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      Visualization of Leukocyte Transendothelial and Interstitial Migration Using Reflected Light Oblique Transillumination in Intravital Video Microscopy

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          Dynamic visualization of the intravascular events leading to the extravasation of leukocytes into tissues by intravital microscopy has significantly expanded our understanding of the underlying molecular processes. In contrast, the detailed observation of leukocyte transendothelial and interstitial migration in vivo has been hampered by the poor image contrast of cells within turbid media that is obtainable by conventional brightfield microscopy. Here we present a microscopic method, termed reflected light oblique transillumination microscopy, that makes use of the optical interference phenomena generated by oblique transillumination to visualize subtle gradients of refractive indices within tissues for enhanced image contrast. Using the mouse cremaster muscle, we demonstrate that this technique makes possible the reliable quantification of extravasated leukocytes as well as the characterization of morphological phenomena of leukocyte transendothelial and interstitial migration.

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          Most cited references 8

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          T-cell function and migration. Two sides of the same coin.

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            An open cremaster muscle preparation for the study of blood vessels by in vivo microscopy.

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              Absence of trauma-induced leukocyte rolling in mice deficient in both P- selectin and intercellular adhesion molecule 1

              Leukocyte recruitment during inflammation is achieved through a multistep paradigm that includes margination, selectin-mediated rolling, beta 2 integrin-mediated firm adhesion, emigration, and migration into the site of inflammation. We have used the mouse cremaster muscle as a model of trauma- and cytokine-induced inflammation to study the possible role of intercellular adhesion molecule (ICAM) 1 in leukocyte rolling using gene-targeted mice deficient in ICAM-1, P-selectin, and a combination of P-selectin and ICAM-1. Rolling flux and average leukocyte rolling velocity in ICAM-1- deficient mice was not different from wild-type mice, but P- selectin/ICAM-1-deficient mice showed a total absence of rolling for at least 2 h after surgical trauma. Rolling in both wild-type and ICAM-1- deficient mice 60-120 min after trauma was significantly inhibited by a P-selectin monoclonal antibody (mAb) (RB40.34). In contrast, an mAb (KAT-1) blocking ICAM-1 binding to leukocyte function-associated antigen 1 did not block residual rolling in P-selectin-deficient mice. TNF-alpha induced leukocyte rolling in P-selectin/ICAM-1-deficient mice, but the rolling flux fraction was significantly lower than in TNF- alpha-treated ICAM-1-deficient mice. Leukocyte rolling in P- selectin/ICAM-1-deficient mice treated with TNF-alpha for 3 h was completely blocked by an E-selectin mAb (9A9E3), and partially by an L- selectin mAb (MEL-14). This clearly demonstrates E-selectin-dependent rolling in vivo. Leukocyte rolling velocities were significantly reduced after TNF-alpha treatment and were similar in wild-type and gene-targeted strains. We conclude that the residual trauma-induced leukocyte rolling seen in P-selectin-deficient mice is completely abolished by concomitant ICAM-1 deficiency. This severe defect in leukocyte rolling may explain the absence of leukocyte recruitment into the inflamed peritoneal cavity of P-selectin/ICAM-1-deficient mice at early time points (< or = 4 h).

                Author and article information

                J Vasc Res
                Journal of Vascular Research
                S. Karger AG
                October 2003
                03 October 2003
                : 40
                : 5
                : 435-441
                Institute for Surgical Research, University of Munich, Munich, Germany
                73902 J Vasc Res 2003;40:435–441
                © 2003 S. Karger AG, Basel

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                Figures: 4, References: 20, Pages: 7
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