Partial Hepatectomy Group Research Articles

Effect of Resveratrol and Melatonin on Oxidative Stress Enzymes, Regeneration, and Hepatocyte Ultrastructure in Rats Subjected to 70% Partial Hepatectomy

Aim We sought to compare the antioxidant effects of resveratrol (R) and melatonin (M) after 70% partial hepatectomy (PH) as evidenced by ultrastructural alterations and effects on hepatocyte proliferation and apoptosis. Methods Twenty-six male Wistar albino rats were randomized into four groups: group A ( n = 8) resveratrol (R); group B ( n = 8) melatonin (M); group C ( n = 5) control PH; group D ( n = 5) sham operated animals. The rats that received either R or M were sacrificed a week after PH. The malondialdehyde, glutathione, glutathione S-transferase, and nitric oxide levels were estimated in liver homogenates. The morphological changes were investigated using light and electron microscopy (EM). Cell proliferation was detected by immunohistochemical staining with monoclonal antibodies to Ki-67. Apoptosis was detected by the transferase-mediated dUTP nick end-labeling method. Results PH induced hepatic LP, decreased GSH and NO, and inhibited GST activity ( P < .05). R and M completely prevented PH-induced lipid peroxidation, decreased hepatic GSH and NO levels ( P < .05). The inhibition of GST activity was prevented by R ( P < .05), but not with M ( P > .05). In the PH group EM showed severe morphological changes: mitochondrial degeneration, vacuoles, lipid droplets, and myelin-like figures. In both the R and M groups, morphological alterations repaired protective effects more prominently in the R group. Ki-67 indices (KI) were increased in the PH group and decreased in both R and M groups ( P < .001). In the M group, KI was the lowest, but the difference compared with R was not significant ( P > .05). Apoptosis was slightly increased in PH, but in either the R or M groups, apoptosis was intensively increased ( P < .001). Increased apoptosis was greatest in the M group and the difference compared with the R group was statistically significant ( P < .05). Conclusion R and M suppressed PH-induced oxidative damage, attenuated proliferation, and stimulated apoptosis. When we compared R and M, R showed more potent antioxidative effects and was morphologically more protective to hepatocytes. Antiproliferative effects of M were more potent. Because of their potent antioxidative effects, R and M can be effective for oxidative damage like ischemia-reperfusion injury; however, because of the adverse effects on proliferation and apoptosis more studies are needed in states in which regeneration is critical.

Expression Patterns of the Cell Junction-associated Genes During Rat Liver Regeneration

To study actions of the genes associated with tight junction, adherent junction, focal adhesion, and gap junction during liver regeneration (LR), these genes were obtained by collecting data from databases and thesis, and their expression profiles in rat regenerating liver were detected employing Rat Genome 230 2.0 array. Next the LR-associated genes were identified by comparing the difference between sham operation (SO) and partial hepatectomy (PH) groups. 79, 53, 109, 53 genes involved in the above four junctions were found to be LR-associated. The initial and total expression numbers of these genes occurring in the initial phase of LR, G0/G1, cell proliferation, cell differentiation, and structure-functional rebuilding were 124, 43, 122, 10, and 249, 145, 957, 306, respectively, illustrating that genes were initially expressed mainly in the initiation stage, and functioned in different phases. Up-regulation and down-regulation to a total of 972 and 540 times, as well as, 41 types of expression patterns showed that the physiological and biochemical activities were diverse and complicated in LR. According to the data, there was an increase in the forepart and prophase, but a decrease in late-metaphase and anaphase for gap junction assembly. Focal adhesion formation displayed an enhancement in forepart, prophase, and anaphase; and formation of tight junctions and adherent junctions last throughout the LR.

