An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

This Choroid assay is highly reproducible and pertinent to Choroidal Angiogenesis research and Age related Macular Degeneration, AMD It can compliment in vivo studies of microvascular behavior. This assay can be used to screen compounds as potential treatments for neovascular AMD or to assess pathways involved in Choroidal Neovascularization using wild type and genetically modified mouse tissue. To begin, add five milliliters of penicillin, streptomycin and 10 and five milliliters of commercially available supplements to 500 milliliters of complete classic medium with serum then aliquot 50 milliliters of the medium thaw the basal membrane extract or BME overnight in a refrigerator at two to eight degrees Celsius.

Put an aliquot of complete classic medium on ice. Clean the dissecting microscope, forceps and scissors with 70%ethanol. Prepare to cell culture dishes and to place one on the dissection microscope and the other on ice.

Then add 10 milliliters of complete classic medium to each dish. Keep the eyes in complete classic medium on ice before dissection. Remove the connective tissue and optic nerve then use a micro scissor to circumferentially cut 0.5 millimeters posterior to the corneal Limbus.

Remove the cornea Iris complex, vitreous and to the lens. Make a one millimeter incision perpendicular to the cut edge towards the optic nerve and to cut a circumferential one millimeter wide band then separate the central and peripheral regions of the complex. Use forceps to peel the retina from the RPE choroid sclera complex.

Keep the peripheral choroid band in complete classic medium on ice. Isolate the other eye and repeat the process to cut a second band. Cut the circular band into five to six approximately equal square pieces.

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 X plant. 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 eight bit with gray scale.

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 in 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. This protocol was used to examine and quantify choroid sprouting in C 57, black six J mice from day three to day six. In a representative case, the choroidal sprouting area was 0.38, 1.47, 5.62 and 10.09 millimeters squared at days three, four, five, and six, respectively.

The effects of loss of FFAR4 on choroidal vascular sprouting were evaluated using the choroid sprouting assay in FFAR4 four knockout and FFAR4 wild type mice. The sprouting area in FFAR4 knockout mice increased choroidal vascular growth compared to FFAR4 wild type at day six. Furthermore, treatment with one micromolar FFAR4 agonist reduced the choroidal sprouting area compared to untreated mice at day six.

This assay allows reproducible evaluation of anti angiogenic potential of pharmacological compounds and the variation of the role of a specific pathways in Choroidal Neovascularization using genetically modified mouses tissue.