Y. Gundersen

The nitric oxide donor sodium nitroprusside protects against hepatic microcirculatory dysfunction in early endotoxaemia.

Objective: Endotoxin rapidly inhibits the activity of the constitutive endothelial nitric oxide synthase (ecNOS); this precedes the production of NO from inducible NOS (iNOS). This leaves a period in early endotoxaemia with a supposed scarcity of NO. The present study was conducted to examine the effects of external supplementation of NO on liver microcirculation and function. Material: 13 male Sprague Dawley rats. Interventions: The rats underwent laparotomy, and the left liver lobe was exteriorised. All animals were given a bolus dose of endotoxin (LPS) 5 mg/kg intraportally. One group (n = 6) had a continuous infusion of sodium nitroprusside (SNP) 1.4 μg/kg per min started concurrently, the other group (n = 7) was treated with normal saline. The study was terminated after 3 h LPS. Measurements and results: Intravital microscopy was performed at baseline, at 2 h and 3 h LPS. Hepatic function was assessed by arterial ketone body ratio, acid base values, and bile flow. At baseline 1 % of the sinusoids were without perfusion. After 2 h LPS this figure had risen to 9.8 ± 1.5 % in the SNP group versus 16.9 ± 1.4 % in the controls (p < 0.05 vs controls). The corresponding values after 3 h LPS were 13.5 ± 1.5 versus 19.3 ± 1.5 % (p < 0.05 vs controls). The leukocyte count in sinusoids and venules had a similar development. Functional parameters were all slightly better preserved in the SNP group, but with no individual significance versus controls. Conclusions: Infusion of the NO donor SNP in early endotoxaemia attenuates the detrimental effects of LPS on liver microcirculation, most probably by alleviating a relative deficit of NO at the microcirculatory level.

Aminoethyl-isothiourea, a selective inhibitor of inducible nitric oxide synthase activity, improves liver circulation and oxygen metabolism in a porcine model of endotoxemia

The role of nitric oxide (NO) in hepatic oxygen transport is unclear. We investigated the effects of aminoethyl-isothiourea (AE-ITU), a selective inhibitor of iNOS activity, on liver blood flow and oxygen consumption (Vo2H) in the pig. Endotoxin (lipopolysaccharide, LPS) was given intraportally (1.7 μg/kg/h), followed by AE-ITU (10 mg/kg) after 3 h (n = 7). LPS controls (n = 8) received LPS for 6 h. AE-ITU controls (n = 6) received saline/AE-ITU. LPS (treatment group) caused significant reductions at 3 h in cardiac output (CO) from 4.4 ± .4 to 2.7 ± .3 L/min, in hepatic artery flow (QHA) from 266 ± 53 to 127 ± 19 mL/min, and in portal venous flow (QPV) from 630 ± 50 to 323 ± 33 mL/min. Hepatic oxygen delivery (Do2H) was reduced from 93 ± 11 to 38 ± 5 mL/min (p < .05), while hepatic oxygen extraction ratio (ERo2H) increased, and Vo2H was maintained. Similar changes were observed in LPS controls. AE-ITU caused no changes in saline controls. After injection of AE-ITU during LPS infusion, CO was unchanged, while QHA increased gradually from 127 ± 20 to 268 ± 40 mL/min over 3 h (p < .05) and Do2H from 38 ± 5 to 60 ± 5 mL/in (p < .05). ERo2H increased from .54 ± .04 to .69 ± .03 in 30 min, while Vo2H increased from 23 ± 4 to 35 ± 3 mL/in in 3 h (p < .05). Thus, AE-ITU restored hepatic arterial blood flow and increased hepatic oxygen consumption in pigs with endotoxemia.

Effects of the nitric oxide synthase inhibitors NG-nitrol-L-arginine methyl ester and aminoethyl-isothiourea on the liver microcirculation in rat endotoxemia

