Effect of the immunosuppressants on hepatocyte proliferation and apoptosis in a young animal model of liver regeneration: An immunohistochemical study using tissue microarrays

Hepatocyte proliferation and apoptosis (programmed cell death) occur during the liver parenchyma regeneration and the liver size modeling is mainly controlled by hepatocyte apoptosis. The purpose of the present study was to verify the influence of immunosuppressant drugs on these phenomena by utilizing tissue microarray techniques. Thirty-six weaning rats (age 21-23 days, weight 30-50 g) were divided into six groups: control, sham, hepatectomy, hepatectomy plus solumedrol, hepatectomy plus CsA, and hepatectomy plus Tac. The animals were killed one day after hepatectomy, and the remnant livers were weighed and harvested for tissue microarray sections. Liver cell proliferation was evaluated by staining for PCNA and apoptosis was detected by the TUNEL method. It was verified that CsA promoted a decrease in the liver weight, Tac and CsA decreased the proliferation index of hepatocytes, and glucocorticoid had no significant effects. The apoptosis index was not altered by hepatectomy or immunosuppressants. Our data indicate that, in the growing rat, CsA and Tac have negative effects on hepatocyte proliferation and have no effect on the hepatocyte apoptosis.

Effects of immunosuppressants on hepatocyte cell mitosis during liver regeneration in growing animal models of partial hepatectomy

Objective. We sought to evaluate the effects of immunosuppressant drugs (corticosteroid, cyclosporine [CsA], and tacrolimus [Tac]) on liver regeneration in growing animals submitted to 70% hepatectomy. Materials and Methods. Newborn and weaning rats were submitted to 70% hepatectomy receiving separately methylprednisolone, CsA, or Tac. All animals were sacrificed 24 hours after the procedure. The remnant liver lobes were subjected to histomorphometric analyses with determination of hepatocyte mitotic index. Results., Administration of immunosuppressants did not change the mitotic index of the regenerating liver in newborn animals. In weaning rats, methylprednisolone reduced the mitotic index (P = .01) and Tac caused a greater increase in this rate (P = .001). CsA had no effect on mitotic index. The number of hepatocyte mitoses in newborn animal livers was greater than that in weaning animal livers (P = .001). Conclusion. In situations in which intense, fast processes of liver regeneration are crucial, the advantages of the use of Tac must be considered, such as in pediatric transplant patients.

Effect of the immunosuppressants on hepatocyte cells proliferation and apoptosis during liver regeneration after hepatectomy - molecular studies

The regeneration and remodeling of the transplanted liver is the result of hepatocyte proliferation and apoptosis (programmed cell death). The purpose of this study was to verify the influence of immunosuppressants on the expression levels of genes: IL-6 (regulator of hepatocyte proliferation), pro-apoptotic (Bak and Bax) and anti-apoptotic (Bcl-Xl and Bcl-2). 36 newborn suckling rats (age 5-7 days, weight 6-10 g) were divided into four groups: hepatectomy, hepatectomy plus methylprednisolone, hepatectomy plus CsA and hepatectomy plus Tac. The same experiments were performed in 24 weaning rats (age 21-23 days, weight 30-50 g). The animals were killed one day after the hepatectomy and the remnant livers were analyzed. The livers of all animals exhibited histological changes of liver regeneration. The immunosuppressants did not promote any alteration on IL-6 gene expression levels. Methylprednisolone and CsA increased the expression levels of Bak gene in newborn rats. However, methylprednisolone and Tac promoted increased expression levels of Bcl-2 in all groups. We hypothesize that these effects explain the efficacy of these drugs on the treatment of acute and chronic liver rejection as the expression of Bcl-2 in cholangiocytes is decreased as a consequence of bile duct lesions.

The role of NFI-C liver regeneration and its potential function as a general regulator of regenerative processes