Effect of partial hepatectomy on the memory ability in rats

Abstract Background Patients suffer from changes of cognitive function postoperatively, which has attracted extensive attentions in clinic. It is still to be investigated whether operation damages both antegrade memory and retrograde memory, or either of them. Objective To observe the effects of operation on the anterograde and retrograde memory with Y maze test. Design A randomly controlled animal trial. Setting Department of Anesthesiology, Pudong New Area Gongli Hospital of Shanghai City. Materials Sixty-three male healthy Sprague-Dawley (SD) rats of clean degree, weighing 200–250 g, 10 weeks old, were provided by the animal center of Chinese Academy of Sciences. The rats were tested by antegrade and retrograde respectively. The rats were randomly divided into normal control group (n =7), anesthesia group (n =14), sham-operated group (n =14); partial hepatectomy group (n =14), short-term retrograde memory group (n =7) and long-term retrograde memory group (n =7). According to the time of Y maze training started, rats in the anesthesia group, sham-operated group and partial hepatectomy group were observed at 1 and 7 days after anesthesia. Methods The experiments were carried out in the central laboratory of Pudong New Area Gongli Hospital of Shanghai City from February to June in 2006. Test for antegrade memory: Rats in the normal control group were only treated with intraperitoneal injection of saline without aesthesia and operation; Those in the anesthesia group were anesthetized with intraperitoneal injection of 10 g/L pentobarbital sodium (40 mg/kg); Those in the sham-operated group were anesthetized, then intraperitoneal exploration was performed without hepatectomy; Those in the partial hepatectomy group were anesthetized, then fixed in a supine position after righting reflex disappeared, and an incision of 1.5–2.0 cm was made along the midline of xiphoid lower abdomen, then left lateral lobe of liver (about 1/3 of total liver) was freed, and ligated at distal pedicel with 1# filament. The abdomen was closed layer by layer after complete hemostasis. The learning and memory abilities were tested after operation. The rats were exposed to Y maze exercise from the 1st or 7th day after surgery, the error numbers, the time to complete whole trials per day and active avoidance numbers were recorded to evaluate the antegrade memory ability. Test for short-term retrograde memory: Rats in the short-term retrograde memory group were trained in Y maze as the above methods for 1 day, then partial hepatectomy was performed immediately, and the effects on short-term memory were evaluated at 24 hours postoperatively. Test for long-term retrograde memory: Rats in the long-term retrograde memory group were trained in Y maze till reached the standard for grasping, then partial hepatectomy was performed in those reached the standard for grasping within 3 days, and the effects on long-term memory were evaluated at 48 hours postoperatively. Main Outcome Measures The error times, the time to complete whole trials per day and active avoidance times were observed every day. Results Totally 63 SD rats were used, including 6 failed in the partial hepatectomy in the partial hepatectomy group, 3 died due to over-anesthesia, and 5 in the long-term retrograde memory group grasped the tasks in Y maze till the 4th day, which were supplemented by other rats. * Impairment of anterograde memory after partial hepatectomy: The results of Y maze test in the partial hepatectomy group were not obviously different from those in the other groups on the 1st day. The error times on the 2nd and 3rd days of training in the partial hepatectomy group at 1 and 7 days after anesthesia were obviously more than those in the normal control group (F =6.08, P Conclusion Partial hepatectomy mainly impaires the anterograde memory abilities of rats, and it has no effect on the retrograde short-term memory and long-term memory.

N‐ACETYLCYSTEINE IMPROVES INTESTINAL BARRIER IN PARTIALLY HEPATECTOMIZED RATS

Translocating enteric bacteria play an important role in the development of infections following partial hepatectomy. The intestine itself is the first line of defence against bacterial translocation (BT). We investigated the effect of N-acetylcysteine (NAC) on BT and the intestinal wall. We compared four groups of Sprague-Dawley male rats (eight in each group): sham, sham plus preoperative single dose of NAC, partial hepatectomy and partial hepatectomy plus preoperative single dose of NAC. Microorganism count in the tissues and the glutathione and malondialdehyte contents of the intestinal wall were studied at the end of the 24th hour. Bacterial growth was observed in the spleen and mesenteric lymph nodes in the sham group. There was bacterial growth in all the samples of the partial hepatectomy group. Differences were significant except in atrial and portal blood counts. In the partial hepatectomy plus NAC treatment group, counts were significantly low in all, except atrial and portal blood samples. The malondialdehyte level in the intestinal wall was 35.38 +/- 10.27 in the sham group, increasing significantly in the partial hepatectomy group (69.50 +/- 21.48), and decreasing in the partial hepatectomy plus NAC treatment group (35.63 +/- 14.12). Glutathione levels decreased significantly in the partial hepatectomy group and increased with preoperative single-dose NAC. Partial hepatectomy resulted in oxidative disturbances in intestinal wall, which in turn gave rise to BT. Parenteral NAC protects the intestinal wall from oxidative injury and attenuates BT.

