Dynamic changes in apoptotic and necrotic cell death correlate with severity of ischemia-reperfusion injury in lung transplantation

Ischemia-reperfusion (IR) injury is a major cause of organ dysfunction following lung transplantation. We have recently described increased apoptosis in transplanted human lungs after graft reperfusion. However, a direct correlation between ischemic time, cell death, and posttransplant lung function has not yet been demonstrated. We hypothesized that an increased ischemic period would lead to an increase in cell death, and that the degree and type of cell death would correlate with lung function. To investigate this, we preserved rat lungs at 4 degrees C for 20 min and 6, 12, 18, and 24 h, and then transplanted the lungs and reperfused them for 2 h. Cell viability was determined with a triple staining technique combining trypan blue, terminal deoxynucleotidyl transferase-uridine nucleotide end-labeling, and propidium iodide nuclear staining. Percentages of apoptotic and necrotic cells were calculated from total cell numbers. Following 20 min and 6 and 12 h of cold preservation, less than 2% of graft cells were dead, whereas after 18 and 24 h of cold preservation, 11% and 27% of cells were dead (p < 0.05), the majority of which were necrotic. After transplantation and reperfusion, the mode of cell death changed significantly. In the 6- and 12-h groups, approximately 30% of cells were apoptotic and < 2% were necrotic, whereas in the 18- and 24-h groups, 21% and 29% of cells, respectively, were necrotic and less than 1% were apoptotic. Lung function (Pa-O2) decreased significantly (p < 0.05) with increasing preservation time. The percentage of necrotic cells was inversely correlated with posttransplant graft function (p < 0.0001). The study demonstrates a significant association among cold preservation time, extent and mode of cell death, and posttransplant lung function, and suggests new potential strategies to prevent and treat IR injury.