Collectively, research aimed to understand the regeneration of certain tissues has unveiled the existence of common key regulators. Knockout studies of the murine Nuclear Factor I-C (NFI-C) transcription factor revealed a misregulation of growth factor signaling, in particular that of transforming growth factor ß-1 (TGF-ßl), which led to alterations of skin wound healing and the growth of its appendages, suggesting it may be a general regulator of regenerative processes. We sought to investigate this further by determining whether NFI-C played a role in liver regeneration. Liver regeneration following two-thirds removal of the liver by partial hepatectomy (PH) is a well-established regenerative model whereby changes elicited in hepatocytes following injury lead to a rapid, phased proliferation. However, mechanisms controlling the action of liver proliferative factors such as transforming growth factor-ßl (TGF-ß1) and plasminogen activator inhibitor-1 (PAI-1) remain largely unknown. We show that the absence of NFI-C impaired hepatocyte proliferation due to an overexpression of PAI-1 and the subsequent suppression of urokinase plasminogen (uPA) activity and hepatocyte growth factor (HGF) signaling, a potent hepatocyte mitogen. This indicated that NFI-C first acts to promote hepatocyte proliferation at the onset of liver regeneration in wildtype mice. The subsequent transient down regulation of NFI-C, as can be explained by a self- regulatory feedback loop with TGF-ßl, may limit the number of hepatocytes entering the first wave of cell division and/or prevent late initiations of mitosis. Overall, we conclude that NFI-C acts as a regulator of the phased hepatocyte proliferation during liver regeneration. Taken together with NFI-C's actions in other in vivo models of (re)generation, it is plausible that NFI-C may be a general regulator of regenerative processes. - L'ensemble des recherches visant à comprendre la régénération de certains tissus a permis de mettre en évidence l'existence de régulateurs-clés communs. L'étude des souris, dépourvues du gène codant pour le facteur de transcription NFI-C (Nuclear Factor I-C), a montré des dérèglements dans la signalisation de certains facteurs croissance, en particulier du TGF-ßl (transforming growth factor-ßl), ce qui conduit à des altérations de la cicatrisation de la peau et de la croissance des poils et des dents chez ces souris, suggérant que NFI-C pourrait être un régulateur général du processus de régénération. Nous avons cherché à approfondir cette question en déterminant si NFI-C joue un rôle dans la régénération du foie. La régénération du foie, induite par une hépatectomie partielle correspondant à l'ablation des deux-tiers du foie, constitue un modèle de régénération bien établi dans lequel la lésion induite conduit à la prolifération rapide des hépatocytes de façon synchronisée. Cependant, les mécanismes contrôlant l'action de facteurs de prolifération du foie, comme le facteur de croissance TGF-ßl et l'inhibiteur de l'activateur du plasminogène PAI-1 (plasminogen activator inhibitor-1), restent encore très méconnus. Nous avons pu montrer que l'absence de NFI-C affecte la prolifération des hépatocytes, occasionnée par la surexpression de PAI-1 et par la subséquente suppression de l'activité de la protéine uPA (urokinase plasminogen) et de la signalisation du facteur de croissance des hépatocytes HGF (hepatocyte growth factor), un mitogène puissant des hépatocytes. Cela indique que NFI-C agit en premier lieu pour promouvoir la prolifération des hépatocytes au début de la régénération du foie chez les souris de type sauvage. La subséquente baisse transitoire de NFI-C, pouvant s'expliquer par une boucle rétroactive d'autorégulation avec le facteur TGF-ßl, pourrait limiter le nombre d'hépatocytes qui entrent dans la première vague de division cellulaire et/ou inhiber l'initiation de la mitose tardive. L'ensemble de ces résultats nous a permis de conclure que NFI-C agit comme un régulateur de la prolifération des hépatocytes synchrones au cours de la régénération du foie.

Nuclear Factor I-C acts as a regulator of hepatocyte proliferation at the onset of liver regeneration.

BACKGROUND & AIMS: Knockout studies of the murine Nuclear Factor I-C (NFI-C) transcription factor revealed abnormal skin wound healing and growth of its appendages, suggesting a role in controlling cell proliferation in adult regenerative processes. Liver regeneration following partial hepatectomy (PH) is a well-established regenerative model whereby changes elicited in hepatocytes lead to their rapid and phased proliferation. Although NFI-C is highly expressed in the liver, no hepatic function was yet established for this transcription factor. This study aimed to determine whether NFI-C may play a role in hepatocyte proliferation and liver regeneration. METHODS: Liver regeneration and cell proliferation pathways following two-thirds PH were investigated in NFI-C knockout (ko) and wild-type (wt) mice. RESULTS: We show that the absence of NFI-C impaired hepatocyte proliferation because of plasminogen activator I (PAI-1) overexpression and the subsequent suppression of urokinase plasminogen activator (uPA) activity and hepatocyte growth factor (HGF) signalling, a potent hepatocyte mitogen. This indicated that NFI-C first acts to promote hepatocyte proliferation at the onset of liver regeneration in wt mice. The subsequent transient down regulation of NFI-C, as can be explained by a self-regulatory feedback loop with transforming growth factor beta 1 (TGF-ß1), may limit the number of hepatocytes entering the first wave of cell division and/or prevent late initiations of mitosis. CONCLUSION: NFI-C acts as a regulator of the phased hepatocyte proliferation during liver regeneration.

