L. Szalay

Epr analysis reveals three tissues responding to endotoxin by increased formation of reactive oxygen and nitrogen species

The excessive formation of reactive oxygen and nitrogen species (RONS) in tissue has been implicated in the development of various diseases. In this study we adopted ex vivo low temperature EPR spectroscopy combined with spin trapping technique to measure local RONS levels in frozen tissue samples. CP-H (1-hydroxy-3-carboxy-pyrrolidine), a new nontoxic spin probe, was used to analyze RONS in vivo. In addition, nitrosyl complexes of hemoglobin were determined to trace nitric oxide released into blood. By this technique we found that RONS formation in tissue of control animals increased in the following order: liver < heart < brain < cerebellum < lung < muscle < blood < ileum < kidney < duodenum < jejunum. We also found that endotoxin challenge, which represents the most common model of septic shock, increased the formation of RONS in rat liver, heart, lung, and blood, but decreased RONS formation in jejunum. We did not find changes in RONS levels in other parts of gut, brain, skeletal muscles, and kidney. Scavenging of RONS by CP-H was accompanied by an increase in blood pressure, indicating that LPS-induced vasodilatation may be due to RONS, but not due to nitric oxide. Experiments with tissue homogenates incubated in vitro with CP-H showed that ONOO(-) and O(2)(*)(-), as well as other not identified RONS, are detectable by CP-H in tissue. In summary, low-temperature EPR combined with CP-H infusion allowed detection of local RONS formation in tissues. Increased formation of RONS in response to endotoxin challenge is organ specific.

Release of S100B differs during ischemia and reperfusion of the liver, the gut, and the kidney in rats

S100B, an acknowledged marker of brain damage, is increased post-traumatically in plasma. The aim of this study was to investigate the diagnostic value of S100B release in experimental local extracranial ischemia and reperfusion. Anesthetized rats underwent laparotomy and ligation of the afferent blood vessels to the liver, gut, or kidney to achieve local ischemia in each organ separately. After 60 min of ischemia, ligatures were removed and resuscitation was performed for 3 h. S100B was determined in plasma by immunoluminometric assay 55, 65, and 240 min after the onset of ischemia (5 min before reperfusion and 5 min and 3 h after the onset of reperfusion). During ischemia of the liver, S100B increased before ligature removal and reperfusion, reaching significance early after the onset of reperfusion and remaining almost unchanged throughout reperfusion. In contrast, S100B did not increase during ischemia of the gut or kidney before ligature removal or during early reperfusion but increased significantly to similar levels as during reperfusion of the liver 240 min after the onset of ischemia (after 3 h of reperfusion). Our findings show for the first time that S100B increases during local extracranial ischemia and reperfusion. These experimental findings support the concept that brain damage is not necessarily the cause of increased S100B. Although S100B has been an acknowledged marker of brain damage for years, our experimental clinically relevant data indicate that S100B is, in fact, not specific as a marker of brain damage in the setting of local ischemia and reperfusion of the liver, gut, and kidney because local ischemia and reperfusion of these organs cause an S100B increase per se.

Hemorrhagic shock induces an S 100 B increase associated with shock severity.

S 100 B is a glial marker of cerebral injury. In a previous clinical study, we found an S 100 B increase within the first 24 h in patients with multiple trauma and hemorrhagic shock but without cerebral trauma. The aim of our current experimental study was to determine whether this posttraumatic S 100 B increase is caused by extracerebral soft tissue injury or by hemorrhagic shock and whether it is associated with the severity of hemorrhagic shock. Hemorrhagic shock was achieved by bleeding anesthetized rats to a mean arterial pressure (MAP) of 30–35 mmHg through a femoral catheter and maintaining this MAP until incipient decompensation. At incipient decompensation, MAP was either increased immediately to 40–45 mmHg (moderate shock) or was maintained until 40% of shed blood had been returned (severe shock), and then increased to 40–45 mmHg. Resuscitation was provided after 40–45 mmHg MAP had been maintained for 40 min. Soft tissue injury was achieved by midline laparotomy performed at the onset of hemorrhagic shock or without shock and was maintained for 30 min. Hemorrhagic shock caused an early S 100 B increase at the onset of decompensation. S 100 B remained increased for 24 h and was significantly higher after severe than after moderate shock. In contrast, soft tissue injury without hemorrhagic shock caused no S 100 B increase. The data presented demonstrate for the first time that the S 100 B increase is induced by hemorrhagic shock and is associated with the severity of shock.

