Intravital microscopy of the cremaster muscle

Eight to twelve-week-old male C57BL/6 mice (purchased from Janvier Labs, Le Genest-Saint-Isle, France) were injected i.p. with 2 mg DHEA (Sigma-Aldrich, Munich, Germany) diluted in PBS containing 4.5% ethanol and 1% BSA or the same amount of control vehicle diluent (4.5% ethanol, 1% BSA in PBS), as previously described (37). Thirty minutes after DHEA injection, 50 ng of LPS (Escherichia coli O111:B4; Sigma-Aldrich) were injected intrascrotally. Intravital microscopy was performed 3.5 h later. The cremaster muscle preparation was performed as previously described (41). Briefly, the scrotum of the mouse was incised, the cremaster muscle was exteriorized, additional tissue was removed, and the muscle was then opened through a longitudinal incision and mounted onto a self-customized stage. During intravital microscopy, the cremaster muscle was constantly superfused with warm superfusion buffer (41). Intravital microscopy was conducted on a BX51WI microscope (Olympus, Center Valley, PA) equipped with a 40× saline immersion objective (MplanFI/RI, 0.8 numerical aperture; Olympus) and a charge-coupled device camera (Kappa CF8 HS). VirtualDub (version 1.9.11) was used for recording of postcapillary venules. Leukocyte rolling was assessed as a percentage of rolling leukocytes relative to the number of leukocytes passing the vessel (rolling flux fraction), and leukocyte adhesion efficiency as was assessed as the number of adherent leukocytes per square millimeter relative to the WBC count per microliter. Adherent leukocytes were defined as nonmoving cells or cells with a displacement smaller than one cell diameter during 1 min of observation (42). Leukocyte rolling velocities were measured as averages over a 1-s time window using the Fiji software (43). Vessel diameters were measured using a digital image processing system (44). Centerline RBC velocity in cremaster muscle microvessels was obtained using a dual photodiode and a digital on-line cross-correlation program (CircuSoft Instrumentation, Hockessin, Germany). Wall shear rates were assessed as 4.9 (8 v/d), where v is the mean blood flow velocity and d is the diameter of the vessel (45). Blood cell numbers were determined through IDEXX ProCyte Dx Hematology Analyzer (IDEXX Europe B.V.). Experiments were approved by the Regierung of Oberbayern, Germany.