**4. Discussion**

166 Liver Regeneration

increase in the quantity of binucleated hepatocytes in the hypertrophic liver lobes in the TGF-β1 group (see figure 14). We observed a larger distribution of the number of binucleated hepatocytes per field in the hypertrophic lobe from TGF-β1 group (1-4 binucleated hepatocytes per field) in contrast to the control group (2-3,5 binucleated hepatocytes per field). The test of normality demonstrated a normal distribution of this parameter in each animal in both experimental groups. The size of hepatocytes was also not proved to be a statistically significant parameter for differences between the TGF-β1 group and the control group (see figure 15). Proliferative activity in both groups was

In case of comparison of histological findings at IL-6 and control groups the differences in lobulus length were not statisticaly significant. The statistical analysis proved significant increased amount of binucleated hepatocytes in hypertrophic liver lobes in IL-6 group (fig. 16). The length of hepatocytes was not proved also as statistically significant parameter. The

Fig. 14. Comparison of the concentration of binucleated hepatocytes in hypertrophic lobes between TGF-β1 and control groups. The binucleated hepatocytes were detected in 20

Fig. 15. Comparison of size of hepatocytes in hypertrophic lobes between TGF-β1 and

proliferative activity in both groups was very decreased (Liska, 2009).

greatly reduced.

microscopic fields.

control groups.

#### **4.1 Functional liver remnant volume**

In the presented chapter the authors want to review all experiences with influence of experimental portal vein ligation with cytokines, monoclonal antibody against growth factor and mesenchymal stem cells. This model closely mimics the situation of human oncological liver surgery, in which portal vein embolization can be utilized to create a larger future liver remnant volume before resection. The clinical goal of portal vein embolization is to increase the number of patients that can undergo extensive liver resections. This porcine experimental model of liver regeneration is especially applicable to the clinical situation since pigs closely resemble human liver anatomy and fysiology (Liska, 2009, Liska, 2009).

After portal venous infusion of MSC in this model, augmented and accelerated regeneration of the non-occluded liver lobes is outlined. This effect was strongest between the third and seventh postoperative day and slowly weaned off thereafter. However, there was no overall volume difference of statistical significance between both groups at the end of the observation period. This result might have been expected, though, since the body weight to liver weight ratio is tightly controlled in most species (Liska, 2009).

Previous studies have tried to clarify the potential contribution of MSC to liver regeneration in rodent models. Most of these approaches used toxic models, in which the intrinsic selfrenewing capacity of the recipient liver was suppressed (Dahlke, 2003, Kang, 2005). Di Bonzo observed only 0,2% human hepatocytes after transplantation of human MSC into immunodeficient mice with chronic liver injury (Di Bonzo, 2008). Overall, whether MSC can significantly contribute to liver regeneration in vivo remains controversial (Dahlke, 2004, Lysy, 2008, Popp, 2006), although MSC can show some features of hepatic differentiation in culture or under immunoprivileged conditions (Najimi, 2007, Ong, 2006). Chamberlain observed differentiation of human MSC into hepatocytes after infusion of MSC into foetal sheep (Chamberlain, 2007) and transplantable hepatocytes were also obtained from heavily growth factor-treated MSC (Banas, 2007, Yamamoto, 2008). Despite the fact that it was not possible to identify transplanted MSC within recipient livers in this model after 14 days, liver hypertrophy was augmented. It is therefore likely that bystander effects of the transplanted

Liver Parenchyma Regeneration in Connection

**4.2 Biochemistry** 

periphery (Liska, 2009).

with Extended Surgical Procedure – Experiment on Large Animal 169

human medicine. The achieved acceleration of growth of the hypertrophic liver lobes after application of monoclonal antibody against TGF-β1between 3rd and 7th postoperative days confirmed the key role of the studied cytokine in terminating the regeneration of liver parenchyma after portal vein ligation. The experimental results could be settings for a clinical study in patients with a low response regeneration of future liver remnant volume

All biochemical parameters, including cytokine levels, that were assessed during the observation period, did not outline any significant differences between the MSC-infused and the control group, indicating that both groups were systemically stable throughout the regenerative period. Concerning the role of cytokines it may also be assumed that these act on a more local level within the liver parenchym and differences cannot be observed in the

