retrorsine and Disease-Models--Animal

Primary human hepatocytes (PHHs) are an essential tool for modeling drug metabolism and liver disease. However, variable plating efficiencies, short lifespan in culture, and resistance to genetic manipulation have limited their use. Here, we show that the pyrrolizidine alkaloid retrorsine improves PHH repopulation of chimeric mice on average 10-fold and rescues the ability of even poorly plateable donor hepatocytes to provide cells for subsequent ex vivo cultures. These mouse-passaged (mp) PHH cultures overcome the marked donor-to-donor variability of cryopreserved PHH and remain functional for months as demonstrated by metabolic assays and infection with hepatitis B virus and

Topics: Animals; Cell Transplantation; Chimera; Disease Models, Animal; Female; Genetic Therapy; Hepatitis B; Hepatitis B virus; Hepatocytes; Homeodomain Proteins; Humans; Hydrolases; Interleukin Receptor Common gamma Subunit; Liver; Liver Diseases; Malaria; Male; Mice; Mice, Inbred NOD; Mice, Knockout; Plasmodium falciparum; Pyrrolizidine Alkaloids

A feasible large animal model to evaluate regenerative medicine techniques is vital for developing clinical applications. One such appropriate model could be to use retrorsine (RS) together with partial hepatectomy (PH). Here, we have developed the first porcine model using RS and PH. RS or saline control was administered intraperitoneally to Göttingen miniature pigs twice, two weeks apart. Four weeks after the second dose, animals underwent PH. Initially, we tested different doses of RS and resection of different amounts of liver, and selected 50 mg/kg RS with 60% hepatectomy as our model for further testing. Treated animals were sacrificed 3, 10, 17 or 28 days after PH. Blood samples and resected liver were collected. Serum and liver RS content was determined by Liquid Chromatograph-tandem Mass Spectrometer. Blood analyses demonstrated liver dysfunction after PH. Liver regeneration was significantly inhibited 10 and 17 days after PH in RS-treated animals, to the extent of 20%. Histological examination indicated hepatic injury and regenerative responses after PH. Immunohistochemical staining demonstrated accumulation of Cyclin D1 and suppression of Ki-67 and PCNA in RS-treated animals. We report the development of the first large animal model of sustained liver injury with suppression of hepatic regeneration.

Topics: Animals; Cyclin D1; Disease Models, Animal; Dose-Response Relationship, Drug; Hepatectomy; Hepatocytes; Ki-67 Antigen; Liver; Liver Regeneration; Pyrrolizidine Alkaloids; Regenerative Medicine; Swine; Swine, Miniature

In the retrorsine (RS)-based model of massive liver repopulation, preexposure to this naturally occurring alkaloid is sufficient to prime normal host parenchymal cells to be slowly replaced by transplanted normal hepatocytes. The basis for this striking effect is yet to be fully elucidated. In the present studies the possible involvement of cell senescence was investigated. Fischer 344 rats were treated according to the RS-based protocol for hepatocyte transplantation, i.e., two doses of RS, 2 weeks apart, and were killed at 4 or 8 weeks after treatment. Control groups were given saline. Expression of senescence-associated beta-galactosidase was greatly induced in hepatocytes exposed to RS. In addition, several other changes that have been related to cell senescence were observed: these included markers of persistent activation of a DNA damage response, an increased expression of mammalian target of rapamycin, and positive regulators of the cell cycle, together with the induction of p21 and p27 cyclin-dependent kinase inhibitors. Furthermore, RS treatment increased levels of interleukin-6 in the liver, consistent with the activation of a senescence-associated secretory phenotype.. These findings indicate that RS induces hepatocyte senescence in vivo. We propose that cell senescence and the associated secretory phenotype can contribute to the selective growth of transplanted hepatocytes in this system.

Topics: Animals; Antineoplastic Agents, Phytogenic; beta-Galactosidase; Cell Cycle; Cell Division; Cell Transplantation; Cellular Senescence; Disease Models, Animal; DNA Damage; Hepatocytes; Interleukin-6; Liver; Liver Regeneration; Male; Pyrrolizidine Alkaloids; Rats; Rats, Inbred F344; Transplantation Conditioning

