The animals were fasted for 8 h before hepatectomy. A mixture of 5 mg/kg xylazine (Rompun Bayer, Turkey) and 30 mg/kg ketamine hydrochloride (Ketalar Panker Davis, Turkey) was administered i.p. for anesthesia/analgesia. 70% hepatectomy was performed on the animals in accordance with the technique described by Higgins and Anderson (Higgins and Anderson 1931 ; Nevzorova et al. 2015 (link)). The rats were randomized into 8 groups (n ≥ 3), which were defined as day-0, -1, -2, -3, -5, -7, -10,-14; denoting the days that the rats were sacrificed after PH. Serum samples were collected and regenerating remnant livers were removed on the day of sacrification. A portion of each liver specimen was fixed in 10% formalin solution for immunohistochemistry; a portion of the liver specimen was dissected in RIPA lysis buffer (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA) for protein analyses and a portion of the liver specimen was kept in RNAlater stabilization solution (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA) for RT-qPCR analyses. mRNA expression levels of Vegf and Pcna were analyzed in addition to immunohistochemical evaluations. Liver tissue protein levels of endostatin, endocan and p-eEF2K/eEF2K were determined with Western blot and serum levels of endostatin and endocan were assessed with ELISA.
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