Impact of machine perfusion after long static cold storage on delayed graft function incidence and duration and time to hospital discharge.

Delayed graft function (DGF) is very high in our center (70%-80%), and we usually receive a kidney for transplant after more than 22 hours of static cold ischemia time (CIT). Also, there is an inadequate care of the donors, contributing to a high rate of DGF. We decided to test whether machine perfusion (MP) after a CIT improved the outcome of our transplant patients. We analyzed the incidence of DGF, its duration, and the length of hospital stay (LOS) in patients who received a kidney preserved with MP after a CIT (hybrid perfusion-HP). We included 54 deceased donors kidneys preserved with HP transplanted from Feb/13 to Jul/14, and compared them to 101 kidney transplants preserved by static cold storage (CS) from Nov/08 to May/12. The median pumping time was 11 hours. DGF incidence was 61.1% vs 79.2% (P = .02), median DGF duration was 5 vs 11 days (P < .001), and median LOS was 13 vs 18 days (P < .011), for the HP compared to CS group. The observed reduction of DGF with machine perfusion did not occur in donors over 50 years old. In the multivariate analysis, risk factors for DGF, adjusted for CIT, were donor age (OR, 1.04; P = .005) and the absence of use of MP (OR, 1.54; P = .051). In conclusion, the use of HP contributed to faster recovery of renal function and to a shorter length of hospital stay.

The effect of n-acetylcysteine on hepatic histomorphology during hypothermic preservation.

PURPOSE: To evaluate the NAC effects on liver hypothermic preservation at different time intervals. METHODS: For this, we used livers of male Wistar rats weighing between 250 and 300 g, undergoing preservation in Ringer solution at 4°C for up to 24 hours. Tissue samples were obtained at four moments of preservation for histological examination by hematoxylin and eosin staining: T0 = beginning of preservation, T12 = 12 hours, T18 = 18 hours and T24 = 24 hours. Will be analyzed vacuolation, hepatic apoptosis by optical microscopy and parenchymal. RESULTS: The results showed a progressive increase in hepatic injury in both groups and showed that NAC was effective at T0. The parenchyma preservation was better in the NAC group and no difference when vacuolization of the cells. CONCLUSION: Hypothermic preservation, over time, causes changes in the hepatic parenchyma with increased apoptosis, loss of architecture, vacuolization, culminating in severe injury. The administration of N-acetylcysteine protects against preservation liver injury.

The effect of n-acetylcysteine on hepatic histomorphology during hypothermic preservation

PURPOSE: To evaluate the NAC effects on liver hypothermic preservation at different time intervals. METHODS: For this, we used livers of male Wistar rats weighing between 250 and 300g, undergoing preservation in Ringer solution at 4°C for up to 24 hours. Tissue samples were obtained at four moments of preservation for histological examination by hematoxylin and eosin staining: T0 = beginning of preservation, T12 = 12 hours, T18 = 18 hours and T24 = 24 hours. Will be analyzed vacuolation, hepatic apoptosis by optical microscopy and parenchymal. RESULTS: The results showed a progressive increase in hepatic injury in both groups and showed that NAC was effective at T0. The parenchyma preservation was better in the NAC group and no difference when vacuolization of the cells. CONCLUSION: Hypothermic preservation, over time, causes changes in the hepatic parenchyma with increased apoptosis, loss of architecture, vacuolization, culminating in severe injury. The administration of N-acetylcysteine protects against preservation liver injury. .

Delivery of the bioactive gas hydrogen sulfide during cold preservation of rat liver: effects on hepatic function in an ex vivo model.

