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

Modifiye Matris Jel Tıkaç Testi ile Anjiyogenez Çalışmalarının Basitleştirilmesi

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.

Yazar Spot Işığı: Modifiye Matris Jel Tıkaç Testi ile Anjiyogenez ve Damar Geçirgenliğinin Araştırılması

Anjiyogenez Çalışmaları için Modifiye In Vivo Matrix Jel Plug Testi

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 Jel Plug Testi için Fareden Dextran-FITC Enjeksiyon ve Matrix Jel Plug Toplama

Fareye Dekstran enjekte etmek için, matris jel enjeksiyonundan yedi gün sonra FITC, 500 mikrolitre çift damıtılmış suda 0.5 miligram Dextran, FITC'yi YENİDEN SÜSPANSE EDİN. Mikrolitre Dekstran, FITC çözeltisi başına bir mikrogram elde etmek için iyice vorteksleyin. 29 gauge şırıngayı 50 mikrolitre Dextran FITC çözeltisi ile doldurun.Fareyi kuyruk damarı enjeksiyon aletine sabitleyin, şırıngadaki 50 mikrolitre Dextran FITC solüsyonunu kuyruk damarına dikkatlice enjekte etmeden önce kuyruğu% 75 alkole batırılmış bir pamuk top ile sterilize edin. Kanamayı durdurmak için pamuklu çubukla enjeksiyon bölgesine bir dakika boyunca basınç uygulayın. Fareyi kafesine geri koyun ve matris jel tapasını toplamadan önce 30 dakika dinlenmesine izin verin.Jel tıkacını toplamak için, ötenazi farenin derisini cerrahi makas kullanarak fişin işaretli kenarı boyunca kesin. Ve matris jel tapasının üzerindeki cildi çıkarın. Tapayı toplayın ve fazla kanı yıkamak için fosfat tamponlu salin içeren bir beherde durulayın.Daha sonra, doku gömme kasetinin altını yaklaşık 0,5 mililitre doku gömme jeli ile örtün. Ardından, matris jel tıkacını planlanan yönde doku gömme jeli üzerine hızlı bir şekilde yerleştirin. Fişi ek doku gömme jeli ile gömün.Kasetleri eksi 80 santigrat derecede 12 saat katılaştırın. Katılaşmış tapa bloğunu çıkardıktan sonra, dondurucu bir mikrotom kullanarak 12 mikron kalınlığında bölümler kesin. Ardından bölümleri mikroskop slaytlarına monte edin.

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 ...