Changes in corneal endothelial apical junctional protein organization after corneal cold storage.
OBJECTIVE
Understanding the mechanisms regulating corneal endothelial permeability during storage and recovery is of critical importance both to improving Eye Banking practices and preventing corneal transplant failure. The goal of this study was to determine the effects of cold storage on the organization of apical junctional complex (AJC) proteins and their relationship to F-actin in corneal endothelium.


METHODS
Immunostaining using antibodies to the AJC proteins, ZO-1, cadherin, and alpha- and beta-catenin was performed on 16 eye bank corneas and four cat corneas after 2-8 days of storage at 4 degrees C in Optisol-GS, and compared with fresh corneas. The 3-D in situ localization of the AJC proteins was then determined by using laser confocal microscopy. AJC organization also was assessed after stored human corneas were further incubated at 37 degrees C in Optisol-GS or in serum-free media.


RESULTS
In normal human and cat corneas, F-actin was organized into dense peripheral bands (DPBs) along the apical cell border. The tight-junction protein, ZO-1, and the adherens junction proteins, cadherin and alpha- and beta-catenin, each formed a uniquely discontinuous hexagonal apical band with the largest gaps occurring at the Y-junctions between adjacent endothelial cells. In stored eye bank and cat corneas, cells lost their normal hexagonal F-actin staining pattern and appeared rounded and distorted, with increased cytoplasmic staining and incomplete and condensed DPBs. Similar distortions were observed in the apical bands of cadherin, catenin, and ZO-1 staining between endothelial cells. Gaps in staining at the endothelial Y-junctions were significantly enlarged; corresponding gaps also were observed with phalloidin staining. These changes were reversed after overnight incubation at 37 degrees C in either serum-free media or Optisol-GS. Quantitative analysis demonstrated a significant increase in the size of the Y-junctional gaps (p < 0.0001) after cold storage of cat corneas as compared with fresh corneas.


CONCLUSIONS
These results suggest that disruption of the F-actin cytoskeleton and AJC may explain, in part, the loss of function (corneal swelling) after prolonged cold storage.
Hsu
J K
JK
Department of Ophthalmology, University of Texas Southwestern Medical Center at Dallas, 75235-9057, USA.
Cavanagh
H D
HD
Jester
J V
JV
Ma
L
L
Petroll
W M
WM
D000818:Animals; D002415:Cats; D015925:Cryopreservation; D004728:Endothelium, Corneal / Q000166:cytology / Q000378:metabolism*; D005125:Eye Banks; D006801:Humans; D007120:Immunochemistry; D008565:Membrane Proteins / Q000378:metabolism*; D018613:Microscopy, Confocal; D019108:Tight Junctions / Q000378:metabolism*
D008565:Membrane Proteins