Search

We have previously demonstrated that fresh or cryopreserved xenogeneic hepatocytes manually macroencapsulated in AN69 polymer and transplanted intraperitoneally in rats were able to improve the survival rate after 95% hepatectomy without immunosuppression. In addition, we developed a semiautomatic device where porcine hepatocytes were coextruded with AN69 hydrogel in order to macroencapsulate large amounts of cells. The purpose of the present study was to 1) test whether transplanted porcine hepatocytes macroencapsulated in this device remained functional as evaluated by their ability to prevent death from acute liver failure, and 2) compare the efficiency of cryopreserved or freshly isolated hepatocytes. Fresh or cryopreserved porcine hepatocytes were macroencapsulated in the semiautomatic device by coextrusion in AN69 polymer in 2-m minitubes containing 6 × 107 cells. Acute liver failure was induced in rats by two-step 95% hepatectomy. At the time of completion of liver resection, rats were either not transplanted with minitubes (control group I, n = 13), or were implanted with two minitubes containing culture medium (control group II, n = 11), hepatocytes killed by heat treatment (control group III, n = 10), coextruded fresh hepatocytes (group IV, n = 11), or coextruded cryopreserved hepatocytes (group V, n = 11), without immunosuppression. The survival rate at day 7 was between 0% and 31% in the three control groups. By contrast, coextruded fresh hepatocytes significantly improved the survival rate (group IV, 82%) as did cryopreserved cells (group V, 91% survival). In surviving rats, minitubes were explanted after 20 days: either fresh or cryopreserved hepatocytes appeared morphologically viable and their ultrastructure was preserved. Their detoxification capacities evaluated by the activity of the cyt P450 CYP3A4 were partly maintained. In conclusion, porcine hepatocytes macroencapsulated by coextrusion using a semiautomatic device and transplanted without immunosuppression were able to prevent death from acute liver failure in rats. Cryopreserved cells were as efficient as fresh hepatocytes.

We have developed a culture model to assess antifibrotic drugs using normal human liver myofibroblasts (HLMFs) obtained from 31 subjects. Activation was evaluated in terms of α-smooth muscle actin (α-SMA) and collagen 1 (Coll1) expression using RT-PCR, and proliferation as the uptake of 5-ethynil-2'-deoxyuridine. Under analysis of variance, between-subject differences accounted for 70% of all variability and inter-experiment differences for 30%. The sensitivity of the model was determined by quantifying the effects in terms of relative expression, which were 0.74±0.03 for cyclosporine A (CsA) and 2.4±0.10 for transforming growth factor-beta (TGF-β) (P<0.0001 vs no treatment) for α-SMA expression. Inter-subject variations in α-SMA and Coll1 expression enabled the classification of subjects as potentially low or high fibrosers. Finally, we observed that pirfenidone (which has beneficial effects in vivo) significantly reduced the expressions of α-SMA and Coll1, whereas the angiotensin-converting enzyme inhibitor losartan (which has no effect in vivo) had no significant effect. Our model may thus detect the antifibrotic properties of drugs. Antifibrotic drugs with promising clinical relevance could possibly be selected using a bank of HLMFs from high fibrosers.

Hepatocellular carcinoma (HCC) displays a striking resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Therefore, the characterization of pharmacological agents that overcome this resistance may provide new therapeutic modalities for HCC. Here, we examined whether glycogen synthase kinase-3 (GSK-3) inhibitors could restore TRAIL sensitivity in hepatoma cells. To this aim, the effects of two GSK-3 inhibitors, lithium and SB-415286, were analyzed on TRAIL apoptotic signaling in human hepatoma cell lines in comparison with normal hepatocytes. We observed that both inhibitors sensitized hepatoma cells, but not normal hepatocytes, to TRAIL-induced apoptosis by enhancing caspase-8 activity and the downstream recruitment of the mitochondrial machinery. GSK-3 inhibitors also stabilized p53 and the down-regulation of p53 by RNA interference abolished the sensitizing effect of lithium on caspase-3 activation. Concomitantly, GSK-3 inhibitors strongly activated c-Jun N-terminal kinases (JNKs). The pharmacological inhibition of JNKs with AS601245 or SP600125 resulted in an earlier and stronger induction of apoptosis indicating that activated JNKs transduced protective signals and provided an anti-apoptotic balance to the pro-apoptotic effects of GSK-3 inhibitors. These findings demonstrate that GSK-3 exerts a negative and complex constraint on TRAIL apoptotic signaling in hepatoma cells, which can be greatly alleviated by GSK-3 inhibitors. Therefore, GSK-3 inhibitors may open new perspectives to enhance the anti-tumor activity of TRAIL in HCC.

