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

Forenkling av angiogenesestudier med en modifisert matrisegelplugganalyse

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: Undersøkelse av angiogenese og karpermeabilitet gjennom en modifisert matrisegelplugganalyse

Modifisert in vivo matriks gelplugganalyse for angiogenesestudier

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.

Dextran-FITC injeksjon og Matrix Gel Plug Collection fra mus for Matrix Gel Plug Assay

For å injisere musen med Dextran, FITC syv dager etter matriksgelinjeksjonen, RESUSPEND 0,5 milligram Dextran, FITC i 500 mikroliter dobbeltdestillert vann. Virvel det grundig for å oppnå en mikrogram per mikroliter Dextran, FITC-løsning. Fyll en 29 gauge sprøyte med 50 mikroliter av Dextran FITC oppløsning.Fest musen på injeksjonsinstrumentet i halevenen, desinfiser halen med en bomullsdott på 75 % alkohol før du forsiktig injiserer 50 mikroliter av Dextran FITC oppløsningen i sprøyten inn i halevenen. Trykk på injeksjonsstedet med bomullspinnen i ett minutt for å stanse blødningen. Sett musen tilbake i buret og la den slappe av i 30 minutter før du samler opp matriksgelpluggen.For å samle gelpluggen, kutt huden på den avlivede musen langs den merkede grensen til pluggen, ved hjelp av kirurgisk saks. Og fjern huden over matriksgelpluggen. Samle pluggen og skyll den i et beger med fosfatbufret saltvann for å vaske av overflødig blod.Deretter dekker du bunnen av vevsembeddingskassetten med ca. 0,5 milliliter vevsembeddingsgel. Plasser deretter matriksgelpluggen raskt på vevets innbyggingsgel i den planlagte retningen. Legg pluggen inn med ekstra gel for vevsinnbygging.Stivne kassettene ved minus 80 grader Celsius i 12 timer. Etter å ha fjernet den størknede pluggblokken, kutt 12 mikron tykke seksjoner ved hjelp av en frysende mikrotom. Monter deretter seksjonene på mikroskoplysbilder.

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 ...

Preparing Vascular Cells for In Vivo Matrix Gel Plug Assay

Klargjøring av vaskulære celler for in vivo matriksgelplugganalyse

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

Klargjøring av Matrix Gel med vaskulære celler for in vivo Matrix Gel Plug-analyse

Matrix Gel Mixture Injection into Mouse for Matrix Gel Plug Assay

Matrix Gel Mix Injeksjon i mus for Matrix Gel Plug Assay