On the contrary application of TNF-α brought the tendency to differences in serum level of AST, ALT, GGT and creatinine in postoperative period, which were not statistically significant. All these biochemical parameters could be due to operative stress and also to occlusion of portal branches of right liver lobes, which represent significant liver function reserves. The postoperative elevation of these parameters could be hypothesized also to atrophic changes in occluded lobes. This reduction of expected increased serum level of discussed biochemical parameters after application of studied cytokine in non occluded portal vein could present also one of its pleiotropic functions – hepatoprotection agains changes after operative stress and reduction of functional parenchyma (Teoh, 2006, Baier, 2006). This hypothesis should be probably examined on non-resection experimental model (Liska, 2012). We did not prove any statistically significant differences between serum levels of studied biochemical parameters in particular time points between IL-6 and control groups. It demonstrates also no unsuitable influence of applied cytokine on the liver function. The changes in serum levels of studied cytokines, growth factors were also not observed as

different between IL-6 and control group in separate time points (Liska, 2009).

**4.3 Histology and immunohistochemistry** 

In case of comparison of TGF-β1 and control group 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 the applied monoclonal antibody on the liver function. This results support our ideas to choose monoclonal antibody against TGF-β1 for future clinical studies in human liver (Liska, 2012).

No histological differences were observed after 14 days between the groups (number of mitotic figures, binucleated hepatocytes, length of lobuli, hepatocyte size), indicating that the initial phase of liver regeneration had been completed in both groups and there was no influence of the infused MSC on liver architecture. Using BrdU staining, only very few of the infused MSC could be detected. Thus, injected MSC do not contribute to liver regeneration through proliferation, but most likely help to establish a micormilieu supportive for intrinsic proliferation of resident hepatocytes. Results acquired by this experimental study confirm the experiences of Furst and Esch, who have previously shown that cell grafts of bone marrow

after portal vein embolization that does not allow surgical treatment (Liska, 2012).

MSC accelerated regeneration in this model. This effect may be well mediated through paracrine effects of the infused MSC on the microenvironment in the periportal regions, where parenchymal regeneration is initiated. Aldeguer et al. suggested that increased production of IL-6 by bone marrow-derived cells, for example, can stimulate intrinsic liver regeneration (Aldeguer, 2002). In addition to being effective in terms of an accelerated regenerative response, the infusion of syngenic MSC into the portal vein also proved to be safe in the preclinical model. Probable side effects of MSC infusions include immunological problems (hypersensitivity, immune complex reactions), metabolic dysregulation and emboli. None of these problems occured in the presented study cohort (Liska, 2009).

The acceleration of growth of hypertrophic liver lobes after application of TNF-α confirmed results gained at in vitro models and in experiments in small laboratory animals (Cornell, 1990). The selected concentration of applied cytokine initiated acceleration of regeneration of liver parenchyma in non-occluded liver lobes (Heinrich, 2006, Teoh, 2006).

The acceleration of growth of hypertrophic liver lobes after application of IL-6 confirmed results gained at in vitro models and in experiments in small laboratory animals (Cornell,1990, Fukuhara, 2003). The selected concentration of applied cytokine initiated acceleration of regeneration of liver parenchyma in non-occluded liver lobes (Baier,2006, Heinrich, 2006).

We presented possibilities of the application of extrinsic MAB TGF-β1 to increase the required future remnant liver volume after partial portal vein ligation. The absolute volume of hypertrophic lobes (left lateral and medial lobes) grew more rapidly after application of MAB TGF-β1, whereas the control group had a slow but continual growth in the hypertrophic liver lobes during the whole follow-up period. The augmentation of growth of the hypertrophic lobes was maximal between the 3rd and 7th postoperative days in comparison with the control group (p<0.05). Nevertheless this growth accelerating effect was lost during the next ultrasonographic controls, and on the 14th postoperative day there were no statistically significant differences. The results of the presented study confirmed in large animal experiment previous findings gained using in vitro models and in experiments with small laboratory animals (Armendariz-Borunda, 1993, Armendariz-Borunda, 1997, Deneme, 2006, Delgado-Rizo, 1998). The selected concentration and timing of the application of monoclonal antibody prolonged acceleration of liver parenchyma regeneration in non-occluded liver lobes of animals (Armendariz-Borunda, 1993, Deneme, 2006). The secondary effects that could be hypothesized after application of the monoclonal antibody against key pleiotropic growth factor (changes in immune reactions and homeostasis) were not observed either during application or in the whole postoperative period. In previous described experimental groups (IL-6 and TNF-α group) we have shown the importance and usefulness of cytokines of the first phase of liver regeneration that increase priming of the hepatocytes – Interleukine-6 and Tumor necrosis factor – α (Liska, 2009, Liska, 2012), but these cytokines have pleitropic functions, which could be altered or changed by application of these cytokines from an extrinsic source. The choice of the monoclonal antibody against growth factor that terminates this first phase of liver regeneration and stimulates hepatocytes to differentiation, production of extracellular matrix and remodelation of liver tissue structure, imitates the same effect of these named cytokines (Kusaka, 2004).

