Masato Isobe

Beneficial effects of inducible nitric oxide synthase inhibitor on reperfusion injury in the pig liver.

BACKGROUND Although inhibition of endothelial nitric oxide synthase (eNOS) has been reported to aggravate hepatic ischemia-reperfusion (I/R) injury, the role of inducible nitric oxide synthase (iNOS) has been still unknown. We investigated the role of NO produced by iNOS, and evaluated the effect of an iNOS inhibitor on prolonged warm I/R injury in the pig liver. METHODS Pigs were subjected to 120 min of hepatic warm I/R under the extracorporeal circulation. We investigated the time course of changes in serum and hepatic microdialysate NO2- + NO3- (NOx) and the cellular distribution of eNOS and iNOS by immunohistochemistry, including a double-immunofluorescence technique in combination with confocal laser scanning microscopy. The effect of iNOS inhibitor was also investigated. RESULTS Hepatic I/R induced new nitric oxide production in serum and hepatic microdialysate NOx after reperfusion and severe hepatic damage in the centrilobular region where nitrotyrosine was strongly expressed. Diffuse eNOS expression in sinusoidal endothelium did not differ before and after reperfusion. In contrast, strong iNOS expression in Kupffer cells and neutrophils appeared strongly in the centrilobular region after reperfusion. Pigs with intraportal administration of N(G)-nitro-L-arginine (10 mg/kg) died during the period of ischemia or early in the period of reperfusion with a high mortality rate (80.0%). Intraportal administration of aminoguanidine hemisulfate (10 mg/kg) significantly suppressed nitric oxide production and serum aspartate aminotransferase after reperfusion, inhibited nitrotyrosine expression, and attenuated hepatic damage. CONCLUSIONS These results indicate that hepatic I/R injury is triggered by centrilobular iNOS expression; and attenuated by inhibition of iNOS.

A novel inhibitor of inducible nitric oxide synthase (ONO-1714) prevents critical warm ischemia-reperfusion injury in the pig liver

Background. Recently, a novel inhibitor of inducible nitric oxide synthase, ONO-1714, was developed. We evaluated the effect of ONO-1714 on a critical warm I/R model of the pig liver. Methods. Pigs were subjected to 180 min of hepatic warm I/R under the extracorporeal circulation. We investigated the time course of changes in the serum NO2− + NO3− (NOx), the cellular distribution of endothelial and inducible nitric oxide synthase, thrombocyte-thrombi, and nitrotyrosine by immunohistochemistry. The hepatic tissue blood flow (HTBF) was measured continuously using a laser-Doppler blood flowmeter. Results. ONO-1714 at 0.05 mg/kg improved the survival rate from 54 (control group) to 100%. The serum NOx levels in the ONO-1714 group were significantly lower than those in the control group at 1, 1.5, 2, 3, and 6 hr after reperfusion. The serum aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) levels of the ONO-1714 group were significantly lower than the control group, and the HTBF of the ONO-1714 group was significantly higher than the control group. The formation of thrombocyte-thrombi and nitrotyrosine after reperfusion was significantly lower in the ONO-1714 group. Conclusions. These results indicated that ONO-1714 improved the survival rates and attenuated I/R injury in a critical hepatic warm I/R model of the pig. ONO-1714 will be beneficial for hepatectomy or liver transplantation in the clinical field.

Role of inducible nitric oxide synthase in pig liver transplantation

BACKGROUND Previously, we clarified the role of inducible nitric oxide synthase (iNOS) and the protective effect of an iNOS inhibitor in warm ischemia and reperfusion model. In this study, we investigated whether the same effects would be obtained by iNOS inhibitor in liver transplantation model. MATERIAL AND METHODS Orthotopic liver transplantation was performed in pigs in the usual manner after about 6 h of cold preservation in University of Wisconsin solution. Aminoguanidine hemisulfate (AG) was used as the iNOS inhibitor and AG was administered intraportally at the dose of 10 mg/kg just after reperfusion. Two experimental groups were subjected, control group (n = 10), and AG group (n = 10). We investigated changes of serum nitrite/nitrate (NOx) and aspartate aminotransferase (AST). Expression of iNOS was examined by immunohistochemistry, including a double immunofluorescnce technique in combination with cofocal laser scanning microscopy. RESULTS Serum NOx and AST were significantly lower in the AG group. Histological hepatic damage and thrombocyte thrombi were attenuated in the AG group. Expression of iNOS was recognized strongly at Kupffer cells and neutrophils in the centrilobular region of liver after reperfusion by cofocal laser scanning microscopy. Moreover, iNOS staining was attenuated in AG group compared with control group. CONCLUSIONS These results indicate that hepatic ischemia and reperfusion injury in liver transplantation might be triggered by iNOS expression of Kupffer cells and neutrophils, and attenuated by administration of an iNOS inhibitor. Moreover, AG showed down regulation of iNOS expression after reperfusion.

