Seventy male albino Wistar rats, weighing 250-320 g, were used in the study. They were housed in stainless-steel cages, three rats per cage, under controlled temperature (23°C) and humidity conditions, with 12 h dark/light cycles, and maintained on standard laboratory diet with tap water ad libitum throughout the experiment, except for an overnight fast before surgery.

The experiments were carried out according to international standards on animal welfare (86/609/EEC) and to the guidelines of the Ethics Committee of Patras University Hospital. The study was approved by the local ethics committee.

Animals were divided randomly into five groups: group I (n = 10): non-operated controls; group II (n = 15): sham operated; group III (n = 15): PHx (70%); group IV (n = 15): PHx and BBS administration; group V (n = 15): PHx and NT administration.

Starting on day 0, the animals of groups IV and V were treated daily with BBS (10 μg/kg, subcutaneously, three times a day) and NT (300 μg/kg, intraperitoneally, once a day) respectively, while the animals of groups I, II and III were divided to receive daily either three subcutaneous or one intraperitoneal injection of 0.5 mL normal saline. Previous studies have shown that the route of saline administration does not affect the results[24]. On the 8th day, animals from groups III, IV and V underwent laparotomy and PHx (almost 70%) as described by Higgins and Andersson[27], while animals in group II underwent laparotomy and mobilization of the liver. The abdominal incision was closed in two layers with chromic 4-0 cat gut and 4-0 silk. All surgical procedures were performed under strict sterile conditions, using light ether anesthesia. Administration of BBS, NT and normal saline was continued for 48 h after surgery. On the 10th day, all animals were operated (group I) or reoperated on (groups II, III, IV and V), again under strict sterile conditions. Samples were obtained according to the experimental protocol, after which the rats were sacrificed by exsanguination.

A stock solution of BBS (Sigma Chemical Co., St. Louis, Missouri, United States) was prepared by first dissolving the amount of peptide needed for the study in 1 mL sterile water containing 0.1% (w/v) bovine serum albumin and then diluted with normal saline containing 1% (w/v) bovine serum albumin to a concentration of 3.5 μg BBS/0.1 mL. This solution was divided into equal aliquots of 0.1 mL that were stored in plastic tubes at -20°C. At the time of administration, a volume corresponding to a dose of 10 μg BBS/kg body weight was taken from each aliquot and was further diluted with sterile saline to a final volume of 0.5 mL that was injected subcutaneously three times daily. Selection of dose and route of administration was based on previous reports[24].

A stock solution of NT (Sigma Chemical Co., St. Louis, Missouri, United States) was prepared by first dissolving the amount of peptide needed for the study in 1 mL sterile water containing 0.1% (w/v) bovine serum albumin and then diluted with normal saline containing 0.1% (w/v) bovine serum albumin to a concentration of 100 μg NT/0.1 mL. This solution was divided into equal aliquots of 0.1 mL that were stored in glass vials at -20°C. At the time of administration, a volume corresponding to a dose of 300 μg NT/kg body weight was taken from each aliquot and was further diluted with sterile saline to a final volume of 0.5 mL that was injected intraperitoneally once daily. Selection of dose and route of administration was based on previous reports[24].

For the determination of endotoxin concentrations in the portal vein, a laparotomy was performed in all groups, the portal vein was punctured and samples of 1 mL of blood were obtained. Endotoxin concentration was determined by the Limulus Amebocyte Lysate test (LAL, QCL-1000, Lonza, Walkersville, MD, United States) according to the manufacturer’s instructions.

After laparotomy, a tissue sample of the liver of each animal was excised, washed in 9 g/L of NaCl and homogenized in sodium phosphate buffer 10 mmol/L, pH = 7.2 (containing 1 mmol/L ethylenediaminetetraacetic acid and 1 mmol/L butylated hydroxyanisole in 0.15% ethanol) by liquid nitrogen for the determination of glutathione redox state. Reduced glutathione was determined spectrophotometrically using Elman’s reagent (DTNB) and oxidized glutathione (GSSG) was quantitated by a standard enzymic assay, as described previously[24].

In situ hybridization forα-fetoprotein expression in paraffin sections: For the detection of α-fetoprotein (AFP) mRNA (oval cell phenotype), a standard non-radioactive in situ hybridization method (ISH) was performed on paraffin sections, as described elsewhere[13]. The Hybridization/Detection Complete System (MBI, Rockville, MD) and the digoxigenin (DIG)-labeled riboprobe for AFP subunit-1 in a 10-fold dilution in hybridization solution were used. Paraffin sections of embryonic rat liver tissue were used as a positive control. To confirm that the positive stain was specific, the slides were processed in an identical way and hybridized with probes known to be complementary to sequences in the test sections (rat genomic DNA probes) (positive control probes). These probes (biotynilated oligonucleotide probes) were similar in length and GC content to AFP probe. For negative control purposes, the slides were processed in the same way but hybridized with heterologous probes. The latter were not complementary to any sequence in the test tissues. These negative control probes were similar in length and GC content to AFP probe.

