Samples were centrifuged at 3500 rpm for 10 minutes at 4��C and plasma was collected and stored frozen at -80��C until assaying. IL-6 and TNF-�� were this site measured in duplicate using a commercially available ELISA kit (Biosource, CA, USA). The sensitivities of the assays were 3 pg/ml for IL-6 and 3 pg/ml for TNF-�� and 3 pg/ml for IL-6.Western blot methodologyAt the end of the experimental period (nine hours for western blot experiments) spleens were harvested (n = four per group). The samples of spleen were then homogenized (Polytron homogenizer by Kinematica, Bethlehem, PA, USA) in ice-cooled lysis buffer (20 mm Tris-HCl, 150 mm NaCl, 1 mm Na2DTA, 1 mm EGTA, 1% Triton, 2.5 mm sodium pyrophosphate, 1 mm ��-glycerophosphate, 1 mm Na3VO4, 2 mm dl-dithiothreitol, 1 mm phenylmethanesulfonyl, and 1 ��g/ml leupeptin; pH 7.

5) and centrifuged at 3000 g for 10 minutes at 4��C. The supernatant was further centrifuged twice, initially at 12,000 g for 15 minutes at 4��C and a second time at 20,000 g for 45 minutes at 4��C. The protein concentration of supernatant was determined with the Bradford protein assay (Bio-Rad, Herts, UK). The supernatant (10 ��g protein per sample) were denaturated in NuPAGE LDS Sample buffer (Invitrogen, Paisley, UK) at 70��C for 10 minutes and then were loaded on a NuPAGE 4 to 12% Bis-Tris Gel (Invitrogen, Paisley, UK). After electrophoresis, the proteins were electrotransferred to a nitrocellulose membrane (Hybond ECL; Amersham Biosciences, Buckinghamshire, UK) and incubated with a blocking solution composed of 5% fat dry milk in Tween-containing Tris-buffered saline (pH 8.

0, 10 mm Tris, 150 mm NaCl, 0.1% Tween). The blocked membrane was incubated overnight at 4��C with the cleaved caspase-3 antibody (New England Biolab, Hitchin, United Kingdom). After washing with Tween-containing Tris-buffered saline for four times, the membrane was incubated for one hour at room temperature with the appropriate horseradish peroxidase-conjugated secondary antibody directed at the primary antibody. The bands were then visualized with enhanced chemiluminescence (New England Biolab, Hitchin, United Kingdom) and exposed onto Hyperfilm ECL film (Amersham Biosciences, Buckinghamshire, United Kingdom). Subsequently, the membrane was re-probed with caspase 3 and beta-action primary antibody respectively and the rest procedures were repeated again as above.

The band density was analyzed densitometrically and normalized with the housekeeping protein beta-actin and then presented as percentage of control.Mortality rateAnimals Batimastat were monitored every two hours via video recording of the animal in its cage following the initial eight-hour sedative infusion period and animal mortality was noted (n = 10 per group). After 16 hours of follow up (i.e., 24 hours post CLIP) all animals were sacrificed by lethal sodium pentobarbital injection.