Structures of the dimeric and monomeric chromanones, gonytolides A-C, isolated from the fungus Gonytrichum sp. and their promoting activities of innate immune responses.

Innate immunity is the front line of self-defense against microbial infection. After searching for natural substances that regulate innate immunity using an ex vivo Drosophila culture system, we identified a novel dimeric chromanone, gonytolide A, as an innate immune promoter from the fungus Gonytrichum sp. along with gonytolides B and C. Gonytolide A also increased TNF-α-stimulated production of IL-8 in human umbilical vein endothelial cells.

Apoptosis and necrosis after warm ischemia-reperfusion injury of the pig liver and their inhibition by ONO-1714.

Background. It is still controversial whether a major mode of cell death during hepatic ischemia-reperfusion (I/R) injuries is apoptosis or necrosis. Moreover, the correlation between these cell deaths and the effects of a novel inducible nitric oxide synthase inhibitor (ONO-1714) has not been studied before. Methods. Pigs were subjected to 180 min of hepatic warm I/R under extracorporeal circulation. The control group was not administered ONO-1714. In the ONO-1714 group, ONO-1714 was administered 5 min before ischemia at a dose of 0.05 mg/kg through a portal vein catheter. The apoptotic and necrotic changes after reperfusion were examined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and hematoxylin-eosin staining. Nitrotyrosine, active caspase-3, and cytochrome c were examined by immunohistochemistry. The plasma NO2- + NO3-, aspartate aminotransferase, and lactate dehydrogenase levels were also examined. Results. In the control group, the frequency of apoptotic cells was only 2.6%; nevertheless, that of necrotic cells was 37% at 24 hr after reperfusion. ONO-1714 significantly attenuated apoptosis and necrosis, the expression of nitrotyrosine, and the increases of the plasma aspartate aminotransferase, lactate dehydrogenase, and NO2- + NO3- levels in the reperfusion phase. Conclusions. A major mode of cell death during hepatic warm I/R injury was necrosis, and apoptosis was not dominant. These necrotic changes were caused by the excess production of peroxynitrite, and ONO-1714 greatly attenuated I/R injuries as the result of inhibition of the peroxynitrite production.

Role of inducible nitric oxide synthase on hepatic ischemia and reperfusion injury

PROTECTION of hepatocytes from ischemia/reperfusion (I/R) injury is an important issue in the field of hepatic surgery, which includes liver resection and transplantation. Nitric oxide (NO), a well-known endotheliumderived relaxing factor, has been implicated in tissue injury as a result of its capacity to react with superoxides and hydroxyl radical, suggesting that it may influence the pathogenesis of I/R injury. However, little is known about the role of inducible nitric oxide synthase (iNOS) in hepatic I/R injury. In the present study, we investigate the role of iNOS on hepatic I/R injury and study the cellular distribution of endothelial nitric oxide synthase (eNOS) and iNOS by immunohistochemistry, including a double-immunofluorescence technique in combination with confocal laser scanning microscopy. The effect of intraportal administration of aminoguanidine (AG), a selective iNOS inhibitor, is also investigated.

New strategy for the antifibrotic therapy with oral administration of FR260330 (a selective inducible nitric oxide synthase inhibitor) in rat experimental liver cirrhosis

