**Liver Parenchyma Regeneration in Connection with Extended Surgical Procedure – Experiment on Large Animal**

Vaclav Liska et al.1,\* *1Department of Surgery Teaching Hospital and Medical School Pilsen, Charles University Prague, Czech Republic* 

#### **1. Introduction**

150 Liver Regeneration

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Liver surgery underwent enormous evolution after development and introduction of new technical skills in surgical praxis. Nevertheless many patients with primary or secondary liver malignancies are not indicated to radical surgical therapy that could reach complete remission of malignant disease because the frontiers of liver surgery are limited today by the functional reserves of remnant parenchyma. The main argument to non surgical treatment is increased risk of acute liver failure after extended liver resection, where retained liver parenchyma is to small to sustain the liver functions (Abdalla, 2001). Portal vein embolization (PVE) can multiply the future liver remnant volume (FLRV) in spite of affection of only one of liver lobes by malignant diseases (Makuuchi, 1984, Makuuchi, 1990, Harada, 1997). This procedure was performed firstly in 1984 by Makuuchi (Abdalla, 2001, Makuuchi, 1984, Makuuchi, 1990). PVE of portal branch of with malignancy afflicted liver lobe initiates compensatory hypertrophy of contralateral non-occluded lobe. The occluded lobe underlies atrophy. The compensatory hypertrophy is supposed to be stimulated by increased flow of portal blood, that contains hepatotrophic substances (Kusaka, 2004, Azoulay, 2000). Liver resection after PVE is performed only in 63-96% of patients (Kokudo, 200, Stefano, 2005, Lagasse, 2000). The main reason for this resolution is unsuccessful hypertrophy of FLRV or progression of malignancy. Liver resection after PVE is performed only in 63-96% of patients (Azoulay, 200, Kokudo, 2001, Stefano, 2005). The main reason for this resolution are unsuccessful hypertrophy of FLRV or progression of malignancy.

<sup>\*</sup> Vladislav Treska1, Hynek Mirka2, Ondrej Vycital1, Jan Bruha1, Pavel Pitule1, Jana Kopalova1,

Tomas Skalicky1, Alan Sutnar1, Jan Benes3, Jiri Kobr4, Alena Chlumska5, Jaroslav Racek6

and Ladislav Trefil6

*<sup>1</sup>Department of Surgery,* 

*<sup>2</sup>Department of Radiology,* 

*<sup>3</sup>Department of Anaesthesiology and Resuscitation, 4Department of Pediatrics,* 

*<sup>5</sup>Institute of Pathology,* 

*<sup>6</sup>Institute of Biochemistry and Haemathology,* 

*Teaching Hospital and Medical School Pilsen, Charles University Prague, Czech Republic* 

Liver Parenchyma Regeneration in Connection

possible with PVE/PVL in human medicine.

group and 7 piglets in MAB TGF-β1 group.

into two doses (before surgery and two hours later).

simplify the postoperative ultrasonography.

Mesenchymal Stromal Cells.

**2.1 Surgical procedure** 

**2. Methods** 

with Extended Surgical Procedure – Experiment on Large Animal 153

regeneration by bystander effects, such as the provision of a beneficial proliferative cytokine

The aim of presented studies was to influence regeneration of liver parenchyma after portal vein embolization/ligation by exogenous cytokines, growth factors and monoclonal antibodies against growth factors with inhibitory funcions or syngeneic Multipotent

All described procedures were prepared and performed after by law of Czech Republic, which is compatible with legislature of European Union. The experimental animal was piglet. The experimental porcine model was established to be as much compatible as

There were no statistical differencies in weight and age of the piglets undergoing portal vein ligation. The animals were housed under same conditions. In this study there were included 9 piglets in control group, 9 piglets in TNF-α group, 8 piglets in IL-6 group, 6 piglets in MSC

The piglets were premedicated intramuscularly with atropine 1,5 mg and azaperon 1,0 mg/kg. The anesthesia was administered continually through central venous catheter in whole average doses: azaperon 1,0 mg/kg/hour, thiopental 10 mg/kg/hour, ketamin 5-10 mg/kg/hour and fentanyl 1-2 ug/kg/hour. The muscle relaxation was provided by bolus administration of pancuronium 0,1-0,2 mg/kg at the begin of surgery. Animals 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. The antibiotic prophylaxis was administered in total dosis of 1,2 g amoxicillin with clavulanic acid divided

The 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 hepatic artery branches. The blood flow in hepatic artery branches and occlusion of portal vein branches were controled by Doppler ultrasonography (Medison Sonoace 9900, linear probe with frequency 7,5 MHz, Fig.1). The borders between atrophic and hypertrophic liver lobes were marked by titanium staples to

The recombinant porcine TNF-α in amount 5μg/kilos (rpTNF-α, ProSpec TechnoGene, Israel, 9 piglets from TNF-α group), the recombinant porcine IL-6 in amount 0,5μg/kg (rpIL-6, ProSpec TechnoGene, Israel, 8 piglets from Il-6 group), MSC (autologous bone marrow stem cells cultured in expansion medium (low glucose DMEM with GlutaMAX, without pyruvate, Biochrom) supplemented with 10% fetal bovine serum (FBS, Biochrom), 1% penicillin/streptomycin and 1% glutamine. After 24, 48 and 72 hours, non-adherent cells were removed by changing the culture medium. Adherent cells were then trypsinized (0.5% trypsin-EDTA), harvested and re-plated into new flasks, each time, when cell confluency reached 60% to 80%. To histologically identify transfused MSC within the recipient liver,

milieu or antiapoptotic effects (Aldeguer, 2002, Dahlke, 2004, Liska, 2009).

