Important Constituents of Heavy Water-containing Solution for Cold Storage and Subsequent Reperfusion on an Isolated Perfused Rat Liver.

The University of Wisconsin (UW) solution is the most effective preservation solution currently used; however, to safely use expanded-criteria donor grafts, a new cold storage solution that alleviates graft injury more effectively is required. We prepared a heavy water (D2O)-containing buffer, Dsol, and observed strong protective effects during extended cold storage of rat hearts and livers. In the current study, we modified Dsol (mDsol) and tested its efficacy. The aim of the present study was to determine whether mDsol could protect the rat liver more effectively than the UW solution and to clarify the roles of D2O and deferoxamine (DFX). Rat livers were subjected to cold storage for 48 hours in test solutions: UW, mDsol, mDsol without D2O or DFX (mDsol-D2O[-], mDsol-DFX[-]), and subsequently reperfused on an isolated perfused rat liver for 90 minutes at 37°C. In the UW group, the liver was dehydrated during cold storage and rapidly expanded during reperfusion. Accordingly, the cumulative weight change was the highest in the UW group, together with augmented portal veinous resistance and ALT leakage and decreased oxygen consumption rate and bile production. These changes were significantly suppressed in the mDsol-treated group. In the mDsol-D2O(-) and mDsol-DFX(-) groups offered partial protection. In conclusion, mDsol appeared to be superior to the UW solution for simple cold storage of the rat liver, presumably due to improved microcirculation in the early phase of reperfusion. Both heavy water and deferoxamine are essential for alleviating seamless organ swelling that occurs during cold storage and subsequent reperfusion.

Exploration of Optimal pH in Hypothermic Machine Perfusion for Rat Liver Grafts Retrieved after Circulatory Death.

Ex vivo hypothermic machine perfusion (HMP) is a strategy for controlling ischemia-reperfusion injury in donation after circulatory death (DCD) liver transplantation. The pH of blood increases with a decrease in temperature and water dissociation, leading to a decrease in [H+]. This study aimed to verify the optimal pH of HMP for DCD livers. Rat livers were retrieved 30 min post-cardiac arrest and subjected to 3-h cold storage (CS) in UW solution (CS group) or HMP with UW-gluconate solution (machine perfusion [MP] group) of pH 7.4 (original), 7.6, 7.8, and 8.0 (MP-pH 7.6, 7.8, 8.0 groups, respectively) at 7-10 °C. The livers were subjected to normothermic perfusion to simulate reperfusion after HMP. All HMP groups showed greater graft protection compared to the CS group due to the lower levels of liver enzymes in the former. The MP-pH 7.8 group showed significant protection, evidenced by bile production, diminished tissue injury, and reduced flavin mononucleotide leakage, and further analysis by scanning electron microscopy revealed a well-preserved structure of the mitochondrial cristae. Therefore, the optimum pH of 7.8 enhanced the protective effect of HMP by preserving the structure and function of the mitochondria, leading to reduced reperfusion injury in the DCD liver.

Warm Ischemia Induces Spatiotemporal Changes in Lysophosphatidylinositol That Affect Post-Reperfusion Injury in Normal and Steatotic Rat Livers.

Warm ischemia-reperfusion injury is a prognostic factor for hepatectomy and liver transplantation. However, its underlying molecular mechanisms are unknown. This study aimed to elucidate these mechanisms and identify the predictive markers of post-reperfusion injury. Rats with normal livers were subjected to 70% hepatic warm ischemia for 15, 30, or 90 min, while those with steatotic livers were subjected to 70% hepatic warm ischemia for only 30 min. The liver and blood were sampled at the end of ischemia and 1, 6, and 24 h after reperfusion. The serum alanine aminotransferase (ALT) activity, Suzuki injury scores, and lipid peroxidation (LPO) products were evaluated. The ALT activity and Suzuki scores increased with ischemic duration and peaked at 1 and 6 h after reperfusion, respectively. Steatotic livers subjected to 30 min ischemia and normal livers subjected to 90 min ischemia showed comparable injury. A similar trend was observed for LPO products. Imaging mass spectrometry of normal livers revealed an increase in lysophosphatidylinositol (LPI (18:0)) and a concomitant decrease in phosphatidylinositol (PI (18:0/20:4)) in Zone 1 (central venous region) with increasing ischemic duration; they returned to their basal values after reperfusion. Similar changes were observed in steatotic livers. Hepatic warm ischemia time-dependent acceleration of PI (18:0/20:4) to LPI (18:0) conversion occurs initially in Zone 1 and is more pronounced in fatty livers. Thus, the LPI (18:0)/PI (18:0/20:4) ratio is a potential predictor of post-reperfusion injury.

