The role of endothelin peptides was evaluated on survival and organ injury in a model of polymicrobial sepsis, induced by caecal ligation and puncture with particular emphasis on the timing of the administration of its blocker bosentan in Swiss albino mice (20–40 g). The cardiovascular response pattern in this experimental model was characterized by an early, “hyperdynamic” phase starting at 5 h, followed by a late but “hypodynamic” phase that commence after 20 h, provided that the animals are “resuscitated” by injecting 1 ml of saline i.p. at the end of the surgery. However, if saline resuscitation is omitted, then only hypodynamic pattern is observed starting at 5 h without any hyperdynamic phase. Thus, mice were first allocated into saline-resuscitated or unresuscitated groups and endothelin receptor antagonist bosentan (30 mg kg−1, i.p., either 5 or 20 h after caecal ligation and puncture) was then administered. The control animals received the solvent of bosentan (i.e., saline: %0.9 NaCl, w/v). The survival rates in each group (n=14) were recorded over the following 144 h. In unresuscitated mice, the overall survival at 144 h was 14.3% in controls while bosentan treatment at 5 h (78.6%, P=0.0018) or 20 h (64.3%, P=0.0183) have both significantly improved the survival. However, in saline-resuscitated mice, bosentan administered at 20 h has significantly improved the survival (71.4%, P=0.0213) while its administration at 5 h has yielded exactly the same percent of survival (i.e., 21.4%) as observed in control animals. The beneficial effects of bosentan in preventing the tissue injury due to caecal ligation and puncture were also observed histopathologically in liver, spleen and kidney. Therefore, we concluded that the blockade of endothelin receptors by using bosentan during the later (hypodynamic) stages of septic shock is a promising therapeutic manoeuvre.

Despite important advances in understanding its pathophysiology, therapy for septic shock remains largely symptomatic and supportive. Aiming to elevate the systemic arterial blood pressure by using vasoconstrictor manoeuvers are preferred without paying much attention to the ischaemia produced at the peripheral tissues. Since, these maneuvers proved no remarkable success in reducing the mortality up to date, we now propose a different perspective in this manuscript. Although it is not always easy to distinguish the different phases of septic shock, at least two fundamentally different phases can be distinguished, i.e. (i) hyperdynamic phase and (ii) hypodynamic phase mandating the adoption of vasodilative and vasoconstrictive interventions, consequently. Additionally, endothelium-derived vasodilator and vasoconstrictor substances such as nitric oxide and endothelin play key roles in systemic inflammatory response syndrome that lead to fatal multiple organ dysfunction. Therefore, we hypothesize that the inhibition of nitric oxide production during earlier phases of septic shock combined with the blockade of endothelin receptors at later stages appear feasible and a novel strategy for the therapy of septic shock.

Background. Circulatory failure in multiple organ dysfunction syndromes (MODS) is characterized with systemic vasodilation, diminished blood flow to various vascular beds. The aim of this study was to investigate the effects of selective inhibition of nitric oxide on the mesenteric arterial blood flow (MABF), survival and organ injury of the liver, kidney, lung and spleen in zymosan-induced MODS.Materials and methods. Forty Swiss albino mice (20-40 g), 7 to 9 weeks old, were obtained. Animals were randomly divided into four groups. The first group were treated intraperitoneally (i.p) with vehicle (saline) and served as a sham group for aminoguanidine (AG) (n = 10). The second group was treated with zymosan (500 mg/kg, suspended in saline solution, i.p). The mice in the third and fourth group received AG (15 mg/kg) 1 h and 6 h after zymosan or saline administration, respectively. Eighteen hours after the administration of zymosan, animals were assessed for MODS described subsequently. The signals from the flowmeter were also recorded on mesenteric arterial blood flow values.Results. In zymosan-treated animals, the MABF was significantly lower than that of solvent (saline)treated controls (ml min(-1), controls: 4.6 +/- 0.6; zymosan: 1.6 +/- 0.9, P < 0.05). When animals were treated with AG, there were no significant differences in MABF values between AG group and solvent (saline)-treated control group. However AG prevented zymosan-induced mesenteric MABF decrease. Treatment with aminoguanidine also decreased mortality.Conclusion. AG is capable of inhibiting both the induction and the activity of the already iNOS; it remains a potential therapeutic agent in patients with MODS. (c) 2005 Elsevier Inc. All rights reserved.