The present study describes a new usage of a monoclonal antibody against TGF-β1 in large animal experimental model of partial portal vein ligation, which simulates the situation in human medicine. The achieved acceleration of growth of the hypertrophic liver lobes after application of monoclonal antibody against TGF-β1between 3rd and 7th postoperative days confirmed the key role of the studied cytokine in terminating the regeneration of liver parenchyma after portal vein ligation. The experimental results could be settings for a clinical study in patients with a low response regeneration of future liver remnant volume after portal vein embolization that does not allow surgical treatment (Liska, 2012).

#### **4.2 Biochemistry**

168 Liver Regeneration

MSC accelerated regeneration in this model. This effect may be well mediated through paracrine effects of the infused MSC on the microenvironment in the periportal regions, where parenchymal regeneration is initiated. Aldeguer et al. suggested that increased production of IL-6 by bone marrow-derived cells, for example, can stimulate intrinsic liver regeneration (Aldeguer, 2002). In addition to being effective in terms of an accelerated regenerative response, the infusion of syngenic MSC into the portal vein also proved to be safe in the preclinical model. Probable side effects of MSC infusions include immunological problems (hypersensitivity, immune complex reactions), metabolic dysregulation and emboli. None of

The acceleration of growth of hypertrophic liver lobes after application of TNF-α confirmed results gained at in vitro models and in experiments in small laboratory animals (Cornell, 1990). The selected concentration of applied cytokine initiated acceleration of regeneration

The acceleration of growth of hypertrophic liver lobes after application of IL-6 confirmed results gained at in vitro models and in experiments in small laboratory animals (Cornell,1990, Fukuhara, 2003). The selected concentration of applied cytokine initiated acceleration of regeneration of liver parenchyma in non-occluded liver lobes (Baier,2006,

We presented possibilities of the application of extrinsic MAB TGF-β1 to increase the required future remnant liver volume after partial portal vein ligation. The absolute volume of hypertrophic lobes (left lateral and medial lobes) grew more rapidly after application of MAB TGF-β1, whereas the control group had a slow but continual growth in the hypertrophic liver lobes during the whole follow-up period. The augmentation of growth of the hypertrophic lobes was maximal between the 3rd and 7th postoperative days in comparison with the control group (p<0.05). Nevertheless this growth accelerating effect was lost during the next ultrasonographic controls, and on the 14th postoperative day there were no statistically significant differences. The results of the presented study confirmed in large animal experiment previous findings gained using in vitro models and in experiments with small laboratory animals (Armendariz-Borunda, 1993, Armendariz-Borunda, 1997, Deneme, 2006, Delgado-Rizo, 1998). The selected concentration and timing of the application of monoclonal antibody prolonged acceleration of liver parenchyma regeneration in non-occluded liver lobes of animals (Armendariz-Borunda, 1993, Deneme, 2006). The secondary effects that could be hypothesized after application of the monoclonal antibody against key pleiotropic growth factor (changes in immune reactions and homeostasis) were not observed either during application or in the whole postoperative period. In previous described experimental groups (IL-6 and TNF-α group) we have shown the importance and usefulness of cytokines of the first phase of liver regeneration that increase priming of the hepatocytes – Interleukine-6 and Tumor necrosis factor – α (Liska, 2009, Liska, 2012), but these cytokines have pleitropic functions, which could be altered or changed by application of these cytokines from an extrinsic source. The choice of the monoclonal antibody against growth factor that terminates this first phase of liver regeneration and stimulates hepatocytes to differentiation, production of extracellular matrix and remodelation of liver tissue structure, imitates the same effect of

The present study describes a new usage of a monoclonal antibody against TGF-β1 in large animal experimental model of partial portal vein ligation, which simulates the situation in

these problems occured in the presented study cohort (Liska, 2009).

