G. Y. Minuk

Liver regeneration: methods for monitoring and their applications

Liver regeneration is an essential component of the reparative process following liver injury and surgical resection. It can be assessed by different tissue-based tests such as liver weights, mitotic counts, DNA contents and synthesis rates, immunohistochemical staining of nuclear antigens, gene expressions and certain protein levels or various serum-based tests that largely consist of specific enzyme determinations or documentation of certain proliferation markers. Although the simplest tissue-based test of liver regeneration is measurement of liver weights, these determinations are influenced by the extent of deposition of various materials not directly related to regeneration, such as lipids, glycogen and blood volumes. Because mitosis constitutes a very short segment of the cell cycle, mitotic counts are infrequently observed by light microscopy. Thymidine and BrdU incorporation into DNA are the reference tools for studying DNA synthesis, but their use requires pre-injection with radioactive isotopes or nucleotides which render them impractical for human studies. Flow cytometry is an accurate and objective method of monitoring hepatic regenerative activity but requires sophisticated equipment that is not generally available in many laboratories. Immunohistochemical staining for nuclear antigens (Ki-67, proliferating cell nuclear antigen [PCNA], DNA polymerase alpha and nucleolar organizer region [NOR] proteins) are acceptable and commonly used methods of monitoring regenerative activity but are subject to inter- and intra-observer variability. Gene expression rates such as Histone-3 mRNA abundance are hampered by the relatively low rates of gene transcription and the need for recombinant DNA technology. Protein and enzyme levels in liver tissues, such as putrescine, ornithine decarboxylase and thymidine kinase, are not precise and are confounded by the nutritional status of the host. While PCNA protein levels measured by immunoblot hold promise as a simple, accurate and reproducible marker of liver regeneration, additional studies are required to determine if this is a valid marker of regenerative activity in various models of hepatic injury and in humans. Of the serum-based determinations: thymidine kinase, ornithine decarboxylase, fibronectin, alpha fetoprotein, and early pregnancy factor offer practical and non-invasive tools to monitor liver regeneration, but the sensitivity and specificity of these tests have yet to be determined. In conclusion, many tissue and serum-based methods have been employed in clinical and experimental studies to assess liver regeneration; however, a gold standard has yet to be identified. Because of the disadvantages inherent in each method, and until a new, more accurate marker is identified, clinicians and scientists should incorporate a minimum of two independent markers in studies of liver regeneration.

Use of proliferating cell nuclear antigen as a marker of liver regeneration after partial hepatectomy in rats

In order to document and compare proliferating cell nuclear antigen (PCNA) mRNA and protein levels with more traditional parameters of hepatic regenerative activity in a rat model, adult male Sprague-Dawley rats (4 to 6 per group) were killed at various times up to 96 hours after 70% partial hepatectomy. At each time interval, tissue PCNA mRNA abundance and protein levels were documented (by Northern and Western blot analysis, respectively) and compared with the results of PCNA immunostaining and 3H-thymidine incorporation into hepatic DNA. Tissue PCNA protein levels were also documented in additional groups of rats 12, 24, 36, and 48 hours after sham or 30% partial hepatectomy. PCNA mRNA expression after partial hepatectomy was variable: almost undetectable at 24 hours, levels returned to baseline at 36 hours, then fell again to low levels at 96 hours. PCNA protein levels remained stable for 36 hours, increased to fourfold above baseline (p < 0.01) at 48 hours, then remained elevated for the duration of the 96-hour study. Changes in PCNA by immunostaining were similar but tended to occur somewhat earlier (significant increases being detectable at 24 hours), whereas 3H-thymidine incorporation detected the earliest increases in DNA synthesis at 12 hours and peaked at 36 hours. Peak PCNA protein levels correlated with the extent (0%, 30%, or 70%) of hepatic resection. The results indicate that PCNA protein level as determined by Western blot analysis, but not PCNA mRNA expression, correlates with PCNA immunostaining and 3H-thymidine incorporation in the regenerating liver. These findings support the use of PCNA protein determinations as an additional quantitative measure of hepatic regenerative activity after partial hepatectomy in rats.