Liver regeneration with l-glutamine supplemented diet: experimental study in rats

OBJECTIVE: To assess liver regeneration in rats after 60% hepatectomy with and without supplementation of L-glutamine through liver weight changes, laboratory parameters and histological study.METHODS: 36 male rats were divided into two groups: glutamine group and control group. Each group was subdivided into three subgroups, with death in 24h, 72h and seven days. The glutamine group received water and standard diet supplemented with L-glutamine, and the control recieved 0.9% saline. In all subgroups analysis of liver regeneration was made by the Kwon formula, study of liver function (AST, ALT, GGT, total bilirubin, indirect and indirect bilirubin and albumin) and analysis of cell mitosis by hematoxylin-eosin.RESULTS: In both groups there was liver regeneration by weight gain. Gamma-GT increased significantly in the control group (p < 0.05); albumin increased in the glutamine group. The other indicators of liver function showed no significant differences. Histological analysis at 72h showed a higher number of mitoses in the glutamine group, with no differences in other subgroups.CONCLUSION: Diet supplementation with L glutamine is beneficial for liver regeneration.

Sirolimus influence on hepatectomy-induced liver regeneration in rats

OBJECTIVE: To evaluate the influence of sirolimus on liver regeneration triggered by resection of 70% of the liver of adult rats.METHODS: we used 40 Wistar rats randomly divided into two groups (study and control), each group was divided into two equal subgroups according to the day of death (24 hours and seven days). Sirolimus was administered at a dose of 1mg/kg in the study group and the control group was given 1 ml of saline. The solutions were administered daily since three days before hepatectomy till the rats death to removal of the regenerated liver, conducted in 24 hours or 7 days after hepatectomy. Liver regeneration was measured by the KWON formula, by thenumber of mitotic figures (hematoxylin-eosin staining) and by the immunohistochemical markers PCNA and Ki-67.RESULTS: there was a statistically significant difference between the 24h and the 7d groups. When comparing the study and control groups in the same period, there was a statistically significant variation only for Ki-67, in which there were increased numbers of hepatocytes in cell multiplication in the 7d study group compared with the 7d control group (p = 0.04).CONCLUSION: there was no negative influence of sirolimus in liver regeneration and there was a positive partial effect at immunohistochemistry with Ki-67.

Evaluation of liver regeneration diet supplemented with omega-3 fatty acids: experimental study in rats

Objective: to evaluate liver regeneration in rats after partial hepatectomy of 60% with and without action diet supplemented with fatty acids through the study of the regenerated liver weight, laboratory parameters of liver function and histological study. Methods: thirty-six Wistar rats, males, adults were used, weighing between 195 and 330 g assigned to control and groups. The supplementation group received the diet by gavage and were killed after 24h, 72h and seven days. Evaluation of regeneration occurred through analysis of weight gain liver, serum aspartate aminotransferase, alanine aminotransferase, gamma-glutamyltranspeptidase, and mitosis of the liver stained with H&E. Results: the diet supplemented group showed no statistical difference (p>0.05) on the evolution of weights. Administration of fatty acids post-hepatectomy had significant reduction in gamma glutamyltransferase levels and may reflect liver regeneration. Referring to mitotic index, it did not differ between period of times among the groups. Conclusion: supplementation with fatty acids in rats undergoing 60% hepatic resection showed no significant interference related to liver regeneration.

Angiotensin-converting enzyme inhibition by lisinopril enhances liver regeneration in rats

Bradykinin has been reported to act as a growth factor for fibroblasts, mesangial cells and keratinocytes. Recently, we reported that bradykinin augments liver regeneration after partial hepatectomy in rats. Angiotensin-converting enzyme (ACE) is also a powerful bradykinin-degrading enzyme. We have investigated the effect of ACE inhibition by lisinopril on liver regeneration after partial hepatectomy. Adult male Wistar rats underwent 70% partial hepatectomy (PH). The animals received lisinopril at a dose of 1 mg kg body weight-1 day-1, or saline solution, intraperitoneally, for 5 days before hepatectomy, and daily after surgery. Four to six animals from the lisinopril and saline groups were sacrificed at 12, 24, 36, 48, 72, and 120 h after PH. Liver regeneration was evaluated by immunohistochemical staining for proliferating cell nuclear antigen using the PC-10 monoclonal antibody. The value for the lisinopril-treated group was three-fold above the corresponding control at 12 h after PH (P<0.001), remaining elevated at approximately two-fold above control values at 24, 36, 48 (P<0.001), and at 72 h (P<0.01) after PH, but values did not reach statistical difference at 120 h after PH. Plasma ACE activity measured by radioenzymatic assay was significantly higher in the saline group than in the lisinopril-treated group (P<0.001), with 81% ACE inhibition. The present study shows that plasma ACE inhibition enhances liver regeneration after PH in rats. Since it was reported that bradykinin also augments liver regeneration after PH, this may explain the liver growth stimulating effect of ACE inhibitors.

