In Vivo Imaging and Quantitative Analysis of Leukocyte Directional Migration and Polarization in Inflamed Tissue Alexander Georg Khandoga1¤*, Andrej Khandoga2, Christoph Andreas Reichel1, Peter Bihari1, Markus Rehberg1, Fritz Krombach1 1 Walter Brendel Centre of Experimental Medicine, Ludwig-Maximilians-Universita¨t M u¨nchen, Munich, Germany, 2 Department of Surgery-Grosshadern, Ludwig- Maximilians-Universita¨t M u¨nchen, Munich, Germany Abstract Directional migration of transmigrated leukocytes to the site of injury is a central event in the inflammatory response. Here, we present an in vivo chemotaxis assay enabling the visualization and quantitative analysis of subtype-specific directional motility and polarization of leukocytes in their natural 3D microenvironment. Our technique comprises the combination of i) semi-automated in situ microinjection of chemoattractants or bacteria as local chemotactic stimulus, ii) in vivo near-infrared reflected-light oblique transillumination (RLOT) microscopy for the visualization of leukocyte motility and morphology, and iii) in vivo fluorescence microscopy for the visualization of different leukocyte subpopulations or fluorescence-labeled bacteria. Leukocyte motility parameters are quantified off-line in digitized video sequences using computer-assisted single cell tracking. Here, we show that perivenular microinjection of chemoattractants [macrophage inflammatory protein-1a (MIP-1a/Ccl3), platelet-activating factor (PAF)] or E. coli into the murine cremaster muscle induces target-oriented intravascular adhesion and transmigration as well as polarization and directional interstitial migration of leukocytes towards the locally administered stimuli. Moreover, we describe a crucial role of Rho kinase for the regulation of directional motility and polarization of transmigrated leukocytes in vivo. Finally, combining in vivo RLOT and fluorescence microscopy in Cx3CR1gfp/gfp mice (mice exhibiting green fluorescent protein-labeled monocytes), we are able to demonstrate differences in the migratory behavior of monocytes and neutrophils. Taken together, we propose a novel approach for investigating the mechanisms and spatiotemporal dynamics of subtype-specific motility and polarization of leukocytes during their directional interstitial migration in vivo. Citation: Khandoga AG, Khandoga A, Reichel CA, Bihari P, Rehberg M, et al. (2009) In Vivo Imaging and Quantitative Analysis of Leukocyte Directional Migration and Polarization in Inflamed Tissue. PLoS ONE 4(3): e4693. doi:10.1371/journal.pone.0004693 Editor: Howard E. Gendelman, University of Nebraska, United States of America Received November 26, 2008; Accepted January 23, 2009; Published March 4, 2009 Copyright: 2009 Khandoga et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by EU FP6 NoE ‘‘MAIN’’ (LSHG-CT-2003-502935). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: alexander.khandoga@med.uni-muenchen.de ¤ Current address: Deutsches Herzzentrum Mu¨ nchen, Technische Universita¨t M u¨nchen, Munich, Germany Introduction The multistep cascade of leukocyte migration into injured or inflamed tissue consists of sequential intercellular adhesion events with endothelial cells [1]. The mechanisms underlying these initial steps of leukocyte rolling and firm adhesion have been studied in detail [1,2,3]. In the last decade, new insights have been gained into signaling pathways triggering post-adhesion events such as leukocyte strength- ening and intraluminal crawling as well as into the mechanisms mediating leukocyte transendothelial migration (rev. in [2,3,4,5]). After having passed the perivenular basement membrane, transmigrated leukocytes are suggested to locomote within the interstitial tissue in a target-oriented manner [6,7]. However, the mechanisms underlying leukocyte directional migration within a (patho)physiological microenvironment remain largely unclear. Whilst 2-photon laser scanning microscopy enabled investigations on the dynamics of lymphocyte and dendritic cell migration in the lymph node [8], the majority of previously published studies on interstitial migration of inflammatory cells is based on in vitro studies in various 2- and 3-dimensional systems [9]. Thereby, the complex architecture of the interstitial tissue as well as the dramatic phenotypic and functional changes leukocytes undergo during their diapedesis are disregarded [4]. Moreover, directional migration of leukocytes in inflamed ‘‘non-lymphatic’’ tissue is poorly understood. The studying of leukocyte interstitial migration in ‘‘non-lymphatic’’ tissues, however, remains limited because of the induction of diffuse inflammation with ‘‘chemotactic chaos’’ in the interstitial tissue after usage of conventional routes of stimulation such as superfusion or intrascrotal injection of chemoattractants [10,11,12]. In addition, adequate in vivo models for evaluating leukocyte migration toward bacteria are still lacking. Here, we suggest perivenular microinjection of chemoattractants or bacteria into the murine M. cremaster using a microinjection technique in order to induce target-oriented leukocyte migration. Using in vivo near-infrared reflected-light oblique transillumination (RLOT) microscopy, we analyzed leukocyte adhesion, transmi- gration, and interstitial migration upon microinjection with relevant chemoattractants including MIP-1a, PAF, or fluores- cent-labeled E. coli. We compared the character of leukocyte migration after microinjection of chemoattractants with that induced by a conventional route of stimulation, intrascrotal injection of chemoattractant. Next, we proved our approach by PLoS ONE | www.plosone.org 1 March 2009 | Volume 4 | Issue 3 | e4693