Flucloxacillin, an isoxazolyl-penicillin, causes cholestasis and biliary epithelium injury. The aim of the study was to determine whether flucloxacillin, either directly or through metabolite formation, may induce cytotoxicity in hepatic or biliary cells. Cytotoxicity was assessed by lactate dehydrogenase release in primary cultures of human hepatocytes and of gallbladder-derived biliary epithelial cells (BEC). Metabolite production in microsome and cell preparations was analyzed by chromatography, nuclear magnetic resonance spectroscopy, and mass spectrometry. While flucloxacillin induced no direct cytotoxicity in any of the hepatocyte (n = 12) and BEC (n = 19) preparations, the conditioned media from cultured hepatocytes preincubated with flucloxacillin (50-500 mg/L) triggered a significant increase in lactate dehydrogenase release over controls in approximately 50% of BEC preparations (7/12), and this effect depended upon flucloxacillin concentration. Remaining BEC preparations exhibited no toxic response. Cytotoxicity in BEC preparations (9/13) was also induced by the supernatants of human liver microsomes and of recombinant human cytochrome P450 (CYP)3A4 preincubated with flucloxacillin (500 mg/L). Supernatants from both liver microsome and CYP3A4 preparations contained one major metabolite which was identified as 5'-hydroxymethylflucloxacillin. The production of this metabolite was inhibited following CYP3A4 inhibition by troleandomycin in human liver microsomes, and markedly enhanced following CYP3A induction by dexamethasone in rat liver microsomes. As opposed to BEC, cultured hepatocytes displayed significant CYP3A activity and produced low amounts of this metabolite. The purified metabolite (0.01-5 mg/L) exerted toxic effects in BEC but not in hepatocytes. In conclusion, hepatocytes mainly via CYP3A4 activity, generate flucloxacillin metabolite(s) including 5'-hydroxymethylflucloxacillin that may induce cytotoxicity in susceptible BEC. These metabolic events may contribute to the pathogenesis of drug-induced cholangiopathies.

Background: This study evaluated the survival and functions of encapsulated porcine hepatocytes after intraperitoneal allotransplantation and xenotransplantation without immunosuppression., Methods: Isolated porcine hepatocytes were encapsulated in AN 69 polymer capsules (45.10(6)/capsule) and transplanted intraperitoneally in 12 rats and 12 pigs. Fifteen, 30, and 60 days after transplantation, capsules were removed and the viability and morphology of explanted hepatocytes were examined under light and electronic microscopy. The potential to produce albumin was assessed by evaluating the level of albumin messenger RNA, using semiquantitative reverse transcription-polymerase chain reaction. 6beta-Hydroxylase activity was measured by high-performance liquid chromatography. In addition, cytochrome P450 3A proteins were detected by Western blot only in allogeneic hepatocytes., Results: Similar results were observed after allotransplantation and xenotransplantation. Histologic studies showed that hepatocytes were well-preserved and arranged in cords for up to 30 days. The expression of porcine albumin gene was maintained up to 15 days. 6beta-Hydroxylase activity was 2.5-fold lower at day 15 than in freshly encapsulated hepatocytes, which were not transplanted. In allogeneic hepatocytes, the expression of CYP 3A protein was detected up to 60 days after transplantation., Conclusions: Encapsulated porcine hepatocytes remain viable and functional for at least 15 days after allotransplantation and xenotransplantation without immunosuppression. The demonstration of maintained hepatic functions in transplanted porcine hepatocytes up to 15 days is a first step toward application in the treatment of acute liver failure.