Heinrich, 2006).

these named cytokines (Kusaka, 2004).

of liver parenchyma in non-occluded liver lobes (Heinrich, 2006, Teoh, 2006).

All biochemical parameters, including cytokine levels, that were assessed during the observation period, did not outline any significant differences between the MSC-infused and the control group, indicating that both groups were systemically stable throughout the regenerative period. Concerning the role of cytokines it may also be assumed that these act on a more local level within the liver parenchym and differences cannot be observed in the periphery (Liska, 2009).

On the contrary application of TNF-α brought the tendency to differences in serum level of AST, ALT, GGT and creatinine in postoperative period, which were not statistically significant. All these biochemical parameters could be due to operative stress and also to occlusion of portal branches of right liver lobes, which represent significant liver function reserves. The postoperative elevation of these parameters could be hypothesized also to atrophic changes in occluded lobes. This reduction of expected increased serum level of discussed biochemical parameters after application of studied cytokine in non occluded portal vein could present also one of its pleiotropic functions – hepatoprotection agains changes after operative stress and reduction of functional parenchyma (Teoh, 2006, Baier, 2006). This hypothesis should be probably examined on non-resection experimental model (Liska, 2012).

We did not prove any statistically significant differences between serum levels of studied biochemical parameters in particular time points between IL-6 and control groups. It demonstrates also no unsuitable influence of applied cytokine on the liver function. The changes in serum levels of studied cytokines, growth factors were also not observed as different between IL-6 and control group in separate time points (Liska, 2009).

In case of comparison of TGF-β1 and control group 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 the applied monoclonal antibody on the liver function. This results support our ideas to choose monoclonal antibody against TGF-β1 for future clinical studies in human liver (Liska, 2012).

#### **4.3 Histology and immunohistochemistry**

No histological differences were observed after 14 days between the groups (number of mitotic figures, binucleated hepatocytes, length of lobuli, hepatocyte size), indicating that the initial phase of liver regeneration had been completed in both groups and there was no influence of the infused MSC on liver architecture. Using BrdU staining, only very few of the infused MSC could be detected. Thus, injected MSC do not contribute to liver regeneration through proliferation, but most likely help to establish a micormilieu supportive for intrinsic proliferation of resident hepatocytes. Results acquired by this experimental study confirm the experiences of Furst and Esch, who have previously shown that cell grafts of bone marrow

Liver Parenchyma Regeneration in Connection

**5. Conclusion** 

extended liver resection.

**6. Summary** 

cells, TGF-β1.

**7. Acknowledgement** 

pp. 165-175, ISSN 1365-2168

1247-1256. ISSN: 0959-8049

2002), pp. 40-48, ISSN 1527-3350

*of some vital organs*).

**8. References** 

with Extended Surgical Procedure – Experiment on Large Animal 171

Application of IL-6 and TNF-α augments hypertrophy of FLRV on 7th postoperative day in comparison to control group. In case of application of MAB TGF-β1 we observed maximal increase of FLRV between 3rd and 7th days. Application of MSC augments hypetrophy of FLRV on 3rd day. The biochemical and histological examinations did not prove any important differencies among the groups. The use of TNF-α, IL-6, MAB TGF-β1, MSC could increase the process of liver regeneration after portal vein ligation.These experimental results could be used in clinical practice in patients with risk of acute liver failure after

The aim of presented studies was to influence regeneration of liver parenchyma after portal vein embolization/ligation. Physiological solution (controls, 9 animals) or TNF-α (9) or interleukin-6 (8) or MSC (6) were applied into non-occluded portal vein branches after ligation of portal vein branches for right lobes. Administration of MAB TGF-β1 was performed 24 hours after ligation (7). Compensatory hypertrophy (FLRV) was followed-up in next 14 days by regular ultrasonography, serum level of biochemical parametres and by histological examinations. Application of IL-6 and TNF-α augments hypertrophy of FLRV on 7th postoperative day in comparison to control group. In case of application of MAB TGF-β1 we observed maximal increase of FLRV between 3rd and 7th days. Application of MSC augments hypetrophy of FLRV on 3rd day. The biochemical and histological examinations did not prove any differences among the groups. The use of TNF-α, IL-6, MAB TGF-β1, MSC

