Wenshi Gao

Antibiotics prevent liver injury in rats following long-term exposure to ethanol.

BACKGROUND/AIMS Kupffers cells participate in alcohol-induced liver injury, and endotoxemia is observed in human alcoholics and in a rat model. This study evaluated the effect of reducing bacterial endotoxin production by intestinal sterilization on alcohol-induced liver injury. METHODS Male Wistar rats were exposed to ethanol continuously for up to 3 weeks via intragastric feeding. The gut was sterilized with polymyxin B and neomycin. RESULTS Fecal culture of stool samples from ethanol-fed rats treated with antibiotics showed virtually no growth of gram-negative bacteria. Endotoxin levels of 80-90 pg/mL in plasma of ethanol-fed rats were reduced to < 25 pg/mL by antibiotics. Antibiotic treatment also completely prevented elevated aspartate aminotransferase levels and significantly reduced the average hepatic pathological score in rats exposed to ethanol. Oxygen tension on the surface of the liver measured in vivo was decreased significantly from control values of 48 +/- 1 to 39 +/- 1 mumol/L in ethanol-treated rats. This hypoxia was prevented by treatment with antibiotics. Moreover, the increase in rates of ethanol elimination due to long-term ethanol treatment was prevented by antibiotic treatment. CONCLUSIONS Intestinal sterilization prevented alcohol-induced liver injury in the rat, supporting the idea that hypermetabolism and consequent hypoxia caused by activation of Kupffers cells by endotoxin is involved in the mechanism.

Leukocyte Adhesion And Cell Death Following Orthotopic Liver Transplantation In The Rat

The purpose of these experiments was to employ video microscopy to identify early pathological changes after orthotopic liver transplantation in the rat. Liver transplantation was performed using the cuff technique. Survival was greater than 90% when livers were stored for 1 hr in Ringers solution prior to transplantation (survival conditions), whereas all rats died following storage of grafts for 4 hr in cold Euro-Collins solution (nonsur-vival conditions). Postoperatively, each recipient animal was anesthetized, the abdomen was opened, and the liver was placed on the stage of an inverted fluorescence microscope equipped with a low-light ISIT video camera. Precautions were taken to prevent the liver surface from drying. The fluorescent dyes fluorescein sodium (1.0 μmol/kg), acridine orange (2.5 μmol/kg), and propidium iodide (1 μmol/kg) were injected intravenously to label hepatocytes, polymorphonuclear leukocytes, and the nuclei of irreversibly damaged cells, respectively. In livers from untransplanted rats, movement of labeled leukocytes through the hepatic sinusoids was smooth and rapid (velocity, 500–550 μn/sec) and margination (adhesion) of cells was minimal (less than 1%). After transplantation, however, velocity was diminished 2–3-fold, and margination was increased to 10% of leukocytes in survival groups and to 40% in nonsurvival groups 4 hr postoperatively. In the nonsurvival groups, irreversible cell death detected by propidium iodide fluorescence was minimal 15 min after transplantation—however, massive cell damage was detected 4 hr postoperatively. In addition, serum transaminases and necrosis were higher at 4 hr than 15 min postoperatively. Taken together, these data demonstrate that leukocyte margination increases following orthotopic liver transplantation and is followed rapidly by cell death. These events likely play a role in the mechanism of early graft failure following transplantation.

Evidence that free radicals are involved in graft failure following orthotopic liver transplantation in the rat : an electron paramagnetic resonance spin trapping study

&NA; The purpose of these studies was to determine whether free radicals were formed as a consequence of reperfusion during orthotopic liver transplantation and whether their formation was related to graft failure. Grafts were stored for 18 hr in Euro-Collins solution or for 48 hr in University of Wisconsin solution (nonsurvival conditions) and reperfused with blood containing the spin trap α-phenyl N-tert-butylnitrone (PBN). Venous blood samples (4–5 ml) were collected, and serum was extracted with chloroform and methanol (2:1) and analyzed for radical adducts by electron paramagnetic resonance (EPR) spectroscopy. In samples from livers stored under nonsurvival conditions, EPR spectra were detected indicating the presence of PBN radical adducts. In contrast, radical adduct formation was 3− to 4-fold lower in similar experiments performed with untransplanted livers or with livers stored under survival conditions (1 hr in Ringers solution or 24 hr in UW solution). Oxygen radicals are most likely involved in the production of radical adducts because formation was nearly completely prevented by superoxide dismutase plus catalase or Carolina rinse, which contains glutathione, desferrioxamine mesylate, and allopurinol. Radical adduct formation was much greater in a blood-free perfusion system where oxygen delivery was high, suggesting that blood elements are not necessary for radical adduct formation. An inverse correlation between survival of livers stored in UW solution and radical adduct signal was observed in this study. Thus, it is concluded that free radicals formed during reperfusion are involved in the mechanism of graft failure following liver transplantation in the rat.