Serum aspartate but not alanine aminotransferase levels help to predict the histological features of chronic hepatitis c viral infections in adults

OBJECTIVES:The aims of this study were to assess the predictive values of age, gender, route of transmission, extent of steatosis, alcohol consumption, and serum aminotransferase values on the histological findings in patients with chronic hepatitis C viral infections.METHODS:We retrospectively reviewed the charts and liver biopsy findings from 79 adult patients with serological evidence of chronic hepatitis C viral infections.RESULTS:The mean (± SD) age of the patient population was 43.5 ± 10.8 yr; 47 patients (60%) were male. The routes of transmission were considered to be parenteral drugs in 44 patients (56%), previous blood transfusions in 25 (32%), and miscellaneous parenteral and nonparenteral routes in 10 (13%). The mean histological activity score of the group as described by Desmet et al. was 3.5 ± 0.8 (maximum possible score, 18) and the fibrosis score 1.5 ± 0.4 (maximum possible score, 4), indicating relatively mild disease in the majority of cases. The extent of inflammation correlated with fibrosis (r = 0.72). By multivariate stepwise regression analyses, serum aspartate aminotransferase (AST) values emerged as the most important predictive variable of histological activity (r = 0.62). When overall histological activity was further divided into portal inflammation, piecemeal necrosis, and lobular activity, correlations were found between AST values and portal inflammation (r = 0.58) and piecemeal necrosis (r = 0.61) but not lobular activity (r = 0.1). A correlation was also observed between AST values and the extent of hepatic fibrosis (r = 0.64). On the other hand, serum ALT values did not correlate with histological activity but did correlate weakly with the extent of hepatic fibrosis (r = 0.39 and 0.51, respectively). There were no significant correlations between age, gender, route of transmission, steatosis, or alcohol consumption with the extent of histological activity or fibrosis.CONCLUSIONS:Serum AST values correlate well with two of three features of hepatic inflammation and with the extent of hepatic fibrosis. These findings suggest that, among other factors, serum AST values should be considered in decisions regarding the need for liver biopsy and treatment in patients with chronic hepatitis C viral infections.

Randomized controlled trial of total immunosuppression withdrawal in liver transplant recipients : Role of ursodeoxycholic acid

Background. Total immunosuppression withdrawal (TIW) without causing rejection has been reported in stable liver recipients. The role of ursodeoxycholic acid (UDCA) and patient characteristics that predict the success of this tolerance are unclear. There are two goals, to determine: 1) whether TIW is frequently associated with rejection; and 2) whether UDCA decreases the risk of liver disease (both rejection and recurrence) after TIW. Methods. Twenty-six liver recipients who had been free of rejection while on immunosuppressive agents for a minimum of 2 years were randomized to receive either (15 mg/kg) of UDCA (n=14) or identical placebo (n=12) followed by sequential withdrawal of their immunosuppressive regimen over several months. Endpoints were defined as biochemical and histological evidence of rejection, graft dysfunction without rejection, recurrence of pretransplant disease, or 6 months without immunosuppression and no rejection or dysfunction on repeat liver biopsy. Results. Rejection occurred in 6 of 14 (43%) of the UDCA group and 9 of 12 (75%) of those receiving placebo (P=0.09). Degree of rejection was mild, moderate, and severe in 73%, 20%, and 7% of patients respectively. All responded to rescue therapy and none developed chronic rejection. Nine of the remaining 11 patients (eight of the UDCA recipients and three of controls) who did not develop rejection developed graft dysfunction which responded to reintroduction of immunosuppressive agents in each case. Disease recurrence was most common in patients with underlying immune-mediated disorders of the liver. One year after withdrawal only two patients were free of immunosuppression, 80% were able to discontinue prednisone therapy (steroid free), and 50% were able to reduce their dose of cyclosporine. Age, underlying cause of liver disease, and regimen of immunosuppression were favorable predictors. Conclusions. The results of this study suggest that TIW: 1) is frequently associated with subsequent rejection, 2) increases the risk of underlying disease recurrence, and 3) is not facilitated by UDCA use and responds properly to the reintroduction of immunosuppressive therapy.

Serum immunoglobulins predict the extent of hepatic fibrosis in patients with chronic hepatitis C virus infection

Summary.  Recently, we documented that immunoglobulins stimulate the proliferative activity of rat hepatic stellate cells in vitro. The aim of the present study was to determine whether there is any association between serum immunoglobulin levels and hepatic fibrosis in patients with chronic hepatitis C virus (HCV) infection. Charts from 116 patients with biochemical, serologic, virologic and histologic evidence of chronic hepatitis C infection and serum immunoglobulin levels (IgA, IgG, IgM and total) were reviewed. The mean (±SD) age of the study population was 46 ± 11 years and 67 (58%) were male. There were significant correlations between serum IgA (r = 0.39, P = 0.00001), IgG (r = 0.49, P = 0.000002) and total (r = 0.51, P = 0.000003) immunoglobulin levels and the stage of hepatic fibrosis. When serum immunoglobulin levels were included into logistic regression analysis with variables known to be associated with advanced disease (male gender, age >40 years at onset of infection, duration of infection beyond 20 years and concurrent alcohol abuse) only IgA, IgG and total immunoglobulin levels (P < 0.05, <0.05 and <0.005, respectively) emerged as independent predictors of hepatic fibrosis. Our data indicate a strong association between serum immunoglobulin levels (IgA, IgG and total) and hepatic fibrosis in patients with HCV infection. This finding supports the need to further investigate whether immunoglobulins independently promote disease progression in patients with chronic HCV infection.