Complete liver regeneration after partial hepatectomy (PH) in rats exposed to the pyrrolizidine alkaloid retrorsine is accomplished through the activation, expansion, and differentiation of a population of small hepatocyte-like progenitor cells (SHPCs). The mechanism(s) governing activation of SHPCs after PH in retrorsine-injured rats has not been investigated. We examined the possibility that SHPCs require cytokine priming prior to becoming growth factor responsive in this model of liver injury and regeneration. Male Fischer 344 rats were treated with retrorsine (30 mg/kg ip) at 6 and 8 weeks of age. Retrorsine-exposed and age-matched control rats were randomized into dexamethasone-treated and no DEX groups. DEX-treated animals were either given a single dose of DEX (2 mg/kg ip) at the time of PH or multiple DEX treatments (2 mg/kg ip each) at 24 and 1 h before PH and 1, 2, and 3 days post-PH. A subset of rats received 10 microg of recombinant IL6 protein, administered intravenously 30 min after PH. Liver tissues were harvested at 7, 14, 21, and 30 days post-PH. Treatment of retrorsine-exposed rats with the cytokine inhibitor dexamethasone (DEX) effectively blocked the emergence of SHPCs resulting in an inhibition of liver regeneration and producing significant short-term mortality. The livers of DEX-treated retrorsine-exposed rats displayed decreased numbers and smaller SHPC clusters compared to retrorsine-exposed rats in the absence of DEX treatment. Administration of recombinant IL6 to DEX-treated retrorsine-exposed rats restored the emergence of SHPCs and SHPC-mediated regenerative response. The livers of DEX-treated retrorsine-exposed rats that received IL6 displayed numbers of expanding SHPC clusters comparable to that of retrorsine-exposed rats in the absence of DEX treatment. These results combine to suggest that SHPC activation after PH in retrorsine-exposed rats is cytokine dependent and may specifically require IL6.

Topics: Animals; Antineoplastic Agents, Phytogenic; Cytokines; Dexamethasone; Disease Models, Animal; Drug Antagonism; Drug Therapy, Combination; Glucocorticoids; Hepatectomy; Hepatocytes; Interleukin-6; Liver; Liver Regeneration; Longevity; Male; Organ Size; Pyrrolizidine Alkaloids; Rats; Rats, Inbred F344; Recombinant Proteins; Stem Cells

The therapeutic effects of bone marrow and hepatocyte transplantation were investigated regarding the treatment of retrorsine-partial hepatectomy-induced liver injury.. Analbuminemic F344alb rats were given two doses of retrorsine 2 wk apart, followed 4 wk later by transplantation with F344 rat bone marrow cells or hepatocytes immediately after a two-thirds hepatectomy. The survival rate, liver regeneration rate, liver functions, albumin-positive hepatocytes, and normal albumin gene sequences in the liver and serum albumin levels were investigated in the recipients.. Although 65% retrorsine/partial hepatectomy-treated F344alb died between 1 and 11 d after the partial hepatectomy, only 27.5% of the animals died following bone marrow transplantation, and 50% with hepatocyte transplantation. Both bone marrow and hepatocyte transplantation ameliorated acute liver injury after a partial hepatectomy. Bone marrow transplantation yielded a very small increase in the number of albumin-positive hepatocytes in the liver, while hepatocyte transplantation resulted in massive replacement of the liver tissues by the donor hepatocytes associated with an elevation of serum albumin after an extended time.. Both bone marrow and hepatocyte transplantation could prevent acute hepatic injury, conceivably due to a paracrine mechanism.

Topics: Acute Disease; Animals; Antineoplastic Agents, Phytogenic; Bone Marrow Transplantation; Chemical and Drug Induced Liver Injury; Disease Models, Animal; Hepatectomy; Hepatocytes; Liver Diseases; Liver Regeneration; Male; Paracrine Communication; Pyrrolizidine Alkaloids; Rats; Rats, Inbred F344; Serum Albumin; Survival Rate

Mature hepatocytes divide to restore liver mass after injury. However, when hepatocyte division is impaired by retrorsine poisoning, regeneration proceeds from another cell type: the small hepatocyte-like progenitor cells (SHPCs). Our aim was to test whether SHPCs could originate from mature hepatocytes.. Mature hepatocytes were genetically labeled using retroviral vectors harboring the beta-galactosidase gene. After labeling, retrorsine was administered to rats followed by a partial hepatectomy to trigger regeneration. A liver biopsy was performed one month after surgery and rats were sacrificed one month later.. We observed the proliferation of small hepatocytes arranged in clusters in liver biopsies. These cells expressed Ki67 antigen and displayed a high mitotic index. At sacrifice, regeneration was completed and clusters had merged. A significant proportion of clusters also expressed beta-galactosidase demonstrating their origin from labeled mature hepatocytes. Finally, the overall proportion of beta-galactosidase positive cells was identical at the time of hepatectomy as well as in liver biopsy and at sacrifice.. The constant proportion of beta-galactosidase positive cells during the regeneration process demonstrates that mature hepatocytes are randomly recruited to proliferate and compensate parenchyma loss in this model. Furthermore, mature hepatocytes are the source of SHPC after retrorsine injury.

