[Exogenous nitric oxide donor in the liver inflammatory and hemodynamic response after hemorrhagic shock]. / Donador exógeno de óxido nítrico en la respuesta inflamatoria hepática y hemodinámica después de choque hemorrágico.

BACKGROUND: Hemorrhagic shock (HS) results in oxidative stress to cells and in the induction of the inflammatory response, with an increased expression of a number of proinflammatory mediators and cytokines. We tested the ability of the nitric oxide (NO) donor sodium nitroprusside (NP) to reduce tissue injury in a rodent model of uncontrolled hemorrhagic shock. METHODS: Seventy two Sprague Dawley rats weighing 250-300 g were subjected to a model of uncontrolled hemorrhagic shock. Four groups of animals were included (n = 18 per group): sham/saline, sham/NP, shock/saline, shock/NP. Experimental design consisted of the development of hemorrhagic shock (3 ml/100 g) in a 15-min period, tail amputation (75%) and drug administration at 30 min, fluid resuscitation (FR) with Ringer's lactate (RL) solution to reach a mean arterial pressure (MAP) of 40 mmHg, a hospital phase of 60 min with hemostasis and FR with LR solution to reach a MAP of 70 mmHg, and a 3-day observation phase. Treatment at the beginning of resuscitation included either normal saline (groups 1, 3) or NP (0.5 mg/kg) (groups 2, 4). The following parameters were evaluated: fluid requirements for resuscitation, liver injury tests, liver tissue myeloperoxidase (MPO), liver histology, and 3-day survival. RESULTS: NP significantly reduced fluid requirements for resuscitation (p = 0.0001). We also observed an improved statistically significant difference in tests demonstrating hepatic injury (p = 0.0001), neutrophil infiltration as evidences by liver MPO (p <0.05), and histology studies (p = 0.001). Survival was also increased from 40% in controls to 60% with NP treatment. CONCLUSIONS: These data suggest that excess NO mediates hemorrhage-induced liver injury, and that the suppression of NO with NP may reduce the pathological consequences of severe hemorrhage, possibly by scavenging superoxide (O(2)(-)), thus limiting the production of more aggressive radicals.

Donador exógeno de óxido nítrico en la respuesta inflamatoria hepática y hemodinámica después de choque hemorrágico / Exogenous nitric oxide donor in the liver inflammatory and hemodynamic response after hemorrhagic shock

BACKGROUND: Hemorrhagic shock (HS) results in oxidative stress to cells and in the induction of the inflammatory response, with an increased expression of a number of proinflammatory mediators and cytokines. We tested the ability of the nitric oxide (NO) donor sodium nitroprusside (NP) to reduce tissue injury in a rodent model of uncontrolled hemorrhagic shock. METHODS: Seventy two Sprague Dawley rats weighing 250-300 g were subjected to a model of uncontrolled hemorrhagic shock. Four groups of animals were included (n = 18 per group): sham/saline, sham/NP, shock/saline, shock/NP. Experimental design consisted of the development of hemorrhagic shock (3 ml/100 g) in a 15-min period, tail amputation (75%) and drug administration at 30 min, fluid resuscitation (FR) with Ringer's lactate (RL) solution to reach a mean arterial pressure (MAP) of 40 mmHg, a hospital phase of 60 min with hemostasis and FR with LR solution to reach a MAP of 70 mmHg, and a 3-day observation phase. Treatment at the beginning of resuscitation included either normal saline (groups 1, 3) or NP (0.5 mg/kg) (groups 2, 4). The following parameters were evaluated: fluid requirements for resuscitation, liver injury tests, liver tissue myeloperoxidase (MPO), liver histology, and 3-day survival. RESULTS: NP significantly reduced fluid requirements for resuscitation (p = 0.0001). We also observed an improved statistically significant difference in tests demonstrating hepatic injury (p = 0.0001), neutrophil infiltration as evidences by liver MPO (p <0.05), and histology studies (p = 0.001). Survival was also increased from 40% in controls to 60% with NP treatment. CONCLUSIONS: These data suggest that excess NO mediates hemorrhage-induced liver injury, and that the suppression of NO with NP may reduce the pathological consequences of severe hemorrhage, possibly by scavenging superoxide (O(2)(-)), thus limiting the production of more aggressive radicals.

Evaluation of endotoxin models for the study of sepsis.

