In this study, we successfully developed a human-to-cat xenograft model. All human donor corneas maintained in preservative (Optisol-GS; Chiron) were clear with normal corneal thickness 1 week after penetrating keratoplasty. The epithelium, presumably from the cat, had healed over the grafts in a manner similar to human allografts and was intact by 1 week in most corneas. The keratocyte density 1 week after surgery was similar to that of human transplants in vivo,
25 which is decreased compared with normal corneas.
20 The mean endothelial cell loss at 1 week was 7%, consistent with clinical studies of postoperative cell loss in corneas preserved at 4°C in solutions that contain chondroitin sulfate.
26 27 28 This model will allow the testing of promising long-term corneal storage techniques and methods for endothelial cell augmentation on human corneas before clinical trials with human subjects. It will also allow investigators to study in more detail the characteristics of wound healing in the human cornea, including cellular apoptosis and keratocyte activation.
Apoptosis was infrequent in these corneas 1 week after transplantation. The median percentage of TUNEL-positive cells was either 0% or 1% in all four histologic regions for each cell type, except keratocytes in the human donor near the wound. Apparently, there was little apoptotic cell death in the cornea at the end of the first postoperative week, except for donor keratocytes near the wound. Higher levels of apoptosis may have been present earlier after keratoplasty, however, just as higher levels are often present during storage at 4°C.
21 In 10 human corneas stored for 0 to 21 days (mean, 10 days) in preservative (Optisol-GS; Chiron) at 4°C, the mean percentages of TUNEL-positive epithelial cells (13%), keratocytes (11%), and endothelial cells (8%)
21 were greater than those in the central human donor corneas 1 week after xenotransplantation (2%, 0%, and 2%, respectively;
Table 3 ). After the corneas returned to an in vivo setting for 1 week (i.e., by xenotransplantation), any increased apoptosis present during storage at 4°C apparently resolved. Although the TUNEL assay can at times be positive for cell death by necrosis,
29 30 31 32 in the human cornea the assay detects apoptotic, but not necrotic, cells.
21 Therefore, we assumed that the TUNEL-positive cells in this study were apoptotic. Not all apoptotic cells are detected by the TUNEL assay in the cornea, however; the percentage of apoptotic cells found by histology is higher than that found by the TUNEL assay.
21
Apoptosis of donor keratocytes, however, was significantly increased to a median rate of 7% near the wound. In the remaining three regions of the stroma, keratocyte apoptosis was 1% or less. In particular, almost no keratocyte apoptosis was found in the central graft, where the recently healed epithelium was intact in 7 of the 10 grafts. An increase of donor keratocyte apoptosis near the wound is consistent with the advent of wound healing in this area.
4 Increased apoptosis was not found in the cat keratocytes on the recipient side of the wound. We have no explanation for this finding; one would expect the early wound-healing process to proceed in both donor and recipient tissues near the wound. The highest full-thickness histologic keratocyte density was in the central donor cornea, where the apoptosis rate was the least. Conversely, the lowest keratocyte density was in the peripheral donor cornea near the wound, where the keratocyte apoptosis rate was the highest. The central full-thickness histologic keratocyte density was somewhat less than that observed by Komuro et al.
21 in donor corneas preserved at 4°C, consistent with further loss of donor keratocytes after transplantation. If the keratocytes lost to programmed cell death are not replaced, these findings are consistent with apoptosis’s lowering the keratocyte density in transplanted human corneas.
25 Cell death by necrosis
21 may also contribute, however.
In all corneas, we observed activated keratocytes, recognized by the visibility of their cell bodies and processes (as well as the nuclei, which are normally visible) in confocal microscopy. These cells probably represent repair fibroblasts,
22 33 which appear 3 days after corneal stromal wounding in the rabbit.
7 The cell bodies and processes are thought to become visible because of a change in their refractive index as the protein composition of the cytosol changes.
34 These cells affect corneal transparency, and they have been found in corneal grafts with late endothelial failure.
25 The amount of cellular activation may have been affected by the depo-corticosteroids given at keratoplasty. The cornea with the greatest amount of keratocyte activation, which extended the full thickness, had a large, persistent epithelial defect. The relationship between keratocyte activation and epithelial healing deserves further study. We did not see another type of corneal stromal cell common in healing wounds, the myofibroblast, although we did not specifically stain for α-smooth muscle actin
35 or search for stress fibers to identify these cells.
36 Myofibroblasts appear in the cornea 14 days after wounding in cats and primates,
3 long after the end of this experiment.
In summary, we developed a human-to-cat xenograft model that allows the histologic study of recently transplanted human corneas. One week after surgery, all three corneal cell types in the central cornea experienced a very low rate of apoptosis, lower than the rate often seen during corneal storage at 4°C.
21 The rate of apoptosis of donor keratocytes near the wound was increased, however. All grafts contained activated keratocytes with visible cell bodies and processes. This model will be useful in the development of new long-term corneal preservation techniques and methods for endothelial cell augmentation, postoperative keratocyte repopulation, and wound healing during the early postoperative period, before xenograft rejection occurs.
The authors thank Cheryl Hann for assistance with transmission electron microscopy.