Topics: Animals; Animals, Genetically Modified; Antineoplastic Agents, Phytogenic; beta-Galactosidase; Chemical and Drug Induced Liver Injury; Disease Models, Animal; Hepatocytes; Lac Operon; Liver Diseases; Liver Regeneration; Male; Pyrrolizidine Alkaloids; Rats; Rats, Sprague-Dawley; Stem Cells

Retrorsine, a naturally occurring pyrrolizidine alkaloid, impairs liver regeneration after partial hepatectomy by mechanisms that are still unclear.. The aim of the study was to clarify the influence of retrorsine on cell cycle progression in the regenerating liver lobes of rats after portal branch ligation (PBL).. Liver weight, protein and DNA contents, DNA synthesis (5'-bromodeoxyuridine (BrdU) incorporation) and cellular levels of Cyclin E, CDK-2, CDK-4 and proliferating cell nuclear antigen (PCNA) were assessed before and 24, 48, 72 and 168 h after PBL.. Before surgery, higher levels of cyclin E, CDK-2, CDK-4 and PCNA as well as BrdU incorporation were found in the liver of retrorsine-treated rats than in untreated rats. Liver weight gain, protein and DNA synthesis as well as induction of cell cycle related proteins were all strongly impaired by retrorsine in the regenerating lobes after PBL.. In conclusion, retrorsine impairs liver regeneration in the PBL model not only by an S or G2/M phase block, but also by a block located before the G1/S transition of the cell cycle.

Topics: Animals; Antineoplastic Agents, Phytogenic; Blotting, Western; Bromodeoxyuridine; CDC2-CDC28 Kinases; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Disease Models, Animal; DNA; Kinetics; Ligation; Liver Diseases; Liver Regeneration; Male; Organ Size; Portal Vein; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins; Pyrrolizidine Alkaloids; Rats; Rats, Wistar; Weight Gain

Adult bone marrow contains progenitors capable of generating hepatocytes. Here a new liver failure model is introduced to assess whether bone marrow-derived progeny contribute to liver regeneration after acute hepatotoxic liver failure.. Retrorsine was used to inhibit endogenous hepatocyte proliferation, before inducing acute liver failure by carbon tetrachloride. Bone marrow chimeras were generated before inducing liver failure to trace bone marrow-derived cells. Therefore, CD45 and major histocompatibility complex (MHC) class I dimorphic rat models were applied.. Early after acute liver failure a multilineage inflammatory infiltrate was observed, mainly consisting of granulocytes. In long-term experiments small numbers of CD90+/CD45- cells of donor origin occurred in clusters associated with portal triads. Bone marrow cell infusion was not able to enhance liver regeneration. Cellular hypertrophy was the predominant way of liver mass regeneration in models applying retrorsine.. Retrorsine pretreatment did not affect sensitivity for carbon tetrachloride. A multilineage inflammatory infiltrate was observed in rats whether pretreated with retrorsine or not. Few donor cells co-expressing CD90 (THY 1) were present in recipient livers, which may resemble donor-derived hematopoietic progenitors or oval cells. No other donor cells within liver parenchyma were detected. This is in contrast to other cell infusion models of acute cell death.

Topics: Animals; Bone Marrow Cells; Bone Marrow Transplantation; Carbon Tetrachloride; Cell Line; Chimera; Disease Models, Animal; Granulocytes; Hematopoietic Stem Cells; Leukocyte Common Antigens; Liver; Liver Failure, Acute; Liver Regeneration; Pyrrolizidine Alkaloids; Rats; Rats, Inbred Lew; Thy-1 Antigens