Hepatic 31P MRS in rat models of chronic liver disease: assessing the extent and progression of disease.

Background: Hepatic adenosine triphosphate (ATP) levels are an accurate reflection of functioning hepatic mass following surgical resections and acute liver injury. Objective: To determine whether hepatic ATP levels can serve as a non-invasive means of documenting progression of chronic liver disease to cirrhosis. Methods: In vivo phosphorus-31 magnetic resonance spectroscopy (31P MRS) was performed in three animal models of chronic liver disease. Sixty six adult Sprague- Dawley rats were subjected to either thioacetamide, carbon tetrachloride (CCl4), or common bile duct ligation (CBDL) to induce liver disease (n=35, 21, and 10, respectively). Serial MRS examinations, blood samples, and liver biopsies (when appropriate) were obtained throughout and/or on completion of the study. Results: Over the course of the chronic liver disease, a progressive decrease in hepatic ATP levels was consistently observed in each model. The findings were most striking when end stage liver disease (cirrhosis) was established. The reduction in hepatic ATP levels correlated with significant changes in serum albumin concentrations (CCl4 and CBDL models) and the extent of hepatocyte loss seen histologically (all models). Conclusion: The results of this study indicate that during progression of chronic liver disease to cirrhosis, there is a progressive reduction in hepatic ATP levels. In addition, changes in hepatic ATP levels correlate with changes in liver function and histology. Thus hepatic 31P MRS provides a non-invasive means of documenting the severity and progression of parenchymal and cholestatic models of chronic liver disease in rats.

Gaba and hepatocellular carcinoma

Data derived from models of hepatic regeneration indicate that transient, recipricol changes in polyamines, potent growth promoters, and gamma aminobutyric acid (GABA), an amino acid neurotransmitter with growth inhibitory properties, play important roles in enhancing and inhibiting respectively regulated hepatocyte proliferation. Based on these findings and supportive data derived from studies of human carcinoma tissues and malignant cell lines we propose that permanent increases in polyamine and decreases in GABAergic activity act in concert to contribute to the pathogenesis of hepatocellular carcinoma.

Immediate-Early Protooncogene Expression and Liver Function Following Various Extents of Partial Hepatectomy in the Rat

Immediate–early protooncogenes (IEP) are thought to play an important role in hepatocyte replication. Whether the extent of their expression correlates with the strength of the proliferative stimulus and subsequent regenerative activity has yet to be documented in vivo. Data are also lacking with respect to the level at which liver disease is associated with biochemical evidence of hepatic dysfunction. Thus, the objectives of this study were to determine whether a correlation exists between IEP gene mRNA expression and varying extents of partial hepatectomy (PHx) and to document the extent of resection required to result in increases in serum bilirubin levels. Eighty-nine adult, male Sprague-Dawley rats underwent either sham surgery or 20%, 35%, 55%, 70% or 90% PHx. Postoperatively, rats were killed (N = 3–6/group) at 15 and 30 mins and 8 and 24 hrs for c-fos, c-jun, and c-myc mRNA expression by northern blot analyses. Rats killed at 24 hrs also had hepatic regenerative activity documented by [3H]thymidine incorporation into hepatic DNA and serum bilirubin determinations. While c-fos mRNA expression at 15 mins and c-myc mRNA expression at 8 hrs after PHx did not correlate with the extent of PHx (r2 = 0.478 and 0.018, respectively), a weak correlation existed between c-jun mRNA expression at 30 mins and the extent of PHx (r2 = 0.662, P < 0.05). In terms of IEP mRNA expression and hepatic regenerative activity, a strong correlation existed between c-fos mRNA expression and [3H]thymidine incorporation (r2 = 0.851, P < 0.01) but not c-jun or c-myc mRNA expression. Compared to sham operated controls, [3H]thymidine incorporation was 2.0×, 3.4×, 3.2×, 7.8×, and 2.2× increased following 20%, 35%, 55%, 70%, and 90% PHx, respectively. Serum bilirubin levels remained unchanged until 70% PHx, when they increased from baseline values of 0.54 ± 0.05 mg/dl to 1.02 ± 0.15 mg/dl (P < 0.05). A further increase occurred following 90% PHx (1.83 ± 0.30 mg/dl, P < 0.01). In conclusion these findings indicate that c-fos mRNA expression 15 mins after PHx correlates with hepatic regenerative activity but not the strength of the regenerative stimulus and that hepatic parenchymal loss of 55–70% must occur prior to the detection of elevated serum bilirubin levels. The results also indicate that relative to a 70% PHx, 90% PHx is associated with decreased rather than increased hepatic regenerative activity.