Immunohistochemistry for the detection of CK19 and Ki67 proteins in paraffin sections: The detection of CK19 protein expression (oval cell phenotype)[5,13] and Ki67 expression (proliferation marker) relied on immunohistochemistry based on a streptavidin biotin peroxidase method (ImmunoCruz™ Staining systems sc-2053; Santa Cruz Biotechnology, Santa Cruz, CA). Briefly, 4-μm thick sections were dewaxed in xylene and hydrated through graded concentrations of alcohol. Endogenous peroxidase activity was blocked with 1% hydrogen peroxide for 15 min. Sections were then processed in a microwave oven twice for 5 min each time at high power, and subsequently stained with anti-CK19 [goat polyclonal (sc-33119) (Santa Cruz Biotechnology, Santa Cruz, CA) in a dilution of 1:150] and anti-Ki67 [goat polyclonal antibody (M-19) (sc-7846) (Santa Cruz Biotechnology, Santa Cruz, CA) in a dilution of 1:100]. All incubations were performed for 30 min at room temperature. Between the steps, sections were washed in TBS. Diaminobenzidine (Sigma Fast DAB tablets-D-4293, St Louis, MO) was used as the chromogen. Cytoplasmic staining for CK19 and nuclear staining for Ki67 were considered as positive. For negative control purposes, the same streptavidin-biotin technique was used on tissue sections in which 1% BSA in PBS was substituted for the primary antibody.

In situ labeling of fragmented DNA for the detection of apoptotic cells: On paraffin sections, a standard terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick-end labeling (TUNEL) method was employed to detect the fragmented nuclear DNA associated with apoptosis[9]. For this purpose, the In Situ Cell Death Detection Kit, POD (Roche, United States) was used according to the manufacturer’s instructions. After standard deparaffinization, hydration, incubation with proteinase K and blocking of endogenous peroxidase, tissue sections were incubated: (1) with TdT and DIG-dUTP (TUNEL reaction mixture) at 37°C for 60 min; and (2) with peroxidase converter anti-fluorescein antibody at 37°C for 30 min. Diaminobenzidine (Sigma Fast DAB tablets, D-4293, Sigma St. Louis, MO, United States) was used as the chromogen. For physiological positive controls, sections of rat small intestine were subjected to the same procedure. For negative controls, some slides were incubated with label solution that did not contain TdT.

Morphometric analysis: (1) Oval cell measurement: morphometric analysis for the evaluation of oval cell presence was performed as described previously[13]. Briefly, sections were screened at low power and areas with increased oval cell staining were determined. Cells were scored when they fulfilled the morphological criteria for oval cells (small cells with ovoid nuclei and scant cytoplasm) and expressed AFP mRNA, cytoplasmic protein CK19 and/or nuclear protein Ki67. Cell counts were performed manually at a × 400 magnification (high power field, HPF) using a 10 × 10-microscope grid. The number of oval cells was determined by visual inspection of five non-overlapping different fields per section. The variance in oval cell counts from section to section in the same biopsy was < 10%. The average of these scores was then taken; and (2) Evaluation of proliferation and apoptosis in hepatocytes: immunohistochemical results regarding Ki67 expression and ISH results (TUNEL+ cells) were estimated for hepatocytes. All Ki67⁺ hepatocytes were considered proliferating cells. Regarding apoptosis, in order to avoid its overestimation by the TUNEL method, hepatocytes were considered apoptotic only if, in addition to positive TUNEL stain, they displayed the morphological features of apoptosis on light microscopy (cytoplasmic fragmentation and nuclear condensation) and were not “surrounded” by inflammatory elements. Estimation of proliferation and apoptosis in each case was performed by a stereological method. Specifically, sections from each liver biopsy were viewed through a light microscope with × 40 flat field objective. A square lattice of 100 points with a total surface area of 0.064 mm² was superimposed onto the tissue. Data were collected from a series of randomly selected 15 adjacent fields extending throughout the biopsy. For each field a percentage value for each parameter (Ki67⁺ cells, apoptotic cells) was obtained by dividing the points falling on stained tissue by the total number of measured points. Also, for each field, the ratio of the obtained values (% Ki67⁺ cells/% apoptotic cells) was calculated as a balancing index expressing net cell turnover. It should be noted that the variance in cell counts from field to field in the same section was < 10%. The average of these scores was then taken and expressed as proliferation and apoptotic indexes and proliferation/apoptosis ratio respectively for each case.

Data were analyzed using the SPSS statistical package (SPSS Inc., 2001, Release 11.0.0, United States). In groups I, II and III, data obtained from subcutaneously and intraperitoneally saline-treated rats were pooled, as there was no significant difference between differentially injected animals for all parameters studied. Results are expressed as mean (SD). Comparisons among multiple groups were performed using the one-way ANOVA, followed by Bonferroni’s post hoc test when variances across groups were equal or by Dunnette’s T3 post hoc test when variances were not equal. Variance equality was tested by Levene statistical analysis. In all cases, differences were considered significant when P < 0.05.