The phosphoinositide 3-kinase/Akt-signal pathway mediates proliferation and secretory function of hepatic sinusoidal endothelial cells in rats after partial hepatectomy

Objective To investigate the role of AKT signaling pathway in hepatic sinusoidal endothelial cells (SECs) early after partial hepatectomy in rats and the regulatory mechanisms involved. Methods The animal model of 70% hepatectomy was made. Hepatic SECs were isolated and cultured according to Braet et al.’s method with some modifications. The cultured hepatic SECs were divided into two groups: 70% partial hepatectomy groups and LY294002 group (LY). We observed the expressions of AKT and NF-κB in cultured hepatic SECs by Western blot, measured the levels of NO, NOs, IL-6, and HGF in the supernatants of hepatic SEC cultures and [ 3H]thymidine incorporation, and analyzed cell cycle of cultured hepatic SECs by flow cytometer. The relationship of the Akt pathway with secretions and proliferation of hepatic SECs after partial hepatectomy was probed. Results The levels of Akt protein expression increased significantly after partial hepatectomy in OG group and with a peak at 24 h post operation. Meanwhile, there was a markedly increase in phosphorylated Akt protein during 2–72 h after operation. But the expression and activity of Akt protein did not change significantly after partial hepatectomy in the LY group. So, partial hepatectomy can marked induce Akt expression and result in rapid and marked phosphorylation of Akt from 2 to 72 h thereafter. The changes of NF-κB expression in cultured hepatic SECs were similar to those of Akt expression after operation. The concentrations of HGF and IL-6 in the supernatants of cultured hepatic SECs were relatively low in the LY group, and were markedly increased after partial hepatectomy, with a peak at 24 h in the OG group. There were significant differences between the OG and LY groups at 6 and 24 h ( P < 0.05). Both NO and NOS secretion was increased in the OG group compared to the LY group within 24 h after partial hepatectomy. But the secretion of NO and NOS was increased more markedly in the LY group than that in the OG beyond 24 h. These findings suggest that the secretion of the cytokines by hepatic SECs is mediated by Akt signaling. Akt signaling pathway in relationship with proliferation of hepatic SECs and suppression of apoptosis. In OG group, the hepatic SECs in S and G2/M obviously increased. The proliferative index of hepatic SECs in OG group had significant differences with that in LY group at 6, 24, and 72 h, P < 0.05. Meanwhile, the cells of apoptosis in OG group were very low, and the cells in LY group gradually increased. Conclusions These results suggest that AKT signaling pathway plays a crucial role in mediating proliferating and secreted signals in hepatic SECs. AKT has been suggested to play a pivotal role in early liver regeneration involved in the induction of secreted cytokines and proliferation of hepatic SECs.

Liver regeneration is a salivary VEGF dependent phenomenon

Introduction: Liver “regeneration” following partial hepatectomy (PH) is associated with VEGF upregulation and involves the formation of a complex network of liver sinusoids through which the blood flows. Previous work in our lab demonstrates the importance of submandibular salivary gland- derived VEGF in small bowel adaptation following massive small bowel resection. We hypothesize that salivary- derived VEGF may be essential to the regenerative stimulus for the compensatory growth after PH. We examined the effect on liver regeneration following PH, of both submandibular gland excision (SAL) and selective blockade of VEGF by Ad-VEGF-trap with and without supplemental Oro-gastric VEGF replacement. Methods: C57BL/6 Male mice underwent either Sham operation (n=5), PH alone (70% liver resection, n=5), PH+SAL (n=5), PH+AdVEGF-trap (1× 108 pfu) via retrograde parotid duct injection (n=4), PH+SAL+VEGF (100ng/ml) (n=5). Animals were harvested after 14 days. Liver regeneration was evaluated by wet liver weights. Hepatic angiogenesis was quantified by intrahepatic microvascualr density using CD31 (PECAM-1) staining of paraffin embedded liver. Statistical analysis was performed with ANOVA. Results: The wet liver weight was significantly lower in SAL treated mice compared to control PH mice (0.996±0.26gms vs.1.2±0.31gms, p=0.005) as it was in selective blockade of VEGF treated mice (1.00±0.48gms vs.1.2±0.31gms,p=0.005). Compared to the PH group microvascualr density was significantly reduced in the SAL (46.04±3.31 vs.59.8±2.32,p=0.005) and selective VEGF blockade (43.6±1.07 vs.59.8±2.32,p=0.005).VEGF supplementation in the PH+SAL+ VEGF group normalized both wet liver weight (1.15±0.46gms vs. 1.2±0.31gms p=ns) and the microvascualr density (59.80±2.32 vs.62.97±2.38,p=ns). Conclusion: Liver regeneration is a salivary VEGF dependent process. SAL and selective blockade of VEGF with Ad VEGF-trap block hepatic regeneration based on wet weight and microvascualr density. Oral VEGF supplementation completely corrects the liver regeneration and vessel density, demonstrating a previously unrecognized role of salivary VEGF in inducing neovascularization dependent liver regeneration. These results may suggest new therapeutic strategies to augment compensatory growth of liver following resection.