**Keywords:** Liver surgery, Liver regeneration, Experimental study, Porcine model, Experimental model, Portal vein embolization, TNF-α, Interleukin-6, Mesenchymal stem

Supported by grants IGA MZ CR 12025 and Research project MSM 0021620819 (*Replacement* 

Abdalla, E. K., Hicks M. E. & Vauthey, J. N. (2001). Portal vein embolization: rationale,

Alison, M.R. et al.(2006): Stem cell plasticity and tumour formation. *Eur J Cancer*, 42, pp

Aldeguer, X. et al. (2002). Interleukin-6 from intrahepatic cells of bone marrow origin is

Armendariz-Borunda, J. et al. (1993). Transforming growth factor beta gene expression is

*Laboratory Investigation*, Vol. 69, No. 3, (1993), pp. 283-294, ISSN 0023-6837

technique and future prospects. *British Journal of Surgery,* Vol. 88, No. 7, (July 2001),

required for normal murine liver regeneration. *Hepatology*, Vol. 35, No. 1, (January

transiently enhanced at a critical stage during liver regeneration after CCl4 treatment.

could increase the process of liver regeneration after portal vein ligation.

origin (CD133 positive cells) applied after PVE in human surgery can increase the FLRV (Furst, 2007). From the present experimental data, however, it cannot be concluded with certainty that the application of stem cells, including MSC, does not support the growth of liver malignancies to the same extent that it supports liver regeneration. Assuming a bystander effect of MSC on the micromilieu makes this even more likely (Alison, 2006). Thus, further animal investigation is necessary before optimized MSC therapies can be applied in the setting of human medicine (Alison, 2006). In conclusion, it has been hereby shown that the intraportal infusion of syngeneic porcine MSC after PVE in a setting of liver regeneration led to an accelerated and augmented compensatory liver hypertrophy. This effect is most likely due to bystander effects of the transplanted MSC (Liska, 2009).

The increased number of larger lobulus in the hypertrophic parenchyma of TNF-α group in comparison with control group could be explained by incomplete liver regeneration. Because there are practically no mitotic figures or the amount is of same quantity as in the normal liver parenchyma without any surgical procedures or toxic insult, we could hypothesize, that the first phase of liver regeneration is finished and the next phase of regeneration proceeds. It means the remodelation phase and the phase, when the liver microstructure is restored. Next would be objective to future study – the detection of intracellular or extracelular matrix changes during the process of liver regeneration. No differences in the amount of binucleated hepatocytes could be discussed also by end of proliferative phase of liver parenchyma at the time of sampling. The same size of hepatocytes and no atypical hepatocytes in the biopsies could also be explained by the same reason. This hypothesis is supported by restitution of all liver function monitored by biochemical parametres at the moment of sacrifying of experimental animals (Liska, 2012).

The increased number of binucleated hepatocytes in the hypertrophic parenchyma of IL-6 group in comparison with control group could be explained by incomplete liver regeneration at the end of experiment. Because there are practically no mitotic figures or the amount is of same quantity as in the normal liver parenchyma without any surgical procedures or toxic insult, we could hypothesize, that the first phase of liver regeneration is finished and the next phase of regeneration proceeds. It means the remodelation phase and the phase, when the liver microstructure is restored. Next would be objective to future study – 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 not proved (Liska, 2009).

The larger distribution of the number of binucleated hepatocytes in the hypertrophic parenchyma of TGF-β1 group in comparison with the control group could be explained by incomplete liver regeneration at the end of experiment. Because there are practically no mitotic figures, or the amount 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, namely the remodelling phase and the phase when the liver microstructure is restored (Mangnall, 2003). The size of hepatocytes and the length of lobuli were not proved to be statistically different between study and control group. The same size of hepatocytes and length of lobuli in the bioptical samples from the hypertrophic parenchyma could be also explained in the same way. This hypothesis is supported by the restitution of all liver functions monitored by biochemical parameters and completion of the proliferative phase of liver regeneration at the moment of sacrificing of the experimental animals (Liska, 2012).