We have previously demonstrated that fresh or cryopreserved xenogeneic hepatocytes manually macroencapsulated in AN69 polymer and transplanted intraperitoneally in rats were able to improve the survival rate after 95% hepatectomy without immunosuppression. In addition, we developed a semiautomatic device where porcine hepatocytes were coextruded with AN69 hydrogel in order to macroencapsulate large amounts of cells. The purpose of the present study was to 1) test whether transplanted porcine hepatocytes macroencapsulated in this device remained functional as evaluated by their ability to prevent death from acute liver failure, and 2) compare the efficiency of cryopreserved or freshly isolated hepatocytes. Fresh or cryopreserved porcine hepatocytes were macroencapsulated in the semiautomatic device by coextrusion in AN69 polymer in 2-m minitubes containing 6 x 10(7) cells. Acute liver failure was induced in rats by two-step 95% hepatectomy. At the time of completion of liver resection, rats were either not transplanted with minitubes (control group I, n = 13), or were implanted with two minitubes containing culture medium (control group II, n = 11), hepatocytes killed by heat treatment (control group III, n = 10), coextruded fresh hepatocytes (group IV, n = 11), or coextruded cryopreserved hepatocytes (group V, n = 11), without immunosuppression. The survival rate at day 7 was between 0% and 31% in the three control groups. By contrast, coextruded fresh hepatocytes significantly improved the survival rate (group IV, 82%) as did cryopreserved cells (group V, 91% survival). In surviving rats, minitubes were explanted after 20 days; either fresh or cryopreserved hepatocytes appeared morphologically viable and their ultrastructure was preserved. Their detoxification capacities evaluated by the activity of the cyt P450 CYP3A4 were partly maintained. In conclusion, porcine hepatocytes macroencapsulated by coextrusion using a semiautomatic device and transplanted without immunosuppression were able to prevent death from acute liver failure in rats. Cryopreserved cells were as efficient as fresh hepatocytes.

Background: An implantable bioartificial liver (BAL) using xenogeneic isolated hepatocytes may be an alternative method to orthotopic liver transplantation for treatment of acute liver failure. The purpose of this study was to demonstrate that not only fresh but also cryopreserved porcine hepatocytes could be used in a BAL to prevent death after the onset of acute liver failure in rats., Methods: Acute liver failure was induced by two-stage 95% hepatectomy. At the time of completion of liver resection, 100 rats were assigned to undergo or not undergo transplantation into the peritoneum of 4 meters of hollow fibers filled with 60 million either fresh or cryopreserved porcine hepatocytes, or syngeneic hepatocytes, or culture medium, or of 60 million nonencapsulated cryopreserved porcine hepatocytes without immunosuppressive therapy. Survival rates at 7 days were compared between the different groups., Results: In the control groups of hepatectomized animals not receiving encapsulated hepatocytes, 69-79% of the rats died from acute liver failure. The mortality rate was reduced to 15% (2 of 13) in rats receiving fresh porcine hepatocytes (P<0.01), 25% (4 of 16) in rats transplanted with either cryopreserved or syngeneic hepatocytes (P<0.05). Survival rates were maintained when hollow fibers were explanted > or =4 days after hepatectomy. In surviving rats, the weight of the remnant native liver increased with time and returned to the initial weight after 1 month., Conclusions: The implantable BAL using xenogeneic porcine hepatocytes was able in preventing death from acute liver failure without immunosuppressive therapy. Encapsulated cryopreserved hepatocytes were as effective as fresh hepatocytes.