Predominance of released mitochondrial enzymes by partial hepatectomy-induced rat regenerating liver is controlled by hemodynamic changes and not related to mitochondrial damage

Objective. Serum activities of assumed organ-specific enzymes are useful protein markers in the diagnosis of necrotic liver diseases. However, after partial hepatectomy (PH) in rats, remaining hepatocytes proliferate to restore the lost liver mass, even when there is a drastic but selective elevation of serum enzyme activities. The aim of the present study was to elucidate the underlying mechanisms involved in this PH-induced enhancement of enzyme release. Material and methods. Routine spectrophotometric methods were used to measure nine “marker” enzyme activities in sera, in effluents from isolated perfused livers, as well as in the incubation media used for liver slices and isolated cells from either sham-operated or 70%-PH rats. Results. PH induced a drastic increase in serum activities of liver enzymes, predominantly of mitochondrial localization. In the control and 70%-PH groups, liver enzymes were differentially released by varying in vitro flow rate/liver mass ratio, using livers perfused at variable flow rates. This event was reversible and not associated with liver structural or functional alterations, but was dependent on the flow-bearing physical forces and independent of production of extra-hepatic factors. Liver slices and isolated cells were used to identify additional flow-independent enzyme release. The 70%-PH-induced drastic release of specific enzymes (predominantly those from mitochondria) could be mimicked in control livers by changing the hepatic blood flow/mass ratio, and closely resembled urea production by these livers. Conclusions. PH-induced effects were not associated with liver necrosis or mitochondrial dysfunction and evidenced previously unrecognized mechanisms controlling the rate of enzyme release into the bloodstream, which might have clear clinical implications.

Alterations of the renal function and oxidative stress in renal tissue from rats chronically treated with aluminium during the initial phase of hepatic regeneration

Various indices of renal functions during the early stage of hepatic injury were studied in rats chronically treated with aluminum (Al) lactate. Tubular and hemodynamic parameters were analyzed four days after producing a 65% partial hepatectomy (PH). Water and sodium balances were also studied. Oxidative stress and the activity of Na–K–ATPase were determined in renal tissue. The rats were distributed in four groups: control, Al, PH, Al + PH. Al did not modify the hemodynamic renal functions and the PH-group reduced the glomerular filtrate rate (GFR). The Al + PH group presented a decrease in the renal blood flow and accentuated the GFR fall as compared with PH. The fractional excretion (FE) of water and sodium increased in the PH group. The rats chronically treated with Al and then submitted to the PH protocol developed a further increase in FE of water but a reduction in FE of sodium. Both PH and Al promoted an increase in the aldosterone. PH and Al induced a similar increase of the lipoperoxidation status with reduction of glutathione (GSH) and the activity of glutathione peroxidase (GSH-Px). The data indicated that Al is an inhibitor of catalase. The GSH and GSH-Px activity in the Al + PH group demonstrated a synergic effect of Al and PH. This work demonstrates that rats treated chronically with Al and submitted to another injury (such as hepatic damage) can aggravate renal functions, probably by increasing the oxidative state, at least in kidneys.

Transcript AA454543 Is a Novel Prognostic Marker for Hepatocellular Carcinoma after Curative Partial Hepatectomy

BACKGROUND: We have previously reported on the cDNA microarray gene expression profiles of hepatocellular carcinomas (HCCs). Among the genes that show prognostic significance and are overexpressed in tumor compared with adjacent nontumorous liver, transcript AA454543 may have potential for practical use. Our aim is to validate the prognostic significance of transcript AA454543 by alternative research methods and in a separate group of HCC patients. METHODS AND RESULTS: The data of transcript AA454543 derived from microarray analysis of 48 patients having curative partial hepatectomy (group 1) were verified by quantitative reverse transcription polymerase chain reaction (r = 0.618, P < .001). A separate sample set of HCCs obtained from 53 patients (group 2) was examined and the association of AA454543 expression level with overall survival was again validated (P = .027). By Cox regression analysis, transcript AA454543 [hazard ratio (HR) = 3.0, P =.01 7] and pathologic tumor node metastasis (pTNM) stage (HR = 3.3, P = .010) were independent prognostic factors for overall survival. The accuracy of prediction for 3-year overall survival for transcript AA454543 (74.2%, P = .001) and pTNM stage (76.4%, P = .001) was comparable as measured by the area under the receiver operating characteristic curve. CONCLUSION: Transcript AA454543 is a potentially useful molecular prognostic marker for overall survival after curative partial hepatectomy for HCC.

