The overall goal of this procedure is to stimulate blood vessel growth in the mouse cornea. This is accomplished by first making pellets containing growth factor. The second step is to perform the surgical implantation of a pellet into the cornea.
The mice are then left for five to six days, depending on the growth factor to allow for vessel development. The final step is the quantification of vessel area, using a slit lamp microscope to measure average vessel length and the distance around the circumference of the eye that the vessels have grown. Ultimately, the corneal micro pocket assay is used as a reproducible and reliable method for the study of in vivo angiogenesis.
Generally, individuals new to this method will struggle because of the technically delicate nature of the procedure. Investigators should anticipate extensive practice prior to experimental trials to ensure consistency in both the surgical and grading techniques. Begin the pellet preparation by weighing out 10 milligrams of suc fate and 60 milligrams of hydron with a sterile un bent spatula and place them in separate micro centrifuge tubes.
Place a one centimeter square piece of nylon mesh into a sterile 10 centimeter dish. Invert it so the mesh rests on the shallow lid and set it aside. Next, add 500 microliters of ethanol to the hydron and vortex it for at least 10 minutes.
Retrieve a vial of growth factor from the minus 80 freezer growth factor. Stock should be at a concentration of one milligram per milliliter. Then add the appropriate amount of growth factor to the suc fate.
The added volume should be a minimum of 20 microliters to ensure the sucre fate is completely moistened, but should not exceed 50 microliters to ensure that it can be evaporated easily. After adding the growth factor vortex briefly place the sucre fate growth factor mixture into the centrifugal evaporator set on low for 30 to 50 minutes, depending on the volume of liquid used. When finished, the mixture should be completely dry.
Check the mixture for residual moisture with the tapered end of a sterile spatula. The mixture should feel crunchy when it is dry. Proceed to use the spatula to break up the salate mixture.
At this point, remove the lid that is covering the nylon mesh so the mesh is ready for the next step. Then prepare to add the hydron by cutting the end from a 200 microliter pipette tip. Due to the viscous nature of hydron, draw up and release 10 microliters of the hydron before pipetting it up again and adding it to the suc fade.
Use a bent spatula to quickly mix the hydron and suc fate. Then gather the mixture along the bottom side of the spatula tip and rotate the tube while drawing the spatula out along the wall of the tube. The mixture dries rapidly, so this step must be done efficiently.
Now holding the prepared mesh in place with forceps spread the hydron suc fate growth factor mixture onto the mesh. The mixture should be an even layer and fill in the holes of the mesh. Immerse the bent spatula into the tube containing the hydron, and use it to coat the top and bottom of the mesh.
Lean the coated mesh on its side against the edge of the dish and allow it to dry at room temperature for 30 to 45 minutes after the mesh has dried, place the 35 millimeter dish containing the mesh inside of a larger Petri dish. Use a pair of forceps to carefully pull the fibers of the mesh apart over the 35 millimeter dish. This action breaks the dried mixture into pellets.
At the end, tap the larger dish to dislodge stray pellets into the smaller dish. Check the pellets for uniformity under a scope. This process yields approximately 250 pellets.
After taping the lid on the pellets can be stored in a 35 millimeter dish in a minus 20 degrees Celsius freezer for up to three months. After anesthetizing a mouse with ton, position the mouse under a surgical microscope so an eye is visible and perform a toe pinch. To ensure a sufficient level of anesthesia, anesthetize the mouse's eye by placing one drop of propane on the eyeball.
Wait 20 to 30 seconds and dab it with a gauze pad. Use a dulled number one jeweler's forceps and being careful to leave skin between the forceps and the eye prop to the eye. It is important to avoid too firm or too loose of a hold.
Use a 30 degree micro knife to make a one to two millimeter incision into the cornea approximately one millimeter from the limbus. The incision should be deep enough to penetrate beyond the epithelial layer into the mids stroma, but not so deep as to rupture the eye. Slide a von graft knife under the corneal layer at the incision site and gently work the knife in and move it along the incision to enlarge the space.
Thus forming a pocket perpendicular to the incision. It helps to move the mouse so as to work with the curvature of the eye. Be careful not to push downward with the tip to avoid rupturing the eye.
After wetting a number five jeweler's forceps with propane, pick up a pellet from the dish and place the pellet on the eye. Next, moisten the von graft knife with propane and transfers some of the liquid onto the pellet to make it rubbery and pliable. Remove any excess moisture with a gauze pad or cotton swab.
Now use the von graft knife to push the pellet inside the pocket beneath the corneal layer. Once the whole pellet is inside, run the flat side of the von graft knife over the site to check that the pellet is secure. At this point, coat the eye with triple antibiotic ointment and turn the mouse over to repeat the surgery in the other eye.
After allowing several days for neovascularization, anesthetize the mouse with ton. A slit lamp microscope is used for grading. One ocular has a ridicule, which are lines within the eyepiece, used as a measuring aid to quantify vessel length and distance around the eye circumference that the vessels have sprouted.
Now hold the mouse in front of the scope, positioning it so the eye is seen in the ocular with the pellet directly ahead. Use a thumb and forefinger to stabilize the head so the skin is tightened on the face and the eye is slightly prop toast. Place the Y axis of the ridicule along the limbal vessel directly beneath the pellet.
Then measure the length of the vessels branching upward toward the pellets. Record this measurement in tens of millimeters and designate it as the vessel length turn either the mouse or the ridicule so that the distance around the eye that these vessels have sprouted becomes clear. It is easiest to think of the eye as a clock face with intervals of one through 12.
Designate the distance that the vessels have sprouted as the clock hour. The clock hour can be a whole number or fractions of 0.25 and is subjective. Continue to make measurements in the same fashion with the other mice.
While it is possible to view the eye from straight on the sideways view tends to give the most information. However, the sideways view only shows about 25%of the circumference, and so the mouse does need to be rotated to get a complete picture. The vessel area is calculated to be the vessel length times the clock, hour times 0.2 pi.
This calculation is based on the area of an oval. In this image, the measured vessel length is 0.9 millimeters and the clock hour is 3.25. So the calculated vessel area is 1.84 millimeters squared.
Growth factor induced corneal neovascularization is shown here. Implantation of an 80 nanogram basic FGF pellet into a low angiogenic black six mouse resulted in a vessel area equal to 2.0 millimeters squared. It is very common for this dose to cause vessels to reach the pellet and grow into it.
A 20 nanogram basic FGF pellet implanted into a black six mouse resulted in a vessel area equal to 1.04 millimeters squared. A 200 nanogram VEGF pellet implanted into a black six mouse resulted in a vessel area equal to 0.71 millimeters squared. The corneal neovascular area in black six mice increased with increasing doses of either VEGF or basic FGF.
When a 20 nanogram basic FGF pellet was implanted in the high angiogenic 1 2 9 strain, an angiogenic potential greater than twice that of the black six mice was observed. The anti-angiogenic drug thalidomide inhibited the basic FGF driven neovascularization. The treatment resulted in a 38%inhibition of vessel growth compared to controls that only received the basic FGF pellet.
Hyphemas can make grading the corneal neovascularization difficult or impossible. A mild hyphema is shown here in this image of a severe hyphema. The entire eye has a reddish tinge and there are pronounced pools of blood closer to the bottom of the iris.
While attempting this procedure, it's important to remember to relax and be patient. The more you practice the surgical technique and grading method, the more reproducible and reliable your data output will be.
