Metformin Preconditioning and Postconditioning to Reduce Ischemia Reperfusion Injury in an IsolatedEx VivoRat and Porcine Kidney Normothermic Machine Perfusion Model

Metformin may act renoprotective prior to kidney transplantation by reducing ischemia-reperfusion injury (IRI). This study examined whether metformin preconditioning and postconditioning duringex vivonormothermic machine perfusion (NMP) of rat and porcine kidneys affect IRI. In the rat study, saline or 300 mg/kg metformin was administered orally twice on the day before nephrectomy. After 15 minutes of warm ischemia, kidneys were preserved with static cold storage for 24 hours. Thereafter, 90 minutes of NMP was performed with the addition of saline or metformin (30 or 300 mg/L). In the porcine study, after 30 minutes of warm ischemia, kidneys were preserved for 3 hours with oxygenated hypothermic machine perfusion. Subsequently, increasing doses of metformin were added during 4 hours of NMP. Metformin preconditioning of rat kidneys led to decreased injury perfusate biomarkers and reduced proteinuria. Postconditioning of rat kidneys resulted, dose-dependently, in less tubular cell necrosis and vacuolation. Heat shock protein 70 expression was increased in metformin-treated porcine kidneys. In all studies, creatinine clearance was not affected. In conclusion, both metformin preconditioning and postconditioning can be done safely and improved rat and porcine kidney quality. Because the effects are minor, it is unknown which strategy might result in improved organ quality after transplantation

Strict Selection Alone of Patients Undergoing Liver Transplantation for Hilar Cholangiocarcinoma is Associated with Improved Survival

Liver transplantation for hilar cholangiocarcinoma (hCCA) has regained attention since the Mayo Clinic reported their favorable results with the use of a neo-adjuvant chemoradiation protocol. However, debate remains whether the success of the protocol should be attributed to the neo-adjuvant therapy or to the strict selection criteria that are being applied. The aim of this study was to investigate the value of patient selection alone on the outcome of liver transplantation for hCCA. In this retrospective study, patients that were transplanted for hCCA between 1990 and 2010 in Europe were identified using the European Liver Transplant Registry (ELTR). Twenty-one centers reported 173 patients (69%) of a total of 249 patients in the ELTR. Twenty-six patients were wrongly coded, resulting in a study group of 147 patients. We identified 28 patients (19%) who met the strict selection criteria of the Mayo Clinic protocol, but had not undergone neo-adjuvant chemoradiation therapy. Five-year survival in this subgroup was 59%, which is comparable to patients with pretreatment pathological confirmed hCCA that were transplanted after completion of the chemoradiation protocol at the Mayo Clinic. In conclusion, although the results should be cautiously interpreted, this study suggests that with strict selection alone, improved survival after transplantation can be achieved, approaching the Mayo Clinic experience

Strict Selection Alone of Patients Undergoing Liver Transplantation for Hilar Cholangiocarcinoma Is Associated with Improved Survival

Liver transplantation for hilar cholangiocarcinoma (hCCA) has regained attention since the Mayo Clinic reported their favorable results with the use of a neo-adjuvant chemoradiation protocol. However, debate remains whether the success of the protocol should be attributed to the neo-adjuvant therapy or to the strict selection criteria that are being applied. The aim of this study was to investigate the value of patient selection alone on the outcome of liver transplantation for hCCA. In this retrospective study, patients that were transplanted for hCCA between1990 and 2010 in Europe were identified using the European Liver Transplant Registry (ELTR). Twenty-one centers reported 173 patients (69%) of a total of 249 patients in the ELTR. Twenty-six patients were wrongly coded, resulting in a study group of 147 patients. We identified 28 patients (19%) who met the strict selection criteria of the Mayo Clinic protocol, but had not undergone neo-adjuvant chemoradiation therapy. Five–year survival in this subgroup was 59%, which is comparable to patients with pretreatment pathological confirmed hCCA that were transplanted after completion of the chemoradiation protocol at the Mayo Clinic. In conclusion, although the results should be cautiously interpreted, this study suggests that with strict selection alone, improved survival after transplantation can be achieved, approaching the Mayo Clinic experience

Metformin Preconditioning Improves Hepatobiliary Function and Reduces Injury in a Rat Model of Normothermic Machine Perfusion and Orthotopic Transplantation