We have previously demonstrated that allogenic and xenogenic hepatocytes macroencapsulated manually in AN-69 polymer and transplanted intra-peritoneally in rats remained viable for several weeks. However, this manual technique is inadequate to encapsulate several billions of hepatocytes which would be required to correct hepatic failure in big animals or humans. In the present study, we developed an original semiautomatic device in which isolated pig hepatocytes and the polymer solution containing 6% poly(acrylonitrile-sodium methallylsulfonate), 91% dimethylsulfoxide and 3% 0.9% NaCl solution were coextruded through a double-lumen spinneret. The extruded minitube (inner diameter: 1.8 mm, wall thickness: 0.07-0.1 mm) containing the encapsulated hepatocytes fell and coiled up in a 0.9% NaCl solution at 4 degrees C and was cut down in 4 m units containing about 120 million hepatocytes. This process allowed to encapsulate 50 million hepatocytes by minute with a preserved immediate cell viability (92 +/- 5%). To test prolonged cell viability after coextrusion, the minitubes were implanted intraperitoneally in rats. Three and seven days after implantation, they were explanted and analyzed. Cells were viable and well-preserved. Therefore, the semiautomatic device appears able to efficiently macroencapsulate in a limited time several billions of porcine hepatocytes which remain viable after transplantation in xenogenic conditions.

Background: To evaluate new therapies for human fulminant hepatic failure, a suitable large animal model is needed. The purpose of this study was to develop a reversible surgical model of acute hepatic liver failure by transient ischemia in pigs., Materials and Methods: Under general anesthesia, an end-to-side portacaval shunt was performed in 17 pigs and tape was laid around the hepatoduodenal ligament. Two days after construction of the functional portacaval shunt, 13 ambulant pigs underwent transient total liver ischemia by tightening of the tape around the hepatoduodenal ligament for 5.5 h. During ischemia, 10% glucose was continuously infused intravenously to prevent hypoglycemia., Results: Ten animals (77%) died with hepatic coma after a mean duration of 22.5 +/- 1.9 h. The 3 remaining animals survived more than 5 days and were sacrificed. In dying animals, encephalopathy was observed 14 +/- 1.7 h after the onset of ischemia. During ischemia, similar progressive decrease of fibrinogen, platelets, prothrombin time, and factors V and VII activities was observed in dying and surviving animals. Just before death, mean prothrombin and factors V and VII activities were respectively 22 +/- 2, 21 +/- 4.4, and 24 +/- 5%. At 22 h, plasma ammonia and lactate levels were respectively 705 +/- 93 micromol/L and 10.5 +/- 0.4 mmol/L in dying animals and 249 +/- 75 micromol/L and 2.9 +/- 0.1 mmol/L in surviving animals (P < 0.01). Estimation of the percentage liver cells necrosed was 74 +/- 4.7% in the survivors and 86 +/- 5.5% in animals who died of hepatic coma (NS)., Conclusions: This model is reproducible and reversible and should allow the quantitative evaluation of new technologies, such as bioartificial liver, for the support of hepatic failure in humans., (Copyright 2000 Academic Press.)

Objective: To demonstrate that a bioartificial liver, using allogeneic or xenogeneic hepatocytes protected from rejection by a semipermeable membrane, could prevent death from acute liver failure., Summary Background Data: An implantable bioartificial liver using isolated hepatocytes could be an alternative to orthotopic liver transplantation to treat patients with acute liver failure. It could serve either as a bridge until liver transplantation or as the main treatment until recovery of the native liver. However, allogeneic or xenogeneic hepatocytes that could be used in clinical applications are spontaneously rejected., Methods: Acute liver failure was induced in rats by 95% liver resection. Twenty-five million hepatocytes harvested in rats (allogeneic) or guinea pigs (xenogeneic) were encapsulated in a semipermeable membrane to protect them from rejection. The hollow fibers containing hepatocytes were transplanted into the peritoneum of recipient rats. Survival rates were compared between rats transplanted or not with hepatocytes., Results: In groups not transplanted with viable hepatocytes, 73% to 93% of rats died after 95% liver resection. The mortality rate was reduced to 39% in rats transplanted with allogeneic hepatocytes and 36% in rats transplanted with xenogeneic hepatocytes. The bioartificial liver could be removed 1 month after transplantation, when regeneration of the native liver was complete. Allogeneic and xenogeneic hepatocytes remained viable., Conclusions: The implantable bioartificial liver was able to prevent death in this model of acute liver failure. This could be an important step toward clinical application of the method.

