Comparison of two chemical models to induce hepatic preneoplasia in male Wistar rats.

BACKGROUND: One established model to induce hepatic preneoplasia (HP) (DEN 150) uses diethylnitrosamine (DEN) as initiator agent and 2-acetylaminofluorene (2-AAF) as a promoter drug. In addition, both chemicals cause liver cholestasis and fibrosis. AIM: We compared DEN 150 model with another adapted by us, DEN 200 to simplify the first one and to evaluate the effectiveness of both treatments to induce HP in rats. MATERIAL AND METHODS: Male Wistar rats were divided in 3 groups: controls; DEN 150 (rats received 2 doses of DEN, 150 mg/kg body weight, 2 weeks apart, and then 2-AAF, 20 mg/kg body weight, 4 doses per week during 3 weeks); and DEN 200 (rats received a single dose of DEN 200 mg/kg body weight, and 2 weeks apart 2-AAF, 20 mg/kg body weight, 2 doses per week during 3 weeks). Four hepatic enzymes, prothrombin time percentage, the number of bile ductules, total collagen amount, the number of altered hepatic foci (AHF) per liver and the percentage of liver occupied by foci were analyzed. Results. There were no differences in the number of AHF per liver between treated groups. Rats from DEN 200 group showed a significant diminution in the volume of liver occupied by foci. DEN 200 group had no fibrosis and better hemostatic conditions than DEN 150 group. Both groups developed cholestasis. CONCLUSION: In conclusion, both protocols are good alternatives to induce HP in rats and the new protocol proposed is an effective and a simple methodology to provide subclinic states of liver cancer.

Structural, ultrastructural and functional studies of human cardiac valve allografts that suffered an increment of the cryostorage temperature.

Human cardiac valve allografts (HVAs) suffer injuries during the cryopreservation period. Here, we described structural, ultrastructural and functional damages suffered by HVAs after an increment of their cryostorage temperature (100 degree C). Two experimental groups of pulmonary and aortic HVAs were compared: cryopreserved (HVAcryo) and cryopreserved with temperature changes (HVAΔT). Transmission electron microscopy (TEM) was used to analyze valve fibroblasts and extracellular matrix morphology. Total collagen amount was estimated using two different methods and fibroblast viability was assessed measuring their oxygen consumption rate. Porcine heart grafts valves were used to set the techniques. Disorganized collagen network was seen in HVAΔT by TEM. Fibroblasts showed damages in the cellular membrane and many secretor vesicles. Mitochondria and chromatin were also altered. HVAΔT had less amount of collagen and fibroblasts showed an oxygen consumption rate markedly diminished compared to HVAcryo. The increment of 100 degree C suffered by HVAs caused damages that made them unsuitable for clinical purposes.

Hypothermic preservation of rat liver microorgans (LMOs) in bes-gluconate solution. Protective effects of polyethyleneglycol (PEG) on total water content and functional viability.

We have reported of an alternative solution to preserve hepatocytes that have three key components: gluconate, sucrose and an aminosulfonic acid (BGS solution). In order to extend the use of this solution to organs as the liver, we evaluate the effect of the addition of PEG of 8, 20 and 35 kDa to BG Solution on the total water content and functional viability of rat liver microorgans (LMOs). LMOs were preserved (48 h 0 ºC) in the following solutions: ViaSpan(®); BGS; BG plus 4% PEG 8000 (BG8); BG plus 4% PEG 20.000 (BG20) and BG plus 4% PEG 35.000 (BG35). LDH Release and Total Water Content showed a marked increase in LMOs preserved in BGS. This indicates that, in the absence of PEG, the tissue showed important cell membrane integrity deterioration and was incapable of regulating cell volume. After the preservation period, all groups were reoxygenated (120 min, 37 ºC, KHR) and Total Water Content, Glycogen Content and Oxygen Consumption were determined. After 120 min LMOs preserved in BG35 showed values of Oxygen Consumption similar to controls. On the other hand, LMOs preserved in BG8, BG20 and ViaSpan(®) showed oxygen consumption rates and glycogen content significantly smaller than controls. In conclusion, BG35 was the most effective preservation solution to protect LMOs against cold preservation injury due to ischemia and reoxygenation. It is a good alternative to ViaSpan(®) because of its higher buffer capacity, its best indexes of respiration activity and for being considerably less expensive.

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.

Morphological and biochemical analysis of human cardiac valve allografts after an increment of the cryostorage temperature.