Increased plasma D-lactate is associated with the severity of hemorrhagic/traumatic shock in rats.

D-lactate is produced by indigenous bacteria in the gastrointestinal tract. Mammals do not have the enzyme systems to metabolize D-lactate rapidly. The present study was designed to determine the kinetics of circulating D-lactate levels and to examine whether the severity of shock affects circulating D-lactate levels in rats subjected to hemorrhagic/traumatic shock. Anesthetized rats underwent midline laparotomy (duration 30 min) and were bled to 30–35 mmHg mean arterial pressure (MAP). After the onset of decompensation, MAP was either increased to 40–45 mmHg immediately by administration of Ringers solution (moderate shock) or after 40% of shed blood volume had been re-infused as Ringers solution (severe shock). MAP was then maintained at 40–45 mmHg for 40 min by further administration of Ringers solution (inadequate resuscitation). Subsequently, adequate resuscitation was performed for 60 min with shed blood and additional Ringers solution. Metabolic acidosis was significantly more pronounced in severe than in moderate hemorrhagic/traumatic shock. Plasma D-lactate levels were already significantly increased at the end of severe hemorrhagic/traumatic shock and remained high during inadequate resuscitation. D-lactate levels were significantly higher after severe than after moderate shock. Endotoxin levels did not correlate with shock severity. Damage to the intestinal mucosa was more profound in severe shock than in moderate shock. Our data suggest that hemorrhagic/traumatic shock is associated with mucosal damage and increased plasma D-lactate levels. The severity of shock affects D-lactate concentrations in plasma. Plasma D-lactate may be a useful marker of intestinal injury after hemorrhagic/traumatic shock.

Prevention of early myocardial depression in hyperdynamic endotoxemia in dogs

In our study the pathomechanism of sepsis-induced early myocardial depression was investigated. We determined the effects of the inducible nitric oxide synthase inhibitor and free radical scavenger mercaptoethylguanidine (MEG) on the myocardial contractility, the endothelial and inducible nitric oxide synthase (eNOS and iNOS) activities, and the activation and tissue accumulation of polymorphonuclear leukocytes in hyperdynamic endotoxemia in dogs. Group 1 served as endotoxemic control. Mean arterial pressure and cardiac output were measured, myocardial contractility was estimated from the end-systolic pressure-diameter relationship. The eNOS, iNOS and myeloperoxidase activities were determined on myocardial biopsy samples, and the free radical-producing capacity of granulocytes was measured from separated cells. The effect of MEG on the in vitro free radical production of isolated granulocytes was measured by chemiluminometry. Endotoxin induced a hyperdynamic circulatory reaction and significant myocardial depression. The myocardial eNOS activity was significantly increased 4 h after induction of endotoxemia and remained elevated, the iNOS activity was increased only 8 h after endotoxemia induction. The free radical-producing capacity and the myocardial accumulation of the granulocytes were significantly increased. In group 2, MEG treatment selectively inhibited the iNOS activity, prolonged the hyperdynamic circulatory reaction, prevented myocardial depression and decreased the activation and tissue accumulation of granulocytes. The compound dose-dependently decreased the in vitro activation of previously resting granulocytes. Our study demonstrates that iNOS do not contribute to the early cardiac failure in endotoxemia. MEG selectively inhibits iNOS in vivo, but its beneficial effects are rather related to the decreases in leukocyte and free radical-mediated myocardial dysfunction during early endotoxemia.

Endothelin-A receptor antagonism improves small bowel graft perfusion and structure after ischemia and reperfusion.