BACKGROUND/METHODSnThe question whether nitric oxide protects or impairs organ perfusion during early endotoxemia has not been completely answered. To evaluate the regulative function of nitric oxide on organ microvascular perfusion and leukocyte accumulation during endotoxemia, we studied the influence of a non-selective nitric oxide inhibitor and a preferential inducible nitric oxide synthase inhibitor (respectively, N(G)-nitro-L-arginine methyl ester and aminoethyl-isothiourea) on liver microcirculation (intravital fluorescence microscopy) in a rat model.nnnRESULTSnTwo hours after intraportal injection of lipopolysaccharide (5 mg/kg in 10 min) the rats were randomly treated and received a bolus dose of N(G)-nitro-L-arginine methyl ester (10 mg/kg, n = 7), aminoethyl-isothiourea (10 mg/kg, n = 6) or normal saline, (n = 7). After 1 h, N(G)-nitro-L-arginine methyl ester blockade yielded a higher rate of non-perfused sinusoids than normal saline (27 +/- 2% vs 19 +/- 5%, p < 0.05). LPS-induced leukocyte stagnation in sinusoids was further increased (p < 0.05) in all groups after 1 h treatment, but N(G)-nitro-L-arginine methyl ester clearly accentuated leukocyte accumulation in sinusoids as compared to normal saline (69 +/- 19% vs 16 +/- 4%, p < 0.05). Both modalities of nitric oxide blockade elicited a significant enhancement in the number of leukocytes adherent to the postsinusoidal venules in contrast to normal saline (N(G)-nitro-L-arginine methyl ester 48 +/- 17%, aminoethyl-isothiourea 33 +/- 9% vs normal saline 1 +/- 5%, p < 0.05).nnnCONCLUSIONSnWe conclude that complete nitric oxide blockade aggravates lipopolysaccharide-induced hepatic microvascular perfusion failure and enhances leukocyte accumulation, in both sinusoids and post-sinusoidal venules. The preferential inducible nitric oxide synthase inhibitor aminoethyl-isothiourea has a moderate negative effect, favoring leukocyte adhesion in postsinusoidal venules, and its usefulness demands further research, especially concerning its late effects.

USE OF SELECTIVE AND NONSELECTIVE NITRIC OXIDE SYNTHASE INHIBITORS IN RAT ENDOTOXEMIA: EFFECTS ON HEPATIC MORPHOLOGY AND FUNCTION

Endotoxin has profound effects on nitric oxide (NO) production, and considerable controversies exist as to whether these alterations are beneficial or deleterious. Increased mortality has been reported from nonselective inhibition of NO synthase. Results from selective inhibition of the inducible isoform (iNOS) appear largely positive. In a model of rat endotoxemia we have compared the early effects on hepatic morphology and function of selective and nonselective NO inhibition. Two hours after endotoxin injection (5 mg/kg intraportally) the rats were treated with either the selective iNOS inhibitor aminoethyl isothiourea (AE-ITU, 10 mg/kg), the nonselective NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg), or normal saline. The animals were observed for another hour. Using an immunohistochemical method, induction of iNOS was demonstrated in various tissues in all slices examined. No unequivocal benefit from NO inhibition was noted. Electron microscopic examination revealed widespread alterations of liver morphology, without obvious differences between the groups. Liver function, as assessed by ketone body ratio, hepatic venous acid base values, and bile production, was generally more adversely affected after NO inhibition. Even with the iNOS selective inhibitor AE-ITU no benefit was noted. We conclude that during the early phases of endotoxemia therapeutic reduction of NO production has no positive effects on liver function or morphology.

Nitric Oxide Administration Restores the Hepatic Artery Buffer Response During Porcine Endotoxemia

The hepatic artery buffer response, which is lost during endotoxemia, plays a central role in the autoregulation of liver perfusion. A temporarily decreased synthesis of nitric oxide during early endotoxemia might be responsible for this dysfunction; hence exogenous administration of nitric oxide could reestablish the autoregulation of hepatic blood flow and help prevent hepatic damage later in septic shock. Fifteen pigs were treated with lipopolysaccharide +/− the nitric oxide donor nitroprusside-sodium via the portal vein. Hemodynamics were measured, and serum chemistry and liver biopsies for nitric oxide synthase expression were obtained. Lipopolysaccharide decreased arterial liver perfusion after 5 hours by 38% (p =. 012), which was reversed by addition of nitroprusside (8%). Administration of nitroprusside preserved an increase of 28% in hepatic arterial upon portal vein flow reduction (p <. 001). Nitroprusside maintained mRNA levels of constitutive nitric oxide synthase in liver tissue which were decreased by lipopolysaccharide (p =. 026 vs. p =. 114) and tempered the burst in inducible nitric oxide synthase expression at t = 3 hours. The early administration of the nitric oxide donor sodium nitroprusside during endotoxemia is able to reestablish the autoregulatory response of the hepatic artery following reduction of hepatic blood flow. This beneficial effect might help to prevent subsequent hepatic damage in the course of abdominal sepsis.

