Exogenous melatonin increases bile production after cold storage and reperfusion in rat liver.

Melatonin, found at extremely high levels in the bile, is thought to protect bile duct cells from bile toxicity and to prevent oxidative damage to the intestinal epithelium by bile acids (1). It is a powerful scavenger of free radicals at both physiological and pharmacological concentrations, both in vitro and in vivo (2). Exogenous melatonin showed to increase bile production by preserving the functional and energetic status during warm ischemia/reperfusion associated with reduced concentration of TNF-α and to inhibit the expression of iNOS and NO production (3). The rapid restoration of biliary secretion is an important index of hepatic functional restoration after cold ischemic injury (4). Although the use of melatonin in the transplantation field was suggested (5) till now it had not been tested in a liver cold preservation and reperfusion model. If storage time exceeds over 10 to 12 hours, late postransplant complications such as biliary stricture occur in more than 25% of liver transplant recipients (6). Based upon the above premises we used the isolated and perfused rat liver model for studying the effect of melatonin added during reperfusion, as an innovative strategy to control liver damage after hepatic cold preservation. Livers were harvested from male Wistar rats and then flushed with two different preservation solutions, Celsior or UW, and stored for 20 hr at 4°C in the respective solution. Reperfusion (120 min) was performed using a non-recirculating system with oxygenated Krebs-Henseleit buffer at 37°C without and with glucose 5 mM. In some experiments melatonin 100 M was added in the perfusate during the reperfusion period. After ischemic storage we evaluated, in the effluent perfusate, lactate dehydrogenase (LDH), as an index of hepatocyte damage, hyaluronic acid (HA) uptake, as an index of sinusoidal endothelial cell viability and thiobarbituric acid-reactive substances (TBARS). Tissue reduced and oxidized glutathione (GSH; GSSG) were also evaluated. Bile secretion analisis was performed measuring melatonin content and -glutamyl transpeptidase (-GT) levels. LDH release was similar during the all reperfusion period using UW or Celsior solution in absence or presence of melatonin. These results were observed also in presence or absence of 5 mM glucose in the perfusate medium. Bile production was higher when melatonin was added during the reperfusion period either with UW and Celsior solutions also in presence or absence of glucose. Release of -GT to the bile from cholangyocytes was higher in Celsior than in UW perfused liver; the addition of melatonin to the perfusate reduced Celsior-induced enzyme release. Melatonin had no effect on HA uptake and TBARS formation in the perfusate. In addition, no statistically significant difference of GSH/GSSG ratio was detected. Bile flow is an indicator of cell energy status even after prolonged ischemia and it reflects the ability of the liver to genarate ATP during reperfusion (6). Our preliminary data indicate that exogenous melatonin improves liver function during cold preservation and reperfusion, thus confirming its potential in liver transplantation. Further research to evaluate the mechanisms by which melatonin treatment increases bile production either using UW or Celsior preservation solutions is in progress.