BACKGROUND We hypothesized that endothelin-A (ET-A) receptor activation plays a central role in intestinal ischemia-reperfusion-induced hemodynamic changes and may trigger the process of mucosal barrier destruction. Our aims were to investigate and compare the effects of systemic and intragraft ET-A receptor antagonist therapy during the early revascularization phase of small bowel transplants. METHODS In Groups 1, 2, and 3 orthotopic small bowel autotransplants were performed in anesthetized dogs. Group 4 served as sham-operated control. Group 2 was treated i.v. with the ET-A receptor antagonist ETR-p1/fl peptide at the onset of reperfusion. In Group 3, intragraft infusion of the ETR-p1/fl peptide was applied during cold ischemia. The mucosal myeloperoxidase activity and the free radical-producing capacity of the granulocytes passing the intestinal graft were determined, and the systemic hemodynamic features were recorded. The extent of the mucosal injury was determined from tissue biopsies taken after 4 hr of reperfusion. RESULTS Reperfusion progressively decreased the mesenteric blood flow, increased the mesenteric vascular resistance, and enhanced the accumulation and free radical production capacity of the leukocytes. These changes were significantly inhibited in Group 2 with systemic (i.v.) administration of the ET-A receptor antagonist. The local, intragraft treatment improved the mesenteric hemodynamic changes and decreased the accumulation but not the activation of the circulating leukocytes. The structural injury of the graft was prevented in both treated groups. CONCLUSIONS Endothelins are involved in the hemodynamic events leading to structural injury of the intestinal graft after ischemia-reperfusion. The antagonism of intestinal ET-A receptors by a combination of local and systemic drug delivery offers a rational treatment modality in these conditions.

The role of endothelin-1 in circulatory changes during hypodynamic sepsis in the rat

Our objective was to investigate the significance of endogenous endothelin-1-induced systemic circulatory reactions during hypodynamic sepsis. In the first part of this study, we observed the changes in global hemodynamic parameters in Wistar rats after exogenous endothelin-1 administration in order to test an intervention strategy aimed at preventing the development of hypodynamic cardiovascular derangement during intraabdominal sepsis. Cardiac output, mean arterial blood pressure, and peripheral vascular resistance were recorded, and the endothelin-A receptor antagonist BQ-610 and the endothelin-B receptor antagonist IRL-1038 were used to investigate the role of receptor subtypes in circulatory changes. In addition, the effects of treatment with the novel endothelin-A receptor inhibitor ETR-P1/fl peptide were examined in endothelin-1-treated anesthetized rats. The injection of 1 nmol/kg endothelin-1 induced a significant rise in peripheral vascular resistance, a transient increase in mean arterial pressure, and a decrease in cardiac output. Administration of the endothelin-A receptor antagonist BQ-610 and ETR-P1/fl peptide increased cardiac output and decreased systemic vascular resistance in the controls and in animals treated with exogenous endothelin. In the second part of the study, the animals were instrumented for hemodynamic monitoring and randomized to undergo cecal ligation and perforation for 8 h or control laparotomy. Septic animals with cecal ligation and puncture were normotensive and hypodynamic, with a significantly increased total peripheral resistance throughout the 8 h observation period. ETR-P1/fl peptide treatment started after the induction of sepsis significantly increased cardiac output and decreased systemic vascular resistance almost to control levels. We conclude that endogenous endothelin-1 contributes significantly to the systemic hemodynamic alterations during hypodynamic circulatory response, and the inhibition of endothelin-A receptors may improve global hemodynamic status in this phase of sepsis.

