The overall goal of this procedure is to use the most cornea pocket assay model to test the angiogenic effect of basic FGF. This is accomplished by making a pocket in the most cornea and inserting a basic FGF pellet into the pocket. Basic FGF is then released from the pellet and induces new blood vessels to grow from the limbal vessels.
Ultimately, this protocol can produce robust new blood vessel growth from the limbal vessels towards the basic FGF pellet. Hi, I'm Shuan from Dr.Shiley lab in National Institute INIH. The main advantage of this model is it has relatively simple model with high reproducibility.
Another advantage is since the normal cornal lacks blood vessels, the background is very long. Now we have the general idea of the model. I will show you step by step how to make the corneal popul disease.
So let's get started. To prepare for this procedure, make the following solutions, 10%weight by volume sate and PBS 12%weight by volume, poly hema and ethanol, and one gram per microliter basic FGF. The basic FGF must be stored at minus 80 degrees Celsius, but the other two solutions can be stored at room temperature.
To make 50 angiogenic factor pellets, combine five microliters of poly hema solution, one microliter of sate solution, and four microliters of basic FGF solution In a 200 microliter PCR tube, then vortex. The mixture thoroughly. Now place a section of parfum in a Petri dish and eject 0.2 microliter drops of the mixture onto the parfum.
Each drop will dry into one pellet. Allow the pellets to dry at room temperature for one to two hours. The dried pellets can be stored at four degrees Celsius for several days, or at minus 80 degrees Celsius for several months.
To use the pellets, pry them from the param using a pair of forceps, be extremely careful not to break them or make them spring away. After anesthetizing a mouse with a mixture of ketamine and xylazine, use a pipette to apply 50 microliters of 0.5%propane hydrochloride to the cornea. Wait five minutes and then position the mouse under a dissecting microscope so that one eye is directly under the objective.
Using a sterile von grave cataract knife, make a very gentle cut in the middle of the cornea about 1.2 to 1.4 millimeters from the corneal limbus. Be extremely careful not to cut through the cornea. Now make a pocket under the epithelial layer of the cornea by horizontally inserting the knife into the incision and carefully extending the knife.
Make the pocket large enough to accommodate one basic FGF pellet. Once again, be extremely careful to avoid puncturing the cornea. Place the pellet on the cornea and fold it twice.
Using forceps, slowly insert the pellet into the pocket, then flatten out the pellet as much as possible using the forceps to finish the procedure, use an ophthalmic tip applicator to cover the whole eye. With topical ophthalmic antibiotic ointment, move the mouse to a warm and dry area. When the mouse is able to maintain an upright posture, return it to its home cage twice daily while the mouse recovers.
Apply systemic analgesics like buprenorphine and topical antibiotic ointment to the eye between four and 10 days after the pellet implantation, anesthetize the mouse and examine the eye. Under slit lamp, bio microscope, blood vessels in the cornea induced by the angiogenic factor were observed under a dissecting microscope and a cornea treated with a vehicle alone. No blood vessel growth was observed in a cornea treated with basic FGF evidence of angiogenesis.
The growth of new blood vessels was robust. Blood vessel growth was assessed by measuring their maximal length between the normal limbal vessels and the top of the new vessels. Likewise, the total blood vessel area was also measured.
You see, this is relatively simple procedure. Once master, it can be done in five minutes for one eye. While ing this procedure, special care need to be taken, especially while try to make the pocket and in insert the pad into the pocket.
The knife must be handled precisely so that it will not puncture the cornea. So thanks for your attention. Good luck.