Background: Hepatocyte transplantation could be an alternative to whole organ transplantation to correct enzymatic disorders. To this end, it would be of major importance to use xenogeneic cells without immunosuppression. The aim of this study was to investigate the survival and metabolic activity of encapsulated xenogeneic hepatocytes in the absence of immunosuppression. For this purpose, we used Gunn rats genetically incapable of bilirubin conjugation., Methods: Xenogeneic (from guinea pigs) and allogeneic (from Lewis rats) hepatocytes (2x10(7)) were isolated, macroencapsulated in hydrogel hollow fibers made with an acrylonitrile-sodium methallyl-sulfonate copolymer, and transplanted into the peritoneum of Gunn rats without any immunosuppression. Plasma bilirubin levels were evaluated weekly. Bilirubin conjugates in bile and cell morphology were studied after 5 and 12 weeks, respectively., Results: In Gunn rats transplanted with xenogeneic hepatocytes, a significant decrease in the serum bilirubin level was observed between 3 and 9 weeks after transplantation when compared with controls transplanted with empty hollow fibers: it fell to 62% of the initial level at weeks 5-7 (P < 0.01). A comparable result was observed in Gunn rats transplanted with encapsulated allogeneic cells. Bilirubin conjugates were observed in bile samples of rats transplanted with encapsulated hepatocytes. After explantation, hollow fibers appeared intact with minimal fibrosis. Cell viability and hepatocyte morphology were preserved., Conclusions: These results indicate that macroencapsulated xenogeneic hepatocytes can survive and remain functional for more than 2 months when transplanted in vivo in the absence of any immunosuppression.

Background: Physiopathology of hepatic encephalopathy remains unclear. Recent studies have suggested that ammonia would not act by itself but through an increase in glutamine in the brain. We have previously demonstrated that transplantation of syngeneic hepatocytes into the spleen was able to correct both behavioral deficits and plasma amino acid changes observed in portacaval shunted rats. The aim of the present work was to show a correlation between the correction of chronic hepatic encephalopathy by means of intrasplenic hepatocyte transplantation and two parameters, brain glutamine concentration and ultrastructural aspects of astrocytes., Methods: Inbred male Wistar Furth rats were divided into three groups: sham-operated rats (n = 10), rats subjected to portacaval shunt (n = 10), and rats subjected to portacaval shunt and intrasplenic hepatocellular transplantation of 10(7) hepatocytes isolated from livers of syngeneic rats (n = 10). Chronic hepatic encephalopathy was quantified 30 and 60 days after operation by means of nose-poke exploration and spontaneous activity. Pathologic examination and measurement of glutamine concentrations in the corpus striatus and in the cerebral cortex were performed 60 days after operation., Results: Portacaval shunt rats showed reduced spontaneous activity and nose-poke exploration scores. After portacaval shunt a significant glutamine increase occurred in the corpus striatus and in the cerebral cortex when compared with sham rats (p < 0.05). Ultrastructural examination showed modification of astrocytes named Alzheimer type II after portacaval shunt. Correction of behavioral abnormalities by means of intrasplenic hepatocyte transplantation was associated with partial correction of striatal glutamine increase and with decrease in astrocyte alterations. Cortex glutamine concentration in portacaval shunt-intrasplenic hepatocyte transplantation group and in portacaval shunt rats did not differ significantly., Conclusions: These data show that intrasplenic hepatocyte transplantation not only prevents neurologic disorders of hepatic encephalopathy but can also decrease glutamine and ultrastructural alterations in the corpus striatus in an experimental model of chronic liver failure. These data are in favor of the involvement of glutamine in chronic hepatic encephalopathy. These results suggest that intrasplenic hepatocyte transplantation might be of therapeutic interest in chronic liver failure.

