Two hundred mg/kg bodyweight

(bw) TAA was injected intr

Two hundred mg/kg bodyweight

(b.w.) TAA was injected intraperitoneally into DPPIV− F344 rats (1.5 to 2 months of age) twice weekly for up to 3 months prior to cell transplantation, followed by 100 or 200 mg/kg b.w. TAA Rucaparib price twice weekly after cell infusion. All animal studies were conducted under protocols approved by the Institutional Animal Care and Use Committees of AECOM and University of Pittsburgh in accordance with National Institutes of Health (NIH) guidelines. Unfractionated fetal liver cells were isolated from ED14/15 fetal livers of pregnant DPPIV+ or DPPIV+/EGFP+ F344 rats, as described.[18, 19] Hepatocytes were isolated from livers of adult DPPIV+ F344 rats. Detailed information concerning the cell isolation procedures can be found in the Supplemental Materials and Methods of Ref. [21]. Fetal liver cells (viability >95%) or adult hepatocytes (viability >80%) were transplanted through the portal vein into DPPIV− F344 rats[22] treated with TAA or untreated recipients with or without 2/3 PH (hepatectomized liver lobes were used to assess liver fibrosis and for other studies). After rats were sacrificed at different

times following cell transplantation, liver repopulation was determined by enzyme histochemistry for DPPIV, as described.[18, Forskolin 19] For engraftment studies, transplanted fetal liver cells were detected by immunohistochemistry for EGFP. Total RNA was extracted from snap-frozen liver tissue derived from TAA-treated DPPIV− F344 rats and untreated age-matched control rats. Qualitative RT-PCR analyses were performed at least twice. Quantitative real-time RT-PCR was performed in doublet/triplicate, as described in the Supplemental Materials and Methods of Ref. [21]. A list of the primers is shown in Supporting

Table 1. Information concerning histochemical and immunohistochemical analyses can be found in the Supporting Materials and Methods. Using two different fragments per liver, the HYP content was determined biochemically, as described.[23] Tissue slides were examined under an AxioObserver Z1 microscope. Images were obtained with an AxioCam ICc3, ICm1, or HRc camera and processed with AxioVision 4.8 or ZEN imaging software (Carl Zeiss MicroImaging). Data were analyzed using SigmaStat 2.01 4-Aminobutyrate aminotransferase (SPSS Scientific), GraphPad Prism5 (GraphPad), and NIS-Elements D (Nikon) software and are reported as mean ± SEM. After chronic TAA administration (200 mg/kg b.w., twice weekly), liver fibrosis was assessed (Fig. 1) using the Laennec classification system.[24] At 6 weeks, the progressive liver injury produces moderate fibrosis and mild cirrhosis, i.e., predominant nodularity caused by narrow fibrous septa bridging portal areas. By 3 months, more advanced fibrosis occurs, leading to moderate to severe cirrhosis in different parts of the liver.

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