An approach for studies of mediator‐induced leukocyte rolling in the undisturbed microcirculation of the rat mesentery

Abstract

1 Although intravital microscopy is the method of choice for observation of in¯ammatory leukocyte rolling and adhesion in small venules in vivo, a problem with this technique is that surgical exposure of suitable tissues per se triggers the rolling mechanism. In this study, we describe an approach to investigate induction of rolling in undisturbed microvessels. For this purpose, intravital microscopic observation of leukocyte rolling and adhesion in the rat mesentery was combined with histological determination of the intravascular concentrations of polymorphonuclear and mononuclear leukocytes (PMNL and MNL). 2 By relating the histologically determined number of intravascular leukocytes to either microvessel volume or to the erythrocyte concentration, the baseline MNL and PMNL content was found to be 3 ± 6 fold higher in venules than in systemic blood. This increase in microvessel leukocyte concentration did not seem to be related to leukocyte-endothelium interactions, because the leukocyte concentration was similarly elevated in arterioles where rolling and adhesion did not take place. 3 Preparation of the rat mesentery for intravital microscopy time-dependently increased the venular PMNL concentration to over 100 fold the systemic PMNL concentration 45 min after exteriorization of the small intestine. The MNLs were much less responsive to the preparative manipulation. By treatment with the polysaccharide fucoidin (inhibits rolling but not ®rm adhesion per se), or by use of intravital microscopy immediately before tissue ®xation, approximately 90% of the accumulated venular PMNLs were found to represent rolling cells. Intraperitoneal injection of 1073 M histamine increased the venular PMNL (but not the MNL) concentration to almost 50 fold the systemic PMNL value. The histamine response did not vary with venular diameter, and the relative contribution of rolling vs ®rmly adherent cells to the PMNL, accumulation was again &90%. Intraperitoneal injection of leukotriene C 4 , but not prostaglandin E 2 , caused a signi®cant increase in venular PMNL concentration. 5 Systemic treatment with the anti-P-selectin monoclonal antibody PB1.3 had no eect on the histamine-induced venular PMNL accumulation (i.e. rolling) in female Wistar or male Sprague-Dawley rats. On the other hand, identical treatment with PB1.3 very eectively inhibited the histamine-induced PMNL response in the mesentery of rabbits. 6 In conclusion, we have shown that a histologically determined increase in leukocyte concentration in rat mesenteric venules may be used as an index of mediator-induced leukocyte rolling if the relative contribution of rolling and ®rm leukocyte adhesion is ®rst determined, for example by the means described in this study. This relatively simple approach may be very useful for studying various aspects of leukocyte rolling when the`spontaneous' rolling triggered by preparation of tissues for intravital microscopy is undesirable.

Advances in small animal mesentery models for in vivo flow cytometry, dynamic microscopy, and drug screening

“…In mesenteric microvessels, the imaging of flowing cells in vivo without any staining or labeling was realized mainly with transmission microscopy for slow moving, rolling (30-70 µm/s), and adhesive WBCs [10,12,14,15,17] . The monitoring of single RBCs and their small aggregates has usually been performed in two modes: 1) in selected microvessels (small venules or capillaries) with slow flow using a frame rate of 20-30 frames per second (fps), or 2) using a short time-exposure mode (∼0.1-1 ms) by which only single images of fast-moving cells can be captured [56][57][58] .…”

“…Nevertheless, some methods used in most experiments have limitations. For example, the majority of the results about platelets [e.g., platelet thrombosis, or their interaction with white blood cells (WBCs) or vessel walls], red blood cells (RBCs), and tumor cells were obtained with what are currently the most powerful techniques, such as fluorescent labe ling [12,13,21,[41][42][43] . However, despite significant progress in the development of new labels [44][45][46] (e.g., quantum dots, fluorescent-specific antibodies [47] ), these techniques in vivo (as in many other experiments in vitro) are potentially subject to photobleaching (despite the short exposure time), or cytotoxicity.…”

Various adhesion molecules impair microvascular leukocyte kinetics in ventilator-induced lung injury

“…However, there has been no reliable study analyzing the contribution of lymphocytes to the initiation and/or aggravation of VILI. Lymphocytes may have a number of functions, including immune response, release of various cytokines, and cytotoxicity (36), all of which are expected to play a pivotal role in the development and pathogenesis of VILI. Although the recruitment of lymphocytes to inflammatory sites in systemic organs and tissues has been shown to be mediated in part through an adhesive mechanism involving ICAM-1-and/or VCAM-1-dependent pathways (2,34), the issue of whether the same holds true for lymphocyte recruitment to the microcirculation of the diseased lung has not been extensively addressed.…”

Microvascular Mechanisms of Polyphosphate-Induced Neutrophil-Endothelial Cell Interactions in vivo

“…1a). Surgical preparation of tissues for intravital microscopy is known to trigger leukocyte rolling [18]. Thus, as expected, local challenge with PolyPs did not significantly enhance the number of rolling leukocytes (Fig.…”