Abstract
Background: Portal vein ligation (PVL) could multiply the future liver remnant volume (FLRV). Interuleukin-6 (IL-6) is a pleiotropic cytokine that is associated with an initial phase of liver regeneration. The aim of this study was to accelerate the regeneration of liver parenchyma after PVL by intraportal cytokine application. Materials and Methods: After ligation of portal branches of caudate and right lateral and right medial liver lobes, recombinant porcine IL-6 (IL-6 group) or physiological solution (control group) were applied into non-occluded portal vein branches. The biochemical and immunoanalytical parameters were assessed. The compensatory hypertrophy was evaluated by periodic ultrasonography. The histological examination of liver was performed. Results: The acceleration of growth of hypertrophic liver lobes was maximal at the 7th postoperative day in comparison with the control group (p<0.05), nevertheless, this stimulating effect was lost at the end of the experiment. Important differences in biochemical or histological studied parametres were not proved. Conclusion: The presented study describes the use of IL-6 for stimulation of the first phase of liver regeneration. The achieved acceleration of growth of hypertrophic liver lobes after application of IL-6 confirmed the key role of the studied cytokine in priming regenerating liver parenchyma after portal vein ligation.
- Interluekin-6
- liver surgery
- portal vein embolization
- portal vein ligation
- liver regeneration
- porcine model
Abbreviations: ALP: Alkaline phosphatase; ALT: alanine aminotransferase; AST: aspartate aminotransferase; CHE: Cholinesterase; CRP: C-reactive protein; FLRV: future liver remnant volume; GGT: gamma glutamyl transpeptidase; HGF: hepatocyte growth factor; IGF: insuline-like growth factor; IL-6: interleukin 6; p.d.: postoperative day; PVE: portal vein embolization; PVL: portal vein ligation; TGF-β: transforming growth factor-beta; TNF-α: tumour necrosis factor-alpha.
The development of new surgical techniques and procedures has enabled liver surgery to achieve admirable progress in last two decades. This has also led to an important expansion of possibilities in perioperative intensive care. Nevertheless, many patients with primary or secondary liver malignancies are not directed to radical surgical therapy that could extend their chance of complete remission of their malignancy. The main argument for non-surgical treatment is the increased risk of acute liver failure after extended liver resection, where the liver remnant is too small to sustain the liver functions (1). The future liver remnant volume (FLRV) could be increased by portal vein embolization (PVE) performed before liver resection (2). The essential condition for indication of this procedure is that only one of the liver lobes is affected by malignant diseases (3, 4). Compensatory hyperplasia of the contralateral liver lobe is initiated by PVE of the portal branch of the liver lobe afflicted by the malignancy which underlies atrophy. Compensatory hyperplasia is purportedly to be stimulated by increased blood flow in the non-occluded portal vein branch or by hepatotrophic substances that are contained in portal blood (5, 6). Liver surgery is performed only in 63-96% of patients after PVE (7-9). Unsuccessful hypertrophy of FLRV or progression of malignancy are the main reasons for resorting to liver resection after PVE.
Partial hepatectomy, portal vein ligation or liver injury evoke an increase in the serum level of interleukin-6 (IL-6). IL-6 has been shown to be involved in priming of hepatocytes and triggers them from G0 to G1 cell cycle phase (10). IL-6 is secreted by liver non-parenchymal cells of extrahepatic origin (Kupffer cells) (11). IL-6 is one of the most important stimuli (together with e.g. TNF-α) that are responsible for the induction of genes of the G1 cell cycle phase (12, 13). Maximum IL-6 serum levels after liver injury are observed in the first twelve hours after partial hepatectomy (12). The decrease in liver weight was observed to be completely restored to control levels on the 7th day after partial hepatectomy (14). The adverse behavior of hepatocytes is observed in non-resection models of liver injury, e.g. PVE. The replication of hepatocytes culminates on the 7th day (14% of hepatocytes) and a return to the quiescent state was observed on the 12th day after PVE in a swine experimental model (15).
The aim of the study presented here was to accelerate and amplify the regeneration of liver parenchyma after portal vein ligation (PVL) by increasing of the amount of quiescent hepatocytes entering the G1 phase by the application of IL-6. The experimental porcine model that was used was designed to be as compatible as possible with PVL in human medicine (16). The results could be used in human liver surgery to increase the number of patients undergoing radical extended liver resection for malignancy. The differences between PVE and PVL were not shown to be statistically significant for the achieved FLRV (17). However, contemporary literature has considered portal vein ligation only on murine experimental model (18).
Materials and Methods
The experimental procedure and use of piglets was certified by the Commission for Work with Experimental Animals at the Pilsen Medical Faculty of the Charles University, Prague, and was under control of the Ministry for Education of the Czech Republic. All the procedures performed were prepared and performed under the law of the Czech Republic, which is compatible with legislation of the European Union.