Hepatectomized animals (group III) presented significantly elevated endotoxin concentrations in portal blood compared with groups I and II (P < 0.001). Treatment with BBS or NT led to significantly lower endotoxin values in portal vein in groups IV and V (P < 0.001 vs group III, respectively) (Table 1).

Evaluation of glutathione redox state showed significantly increased levels of reduced glutathione hormone (GSH):GSSG in hepatectomized rats of group III (P < 0.001 vs groups I and II). Administration of BBS or NT resulted in further increase of GSH:GSSG ratio in groups IV and V (P < 0.05 and P < 0.001 vs group III, respectively) (Table 1).

Oval cells were present in all specimens studied. In the control and sham operated groups, oval cells were significantly less compared to groups III, IV and V (P < 0.001, Table 2). In PHx rats (group III) they were located in periportal areas and the formation of small ducts was occasionally recorded. Oval cells expressed CK19 (Figure 1A and B), AFP mRNA (Figure 1C and D) and Ki67. When rats subjected to PHx were treated with either BBS (group IV) or NT (group V), the levels of expression of all three molecules were significantly increased compared to group III (P < 0.001, Table 2).

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Figure 1 Microphotographs showing expression of CK19 from oval cells (arrows) in livers of group III (A) and group IV (B) and α-fetoprotein mRNA expression from oval cells (arrows) in group III (C) and group IV (D). In group IV there is higher oval cell presence as demonstrated by both markers [CK19: streptavidin biotin peroxidase (A) × 250, (B) × 100, α-fetoprotein mRNA: in situ hybridization × 250]. PT: Portal tract.

The proliferation index of hepatocytes was significantly higher in group III as compared to groups I and II (P < 0.001, respectively). Administration of BBS or NT in PHx rats induced a significant increase of hepatocyte proliferation in groups IV and V compared to group III (P < 0.001, respectively, Table 3) (Figure 2A and B). In control and sham operated rats, no apoptotic bodies were detected in hepatocytes. After PHx (group III), increased apoptotic activity was detected in lobules, whilst administration of BBS or NT significantly decreased the apoptotic index (P < 0.001, for groups IV and V compared with group III, Table 3) (Figure 2C and D). The proliferation/apoptosis ratio was significantly increased in groups IV and V compared to group III (P < 0.001, respectively).

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Figure 2 Microphotographs showing Ki67 (+) hepatocytes (arrows) from livers of group III (A) and group IV (B) and TUNEL (+) hepatocytes (arrows) in group III (C) and group IV (D). In group IV there are more Ki67 (+) hepatocytes (streptavidin biotin peroxidase × 100) and less TUNEL (+) hepatocytes (streptavidin biotin peroxidase and hematoxylin eosin × 250). PT: Portal tract.

The regenerative response of human liver is of major clinical importance for patients’ outcome in a number of diverse clinical conditions. The hepatocyte is the most efficient cell for liver repopulation after injury; however, oval cells participate, possibly as an amplifying transit compartment for hepatocyte differentiation, in processes in which hepatocytes do not respond quickly enough or are unable to respond to proliferative stimuli.

Pharmacological augmentation of the hepatic regenerative response in diverse types of liver injury has been the topic of intense research for several decades. Improving the efficiency of the regenerative response of liver progenitor cells might have a substantial clinical impact, especially in cases of coexisting inhibition of mature hepatocyte proliferation, such as in viral hepatitis, chemical toxicity and obstructive cholestasis. Up until now, most experimental trials of pharmaceutical expansion of oval cell compartment have been conducted in animal models of mature hepatocyte proliferation inhibition by toxic chemical compounds. However, for therapeutic application a non-toxic activation of this stem cell compartment is required.

The present study evaluated the effect of the neuropeptides bombesin (BBS) and neurotensin (NT) on oval cells and hepatocytes in partially hepatectomized rats, a widely applied model of liver regeneration. The experimental design did not include any treatment with known inhibitors of hepatocyte proliferation as a method for oval cell compartment activation. Therefore, we aimed at evaluating in vivo the potential net proliferative effect of the tested peptides on oval cells for the first time. The results presented clearly demonstrate the proliferative effect of BBS and NT on oval cells in partially hepatectomized rats in conjunction with improvement of the regenerative response of mature hepatocytes, evidenced by promotion of hepatocytes’ proliferation and prevention of their apoptosis.

Promotion of oval cell proliferation is of great clinical importance profoundly in cases of inhibition of hepatocyte proliferation as this cell type carries on the process of liver repopulation, as well as in cases of effective hepatocyte proliferation, providing a ready cell compartment to continue liver regeneration if a hepatocytic inhibitory insult arises. BBS and NT promoting oval cell and hepatocyte proliferation could be a potent therapeutic modality in the treatment of patients with several liver diseases, such as fulminant hepatic failure.

This is an interesting observational study. The concept is new, the results are robust and may provide potential target for clinical patient care.