A device to measure oxygen consumption during the hypothermic perfusion of the liver.

This work deals with the construction and performance of a device designed to measure the oxygen consumption by the liver during hypothermic perfusion in the rat model. Due to its simple design and the utilization of standard materials, it could serve to determine the role of oxygenation during hypothermic perfusion of the liver. The system consists of a reservoir containing the preservation solution, a peristaltic pump and an internal oxygenator made of silicone tube. A five ports manifold connects the circulation to the liver (inflow), to a hydrostatic manometer and to two sample ports; the liver outflow and temperature sensor or gas calibration. Finally the exit port connects the circulation fluid with an oxygen electrode. The preservation solution is pumped through the liver at a constant pressure (77 i 15 mm H2O) and a perfusion flow of 0.39 - 0.49 mL per min per g liver. To test the system, two to four hours perfusion experiments were performed, at temperatures of 5 and 10 degree C. Two preservation solutions were evaluated: Custodiol and Bes-Gluconate-Sucrose. The solubility of oxygen in the preservation solutions was determined, and the oxygen consumption by preserved rat livers was measured.

Construction and performance of a minibioreactor suitable as experimental bioartificial liver.

This work deals with the construction and performance of a hollow fiber-based minibioreactor (MBR). Due to its simple design and the utilization of standard materials, it could serve as a suitable tool to evaluate the behavior and performance of cold preserved or cultured hepatocytes in bioartificial liver devices. The system consists of 140 fiber capillaries through which goat blood is pumped at a flow of 9 mL/min. The cell compartment contains 90 x 10(6) rat hepatocytes (volume 10 mL) and an internal oxygenator made of silicone tubing. To test the in vitro function of the system, 2-h perfusion experiments were performed, the evolution of hematocrit, plasma and extra-fiber fluid osmolality, and plasma urea and creatinine concentrations were evaluated. The detoxication efficiency of an ammonia overload was tested, showing that the system has enough capacity to remove ammonium. Also, the MBR oxygen transfer capacity to hepatocytes was tested, showing that the cells received an adequate oxygen supply.

Cold preservation of isolated hepatocytes in UW solution: experimental studies on the respiratory activity at 0 degrees C.

To date, little attention has been paid to the role of the gas milieu in preservation solutions and its effect on cell viability. Dissolved O2 in the preservation media may be an important parameter to consider. In this study we polarographically measured the O2 concentration in air-equilibrated UW solution at 0 degrees C, as well as the respiratory activity of isolated hepatocytes cold-preserved in this solution up to 72 hours. To perform measurements at 0 degrees C, it was first necessary to characterize the sensor behavior at low temperatures. We verified that the sensor response is still linear at this temperature but the rate of response is significantly slower. The O2 solubility in UW-air solution at 0 degrees C was determined using a modified physical method and it was 410 microM O2, which, as expected, is lower than the solubility in water at the same temperature (453 microM O2). Isolated hepatocytes cold-stored in UW-air solution retained a measurable respiratory activity during a period of 72 hours. The O2 consumption rate was 0.48 +/- 0.13 nmol/O2/min/10(6) cells, which represents 1% of the control value at 36 degrees C (61.46 +/- 14.61 nmol/O2/min/10(6) cells). The respiratory activity and cell viability were well maintained during the preservation period. At present, preservation conditions need to be improved for cells to remain functionally active. Dissolved O2 may be required for energy re-synthesis but it also leads to an increment in reactive oxygen species. The O2 concentration in the preservation solution should be carefully controlled, reaching a compromise between cell requirement and toxicity.

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.

The benefit of adding sodium nitroprusside (NPNa) or S-nitrosoglutathion (GSNO) to the University of Wisconsin solution (UW) to prevent morphological alterations during cold preservation/reperfusion of rat livers.

Cold liver preservation in the University of Wisconsin solution (UW) followed by reperfusion alters hepatic parenchyma and extra cellular matrix. In this study we analyzed the benefit of adding either 500 microM Sodium Nitroprusside (NPNa) or 100 microM S-nitrosoglutathione (GSNO) as Nitric Oxide (NO) donors to the UW solution to prevent hepatic injury. Wistar adult rat livers were stored in UW solution (0 degrees C) for 48Hs and reperfused (60 minutes) in the isolated perfused rat liver model (IPRL). Untreated livers were used as normal controls. Livers perfused but not preserved were used as controls of reperfusion. Parenchyma damages were evaluated by Hematoxylin-Eosin stain. Picrosirius Red and Gordon-Sweets stains were used for collagen and reticulin networks, respectively. An inmunohistochemistry assay for albumin was used as functional test. Cold preservation step was followed by swollen hepatocytes with "light empty halos" surrounding the nucleus, conserved hepatocyte cords and many rounded endothelial cells. The addition of NPNa or GSNO into UW solution, avoid these alterations. Livers preserved for 48 Hs and then reperfused showed extended areas of vacuolation around central veins, and many endothelial cells were rounded and located inside sinusoidal lumens. The collagen network was disorganized while the reticulin one was less altered. Albumin was distributed preferentially in pericentral areas. On the contrary, livers preserved in presence of NPNa or GSNO did not show vacuolation and both collagen and reticulin networks were unchanged. Albumin was more homogeneously distributed in both groups. In conclusion, the addition of 500 microM NPNa or 100 microM GSNO as a NO donor, improves UW solution properties to preserve rat livers by maintaining the hepatic morphology and avoiding hepatic injury post-cold preservation/reperfusion.

Effect of S-nitrosoglutathione (GSNO) added to the University of Wisconsin solution (UW): II) Functional response to cold preservation/reperfusion of rat liver.

Livers cold preserved in University of Wisconsin (UW) solution followed by reperfusion suffer ischemia/reperfusion injuries. Microcirculation is the primary target of damage, characterized by sinusoidal perfusion failure due, mainly, to morphological changes of sinusoidal endothelial cells. Here, we demonstrated that the addition of S-nitrosoglutathione (GSNO) to the UW solution before cold storage, as a nitric oxide (NO) donor, attenuated hepatic injuries.Wistar adult rat livers were stored in UW solution (0 degrees C-48 hs) and then reperfused during 60 minutes using the Isolated Perfused Rat Model (IPRL). We assayed four GSNO concentration (50, 100, 250 and 500 mM). NO concentration was estimated calculating the amount of nitrite (NO2-) generated in the UW solution. Injuries during cold preservation were established measuring lactate dehydrogenase (LDH) released to the UW solution. Meanwhile, intrahepatic resistance (IR), LDH released to the perfusate, the effluent/perfusate ratio for K+, bile flow, liver glycogen content and sinusoidal endothelial cell morphology were studied after 1 hour of reperfusion in the IPRL system. In cold preserved livers without GSNO, glycogen content was dramatically reduced, IR increased markedly, LDH released was high, bile flow diminished and sinusoidal endothelial cells appeared rounded and detached from perisinusoidal matrix after reperfusion. The presence of 100 mM GSNO prevented the IR rise and LDH release, improved bile production and partially reduced endothelial cells damages. In conclusion, the addition of 100 mM GSNO to UW solution improved hemodynamic and function capacity of cold preserved/reperfused livers.