semiautomatic micromanipulator (InjectMan NI 2H, Eppendorf, Hamburg, Germany) and a microinjector (FemtoJetH, Eppendorf). A successful microinjection was verified by the observation of visible swelling of the interstitial tissue during injection. The vessel and the surrounding tissue were visualized during a time period of 5 min at baseline conditions before stimulation as well within 60 min after microinjection. In an additional group (n = 3), the character of leukocyte migration was analyzed 60 min after intrascrotal application of PAF (100 nM in 0.3 ml PBS) two hours prior to the in vivo microscopic observation. In additional experiments (n = 2), an intrascrotal injection of MIP-1a was performed in order to better compare the character of leukocyte interstitial migration upon intrascrotal injection of MIP-1a with that after microinjection of MIP-1a. In a separate set of experiments, leukocyte motility was analyzed after inhibition of Rho kinase with a selective inhibitor (Y-27632, 50 mM) [38,30]. Leukocyte migration was initiated by microin- jection of MIP-1a, as described above. Sixty min after microin- jection of MIP-1a, the exteriorized cremaster muscle was superfused with Y-27632 for either 5 or 30 min. In vivo microscopic analysis was performed upon either 5 min or 30 min of Y-27632 superfusion in two separate groups. For both inhibitor-treated groups, corresponding time controls were performed with saline superfusion. In vivo microscopy The set-up for in vivo microscopy was centered around an Olympus BX 50 upright microscope (Olympus Microscopy, Hamburg, Germany), equipped for stroboscopic fluorescence epi-illumination microscopy. Light from a 75-W xenon source was narrowed to a near monochromatic beam of a wavelength of 700 nm by a galvanometric scanner (Polychrome II, TILL Photonics, Gra¨felfing, Germany) and directed onto the specimen via a fluorescein isothiocyanate (FITC) filter cube equipped with dichroic and emission filters (DCLP 500, LP515, Olympus Microscopy). Microscopic images were obtained with Olympus water immersion lenses [206/numerical aperture (NA) 0.5 and 406/NA 0.8] and recorded with an analog black and white charged-coupled device video camera (Cohu 4920, Cohu, San Diego, CA) and an analog video recorder (AG-7350-E, Panasonic, Tokyo, Japan). Oblique illumination was obtained by positioning a mirroring surface (reflector) directly below the specimen and tilting its angle relative to the horizontal plane. The reflector consisted of a round coverglass (thickness 0.19–0.22 mm, diameter 11.8 mm) which was coated with aluminum vapor (Freichel, Kaufbeuren, Germany) and brought into direct contact with the overlying specimen. For measurement of centerline blood flow velocity, green fluorescent microspheres (2 mm diameter, Molecular Probes, Leiden, The Netherlands) were injected via an arterial catheter, and their passage through the vessels of interest was recorded using the FITC filter cube under appropriate stroboscopic illumination (exposure 1 ms, cycle time 10 ms, l~488 nm), integrating video images for sufficient time (.80 ms) to allow for the recording of several images of the same bead on one frame. Beads that were flowing freely along the vessels were used to determine centerline blood flow velocity (see below). Tissue distribution of applied fluorescent dye per time In an attempt to get information about the character of tissue distribution of injected mediators, microinjection of the fluorescent dye rhodamine 6G (130 pl, 0.05%, Sigma Aldrich) was performed in the cremaster muscle according to the above described technique in a separate set of experiments (n = 7). The distribution of rhodamin 6G in the interstitium was analyzed in the regions of Leukocyte Migration In Vivo interests using in vivo fluorescence microscopy (excitation: 530 to 560 nm, emission: .580 nm, Olympus). Light from a 75-watt xenon source was narrowed to a near monochromatic beam by a digitally controlled galvanometric scanner (Polychrome II, TILL Photonics, Gra¨felfing, Germany). Fluorescence emission was collected by a CCD camera (Sensicam, PCO, Kelheim, Germany) and subjected to digital image analysis (TILL Vision 4.0; TILL Photonics). Spatial dynamics of the fluorescence intensity were measured before and within 60 min after microinjection and expressed as mean gray value [39]. Mean gray values of three regions of interests (ROIs; 100675 mm) were analyzed: 1) on the vessel side of the postcapillary venule ipsilateral to the microin- jection site, 2) on the contralateral side as well as 3) in the interstitial tissue 350 mm from the site of microinjection (consid- ered as background) (depicted in the Fig 2B). Microhemodynamic parameters Centerline blood flow velocity was measured using micro- spheres administered intraarterially. Quantitative analysis of velocity was performed off-line using CAPImageH by measuring the distance between several images of one fluorescent bead under stroboscopic illumination. From measurement of the vessel diameters and centerline blood flow velocity, the Newtonian wall shear rate [s21] was estimated as 86[Vb/d], where Vb is the mean blood flow velocity, d is the diameter of the vessel. Mean blood velocity, Vb, was approximated by multiplying the centerline blood velocity by 0.625. The interfacial shear rate is the slope of the velocity profile at the interface of the endothelial surface layer and the vessel lumen, and it was calculated as 4.9686[Vb/d], where 4.9 is a mean empirical correction factor [40]. Parameters of leukocyte recruitment Quantitative analysis of leukocyte-endothelial cell interactions was performed off-line using CAPImageH. Rolling leukocytes were defined as those moving slower than the associated blood flow and quantified during 30 seconds. Leukocyte rolling flux fraction was determined from video recordings by counting all visible cells passing through a plane perpendicular to the vessel axis and dividing this number by the total leukocyte flux through the vessel, which can be estimated by the product of the systemic leukocyte count, mean blood flow velocity, and estimated vessel cross- sectional area. Firmly adherent cells were determined as those resting in the associated blood flow for more than 30 sec and related to the luminal surface per 100 mm vessel length. Emigrated cells were counted in regions of interests (ROIs) reaching out 75 mm to each side of a vessel over a distance of 100 mm vessel length and are presented per 104 mm2 tissue area. Single cell tracking of interstitially migrating leukocytes Intravital microscopic video recordings were transferred into a computer system using a frame grabber. Digital video sequences were analyzed using the imaging software ‘‘Simple PCI’’ (Hamamatsu Corporation/Compix Inc., Cranberry Twp, PA). On each side of analyzed vessel, at least 15 emigrated leukocytes were identified within ROIs and tracked in the perivascular space within a time period of 5 min. Parameters of leukocyte motility such as curve-line and straight-line velocities were automatically calculated by the software. Curve-line/straight-line velocities are the speeds along the curve-line distance or straight-line distance. Curve-line distance is a total (accumulated) distance presented as a line connecting the position of migrating leukocyte at each time point. Straight-line distance represents the shortest line connecting the start and end point of the leukocyte migration track. To quantify the directionality of migration, the chemotactic index PLoS ONE | www.plosone.org 10 March 2009 | Volume 4 | Issue 3 | e4693