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BACKGROUND: Portal vein ligation (PVL) combined with in situ splitting (ISS) has been shown to induce remarkable liver regeneration in patients. The purpose of this study was to establish a model of PVL+ISS in rats for exploring the possible mechanisms of liver regeneration using these techniques. MATERIALS AND METHODS: Rats were randomly assigned to three experimental groups: selective PVL, selective PVL+ISS and sham operation. The hepatic regeneration rate (HRR), Ki-67, liver biochemical determinations and histopathology were assessed at 24, 48, and 72 h and 7 days after the operation. The microcirculation of the median lobes before and after ISS was examined by laser speckle contrast imaging. Meanwhile, cytokines such as TNF-α, IL-6, HGF and HSP70 in regenerating liver lobes at 24 h was investigated by RT-PCR and ELISA. RESULTS: The HRR of PVL+ISS was much higher than that of the PVL at 72 h and 7 days after surgery (p<0.01). The expression of Ki-67 in hepatocytes in the regenerating liver lobe was stronger in the PVL+ISS group than in the PVL group at 48 and 72 h (p<0.01). There was a significant reduction in microcirculation blood perfusion of the left median lobe before and after ISS. Liver biochemical determinations and histopathology demonstrated more severe hepatocyte injury in the PVL+ISS group. Both the mRNA levels of TNF-α and IL-6 and the protein levels of TNF-α, IL-6 and HGF in regenerating liver lobes were higher in the PVL+ISS than the PVL alone. CONCLUSIONS: The higher HRR in the PVL+ISS compared with the PVL confirmed that we had successfully established a PVL+ISS model in rats. The possible mechanisms included the reduced microcirculation blood perfusion of the left median lobe and up-regulation of cytokines in the regenerating lobes after ISS.