dextran-fitc injection and matrix gel plug collection from mouse for matrix gel plug assay

使用改良基质凝胶塞测定简化血管生成研究

Angiogenesis, the process of formation of neo-vessels from pre-existing vessels, plays a critical role in many physiological and pathological processes, such as embryonic development, wound healing, atherosclerosis, tumor development, etc.1,2,3,4,5. This dynamic process involves several steps, including the degradation of the matrix, vascular cell proliferation, migration and self-organization to form tubular structures and the stabilization of the neo-vessels6. Promoting angiogenesis has been demonstrated to be critical in the treatment of myocardial infarction, stroke and other kinds of ischemic diseases7 while inhibiting angiogenesis has been considered a promising strategy in the treatment of cancers8 and rheumatoid diseases9. Angiogenesis has been considered an organizing principle for drug discovery10. Thus, the construction of a reliable and convenient method to assess the extent of angiogenesis is critical for mechanical research or drug discovery in angiogenesis-dependent diseases.
Several in vitro and in vivo models have been developed to evaluate angiogenesis11. Among these, two-dimensional (2-D) models, like matrix gel tube formation assay12, cannot form functional tubular structures. The animal models, such as the hind limb ischemia model13,14, can reproduce the angiogenesis process but are complex and require a laser speckle blood flow imaging system. 3D models of vascular morphogenesis, like matrix gel plug assay, provide a simple platform that can mimic the process of angiogenesis in vivo15, but the detection of angiogenesis requires immunohistochemistry or immunofluorescence staining16,17,18, which are variable and poorly visualized.
Here, we describe a protocol for a modified matrix gel plug assay where vascular cells were mixed with matrix gel and subcutaneously injected into the back of mice to form a plug. In the plug, vascular cells need to degrade the matrix, proliferate, migrate, and self-organize to finally form functional vessels with blood flow in the internal environment. Thereafter, fluorescent-labeled dextran is injected via the tail vein, to flow through the plug, and the label is visualized to indicate neo-vessels. The content of angiogenesis can be quantitatively evaluated by the length of the vessels. This method can form functional vessels that cannot be produced in 2-D angiogenesis models12, and does not need complex stain process as in ordinary matrix gel plug assay11. It also does not require expensive specific instruments like laser speckle blood flow imaging system in hind limb ischemia model13,14,19. This method is versatile, low-cost, quantifiable, and easy to perform, and can be used to determine the pro- or anti-angiogenic capability of drugs or be used in mechanical research involved in angiogenesis.

作者聚焦：通过改良基质凝胶塞测定研究血管生成和血管通透性

用于血管生成研究 的改良体内 基质凝胶塞测定

Our research deals with angiogenesis, the process of formation of new vessels from preexisting vessels. It plays a critical role in many physiological and pathological processes, such as embryonic development or curing atherosclerosis, tumor development, etc. The construction of a reliable model to evaluate the extent of angiogenesis is critical for myocardial research or the discovery of medicines for angiogenesis-depend diseases.However, most of the existing models are complex, expensive, and require specialized equipment. Here, we present a modified matrix gel plug assay that can be used to investigate angiogenesis and vessel permeability without staining in a simple, more intuitive, as well as efficient manner.

来自小鼠的葡聚糖-FITC 注射液和基质凝胶塞集合，用于基质凝胶塞测定

为了给小鼠注射葡聚糖，在基质凝胶注射后7天，将0.5毫克葡聚糖，FITC重悬于500微升双蒸水中。彻底涡旋以获得每微升 1 微克的葡聚糖、FITC 溶液。用 29 微升葡聚糖 FITC 溶液填充 50 号注射器。将鼠标固定在尾静脉注射器上，用75%酒精浸泡的棉球对尾部进行消毒，然后小心地将注射器中的50微升葡聚糖FITC溶液注射到尾静脉中。用棉签对注射部位施加压力一分钟以止血。将鼠标放回笼子中，让它放松30分钟，然后收集基质凝胶塞。要收集凝胶塞，请使用外科剪刀沿着塞子的标记边缘切割安乐死小鼠的皮肤。并去除基质凝胶塞上方的皮肤。收集塞子并在烧杯中用磷酸盐缓冲盐水冲洗，以洗掉多余的血液。接下来，用约0.5毫升组织包埋凝胶覆盖组织包埋盒的底部。然后以计划的方向迅速将基质凝胶塞放在组织包埋凝胶上。用额外的组织包埋凝胶包埋塞子。在零下 80 摄氏度下凝固盒 12 小时。取出凝固的塞块后，使用冷冻切片机切割 12 微米厚的部分。然后将切片安装到显微镜载玻片上。

Research

JoVE Journal

Biology

Dextran-FITC Injection and Matrix Gel Plug Collection from Mouse for Matrix Gel Plug Assay

Modified In Vivo Matrix Gel Plug Assay for Angiogenesis Studies

Several models have been developed to investigate angiogenesis in vivo. However, most of these models are complex and expensive, require specialized ...