Alternatively, platelet�Cleukocyte aggregates may be trapped, mechanically, at the narrow sites in the organ microvasculature. Mechanistic selleck chemical Enzastaurin studies have revealed that P-selectin is an adhesive link between platelets and leukocytes in aggregate formation. Interestingly, Singer et al. (2006) have recently reported that neutrophils can facilitate platelet adhesion in septic liver injury. However, a role of platelets in leukocyte recruitment and cholestatic liver injury remains to be demonstrated. Based on the considerations above, the aim of the present study was to determine the role of platelets and P-selectin in cholestasis-induced leukocyte recruitment and hepatocellular damage. For this purpose, intravital fluorescence microscopy of the hepatic microcirculation was examined after ligation of the common bile duct in mice.

Methods Animals All animal procedures were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council, WA, USA), and were approved by the local ethics committee at Lund University. Adult male C57BL/6 mice with a body weight of 22�C27g were used for the study. The animals were housed one per cage on a 12�C12h light�Cdark cycle and had free access to standard pellet food and tap water throughout the experiment. Animals were anaesthetized by i.p. administration of 7.5mg ketamine hydrochloride (Hoffman-La Roche, Basel, Switzerland) and 2.5mg xylazine (Janssen Pharmaceutica, Beerse, Belgium) per 100g body weight. Test substances and fluorescent dyes were administered i.

v. via retroorbital injection. Experimental protocol Animals underwent bile duct ligation (BDL) to induce obstructive cholestasis. BDL was performed under ketamine/xylazine anaesthesia via a midline laparotomy. By means of a surgical microscope, the common bile duct was prepared and carefully ligated with a 7�C0 prolene suture. Subsequently, the laparotomy was closed again by a 5�C0 running suture and the animals were allowed to recover from anaesthesia and surgery for 12h. Sham-operated animals received phosphate-buffered saline (PBS) i.v. and underwent an identical laparotomy and liver manipulation without BDL. To delineate the role of platelets and P-selectin in the pathogenesis of obstructive cholestasis, animals were pretreated i.v.

2h prior to BDL with an anti-GP1b�� antibody (ab) (rat IgG, 1mgkg?1, Emfret Analytics GmbH & Co., Eibelstadt, Germany), which depletes mice of platelets, an anti-P-selectin ab (RB40.34, rat IgG, 1.5mgkg?1, Pharmingen, San Diego, CA, USA) or an isotype-matched control ab (rat IgG, R3�C34, 1.5mgkg?1, Pharmingen). Intravital fluorescence microscopy Twelve hours after BDL, the hepatic microcirculation was examined Carfilzomib by intravital fluorescence microscopy.