Subsequently, the mice were positioned on their left side and the left liver lobe was carefully exteriorized onto an adjustable stage for microscopic analysis. An equilibration period of 5min was allowed before starting the microscopical selleckchem observation. For intravital fluorescence microscopy, we used a modified Olympus microscope (BX50WI, Olympus Optical Co. GmbH, Hamburg, Germany) equipped with different water immersion lenses ( �� 40 NA 0.75/ �� 63 NA 0.9). The microscopic images were recorded by a charge-coupled device video camera (FK 6990 Cohu, Pieper GmbH, Schwerte, Germany) and transferred to CD-ROM for off-line evaluation. Blood perfusion within individual microvessels was studied after i.v. injection of 0.1ml 5% fluorescein isothiocyanate-labeled dextran 150000 (contrast enhancement by intravascular staining of plasma; Sigma Chemical Co.
, St Louis, MO, USA). In vivo labelling of leukocytes and platelets with 0.1% rhodamine-6G (0.1ml i.v., Sigma Chemical Co.) enabled quantitative analysis of leukocyte and platelet-flow behaviour in both sinusoids and postsinusoidal venules. Five postsinusoidal venules with connecting sinusoids were evaluated in each animal. Microcirculatory analysis included determination of sinusoidal perfusion by measuring the number of non-perfused sinusoids given as a percentage of the total number of sinusoids observed. Within sinusoids and postsinusoidal venules, leukocyte and platelet adhesion were measured by counting the number of cells adhering along the venular endothelium and remaining stationary during an observation period of 20s.
Cell adhesion is expressed as number of cells per 10 high-power field (HPF) and cellsmm?2, respectively. In addition, platelet aggregates (that is more than three platelets) in sinusoids and postsinusoidal venules were determined in each animal and are expressed as cells per 10 HPF and cellsmm?2, respectively. After intravital microscopic observations, animals were killed and blood was drawn from the inferior vena cava for standard spectrophotometric analysis of bilirubin and liver enzymes, including alanine aminotransferase and aspartate aminotransferase. In addition, systemic platelet and leukocyte counts, including polymorphonuclear leukocytes, were determined Carfilzomib with a haematocytometer. Measurement of myeloperoxidase activity Liver tissue was collected, weighed and homogenized in 10ml 0.5% hexadecyltrimethylammonium bromide. Subsequently, the sample was freeze-thawed, after which the myeloperoxidase (MPO) activity of the supernatant was assessed. The MPO activity was determined spectrophotometrically as the MPO-catalysed change in absorbance occurring in the redox reaction of H2O2 (460nm, 25��C). Values are expressed as MPO units per g liver tissue.