matrix gel mixture injection into mouse for matrix gel plug assay

Forenkling af angiogeneseundersøgelser med et modificeret matrix gelstik-assay

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.

Forfatter Spotlight: Undersøgelse af angiogenese og karpermeabilitet gennem et modificeret matrix gelstik assay

Modificeret In vivo Matrix Gel Plug Assay til angiogenese undersøgelser

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 gelblanding injektion i mus til matrix gel plug assay

Til at begynde med skal du placere de 30 gauge en milliliter insulinsprøjter i et køleskab med fire grader Celsius til afkøling. Opbevar den frisklavede matrixgelblanding indeholdende vaskulære celler på is, indtil musen er klar til injektionen. Placer derefter den korrekt bedøvede mus på betjeningskortet i en udsat position og fastgør dens lemmer på brættet ved hjælp af tape.Læg en forkølet insulinsprøjte med 300 mikroliter matrixgelblanding, undgå dannelse af bobler. Desinficer huden på bagsiden af musen med 75% alkoholpuder, og injicer derefter subkutant 300 mikroliter matrixgelblandingen i sprøjten i den ene side af dyrets ryg. Fjern forsigtigt kanylen fra injektionsstedet for at forhindre lækage af gelblandingen.Sørg for dannelsen af en lille pukkel på injektionsstedet. Placer musen på en varmepude i to minutter for at lade matrixgelblandingen koagulere og danne et stik. Gentag de demonstrerede trin for at oprette et stik på den anden side af musens bagside.Marker pukkelens kanter ved hjælp af en markørpen. Opstald dyret i et specifikt patogenfrit forsøgsdyrlaboratorium ved 20 til 24 grader med 12 timers lys og 12 timers mørke cyklusser i syv dage.

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 ...

Preparing Vascular Cells for In Vivo Matrix Gel Plug Assay

Forberedelse af vaskulære celler til in vivo matrix gel plug assay

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

Forberedelse af matrixgel med vaskulære celler til in vivo matrixgelpropanalyse

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

Dextran-FITC injektion og matrix gel plug samling fra mus til matrix gel plug assay