Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

The overall goal of the following procedure is to introduce a model for acute corneal allograft rejection. This is accomplished by first removing the donor cornea. The next steps of the procedure are to prepare the recipient graft bed and suture the donor cornea into place.

Then the integrity of the graft is tested, and if there are no leaks, the eyelid is sutured closed. After a week, the opacity of the corneal graft is evaluated to measure rejection of the allograft. Ultimately, the model can be used, can test a variety of therapeutic strategies to define the most important factors linked to allograft rejection Of the solid organ transplants.

Corneal transplantation is the most common in the United States. There are several animal models available for this procedure, but mice are the most commonly used. This model has been used to define factors, involve both the rejection and acceptance of the corneal transplant.

After anesthetizing the donor and recipient mice and confirming their anesthetized states by a toe pinch, apply pure lube ointment to the eyes not undergoing surgery. Then secure the head with the donor eye horizontal to a sturdy, movable support using a strip of tape across the neck. Now using a two millimeter diameter tine whose tip was dyed with methyl blue, outline the central cornea graft site.

Then with a 15 degree ophthalmic knife blade penetrate the cornea to help avoid damage to the underlying lens. Inject the viscoelastic solution into the anterior chamber so the chamber becomes deeper. Now excise the donor graft with Venice scissors and transfer the graft to a dish of HBSS on ice until use to achieve madres, administer a couple of drops of 1%tpic aide and 2.5%efrin hydrochloride to the recipient eye.

Then using a 1.5 millimeter tine outline the graft site as described earlier, using a 15 degree ophthalmic knife blade, penetrate the cornea and inject a viscoelastic solution into the anterior chamber to deepen it. In order to maintain space between the donor cornea and the lens, this reduces the chance of damage to the underlying lens and to the donor endothelium. With Venice scissors, remove the outlined central corneal button from the recipient and discard.

Add viscoelastic medium to the area vacated by removal of the corneal button to protect the corneal endothelial cells from damage by direct contact with the lens. Now place the donor cornea over the graft bed using super fine tipped micro forceps. Place the first bite of 11.0 nylon suture into the donor side through the donor cornea to approximately 90%of full thickness.

Then to the recipient side at the same depth, then tie off. Continue securing the transplanted cornea using mid cardinal interrupted sutures. A total of eight to 10 sutures are all that are needed.

Now reform and wash the anterior chamber with an injection of saline. Alternatively, inject air into the anterior chamber to reform it. After the injection, gently test the integrity of the graft using a cellulose sponge.

Finally, evaluate the eye. Determine if the pupil round and if the anterior chamber appear normal. If the graft appears good, apply antibiotic ointment and optionally close the eyelid with running 7.0 silk suture postoperatively.

Monitor the mouse until it is fully awake and then continue observing it in its home cage for two days. Remove the sutures from the anesthetized mouse between four and seven days following the operation. After removing the sutures, observe the mouse for several days.

Using an opacity scale, evaluate every cornea with a score from zero to five. A zero indicates no opacity. A score of one corresponds to minimal superficial opacity.

A score of two indicates mild to deeper opacity with a discernible underlying pupil. And iris a sign a score of three to corneas with stromal opacity wherein the iris cannot be seen in detail with the exception of the pupil margins. If there is dense stromal opacity and no underlying structures can be seen, give the graft a score of four.

If, if there is complete opacity with intensive stromal edema and both the pupil and iris are completely obscured, then score the graft a five. The text protocol also suggests a zero to eight scale on blood vessel infiltration. A wide variety of manipulations can be made using the graft model.

For instance, spleen cells can be added to the anterior chamber from the donor mouse. After the donor is euthanized, remove the spleen and transfer it to a cell strainer using a three milliliter syringe plunger, disrupt the spleen, then wash the spleen cells in 10 milliliters of HBSS, resuspend the washed cells in 10 milliliters of HBSS and count the cells with a hemo cytometer. Keep the cells at room temperature until they are used later.

Prior to performing the corneal transplantation, inject the cells into the anterior chamber in the contralateral eye using a dissection microscope, inject 1 million spleen cells in five microliters of HBSS from a 33 gauge needle. The model was used to test whether systemic allo antigen T-cell tolerance to allo antigens expressed by the donor corneal allograft might improve graft survival. BC mice were tolerized to C 57 black 10 allo antigens by injection of B 10 spleen cells into the anterior chamber.

This did not improve corneal allograft acceptance. Studies demonstrate that establishing antigen specific tolerance to allo antigen as measured by DTH responses does not affect the outcome of either skin or corneal allografts. A similar study showed that when DTH tolerance was established in BAB c mice towards B 10 allo antigens, skin graft sparing some or all of B 10 allo antigens did not increase survival.

Thus bowel C with established antigen-specific DTH did not measurably benefit from corneal or skin allografts. As you can see from today's experiment, corneal transplantation is a technically challenging procedure. However, this is the best model to study the cells and factors that determines the graph success or failure.