Morphologic and Time Dependent Changes of Nitric Oxide Synthesis Following Anoxia and Reoxygenation of the Endothelial Cells of the Umbilical Vein

ABSTRACT

PURPOSE: In order to clarify the exact role of nitric oxide for a ischemia-reperfusion (I/R) injury, we observed morphologic change of endothelial cells and a time dependent change of nitric oxide synthesis following anoxia and reperfusion injuries. METHODS: The experimental groups were divided into 4 sub-groups a control group without any treatment, an anoxia group treated with anoxic air (93% N2, 5% CO2, 2% H2) for 50 minutes, a reoxygenation group treated with 100% O2 for 480 minutes, and an allopurinol group treated with allopurinol immediately prior to reoxygenation. Endothelial cells were isolated from a human fetal umbilical vein and cultured in M-199 medium. We observed a morphological change of the endothelial cells with inverted light microscopy and we studied the time dependent change of nitric oxide synthesis with microelectrode following anoxia and reperfusion injuries. RESULTS: Most significantly, the endothelial cells of the anoxia group were more flattened and detached than those of the control group. A more severe detachment of endothelial cells was found in the reoxygenation group than in the anoxia group. There was no significant difference in the morphological change between the allopurinol group and the anoxia group. Additionally, the concentration of NO in the anoxia group (2511.62 428.60 nM/1 105 cells/ml) was lower than that of the control group (3505.14 192.95 nM/1 105 cells/ml) (P<0.005). The NO concentration of thereoxygenation group reached its highest level of 2953.14 90.98 nM/1 105 cells/ml at 180 minutes (P<0.05) and decreased thereafter. There was no significant difference in the NO concentration between the allopurinol and anoxia groups. CONCLUSION: The morphologic damage of endothelial cells in the reoxygenation group was significantly increased as compared with the anoxia group. Nitric oxide syntheses in the reoxygenation and anoxia groups was decreased as compared with the control group.