The insults sustained by transplanted livers (hepatectomy, hypothermic preservation, and normothermic reperfusion) could compromise hepatic function. Hydrogen sulfide (H2S) is a physiologic gaseous signaling molecule, like nitric oxide (NO) and carbon monoxide (CO). We examined the effect of diallyl disulfide as a H2S donor during hypothermic preservation and reperfusion on intrahepatic resistance (IVR), lactate dehydrogenase (LDH) release, bile production, oxygen consumption, bromosulfophthalein (BSP) depuration and histology in an isolated perfused rat liver model (IPRL), after 48 h of hypothermic storage (4 °C) in University of Wisconsin solution (UW, Viaspan). Livers were retrieved from male Wistar rats. Three experimental groups were analyzed: Control group (CON): IPRL was performed after surgery; UW: IPRL was performed in livers preserved (48 h-4 °C) in UW; and UWS: IPRL was performed in livers preserved (48 h-4 °C) in UW in the presence of 3.4 mM diallyl disulfide. Hypothermic preservation injuries were manifested at reperfusion by a slight increment in IHR and LDH release compared with the control group. Also, bile production for the control group (1.32 µL/min/g of liver) seemed to be diminished after preservation by 73% in UW and 69% in UW H2S group at the end of normothermic reperfusion. Liver samples analyzed by hematoxylin/eosin clearly showed the deleterious effect of cold storage process, partially reversed (dilated sinusoids and vacuolization attenuation) by the addition of a H2S delivery compound to the preservation solution. Hepatic clearance (HC) of BSP was affected by cold storage of livers, but there were no noticeable differences between livers preserved with or without diallyl disulfide. Meanwhile, livers preserved in the presence of H2S donor showed an enhanced capacity for BSP uptake (k(A) CON = 0.29 min⁻¹; k(A) UW = 0.29 min⁻¹ ; k(A) UWS = 0.36 min ⁻¹). In summary, our animal model suggests that hepatic hypothermic preservation for transplantation affects liver function and hepatic depuration of BSP, and implies that the inclusion of an H2S donor during hypothermic preservation could improve standard methods of preparing livers for transplant.

Analysis of the liver effluent as a marker of preservation injury and early graft performance.

In liver transplantation, the effluent solution, which represents the washout of residual preservation solution, can be collected before reperfusion to determine the release of the markers of endothelial cell injury and damage to the liver. The enzyme activities detected in the washout solution may allow the development of an index that could be clinically valuable for the prediction of early posttransplant graft function. In the present study, we collected liver effluents from 47 livers at the time of graft rinsing to measure liver enzymes (aminotransferases and lactate dehydrogenase) as well as the serum enzyme levels of the recipients for correlation with early postoperative graft viability (1-month survival). The patients were divided into two groups: death (D) and survival (S). Nonparametric statistical analysis was used with the level of significance set at P < .05. Aminotransferases and lactate dehydrogenase levels higher among the D group (P < .05 for all measurements), leading us to conclude that the effluent represents a good marker of preservation injury and early graft performance.

Autonomic dysfunction of rat jejunum submitted to cold ischemic preservation is prevented by heparin.

Previous studies suggest that cold ischemic preservation (CIP) employed in small bowel transplantation promotes loss of intestinal motility due to severe lesions in autonomic enteric nerves. This autonomic dysfunction may be prevented by antioxidant agents. In this work, we investigated whether preservation with heparin prevented autonomic dysfunction of rat jejunum submitted to CIP for a long time. Jejunal segments (2 cm) of Wistar rats (12 to 16 weeks old) were preserved at 4 degrees C in Ringer's lactate solution without (-) or with (+) 100 UI/mL heparin (H). After preservation for 12 hours, H+ and H- preparations were mounted in parallel in isolated organ baths containing 10 mL Tyrode's solution at 37 degrees C for the study of neurogenic contractions evoked by electrical field stimulation (EFS; 10-30 Hz, 1-ms duration, 60 V) or by stimulation of nicotinic (NIC) or muscarinic (carbachol, CCh) cholinoceptors. The effects of NIC (hexamethonium, HEX) and muscarinic (atropine, ATR) antagonists were studied on these contractions. Contractions induced by EFS (30 Hz) were four times greater in H+ (1.02 +/- 0.12 g) versus H- (0.26 +/- 0.07 g), while contractions induced by NIC (1 mmol/L) were also four times higher in H+ (1.07 +/- 0.10 g) than H- (0.25 +/- 0.09 g) preparations. In addition, contractions induced by CCh (1 mmol/L) were two times higher in H+ (1.21 +/- 0.13 g) than in H- (0.65 +/- 0.10 g). EFS, NIC, and CCh contractions were inhibited by pretreatment of jejunum with HEX or ATR (1 mumol/L/30 min), in H+ and H-. These results indicated that addition of heparin to a preservation solution attenuated the autonomic dysfunction of rat jejunum submitted to CIP for a long time.