Carolina rinse solution-a new strategy to increase survival time after orthotopic liver transplantation in the rat

Recently, we described a new solution, Carolina rinse, that prevents nonparenchymal cell injury in vitro after reperfusion of livers stored in University of Wisconsin cold solution (Currin RT, Toole JG, Thurman RG, Lemasters JJ. Transplantation 1990; 50: 1076). The present study was designed to examine the effect of Carolina rinse on graft survival in vivo. Unlike UW cold storage solution, which is high in potassium, Carolina rinse contains extracellular inorganic ions at levels similar to blood, a calcium channel blocker and a radical scavenger. Carolina rinse also contains fructose and mildly acidotic pH to reduce hypoxic cell death. Livers from Lewis rats were explanted, stored in UW cold storage solution under nonsurvival conditions, and rinsed with either 15 ml of Ringers, UW solution, Carolina rinse, or Carolina rinse saturated with nitrogen prior to completion of implantation surgery. In the Ringers rinse group, only 4% of recipients survived 30 days postop-eratively. In this group, SGOT levels reached maximal values of about 5000 U/L. Survival was also poor (25%) when grafts were rinsed with UW solution. In the Carolina rinse group, however, 9 of 16 rats (56%) survived indefinitely, and maximal postoperative SGOT levels were reduced 3-fold. Liver injury indexed histologically was also decreased about 3-fold by Carolina rinse compared with the control group rinsed with Ringers solution. Carolina rinse diminished postoperative sinusoidal endothelial cell damage assessed by electron microscopy and reduced carbon particle phagocytosis due to Kupffer cells significantly. Moreover, Carolina rinse diminished graft swelling and improved postoperative hepatic microcirculation compared with the Ringers rinse group. Taken together, these results indicate that Carolina rinse is a superior alternative to Ringers solution in vivo to protect liver grafts from reperfusion injury when removing high-potassium-containing cold storage solutions clinically prior to implantation.

Development of a new method for hepatic rearterialization in rat orthotopic liver transplantation : reduction of liver injury and improvement of surgical outcome by arterialization

The purpose of this study was to develop a simple new method for rearterialization to compare rearterialized and nonarterialized models of liver transplantation in the rat in the same laboratory with the same surgeon. Hepatic rearterialization in the rat was completed by connecting the proper hepatic artery with a splint of polyethylene tubing (PE 10). With this technique, no extrahepatic arteries or modification of other organs was required. Further, the recipients gastroduodenalartery remained intact to minimize bile duct hypoxia, and the anastomosis was completed rapidly (<3 min). Postoperative arterial thrombosis and bile duct necrosis occurred with low frequencies with this model (<15%). For comparison, the nonarterialized model of Kamada was used. With the nonarterialized method, survival of livers stored for 24 hr in cold University of Wisconsin solution was approximately 85% (6/7), whereas survival of livers stored for 48 hr was poor (<10%; 1/16). Rearterialization improved survival following 48 hr of storage in UW solution from less than 10% to 50% (3/6), and reduced early enzyme release (AST) by about 50%. Rearterialization also reduced enzyme release following 24 hr of cold storage by about 50%. We conclude that this new technique is a simple and reliable method for hepatic artery reconstruction that may be particularly useful in studying the mechanism of primary graft nonfunction. These data are consistent with the hypothesis

Primary nonfunction of fatty livers produced by alcohol is associated with a new, antioxidant-insensitive free radical species

The formation of free radicals after orthotopic liver transplantation in the rat correlates with graft failure. Fatty livers from alcoholics transplant poorly, so these studies were designed to examine the effect of alcohol on free radical formation in a rearterialized rat liver transplantation model. Treatment of rats for 3-5 weeks with either a high-fat or an ethanol-containing liquid diet caused characteristic pericentral lipid accumulation. After storage in University of Wisconsin cold storage solution (UW) and transplantation, a reperfusion injury characterized by increased postoperative AST levels (greater than 1500 U/l in about 3 hours) was observed in rats fed high-fat or alcohol-containing diets, whereas parenchymal cell injury was seen much less in low-fat controls. Survival was around 63% in the low-fat group but decreased to 12 and 18% in the high-fat and alcohol groups, respectively. Furthermore, intracellular lipid content correlated inversely with survival. In untransplanted livers, the spin trap alpha-phenyl N-tert-butylnitrone (PBN) was infused, and blood samples were collected and extracted with chloroform:methanol. Signals indicative of carbon-centered PBN radical adducts were barely detectable in all untransplanted groups studied by electron paramagnetic resonance. In contrast, a robust 6-line complex spectrum was obtained from all groups studied immediately after 48 hours of cold storage in UW solution and transplantation. A mixture of 3 radical species was identified. Two had coupling constants similar to lipid-derived free radicals, whereas the third is a new species with unique coupling constants and is most likely oxygen derived. In low-fat controls, the signal was reduced significantly by superoxide dismutase (SOD)/catalase; however, SOD/catalase had no effect on free radicals in lipid-loaded livers. Thus, both dietary high fat and alcohol exposure produce a unique SOD/catalase-insensitive free radical species that may be involved in the mechanism of failure of fatty livers after orthotopic liver transplantation.

