Figure 6. Effect of Rho kinase inhibition on leukocyte motility. Leukocyte curve-line velocity (A), straight-line velocity (B), and directionality (C) were analyzed in the cremaster muscle at 60 min after microinjection of MIP-1a (black bars) followed by superfusion of the Rho kinase inhibitor Y27632 (gray bars) for either 0–5 min or 30– 35 min. In control experiments (black bars), the cremaster muscle was superfused with saline upon microinjection of MIP-1a; mean6SEM; *p,0.05 vs. control, # p,0.05 vs. control at 60–65 min; n = 15. doi:10.1371/journal.pone.0004693.g006 Leukocyte Migration In Vivo Microhemodynamic parameters and systemic leukocyte counts To assure intergroup comparability, diameters of analyzed microvessels, centerline blood flow velocity, wall shear rate, and systemic leukocyte counts were measured. No significant differ- ences were detected among the experimental groups (data not shown). Discussion The mechanisms triggering interstitial migration of extravasated leukocytes remain poorly investigated. The vast majority of studies on leukocyte directional migration are performed in vitro. However, the mechanisms underlying leukocyte migration differ between in vitro and in vivo settings [4,13]. In vitro studies do not take into account the phenotypic and functional changes of leukocytes that result from their interactions with endothelial cells and basement membrane during adhesion and transendothelial migration. These changes include increased leukocyte polariza- tion, phagocytosis, release of mediators, enhanced survival as well as upregulation of neutrophil elastase, matrix-metalloproteinases, integrins, etc. [4]. In addition, the pro-emigratory action of chemokines in vivo is dramatically different from their capacity to induce chemotaxis in vitro [13]. Although 2D substrates were preferentially used for in vitro studies on leukocyte chemotaxis, the mechanisms mediating this process seem to be rather different in 2D vs. 3D settings [9,14]. In contrast to 2D migration, the 3D tissue network confines and mechanically anchors cells from all sides so that they intercalate alongside and perpendicular to tissue structures [14]. In this context, 2D but not 3D leukocyte migration seems to be integrin-dependent [9,14]. In the present study, we have designed an approach allowing in vivo imaging and quantitative analysis of directional subtype-specific leukocyte migration and polarization in inflamed tissue. Our technique comprises the combination of in vivo near-infrared RLOT [10] and fluorescence microscopy in the murine cremaster muscle with a microinjection technique for induction of directional leukocyte migration. Although in chemotaxis assays in vitro the mechanisms of leukocyte migration are investigated in the focus of target-oriented cell movement towards a distinct chemoattractant, the study of leukocyte interstitial migration in vivo is limited because of induction of diffuse inflammation in the interstitial tissue [10,11]. An assay allowing the analysis of leukocyte chemotaxis in vivo in the murine cremaster muscle has been already described in the literature. It is based on the slow release of chemokines from an agarose gel placed on the interstitial tissue adjacent to a postcapillary venule [15]. A potential disadvantage of this method is that it seems to not consider the distribution of a chemoat- tractant in the 3D tissue, since the stimulus is applied on the tissue’s surface but not in the interstitial milieu. Moreover, although leukocyte chemotaxis towards bacteria in vitro and bacterial clearance assays in vivo are well described in the literature [16,6,17], there is still no in vivo approach allowing the analysis of leukocyte directional interstitial migration towards applied bacte- ria. As a solution, we suggest here to use perivenular microinjec- tion of relevant chemoattractants or bacteria via a microinjection technique. A similar experimental design was previously used in the cremaster model for local administration of chemokines for studying the mechanisms mediating leukocyte rolling and adhesion in vivo [18]. Here, we used perivenular microinjection of chemoattractants or bacteria in order to investigate leukocyte interstitial migration. We assume that microinjection of a chemoattractant or bacteria into the interstitial tissue would PLoS ONE | www.plosone.org 6 March 2009 | Volume 4 | Issue 3 | e4693