velocity and directionality on both vessel sides. In contrast, microinjection of MIP-1a, PAF, or E. coli induced leukocyte adhesion and transmigration preferentially on the ipsilateral vessel side. Moreover, upon microinjection of chemoattractants or bacteria, leukocyte straight-line velocity and directionality were several times higher on the vessel side ipsilateral to microinjection as compared to those on the contralateral side. In addition, leukocytes moving directly upon microinjection of chemoattrac- tants/bacteria were more polarized as compared to those after intrascrotal injection of PAF. Taken together, these findings demonstrate that microinjection of inflammatory mediators or bacteria induces directional leukocyte migration toward the chemotactic stimuli. Leukocyte motility was comparable in tissues stimulated with MIP-1a, PAF, or E. coli. These data support our previous observation that intrascrotal injection or superfusion of these mediators initiates a comparable extent of leukocyte recruitment and transmigration [19]. How could microinjection of chemoattractants/bacteria induce directional leukocyte migration? On the one hand, chemoattrac- tants administered via microinjection into a perivascular region of the interstitium may diffuse through the extracellular matrix and directly activate the endothelium on the ipsilateral vessel side via G-protein-coupled receptors (GPCRs) on endothelial cells. So called ‘interceptors’ such as DARC (Duffy antigen receptor for chemokines) and D6 have been shown to transport PAF or MIP- 1a and present them to the apical side of the endothelium [13,20]. Bacterial products, mainly N-formylpeptides, can also directly elicit chemotaxis by binding to GPCRs or N-formylpeptide receptor [21]. Alternatively, the endothelium might be stimulated via indirect mechanisms involving the release of mediators derived from cells in the interstitium (e.g., myocytes, fibroblasts, mast cells, smooth muscle cells). Interestingly, glycosaminoglycans have been shown to bind to chemokines and retain them locally in the interstitium, creating a chemotactic gradient and avoiding its rapid distribution in the tissue [22,23]. Next, we addressed the question of whether specific inhibition of Rho kinase with Y27632 would influence leukocyte interstitial migration in our in vivo assay. Rho kinase acts as a key mediator in cytoskeleton reorganization during leukocyte migration and is involved in T cell polarization [24], neutrophil motility [25], as well as chemoattractant-mediated actin assembly during neutro- phil chemotaxis [26]. In our study, inhibition of Rho kinase not only attenuated the directional movement and polarization of emigrated leukocytes upon microinjection of chemoattractant but also blocked common migration ability. Therefore, our study provides in vivo evidence that Rho kinase plays a critical role for the motility and polarization of emigrated leukocytes toward local chemokine stimulation and supports in vitro data from the literature [27,28]. However, several studies demonstrate that Y27632 induces lamellipodia protrusions in monocytes, inhibits tail retractions and has no effect on forward movement [29,30]. These controversial data can be explained by stimulus-, tissue-, or leukocyte subtype-specificity of Rho kinase activity. Finally, we combined in vivo RLOT microscopy with fluores- cence microscopy in order to analyze MCP-1-induced interstitial migration of monocytes and neutrophils in Cx3CR1gfp/gfp mice in which blood monocytes express GFP [31]. Since neutrophils comprise more than 85% of the leukocyte response to MCP-1 [32], we considered GFP-negative cells as neutrophils. We found that monocytes started their target-oriented interstitial migration later than neutrophils and migrated rather slower than neutro- phils. Interestingly, monocytes have been reported to move slower than neutrophils along a stable chemotactic gradient in vitro [33,34]. It seems possible that the ability of neutrophils for fast Leukocyte Migration In Vivo movement would enable them to accumulate more rapidly at the site of inflammation. Moreover, it has been shown that neutrophils produce chemotactic factors for monocytes under certain inflammatory conditions [35,36]. In conclusion, we have established an in vivo 3D leukocyte chemotaxis assay that opens new avenues for in vivo investigations on the mechanisms and spatiotemporal dynamics of target- oriented interstitial migration of single leukocytes. Materials and Methods Animals The experiments were performed on male C57BL/6 mice weighing 22 to 28 g (Charles River, Sulzfeld, Germany). For the analysis of migration patterns of monocytes in separate set of experiments, mice expressing green fluorescence protein (GFP) at the locus of the Cx3CR1 gene (Cx3cr1gfp/gfp) were obtained from European Mouse Mutant Archive (EMMA), Monterotondo, Italy. The animals had free access to tap water and pellet food. All experiments were performed according to German legislation on the protection of animals. Reagents and inhibitors Recombinant murine macrophage inflammatory protein-1a (MIP-1a/Ccl3) and monocyte chemotactic protein-1 (MCP-1/ Ccl2) were purchased from R&D SystemsH (Wiesbaden-Norden- stadt, Germany). Phospholipid platelet-activating factor (PAF), FITC-labeled E. coli particles, and Rho-kinase inhibitor Y27632 were purchased from Sigma Aldrich (Deisenhofen, Germany). Surgical preparation The surgical procedure was made as described in detail elsewhere with slight modifications [37]. Mice were anesthetized by an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). The left femoral artery was catheterized in a retrograde manner for the administration of microspheres to the cremasteric vasculature. The right cremaster muscle was exposed through a ventral incision of the scrotum. The muscle was opened ventrally in a relatively avascular zone, using careful electrocautery to stop any bleeding, and spread over the transparent pedestal of a custom-made microscopy stage. Epididymis and testicle were detached from the cremaster muscle and placed into the abdominal cavity. Throughout the surgical preparation and during in vivo microscopy, the muscle was superfused with warm buffered saline. Tissue temperature was kept at 37uC using a temperature probe (TFN1093, Ebro, Ingolstadt, Germany) throughout the entire experiment. Microinjection of inflammatory mediators and experimental protocol Leukocyte recruitment and interstitial migration were analyzed in the cremaster muscle after microinjection of 130630 pl of macrophage inflammatory protein-1a (MIP-1a; 250 nM), the phospholipid mediator platelet-activating factor (PAF; 100 nM), in murine serum opsonized fluorescent-labeled E. coli (approxim. 4000), or saline in perivascular regions at a distance of 25–50 mm from a postcapillary venule (6 animals in each group). Single unbranched venules with diameters ranging between 25 and 40 mm and lengths .150 mm were selected for this study. Microinjection was performed under control of the intravital microscope with a water immersion lens (46/NA 0.12, Leitz, Wetzlar, Germany) using a borosilicate micropipette (tip pressure of 120 hPa (2000 hPa for microinjection of E. coli) for 0.5 sec, tip diameter ,1 mm) connected to the injecting system involving a PLoS ONE | www.plosone.org 9 March 2009 | Volume 4 | Issue 3 | e4693