Inducible nitric oxide synthase (iNOS) activity is significantly elevated in viral hepatitis, alcoholic cirrhosis, and cholestasis. However, there are few reports on the relationship between iNOS and cirrhosis. Here, we investigated the effects of a new iNOS inhibitor that has been developed for oral administration in an experimental rat liver cirrhosis model. A cirrhotic rat model was developed by long‐term administration of thioacetamide injections. FR260330 is a new, rationally designed, selective iNOS inhibitor that can be administered orally. After 12 weeks of treatment with FR260330, the rats showed inhibition of progressions of cirrhosis, ascites, and splenomegaly as well as a significant reduction in the proportions of connective tissue in the liver. The expression of nitrotyrosine, which indicates the existence of peroxynitrite and nuclear factor‐κB activation, was remarkably decreased in the FR260330 treatment group. In addition, immunohistochemical and Western blot analyses showed that the expression of transforming growth factor‐β1 was remarkably decreased in this group. The present study demonstrates that FR260330 reduces liver fibrosis by the inhibition of transforming growth factor‐β1 and retards the development of cirrhosis. This oral iNOS inhibitor will be a new strategy for the treatment of cirrhosis.

Changes in hepatic venous oxygen saturation in hepatic warm ischemia/reperfusion injury in pigs.

Abstract: To clarify the changes that occur in hepatic venous oxygen saturation (Shvo 2 ) during hepatic ischemia/reperfusion (I/R) injury, we examined the relationship between Shvo 2 , hepatic tissue blood flow (HTBF), and portal vein pressure (PVP) in a warm I/R model using pig livers. Female pigs weighing 18–23 kg were subjected to warm I/R under extracorporeal circulation between the superior mesentric vein and the left jugular vein to avoid portal congestion. The warm ischemic times were 120 min ( n = 4), 180 min ( n = 14), and 240 min ( n = 4). Shvo 2 , HTBF, and PVP were measured after reperfusion. The survival rates of the pigs 3 days after reperfusion were 100% in the 120-min group, 57% in the 180-min group, and 25% in the 240-min group. In the 180-min group, the Shvo 2 was lower in the pigs that died than in those that survived. There was a significant correlation between Shvo 2 and both PVP and HTBF after reperfusion. Histological examination revealed findings of severe I/R injury in pigs with a low Shvo 2 , and mild I/R injury in pigs with a stable Shvo 2 . These observations suggest that the changes in Shvo 2 could reflect the degree of hepatic I/R injury, especially that related to microcirculatory disturbances occurring at the sinusoid levels.

Early detection of graft function using hepatic venous oxygen saturation in pig liver transplantation

In this study, we measured hepatic venous oxygen saturation (Shvo2) in pig liver transplantations in order to evaluate its usefulness as a predictor of early postoperative graft function. Shov2 of the grafts with good function was over 60% after reperfusion, and the mean Shov2 at end of the operations was 69.8+/-6.9%. Shov2 of the grafts with poor function never increased over 60%, and for most of the operation until its end, Shov2 was under 50%. At the end of the operations, the mean Shov2 was 39.7+/-5.5%. Shov2 levels of the grafts with good function were significantly higher than those of the grafts with poor function (P=0.0016). Corresponding with these Shov2 data, glutamic-oxaloacetic transaminase and lactate dehydrogenase levels of grafts with poor function were significantly higher than those of the grafts with good function. Shov2 represents a summation of the hemoglobin oxygen saturation at the venous end of the sinusoids of the liver and indicates adequate hepatic blood flow if the hepatic oxygen is constant. A decrease of Shov2 in poor graft function might indicate a disturbance of microcirculation in the sinusoids. Through the use of Shov2, we are able to recognize conditions of microcirculatory disturbance more quickly than with any other system. In conclusion, Shov2 is a useful indicator for an early and reliable prediction of outcome in liver transplantation.

Successful ex vivo normothermic liver perfusion with purely artificial products using artificial blood

We tried to make an ex vivo functioning liver with an artificial perfusate that consisted of artificial blood in the pig liver. A liver graft from a female pig weighing 20 kg was harvested in the usual manner. The perfusion solution consisted of artificial blood, L-15 medium, distilled water, bovine serum albumin, NaHCO3, NaOH, KCl, human regular insulin, 50% glucose solution, and dexamethasone. The isolated liver was perfused with this oxygenated perfusate through the portal vein at a rate of 300 ml/min for 9 hours. Seven livers were perfused for 9 hours in this system. Five of the livers showed mean oxygen consumption of over 8 ml-O2/min during perfusion. Histological findings showed that the hepatic architecture was almost completely preserved and numerous hepatocytes exhibited PAS-positive cytoplasmic glycogen deposits in these livers. These observations indicate that we have succeeded in developing an ex vivo functioning liver with an artificial perfusate employing artificial blood.