the small size of the mouse pups and the associated technical difficulties. One of the most important challenges is that the short length of the pups&#39; limbs prevents them from reaching the electrodes in the echocardiography platform. Body temperature is the other challenge, as pups are very susceptible to changes in temperature. Therefore, it is important to establish a practical guide for performing echocardiographic studies in small mouse pups to help researchers detect early pathological changes and study the progression of cardiovascular disease over time. The current work describes a protocol for performing echocardiography in mouse pups at the early age of 7 days old. The echocardiographic characterization of cardiac morphology, function, and coronary flow in neonatal mice is also described.

The present protocol describes the echocardiographic assessment of left ventricular morphology, function, and coronary blood flow in 7-day old neonate mice.

新生小鼠左心室结构、功能和冠状动脉血流的超声心动图表征

Echocardiography is a non-invasive procedure that enables the evaluation of structural and functional parameters in animal models of cardiovascular ...