A new high-water-content (83%) and highly permeable anionic polyelectrolyte hydrogel was obtained by phase inversion of a polymer solution containing 6% polyacrylonitrile-sodium methallylsulphonate, 91% dimethylsulphoxide and 3% physiological saline solution. Hydrogel-based hollow fibres (HFs) were fabricated with a co-extrusion apparatus in collaboration with Hospal (France). HFs have an internal diameter of 800 microns and a wall thickness of 100 microns. Experimental results demonstrated that hydrogel-based HFs were permeable to albumin (mol. wt 69,000) and human immunoglobulin G (150,000), but were impermeable to immunoglobulins A (170,000) and M (900,000) after 24 h of diffusion. In vitro, the viability of isolated rat hepatocytes injected into the HFs was 64 +/- 6% after 10 d versus 30 +/- 5% for hepatocytes cultured in Petri dishes (P = 0.0001). Under these conditions, the amount of albumin released by encapsulated hepatocytes was 12 +/- 3 micrograms/24 h/10(6) cells at day 10, whereas at that time no albumin was released by hepatocytes cultured in Petri dishes. In vivo, histological study of hydrogel HFs implanted up to 6 wk in the peritoneum of rats revealed a low inflammatory tissue reaction without giant multinucleate cells in the foreign tissue, which decreased after the third week. The survival rate of encapsulated hepatocytes was over 85% 45 d after transplantation in the peritoneum of syngeneic Lewis rats. Therefore, this hydrogel demonstrates highly favourable properties for encapsulation of hepatocytes with regard to its biocompatibility, permeability and ability to maintain hepatocytes in a functional state for prolonged periods.

Background: Hepatocyte transplantation could be an alternative to whole liver transplantation. Allogeneic hepatocytes are rejected if transplanted without immunosuppression. The aim of this study was to transplant allogeneic hepatocytes in the peritoneum and to protect them from rejection by encapsulation in a new semipermeable membrane., Methods: Rat hepatocytes were encapsulated in hydrogel-based hollow fibers, obtained from AN69 copolymer, before being transplanted into the peritoneum of rats. Outcome of allogeneic hepatocytes encapsulated in hollow fibers was compared with that of syngeneic hepatocytes encapsulated in hollow fibers, with that of free allogeneic hepatocytes, and with allogeneic hepatocytes encapsulated in hollow fibers left open. Cell viability was assessed by erythrosin exclusion, structure by electron microscopy, and function by albumin release., Results: Up to 90 days, viability of allogeneic hepatocytes in hollow fibers was greater than 80%. The structure remained normal at electron microscopy. Albumin release was 16.5 +/- 0.3 micrograms/24 hr/10(6) hepatocytes (day 15), 14.2 +/- 2.0 micrograms/24 hr/10(6) hepatocytes (day 30), 8.8 +/- 0.1 micrograms/24 hr/10(6) hepatocytes (day 60), and 11.4 +/- 0.3 micrograms/24 hr/10(6) hepatocytes (day 90). Free hepatocytes and hepatocytes in hollow fibers left open did not survive at day 15., Conclusions: Viability and function of encapsulated allogeneic hepatocytes were maintained up to 90 days after transplantation, without immunosuppression.