Comparison of different initiation protocols in the resistant hepatocyte model

Several models in rat liver have been developed to study multistage carcinogenesis, including the Solt-Farber resistant hepatocyte model. In this model, initiation consists of either a necrogenic dose of a hepatocarcinogen or a non-necrogenic dose in conjunction with partial hepatectomy (PH). As an alternative to PH, we investigated two different procedures: fasting for 96 h followed by refeeding, or the use of one-day-old neonates. Male Fisher 344 rats were injected p.o. with diethylnitrosamine (DEN) (0, 20, or 100 mg/kg) 24 h after refeeding or PH (controls received DEN alone with no proliferative stimulus). For the neonatal group, male and female Fisher 344 rats were treated with DEN (0 or 20 mg/kg, i.p.) at one day of age. All initiated animals were treated at the same age (11 weeks) with the following selection agents: three daily doses of 2-acetylaminofluorene (AAF) (30 mg/kg), followed by a single dose of carbon tetrachloride (2 ml/kg), followed by three additional daily treatments of AAF (30 mg/kg). Rats were euthanized 2 weeks after the last AAF injection. The PH, neonatal male, and neonatal female groups receiving DEN developed more gamma-glutamyl transpeptidase (GGT)-positive foci per cubic centimeter and foci per liver as compared to untreated rats receiving the same proliferative stimulus, whereas the fasting/refeeding group and the group receiving no proliferative stimulus did not. All DEN-treated groups receiving one of the proliferative stimuli had more foci per cubic centimeter than the DEN-treated group receiving no proliferative stimulus. The volume fractions of GGT-positive foci in the PH/DEN and neonatal male/DEN groups were higher than those of both the DEN-treated group receiving no proliferative stimulus and the groups receiving the same proliferative stimulus without DEN. In neonatal females-receiving DEN, the volume fraction was not different from either neonatal females not receiving DEN or DEN-treated rats receiving no proliferative stimulus. The volume fraction in the fasting/refeeding group was increased when DEN was administered at 100 mg/kg but not at 20 mg/kg. We conclude that the use of male neonatal rats can replace the PH in the Solt-Farber protocol. Fasting/refeeding was also effective at increasing the volume fraction of GGT-positive foci, but only at the higher dose of DEN.

Cyclin expression in the atrophying and proliferating lobes of the liver after portal vein branch ligation and hepatectomy in rats

Background Portal vein branch ligation causes atrophy of the portal vein ligated lobes (PVL) and proliferation of the nonligated lobes (PVNL) of the liver. However, the mechanisms underlying atrophy of the PVL and proliferation of PVNL after portal vein branch ligation have not been clarified except that interleukin-6 (IL-6), nuclear factor kappa B (NF-κB), signal transducer and activator of transcription 3 (STAT3), and immediate-early gene expression are similarly induced in both the PVL and the PVNL. Thus, it is still unclear what factors cause the subsequent atrophy and proliferation. Materials and methods Male Wistar rats were randomly separated into a portal vein branch ligation group and partial hepatectomy group. In the portal vein branch ligation group, the branch of portal vein supplying the median and left lateral lobes of the liver was ligated. In the partial hepatectomy group, the correspondent lobes of the liver were excised. We examined cyclin expression in the PVL and PVNL after portal vein branch ligation in comparison to cyclin expression in the remaining liver (HEP) after partial hepatectomy. Cyclin D1, E, and A mRNA and protein expressions were analyzed by RT-PCR and Western blotting, respectively. Results The mRNA and protein expressions of cyclin D1 and A were not up-regulated in the PVL, whereas those in the PVNL and HEP were up-regulated. Cyclin D1 mRNA and protein expressions were significantly lower in the PVL than in the PVNL and HEP at 18 h. The levels of mRNA and protein expression of cyclin A were significantly lower in the PVL than in the PVNL and HEP at 36 h. Liver regeneration, assessed by the relative liver weight, thymidine incorporation into DNA, and proliferating cell nuclear antigen (PCNA) labeling index was delayed significantly in the PVNL compared to that in the HEP. Cyclin D1 mRNA and protein expressions were significantly lower in the PVNL than in the HEP at 12 and 18 h, respectively. Conclusions Cell-cycle progression might be inhibited at G 1-phase accompanied by impaired cyclin D1 expression in the PVL, which results in atrophy. The fact that liver proliferation of the PVNL is delayed in comparison to that of the HEP is likely due to delayed expression of cyclin D1.

Cell apoptosis and regeneration of hepatocellular carcinoma after transarterial chemoembolization.

