Quantification of allospecific and nonspecific corneal endothelial cell damage after corneal transplantation

Abstract

Purpose To investigate the effect of host immunity (allospecific) and surgical manipulation (non-allospecific) on corneal endothelial cells (CECs) in corneal transplantation. Methods Draining lymph nodes and grafted C57BL/6 corneas were harvested from syngeneic recipients, allograft acceptors, and allograft rejectors (BALB/c) 1, 3, and 8 weeks after transplantation. We analyzed CEC apoptosis using an ex vivo cornea-in-the-cup assay, and visualized cell-to-cell junctions using immunohistochemical staining (ZO-1). Automatic cell analysis using Confoscan software was used to measure CEC density as well as changes in CEC morphology by quantifying the coefficient of variation in cell size (polymegethism) and shape (pleomorphism). Results The cornea-in-the-cup assay showed that allogeneic acceptor T cells and to an even greater extent rejector T cells (but not syngeneic T cells) induced CEC apoptosis. CEC density after corneal transplantation was significantly reduced in allogeneic acceptors compared with syngeneic grafts (Po0.001), and CEC density was even further reduced in the allo-rejector group compared with the allo-acceptor group. Allogeneic grafts showed a greater increase in the coefficient of variation in cell size (polymegethism) when compared with syngeneic grafts 1 week after transplantation (P ¼ Po0.001). However, pleomorphism was not significantly different between syngeneic and allo-acceptor grafts, indicating that polymegethism (but not pleomorphism or cell density) is a sensitive indicator of the effect of alloimmunity on CECs. Conclusions Our data demonstrate that host alloimmunity rather than surgical manipulation alone is the major cause of CEC damage in corneal transplantation, and such morphologic changes of CECs can be detected before the clinically visible onset of allograft rejection.

Alloimmunity and Tolerance in Corneal Transplantation

“…Although in vitro studies have demonstrated that alloreactive CD4 + T cells induce apoptosis in corneal endothelial cells (84), in vivo application of anti-FasL antibody or Fas-Fc protein does not inhibit CD4 T-cell mediated apoptosis of corneal cells (82). High expression of IFNγ and IL-2 in corneas undergoing rejection further supports the central role of Th1 cells in corneal allograft rejection (85, 86).…”

Vasoactive Intestinal Peptide Promotes Corneal Allograft Survival

“…Such high-risk corneal grafting is associated with a high rate of graft rejection and subsequent failure attributable to endothelial cell loss and decompensation. 10,56 Administration of VIP after transplantation in this high-risk model was associated with a significant decrease in graft opacity scores and a significant increase in long-term corneal allograft survival (85% versus 0% at week 8). The main mechanism of graft protection in this context is likely related to the antiapoptotic effects of VIP, which helps maintain CEnCs.…”

Longitudinal Changes to Tight Junction Expression and Endothelial Cell Integrity in a Mouse Model of Sterile Corneal Inflammation

“…This value corresponds closely with other reports of normal CoV in the mouse cornea. 16 At 24 hours, only CpG-ODN eyes showed a relatively higher proportion of Compared with the naïve group, a reduction in ZO-1 expression was evident in CpG-treated eyes only at 24 hours (P < 0.05); however, this reduction was not statistically significant when compared to the saline group. This effect was attenuated by 1 week (F) and remained unchanged at 4 weeks (I).…”

“…13 For all conditions, the absolute degree of endothelial cell hexagonality was unchanged, supporting the concept that polymegethism is a more sensitive index of corneal endothelial responses to inflammation/injury than the degree of hexagonality (pleomorphism). 16 We recently reported the presence of large, multinucleated giant macrophages in the mouse cornea 1 week after CpG-ODN treatment; however, this was not associated with any overt corneal endothelial dysfunction. 13 In the present study, intact ZO-1 expression, normal CCT, and normal ECD were observed in all groups at 1 week post treatment; however, there was reduced ECD and increased CCT at 4 weeks after CpG-ODN treatment.…”

“…The mean cell area within the ROI was divided by the standard deviation to determine the coefficient of variation (CoV). 16 At each time point, the mean CoV of naïve (untreated) eyes was used to define a threshold value for ''physiological polymegethism.'' The proportion of eyes with a CoV above this criterion in each group was analyzed to quantify the amount of nonphysiological corneal endothelial cell polymegethism.…”