Hepatocytes normally express few major histocompatibility complex (MHC) class I and no MHC class II molecules, a phenomenon which could explain their low immunogenicity. However, in pathological situations, such as allograft rejection and cholestasis, hepatocytes strongly express MHC class I molecules and their immunogenicity could be different. The aim of this study was to assess the role of MHC expression on the immunogenicity of hepatocytes in vivo. Hepatocytes were obtained from normal and cholestatic DA rats by whole-liver perfusion with EDTA. Cholestasis was induced by ligation-section of the common bile duct. MHC expression on hepatocytes was assessed by cytofluorimetry after labelling with monoclonal antibodies against MHC class I and class II antigens. The percentage of hepatocytes expressing MHC class I was 9.8 +/- 2.2% in normal rats and 77.2 +/- 3.3% in cholestatic rats (P = 2 x 10(-4)); MHC class II expression was present on 1 +/- 0.5% of normal hepatocytes and 0.4% +/- 0.1% of cholestatic hepatocytes (P > 0.05). Lewis rats received a DA or Wistar-Furth heart allograft 7 days after intravenous injection of 2 x 10(7) hepatocytes from normal or cholestatic DA rats. The DA heart allograft was rejected in 6.3 +/- 0.4 days in Lewis controls, 8.8 +/- 1.1 days (N.S.) in Lewis recipients that received normal DA hepatocytes and 17.6 +/- 3.0 days (P = 2 x 10(-4)) in Lewis recipients that received hepatocytes from cholestatic DA rats.(ABSTRACT TRUNCATED AT 250 WORDS)

The aim of this work was to evaluate the effect of intrasplenic hepatocellular transplantation on hepatic encephalopathy in an experimental model of chronic liver failure induced by end-to-side portacaval shunt in the rat. Inbred male Wistar Furth rats were divided into three groups: rats subjected to portacaval shunt (n = 10), rats subjected to portacaval shunt and intrasplenic hepatocellular transplantation of 10(7) hepatocytes isolated from livers of syngeneic rats (n = 10) and sham-operated rats (n = 10). Behavior tests were performed in a blind fashion at 3 wk, at 2 mo and at 3 mo after surgery. Spontaneous activity and nose-poke exploration by individual rats were studied in automated open field boxes equipped with infrared cells. Each cell beam interruption was automatically recorded on a microcomputer and transformed into a score index (counts/hour). Plasma levels of amino acids, ammonia and total biliary acids were measured. Portacaval shunt rats showed reduced spontaneous activity and nose-poke exploration scores. Intrasplenic hepatocellular transplantation significantly increased spontaneous activity after 2 mo and improved nose-poke exploration after 3 wk. At 3 mo, spontaneous activity and nose-poke exploration in portacaval shunt/intrasplenic hepatocellular transplantation rats were not significantly different from those of sham rats. Increases in plasma ammonia levels after portacaval shunt were not corrected. Amino acid imbalance and bile acid concentration in plasma were partially corrected by intrasplenic hepatocellular transplantation. These data show that intrasplenic hepatocellular transplantation can correct the neurological symptoms of hepatic encephalopathy in an experimental model of chronic liver failure and suggest that intrasplenic hepatocellular transplantation might be of therapeutic interest in chronic liver failure.

The liver is the most frequent site of metastases in colon cancer. No good animal model has been available to help improve the treatment of liver metastases or their prevention after resection of a primary colon cancer. The aim of this study was to develop a model of colon cancer induced by azoxymethane in the rat and to study the outcome after surgical resection alone or in association with intraperitoneal chemotherapy (5-fluorouracil (5-FU). Three hundred male Wistar rats received subcutaneous azoxymethane (10 mg/kg body weight/week) for 12 weeks. Eighty-three rats with isolated colon cancer underwent total colectomy; 40 of these rats with no metastases were randomized into two groups: surgery alone or surgery plus 5-FU (5 mg/kg body weight/day) for 5 days after surgery. Thirty rats were able to be evaluated. At autopsy, peritoneal carcinomatosis and liver metastases were more frequent in the control group than after adjuvant treatment with 5-FU (27.7 percent vs. 0, P less than 0.05; and 22.2 percent vs. 0, P less than 0.05, respectively). The rates of peritoneal and hepatic recurrence after resection of the primary cancer indicate that the model mimics the natural history of human colon cancer. In this model, 5-FU reduced the rate of peritoneal carcinomatosis and liver metastases but did not influence survival.

