Early Endotoxaemia Research Articles

RhoA/Rho-Kinase and Nitric Oxide in Vascular Reactivity in Rats with Endotoxaemia

RhoA/Rho-kinase (RhoA/ROK) pathway promotes vasoconstriction by calcium sensitivity mechanism. LPS causes nitric oxide (NO) overproduction to induce vascular hyporeactivity. Thus, we tried to examine the role of RhoA/ROK and NO in the regulation of vascular reactivity in different time-point of endotoxaemia. Male Wistar rats were intravenously infused for 10 min with saline or E. coli endotoxin (lipopolysaccharide, LPS, 10 mg/kg) and divided to five groups (n = 8 in each group): (i) Control, sacrificed at 6 h after saline infusion; (ii) LPS1h, sacrificed at 1 h after LPS infusion; (iii) LPS2h, sacrificed at 2 h after LPS infusion; (iv) LPS4h, sacrificed at 4 h after LPS infusion; and (v) LPS6h, sacrificed at 6 h after LPS infusion. LPS1h and LPS2h were regarded as early endotoxaemia, whereas LPS4h and LPS6h were regarded as late endotoxaemia. Indeed, our results showed that LPS reproduced a biphasic hypotension and sustained vascular hyporeactivity to noradrenaline (NA) in vivo. Interestingly, this hyporeactivity did not occur in ex vivo during early endotoxaemia. This could be due to increases of aortic RhoA activity (n = 5, P<0.05) and myosin phosphatase targeting subunit 1 phosphorylation (n = 3, P<0.05). In addition, pressor response to NA and vascular reactivity in early endotoxaemia were inhibited by ROK inhibitor, Y27632. Furthermore, plasma bradykinin was increased at 10 min (24.6±13.7 ng/mL, n = 5, P<0.05) and aortic endothelial NO synthase expression was increased at 1 h (+200%. n = 3, P<0.05) after LPS. In late endotoxaemia, the vascular hyporeactivity was associated with aortic inducible NO synthase expression (n = 3, P<0.05) and an increased serum NO level (n = 8, P<0.05). Thus, an increased RhoA activity could compensate vascular hyporeactivity in early endotoxaemia, and the large NO production inhibiting RhoA activity would lead to vascular hyporeactivity eventually.

RhoA/ROK and NO contribute to vascular hyporeactivity in endotoxaemic rats

Hypotension and vascular hyporeactivity are significant characteristics of septic shock. The RhoA/Rho-kinase (RhoA/ROK) pathway is a major target for regulating calcium sensitivity of agonist-induced contraction. Here, we investigated the role of RhoA/ROK pathway in vascular hyporeactivity in endotoxaemic animals. Male Wistar rats were intravenously infused with E. coli lipopolysaccharide (LPS, 10 mg/kg) to induce endotoxaemia. Experimental animals were assigned to five groups: (i) 6 h after saline (Control); (ii) 1 h after LPS (LPS1h); (iii) 2 h after LPS (LPS2h); (iv) 4 h after LPS (LPS4h); (v) 6 h after LPS (LPS6h). LPS1h and LPS2h were regarded as early endotoxaemia, whereas LPS4h and LPS6h were regarded as late endotoxaemia. Results showed that LPS induced a biphasic hypotension and sustained vascular hyporeactivity in vivo, and this hyporeactivity only occurred in late endotoxaemia ex vivo. Meanwhile, the aortic inducible nitric oxide (NO) synthase expression and the serum NO level were time-dependently increased after LPS. However, the aortic RhoA activity and myosin phosphatase targeting subunit 1 phosphorylation were increased in early endotoxaemia and this vascular reactivity was inhibited by ROK inhibitor, Y27632. In addition, the plasma bradykinin level was increased at 15 min and aortic endothelial NO synthase expression was increased 1 h after LPS. In conclusion, an increased RhoA activity may compensate vascular hyporeactivity in early endotoxaemia, whereas in late endotoxaemia, the large amount of NO produced causes inhibition of RhoA activity leading to vascular hyporeactivity in vivo and ex vivo.

Effects of endotoxaemia on protein metabolism in rat fast-twitch skeletal muscle and myocardium.

