matrix gel mixture injection into mouse for matrix gel plug assay

Simplifying angiogenesis studies with a modified matrix gel plug assay(수정된 매트릭스 겔 플러그 어세이를 통한 혈관신생 연구 간소화)

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.

저자 스포트라이트: Investigating Angiogenesis and Vessel Permeability Through a Modified Matrix Gel Plug Assay (수정된 매트릭스 겔 플러그 분석을 통한 혈관신생 및 혈관 투과성 조사)

혈관신생 연구를 위한 수정된 In Vivo Matrix Gel Plug Assay

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.

Matrix Gel Plug Assay를 위해 마우스에 Matrix Gel Mixture 주입

시작하려면 30게이지 1밀리리터 인슐린 주사기를 섭씨 4도 냉장고에 넣어 냉각합니다. 생쥐가 주사할 준비가 될 때까지 혈관 세포가 포함된 갓 준비된 매트릭스 젤 혼합물을 얼음 위에 보관하십시오. 그런 다음 적절하게 마취된 마우스를 작동 보드에 엎드린 자세로 놓고 테이프를 사용하여 팔다리를 보드에 고정합니다.미리 냉각된 인슐린 주사기에 300마이크로리터의 매트릭스 겔 혼합물을 로드하여 기포가 형성되지 않도록 합니다. 75% 알코올 패드를 사용하여 쥐 뒷면의 피부를 소독한 다음 주사기에 있는 매트릭스 젤 혼합물 300마이크로리터를 동물의 등 한쪽에 피하로 주입합니다. 겔 혼합물의 누출을 방지하기 위해 주사 부위에서 바늘을 부드럽게 제거합니다.주입 부위에 작은 혹이 형성되도록 하십시오. 마우스를 가열 패드에 2분 동안 올려 놓으면 매트릭스 겔 혼합물이 응고되어 플러그가 형성됩니다. 시연된 단계를 반복하여 마우스 뒷면의 다른 쪽에 플러그를 만듭니다.마커 펜을 사용하여 혹의 가장자리를 표시하십시오. 20-24도의 특정 병원균이 없는 실험용 동물 실험실에서 7일 동안 12시간의 빛과 12시간의 어두운 주기로 동물을 수용합니다.

Research

JoVE Journal

Biology

Matrix Gel Mixture Injection into 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 ...

연구

생물학

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In Vivo Matrix Gel Plug Assay를 위한 혈관 세포 준비

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

In Vivo Matrix Gel Plug Assay를 위한 혈관 세포가 있는 Matrix Gel 준비

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

Dextran-FITC 주입 및 매트릭스 겔 플러그 수집(Matrix Gel Plug Assay)을 위한 마우스의 Matrix Gel Plug Collection