Surgical procedure. Seventeen piglets were included in this study (8 in the IL-6 group, 9 in the control group). No piglets were excluded because of untimely death or any type of surgical complication. The piglets were premedicated intramuscularly with atropine 1.5 mg and azaperon 1.0 mg/kg. Anesthesia was administered continually through central venous catheter in the following total average doses: azaperon 1.0 mg/kg/hour, thiopental 10 mg/kg/hour, ketamin 5-10 mg/kg/hour and fentanyl 1-2 μg/kg/hour. Muscle relaxation was ensured by bolus administration of pancuronium 0.1-0.2 mg/kg at the begin of surgery. The piglets were intubated and mechanically ventilated during surgical procedure. The monitoring of electrocardiogram, oxygen saturation and central venous pressure was performed. The surgical procedure was performed under aseptic and antiseptic conditions. An antibiotic prophylaxis was administered in total dose of 1.2 g amoxicillin with clavulanic acid divided into two doses (before surgery and two hours later).
First a middle laparotomy was performed. The portal vein branches for caudate, right lateral and right medial lobes (50-60 per cent of supposed liver parenchyma) were prepared and ligated without injury or ligation of the hepatic artery branches. The blood flow in the hepatic artery branches and occlusion of the portal vein branches were controlled by Doppler ultrasonography (Medison Sonoace 9900, linear probe with frequency 7.5 MHz). Recombinant porcine IL-6 (rpIL-6, ProSpec TechnoGene, Israel) at 0.5 μg/kg or a physiological solution (control group) was applied into non-occluded portal vein branches (19). The borders between atrophic and hypertrophic liver lobes were marked by titanium staples to simplify the postoperative ultrasonography. The laparotomy was closed in anatomical layers. At the end of operation the port-a-cath was introduced into the superior caval vein. The animals were extubated and monitored for the next fourteen days.
Biochemistry and immunoanalysis. Blood samples were collected from the central vein catheter before the operation, after ligation of the last portal branch, during application of cytokine, 2 hours after the application of cytokine, on the 1st, 3rd, 7th, 10th, 14th postoperative days. Biochemical serum parameters focused on liver functions to detect the influence of the applied cytokine on the animal organism and to recognise possible differences between IL-6 and the control group. Serum levels of bilirubin, urea, creatinine, alkaline phosphatase (ALP), gammaglutamyltransferase (GGT), cholinesterase (CHE), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and albumin were assessed by biochemical analyser Olympus 2700.
Serum levels of TNF-α, growth factors (TGF-β1 and IGF) and C-reactive protein (CRP) were meassured by enzyme-linked immunosorbent assay (ELISA) by Auto-EIA II Analyzer (Lasystems Oy Helsinki, Finland). The immunoassay kits were produced by Biosource California (TGF-β1, KAC1688/1689), Immunodiagnostic Systems Britain (IGF, Octeia IGF-1), RD Systems Minnesota (IL-6, Quantikine) and Tridelta Development Ireland (CRP, Phase Range).
Ultrasonography. The ultrasonographic controls were performed immediately after operation and on 3rd, 7th, 10th and 14th postoperative days (ultrasound machine Medison Sonoace 9900, convex probe with frequency 3.5 MHz). The diameters of the atrophic and hypertrophic lobes were meassured in B-modus in all three basic planes (axial, sagittal and coronary). The volume of the lobes was assessed by using the standard ultrasonographic formula that is used also in human medicine: axial × sagittal × coronary / 2. The volumes are presented as a percentage to provide better information about achieved changes in volumes.
Termination of experiment. The piglets were examined clinically each day, with particular attention to wound healing, infection of the port-a-cath and function of the gastrointestinal system to diagnose possible surgical complitations. The piglets were slaughthered on the 14th day under deep general anaesthesia with a concentrated solution of potassium chloride administered into a central venous catheter. The piglets were dissected and the measurement of the atrophic and hypertrophic liver lobes was performed. These data were compared with the proportions estimated by ultrasonography. Bioptic samples from atrophic and hypertrophic liver lobes were taken and stored in 10% formaldehyde (methanal) and also frozen below -70°C.
Histology. The histological material from atrophic and hypertrophic parenchyma was examined after staining with hematoxylin-eosin and periodic acid-Schiff (PAS) staining after digestion of preparations with diastase. The proliferation activity was examined using antibody Ki-67 (MIB 1 MW, 1:1000; DakoCytomation, Denmark). Bone marrow stem cells were detected by CD 117 antibody (1:150; Dako Glostrup, Denmark) and stem cells of hepatic origin were studied using CK7 (1:200; Dako Glostrup, Denmark) and CK19 antibody (1:1000; Neomarkers Westinghouse, USA). The primary antibodies were visualised using a streptavidin - biotin - peroxidase complex (DakoCytomation). The lobulus length and binucleated hepatocytes were measured in 20 microscopical fields. The length of the hepatocytes was examined by eyepiece micrometer (Olympus).