To establish the efficacy of cell therapy in Wilson's disease, we used the Long-Evans Cinnamon (LEC) rat model with atp7b gene mutation and copper toxicosis. Several groups of LEC rats were established, including animals pretreated with retrorsine to exacerbate copper toxicosis and inhibit proliferation in native hepatocytes followed by partial hepatectomy to promote liver repopulation. Hepatocytes from normal, syngeneic LEA rats were transplanted intrasplenically. Animal survival, biliary copper excretion, and hepatic copper were determined. The magnitude of liver repopulation was demonstrated by measuring serum ceruloplasmin and hepatic atp7b mRNA. Long-term survival in LEC rats treated with retrorsine, partial hepatectomy, and cell transplantation was up to 90%, whereas fewer than 10% of animals pretreated with retrorsine, without cell therapy, survived, P < 0.001. Liver repopulation occurred gradually after cell transplantation, ranging from <25% at 6 weeks, 26 to 40% at 4 months, and 74 to 100% at 6 months or beyond. Liver repopulation restored biliary copper excretion capacity and lowered liver copper levels. Remarkably, liver histology was completely normal in LEC rats with extensive liver repopulation, compared with widespread megalocytosis, apoptosis, oval cell proliferation, and cholangiofibrosis in untreated animals. These data indicate that liver repopulation with functionally intact cells can reverse pathophysiological perturbations and cure Wilson's disease.

Topics: Adenosine Triphosphatases; Animals; Bile; Carrier Proteins; Cation Transport Proteins; Copper; Copper-Transporting ATPases; Disease Models, Animal; Hepatectomy; Hepatocytes; Hepatolenticular Degeneration; Liver Regeneration; Pyrrolizidine Alkaloids; Rats; Rats, Inbred LEC; Transplantation Conditioning

We have recently developed a new model of extensive liver repopulation by transplanted hepatocytes following exposure to pyrrolizidine alkaloids. In the present study, the effect of 2/3 partial hepatectomy (PH) and that of a potent direct liver mitogen, lead nitrate, were compared in their ability to modulate the kinetics of liver repopulation.. Fischer 344 rats deficient in enzymatic activity for dipeptidyl-peptidase IV (DPPIV-) were used as cell transplantation recipients. They were given 2 doses of the pyrrolizidine alkaloid retrorsine (30 mg/kg, i.p.), 2 weeks apart, followed 2 weeks later by transplantation of 2 x 10(6) hepatocytes (via the portal vein), freshly isolated from a normal congeneic DPPIV+ donor. PH was carried out or a single injection of lead nitrate (100 micromol/kg, i.v.) was administered 2 weeks post-transplantation. Liver samples obtained at different time points post-treatment were processed histochemically for DPPIV activity.. The percent of liver sections occupied by DPPIV+ hepatocytes was <1% at the time of PH or lead nitrate administration. In animals which underwent PH, it increased to 33.4+/-5.7% at 2 weeks and to 55.6+/-8.5% at 1 month. However, in animals receiving lead nitrate, these percentages were only 3.3+/-1.3% at 2 weeks and 16.2+/-3.9% at 1 month. Repeated injections of lead nitrate had no additional effect. Further experiments indicated that an acute mitogenic response to lead nitrate was present in transplanted cells, while resident hepatocytes were inhibited by retrorsine.. These results indicate that direct mitogenic signals (such as those induced by lead nitrate), and compensatory signals (such as those elicited by PH), are not equally effective on kinetics of liver repopulation in this system. The possible reasons for these differential effects are discussed.

Topics: Animals; Antineoplastic Agents, Phytogenic; Cell Transplantation; Dipeptidyl Peptidase 4; Disease Models, Animal; Female; Hepatectomy; Hyperplasia; Lead; Liver; Liver Regeneration; Male; Mitogens; Nitrates; Pyrrolizidine Alkaloids; Rats; Rats, Inbred F344

In Indian childhood cirrhosis (ICC) and related disorders of infancy, hepatic copper overload is associated with cirrhosis. Since copper administration alone has not been shown to induce cirrhosis in animals, synergy between copper and a second hepatotoxin has been suggested. This study investigates the ability of long-term exposure to copper and a pyrrolizidine alkaloid, retrorsine, to produce a model of copper-associated cirrhosis in rats. Groups of rat pups suckled on mothers fed 25 mg/kg diet retrorsine were weaned onto a diet containing 0.5 g/kg diet copper and retrorsine in varying dosage for 13 weeks. Histological similarities between the human disease and rats given copper with retrorsine 5 mg/kg diet included parenchymal destruction, fibrosis, nodular regeneration, and copper accumulation. There were significant histological differences from the human disorder, possibly attributable to inter-species variability or the critical timing or duration of exposure to hepatotoxins in the neonatal period. The hypothesis that ICC results from copper and a second hepatotoxin has not been disproved.

Topics: Animals; Copper; Disease Models, Animal; Drug Synergism; Female; Liver; Liver Cirrhosis, Experimental; Liver Function Tests; Organ Size; Pyrrolizidine Alkaloids; Rats; Rats, Wistar