Copyright @ 2006 by the Shock Society. Unauthorized reproduction of this article is prohibited. LEUKOCYTE-ENDOTHELIUM INTERACTIONS AFTER HEMORRHAGIC SHOCK/REPERFUSION AND CECAL LIGATION/PUNCTURE: AN INTRAVITAL MICROSCOPIC STUDY IN RAT MESENTERY Naomi Kondo Nakagawa,* † Rafael Aydar Nogueira,* Cristiano Jesus Correia,* Sı ´lvia Regina Shiwa, † Jose ´ Wa ´ lber Miranda Costa Cruz, ‡ Luiz Francisco Poli de Figueiredo,* Maurı ´cio Rocha e Silva,* and Paulina Sannomiya* *Research Division, Heart Institute (InCor), LIM 11, University of Sao Paulo Medical School, ‡ Institute of Biomedical Sciences, University of Sao Paulo, † UNICID, Sao Paulo, Brazil Received 19 Jan 2006; first review completed 3 Feb 2006; accepted in final form 2 Mar 2006 ABSTRACT—Hemorrhagic shock/reperfusion (HS/R) followed by sepsis triggers systemic microcirculatory disturbances that may induce multiple organ failure. The present study evaluated the effects of HS/R and cecal ligation and puncture, followed by necrotic cecal resection/peritoneal lavage (REL) on leukocyte-endothelium interactions at the mesentery. Eighty-one anesthetized Wistar rats (200Y250 g) were randomly assigned to a first injury: (1) control-HSVno hemorrhagic shock/no reperfusion group, (2) HS/bloodVHS/R with 25% shed blood, and (3) HS/blood + LRVHS/R with 25% of the shed blood + lactated Ringer’s solution, 3Â shed blood volume. Twenty-four hours post-HS/R, animals were submitted to cecal ligation and puncture and, 24 h thereafter, to REL. Leukocyte-endothelium interactions were assessed by intravital microscopy and intercellular adhesion molecule (ICAM) 1 and P-selectin expression by immunohistochemistry. Lungs were observed for ICAM-1 expression and neutrophil infiltration. Single and double injury induced significant increases in rolling ( ˜ 2-fold), adherent ( ˜ 5-fold), and migrated leukocytes ( ˜ 7-fold); ICAM-1 expression ( ˜ 1/2-fold), and P-selectin expression ( ˜ 1/2-fold) at the mesentery compared with control-HS group. REL normalized leukocyte-endothelium interactions at the mesentery in single-injured animals. However, in double-injured rats, adherence and migration of leukocytes decreased but did not normalize. Similar results were observed on ICAM-1 expression and neutrophil infiltration in the lungs from these animals. In conclusion, the current in vivo observation of the mesenteric microcirculation after a double injury followed by REL is a suitable model for the systematic evaluation of the inflammatory reaction at local and distant sites. In addition, data presented herein emphasized the importance of surgical removal of the septic focus in controlling the otherwise lethal sepsis-induced multiple organ dysfunction syndrome. KEYWORDS—Microcirculation, double injury, mesentery, lung, P-selectin, ICAM-1 INTRODUCTION Hemorrhagic shock/reperfusion (HS/R) and sepsis are high- risk factors for development of multiple organ failure, the major cause of death in emergency and critical care settings (1Y3). These events induce microvascular dysfunction involv- ing arterioles, capillaries, and venules, adversely affecting leukocyte-endothelium interactions, leukocytes accumulation, vascular autoregulatory mechanisms, and rheologic properties of blood, thereby causing heterogeneous flow distribution or/ and capillary obstruction, impairing oxygen diffusion, and producing mismatch between cellular oxygen need and oxygen supply (4 Y 6). HS/R and sepsis have been associated with deleterious functional and structural changes in many organs, particularly in the gastrointestinal tract (7, 8) and lungs (9, 10). Sakr et al. (5) have recently demonstrated that different patterns of microvascular dysfunctions in septic shock can characterize patients’ outcome. Therefore, microcirculation is an impor- tant Btarget organ^ in the pathophysiology of multiple organ failure (4 Y7). Leukocytes and the endothelium are believed to be key ef- fectors of the tissue damage in severe systemic inflammatory conditions that may herald the onset of multiple organ failure (9Y11). During HS/R and sepsis, mesenteric microcirculation becomes a priming bed for circulating neutrophils (7, 8). Leukocyte recruitment and accumulation can be triggered by a reduction in blood flow velocity, release of inflammatory mediators, and activation of leukocytes and the endothelium (12Y19). Leukocyte migration to a site of injury involves a series of leukocyte-endothelial interactions in postcapillary venules, mediated by the expression of adhesion molecules on the vascular endothelium and leukocytes. Rolling is the first step of leukocyte trafficking; and it is mediated by the E-, P-, and L-selectins. Activation of rolling leukocytes is associated with the upregulation of surface integrins and shedding of L-selectin. The increase in surface " 2 integrins induces a firm stationary bond (adherence) between the leukocyte and the endothelial cell by interacting with immunoglobulin super- family of intercellular adhesion molecules (ICAM-1), which are expressed on the endothelial surface. Subsequently, leu- kocytes eventually transmigrate into the interstitial compart- ment and toward the site of injury where degranulation occurs 180 SHOCK, Vol. 26, No. 2, pp. 180Y186, 2006 Address reprint requests to Naomi Kondo Nakagawa, PhD, University of Sao Paulo Medical School, Av. Dr. Arnaldo, 445-2- andar, sala 2148, Bairro de Cerqueira Ce ´sar- Sa ˜o Paulo-SP-Brazil, CEP: 01246-090. E-mail: naomikondo@uol.com.br. Supported by grants of PRONEX and FAPESP. Presented at (preliminary data) the 28th Annual Conference on Shock, 2005, Marco Island, FL. DOI: 10.1097/01.shk.0000223133.10254.82 Copyright Ó 2006 by the Shock Society