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

Modified Matrix Gel Plug Assayによる血管新生研究の簡素化

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.

著者スポットライト:Investigating Angiogenesis and Vessel Permeability Through a Modified Matrix Gel Plug Assay

血管新生研究のための改変 In Vivo マトリックスゲルプラグアッセイ

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.

マウスからのデキストラン-FITC注入およびマトリックスゲルプラグ採取によるマトリックスゲルプラグアッセイ

マウスにデキストランを注射するには、マトリックスゲル注射の7日後にFITCで、0.5ミリグラムのデキストラン、FITCを500マイクロリットルの二重蒸留水に再懸濁します。それを完全にボルテックスして、1マイクロリットルあたり1マイクログラムのデキストラン、FITC溶液を得る。29ゲージのシリンジに50マイクロリットルのデキストランFITC溶液を充填します。マウスを尾静脈注射器具に固定し、75%アルコールに浸した綿球で尾部を消毒してから、シリンジに50マイクロリットルのデキストランFITC溶液を尾静脈に慎重に注入します。.綿棒で注射部位に1分間圧力をかけ、出血を止めます。マウスをケージに戻し、30分間リラックスさせてから、マトリックスゲルプラグを回収します。ゲルプラグを回収するには、手術用ハサミを使用して、プラグのマークされた境界に沿って安楽死させたマウスの皮膚を切断します。そして、マトリックスジェルプラグの上の皮膚を取り除きます。プラグを回収し、リン酸緩衝生理食塩水を入れたビーカーですすぎ、余分な血液を洗い流します。次に、組織包埋カセットの底部を約0.5ミリリットルの組織包埋ゲルで覆います。次に、マトリックスゲルプラグを組織包埋ゲルに計画した向きで迅速に配置します。追加の組織包埋ゲルでプラグを埋め込みます。カセットを摂氏マイナス80度で12時間固化させます。固化したプラグブロックを取り外した後、凍結ミクロトームを使用して12ミクロンの厚さの切片を切断します。次に、切片を顕微鏡スライドに取り付けます。

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 ...

生物学

Preparing Vascular Cells for In Vivo Matrix Gel Plug Assay

In Vivo Matrix Gel Plug Assay 用の血管細胞の調製

Preparing Matrix Gel with Vascular Cells for In Vivo Matrix Gel Plug Assay

In Vivo Matrix Gel Plug Assay のための血管細胞を用いたマトリックスゲルの調製(英語)

Matrix Gel Mixture Injection into Mouse for Matrix Gel Plug Assay

マトリックスゲルプラグアッセイのためのマウスへのマトリックスゲル混合物注入