Combination of Cold Storage in a Heavy Water-Containing Solution and Post-Reperfusion Hydrogen Gas Treatment Reduces Ischemia-Reperfusion Injury in Rat Livers.

We previously reported the efficacy of cold storage (CS) using a heavy water-containing solution (Dsol) and post-reperfusion hydrogen gas treatment separately. This study aimed to clarify the combined effects of these treatments. Rat livers were subjected to 48-hour CS and a subsequent 90-minute reperfusion in an isolated perfused rat liver system. The experimental groups were the immediately reperfused control group (CT), the CS with University of Wisconsin solution (UW) group, the CS with Dsol group, the CS with UW and post-reperfusion H2 treatment group (UW-H2), and the CS with Dsol and post-reperfusion H2 group (Dsol-H2). We first compared the Dsol-H2, UW, and CT groups to evaluate this alternative method to conventional CS. The protective potential of the Dsol-H2 group was superior to that of the UW group, as evidenced by lower portal venous resistance and lactate dehydrogenase leakage, a higher oxygen consumption rate, and increased bile production. Multiple comparison tests among the UW, Dsol, UW-H2, and Dsol-H2 groups revealed that both treatments, during CS and after reperfusion, conferred a similar extent of protection and showed additive effects in combination therapy. Furthermore, the variance in all treatment groups appeared smaller than that in the no-treatment or no-stress groups, with excellent reproducibility. In conclusion, combination therapy with Dsol during CS and hydrogen gas after reperfusion additively protects against graft injury.

Rapid and Reliable Steatosis Rat Model Shrsp5-Dmcr for Cold Storage Experiment.

Interventions for liver grafts with moderate macrovesicular steatosis have been important in enlarging donor pools. Here, we tested a high-fat and cholesterol (HFC) diet to create a steatosis model for cold hepatic preservation and reperfusion experiments. The aim of the present study was to assess the steatosis model's reliability and to show the resulting graft's quality for cold preservation and reperfusion experiment. Male SHRSP5-Dmcr rats were raised with an HFC diet for up to 2 weeks. The fat content was evaluated using magnetic resonance imaging (MRI) proton density fat fraction (PDFF). The nonalcoholic fatty liver disease activity score (NAS) was evaluated after excision. Steatosis created by 2 weeks of HFC diet was subjected to 24-hour cold storage in the University of Wisconsin and the original test solution (new sol.). Grafts were applied to isolated perfused rat livers for simulating reperfusion. The NAS were 2.2 (HFC 5 days), 3.3 (HFC 1 week), and 5.0 (HFC 2 weeks). Ballooning and fibrosis were not observed in any group. An MRI-PDFF showed 0.2 (HFC 0 days), 12.0 (HFC 1 week), and 18.9 (HFC 2 weeks). The NAS and MRI-PDFF values correlated. Many indices in the isolated perfused rat liver experiment tended to improve in the new sol. group but were insufficient. Although the new sol. failed to be effective, it acted at multiple sites under difficult conditions. In conclusion, the HFC diet for 2 weeks in SHRSP5-Dmcr rats, together with MRI-PDFF evaluation, is a reliable method for creating simple steatosis and provides good-quality cold preservation and reperfusion experiments.

Hypothermic Machine Perfusion with Hydrogen Gas Reduces Focal Injury in Rat Livers but Fails to Restore Organ Function.

BACKGROUND: We have previously reported the efficacy of post-reperfusion H2 gas treatment in cold storage (CS) and subsequent reperfusion of the rat liver. The present study aimed to evaluate the effect of H2 gas treatment during hypothermic machine perfusion (HMP) in rat livers retrieved from donation after circulatory death (DCD) and elucidate the mechanism of action of H2 gas. METHODS: Liver grafts were procured from rats after 30 min of cardiopulmonary arrest. The graft was subjected to HMP for 3 hours at 7°C using Belzer MPS with or without dissolved H2 gas. The graft was reperfused using an isolated perfused rat liver apparatus at 37°C for 90 minutes. Perfusion kinetics, liver damage, function, apoptosis, and ultrastructure were evaluated. RESULTS: Portal venous resistance, bile production, and oxygen consumption rates were identical in the CS, MP, and MP-H2 groups. Liver enzyme leakage was suppressed by MP (vs control), whereas H2 treatment did not show a combination effect. Histopathology revealed poorly stained areas with a structural deformity just below the liver surface in the CS and MP groups, whereas these findings disappeared in the MP-H2 group. The apoptotic index in the CS and MP groups was high but decreased in the MP-H2 group. Mitochondrial cristae were damaged in the CS group but preserved in the MP and MP-H2 groups. CONCLUSIONS: In conclusion, HMP and H2 gas treatment are partly effective in DCD rat livers but insufficient. Hypothermic machine perfusion can improve focal microcirculation and preserve mitochondrial ultrastructure.

