Date of Award
Dissertation - Restricted
Doctor of Philosophy (PhD)
Henry F. Edelhauser
William J. Stekiel
Diane L. Van Horn
The cornea remains clear and transparent because of a proper balance in its tissue hydration. A single layer of cells, the endothelium, maintains the proper amount of hydration via an active pumping mechanism. Thus, a viable endothelium is important for successful corneal transplantation. The supply of human donor tissue is limited by rigid eye bank requirements and the short-term shortage of 48 hours at 4°C. A procedure of long-term storage by corneal preservation (Kaufman and Capella, J. Cryosurg. 1:125, 1968) enables viable corneas to be stored in liquid nitrogen for years. However, this method has not been completely accepted by ophthalmic surgeons, because of an insufficient knowledge of the physiology of cryopreserved corneas. Therefore, it was the purpose of this study to evaluate the corneal hydration and endothelial morphology of rabbit corneas while being exposed to cryoprotective solutions and while being rehydrated following control rate freezing, storage at -196°C and thawing. Prior to the cryopreservation studies, an in vitro perfusion technique and baseline data for the maintenance of fresh isolated corneas had to be established.
The technique of specular microscopy (Maurice, Experimentata, 24:19D4, 1968) for isolating and perfusing in vitro corneas was used. This method enables measurement of corneal hydration, observation of the endothelial cell pattern throughout the perfusion period and provides a means of tissue fixation for electron microscopy, while maintaining the cornea's natural curvature with 15 mmHg pressure. Progressive damage of the endothelial cells at the light and electron microscope magnifications correlated directly with, increasing corneal hydration.
A bicarbonate Ringers was not adequate for maintaining a stable hydration of in vitro corneas (60 u/hr swelling rate). However, the Kinsey medium (Kinsey and Reddy, Invest. 0phth. 4:104, 1965) was an adequate maintenance medium providing the perfusion was limited to a 3 hour period (4 u /hr swelling rate). A bicarbonate Ringers which included 0. 3 mM reduced glutathione and 5 mM adenosine (Dikstein and Maurice, in The Biochemistry of the Eye, 1970) was shown to be an excellent maintenance medium, for up to ten hours of corneal stability (1 u/hr swelling rate).
The second phase of this study pertains to assessing the physiological effects of the cryoprotective solutions and the cryopreservation procedure on endothelial function. Dimethylsulfoxide (DMSO), a cryoprotective agent, was added to the perfusion solution in concentrations paralleling those used in the cryoprotective solutions. These studies showed that DMSO perfused to the corneal endothelium at 34°C resulted in an increase in corneal swelling and structural damage to the endothelial cell pattern; whereas 11150 perfused at 4°C and rinsed from the cornea at 4° C did not result in corneal swelling or endothelial cell damage. The complete cryoprotective solutions did not alter the endothelial cell pattern or the stable corneal hydration when used at 4°C.
When the cornea was cryopreserved according to the method of Kaufman and Capella, marked dehydration occurred during freezing which was probably caused by the physical events of freezing. A bicarbonate Ringers with glutathione was found to be insufficient for maintaining the post-thawed tissue at a constant hydration.
However, the endothelial cells remained intact, with some electron microscopic evidence of freeze-thaw-induced injury. This data does not necessarily indicate that the tissue would be irreversibly damaged, since cryopreserved corneas perfused in aqueous humor, within the human eye, yield clear graphs.