The use of light-emitting diodes to stimulate mitochondrial function and liver regeneration of partially hepatectomized rats

The biostimulating effect of laser radiation has been observed in many areas of Medicine. However, there are still several questions to be answered, among them the importance of light coherence in the stimulatory process. In the present study, we used light-emitting diodes (LED) to promote the stimulation of liver regeneration after partial hepatectomy in rats. Fourteen male Wistar rats weighing 200-250 g were submitted to partial hepatectomy (70%) followed by LED light irradiation (630 nm) of the remaining part of the liver at two doses, i.e., 10 (N = 7) and 140 (N = 7) J/cm². A group irradiated with laser, 590 nm (N = 7, 15 J/cm²) was performed for the study of proliferating cell nuclear antigen-labeling index. Data are reported as mean ± SEM. Statistical comparisons of the groups were performed by analysis of variance for parametric measurements followed by the Bonferroni post-test, with the level of significance set at P < 0.05. Respiratory mitochondrial activity was increased in the irradiated groups (states 3 and 4; P < 0.05), with better results for the group exposed to the lower LED dose (10 J/cm²). The proliferating cell nuclear antigen-labeling index, by immunohistochemical staining, was similar for both LED-exposed groups (P > 0.05) and higher than for the control group (P < 0.05). The cell proliferation index obtained with LED and laser were similar (P > 0.05). In conclusion, the present results suggest that LED irradiation promotes biological stimulatory effects during the early stage of liver regeneration and that LED is as effective as laser light, independent of the coherence, divergence and cromaticity.

Kinetic Parameters of Thymidine Kinase and DNA Synthesis During Liver Regeneration: Role Ofthyroid Hormones

The role of thyroid hormones in DNA synthesis and in the activity of Thymidille kinase (TK), a key regulatory enzyme of DNA synthesis was studied in proliferating hepatocytes in vivo. Liver regeneration after partial hepatectomy was used as a model for controlled cell division in rats having different thyroid status - euthyroid, hypothyroid and 3,3',5'-triiodo-L-thyronine (T))-heated hypothyroid. Partial hepatectomy caused a significant elevation of DNA synthesis (p<0.01) in all the three groups compared to their sham-operated counterparts. Hypothyroid liepatectomised animals showed significantly lower (p<0.01) level of DNA synthesis than euthyroid hepatectomised animals. A single subcutaneous close of 1'3 to hypothyroid shamoperated animals resulted in a significant increase (p<0.01) of DNA synthesis in the intact liver. 17tis was comparable to the level of DNA synthesis occurring in regenerating liver of euthyroid animals. In hypothyroid hepatectomised animals, "1'3 showed an additive effect on l)NA synthesis and this group exhibited maximum level of DNA synthesis (p<0.0I ). Studies of the kinetic parameters of TK show that the Michelis-Menten constant, (K111) of TK for thymidine was altered by the thyroid status. K11 increased significantly (p<0.01) in untreated hypothyroid animals when compared to the euthyroid rats. '13 treatment of hypothyroid animals reversed this effect and this group showed the lowest value for K111 (p<0.01). Thus our results indicate that thyroid hormones can influence DNA synthesis during liver regeneration and they may regulate the activity of enzymes such as 17rymidine kinase which are important for DNA synthesis and hence cell division.

Increased 5-HT2C Receptor Binding in the Brain Stem And Cerebral Cortex During Liver Regeneration And Hepatic Neoplasia In Rats

In the present study, serotonin 2C (5-HT2c) receptor binding parameters in the brainstem and cerebral cortex were investigated during liver generation after partial hepatectomy (PH) and N-nitrosodiethylamine (NDEA) induced hepatic neoplasia in male Wistar rats. The serotonin content increased significantly (p<0.01) in the cerebral cortex after PH and in NDEA induced hepatic neoplasia. Brain stem serotonin content increased significantly (p<0.05) after PH and (p<0.001) in NDEA induced hepatic neoplasia. The number and affinity of the 5-HT2c receptors in the crude synaptic membrane preparations of the brain stem showed a significant (p<0.001) increase after PH and in NDEA induced hepatic neoplasia. The number and affinity of 5-HT2c receptors increased significantly (p<0.001) in NDEA induced hepatic neoplasia in the crude synaptic membrane preparations of the cerebral cortex. There was a significant (p<0.01) increase in plasma norepinephrine in PH and (p<0.001) in NDEA induced hepatic neoplasia, indicating sympathetic stimulation. Thus, our results suggest that during active hepatocyte proliferation 5-HT2c receptor in the brain stem and cerebral cortex are up-regulated which in turn induce hepatocyte proliferation mediated through sympathetic stimulation.

