Inhibition of haem oxygenase activity increases leukocyte accumulation in the liver following limb ischaemia-reperfusion in mice

Abstract

Although the consequences of trauma or infection are normally controlled at the site of injury, loss of local control may lead to a whole body response, which has been identified clinically as the systemic inflammatory response syndrome (SIRS). If this systemic inflammatory process involves whole body infection, the condition is termed sepsis. In its worst case, SIRS results in multiple organ dysfunction occurring in approximately 30 % of septic patients, in 24 % of patients suffering from pancreatitis, in over 30 % of trauma and in 40 % of burn patients (Beal & Cerra, 1994; Davies & Hagen, 1997).Liver injury and dysfunction occurring during SIRS has often been overshadowed by concerns regarding cardiac, renal and respiratory function. The fact that liver dysfunction plays a central role in remote injury during SIRS has been supported by studies identifying liver dysfunction as one of the major factors contributing to the mortality of surgical patients suffering from multiple organ dysfunction following infrarenal aortic reconstruction (Huber et al. 1995;Maziak et al. 1998). Currently, pharmacologic protocols and mechanical devices are available to support most vital organ functions but none exists for the liver. The role of haem oxygenase (HO) in the hepatic accumulation of leukocytes in mice during the initiation of remote organ injury following normotensive limb ischaemia-reperfusion (I-R) was investigated. Remote organ injury was initiated by 1 h bilateral hindlimb ischaemia followed by either 1 or 1.5 h reperfusion (I-R) in male C57BL/6 mice. Mice were randomly assigned to either sham (no I-R, n = 4), I-R (n = 4 for both time points), I-R plus chromium mesoporphyrin (CrMP, n = 4) to inhibit HO or I-R plus haemin (n = 4) to increase HO. Leukocyte accumulation and leukocyte-endothelial interaction were directly measured using fluorescence intravital microscopy. Leukocytes were labelled via an injection of rhodamine 6G. In sinusoids the total number and the number of stationary leukocytes were assessed. In postsinusoidal venules the number of adherent and rolling leukocytes and the velocities of both red blood cells and leukocytes were measured. The total number of leukocytes increased in sinusoids of I-R mice reaching a plateau within 1 h compared with sham animals, while the number of stationary leukocytes progressively increased over the entire study period. Stationary leukocytes in sinusoids increased after 1 and 1.5 h of I-R following CrMP, while they were significantly reduced following haemin treatment compared to animals treated with I-R only. In postsinusoidal venules a progressive increase in adherent leukocytes also occurred. As observed in sinusoids, CrMP significantly increased, while haemin significantly reduced leukocyte adhesion. The number of rolling leukocytes increased after CrMP in both I-R groups (1 and 1.5 h). The velocities of rolling leukocytes declined following 1.5 h of I-R compared with sham. Haemin treatment of 1.5 h I-R animals restored the velocities back to sham levels. The calcu...

Bucillamine improves hepatic microcirculation and reduces hepatocellular injury after liver warm ischaemia-reperfusion injury

“…Leukocyte–endothelial interactions were studied by labelling leukocytes in vivo using administering rhodamine 6G (Sigma, Rodermark, Germany) 0.3 mg/kg intra‐arterially 22 at 150 min post‐reperfusion. Ten randomly chosen post‐sinusoidal venules were visualized for 20 s each under green filtered light.…”

Carbon monoxide has antioxidative properties in the liver involving p38 MAP kinase pathway in a murine model of systemic inflammation

“…For the in vivo work we used a normotensive murine model of early systemic inflammation. The hindlimb I/R in this model results in a similar progression to that described for the systemic inflammatory response syndrome (SIRS): an initial spike of TNF‐α, followed by increased plasma concentrations of interleukin‐6 and systemic leukocytosis, hypotension (without fluid resuscitation), and other signs of systemic inflammation [12,13,26,27]. HUVEC stimulated with TNF‐α were used for the in vitro studies.…”

“…Mice ( n = 7/group) were randomly assigned to a naïve (no treatment), sham, or a hindlimb ischemia‐reperfusion (I/R) group. Mice in each of the I/R groups were treated with 60‐minutes bilateral hindlimb tourniquet ischemia as described previously [12–14]. Sham mice were not subjected to ischemia, but remained anesthetized for the same period of time.…”

“…Mice were anesthetized by inhalational isoflurane as described previously [13]. Details of the preparation for microscopy have been described previously [12]. An equilibration period of 10‐minutes was allowed before data collection.…”

Therapeutic roles of carbon monoxide in intestinal ischemia‐reperfusion injury

“…PMN recruitment to the affected organs is a complex multistep process and involves the activation of both vascular endothelium and leukocytes, with subsequent upregulation of the pro‐adhesive phenotype, which, in turn, results in the initiation of adhesive interactions (rolling, firm adhesion, and migration) between PMNs and vascular ECs . Originally, the induction of HO‐1 was shown to suppress both venular leukocyte adhesion elicited by oxidative stress and leukocyte accumulation in liver . Since then, CO inhalation has been shown to attenuate inflammatory responses via the inhibition of PMN accumulation .…”