Cryopreserved human cardiac valve allografts could suffer lethal damages if the temperature is elevated during cryostorage. This work describes the functional and morphological alterations suffered by human cardiac valve allografts after a gradual increment of the cryostorage temperature from -147 degrees C to -47 degrees C due to a technical failure. Three experimental groups of human pulmonary and aortic allografts were compared: fresh, cryopreserved (-147 degrees C) and cryopreserved with temperature changes from -147 degrees C up to -47 degrees C and back to -147 degrees C. Fibroblast functionality was studied to asses the degree of valvular damages. Collagen network was also analyzed with bright light field and polarized microscopy; an immunohistochemistry for procollagen I was performed and the MTT colorimetric assay was used to evaluate fibroblast mitochondrial enzymatic activity. Porcine heart grafts valves were used to set the MTT colorimetric assay. With bright light field microscopy, disorganized collagen network was seen together with interstitial edema in cryopreserved groups. Polarized microscopy showed that fresh allografts had abundant collagen type I and III, cryopreserved group had less amount of collagen type I and in allografts that suffered cryopreservation temperature elevation collagen type I synthesis could not been demonstrated. Procollagen I was present in fibroblast cytoplasm of fresh group, but it was diminished in cryopreserved group and was absent in the group that suffered temperature elevation. Temperature changes during the cryopreservation period of human cardiac valve allografts induced fibroblast activity reduction. When the cryopreservation temperature is elevated during cryostorage, fibroblasts lost their functionality and the allografts may be not suitable for transplant.

Effect of cold preservation/reperfusion on glycogen content of liver. Concise review.

Livers cold preserved during variable periods of ischemia suffer functional, morphological and hemodynamic alteration, which are exacerbated when they are reperfused. One important injury is glycogen depletion during cold ischemia/ reperfusion. How liver can restore their energy during reperfusion is related with the preservation time, nutritional status of the donor, and the preservation solution used. However, there are some treatments that help livers to preserve their energy storage. These procedures used drugs or metabolites, which are added to the liver to maintain their glycogen storage during preservation, time and allow the organ to restore its energy during reperfusion. There are several publications where the nutritional status of the donor was studied. There is controversy about the quality or the donor organ. Some authors say that fasted animals are better donors because this condition reduced hepatic injuries; others think that fed animals provide the necessary glycogen (energy) to improve liver preservation, reducing morphological and functional damages. Many others are convinced that nutritional status of the donor is not relevant because hepatic injuries will occurred even though the donor was fed or not. The preservation solution has an important role in reducing liver damages during cold ischemia/reperfusion and in restoring liver energy storage. Storage in HLR (histidine, lactobionate and raffinose) solution facilitated the resuscitation of energetic status and preserved adenine nucleotide levels significantly greater than Marshall's citrate or Bretschneider's histidine-based solution (HTK). University of Wisconsin (UW) proved to be suitable for energy recovery during reperfusion. In conclusion, the aim of this review is to present studies performed by different authors where they analyzed preservation/reperfusion injuries, how the liver restores its energy storage during reperfusion time, different strategies to avoid glycogen depletion during cold ischemia/reperfusion, the efficacy of preservation solutions and the effect of nutritional status of the donor to prevent functional alteration of the liver during 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.

Engraftment and function of intrasplenically transplanted cold stored rat hepatocytes.

Hepatocellular transplant may potentially be efficacious for the treatment of selected liver metabolic disorders and acute hepatic failure. On the other hand, the use of hepatocyte cold preservation techniques in these transplantation protocols would allow to have available cells at the right time and place and, consequently, make an optimal use of scarce human hepatocytes. In our experiments we evaluated the biodistribution and functionality of cold preserved hepatocytes transplanted in the spleen of syngeneic rats. Isolated hepatocytes were labeled with the fluorescent dye 5(6)-carboxyfluorescein diacetate succinimidyl-ester, cold-preserved in modified University of Wisconsin (UW) solution for 48 or 96 h, and then transplanted into the spleen. Recipient animals were euthanized at 0 and 3 h, and at 1, 2, 3, 5, 10, and 14 days after transplantation for tissue analysis. Labeled hepatocytes were clearly identifiable in the recipient tissues up to 14 days later. Fluorescence microscopy also showed no significant differences in biodistribution when either cold stored or freshly isolated hepatocytes were transplanted. In addition, functional activity of transplanted cells was demonstrated by immunohistochemical detection of albumin at levels comparable to those found in normal hepatocytes. Our findings establish that cold preserved hepatocytes appear morphologically and biochemically normal after intrasplenic transplantation. Consequently, it indicates that modified UW solution makes it possible to safety preserve hepatocytes for up to 96 h before transplantation, perhaps providing sufficient time for hepatocyte allocation and potential recipient preparation, if applicable clinically.

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.