Increase in survival of liver grafts after rinsing with warm Ringer's solution due to improvement of hepatic microcirculation.

Temperature increases membrane fluidity and decreases vascular resistance in isolated organs. Therefore, these studies were designed to determine if a rinse with warm buffer could increase survival time in the rat model of orthotopic liver transplantation by improving hepatic microcirculation. Brief periods of warm ischemia (3-8 min) did not damage the liver as indexed by minimal release of LDH. Survival of rats for 30 days was greater than 90% in this model when livers were stored for 1 hr in Ringers solution; yet grafts stored for 8 hr in Euro-Collins solution and rinsed with 20 ml of cold (0-4 degrees C) Ringers solution survived postoperatively only around 3 days. However, livers stored for 8 hr in Euro-Collins and rinsed with 20 ml of warm (37 degrees C) Ringers survived longer than 30 days (i.e., permanently). Serum transaminase levels reached peak values around 6000 U/L one day postoperatively in the cold-rinsed group, and liver injury assessed histologically was substantial. Under these conditions, pulmonary infiltration of inflammatory cells was observed in about 23% of lung tissue examined and was associated with massive bleeding. Following a warm rinse, however, maximal SGOT levels and injury to both liver and lung were reduced significantly by 80-90% 24 hr postoperatively. Moreover, the warm rinse improved hepatic microcirculation. It accelerated blood flow into the liver approximately two-fold, as indexed by the half-time of changes in hemoglobin reflectance from the liver surface, improved the distribution of colloidal carbon in the organ observed macroscopically, and decreased vascular resistance by over 50%. These data support the hypothesis that a brief rinse of liver grafts with warm buffer markedly improves the hepatic microcirculation, leading to dramatic improvement in graft survival. This work demonstrates clearly that a brief warm rinse may be useful clinically in liver transplantation.

Evidence that adenosine is a key component in Carolina rinse responsible for reducing graft failure after orthotopic liver transplantation in the rat.

Recently, a rinse solution, Carolina rinse, designed to minimize reperfusion injury following liver transplantation in the rat has been developed. When used to rinse cold-stored grafts prior to completion of implantation surgery, Carolina rinse improved postoperative survival dramatically. Here we report the results of studies designed to determine the key components of Carolina rinse. Livers were explanted, stored for 12 hr in cold UW solution (0–4°C), and rinsed with 15 ml of Ringers solution immediately prior to completion of implantation surgery. In this group, 12/13 rats died within 2 days (nonsurvival conditions). In a second group, explants stored under identical conditions were rinsed with 15 ml of Carolina rinse. Carolina rinse increased average 30-day survival time significantly to over 75%. Furthermore, when grafts were rinsed with Carolina rinse lacking nicardipine or with the pH increased to 7.4, long-term survival of recipient rats was also about 75% (i.e., the modifications did not affect survival). However, Carolina rinse lost its efficacy (12% survival) when adenosine was omitted. In addition, when donor livers were rinsed with Ringers solution containing adenosine (0.1 mM), average survival time was increased from 8% to 63%. Rinsing with Ringers solution containing higher concentrations of adenosine (5 mM), however, did not improve survival significantly. Survival was also not improved by rinsing with Ringers containing 0.1 mM ribose and 0.1 mM adenine, substrates for ATP synthesis that are not vasoactive. SGOT values were around 3000 U/L 1–3 days postoperatively in the nonsurviving group rinsed with Ringers solution alone. Values were decreased over 6-fold by Carolina rinse but were not reduced significantly by Ringers solution containing adenosine. Thus, adenosine improves survival following liver transplantation without preventing parenchymal cell injury, indicating that adenosine may work via nonhepatic mechanisms. Liver injury was also assessed by electron microscopy. After either adenosine or Ringers rinse, sinusoidal thrombi and polymorphonuclear margination were observed to gether with a pattern of pericentral hepatocellular vascuolization and disruption of the sinusoidal lining characteristic of changes observed following hypoxia. With Ringers rinse, Kupffer cells exhibited surface irregularity in pericentral regions indicative of activation. Following adenosine rinse, however, Kupffer cells appeared more flattened with less ruffling and reduced surface debris (i.e., they were less activated). Carbon uptake by Kupffer cells was also decreased significantly by Ringers rinse when adenosine was present. Further more, adenosine lowered intracellular free calcium concentration in cultured Kupffer cells and improved hepatic microcirculation postoperatively. Adenosine rinse also affected extrahepatic systems: it reduced postoperative clotting time and diminished lung injury significantly. Therefore, we designed Carolina rinse solution II that contains antioxidants to minimize liver injury and adenosine to improve survival. This new formulation was as effective as the original Carolina rinse at improving survival and minimizing graft injury.

