Aorta Ring Assay
- Aortas were removed from 6-week-old mice killed by CO2 asphyxiation and immediately transferred
to a culture dish containing ice-cold endothelial cell basal medium (EGM-2; Cambrex Bio Science, Walkersville, MD).
- The periaortic fibroadipose
tissue was carefully removed, paying special attention not to damage the aortic wall.
- 1 mm long aortic rings were sectioned
and rinsed extensively in 8 consecutive washes of EGM-2.
- The rings were then individually embedded in 48-well plates previously
coated with 50 uL synthetic basement membrane (Matrigel; BD Biosciences, Bedford, MA) per well.
- Next, an additional 50 uL of
Matrigel was placed over each ring. After 1 hour, 500 uL EGM-2 was added to each well, and the cultures were incubated at 37°C for
5 days.
- The culture medium was changed on day 3 and the test compounds and vehicle were added.
- The aortic rings were photographed on
day 5 at 4x magnification with an inverted microscope. For neovessel-regression experiments, the rings were cultured without drugs
until day 6, after which the rings were treated with the test compound and allowed to grow until day 7.
- The angiogenic response
was determined by measuring the area of neovessel formation on computer (Image Pro Plus software; Media Cybernetics, Inc.).
References:
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Activation of CD36 inhibits and induces regression of inflammatory corneal neovascularization.
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Endostatin inhibits microvessel formation in the ex vivo rat aortic ring angiogenesis assay.Biochem Biophys Res
Commun. 2000 Feb 5;268(1):183-91.