Statistical analysis. Statistical analysis was performed with CRAN 2.4.0 and STATISTICA (98 Edition) software. The assessed parameters (biochemistry, ultrasonography, histology) were analysed by the following statistical tests: the comparison of distribution of the studied parameters over the groups was made using a distribution-free test (Wilcoxon). The Spearman rank correlation coefficient was used because of the non-Gaussian distribution of parameter values. The whole development of studied parameters over time was compared between the groups using the ANOVA test.
Results
The results achieved by measurement of liver lobes volume by ultrasonography and physical examination during necropsy were compatible on the 14th postoperative day. The absolute volume of hypertrophic lobes grew more rapidly after application of IL-6, wheareas the control group had a slow inception of growth in the hypertrophic liver lobes during the first three days. The acceleration of growth of the hypertrophic liver lobes in the IL-6 group was maximal on 7th postoperative day in comparisson with the control group (p<0.05). Nevertheless this stimulating effect was lost during the follow-up period and on the 14th postoperative day there were no statistically significant differencies (Figure 1).
The serum levels of all the studied biochemical and immunological parameters are presented in Figures 2 and 3. All the studied serum biochemical parameters were comparable in both experimental groups and the differences did not show any statistical significance between the IL-6 and the control group at each point in time (Figure 2). The serum levels of all the studied cytokines and the growth factors expressed no statistically significant differencies between the IL-6 and the control group at each point in time (Figure 3). None of the secondary effects mostly found in immune response were observed after application of pleiotropic cytokine. There was no detection of serum level changes of CRP.
The histological examination of biopsies was burdened by the time needed for the collection of specimens, when the proliferative phase of liver regeneration had in fact finished. The differences in lobulus length were not statistically significant. Statistical analysis showed a significant increased in the amount of binucleated hepatocytes in hypertrophic liver lobes in the IL-6 group (Figure 4). The length of hepatocytes was also not proved to be a statistically significant parameter. Proliferative activity in both groups was very reduced. Neither bone marrow nor hepatic stem cells or progenitors were detected in the bioptical material acquired on the 14th postoperative day from the liver, both from hypertrophic and atrophic parenchyma.
Discussion
The study presented here illustrates the importance and possibilities of application of extrinsic IL-6 to increase the required future remnant liver volume after portal vein ligation. The use of piglets in this experimental study is very appropriate in relation to human medicine and surgery because of the similar physiology of piglets and humans. The acceleration of growth of hypertrophic liver lobes after application of IL-6 confirmed results gained using in vitro models and in experiments with small laboratory animals (10, 20). The selected concentration of applied cytokine initiated acceleration of regeneration of liver parenchyma in non-occluded liver lobes (18, 21). The secondary effects that could be hypothesized after application of key pleiotropic cytokine (changes in immune reactions and homeostasis) were not observed either during application or in the whole postoperative period.
No statistically significant differences were shown between serum levels of the studied biochemical parameters at particular points in time. This also demonstrates that there was no unsuitable influence of applied cytokine on the liver function. The changes in serum levels of the studied cytokines and growth factors were also not observed to be different between the IL-6 and the control group at different time points. The increased number of binucleated hepatocytes in the hypertrophic parenchyma of cytokine group in comparison with the control group could be explained by incomplete liver regeneration at the end of the experiment. Because there were practically no mitotic figures, or the number was is the same as in the normal liver parenchyma without any surgical procedures or toxic insult, it was possible to hypothesize that the first phase of liver regeneration was finished and the next phase of regeneration was proceeding (22), namely the remodelling phase and the phase when the liver microstructure is restored. The next objective for future study would be the detection of intracellular or extracellular matrix changes during the process of liver regeneration. No significant differences in other histological parameters (diameter of lobulus and hepatocytes) were shown. The same size of hepatocytes in the biopsies could also be explained in the same way. This hypothesis is supported by the restitution of all liver functions monitored by biochemical parameters at the moment of slaughter of the experimental animals. No stem cells or progenitor cells were detected, not only because of completion of the proliferative phase of liver regeneration but also because of the presumed poor participation in this type of experiment (23).
This presented experimental model of liver regeneration is considered to be more compatible with the human organism than other models in small animals. The performed PVL is also very similar to PVE (17). The results of this experiment could be useful for the support of liver regeneration during complicated liver procedures with high risk of liver failure and larger FLRV. These results have encouraged the preparation of the next studies in human medicine and the initiation of hypertrophy after PVE in at-risk patients, where a weak hypertrophic reaction after PVE can be expected. The application of IL-6 could result in an increase in FLRV after performance of PVE.
Acknowledgements
This article was supported by grant IGA CR NR 8860/1-3 and research project MSM 0021620819 (Replacement and support of some vital organs). Special acknowledgement belongs to Professor Jenner for excellent linguistic correction and to Mrs. Renata Michalkova, Vladka Mlejnkova and Renata Jirkova for wonderful nursing work. This study also had approval of the local Ethical Committee.
- Received September 2, 2008.
- Revision received December 24, 2008.
- Accepted March 11, 2009.
- Copyright© 2009 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved