Viability of the Organ–Cultured Human Cornea Dependent on the Dehydrating Agent, Dextran 500 versus HES 130, a Comparative Study

Abstract

Abstract: : Purpose: The negative influence of Dextran 500 on corneal metabolism and endothelial cell density could be demonstrated in the past. In spite of that it is still used for dehydration of corneal tissue prior to transplantation in European Eye Banks. Taking up HES 130 as a postulated alternative to Dextran 500 the current study was undertaken to investigate possible cytotoxic effects of both agents. Methods: In a paired study 24 human corneas were stored under standardized European Eye Bank conditions for 15 days and dehydrated in Dextran 500 or HES 130 for one day each. Afterwards the corneae were snap–frozen in liquid nitrogen and 3µm cryosections were prepared. Keratocyte death was analyzed immunohistochemically using DNA fragmentation–detecting terminal deoxynucleotidyl transferase deoxy–UTR–nick end labeling (TUNEL) assay. For light–microscopic examination Hematoxylin–Eosin–staining and PAS–procedure were carried out additionally. Results: A strong Tunel–positive reaction could be found throughout all cellular layers of corneas dehydrated in Dextran 500 whereas in corresponding corneas deswollen in HES 130 revealed negative or only delicate staining reaction indicating more viable keratocytes. Beside a distinct corneal edema, the specimens showed a strong unregularity of the corneal lamella, vacuolisation and vesicle–formation of the keratocytes due to dehydration with Dextran 500. In contrast to these findings corneas deswollen in HES 130 showed more physiological conditions and no vacuolisation and vesicle–formation regarding the keratocytes. No differences could be determined for the epithelial–and endothelial–layers Conclusions: The negative effect on corneal metabolism due to deswelling in Dextran 500 could be shown by biochemical data in earlier studies. In the present investigation a real cytotoxic effect of Dextran 500 yielding in DNA–fragmentation indicates a severe deterioration of the corneal keratocytes. This finding arises the question if repairing pathways could compensate this damage long–dated and requires a very close look to the capabilities of the keratocytes. Taking all data into account HES 130 leads to significantly better conditions of the corneal tissue prior to transplantation and should be introduced in eye bank routine in consequence.

Keywords: cornea: storage • cornea: endothelium • cornea: stroma and keratocytes