Endothelin-1 induces mucosal mast cell degranulation in the rat small intestine

The enhanced production of endothelial cell-derived vasoactive mediators and the activation of mast cells (MCs) have been implicated in the pathogenesis of mucosal damage during ischemia and reperfusion injuries. The first objective of our study was to define the in vivo relation between endothelin-1 (ET-1) and the MC system. Secondly, we determined whether pretreatment with ET receptor antagonists would attenuate MC responses to exogenous ET-1. In the first series of experiments, increasing doses of ET-1 (0. 1, 1 and 3 nmol/kg i.v.) were administered to anesthetized rats. In the second series, the animals were pretreated with equimolar doses of the ET-A receptor antagonist BQ-610 or ETR-P1/fl peptide, and the ET-B receptor antagonist IRL-1038. Intestinal perfusion changes and macrohemodynamics were recorded, and the proportion of degranulated MCs was determined in ileal biopsies. The average mucosal thickness was recorded with an image analysis system. ET-1 induced dose-dependent alterations in the hemodynamic and morphological parameters and caused pronounced mucosal injury, with a significant reduction in villus height. The ratio of degranulated MCs was similar in all ET-treated groups (77%, 82% and 86%) to that observed in animals subjected to 15-min ischemia and 60-min reperfusion (85% degranulation). Pretreatment with BQ-610 and ETR-P1/fl peptide attenuated the ET-1 induced alterations in the hemodynamic parameters and decreased structural injury to the mucosa. ET-induced MC degranulation was significantly inhibited by the ET-A receptor antagonists, but not by IRL-1038. These results indicate that elevated levels of circulating ET-1 might induce intestinal mucosal tissue injury and MC degranulation via activation of ET-A receptors, and raise the possibility that ET-A receptor antagonist administration could exert a potentially beneficial effect through a mechanism other than the blockade of vasoconstriction in pathologies associated with an increased ET-1 release.

A Role of Ppar-γ in Androstenediol-mediated Salutary Effects on Cardiac Function Following Trauma-hemorrhage

Objective:To examine the mechanism by which androstenediol improves cardiac function following trauma-hemorrhage (T-H). Summary Background Data:Androstenediol administration improves cardiovascular function and attenuates proinflammatory cytokine production following T-H. Activation of the peroxisome proliferator-activated receptor-gamma (PPAR-γ) has been shown to be protective following ischemic conditions. We hypothesized that PPAR-γ activation plays a role in the androstenediol-mediated salutary effects on cardiac function following T-H. Methods:Male rats underwent laparotomy and hemorrhagic shock (40 mm Hg for 90 minutes), followed by resuscitation with 4 times the shed blood volume in the form of Ringers lactate. Androstenediol (1 mg/kg body weight, i.v.) was administrated at the end of resuscitation. In a separate group of animals, a PPAR-γ antagonist (GW9662) was administered simultaneously with androstenediol and animals were killed at 5 hours thereafter. Results:A decrease in cardiac function and an increase in IL-6 and iNOS gene expression were observed following T-H. Androstenediol treatment normalized cardiac function, increased PPAR-γ DNA binding activity, attenuated IL-6 and iNOS gene expressions, and reduced plasma IL-6. Plasma 15-deoxy-Δ12, 14-prostaglandin J2 (PGJ2, an endogenous PPAR-γ agonist) levels were also increased in androstenediol-treated T-H rats, but these levels were lower than those observed in shams. Coadministration of PPAR-γ antagonist along with androstenediol, however, prevented the androstenediol-mediated reduction in cardiac iNOS and IL-6 expressions and abolished the improvements in cardiac function. Conclusion:The androstenediol-mediated salutary effects on cardiac function following T-H appear to be mediated at least in part via PPAR-γ activation, which down-regulates IL-6 and iNOS gene expression in the heart.

Dynamic in vivo observation of villus microcirculation during small bowel autotransplantation: Effects of endothelin-A receptor inhibition

Background. The aims of this study were to characterize the structural and microcirculatory changes in single intestinal villi during ischemia and reperfusion and determine the site of action of endothelin (ET)-A receptor inhibition during compromised mucosal perfusion. Methods. Small bowel autotransplantation was performed in anesthetized dogs. One group was treated with the ET-A receptor antagonist ETR-p1/fl peptide. The epithelial thickness and villus microcirculatory parameters were observed by orthogonal polarization spectral imaging; the leukocyte-endothelial cell interactions were quantified with fluorescence videomicroscopy. Results. Sixty-minute cold ischemia and 240-min reperfusion induced a decrease in villus functional capillary density and leukocyte-endothelial cell interactions. The epithelial layer was gradually removed, but denuded villi were not observed. ET-A receptor inhibition reduced the leukocyte adherence and attenuated epithelial exfoliation and the decrease in villus functional capillary density. Conclusions. ET-A receptor activation mediates microvascular dysfunction through precapillary blockades and leukocyte-endothelial cell interactions after cold ischemia and reperfusion in the canine small bowel.