Many strategies have been proposed for the treatment of sepsis, and most of the proposed treatment modalities have failed in clinical trials. Many of the previous treatment protocols called for blocking the activity of a single, clearly defined mediator. The underlying hypothesis was that sepsis induced a specific mediator that then caused organ injury and death. This simple, linear reasoning was frequently based on cytokines that were defined using endotoxin models of sepsis. The endotoxin models were widely used to study the pathophysiology of sepsis and were felt to adequately reproduce the full spectrum of inflammatory changes observed in patients with sepsis. Based on mortality and hematologic changes, these assumptions appeared justified. As the models were examined more closely, and directly compared with focus of infection models that more accurately portray the changes in sepsis, it became apparent that the endotoxin models did not accurately mimic the patient with sepsis. In the endotoxin models, the explosive release of cytokines into the circulating blood volume was reproducibly found regardless of the species studied (human, primate, pig, rat, or mouse). This lead to a series of anticytokine sepsis trials, all of which failed. The cytokine response in focus of infection models, such as that induced by cecal ligation and puncture, was examined and found to be more similar to that observed in patients with sepsis. When cytokine inhibitor strategies were used in the cecal ligation and puncture model, they were also generally found to lack efficacy. Compounds that have been shown to be effective at reducing mortality in endotoxin models should be re-evaluated in more clinically relevant models of sepsis.

The attenuation of hemorrhage-induced liver injury by exogenous nitric oxide, L-arginine, and inhibition of inducible nitric oxide synthase.

We investigated the role of nitric oxide (NO) in its ability to reduce liver injury in an animal model of hemorrhagic shock (HS). Ninety-six Sprague-Dawley rats weighing 250 to 300 g were divided in 6 groups (n = 16 per group) that included treatment at the beginning of resuscitation with normal saline (groups 1, 3) sodium nitroprusside (NP) (0.5 mg/kg) (groups 2, 4) L-arginine (300 mg/kg) (group 5), and L-N6-(1-iminoethyl) lysine (L-NIL, 40 mg/kg) (group 6). The experimental model of HS consisted of the withdrawal of 3 mL blood per 100 g in a 15-min period, tail amputation (75%), and drug administration at 30 min. This was followed by fluid resuscitation (FR) with lactated Ringer's (LR) solution to reach a mean arterial pressure (MAP) of 40 mm Hg, then a hospital phase of 60 min with hemostasis and FR with LR solution to reach a MAP of 70 mm Hg with a 3-day observation phase. NP, L-Arginine, and L-NIL significantly reduced fluid requirements for resuscitation (p =.0001) as well as significantly increased MAP after resuscitation from hemorrhage. We also observed an improved statistically significant difference (p =.001) in tests demonstrating less hepatic injury and histology damage. The mRNA expression of cytokines in the liver (interleukin [IL]-1alpha, IL-beta1, tumor necrosis factor [TNF]beta, IL-3, IL-4, IL-5, IL-6, IL-10, TNFalpha, IL-2, interferon [IFN]gamma) was reduced by NP treatment, L-arginine, and L-NIL. These data suggest that excess NO mediates hemorrhage-induced liver injury and that the suppression of inducible nitric oxide synthase (iNOS)-generated NO bioavailability with the NO donor sodium nitroprusside may reduce the pathophysiologic consequences of severe hemorrhage. This effect could be possibly related to the scavenging of to superoxide radicals (O2-) or the blockade of the deleterious effects of TNF and other inflammatory cytokines. The protective action noted with L-arginine cannot be fully explained within the context of this article, although it could be most likely associated with the supplementation of eNOS-generated NO.

Expression and function of C5a receptor in mouse microvascular endothelial cells.

The complement-derived anaphylatoxin, C5a, is a potent phlogistic molecule that mediates its effects by binding to C5a receptor (C5aR; CD88). We now demonstrate specific binding of radiolabeled recombinant mouse C5a to mouse dermal microvascular endothelial cells (MDMEC) with a K(d50) of 3.6 nM and to approximately 15,000-20,000 receptors/cell. Recombinant mC5a competed effectively with binding of [(125)I]rmC5a to MDMEC. Enhanced binding of C5a occurred, as well as increased mRNA for C5aR, after in vitro exposure of MDMEC to LPS, IFN-gamma, or IL-6 in a time- and dose-dependent manner. By confocal microscopy, C5aR could be detected on surfaces of MDMEC using anti-C5aR Ab. In vitro expression of macrophage inflammatory protein-2 (MIP-2) and monocyte chemoattractant protein-1 (MCP-1) by MDMEC was also measured. Exposure of MDMEC to C5a or IL-6 did not result in changes in MIP-2 or MCP-1 production, but initial exposure of MDMEC to IL-6, followed by exposure to C5a, resulted in significantly enhanced production of MIP-2 and MCP-1 (but not TNF-alpha and MIP-1alpha). Although LPS or IFN-gamma alone induced some release of MCP-1 and MIP-2, pre-exposure of these monolayers to LPS or IFN-gamma, followed by addition of C5a, resulted in synergistic production of MIP-2 and MCP-1. Following i.v. infusion of LPS into mice, up-regulation of C5aR occurred in the capillary endothelium of mouse lung, as determined by immunostaining. These results support the hypothesis that C5aR expression on MDMEC and on the microvascular endothelium of lung can be up-regulated, suggesting that C5a in the co-presence of additional agonists may mediate pro-inflammatory effects of endothelial cells.