We investigated the contribution of NO-cyclic GMP (cGMP) pathway to the antinociceptive effects of ketamine in mice by using the nitric oxide synthase inhibitor, nitro(g)- L-arginine methyl ester (L-NAME). Intraperitoneal (i.p.) (1, 5 or 10 mg/kg) or intrathecal (i.th.) (10, 30 or 60 microg/mouse) administration of ketamine produced dose-dependent antinociceptive effects in the acetic acid-induced writhing and formalin tests but not in the tail-flick nor in hot-plate tests. Pretreatment of mice with L-NAME (10 mg/kg, i.p.) which produced no antinociception on its own, significantly inhibited the antinociceptive effect of ketamine (1, 5 or 10 mg/kg, i.p.). However, L-NAME (30 microg/mouse) was given intrathecally, it neither modified the antinociceptive effect of i.th. ketamine (10, 30 or 60 microg/mouse) nor did it produce an antinociceptive effect alone. These data suggest that the activation of the NO-cGMP pathway probably at the supraspinal level, but not spinal level, contributes to the antinociceptive effects of ketamine.

The association between Escherichia coli endotoxin-induced organ damage and nitric oxide-related mechanisms was investigated in the spleen of male Swiss albino mice (20-40 g) by using (1) Pt/Ir electrochemical sensor connected to an amperometric detection system (NO-501, InterMedical Co., Japan), (2) nitrotyrosine immunohistochemistry, (3) conventional light microscopy and (4) immunoblotting techniques in parallel. 1 h before endotoxin injection, animals were pretreated with either nitric oxide synthase inhibitor, L-N(G)-nitroarginine methyl ester (L-NAME, 20 mg kg(-1), i.p.) or inducible nitric oxide synthase expression inhibitor, dexamethasone (5 mg kg(-1), i.p.) or the inhibitor of murine inducible nitric oxide synthase in vivo, 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT, 1 mg kg(-1), i.p.). 5 h after endotoxin treatment, electrochemically detected concentration of nitric oxide was significantly elevated (nM, endotoxin: 716.6 +/- 178.2, n = 10 vs saline: 209.4 +/- 127.8, n = 9, P = 0.0312, unpaired Student's t-test) and remained so throughout the 30 min monitorization period. Neither dexamethasone nor AMT blocked the endotoxin-induced overproduction of nitric oxide indicating that the enhanced inducible nitric oxide synthase activity cannot be the only explanation. When dexamethasone and L-NAME combination was used to block both the constitutive and the inducible isoforms, nitric oxide production was virtually abolished, indicating a significant contribution from the constitutive isoform of nitric oxide synthase. The results of nitrotyrosine immunohistochemistry and the conventional light microscopy were also in agreement with the amperometric method while immunoblotting revealed the expression of both the endothelial and the inducible isoforms of nitric oxide synthase were induced endotoxaemic animals. Thus, conclude that endotoxin-induced splenic damage in endotoxaemia can be explained by enhanced production of nitric oxide due to the induction of both endothelial and inducible nitric oxide synthases while causal relationship and the roles of other deleterious mediators such as oxygen-derived free radicals are yet to be established.

The aim of this study was to investigate the effects of inducible nitric oxide synthase inhibition by aminoguanidine on endotoxin‐induced reduction in mesenteric blood flow.

Burn injury causes mesenteric vasoconstriction and bacterial translocation. Since catecholamines are powerful vasoconstrictors and elevated immediately after burn injury, we hypothesised that adrenaline tolerance might decrease burn-induced mesenteric vasoconstriction and bacterial translocation. Adrenaline tolerance was developed in Swiss albino mice. Adrenaline tolerant and control animals were subdivided into sham-burn and burn subgroups. 24 h after sham-burn or burn injury, specimens were obtained for microbiological evaluation. Also, in a separate group of adrenaline tolerant and control animals, superior mesenteric blood flow was measured. Burn injury increased bacterial translocation rate in both control (P = 0.001) and adrenaline tolerant groups (P = 0.0351). The caecal bacterial level increase was significant after burn injury in control groups (P = 0.0004) but was not significant in adrenaline tolerant animals (P = 0.743). Mesenteric blood flow was decreased significantly by burn injury in both control and adrenaline tolerant animals (P < 0.00001). The results showed that catecholamines do not mediate postburn mesenteric vasoconstriction or bacterial translocation.

