Liver presents a remarkable capacity for proliferation after a partial hepatectomy and can precisely regulate its growth and mass to adjust its size. The exact mechanisms of stimulation and regulation of hepatic regeneration remain unclear. It is well known that various cytokines and growth factors and perhaps cell populations, other than hepatocytes are involved. Many different substances have been reported to stimulate liver cell growth in vivo and in vitro, including a number of known hormones, serum factors and some small nutrient molecules [1, 2, 4].
The discovery that EPO and its receptor play a significant biological role in tissues outside of the hematopoietic system has provoked significant experimental interest and fueled the exploration of additional actions of the hormone [20, 21]. It is a member of the class I cytokines family and is considered a pleiotropic hormone. The EPO-specific receptor has been recognized in different cells, such as endothelial cells, epicardium, placenta, pancreatic islets, renal cells and defined areas of the brain . Previous studies suggested that erythropoietin (EPO) was produced in rats by the regenerating liver  following partial hepatectomy and erythropoietic foci have been recognized 24-72 hours after subtotal hepatectomy in rats . Kupffer cells seem to be the site of erythropoietin production after hepatectomy .
Angiogenesis seems to be a fundamental requirement for liver regeneration and its regulation. The modulation of endothelial cell proliferation or apoptosis has been shown to affect liver regeneration after partial hepatectomy in mice . During liver regeneration the expression and activity of proapoptotic pathways is inhibited and after massive liver resection the activation of apoptosis is a major cause for failure of regeneration . Recently EPO has been found to inhibit apoptosis after injury in various organs, like the brain , kidney  and the myocardium .
GM-CSF is a haematopoietic growth factor that apart from stimulating the proliferation and differentiation of myeloid bone marrow progenitor cells, also enhances the function of mature macrophages that are induced to secrete various cytokines including IL-6 and TNF-a . It has been found to be a very potent immunostimulating agent by priming macrophages to produce cytokines, like TNF-a and IL-6 in blood of healthy humans as well as in blood of immunosupressed patients with sepsis and after cardiopulmonary bypass . Within minutes after PH, Kupffer cells release cytokines, specifically TNF-a and IL-6 that are substantial for hepatocytes priming and preparation for replication . Eroglu et al have already shown that GM-CSF promotes liver regeneration after hepatectomy in normal and cirrhotic livers .
The above mentioned experimental evidences prompted us to compare the effects of the administration of rhEPO, GM-CSF and their combination on liver regeneration following major hepatectomy. Although the effect of EPO in this setting has already been reported, there are few data on the effect of GM-CSF. In addition there are no data on the effect of their combination on liver regeneration. These two factors are thought to be mitogens and their combination should have a cumulative regenerating effect on the liver. However, Fatouros et al have reported that their combined administration seems to attenuate the beneficial role of EPO on intestinal anastomosis healing, which is similarly a mitotic process . The major end-point of this study was to investigate if their combination has a synergistic or antagonistic effect on liver regeneration after major hepatectomy.
In our study we chose to evaluate the expression of two proliferation markers -PCNA and Ki 67-, as these have been shown to peak at different timepoints of the cell cycle, and their expression could vary depending on the stage of cellular duplication. They are sensitive markers of hepatocyte proliferation, which correlate well with the extent of regeneration . In addition, they are have already been widely used for the study of liver regeneration and in particular for the study of the effects of EPO on liver regeneration.
Our study demonstrates that EPO administration had a positive effect on liver regeneration process after 70% hepatectomy by augmenting nuclear activity. This effect is noted even before any "triggering" for regeneration took place, as rats pre-treated with rhEPO showed increased expression of both Ki 67 and PCNA before hepatectomy was performed. This is in accordance with the literature, as Bockhorn et al have also demonstrated similar results. They reported that EPO preconditioning for three days can raise significantly the Ki-67 proliferation index and liver-to-body weight ratio of the normal liver . In addition proliferation markers were increased after hepatectomy until 3 days on rats treated with rhEPO, similarly to our results . Although the increase in our study is substantial, it is the result of a prolonged EPO pretreatment period (8 days). In addition, our results represent to total amount of hepatocytes stained, whether the staining was moderate or intense. As Ki 67 antigen is expressed during the whole cell cycle, it is uncertain whether the moderately stained cells are in the process of mitosis, or the antigen is still expressed in the cell after mitosis.
The dose of EPO administered in our study was 500 IU/kg and was administered subcutaneously. A wide variety of doses have been used by other authors [7–9, 29, 30]. We used the doses used by Fatouros et al in a study trying to compare the combined effect of EPO and GM-CSF on colonic anastomoses healing [15, 16]. Generally they are considered low doses in this experimental setting. However we did not want to use higher doses as they have been shown to inhibit liver regeneration .
In our study, pre-operative GM-CSF administration resulted in increased hepatocyte proliferation before hepatectomy, as well as at postoperative days 1 and 7. Preoperatively only PCNA was over-expressed, and not Ki 67. This can be explained by the fact that these two markers of cellular proliferation do not correspond to the same cell cycle phase, as PCNA concentration seems to peak at the at the S phase of the cell cycle, while Ki 67 peaks later, during mitosis, in the M phase . Eroglu et al showed increased hepatocyte proliferation 2 days after hepatectomy in rats were GM-CSF was administered. This effect however faded at the 7th postoperative day. However in their study GM-CSF was administered immediately after hepatectomy, while in our study we pretreated animals for 8 days before hepatectomy and 2 days after . The dose of GM-CSF administered was 20 mcg/kg as used by other authors .
On the other hand pretreatment with the combination of EPO and GM-CSF resulted in a weaker proliferative response compared with animals that were treated with EPO alone. Since EPO alone increased nuclear activity, it would seem logical that the combination group would have the same results. The fact that this group showed less nuclear activity than the EPO group, suggests perhaps a competitive action between the two growth factors. This is in accordance with the findings of other studies, where although EPO administration increased the tensile strength of colonic anastomoses postoperatively in rats, the combined administration of EPO and GM-CSF failed to show the same results [15, 16]. Many possible mechanisms have been proposed in the literature. GM-CSF may play an antagonistic role on the EPO receptor as these hemopoietins have a high homology [31, 32]. A competition between EPO and GM-CSF has been reported in cells of the marrow . In addition, it has been shown that GM-CSF can modulate EPO effects in certain leukemic cell line models of hematopoiesis, modulating events at the transcriptional and signal transduction level, or decreasing mRNA levels of EPO receptor . Finally concentrations of hemopoietins have been found to play a key role in the final effect on cellular response .