Atenolol attenuates autonomic dysfunction of rat jejunum submitted to cold ischemic preservation.

In vitro studies have demonstrated that cold ischemic preservation (CIP) employed in small bowel transplantation produces loss of intestinal motility due to severe lesions of autonomic enteric nerves and that this autonomic dysfunction is attenuated by antioxidant agents. In this work, we investigated whether preservation with atenolol attenuated autonomic dysfunction of rat jejunum submitted to long-term CIP. Jejunal segments (2 cm) of Wistar rats (12 to 16 weeks old) were surgically isolated and preserved at 4 degrees C in Ringer's lactate solution without (-) or with (+) 1 mumol/L atenolol (AT). After preservation for 12 hours, AT+ and AT- preparations were mounted in parallel in isolated organ baths containing 10 mL Tyrode's solution at 37 degrees C for the study of neurogenic contractions evoked by electrical field stimulation (EFS; 10 to 30 Hz, 1-ms duration, 60 V) or by stimulation with nicotinic (nicotine, NIC) or muscarinic (carbachol, CCh) cholinoceptor agents as well as nicotine (hexamethonium, HEX) and muscarinic (atropine, ATR) antagonists. Contractions induced by EFS (30 Hz) were 46% higher in AT+ (0.38 +/- 0.02 g) than AT- (0.26 +/- 0.01 g), while contractions induced by NIC (1 mmol/L) were 84% higher in AT+ (0.46 +/- 0.03 g) than in AT- (0.25 +/- 0.02 g). In addition, contractions induced by CCh (1 mmol/L) were 34% higher in AT+ (0.87 +/- 0.06 g) than in AT- (0.65 +/- 0.08 g). EFS-, NIC-, and CCh-induced contractions were inhibited by pretreatment of jejunum with HEX or ATR (1 mumol/30 min), in AT+ and AT-. These results suggest that addition of atenolol in the preservation solution attenuated autonomic dysfunction of rat jejunum submitted to long-term CIP.

Biliary inorganic phosphate as a tool for assessing cold preservation-reperfusion injury: a study in the isolated perfused rat liver model.

Ischemia-reperfusion injury is a major cause of early graft dysfunction after liver transplantation. The bile flow has been suggested as an index of ischemic damage, and severely impaired bile flow seems to be predictive of poor survival in experimental studies. Looking for injury markers, biliary inorganic phosphate has the potential of being a useful endogenous marker of diminished hepatobiliary function because this anion is excreted in the bile by a paracellular pathway and it can detect changes in permeability. The goal of this study was to evaluate the effects of cold preservation-reperfusion of the liver on bile flow and bile inorganic phosphate and their relationship with storage-related graft failure. The isolated and perfused rat liver was used to evaluate the injury for ischemia-reperfusion. The intrahepatic resistance, lactate dehydrogenase release, and potassium and biliary inorganic phosphate concentration were used to estimate viability and function of freshly isolated or cold-preserved livers. The intrahepatic resistance and the bile flow were consistent and significantly decreased throughout the perfusion time in relation to the increment in storage. Inorganic phosphate is more concentrated in bile from preserved livers, showing an alteration in paracellular pathway, confirmed by the biliary excretion of horseradish peroxidase. After preservation, concentration and excretion of the paracellular marker were increased during the first peak. The second peak appears earlier in preserved livers (10 minutes) with a different shape but without changes in concentration. In conclusion, inorganic phosphate in bile shows changes in paracellular permeability as occurs in livers after 48 hours of cold preservation.