BackgroundIt is unclear if the rat myocardium undergoes the same rapid reductions in protein content that are classically observed in fast-twitch skeletal muscle during endotoxaemia.Methodology/Principal FindingsTo investigate this further, and to determine if there is any divergence in the response of skeletal muscle and myocardium in the mechanisms that are thought to be largely responsible for eliciting changes in protein content, Sprague Dawley rats were implanted with vascular catheters and administered lipopolysaccharide (LPS; 150 µg kg−1 h−1) intravenously for 2 h, 6 h or 24 h (saline administered control animals were also included), after which the extensor digitorum longus (EDL) and myocardium were removed under terminal anaesthesia. The protein-to-DNA ratio, a marker of protein content, was significantly reduced in the EDL following 24 h LPS administration (23%; P<0.05), but was no different from controls in the myocardium. At the same time point, a significant increase in MAFbx/atrogin-1 and MuRF1 mRNA (3.7±0.7- and 19.5±1.9-fold increase vs. controls, respectively; P<0.05), in addition to protein levels of α1-3, 5–7 subunits of the 20S proteasome, were observed in EDL but not myocardium. In contrast, elevations in phosphorylation of p70 S6K residues Thr421/Ser424, and 4E-BP1 residues Thr37/Thr46 (P<0.05), consistent with an elevation in translation initiation, were seen exclusively in the myocardium of LPS-treated animals.Conclusions/SignificanceIn summary, these findings suggest that the myocardium does not undergo the same catabolic response as skeletal muscle during early endotoxaemia, partly due to the absence of transcriptional and signalling events in the myocardium typically associated with increased muscle proteolysis and the suppression of protein synthesis.

Kinetics of increased generation of (.)NO in endotoxaemic rats as measured by EPR.

Ferrous-diethyldithiocarbamate (Fe(DETC)(2)) chelate is a lipophilic spin trap developed for (.)NO detection by electron paramagnetic resonance (EPR) spectroscopy. Using this spin trap we investigated the kinetics of (.)NO production in endotoxaemia in rats induced by lipopolysaccharide (Escherichia coli, 10 mg/kg). The NO-Fe(DETC)(2) complex was found to give a characteristic EPR signal, and the amplitude of the 3rd (high-field) component of its hyperfine splitting was used to monitor the level of (.)NO. We found that in blood, kidney, liver, heart and lung (.)NO production starts to increase as early as 2 h after LPS injection, reaches the maximum 6 h after LPS injection and then returns to basal level within further 12-18 h. Interestingly, in the eye bulb the maximum of (.)NO production was detected 12 h after LPS, and the signal was still pronounced 24 h after LPS. In brief, the highly lipophilic exogenous spin trap, Fe(DETC)(2) is well suited for assessment of (.)NO production in endotoxaemia. We demonstrated that the kinetics of increased production of (.)NO in endotoxaemic organs, with the notable exception of the eye, do not follow the known pattern of NOS-2 induction under those conditions. Accordingly, only in early endotoxaemia a high level of (.)NO is detected, while in late endotoxaemia (.)NO detectability is diminished most probably due to concomitant oxidant stress.

Kinetics of increased generation of (.)NO in endotoxaemic rats as measured by EPR.

Ferrous-diethyldithiocarbamate (Fe(DETC)(2)) chelate is a lipophilic spin trap developed for (.)NO detection by electron paramagnetic resonance (EPR) spectroscopy. Using this spin trap we investigated the kinetics of (.)NO production in endotoxaemia in rats induced by lipopolysaccharide (Escherichia coli, 10 mg/kg). The NO-Fe(DETC)(2) complex was found to give a characteristic EPR signal, and the amplitude of the 3rd (high-field) component of its hyperfine splitting was used to monitor the level of (.)NO. We found that in blood, kidney, liver, heart and lung (.)NO production starts to increase as early as 2 h after LPS injection, reaches the maximum 6 h after LPS injection and then returns to basal level within further 12-18 h. Interestingly, in the eye bulb the maximum of (.)NO production was detected 12 h after LPS, and the signal was still pronounced 24 h after LPS. In brief, the highly lipophilic exogenous spin trap, Fe(DETC)(2) is well suited for assessment of (.)NO production in endotoxaemia. We demonstrated that the kinetics of increased production of (.)NO in endotoxaemic organs, with the notable exception of the eye, do not follow the known pattern of NOS-2 induction under those conditions. Accordingly, only in early endotoxaemia a high level of (.)NO is detected, while in late endotoxaemia (.)NO detectability is diminished most probably due to concomitant oxidant stress.

