Effects of Warm Ischemia on Valve Endothelium

Abstract

Background. This study investigates the time-dependent resistance of the endothelium of porcine aortic and pulmonary valves to different periods of warm ischemia (WIT). Methods. Twenty-five 9-month-old swine were divided after death into five groups of WIT (0, 6, 12, 24, and 36 hours). Aortic and pulmonary valves were removed and a total of 15 aortic and 15 pulmonary valve specimens were obtained for each WIT interval. Valves were then examined for (1) their viability rate by the trypan blue dye exclusion method at light microscopy (percent of viability compared with 0 hours of WIT); (2) ultrastructural signs of irreversible or reversible ischemic damage by transmission electron microscopy (cell disruption, dilation of endoplasmic reticulum, cytoplasmic edema, nuclear and mitochondrial changes); (3) endothelial function by pharmacologic evaluation of both the endothelial-releasing capacity of prostacyclin and the endothelial-dependent dynamic responses to relaxing (acetylcholine from 1 × 10 −10 mol/L to 1 × 10 −4 mol/L) in aortic and pulmonary valve segments precontracted with norepinephrine (1 × 10 −6 mol/L) and contracting ( N G -monomethyl- l-arginine, 1 × 10 −4 mol/L) drugs. Results. Our results showed an endothelial progressive time-dependent ischemic injury, which reached significance after 12 hours of exposure. Viability and functional data indicated that 6 hours of WIT only provoked slight endothelial damage ( p > 0.05 respect to time 0 hours), with signs at transmission electron microscopy consistent with a reversible injury. At 12 hours of exposure, we observed a significant reduction ( p < 0.05) with respect to time 0 of the viability rate of prostacyclin production and of the endothelium-dependent dynamic responses to acetylcholine and N G -monomethyl- l-arginine. These functional impairments, although significant, were not consistent, however, with a complete loss of viability. Transmission electron microscopic observations confirmed the appearance of signs of irreversible injury; nevertheless, some elements were found to be well preserved or presented reversible damage. After 24 hours of WIT, ultrastructural and functional data were consistent with a dramatic decrease compared with controls in endothelial viability and functions ( p < 0.01). Finally, after 36 hours of WIT, there was a subtotal loss of viability, of functions ( p < 0.001) and, at transmission electron microscopic observations, of the endothelial layer of the valves. Conclusions. Our data show that the endothelial cells are resistant to short periods of WIT (up to 6 hours), and suggest that these cells can endure longer exposures, up to 12 hours of warm ischemia. Periods of 24 and 36 hours of WIT provoke progressive irreversible damage.