To evaluate whether cell apoptosis and regeneration were existed in normal liver cells adjacent to carcinoma after transarterial chemoembolization (TACE). Fifty rabbits with hepatic carcinoma were divided into 5 groups at random: group A (control group), groups B and C (TACE treatment groups), groups D and E (partial hepatectomy groups). There were 10 rabbits in each group. Rabbits in groups B-E were treated by transarterial chemoembolization (TACE) and partial hepatectomy (PH) respectively. The changes of S-phase cell fraction (SPF), proliferation index (PI) and cell apoptosis in the normal liver tissue were determined with flow cytometry (FCM) after operations on the first and third days. We determined the mitosis index (MI) with histo-pathological method and the apoptosis index (AI) with TUNEL method at the same time. Twenty-four hours after operations, compared with control group, the rabbits in TACE group had much higher index of SPF, PI and MI (MI: t=4.89, P<0.001; SPF:t=5.27, P<0.001; PI: t=4.87, P<0.001). Moreover, the proliferation of liver cells in TACE group was much weaker than that of the cells treated by partial hepatectomy, and the differences were significant (MI: t=7.02, P<0.001; SPF: t=4.06, P<0.001; PI: t=2.70, P<0.05). Seventy-two h after operations, FCM showed a small sub-G1 peak in TACE group and PH group, compared with the control group, but there was no difference between them (t=0.41, P>0.05). TACE showed that AI in the treated rabbits was higher than that in control group (t=3.07, P<0.05), and there were no differences between TACE group and PH group, either (t=0.93, P>0.05). Cell apoptosis and regeneration exist in rabbit liver tissues after TACE in some degree, which may be associated with the selective embolization of iodised oil, chemotherapeutic drug and free radical damage.

Antiproliferative activity of Copaifera duckei oleoresin on liver regeneration in rats.

Copaiba oleoresin is widely used in folk medicine as an antiinflammatory, healing and antiseptic agent. Wistar rats received Copaifera duckei oleoresin (CDO) or saline solution (SS) from 7 days before surgery until killing. Partial hepatectomy (PH) or sham-operation was performed. Hepatocellular proliferation and liver mitochondrial respiration were evaluated 24 h after the procedure. The proliferating cell nuclear antigen labelling index was significantly lower in CDO-treated plus PH rats than in SS-treated plus PH rats, suggesting that CDO inhibited cell proliferation. The state 3 and state 4 respiration rates were greater for CDO-treated groups, and the stimulation of state 4 respiration was more expressive. These data resulted in a significant decrease in the respiratory control ratio observed for CDO-treated groups, demonstrating the ability of CDO to uncouple oxidative phosphorylation in mitochondria, a fact that may be one explanation for the inhibition of hepatocellular proliferation observed in the CDO-treated plus PH group.

Gene and protein expressions of p28GANK in rat with liver regeneration.

To observe the gene and protein expression changes of p28GANK in regenerating liver tissues, and to reveal the biological function of p28GANK on the regulation of liver regeneration. One hundred and thirty two adult male Sprague-Dawley rats were selected, weighing 200-250 g, and divided randomly into sham operation (SO) group and partial hepatectomy (PH) group. Each group had eleven time points: 0, 2, 6, 12, 24, 30, 48, 72, 120, 168 and 240 h, six rats were in each time point. The rats were undergone 70% PH under methoxyflurane anesthesia by resection of the anterior and left lateral lobes of the liver. SO was conducted by laparotomy plus slight mobilization of the liver without resection. Liver specimens were collected at the indicated time points after PH or SO. The expression level of p28GANK mRNA was determined by Northern blot as well as at protein level via immunohistochemical staining. The expressions of p28GANK mRNA in these tissues were analyzed by imaging analysis system of FLA-2000 FUJIFILM and one way analysis of variance. The protein expressions of p28GANK in these tissues were analyzed with Fromowitz' method and Rank sum test. The expression of p28GANK mRNA in the regenerating liver tissues possessed two transcripts, which were 1.5 kb and 1.0 kb. There was a significantly different expression patterns of p28GANK mRNA between SO and PH groups (P<0.01). The expression of p28GANK mRNA increased 2 h after PH, the peak time was 72 h (SO group: 163.83+/-1.4720; PH group: 510.5+/-17.0499, P<0.01). There was a significant difference in the 1.5 kb transcript, which decreased gradually after 72 hours. The protein expression of p28GANK was mainly in the cytoplasm of regenerating hepatocytes, and increased near the central region 24 h after PH, and became strongly positive at 48 h (+++, vs the other time points P<0.05), but decreased 72 h after PH. The expression of p28GANK mRNA increases in the early stage of rat liver regeneration, the protein expression of p28GANK is mainly in the cytoplasm of regenerating liver cells. It suggests that the gene of p28GANK may be an important regulatory and controlled factor involved in hepatocyte proliferation during liver regeneration.

Role of hepatocyte direct hyperplasia in lentivirus-mediated liver transduction in vivo.

