Choroid Sprouting Assay to Study Ocular Microvascular Proliferation Ex Vivo

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.

Add 30 microliters of the thawed BME into the center of each well of a 24-well tissue culture plate. Make sure that the droplet of BME forms a convex dome at the bottom of the plate without touching the edges.

Place the tissue in the middle of the BME. Do not flatten the choroid explant. Let the tissue expand within the BME. Incubate the plate at 37 degrees celsius for 10 minutes to let the gel solidify. Then, add 500 microliters of complete classic medium into each well. Change the classic medium every other day.

Choroid sprouting can be observed after three days with a microscope. Open the choroid sprouting image with Image J and check "Image | Type | and 8-bit" with grayscale. Then optimize the contrast by selecting "Image | Adjust | Brightness/Contrast" and adjusting it.

Use the "Magic Wand" function to outline and remove the choroid tissue, which are present in the center of the sprouts. Remove the background of the image with the free selection tools.

Next, go to "Image | Adjust | Threshold " and use the "Threshold" function to define the microvascular sprouts against the background and periphery. Click "F2" and a summary will appear. Click "Save" to save an image of the selected area in the same folder as the original image for future reference.

After a group of samples is measured, copy the recorded data for analysis.