Can the corneal endothelium of the pig proliferate in vivo

Purpose To further validate a recently developed pre-clinical model of corneal graft rejection in the inbred minipig, we have tested whether the corneal endothelium of the pig has proliferative capacity in situ. Methods Eyes of pigs were enucleated at slaughter, corneas were removed aseptically and subjected to freezing injury. They were cultured in vitro in OPTI-MEM with 5% serum, endothelial side up, for up to 8 days. On days 0, 2, 4 or 8, corneas were washed, fixed and subjected to a silver stain to determine endothelial cell migration into the wound, or to Ki67 immuno-labelling to determine whether endothelial cells had entered the cell cycle. Corneas of transplanted minipigs were monitored post-rejection to determine whether graft opacity and oedema diminished. Results By day 8 of in vitro culture there was up to 6-fold enlargement of endothelial cells on the periphery of the wound, but minimal migration of cells into the wound. Ki67+ endothelial cells were absent at harvest (n=5) and on day 1 of culture (n=3). Scattered isolated and occasional small clumps of Ki67+ cells were evident on day 2 (n=6), with no difference between injured and non-injured corneas. Such cells were interpreted to be detached epithelial or endothelial cells, because they were not associated with the wound margin, appeared to be above the cells in the endothelial monolayer and had smaller nuclei. Results to date of clinical post-graft monitoring reveal that corneal opacity and oedema did not improve by 37 days after maximal leukocyte infiltration, i.e. 50 days after onset of clinical rejection. Conclusion The pig corneal endothelium appears to have minimal capacity to proliferate in vivo. This model would therefore be valuable for testing anti-rejection therapies.