The Mouse Cremaster Muscle Preparation for Intravital Imaging of the Microcirculation
The Mouse Cremaster Muscle Preparation for Intravital Imaging of the Microcirculation
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DOI：
10.3791/2874
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发表时间：
2011-06-01
影响因子：
1.2
通讯作者：
Segal, Steven S.
中科院分区：
文献类型：
--
作者：
Bagher, Pooneh;Segal, Steven S.
在整个身体中，维持体内平衡需要不断供应氧气和营养物质，同时清除代谢副产物。这种平衡是通过血液在微循环中的运动来实现的，微循环包括所有组织和器官中血管供应的最小分支。小动脉从动脉分支形成网络，控制流入与实质细胞密切相关的大量毛细血管的含氧血液的分布和量。毛细血管为组织细胞和血液供应之间的扩散交换提供了大的表面积。小静脉收集毛细血管流出物，当它们将脱氧血液返回心脏时会聚。为了在真实的时间内观察这些过程，需要一种可视化和操纵活体微循环的实验方法。大鼠的提睾肌首先被用作死后使用组织学和电子显微镜研究炎症的模型（1，2）。第一份关于暴露的完整大鼠提睾肌的体内报告使用反射光研究了微血管对血管活性药物的反应（3）。然而，肌肉的弯曲和缺乏聚焦照明限制了这种制备的有用性。主要的突破是打开肌肉，将其从睾丸上分离出来，并将其放射状地展开，作为一个平面，以便在复合显微镜下透照（4）。虽然显示是研究大鼠（5）和仓鼠（6）微循环生理学的有价值的制剂，小鼠的提睾肌（7）已被证明在解剖参与调节微血管功能的细胞通路（8-11）和细胞间信号传导的实时成像（12）中特别有用随着睾丸通过腹股沟管下降，提睾肌来源于腹内斜肌和腹横肌（13）。它的作用是支撑（希腊语：cremaster = suspender）和保持睾丸的温度。正如这里所描述的，提睾肌被制备成一个薄的平面片，以获得出色的光学分辨率。在小鼠保持稳定体温和麻醉平面的情况下，手术准备包括将肌肉从周围组织和睾丸中释放出来，将其铺在透明的硅橡胶基座上，并用昆虫针固定边缘，同时用生理盐溶液连续冲洗。本方案利用在小动脉内皮细胞中表达GCaMP 2的转基因小鼠。GCaMP 2是一种遗传编码的荧光钙指示剂分子（12）。宽视野成像和增强的电荷耦合器件照相机使得能够在体内研究小动脉内皮中的钙信号。
Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic by-products. This balance is achieved by the movement of blood through the microcirculation, which encompasses the smallest branches of the vascular supply throughout all tissues and organs. Arterioles branch from arteries to form networks that control the distribution and magnitude of oxygenated blood flowing into the multitude of capillaries intimately associated with parenchymal cells. Capillaries provide a large surface area for diffusional exchange between tissue cells and the blood supply. Venules collect capillary effluent and converge as they return deoxygenated blood towards the heart. To observe these processes in real time requires an experimental approach for visualizing and manipulating the living microcirculation.The cremaster muscle of rats was first used as a model for studying inflammation using histology and electron microscopy post mortem(1,2). The first in vivo report of the exposed intact rat cremaster muscle investigated microvascular responses to vasoactive drugs using reflected light(3). However curvature of the muscle and lack of focused illumination limited the usefulness of this preparation. The major breakthrough entailed opening the muscle, detaching it from the testicle and spreading it radially as a flat sheet for transillumination under a compound microscope(4). While shown to be a valuable preparation to study the physiology of the microcirculation in rats(5) and hamsters(6), the cremaster muscle in mice(7) has proven particularly useful in dissecting cellular pathways involved in regulating microvascular function(8-11) and real-time imaging of intercellular signaling(12).The cremaster muscle is derived from the internal oblique and transverse abdominus muscles as the testes descend through the inguinal canal(13). It serves to support (Greek: cremaster = suspender) and maintain temperature of the testes. As described here, the cremaster muscle is prepared as a thin flat sheet for outstanding optical resolution. With the mouse maintained at a stable body temperature and plane of anesthesia, surgical preparation involves freeing the muscle from surrounding tissue and the testes, spreading it onto transparent pedestal of silastic rubber and securing the edges with insect pins while irrigating it continuously with physiological salt solution. The present protocol utilizes transgenic mice expressing GCaMP2 in arteriolar endothelial cells. GCaMP2 is a genetically encoded fluorescent calcium indicator molecule(12). Widefield imaging and an intensified charge-coupled device camera enable in vivo study of calcium signaling in the arteriolar endothelium.