The liver is the most frequent site of metastases from colon cancer. To improve our knowledge of liver metastases and to develop new adjuvant therapies, a good animal model is necessary. The aims of this study were to obtain a model of liver metastases with intraportal injection of colon adrenocarcinoma cell aggregates (DHDK12 cell line) and to study the effect of various factors, i.e., sex, liver regeneration and immunosuppression, on the development of liver metastasis. Cell aggregates were injected into the portal vein of 59 syngenic male and female BD IX rats following randomization into three groups. Group 1, (control 12 males and 10 females) received only cell aggregates; group 2 (12 males and 10 females) underwent a 70% hepatectomy before cell injection; group 3 (15 males and 10 females) received cyclosporin A injections at a dose of 10 mg/kg per day for 28 days following cell injection. Autopsy was performed at 10 weeks. Liver metastases were more frequent in the male rats in group 3 than in those in group 1 (80% vs. 30%, p less than 0.04). The rate of liver metastases in females was not increased by immunosuppression (22.2% vs. 12.5%, N.S.). Liver resection (group 2) did not significantly modify the incidence of liver metastasis. No female had liver metastases in this group. This relatively simple model rapidly produces liver metastasis with a high yield, but only in male rats. Besides sexual factors, immunosuppression also increased the rate of experimental liver metastasis, while liver regeneration failed to do so.

The aim of this study was to assess directly the function of isolated hepatocytes 1 year after transplantation into the spleen, using an original model of isolated rat-spleen perfusion. Three specific liver functions, albumin synthesis, indocyanine-green clearance, and antipyrine oxidation, were studied. Five x 10(6) isolated hepatocytes were injected into the spleen of syngenic Wistar-Furth rats. One year later, splenectomy was performed, and the splenic pedicle was carefully isolated in order to allow a selective ex vivo perfusion for 3 hr. De novo albumin synthesis was studied by qualitatively using immunoelectrophoresis and autoradiography, and quantitatively using (35S)-methionine incorporation in albumin. De novo albumin synthesis was observed in spleens containing transplanted hepatocytes but not in controls (P less than 0.001); (35S)-methionine incorporation was significantly higher in spleens containing transplanted hepatocytes than in controls (132 +/- 67 cpm/spleen/hr vs. 14 +/- 6 cpm/spleen/hr, P less than 0.001). Antipyrine clearance was significantly higher in spleens with transplanted hepatocytes than in controls (67.4 +/- 4.9 microliters/min/g vs. 0.2 +/- 0.4 microliters/min/g, P less than 0.01). No statistically significant difference was observed with indocyanine-green clearance (4.2 +/- 6.0 microliters/min/g, vs. 5.2 +/- 5.1 microliters/min/g, P greater than 0.05); this was probably due to the absence of compartmentation between the sinusoid and biliary sectors in this model. In conclusion, using this original isolated rat-spleen perfusion model, it was directly observed that 1 year after transplantation, intrasplenic hepatocytes can perform two liver-specific functions, i.e., de novo albumin synthesis and antipyrine clearance.

The aim of this work was to study hepatocyte multiplication after transplantation into the spleen, in order to apply this technique to the treatment of chronic liver disease. Hepatocytes isolated by an in situ collagenase perfusion technique in Wistar Furth rats were injected into the splenic parenchyma of three groups of syngeneic rats: controls with normal liver (group 1), 75% hepatectomies (group 2), and end-to-side portacaval shunts (group 3). The proliferation of transplanted hepatocytes was studied by autohistoradiography after the intraperitoneal administration of 0.6 microCi/g body weight of [3H]-thymidine, at 1, 3, 7 and 15 days after transplantation of hepatocytes. Significant incorporation of [3H]-thymidine by the transplanted hepatocytes during the study period was observed mostly in groups 2 and 3. The incorporation, although delayed was sustained and of greatest magnitude in the portacaval-shunted animals. The ability of transplanted hepatocytes to proliferate in the spleen, particularly after a portacaval shunt, indicates that this procedure may have therapeutic applications in the treatment of chronic liver disease.