Figure 2. Tissue distribution of rhodamine 6G after microin- jection. A-C: In vivo microscopy images show tissue distribution of rhodamine 6G at 1 (A), 30 (B), and 60 min after microinjection (C). Three regions of interests 100675 mm (depicted in B) within the cremasteric interstitial tissue were analyzed: on the vessel side ipsilateral to the microinjection site, on the contralateral side, and in the interstitial tissue at a distance of 350 mm from the site of microinjection (considered as background). Images are shown with different original color scales to emphasize the fluorescent intensity within 60 min after microinjection. The fluorescence intensity was determined within 60 min; the quantitative data are presented in D; n = 7. doi:10.1371/journal.pone.0004693.g002 Leukocyte Migration In Vivo intrascrotal injection of PAF. In additional experiments, leukocyte interstitial migration was also studied upon intrascrotal injection of MIP-1a. Microinjection of the inflammatory mediators and bacteria induced interstitial migration of leukocytes (Fig. 4Aa–d; Fig. 5A, B, C). On the ipsilateral side, leukocyte interstitial migration was target-oriented towards the site of microinjection (Fig. 4Aa–d) and characterized by significantly increased curve-/ straight-line migration velocity and directionality as compared to leukocytes migrating in saline-treated cremaster muscles or to leukocytes migrating on the contralateral vessel side (Fig. 5A, B, C). Curve-line velocity, a measure of leukocyte migration velocity independent of cell directionality, was just slightly increased upon microinjection of chemoattractants or E. coli. By contrast, the effect of microinjection was stronger at the level of straight-line velocity and directionality, parameters that demonstrate how far and how directly leukocytes move toward the stimuli within the time period analyzed. The target-oriented character of interstitial leukocyte migration after microinjection of chemoattractants or bacteria is underlined by the finding that the elevation of straight- line migration velocity and directionality was several times higher than the increase in curve-line velocity (Fig. 5A, B, C). It is worth to be noted that leukocyte motility did not significantly differ between the groups receiving MIP-1a, PAF or E. coli in almost all migration parameters, with exception of curve-line migration velocity, which was significantly higher upon microinjection of MIP-1a as compared to that after microinjection of PAF. On the contralateral vessel side, however, the differences in the migration parameters between animals undergoing microinjection and animals from the control group were very weak. Interestingly, in contrast to microinjection, leukocyte interstitial migration was rather random upon intrascrotal injection of PAF (Fig. 4Ba–d) or MIP-1a (Fig. 4Ca–d) as shown by significantly lower straight-line migration velocity and directionality (Fig. 5B, C). Effect of the Rho kinase inhibitor Y-27632 on leukocyte motility In a separate set of experiments, the effect of the Rho kinase inhibitor Y-27632 on leukocyte motility was analyzed at 60 and 90 min after perivenular microinjection of MIP-1a. After 5 min of superfusion of Y-27632, motility of transmigrated leukocytes was significantly reduced by approximately 45% as compared to controls (Fig. 6) and completely abolished after 30 min of exposure (Fig. 4Da–d; Fig. 6). Morphological changes and polarization of interstitially migrating leukocytes In this part of the study, leukocyte morphological changes and polarization were evaluated after microinjection of chemoattrac- tants or bacteria as well as upon intrascrotal injection of PAF and after Rho kinase inhibition with Y27632. Upon microinjection of MIP-a, PAF, or E. coli particles, leukocytes moved toward the applied chemoattractant or bacteria and formed ruffles (Fig. 7). Then, leukocytes adopted an elongated polarized shape change with a contracted tail and lamellipodia protrusions at the front edge (Fig. 7A). Similar shape changes were observed in interstitially migrating leukocytes moving randomly in animals receiving the chemoattractant via intrascrotal injection (Fig. 7B). After application of the Rho kinase inhibitor, however, intersti- tially migrating leukocytes lost their ability to locomote toward the applied chemoattractant (Fig. 7C). Here, leukocytes became less elongated and more spherical, non-polar with single small protrusions. After microinjection of MIP-a, PAF, or E. coli, leukocytes become strongly polarized with an eccentricity of about PLoS ONE | www.plosone.org 3 March 2009 | Volume 4 | Issue 3 | e4693