Two-time perforation of the ileal J-pouch 6 and 18 years after restorative proctocolectomy and ileal pouch-anal anastomosis for familial adenomatous polyposis: a case report.

BACKGROUND: Perforation of the ileal J-pouch after restorative proctocolectomy and ileal pouch-anal anastomosis are extremely rare. There has been no report of perforation of the ileal J-pouch occurring twice over several years. We report the first case of perforation at 6 and 18 years following restorative proctocolectomy. CASE PRESENTATION: The patient was a 52-year-old man who underwent a two-stage restorative proctocolectomy with a hand-sewn ileal J-pouch anal anastomosis due to familial adenomatous polyposis and sigmoid colon cancer at 34 years of age. At the age of 40, he underwent ileal pouch resection at its blind end, abdominal drainage, and anastomotic dilatation. The patient had a perforation of the blind end of the ileal J-pouch from increased intraluminal pressure, with anastomotic stricture and pervasive peritonitis. The patient had no symptoms for a few years; however, 18 years after the initial surgery and 12 years after the first perforation, the patient presented with severe abdominal pain. Computed tomography demonstrated pneumoperitoneum; accordingly, laparotomy was performed. Upon opening the abdominal cavity, contaminated ascites and inflammatory changes were documented involving the ileum. A 2-mm perforation involving the blind end of the ileal J-pouch was also observed and repaired, followed by temporary loop ileostomy creation. Postoperative endoscopy revealed an ulcer in the ileal J-pouch and a stricture located directly at the anastomosis. CONCLUSIONS: The blind end of the J-pouch repeatedly perforated over the years due to recurrent anastomotic stricture. Regular surveillance is, therefore, considered necessary for the release of stricture, maintenance of anastomotic patency, and prevention of ileal J-pouch perforation.

Imaging Mass Spectrometry Reveals the Changes in the Taurine Conjugates of Dihydroxycholanoic Acid During Hepatic Warm Ischemia and Reperfusion in a Rat Model.

Warm ischemia and reperfusion injury (IRI) is a prognostic factor in donation after cardiac death donor transplantation. However, a reliable method to predict IRI before transplantation has not been established. The aim of this study was to identify predictive markers of hepatic IRI by simultaneous measurement of endogenous molecules using matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS). Rats were subjected to hepatic warm ischemia (70%) for 30 or 90 minutes and subsequent reperfusion. The livers were collected at the end of ischemia and 1 hour, 6 hours, and 24 hours after reperfusion. The liver tissue sections were applied to IMS (m/z 200-2000). Candidate molecules were identified by tandem mass spectrometry. Imaging mass spectrometry (IMS) revealed a significant increase in the taurine conjugates of dihydroxycholanoic acid (TDHCA) during ischemia and a tendency to return to the basal level after reperfusion. Notably, high-resolution measurements revealed focal accumulation of TDHCA in the intrahepatic bile duct with ischemic time. In conclusion, IMS is a useful method to detect minute changes provoked by ischemia, which are barely detectable in assays involving homogenization. Accordingly, focal accumulation of TDHCA during ischemia may be a candidate marker for predicting later IRI.

Heavy Water (D2O) Containing Preservation Solution Reduces Hepatic Cold Preservation and Reperfusion Injury in an Isolated Perfused Rat Liver (IPRL) Model.