Dual role of Kupffer cell activation and endothelial cell damage in reperfusion injury to livers stored for transplantation surgery.

In rat models of liver preservation, the primary event leading to liver graft failure after cold storage is a reperfusion injury causing damage to sinusoidal endothelial cells and activation of Kupffer cells (KC). After storage for longer than 16 h in University of Wisconsin solution, reperfusion induces rapid endothelial cell killing. Kupffer cell activation also occurs as indicated by cell surface ruffling, degranulation, release of hydrolytic enzymes, generation of oxygen radicals, and increased phagocytosis. Down‐regulation of KC activity with nisoldipine or pentoxifylline improves graft survival. Moreover, pretreatment of donors with small amounts of endotoxin to activate KC causes a drastic reduction of graft survival. Together, KC activation and endothelial damage cause marked microcirculatory disturbances after transplantation characterized by reduced and uneven blood flow and increased leucocyte and platelet adhesion. Such events culminate in inflammation, necrosis and fulminant graft failure. Modification of reperfusion conditions can reduce the extent of injury. In particular, flushing livers with Carolina rinse solution (CRS) at the end of storage reduces endothelial cell killing, suppresses KC activation, improves the microcirculation, and increases graft survival. Active ingredients in CRS include antioxidants (allopurinol, desferrioxamine and glutathione), adenosine and slightly acidic pH (6.5). Other potentially important ingredients are nicardipine, a calcium channel blocker, and fructose, glucose and insulin to promote glycolysis. The cytoprotective amino acid, glycine, further improves the performance of Carolina rinse solution. Reperfusion‐induced changes to nonparenchymal cells play an essential role in damage to livers preserved for transplantation surgery. Understanding the role of sinusoidal endothelial cells and KC in this injury has led to promising new strategies to prolong organ storage and reduce graft failure.

Reperfusion rather than storage injury predominates following long-term (48 h) cold storage of grafts in UW solution: studies with Carolina Rinse in transplanted rat liver.

Both storage injury and reperfusion injury have been reported in association with liver transplantation; however, which predominates is not clear. Therefore, these studies were designed to evaluate whether Carolina Rinse, which minimizes reperfusion injury following orthotopic liver transplantation in the rat, would be effective after long-term (48 h) storage of grafts in University of Wisconsin (UW) cold storage solution where sufficient time for development of storage injury exists. Livers were rinsed with either Ringers solution or Carolina Rinse solution immediately prior to completion of implantation surgery. In the Ringers group, 30-day survival was high following 24 h of cold storage (4/5) but was very low after 48 h (1/16). Importantly, survival was increased significantly (5/14) when grafts were rinsed with Carolina Rinse following 48 h of cold storage. In both groups, parenchymal cells appeared normal by scanning electron microscopy, excluded trypan blue, and released SGOT at values only slightly above the normal range immediately (i.e., less than 5 min) after 48 h of cold storage. However, SGOT values rose steadily during the 1st hour postoperatively following reperfusion in the Ringers rinse group and reached levels around 1,000 U/l. In addition, nonparenchymal cells were not labelled with trypan blue following storage, but significant labelling occurred within 1 h. Both SGOT release and nonparenchymal cell injury were reduced significantly when grafts were rinsed with Carolina Rinse prior to completion of surgery. Liver injury assessed histologically 24 h postoperatively was also reduced about 50% by Carolina Rinse. Oxidative stress appeared to be involved, since radical adducts, most likely of lipid origin, were trapped during the first 5 min after reperfusion with the spin trapping technique and detected by electron paramagnetic resonance spectroscopy. Lipid radical formation was reduced nearly completely on reperfusion by Carolina Rinse. Since Carolina Rinse improved survival of liver grafts following long periods of cold storage and reduced lipid radical formation and hepatocellular injury, we concluded that a reperfusion injury rather than a storage injury predominates following orthotopic transplantation of livers stored for long periods of time in cold UW solution.