Adrenergic and serotonergic function in dna synthesis during rat liver regeneration and in hepatocyte cultures

The adult mammalian liver is predominantly in a quiescent state with respect to cell division. This quiescent state changes dramatically, however, if the liver is injured by toxic, infectious or mechanic agents (Ponder, 1996). Partial hepatectomy (PH) which consists of surgical removal of two-thirds of the liver, has been used to stimulate hepatocyte proliferation (Higgins & Anderson 1931). This experimental model of liver regeneration has been the target of many studies to probe the mechanisms responsible for liver cell growth control (Michalopoulos, 1990; Taub, 1996). After PH most of the remaining cells in the renmant liver respond with co-ordinated waves of DNA synthesis and divide in a process called compensatory hyperplasia. Hence, liver regeneration is a model of relatively synchronous cell cycle progression in vivo. In contrast to hepatomas, cell division is terminated under some intrinsic control when the original cellular mass has been regained. This has made liver regeneration a useful model to dissect the biochemical and molecular mechanisms of cell division regulation. The liver is thus, one of the few adult organs that demonstrates a physiological growth rewonse (Fausto & Mead, 1989; Fausto & Webber, 1994). The regulation of liver cell proliferation involves circulating or intrahepatic factors that are involved in either the priming of hepatocytes to enter the cell cycle (Go to G1) or progression through the cell cycle. In order to understand the basis of liver regeneration it is mandatory to define the mechanisms which (a) trigger division, (b) allow the liver to concurrently grow and maintain dilferentiated fimction and (c) terminate cell proliferation once the liver has reached the appropriate mass. Studies on these aspects of liver regeneration will provide basic insight of cell growth and dilferentiation, liver diseases like viral hepatitis, toxic damage and liver transplant where regeneration of the liver is essential. In the present study, Go/G1/S transition of hepatocytes re-entering the cell cycle after PH was studied with special emphasis on the involvement of neurotransmitters, their receptors and second messenger function in the control of cell division during liver regeneration

Effect of sorafenib on murine liver regeneration

Hepatocellular carcinoma (HCC) is a common cause of cancer-related death. Sorafenib prolongs survival of patients with advanced disease and is approved for the systemic treatment of unresectable HCC. It possesses antiangiogenic and antiproliferative properties by way of inhibition of the receptor tyrosine kinases vascular endothelial growth factor receptor 2 (VEGFR-2) and platelet-derived growth factor receptor-beta 1/2 (PDGFR-β) and the kinase RAF. Sorafenib represents a candidate compound for adjuvant therapy in HCC patients. The aim of our study was to investigate whether sorafenib affects liver regeneration. C57BL6 mice received sorafenib orally at 30 mg/kg/day or its vehicle either for 14 days until the day before hepatectomy or starting the day after surgery or both. Animals were sacrificed 24, 72, and 120 hours after hepatectomy. Liver regeneration was calculated as a percent of initial liver weight. Bromodeoxyuridine (BrdU) incorporation and phospho-extracellular signal-regulated kinase (pERK1/2) were determined by immunohistochemistry on liver sections. VEGF-A, PDGF-BB, and hepatocyte growth factor (HGF) levels were measured in liver tissue homogenates. Histological analysis of scar tissue was performed. Treatment stopped 1 day before surgery had no impact on liver regeneration. Continuous sorafenib treatment and treatment started 1 day after surgery had statistically significant effects on liver regeneration at 120 hours compared to vehicle-treated control animals (72% ± 12 versus control 88% ± 15 and 70% ± 13 versus control 86% ± 5 at 120 hours, both P ≤ 0.02). BrdU incorporation showed decreased numbers of positive nuclei in both groups receiving sorafenib after surgery. Phospho-ERK levels were reduced in sorafenib-treated animals. An increase of VEGF-A levels was observed in mice receiving sorafenib. Wound-healing complications were observed in animals receiving sorafenib after surgery and confirmed on histological sections. CONCLUSION: This preclinical study shows that sorafenib did not impact on liver regeneration when ceased before surgery; however, administration after hepatectomy affected late liver regeneration.