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Priyatham Mettu, Tiffany Pridgen, M. Grazia Spiga, Peter Saloupis, Scott Cousins; Aged Mice Manifest More Mature Neovascularization in a Novel Model of Injury-Induced Angiogenesis. Invest. Ophthalmol. Vis. Sci. 2013;54(15):320.
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To develop a novel model of injury-induced neovascularization to analyze the biological factors responsible for severity and vessel maturation in aged as compared to young mice. Previously, our group has demonstrated that aged mice develop larger, more mature, and more fibrotic neovessels after laser-induced choroidal neovascularization (CNV).
Implants were constructed with 6 mm diameter x 1.5 mm height silicone rings filled with growth-factor depleted Matrigel. Following anesthesia, C57/BL6 mice underwent mechanical injury to the subcutaneous perineal fascia. Matrigel implants were secured over the fascia using fibrin/thrombin at the fascial interface and cyanoacrylate at the rim of the chamber, and the overlying skin was securely closed. We compared the severity and maturation of new vessel ingrowth in aged (16 month-old) as compared to young (2 month-old) mice. In vivo assessment of neovessel morphology and flow was performed using indocyanine green (ICGA) or fluorescein (FA) angiography of dye transit observed within surgically exposed chambers in situ. Immunohistochemistry was performed on frozen sections of excised chambers.
The optimal time point for analysis of implant vascularization was 2 weeks. Definite evidence (by FA and ICGA) of new vessel growth in to the Matrigel chamber was observed in 13/15 young animals (87%). In young mice (n=8), FA/ICGA revealed small caliber and relatively localized vascular networks. Immunohistochemistry demonstrated a predominance of capillaries as well as small-caliber arterioles and relatively few large smooth muscle actin (SMA) (+) vessels (average 12 SMA+ vessels/section); the average distance of vascular ingrowth into the chamber was 350 µm. In aged mice (n=8), FA/ICGA revealed vessel morphology of large caliber feeder vessels, branching arterioles and extensive capillary networks. Immunohistochemistry demonstrated a predominance of large, SMA (+) vessels (65 SMA+ vessels/section), and the average distance of vascular ingrowth was 750 µm.
Consistent with observations in the laser-induced CNV model, aged mice demonstrate more mature, larger caliber vessels with larger surface area than young mice in the Matrigel chamber assay. This assay will facilitate mechanistic pathobiological studies of how specific cells, in particular macrophages and bone marrow derived vascular progenitor cells, regulate vascular maturation.
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