超声心动图与小鼠心脏形态学的组织学检查
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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 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。 麻醉通过腹腔 (ip) 注射50毫克/千克戊巴比妥 25 , <sup cl…
Representative Results
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…
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Wheat germ agglutinin (WGA) conjugated tetramethylrhodamine | Life Technologies, Molecular Probes | W849 | |
| Biotinylated Goat Anti-Rabbit IgG Antibody | Vectorlabs | BA-1000 | |
| Avidin/Biotin Blocking Kit | Vectorlabs | SP-2001 | |
| VECTASTAIN Elite ABC HRP Kit (Peroxidase, Standard) | Vectorlabs | PK-6100 | |
| VECTASHIELD Antifade Mounting Medium with DAPI | Vectorlabs | H-1200 | |
| SIGMAFAST 3,3'-Diaminobenzidine tablets | Sigma | D4168 | |
| Hydrogen peroxide solution | Sigma | H1009 | |
| Anti-Pecam-1 (CD31) antibody | Abcam | ab28364 | |
| Ultrasound transmission gel, Gel Aquasonic 100 | Parker | ||
| Linear ultrasound probe, L15-7io | Philips Healthcare | ||
| Echocardiograph, IE33 xMATRIX | Philips Healthcare | ||
| Microscope, Leica DMi8 | Leica | ||
| Fluorescence Filterset DAPI | Leica | 11525304 | |
| Filterset TxR | Leica | 11525310 | |
| Digital Camera, SPOT RT3 Color Slider | Spot Imaging | ||
| Imaging Software, SPOT 5.2 Advanced and Basic Software | Spot Imaging | ||
| Imaging Computer | Dell | ||
| Fine Scissors | Fine Science Tools | 14028-10 | |
| Large Scissors | Fine Science Tools | 14501-14 | |
| Scalpel blades | Fine Science Tools | 10023-00 | |
| Graefe Forceps | Fine Science Tools | 11650-10 | |
| Rodent shaver | Harvard Apparatus | 34-0243 | |
| cassettes for paraffin embedding | Sakura | 4155F | |
| neutral buffered Formalin | Sakura | 8727 | |
| Xylene | Sakura | 8733 | |
| Paraffine TEK III | Sakura | 4511 | |
| automated embedding apparatus, Tissue-Tek VIP | Sakura | 6032 | |
| paraffin-embedding station Tissue-Tek TEC 5 | Sakura | 5229 | |
| microtome blades,Accu-Edge S35 | Sakura | 4685 | |
| microscopy slides, Tissue-Tek | Sakura | 9533 | |
| cover slips, Tissue-Tek | Sakura | 9582 | |
| Mounting medium Tissue-Tek | Sakura | 1408 | |
| slide boxes | Sakura | 3958 | |
| eosine solution | Sakura | 8703 | |
| hematoxyline solution | Sakura | 8711 | |
| microtome, RM2125RT | Leica | 720-1880 (VWR) | |
| water bath, Leica HI1210 | Leica | 720-0113(VWR) | |
| Ethanol | VWR | ACRO444220050 | |
| 15 ml tubes | VWR | 734-0451 | |
| staining glass dish | VWR | MARI4220004 | |
| staining jars | VWR | MARI4200005 | |
| Incubator | Binder | 9010-0012 | |
| DAB and urea hydrogen peroxide tablets, SIGMAFAST 3,3′-Diaminobenzidine tablets | Sigma | D4293 | |
| PBS (10X) | Thermo Fisher Scientific | 70011044 |
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Cite This Article
Baudouy, D., Michiels, J., Vukolic, A., Wagner, K., Wagner, N. Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse. J. Vis. Exp. (128), e55843, doi:10.3791/55843 (2017).