OBJECTIVE To investigate the effect of various doses of melatonin on reduction in mesenteric blood flow (MBF) and increase in tumour necrosis factor alpha (TNFalpha) concentration caused by injection of lipopolysaccharide (LPS). DESIGN University Hospital, Turkey. SETTING Open experimental study. ANIMALS 59 Swiss albino mice. INTERVENTIONS Animals were injected with melatonin solvent or 1, 10, 100, or 500 mg/kg melatonin. Ten minutes later control animals were injected with saline, and the experimental group with LPS. MAIN OUTCOME MEASURES Mesenteric blood flow and serum TNFalpha concentration. RESULTS In control animals, 100 and 500 mg/kg melatonin reduced MBF. LPS reduced MBF in solvent, 1, and 10 mg/kg melatonin groups. The concentration of TNFalpha was considerably increased in the mice given LPS. Melatonin reduced this response significantly. CONCLUSION In high doses melatonin directly reduces MBF. It has no protective effect on the LPS-induced decrease in MBF. In lower doses it blocks, but at higher doses reduces, LPS-induced TNFalpha production.

LPS/endotoxin provokes a plethora of pathological events some of which may be considered as examples of "low perfusion state". These are discussed here. It is well known that hypotension and refractoriness to vasocostrictors are the hallmark of endotoxic shock. Nevertheless, there are some vascular beds, such as mesenteric circulation, that respond with vasoconstriction - not vasodilation to endotoxin. Aminoguanidine, an inhibitor of NOS-2, blocks endotoxin- induced increase of resistance in mesenteric bed and endotoxin-induced translocation of bacteria through the gut wall. It is postulatede that endotoxin has antiarrythmogenic action due to the release of nitric oxide and increase in intracellular cGMP levels. Although we demonstrate that endotoxin increases nitric oxide formation in spleen and liver, its contribution to the injury of these organs by endotoxin is not fully established. In addition, we present our immunochemistry data on nitrotyrosine formation in the liver and spleen of endotoxin-treated animals.

The modulatory effects of a non-selective endothelin receptor antagonist, bosentan, were investigated together with those of relatively selective inducible nitric oxide synthase inhibitors, aminoguanidine and l-canavanine, on mesenteric blood flow decrease, liver and spleen injury elicited by endotoxaemia. Swiss albino mice (20–40 g) were administered intraperitoneally bosentan (3, 10 or 30 mg kg−1), aminoguanidine (15 mg kg−1) or l-canavanine (20 or 100 mg kg−1) 10 min before they received saline or Escherichia coli endotoxin (10 mg kg−1). After 4 h, the mice were anaesthetized, mesenteric blood flow values were measured, spleen and liver weight/body weight ratios were determined and the organs were examined histopathologically. Endotoxin decreased mesenteric blood flow (ml min−1, saline: 3.0±0.2; endotoxin: 2.2±0.2; n=10, P<0.05), increased the weight of liver (g per kg body weight, saline: 47.5±2.0; endotoxin: 60.8±1.9; n=10, P<0.05) and spleen (g per kg body weight, saline: 3.9±0.5; endotoxin: 8.6±0.9; n=10, P<0.01) while it inflicted significant histopathological injury to both organs. Bosentan was ineffective at 3 mg kg−1 but at 10 and 30 mg kg−1 doses, it abolished all the deleterious effects of endotoxin without exception. Aminoguanidine blocked most of the effects of endotoxin except those on spleen. In contrast, l-canavanine blocked only the endotoxin-induced increase in liver weight but itself increased spleen weight and failed to block any other effects of endotoxin. Thus, it can be speculated that the beneficial effects of aminoguanidine are produced largely by mechanisms other than selective inducible nitric oxide synthase inhibition since l-canavanine was not fully effective. The beneficial effects of endothelin inhibition by using bosentan in endotoxaemia can be further exploited for the understanding and the therapy of sepsis-related syndromes.

