The main degenerative change observed with light microscopy in control IPRL is cytoplasmic vacuolation. This is usually mild with a centrilobular distribution. Methods Isolated Perfused Rat Liver (IPRL) These studies were approved by the Animal Ethics Committee of The University of Sydney. The IPRL procedure was performed as described previously [23]. After a

Selleckchem MLN4924 midline incision, 1 ml blood was collected from the caudal vena cava for serum transaminase measurements, and then 500 IU heparin in 0.5 ml (Pfizer, West Ryde, NSW, Australia) was injected. Liver perfusion was commenced with non-recirculating, lactated Ringer’s solution (compound sodium lactate = Hartmann’s solution – Baxter, Old Toongabbie, NSW, Australia) until the first lobe biopsy (ICL) was obtained. This was performed

by infusion from sterile bags manufactured for intravenous fluid therapy and had no additional oxygenation. Once the ICL biopsy was obtained, the perfusate was switched to 100 ml acellular, recirculating Krebs-Henseleit buffer. The composition of the buffer was as follows: 118 mM NaCl, 25 mM NaCO3, 4.7 mM KCl, 2.5 mM CaCl2.2H2O, 1.3 mM NaH2PO4.2H2O, 1.2 mM MgSO4.7H2O, 2% bovine serum albumin (BSA, fraction V, Sigma, Sydney, Australia) and 0.2% glucose [2]. Acellular perfusate is commonly used in IPRL experiments and avoids additional complications and variables check details associated with blood components [24–28]. This was continuously mixed Avelestat (AZD9668) in a reservoir on a magnetic stirrer and aerated with Carbogen (95% O2 + 5% CO2), which was bubbled into the reservoir rather than using an oxygenator to avoid kavalactone adsorption onto oxygen permeable tubing. This solution was recirculated at a constant flow of 16 ml/min using a peristaltic pump (MasterFlex, Cole-Parmer Instrument Company, Chicago, IL). To support bile flow, 60 mM taurocholic acid (Sigma, Castle Hill, NSW, Australia) in Krebs-Henseleit buffer was pumped into the perfusate reservoir at 1 ml h-1 using a syringe infusion pump

(Harvard Apparatus, Holliston, MA). Liver viability was judged on the basis of gross appearance, histology, liver transaminases and bile flow. Liver histology All reagents used for histopathology processing were Fronine brand (Lomb Scientific, Taren Point, NSW, Australia). Liver lobe biopsies were fixed by overnight immersion in 10% neutral-buffered formalin. Tissues were then placed in embedding cassettes (ProSciTech, Thuringowa Queensland, Australia) dehydrated through graded ethanol, cleared in xylene and infiltrated with paraffin wax in an Excelsior ES Tissue Processor (Thermo Fisher Scientific Australia, Scoresby, Victoria, Australia). Processed tissues were embedded in paraffin using a AG 14699 Shandon Histocentre 3 (Thermo). Five micron tissue sections were cut using a Leica RM2235 manual rotary microtome (North Ryde, NSW, Australia), stained with haematoxylin and eosin, and mounted on glass slides.