BACKGROUND: Heavy water (D2O) has many biological effects due to the isotope effect of deuterium. We previously reported the efficacy of D2O containing solution (Dsol) in the cold preservation of rat hearts. Here, we evaluated whether Dsol reduced hepatic cold preservation and reperfusion injury. METHODS: Rat livers were subjected to 48-hour cold storage in University of Wisconsin (UW) solution or Dsol, and subsequently reperfused on an isolated perfused rat liver. Graft function, injury, perfusion kinetics, oxidative stress, and cytoskeletal integrity were assessed. RESULTS: In the UW group, severe ischemia and reperfusion injury (IRI) was shown by histopathology, higher liver enzymes leakage, portal resistance, and apoptotic index, oxygen consumption, less bile production, energy charge, and reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio (versus control). The Dsol group showed that these injuries were significantly ameliorated (versus the UW group). Furthermore, cytoskeletal derangement was progressed in the UW group, as shown by less degradation of α-Fodrin and by the inactivation of the actin depolymerization pathway, whereas these changes were significantly suppressed in the Dsol group. CONCLUSION: Dsol reduced hepatic IRI after extended cold preservation and subsequent reperfusion. The protection was primarily due to the maintenance of mitochondrial function, cytoskeletal integrity, leading to limiting oxidative stress, apoptosis, and necrosis pathways.

Intraductal tubulopapillary neoplasm of the pancreas presenting as recurrent acute pancreatitis: A case report.

INTRODUCTION: The 2010 World Health Organization classification of intraductal neoplasms of the pancreas includes intraductal tubulopapillary neoplasms (ITPNs) and intraductal papillary mucinous neoplasms, the latter being a rare and new concept. ITPN sometimes cause acute pancreatitis; therefore, distinguishing ITPN from idiopathic acute pancreatitis is important but challenging. PRESENTATION OF CASE: We present the case of a 72-year-old male who had recurrent pancreatitis for the past 2 years, his diagnosis was idiopathic acute pancreatitis. He was admitted to our hospital with severe acute pancreatitis and cholangitis due to intrapancreatic bile duct stenosis. After the treatment of cholangitis, contrast-enhanced computed tomography revealed a tumor at the pancreatic head. Endoscopic retrograde cholangiopancreatography (ERCP) showed stenosis of the main pancreatic duct and distal bile duct, and adenocarcinoma was detected using brush cytology of the bile duct stricture and pancreatic juice. The patient was diagnosed with invasive ductal carcinoma and pancreaticoduodenectomy was performed. Histopathological findings revealed dilation of the pancreatic duct, and proliferation of columnar cells and cuboid epithelial cells in the main pancreatic duct of the pancreatic head. Mucus production was poor, and immunostaining results revealed ITPN. The patient is alive and do not exhibit signs of recurrence for 12 months. DISCUSSION: ITPNs can cause acute pancreatitis, which can be challenging to preoperatively diagnose. ITPNs presenting as acute pancreatitis are rare, with reported only 5 cases. CONCLUSION: It is important to be keep in mind that there is a possibility of ITPN after diagnosis of idiopathic acute pancreatitis.

Petersen's hernia after living donor liver transplantation.

BACKGROUND: Hepaticojejunostomy may be used for biliary reconstruction in certain cases of liver transplantation. In this occasion, Roux-en-Y biliary reconstruction is predominantly performed. Petersen's hernia is an internal hernia that can occur after Roux-en-Y reconstruction, and it may lead to extensive ischemic changes affecting incarcerated portions of the small bowel or Roux limb resulting in severe complications with a poor prognosis. CASE PRESENTATION: The present case was a 44-year-old male who underwent living donor liver transplantation (LDLT) for familial amyloid polyneuropathy and in whom biliary reconstruction was performed with Roux-en-Y hepaticojejunostomy. Two years after liver transplantation, symptomatic bowel strangulation was diagnosed by CT examination and emergent surgery was performed accordingly. On exploration, an ischemic limb associated with Petersen's hernia was observed. Although repositioning of the incarcerated bowel loop gradually improved the color of the limb, the limb failed to completely recover to a normal color. To allow accurate evaluation for the viability of the limb, we decided to perform a second-look operation after 48 h. On re-exploration, the surface of the limb remained a dark color; however, intraoperative endoscopic findings revealed only partial necrosis of the mucosa. Next, we resected the portion of ischemic damaged limb only following side-to-side jejunojejunostomy. Consequently, redoing of biliary reconstruction could be avoided and the original hepaticojejunostomy site was preserved. Although the stricture of the remnant Roux limb occurred 1 month thereafter, it was successfully managed by balloon dilation via percutaneous transhepatic biliary drainage route. CONCLUSIONS: The occurrence of Petersen's hernia should always be considered in cases of liver transplantation with Roux-en-Y biliary reconstruction. On the basis of an accurate assessment of the extent of jejunal limb injury, reanastomosis of the hepaticojejunostomy, a potentially high-risk surgical procedure, can be avoided in emergent situations.