Animal procedures were conducted in accordance with French government policies (Services Vétérinaires de la Santé et de la Production Animale, Ministère de l’Agriculture). Acute liver injury was induced by a single intraperitoneal (ip) injection of CCl4 (0.5 mL/kg body weight, 1:5 dilution in MO), Cilomilast supplier as in Serriere-Lanneau et al.21 Control animals received MO. When indicated, mice were treated either with respective vehicle,
IL-6 (0.5 mg/kg, subcutaneous), the CB2 agonist JWH-133 (3 mg/kg, ip), the NO donor SIN-1 (10 mg/kg, ip), or the MMP-2/MMP-9 inhibitor CTTHWGFTLC (13 mg/kg, ip), administered before CCl4 administration. No mortality was observed throughout treatments. Liver samples were taken from several lobes and either fixed in buffered formalin or snap frozen in liquid nitrogen and stored at −80°C until use. Experiments were performed on LDE225 ic50 4-9 animals/group. Two-thirds hepatectomy was performed as previously described,22 while animals were
under isoflurane anesthesia. After ventral laparotomy, the left lateral, left median, and right median lobes were ligated and excised. The removed liver specimens were weighed and snap-frozen in liquid nitrogen to serve as control. Alanine and aspartate aminotransferase activity was measured on an automated analyzer in the Biochemistry Department of Mondor Hospital. Results are the mean from 15 animals/group. Liver cells were digested by two-step collagenase perfusion. Cell suspension was centrifuged at 50 g rpm for 2 minutes. The pellet contained hepatocytes, and nonparenchymal cells were purified from the supernatant by density gradient centrifugation with 25%-50% Percoll. Terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining was performed on paraffin-embedded tissue sections, using the In Situ Cell Death Detection Kit, POD (Roche). TUNEL-positive area from two to three fields (magnification ×100)/animal were quantified with ImageJ. Results are expressed as percent of total area, and were quantified from seven to eight animals/group. Immunohistochemistry was carried out on paraffin-embedded liver tissue sections as previously described.10 Immunohistochemical
detection of proliferating cell nuclear antigen (PCNA) was performed using the MOM immunodetection kit (Vector) and a mouse monoclonal selleck inhibitor anti-PCNA (1/1750; Santa Cruz Biotechnology). The number of labeled hepatocytes per 2000 hepatocytes was quantified in tissue sections (magnification ×200) from 4-6 mice/group. Immunohistochemical detection of F4/80 and myeloperoxidase was performed using rat anti-mouse F4/80 (1:20; Serotec) and rabbit anti-human myeloperoxidase (1:750; Dako), respectively, followed by biotinylated secondary antibody (anti-rat, 1/50; Serotec or anti-rabbit, 1/100; Santa Cruz Biotechnology). The signal was amplified with alkaline phosphatase–conjugated streptavidin (1/20; Serotec), and revealed using the liquid permanent red chromogen system (Dako).