Modulators of Nitric Oxide in Porcine Endotoxemia: Effects on Hepatic Oxygen Delivery and Consumption

In a porcine model of endotoxemia we have studied the effects of nitric oxide (NO) on hepatic oxygen delivery and consumption. After 3 h of endotoxemia, NO biosynthesis was modulated by a bolus dose of the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME). Fifteen minutes thereafter a continuous infusion of the NO donor sodium nitroprusside (SNP) was started. Endotoxin significantly reduced hepatic oxygen delivery from 3.4 +/- 0.6 to 2.2 +/- 0.3 ml/kg/min at 3 h. Due to an increased extraction ratio (ER), oxygen consumption was nearly unaffected. L-NAME further diminished oxygen delivery to 1.0 +/- 0.2 ml/kg/min within 15 min (p < 0.05), but despite an increase in ER from 47 to 68% (p < 0.05), oxygen consumption tended to decrease (from 1.0 to 0.7 ml/ kg/min, nonsignificant). A similar tendency was observed in a control group of 9 pigs which was treated in the same way as the study group, except for the SNP infusion. SNP induced an almost selective increase in hepatic arterial flow, with a corresponding increase in oxygen delivery to 1.8 +/- 0.3 ml/kg/min (p < 0.05). At the same time ER was reduced from 68 to 42% (p < 0.05). Oxygen consumption remained unaltered. The control group exhibited no change in either oxygen delivery or consumption. The study shows that nonselective inhibition of NO synthesis is detrimental to hepatic perfusion and oxygen transport. The NO donor SNP increased oxygen delivery via a selective increase in hepatic arterial flow, but failed to influence oxygen consumption. This was probably mainly due to a massive shutdown of sinusoids, which did not reopen when flow was increased. A functioning microcirculation thus seems to be a prerequisite for the stimulation of organ blood flow to be effective.

SYSTEMIC PETIDOGLYCAN (PEPG) STIMULATES PROINFLAMMATORY RESPONSES IN SYNERGY WITH LIPOPOLYSACCHARIDE (LPS) IN AN IN VIVO AND IN VITRO PORCINE MODEL

TOWARDS RESOLVING THE CHALLENGE OF SEPSIS DIAGNOSTIC. Thomas Herget* and Thomas Joos . *Merck KGaA, Darmstadt, Germany; NMI Natural and Medical Sciences Institute at the University of Tübingen, Reutlingen, Germany Biomarkers have proven to be very useful in clinical conditions such as heart attack, stroke and cancer. There are characteristics linked to sepsis like in blood pressure, body temperature and heart rate. Efforts over the last decade to improve diagnosis for infectious inflammation have been unsuccessful in identifying a single and universal biomarker that provides sufficiently high sensitivity and specificity. In gramnegative septicemia and following major abdominal trauma, the determination of endotoxin continues to be a leading candidate which could become adopted into clinical practice. The importance of endotoxin measurement continues to grow as more clinicians recognize the added value of measuring endotoxin in critically ill patients and with the emergence of major pharmaceutical trials directly targeting endotoxin in the bloodstream. However, hundreds of other candidates potentially serving as biomarker for sepsis have been recently described, e.g. cysteinyl-leukotriene (LTC4) generation, procalcitonin (PCT) and C-reactive protein (CRP). However, none of them fulfils the criteria requested by clinicians, namely being specific and sensitive. The presentation will discuss criteria for a sepsis biomarker, will give an overview of obtaining samples from appropriate cell systems and from patients. Furthermore, tools will be described to identify marker candidates on genetic-, proteinand metabolite level. The integration of these data sets covering e.g. signal transduction, protein : protein interaction, gene expression with the help of bioinformatics and systems biology will help to validate such candidates. The final goal is manufacturing a robust diagnostic device for clinical routine work. A solid sepsis diagnostics method will be beneficial for patients, but also for the healthcare systems and will open challenges for the pharmaceutical industry.

BLOQUEIO DO ÓXIDO NÍTRICO COM L-NAME COMPROMETE A MICROCIRCULAÇÃO HEPÁTICA DURANTE ENDOTOXINEMIA

The blockade of NO production during endotoxemia remains controversial. To evaluate the effect of NO blockade on the liver microcirculation, Sprague-Dawley male rats received LPS and 2h after they were treated by injections of L-NAME (10 mg/kg BW, n=6) or normal saline (NS, n=7). Intravital microscopy (IVM) assessed sinusoidal perfusion, blood samples were taken from the hepatic vein to determine base excess, and bile was collected. After 1h treatment L-NAME increased the LPS-induced sinusoidal perfusion failure (p < 0.05 vs NS), accentuated the acidosis in the hepatic blood effluent (p < 0.05 vs NS), while bile flow was further reduced (L-NAME 2.0 ± 0.5 vs NS 2.4 ± 0.1 ml/g/min). Non-selective NO blockade in endotoxemia enhances the sinusoidal perfusion failure, impairs acid-basic status of the liver and shows a tendency of impairment of the excretory function.