Lentiviral vectors have been used for gene transfer into the liver, but the ability of these vectors to efficiently transduce quiescent hepatocytes remains controversial. Regardless, lentivirus-mediated gene transfer is greatly enhanced when delivered during hepatocellular cycling. For this reason, the present study was designed to determine the role of hepatocyte proliferation in the enhancement of lentiviral transduction by using three different modes of liver regeneration: (1) compensatory regeneration stimulated by two-thirds partial hepatectomy, (2) direct hyperplasia after intragastric administration of the primary mitogen 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), and (3) a combination of modes 1 and 2. Vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped lentiviral vector expressing beta-galactosidase was administered to mice via the peripheral circulation after a regeneration stimulus. Gene transfer as measured by 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-Gal) staining showed 30-fold higher levels of liver transduction in groups 1 and 2 as compared with the non-liver-manipulated control group (p < 0.005). The combination of TCPOBOP and partial hepatectomy (group 3) resulted in an ~80-fold increase in transduction efficiency compared with the control animals. The enhanced transduction was consistent with higher levels of hepatocellular proliferation observed in animals that received both treatments compared with either single treatment alone. Importantly, the hepatocytes were the predominant cell type transduced, although transgene expression was observed in a low number of nonparenchymal cells regardless of which liver stimulus was received. Biodistribution studies confirmed that most of the gene transfer was limited to the liver and spleen. Taken together, this study suggests that disease-induced cellular proliferation in the liver will enhance the utility of this vector in treating diseases such as viral hepatitis, liver cirrhosis, and cancer.

Differential Effects of Partial Hepatectomy and Carbon Tetrachloride Administration on Induction of Liver Cell Foci in a Model for Detection of Initiation Activity

Differential effects of partial hepatectomy (PH) and carbon tetrachloride (CC14) administration on induction of glutathione S‐transferase placental form (GST‐P)‐positive foci were investigated in a model for detection of initiation activity. Firstly, we surveyed cell proliferation kinetics and fluctuation in cytochrome P450 (CYP) mRNA levels by means of relative‐quantitative real‐time reverse transcriptase‐polymerase chain reaction (RT‐PCR) and CYP 2E1 apoprotein amount by immuno‐blotting (experiment I) after PH or CC14 administration. Next, to assess the interrelationships among cell proliferation, fluctuation of CYPs after PH or CC14 administration and induction of liver cell foci, the non‐hepatocarcinogen, 1,2‐dimethylhydrazine (DMH) was administered to 7‐week‐old male F344 rats and initiated populations were selected using the resistant hepatocyte model (experiment II). In experiment I, the values of all CYP isozyme mRNAs after PH or CC14 administration were drastically decreased at the 12‐h tune point. From 72 h, mRNAs for all CYP isozymes began increasing, with complete recovery after 7 days. The CYP 2E1 apoprotein content in the PH group fluctuated weakly, whereas in the CC14 group it had decreased rapidly after 12 h and was still low at the 48 h point. In experiment II, induction of GST‐P‐positive foci was related to cell kinetics in the PH group, with about a 6‐h time lag between tune for carcinogen administration giving greatest induction of GST‐P‐positive foci and peaks in bromodeoxyuridine (BrdU) labeling, presumably due to the necessity for bioactivation of DMH. With CC14 administration, induction of foci appeared dependent on the recovery of CYP 2E1. In conclusion, PH was able to induce cell proliferation with maintenance of CYP 2E1, therefore being advantageous for induction of liver cell foci in models to detect initiation activity.

Evaluation on factors influencing liver cancer metastasis after liver surgery by a mouse model

Objective: To evaluate the influence of surgical trauma on liver cancer metastasis. Methods: A mouse model of experimental liver cancer metastasis was established by subcapsule injecting hepatoma ascites tumor cells (H22) into spleen of NIH mice. Simple intrasplenic inoculation, with sham operation, partial hepatectomy, total occlusion of hepatic blood inflow and blood loss and re-perfusion were performed and metastatic effects were observed. Results: There were significant higher metastasis-augmenting effects in sham operation and partial hepatectomy groups. Compared with no-blood transfusion, blood transfusion group was found to be potent to increase intrahepatic metastases. But, neither inhibition nor enhancement with total occlusion of hepatic blood inflow for 20 and 30 minutes was seen. Conclusions: Surgical trauma, especially partial hepatectomy and blood transfusion, are involved in enhancing metastasis, but total occlusion of hepatic blood inflow is not responsible for enhanced liver metastasis in the experimental metastasis model.