The proper regeneration of liver parenchyma depends on proliferation of parenchymal and non-parenchymal liver cells. The importance of stem cells or liver oval cells is still under discussion and this mechanism provokes many questions (Lagasse, 2000, Petersen, 1999, Vassilopoulos, 2003). After partial hepatectomy or portal vein ligation increase serum levels of Tumour necrosis factor alpha (TNF-α) and Interleukin-6 (IL-6), which were demonstrated to be involved in priming of hepatocytes and trigger them from G0 to G1 cell cycle phase(Cornell, 1990). These cytokines induce gene activation, which are responsible for G1 phase. Both pleiotrophic cytokines are secreted by non-parenchymal liver cells (mostly Kupffer cells) (Fausto, 2000, Fausto, 2005). TNF-α is superior to IL-6 and stimulates increased secretion of IL-6. In hepatectomized regenerating liver, it is known that this signaling pathway follows the sequence TNF-α → TNFR-1 → NFκB → IL-6 → STAT3 (Michalopoulos, 1997). The proliferation of primed hepatocytes is regulated positively by Hepatocyte growth factor (HGF), Epidermal growth factor (EGF), Insulin-like growth factor (IGF), Transforming growth factor-alpha (TGF-α), etc. The termination of proliferation and stimulation of hepatocyte to differentiation, final remodelation of liver tissue and production of extracellular matrix is controled by Transforming growth factor-beta (TGF-β) (Fukuhara, 2003, Mangnall, 2003, Zimmermann, 2004). TGF-β1 plays the most important role in starting the remodelling of the extracellular matrix and restoration of the original structure of the liver parenchyma. TGF-β1 inhibits DNA synthesis and plays a pivotal role in the down-regulation of liver regeneration as has been demonstrated in toxic models of liver regeneration (Armendariz-Borunda, 1993, Armendariz-Borunda, 1997). TGF-β1 also down-regulates the production of the Hepatocyte growth factor that sustains hepatocyte proliferation (Bustos, 2000). Increased expression of TGF-β1 prevents uncontrolled growth during liver regeneration by the regulation of hepatocyte transition from the G1 to the S phase of the cell cycle (Kusaka, 2006, Oe, 2004). TGF-β1 helps maintain the differentiation of hepatocytes and non-parenchymal liver cells. The proposed HGF/ TGF-β1 ratio could reflect the proliferation/differentiation status of hepatocytes (Lilja, 1999). Increased expression of TGF-β1 was also shown to be a crucial factor for the progression of hepatic fibrosis (Friedman, 2008). This could be explained by increased or prolonged production of the liver extracellular matrix (Viebahn, 2008).

The replication of hepatocytes culminates on 7th days (14per cent of hepatocytes) and the return to quiscent status was observed on 12th day after PVE in swine experimental model (Coelho, 2007, Duncan, 1999). The differencies between PVE and PVL (portal vein ligation) were not proved as statistical significant for reached FLRV (Broering, 2002).

Multipotent mesenchymal stromal cells (MSC) are a fraction of the adult bone marrow stem cell compartment. They contain a subpopulation of mesenchymal stem cells that can differentiate into mesenchymal adult tissues under specified conditions. Differentiation into adipocytes, chondrocytes, osteoblasts and myocytes has been demonstrated in vitro and in vivo (Barry, 2003). Whether and how bone marrow-derived stem cells, and MSC in particular, can contribute to liver regeneration is not entirely clear. Initital reports outlined that bone marrow-derived (stem) cells can transdifferentiate into liver cells or their progenitors after bone marrow transplantation (Jiang, 2002, Lagasse, 2000, Petersen, 1999, Ringe, 2002). Later investigations clarified and complemented these observations, outlining that bone marrow cells can also fuse with resident liver cells (Vassilopoulos, 2003, Wang, 2003). In addition to direct cellular effects, transplanted bone marrow cells can also contribute to liver regeneration by bystander effects, such as the provision of a beneficial proliferative cytokine milieu or antiapoptotic effects (Aldeguer, 2002, Dahlke, 2004, Liska, 2009).

The aim of presented studies was to influence regeneration of liver parenchyma after portal vein embolization/ligation by exogenous cytokines, growth factors and monoclonal antibodies against growth factors with inhibitory funcions or syngeneic Multipotent Mesenchymal Stromal Cells.
