BACKGROUNDThough there have been various methods for harvesting and preserving descemet membrane (DM) and intact endothelium, there is no literature about the morphological evaluation of endothelium after graft preparation for descemet membrane endothelial keratoplasty (DMEK). The aim of this study was to establish and improve a simple method for preparing, preserving, and morphologically evaluating the donor graft for DMEK.

METHODSTo obtain a donor graft, an air bubble was formed by injecting a 29 G needle with 1 ml sterile air into a small edge created outside the Schwalbe line. Another needle was inserted into the bubble through the stroma to aspirate the air or replace half the air with organ culture medium. Trypan blue was used to mark the location for small incision to improve the success rate. Frozen sections were stained with hematoxylin and eosin (HE). Based on the air bubble, DM grafts were divided into four groups: group A (normal control), graft without any operative technique; group B, graft with zero-pressure air bubble; group C, graft with full-pressure air bubble; group D, graft with half-pressure air bubble. The four groups of grafts were preserved for 24 hours to observe the effect of bubbles on cells. The gross and ultrastructure morphologies were evaluated using alizarin red and scanning electron microscopy (SEM), respectively.

RESULTSDonor grafts were harvested via the air bubble technique, facilitated by prior trypan blue staining. HE-stained sections revealed a pure graft without stroma. There were no significant changes under light microscope. In group A, SEM revealed a confluent layer of polygonal endothelium with distributed microvilli exhibiting characteristics of interdigitating junctions. In group B, intercellular borders became thinner. In group C, interdigitations were almost flat and microvilli were observed less frequently. In group D, other than less microvilli, there were minimal changes.

CONCLUSIONSThe donor graft preparation method appears to be effective and convenient. Properly decreasing the air pressure could protect and preserve the endothelium.