Proliferative and nutritional dependent regulation of glyceraldehyde-3-phosphate dehydrogenase expression in the rat liver

Abstract. Glyceraldehyde‐3‐phosphate dehydrogenase is a multifunctional protein possessing numerous cytoplasmic and nuclear functions associated with cellular proliferation. Despite the emerging role of glyceraldehyde‐3‐phosphate dehydrogenase in regulating the proliferative process, there is a paucity of data regarding its expression and intracellular distribution in non‐malignant proliferating hepatocytes. Thus the aim of the present study was to document the intracellular distribution of glyceraldehyde‐3‐phosphate dehydrogenase protein in proliferating hepatocytes derived from regenerating rat livers, and glyceraldehyde‐3‐phosphate dehydrogenase gene expression in fasted and re‐fed rats following partial hepatectomy (PHx). Glyceraldehyde‐3‐phosphate dehydrogenase mRNA and protein expression were documented by Northern and Western blot analyses, respectively, at various times following 70% PHx in adult Sprague–Dawley rats. At 24 h post‐surgery, glyceraldehyde‐3‐phosphate dehydrogenase mRNA expression was significantly increased in both PHx and sham operated rats (P < 0.001), respectively. Despite the increase in glyceraldehyde‐3‐phosphate dehydrogenase mRNA expression in both groups, only PHx rats had a significant increase in the nuclear fraction of glyceraldehyde‐3‐phosphate dehydrogenase protein (threefold increase compared to sham and baseline levels, P < 0.01), cytoplasmic levels of glyceraldehyde‐3‐phosphate dehydrogenase protein remained unaltered in both groups. In terms of the effects of feeding and fasting on rats there were no significant differences in glyceraldehyde‐3‐phosphate dehydrogenase mRNA levels, whether fasted or refed, in rats that had undergone PHx, 8 h earlier. On the other hand, glyceraldehyde‐3‐phosphate dehydrogenase mRNA levels were significantly increased in refed compared to fasted sham operated rats 8 h following surgery. Serum insulin concentrations were higher in the refed PHx and sham groups compared to their fasted counterparts. The results of this study indicate that although glyceraldehyde‐3‐phosphate dehydrogenase mRNA are altered to the same extent in PHx and sham‐operated rats following surgery, increases in the nuclear fraction of glyceraldehyde‐3‐phosphate dehydrogenase protein only occur in PHx rats. The results also indicate that glyceraldehyde‐3‐phosphate dehydrogenase expression is affected by the nutritional status of animals undergoing abdominal sham surgery.

The effects of ethanol and gamma aminobutyric acid alone and in combination on hepatic regenerative activity in the rat

BACKGROUND/AIMS Both ethanol and gamma aminobutyric acid (GABA) have been reported to inhibit hepatic regenerative activity in the rat. Because alcoholic beverages contain appreciable amounts of GABA, we documented whether the inhibitory effects of alcohol on the liver are derived from ethanol alone or the combination of ethanol plus GABA. METHODS Adult male Sprague-Dawley rats (n=6/group) were treated with either ethanol (3 g/kg), GABA (500 mg/kg) or ethanol plus GABA (3 kg and 500 mg/kg, respectively), beginning 1 h prior to a 70% partial hepatectomy and continued every 4 h thereafter for a total of 24 h. Rats were then sacrificed and hepatic regenerative activity was documented by 3H-thymidine incorporation into hepatic DNA. RESULTS DNA synthesis was significantly inhibited by ethanol (-37%, p<0.005) and GABA (-19%, p<0.05). Maximum inhibition was achieved with the combination of ethanol plus GABA (-52%, p<0.001). To determine whether the additive effects of ethanol plus GABA were mediated by ethanol-induced enhancement of hepatic GABA(A) receptor activity, additional rats (n=6/group) receiving the combination of ethanol plus GABA were pre-treated with a single injection of either ciprofloxacin (50 mg/kg), a GABA(A) receptor antagonist, or an equal volume of saline. In these experiments, ciprofloxacin pre-treatment prevented the inhibitory effects of the ethanol plus GABA combination. CONCLUSIONS The results of this study indicate that the combination of ethanol plus GABA has a greater inhibitory effect on hepatic DNA synthesis following partial hepatectomy than ethanol alone. The clinical implication of this finding is that, when standardized for ethanol content, not all alcoholic beverages would be expected to have the same inhibitory effect on hepatic regeneration.