Background. Preconditioning of donor livers before organ retrieval may improve organ quality after transplantation. We investigated whether preconditioning with metformin reduces preservation injury and improves hepatobiliary function in rat donor livers during ex situ normothermic machine perfusion (NMP) and after orthotopic liver transplantation. Methods. Lewis rats were administered metformin via oral gavage, after which a donor hepatectomy was performed followed by a standardized cold storage period of 4 hours. Graft assessment was performed using NMP via double perfusion of the hepatic artery and portal vein. In an additional experiment, rat donor livers preconditioned with metformin were stored on ice for 4 hours and transplanted to confirm postoperative liver function and survival. Data were analyzed and compared with sham-fed controls. Results. Graft assessment using NMP confirmed that preconditioning significantly improved ATP production, markers for hepatobiliary function (total bile production, biliary bilirubin, and bicarbonate), and significantly lowered levels of lactate, glucose, and apoptosis. After orthotopic liver transplantation, metformin preconditioning significantly reduced transaminase levels. Conclusions. Preconditioning with metformin lowers hepatobiliary injury and improves hepatobiliary function in an in situ and ex situ model of rat donor liver transplantation

Diffuse reflectance spectroscopy accurately quantifies various degrees of liver steatosis in murine models of fatty liver disease

Background: A real-time objective evaluation for the extent of liver steatosis during liver transplantation is currently not available. Diffuse reflectance spectroscopy (DRS) rapidly and accurately assesses the extent of steatosis in human livers with mild steatosis. However, it is yet unknown whether DRS accurately quantifies moderate/severe steatosis and is able to distinguish between micro-and macrovesicular steatosis. Methods: C57BL/6JolaHsd mice were fed wit a choline-deficient l-amino acid-defined diet (CD-AA) or a choline-sufficient l-amino acid-defined control diet (CS-AA) for 3, 8, and 20 weeks. In addition B6. V-Lepob/OlaHsd (ob/ob) mice and their lean controls were studied. A total of 104 DRS measurements were performed in liver tissue ex vivo. The degree of steatosis was quantified from the DRS data and compared with histopathological analysis. Results: When assessed by histology, livers of mice fed with a CD-AA and CS-AA diet displayed macrovesicular steatosis (range 0-74 %), ob/ob mice revealed only microvesicular steatosis (range 75-80 %), and their lean controls showed no steatosis. The quantification of steatosis by DRS correlated well with pathology (correlation of 0.76 in CD-AA/CS-AA fed mice and a correlation of 0.75 in ob/ob mice). DRS spectra did not distinguish between micro-and macrovesicular steatosis. In samples from CD-AA/CS-AA fed mice, the DRS was able to distinguish between mild and moderate/severe steatosis with a sensitivity and specificity of 86 and 81 %, respectively. Conclusion: DRS can quantify steatosis with good agreement to histopathological analysis. DRS may be useful for real-time objective evaluation of liver steatosis during liver transplantation, especially to differentiate between mild and moderate/severe steatosis

Increasing metformin concentrations and its excretion in both rat and porcine ex vivo normothermic kidney perfusion model

INTRODUCTION: Metformin can accumulate and cause lactic acidosis in patients with renal insufficiency. Metformin is known to inhibit mitochondria, while renal secretion of the drug by proximal tubules indirectly requires energy. We investigated whether addition of metformin before or during ex vivo isolated normothermic machine perfusion (NMP) of porcine and rat kidneys affects its elimination.RESEARCH DESIGN AND METHODS: First, Lewis rats were pretreated with metformin or saline the day before nephrectomy. Subsequently, NMP of the kidney was performed for 90 min. Metformin was added to the perfusion fluid in one of three different concentrations (none, 30 mg/L or 300 mg/L). Second, metformin was added in increasing doses to the perfusion fluid during 4 hours of NMP of porcine kidneys. Metformin concentration was determined in the perfusion fluid and urine by liquid chromatography-tandem mass spectrometry.RESULTS: Metformin clearance was approximately 4-5 times higher than creatinine clearance in both models, underscoring secretion of the drug. Metformin clearance at the end of NMP in rat kidneys perfused with 30 mg/L was lower than in metformin pretreated rats without the addition of metformin during perfusion (both p≤0.05), but kidneys perfused with 300 mg/L trended toward lower metformin clearance (p=0.06). Creatinine clearance was not different between treatment groups. During NMP of porcine kidneys, metformin clearance peaked at 90 min of NMP (18.2±13.7 mL/min/100 g). Thereafter, metformin clearance declined, while creatinine clearance remained stable. This observation can be explained by saturation of metformin transporters with a Michaelis-Menten constant (95% CI) of 23.0 (10.0 to 52.3) mg/L.CONCLUSIONS: Metformin was secreted during NMP of both rat and porcine kidneys. Excretion of metformin decreased under increasing concentrations of metformin, which might be explained by saturation of metformin transporters rather than a self-inhibitory effect. It remains unknown whether a self-inhibitory effect contributes to metformin accumulation in humans with longer exposure times.</p

