Apoptosis pathways and role of nitric oxide incorneal endothelial cell death during acute graft rejection

Abstract

Abstract: : Purpose:To look for apoptosis pathways involved in acute corneal endothelial cell death during graft rejection and to evaluate the potential role of nitric oxide in this process. Methods:Corneal buttons (3mm) from Brown–Norway rats were transplanted into Lewis rat recipients. All corneal grafts were rejected 14 days after transplantation. On days 4, 5, 7, 10, 13 and 14 after transplantation, animals were sacrificed and the eyes processed for either flat–mount or for cryo–section of the corneas including the recipient bed and the transplanted graft. DAPI staining and annexin V binding were used on flat–mount corneas to evaluate the integrity of nuclear and cytoplasmic membranes of endothelial cells. Apoptosis was assessed by TUNEL, caspase–3 dependent and LEI(Leucocyte elastase inhibitor) /LDNaseII caspase independent pathways. Identification of infiltrating cells was carried out by immunohistochemical detection of ED1, OX42, NOSII and nitrotyrosine. Results:The graft endothelial cells density remained stable during the first 7 days decreasing markedly from day 10 onward after transplantation. Nuclear fragmentation, annexin–V binding and bleb formation of these endothelial cells were initially detected on day 10 with increased intensity observed later. Apoptosis associated with caspase–3 activity or TUNEL positive reaction was not observed at any time after transplantation in corneal endothelial cells. Nuclear translocation of LEI was initially observed in apoptotic endothelial cells on day 10 after transplantation. The recipient endothelial cells did not show any remarkable changes and their number remained steady in all studied corneas. NOS–II was expressed in infiltrating cells (OX–42 and ED1 positive cells) present within the graft. This expression was closely associated with nitrotyrosine formation in infiltrating cells and in corneal endothelial cells. Conclusions:During the time course of corneal graft rejection, the graft endothelial cells undergo apoptosis. This reaction is caspase 3 independent and TUNEL negative. It is carried out by an alternative pathway driven by an LEI/L–Dnase II, caspase independent, pathway. Peroxynitrite formation through NOS–II induction in infiltrating cells contribute to cell toxicity and programmed cell death of the graft endothelial cells during corneal graft rejection in this model.

Keywords: cornea: endothelium • cornea: basic science • cell death/apoptosis