Choroid Sprouting Assay to Study Ocular Microvascular Proliferation Ex Vivo
Transcripción
In the mammalian eye, the choroid, located between the sclera and retina, contains perivascular macrophages and the choriocapillaris — a layer of blood capillaries lined by pericytes that supplies oxygen and nutrients to the retina. Abnormal blood vessel sprouting from the choriocapillaris occurs in several vascular eye diseases.
To study dysregulated choriocapillaris sprouting ex vivo, take harvested mouse eye explants containing the choroid attached to the sclera and retinal pigment epithelium, or RPE.
Add droplets of basal membrane extract, or BME — a hydrogel enriched in extracellular matrix proteins — to the wells of a multi-well plate. Place the explants in the droplet, avoiding tissue flattening. Allow the BME to solidify.
Add medium supplemented with endothelial cell, or EC-specific growth factors. Incubate. The RPE secretes vascular endothelial growth factor, which, alongside the EC-specific growth factors, stimulates capillary EC proliferation.
The solidified BME forms an extracellular matrix that promotes the proliferating ECs to form sprouting capillary-like tubules. Pericytes migrate and attach to these sprouting capillaries, facilitating the maturation of the capillary ECs.
Activated macrophages in the sprouting region secrete enzymes to digest the Bruch's membrane — the innermost choroidal layer — which enables the sprouts to migrate into the RPE.
Under a microscope, visualize the sprouting capillaries. Using suitable software, measure the area covered by the choroid sprout.
A larger choroidal sprouting area indicates progressive choriocapillaris sprouting in the explant.