Partial hepatectomy strongly increased the mutagenicity ofN-ethyl-N-nitrosourea in Muta?Mouse liver

The relationship between cell proliferation and mutagenesis can be investigated in vivo due to the advent of transgenic animal mutation assays. In these assays, slowly proliferating tissues, such as mammary gland and liver, that are exposed to mutagens generally have longer manifestation times for mutations and lower mutant frequencies. This may be because the cells have enough time prior to cell division to repair DNA damage. We carried out this study using the MutaMouse positive selection system to investigate the effect of a high rate of cell proliferation induced by partial hepatectomy (PH) on mutation induction. We used a 2 x 2 experimental design for PH (or no PH) and 50 mg/kg N-ethyl-N-nitrosourea (ENU) (or phosphate buffer), with the chemical injected 16-19 hr after PH. In the non-ENU groups, the mean MF was slightly but not significantly higher in the PH group than in the non-PH group. In the ENU non-PH group, the MF was also slightly but not significantly increased. In the ENU PH group, in contrast, the mean MF was 7 times the mean MF of the group that received either treatment alone. These results strongly support the hypothesis that ENU induced pre-mutational DNA lesions in liver are completely repaired prior to cell division, and PH increases the mutagen-induced MF by reducing the amount of time available for such repair.

Regenerative signals for intestinal epithelial organoid units transplanted on biodegradable polymer scaffolds for tissue engineering of small intestine.

Our laboratory is investigating the tissue engineering of small intestine using intestinal epithelial organoid units seeded onto highly porous biodegradable polymer tubes. This study investigated methods of stimulation for optimizing neointestinal regeneration. Intestinal epithelial organoid units harvested from neonatal Lewis rats were seeded onto porous biodegradable polymer tubes and implanted into the omentum of adult Lewis rats in the following groups: (1) the control group (group C), implantation alone (n=9); (2) the small bowel resection (SBr) group, after 75% SBr (n=9); (3) the portacaval shunt (PCS) group, after PCS (n=8); and (4) the partial hepatectomy (PH) group, after 75% PH (n=8). Neointestinal cyst size was recorded using ultrasonography. Constructs were harvested at 10 weeks and were examined using histology. Morphometric analysis of the neomucosa was obtained using a computer image analysis program (NIH Image, version 1.59). Cyst development was noted in all animals. Cyst lengths and diameters were significantly larger in the SBr group at 7 and 10 weeks compared with the other three groups (P<0.05; analysis of variance [ANOVA], Fisher's protected least significant difference). Histology revealed a well-vascularized tissue with a neomucosa lining the lumen with invaginations resembling crypt-villus structures. Morphometric analysis demonstrated a significantly greater villus number, height, area, and mucosal surface in the SBr group compared with the other three groups and a significantly greater crypt number and area in the PCS group compared with group C (P<0.05; ANOVA, Fisher's protected least significant difference). Intestinal epithelial organoid units transplanted on porous biodegradable polymer tubes can successfully vascularize, survive, and regenerate into complex tissue resembling small intestine. SBr and, to a lesser extent, PCS provide significant regenerative stimuli for the morphogenesis and differentiation of tissue-engineered small intestine.

Thyroxine accelerates proliferation of injured liver cells

Background/Aims: Long-term gene transfer into hepatocytes requires DNA synthesis. Although this can be achieved in vitro, using various hepatic mitrogens, marked proliferative response is not seen in vivo in the quiescent liver. We have speculated that controlled reversible liver damage might change the steady state of the liver, and thus render it susceptible to manipulations by growth factors and cytokines. Therefore, the influence of thyroxine on proliferation of hepatocytes and of bile duct epithelial cells was investigated, using an in vivo model of thioacetamide-induced liver insult. Methods: Five groups of ten rats each were studied: normal rats, thioacetamide-treated, thyroxine-treated, both thioacetamide and thyroxine-treated, and a 70% partial hepatectomy group. DNA synthesis was looked at by PCNA labeling. Results: The PCNA labeling indexes of hepatocytes and of bile duct epithelial cells in rats treated with both thioacetamide and thyroxine (9.5±1.2 and 33.8±5.7% respectively) were significantly ( p<0.0002) higher than those of the normal (0.84±0.2 and 4.4±0.5%), thioacetamide-treated (2.1±0.3 and 7.1±2.3%) and thyroxine-treated animals (0.6±0.3 and 11±5.6%). The labeling index in the hepatectomized animals was significantly higher for hepatocytes (18.3±1.2%, p<0.03), but lower for biliary cells (15±2.6, p<0.05) than that observed in thioacetamide and thyroxine-treated rats. Hypothyroid rats had significantly lower PCNA labeling index, as compared to the thioacetamide-thyroxine-treated group or the partial hepatectomy group. Conclusions: Following controlled liver damage, thyroxine is a potent mitogen for both hepatocytes and bile duct epithelial cells.