the analysis of the role of Rho kinase for leukocyte motility and polarization in vivo. Finally, we combined in vivo RLOT and fluorescence microscopy in order to evaluate migration patterns of neutrophils and monocytes in Cx3CR1gfp/gfp mice. Results Determination of the optimal distance for microinjection of chemoattractants In order to establish an optimal protocol for the perivenular microinjection of chemoattractants, we first analyzed the extent of local inflammation in the cremasteric tissue after microinjection of the chemokine MIP-1a performed at three different distances from a venule: 25–50 mm, 75–100 mm, and 175–200 mm. Sixty minutes after microinjection of MIP-1a, leukocyte adhesion and transmi- gration were analyzed. The highest number of adherent and transmigrated leukocytes was found when the microinjection was performed at a distance of 25–50 mm (Fig. 1). By contrast, the lowest numbers were measured after microinjection performed at a distance of 175–200 mm. Therefore, these data show that for the microinjection of chemoattractants a distance of 25–50 mm from the postcapillary venule is optimal, since the inflammatory response is stronger than after microinjections at the two longer distances analyzed. Consequently, microinjection was performed at a distance of 25–50 mm from the postcapillary venule under investigation in all further experiments. Tissue distribution of rhodamine 6G after microinjection In a next step, we sought to evaluate how chemoattractants are distributed within the cremaster tissue after microinjection. In an attempt to answer this question, microinjection (25–50 mm from the venule) of the fluorescent dye rhodamine 6G was performed. Alterations of fluorescence intensity of rhodamine 6G were analyzed within a time period of 60 min in three ROIs (100675 mm): 1) on the vessel side ipsilateral to the microinjection site, 2) on the contralateral side, and 3) at a distance of 350 mm from the venule (considered as background; Fig. 2B). At baseline conditions prior to microinjection, mean gray values on both the ipsi- and the contralateral side did not differ from background levels (Fig. 2D). Immediately after microinjection, fluorescence intensity was dramatically increased on the vessel side ipsilateral to the microinjection site as compared to baseline levels (Fig. 2A, D). The fluorescence intensity of rhodamine 6G decreased within 60 min after microinjection on the ipsilateral side; however, its levels remained higher in comparison to background values as well as the values measured on the contralateral side (Fig. 2D). Forty minutes after microinjection of rhodamine 6G, the fluorescent dye reached the contralateral vessel side as indicated by a slight elevation of mean gray values (Fig. 2C, D). Hence, these data suggest that microinjection of chemoattractants forms a stable source of chemoattractant in the perivenular region of cremaster muscle with slow distribution in the interstitium during 60 min. Leukocyte adhesion and transmigration In this part of the study, leukocyte adhesion and transmigration were analyzed after microinjection of the chemokine MIP-1a, the phospholipid PAF, and E. coli particles. In an additional group, intrascrotal injection of PAF was performed. Leukocyte adhesion and transmigration were observed on the vessel sides ipsi- and contralateral to the microinjection site (Fig. 3). The data show that the numbers of adherent and transmigrated leukocytes were dramatically increased on both vessel sides upon microinjection of MIP-1a, PAF, and E. coli particles at 60 min after microinjection as compared to microinjection of saline (Fig. 3). The extent of leukocyte adhesion and transmigration Leukocyte Migration In Vivo Figure 1. Dependency of leukocyte adhesion and transmigra- tion on the distance of microinjection from the vessel. Leukocyte adhesion (A) and transmigration (B) were analyzed 60 min after microinjection of MIP-1a performed at distances of 25–50, 75–100, and 175–200 mm from the postcapillary venule. Both parameters were observed in ROIs (100650 mm) along the venule (mean6SEM; n = 3). doi:10.1371/journal.pone.0004693.g001 did not significantly differ between the groups undergoing microin- jection of MIP-1a, PAF, and E. coli (Fig. 3A, B). Next, we compared leukocyte adhesion and transmigration on the vessel side ipsilateral to the microinjection site with those on the contralateral side (Fig. 3). Upon microinjection of the inflammatory mediators as well as bacteria, 65–70% of all adherent and extravasated leukocytes were localized on the ipsilateral vessel side (Fig. 3A, B, C, E). In contrast, intrascrotal microinjection of PAF was associated with a diffuse character of leukocyte adhesion and transmigration as shown by comparable amounts of adherent and transmigrated leukocytes on both the ipsi- and contralateral vessel sides (Fig. 3A, B, D, F). Leukocyte interstitial migration Motility of single interstitially migrating leukocytes was analyzed after microinjection of MIP-1a, PAF, or E. coli as well as after PLoS ONE | www.plosone.org 2 March 2009 | Volume 4 | Issue 3 | e4693