Biliary complications after orthotopic liver transplantation

Purpose of reviewThe incidence, pathogenesis and management of the most common biliary complications are summarized, with an emphasis on nonanastomotic biliary strictures (NAS) and potential strategies to prevent NAS after liver transplantation.Recent findingsNAS have variable presentations in time and localization, suggesting various underlying pathogeneses. Early-onset NAS (presentation within 1 year) have shown to be largely related to ischemia-induced bile duct injury, whereas late-onset NAS [>1 year after orthotopic liver transplantation (OLT)] have more immune-mediated causes. Cytotoxic hydrophobic bile salts and impaired biliary HCO3- secretion may also play a role in the occurrence of NAS. Recently, insufficient biliary epithelial regeneration capacity after transplantation has also been suggested to play a major role in the pathogenesis of NAS. A potential strategy to prevent NAS has been proposed to be preservation by machine perfusion instead of classical static cold storage. Although machine perfusion has been shown to be a better preservation method for the liver parenchyma, efficacy in preventing ischemic injury of the biliary epithelium is largely unknown.SummaryThe potential advantages of machine perfusion are very promising as it may provide better protection of the vulnerable bile ducts against ischemia-reperfusion injury. Clinical trials will be needed to demonstrate the impact of machine perfusion in reducing the incidence of biliary complications, especially NAS, after OLT

Open-book tests:Search behaviour, time used and test scores

<p>Background: Because of the increasing medical knowledge and the focus of medical education on acquiring competencies, the use of open-book tests seems inevitable. Dealing with a large body of information, indicating which kind of information is needed to solve a problem, and finding and understanding that knowledge at the right moment are behaviours that cannot be assessed during closed-book tests.</p><p>Aims: To examine whether there is a relationship between students' search behaviour - using references or not when answering a question - during open-book tests and their test scores.</p><p>Method: Second- (N = 491) and third-year medical students (N = 325) participated in this study. Search behaviour was operationalized as the number of questions for which students consulted their references. Furthermore, we collected data on the time students spent on answering all open-book questions and their test scores. To determine the relations, we calculated Spearman's and Pearson's correlations.</p><p>Results: Second-and third-year students consulted their references for 87% and 73% of the questions and spent 5.0 and 4.3 min on answering an open-book question, respectively. We did not find significant correlations between search behaviour and test scores.</p><p>Conclusion: Both 'well' and 'poorer performing' students often consulted their references. Spending almost 5 min per open-book question in multiple choice format seems to be too much. More research is needed to establish optimal open-book test time and to explore how 'well performing' students use their references during open-book tests.</p>

Similar outcome after transplantation of moderate macrovesicular steatotic and nonsteatotic livers when the cold ischemia time is kept very short

Background: Livers with moderate (30-60%) macrovesicular steatosis have been associated with poor outcome after transplantation. Aim of this study was to examine the outcome after transplantation of livers with moderate macrovesicular steatosis when the cold ischemia time (CIT) is kept very short. Methods: Postoperative outcome of 19 recipients of a moderate steatotic liver were compared with a matched control group of 95 recipients of a nonsteatotic liver graft (1:5 ratio). We studied graft/patient survival rates, incidences of primary nonfunction, postoperative complications (classified according to the Clavien-Dindo classification), first-week postoperative hepatic injury serum markers (AST/ALT), and liver function tests (PT time/bilirubin/lactate). In addition, we studied reversal of graft steatosis in follow-up biopsies. Results: Median CIT in livers with moderate steatosis and in controls was below 8 h in both groups. Although short-and long-term patient/graft survival rates and results of liver function tests were similar, serum markers of hepatic injury and postoperative complications (especially grade IVa) were significantly higher in recipients of a moderate steatotic liver. Reversal of steatosis was seen in 9 of the 11 (82%) recipients with follow-up liver biopsies. Conclusion: Despite the association with severe postoperative complications, moderate macrovesicular steatotic livers can be used successfully for transplantation if the CIT is kept very short