Intravital Microscopy of Liver Leukocytes

For intravital microscopy of the liver [30 (link),31 (link)], overnight-fastened animals were anesthetized via intraperitoneal (i.p.) injection with ketamine (100 mg/kg) and xylazine (10 mg/kg). A midline and a left subcostal incision were made to exteriorize the liver. The hepatic ligaments were dissected and the intestine was covered with a saline-soaked gauze to minimize tissue dehydration. The left liver lobe was then exteriorized and placed on a glass disk and covered with a glass slide for microcirculation analyses. With the use of a 10X ocular and 10X objective (Olympus BX150WI; Center Valley, PA, USA) images were displayed on a television monitor and recorded by a digital video recorder (DP73; Olympus, MA, USA) for off-line analysis with the Cellsens standard 1.9 software program (Olympus, MA, USA). The leukocyte–endothelial interaction was evaluated by counting the number of labelled leukocytes (0.3 mg/kg rhodamine 6G, i.v.) rolling or adhering to sinusoids and post-sinusoidal venules. Rolling leukocytes were defined as white blood cells with a slower velocity than the erythrocytes and a detectable rolling motion. Leukocytes that remained stationary on the sinusoidal or venular endothelium for 30 s or longer were considered adherent cells. Rolling and adherent cells were counted in a 170 μm² area comprising sinusoids and post-sinusoidal venules.