BACKGROUND:Endotoxic shock is associated with release of catecholamines as well as decreased mesenteric vascular perfusion, which is thought to cause remote organ injury. Adrenaline tolerance was reported to decrease mortality in endotoxic shock and have cross-tolerance with endotoxin tolerance. Our aim was to investigate the effect of these two tolerance conditions on the lipopolysaccharide (LPS)-induced decrease in mesenteric blood flow (MBF).METHODS:Adrenaline tolerance was developed by injecting 0.03 mg/kg adrenaline to Swiss-albino mice, gradually increasing the dose to 2 mg/kg over 5 days. Endotoxin tolerance was developed by injecting saline for 4 days and LPS 1 mg/kg at the fifth day. Control animals were injected with saline for 5 days. At 72 h after completion of injections, half of the animals in each group were challenged with saline and the other half with 20 mg/kg LPS, at 0 h. Mesenteric blood flow was measured at 4 and 24 h.RESULTS:Neither endotoxin nor adrenaline tolerance prevented an LPS-induced decrease in MBF.CONCLUSION:A low dose of LPS prior to a higher dose does not prevent an LPS-induced decrease in MBF and may actually prime for a decrease. Also, catecholamines are not primary mediators of LPS-induced decreases in MBF.

The inhibitory effect of the selective M3 musarinic acetylcholine receptor antagonist, 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP, 0.1–10 μM) on nicotine (100 μM)-induced nitrergic relaxation was investigated in comparison to d-tubocurarine (0.1–10 μM) and hexamethonium (0.1–10 μM) by using phenylephrine (1 μM)-precontracted rat anococcygeus muscles in vitro. Nicotine produced a 60.1± 2.4% (n = 40) inhibition of phenylephrine precontractions. But this relaxant response was at a significantly lower magnitude of 20.2± 4.6% (n = 18, P < 0.01 vs. control) in the presence of the nitric oxide synthase (NOS) blocker NG-nitro-L-arginine methyl ester (L-NAME, 30 μM), and it was 26.5± 5.5% (n = 8, P < 0.01 vs. control) in the presence of the soluble guanylate cyclase inhibitor methylene blue (30 μM). However, aminoguanidine (100 μM), a relatively selective blocker of the inducible nitric oxide synthase (iNOS), had no significant effect. Similarly, other iNOS inhibitors such as dexamethasone (5 mg/kg) or L-canavanine (100 mg/kg) did not modify contractile nor relaxant responses when they were given in vivo, concomitantly with Escherichia coli endotoxin (1 mg/kg, ip) 4 h before the isolation of the tissues. 4-DAMP, hexamethonium, and d-tubocurarine inhibited nicotine-induced relaxation in a concentration-dependent manner with the following order of potency: 4-DAMP > hexamethonium > d-tubocurarine with IC50 values being 0.47± 0.04 μM, 0.75± 0.06 μM, and 1.02± 0.05 μM, respectively. Therefore, it was concluded that the selective M3 muscarinic acetylcholine receptor antagonist 4-DAMP also possesses potent antagonistic action on nicotinic receptors of peripheral nitrergic neurons that innervate the rat anococcygeus muscle. Drug Dev. Res. 46:148–154, 1999. © 1999 Wiley-Liss, Inc.

BACKGROUND:Bacterial translocation is thought to be responsible for infectious complications after hemorrhagic shock. The aim of this study is to investigate the effects of granulocyte colony-stimulating factor (G-CSF) treatment on bacterial translocation in starved or fed animals subjected to hemorrhagic shock.MATERIALS AND METHODS:Fifty Wistar albino rats (200-275 g) were divided into six groups such as naive control (n = 7), G-CSF treatment (n = 7), hemorrhagic shock in starved rats (n = 9), hemorrhagic shock in fed rats (n = 9), G-CSF treatment 24 h before hemorrhagic shock in starved rats (n = 9), and G-CSF treatment 20 min after hemorrhagic shock in fed rats (n = 9). Hemorrhagic shock was induced by withdrawal of 2.1 ml/100 g blood via a carotid arterial cannulae placed under sodium pentobarbital anesthesia. Twenty-four hours later, mesenteric lymph nodes, liver, spleen, and peripheral blood samples were evaluated by using a quantitative microbiological technique and the numbers of colony-forming units were compared between groups.RESULTS:No bacteria was detected in samples from naive controls or G-CSF-treated unshocked rats. In animals subjected to hemorrhage, Escherichia coli was the predominant pathogen together with Streptococcus faecalis, Pseudomonas, and Lactobacillus species. In this model, starvation augmented the magnitude of bacterial translocation while G-CSF treatment has virtually abolished it.CONCLUSION:Under experimental conditions, preshock starvation increases gut-derived bacterial translocation and administration of G-CSF before or after hemorrhagic insult significantly reduces it.

Background The role of inducible nitric oxide synthase (iNOS) in endotoxin-induced bacterial translocation was investigated by using its specific blocker aminoguanidine in 46 albino mice (25-35 g) allocated into four groups.Methods The first group received intraperitoneal saline (control; 0.9 per cent w v(-1) sodium chloride 1 ml kg(-1); n = 6), the second group intraperitoneal endotoxin (Escherichia coli lipopolysaccharide 055:B5 20 mg kg(-1); n = 19), the third group intraperitoneal aminoguanidine (20 mg kg(-1), 20 min before and 12 h after saline; n = 6) and the fourth group both endotoxin and aminoguanidine intraperitoneally (n = 15). Some 24 h later, the animals were anaesthetized with ether and blood samples were collected by cardiac puncture together with mesenteric lymph node (MLN), spleen and liver specimens under aseptic conditions. Specimens were then cultured to determine the presence of colony-forming units as an index of bacterial translocation.Results No bacterial growth was detected in samples from the first and third groups. Colony-forming bacteria were found in ten of 14 MLN samples, eight of 14 spleens, four of 14 livers and three of 14 peripheral blood samples in the second group, with E. coli being the predominant pathogen. In contrast, in the fourth group, colony-forming bacteria were found in only three of 14 MLN samples (P = 0.02 versus the second group), three of 14 spleens and one of 14 liver specimens. None of the values in the fourth group was significantly different from those in the saline control group.Conclusion The inhibition of iNOS during endotoxaemia by its specific blocker aminoguanidine attenuates the incidence of bacterial translocation in mice. These results may be exploited clinically for the prophylaxis and treatment of septic states.

Contralateral testicular perfusion during unilateral testicular torsion was evaluated using simultaneous blood flow and O2 content determinations. Two groups, each consisting of 7 rats, were studied. Sham operation or 720 degrees clockwise twisting was performed on the left testes, and blood flow, O2 content and temperatures were monitored in the right testes for 180 min. An ultrasonic perivascular Doppler flowmeter system, an electronic thermometer and an O2 electrode were used for the monitoring. The contralateral testicular blood flow and relative O2 contents were stable in the control group. However, the initial and 180 min blood flow values decreased from 0.21 +/- 0.04 to 0.11 +/- 0.02 ml/min (p < 0.001), and the O2 contents from 0.857 +/- 0.123 to 0.319 +/- 0.037 (1.0 corresponds to 19.6 mm Hg pO2, p < 0.05) in the experimental group. Unilateral testicular torsion decreases the blood flow and O2 content of the contralateral testis. The contralateral testicular injury encountered following unilateral testicular torsion might result from hypoxia following the decrease in blood flow.

The role of inducible nitric oxide synthase in the antiarrhythmic effects of Escherichia coli endotoxin was examined in an anaesthetised rat model of myocardial ischaemia (7 min occlusion) and reperfusion (7 min) arrhythmias by using its specific blocker L-canavanine (100 mg/kg) and dexamethasone (5 mg/kg), which inhibits its expression. Endotoxin(1 mg/kg) or its solvent saline was administered intraperitoneally 4 h before the occlusion of the left coronary artery and L-canavanine or dexamethasone was administered 1 h before endotoxin or saline injection. The mean arterial blood pressure of rats receiving endotoxin was significantly lower than that of saline-treated controls, and neither L-canavanine nor dexamethasone prevented the hypotension exerted by endotoxin. However, during both the occlusion and reperfusion periods, endotoxin significantly reduced the total number of ectopic beats (e.g., during reperfusion, saline: 1177 +/- 183, n = 11. endotoxin: 248 +/- 91, n = 9; P < 0.005) and the duration of ventricular tachycardia (e.g., during occlusion, saline: 30.9 +/- 5.7 s; endotoxin: 1.8 +/- 0.9 s; P < 0.0001) while L-canavanine or dexamethasone treatment abolished the reduction exerted by endotoxin. Therefore we conclude that endotoxin possesses significant antiarrhythmic (protectant) effects in this rat model of ischaemia-reperfusion arrhythmias, and that its mechanism appears to involve the inducible nitric oxide synthase since both L-canavanine and dexamethasone inhibited this phenomenon.

The effects of acyl-coenzyme a: lysolecithin acyltransferase (LAT) inhibitor thimerosal (20, 30 and 40 mg kg-1), anti-ischaemic and free radical scavenging drug trimetazidine (10 mg kg-1) and endothelin ETA-ETB receptor blocker bosentan [30 mg kg-1) were investigated in an anaesthetized rat model of coronary artery ligation (7 min) and release (7 min) induced myocardial ischaemia-reperfusion arrhythmias. Neither of the drugs produced significant effects on blood pressure except thimerosal which induced a transient fall lasting 5-8 min. The total number of ectopic beats during occlusion (controls 141.8 +/- 73.5, n = 12) and reperfusion (controls 1151.1 +/- 199.1, n = 10) was not modified by either trimetazidine (occlusion 452.5 +/- 128.6, n = 10; reperfusion 1002.0 +/- 198.4, n = 6) or bosentan (occlusion 431.2 +/- 112.8, n = 10; reperfusion 1530.7 +/- 296.8, n = 9) while thimerosal attenuated them (occlusion 50.2 +/- 14.9, n = 10, P < 0.01 vs controls; reperfusion 345.1 +/- 83.8, n = 8, P < 0.01 vs controls). The durations (in seconds) of ventricular tachycardia (occlusion 33.9 +/- 6.1, n = 12; reperfusion 94.2 +/- 18.1, n = 10) were also shortened by thimerosal (occlusion 2.6 +/- 1.0, n = 10, P < 0.01; reperfusion 28.5 +/- 7.8, n = 8, P < 0.01) while trimetazidine or bosentan did not significantly modify them. The incidences of ventricular fibrillation or mortality were not significantly modified by either of the drugs. Besides the lack of any effect of trimetazidine, we conclude that endogenous endothelin has no pathophysiological significance in this experimental model while LAT inhibition does.

The difference between the responses of phenylephrine (1 microM)-precontracted vascular (endothelium-denuded rat or rabbit aortic strips) and nonvascular (rat anococcygeus muscle) smooth muscles to acetylcholine (0.1-100 microM) was investigated when they were mounted co-axially inside the tracheas isolated from normal or ovalbumin-sensitized guinea-pigs. Acetylcholine produced concentration-dependent relaxations in both types of bioassay tissues. These relaxations, previously shown to be due to the release of airway epithelium-derived relaxing factor(s), were significantly attenuated when the epithelial layer of the tracheas was removed mechanically (as confirmed by histological examination). There were no significant differences in responsiveness to acetylcholine between vascular strips mounted inside the epithelium-intact normal or sensitized tracheas. The phenylephrine-induced precontraction was significantly more pronounced in rat anococcygeus muscles mounted inside sensitized tracheas as compared to tissues mounted inside control tracheas. The acetylcholine-induced relaxations were significantly decreased but this effect disappeared when the concentration of phenylephrine was reduced to obtain a similar precontraction level as in tissues mounted inside control tracheas. The responsiveness of both vascular strips and anococcygeus muscles to acetylcholine was attenuated when they were mounted inside sensitized tracheas and incubated with ovalbumin for 20 min, which may be explained by the epithelial damage induced by ovalbumin challenge. This attenuation was absent when co-axial pairs, utilizing normal tracheas, were used. These results indicate a difference in response patterns of the rat anococcygeus muscle and vascular strips in ovalbumin-sensitized tracheas, which should be taken into consideration in co-axial bioassay studies.