Administration of nitric oxide restores the abolished hepatic artery buffer responses in early porcine endotoxaemia

Abstract Background Among other organs, the liver may be severely compromised by the altered haemodynamic conditions of septic shock. The hepatic artery buffer response (HABR), which is mediated mainly by adenosine 5′-triphosphate and nitric oxide as the second messenger, and is ablated during endotoxaemia, plays a central role in the autoregulation of liver perfusion. It was hypothesized that a temporarily decreased synthesis of nitric oxide during early endotoxaemia might be responsible for the dysfunction of the HABR and that administration of nitric oxide could re-establish the autoregulation of hepatic blood flow. Methods The experiment was performed in 15 juvenile Landrace pigs, which were randomly assigned to receive endotoxin (ET) 1·7 μg kg−1 min−1 (Escherichia coli) and the non-enzymatic nitric oxide donor sodium nitroprusside (SNP) 3 μg kg−1 min−1 (ET-SNP group; n = 7) or ET 1·7 μg kg−1 min−1 (E. coli) and vehicle (ET group; n = 8). ET and nitric oxide were administered into the portal vein. Study measurements (haemodynamic measurements, liver perfusion) were performed for 5 h. Results ET caused a significant decrease in liver perfusion after 5 h. Hepatic arterial flow (Qha) was reduced by 50 per cent and portal vein flow (Qpv) by 38 per cent. The additional administration of nitric oxide ameliorated liver perfusion; Qha and Qpv were not altered significantly in the ET-SNP group. The mechanical reduction of Qpv to approximately 50 per cent of baseline increased Qha in all animals before administration of ET. Flow increase in the hepatic artery due to partial Qpv occlusion (HABR) was lost in the ET group, while nitric oxide preserved a mean increase of 28 per cent in Qha (P &amp;lt; 0·01). At time zero the Qpv reduction decreased mean hepatic artery resistance (HAR) in both groups. Thereafter ET infusion led to a continuous loss of the response of HAR to reduction of Qpv, while nitric oxide administration kept the response of HAR at pre-ET levels throughout the experiment. At 4 and 5 h the reactivity of HAR to manipulations of Qpv had returned to baseline levels in the ET group. Conclusion Early administration of the non-enzymatic nitric oxide donor SNP during endotoxaemia is able to re-establish an autoregulatory response of the hepatic artery following reduction of hepatic blood flow.

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

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. 13 male Sprague Dawley rats. 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 microg/kg per min started concurrently, the other group (n = 7) was treated with normal saline. The study was terminated after 3 h LPS. 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. 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.

Different effects of early endotoxaemia on hepatic and small intestinal oxygenation in pigs

Study on simultaneous O2 supply/uptake relationships in liver and gut during endotoxaemia, to determine whether signs of dysoxia develop uniformly in the splanchnic region. Animal study to assess the early effects of endotoxaemia on oxygenation of both liver and small intestine. Eight anaesthetized pigs received a continuous portal venous infusion of lipopolysaccharide (0.5 microgram.kg-1.h-1) for 6 h. Systemic, pulmonary and splanchnic haemodynamics as well as systemic and splanchnic O2 supply/uptake relationships were determined. There was a multiphasic haemodynamic response pattern characterized by an early (within the 1st h) and a subsequent more prolonged phase (between the 2nd and 6th h) of decreases and recovery of hepatic arterial, portal venous and superior mesenteric arterial blood flows (electromagnetic flow probes) and splanchnic O2 deliveries. Unrelated to perfusion pressure and O2 delivery, there were early and sustained decreases in ileal mucosal surface partial pressure of oxygen (PO2) (multiwire PO2 electrode) and pH (tonometry). This was not reflected by ileal serosal surface PO2, O2 uptake and arteriomesenteric venous pH and partial pressure of carbon dioxide (PCO2) gradients. There was little evidence of concomitant hepatic dysoxia as evaluated by surface PO2. The study demonstrates early and sustained regional (mucosa) intestinal hypoxia with little evidence of simultaneous hepatic dysoxia during initial endotoxaemia.

Different effects of early endotoxaemia on hepatic and small intestinal oxygenation in pigs

ObjectiveStudy on simultaneous O2 supply/uptake relationships in liver and gut during endotoxaemia, to determine whether signs of dysoxia develop uniformly in the splanchnic region.DesignAnimal study to assess the early effects of endotoxaemia on oxygenation of both liver and small intestine.InterventionsEight anaesthetized pigs received a continuous portal venous infusion of lipopolysaccharide (0.5 μg·kg−1·h−1) for 6 h. Systemic, pulmonary and splanchnic haemodynamics as well as systemic and splanchnic O2 supply/uptake relationships were determined.ResultsThere was a multiphasic haemodynamic response pattern characterized by an early (within the 1st h) and a subsequent more prolonged phase (between the 2nd and 6th h) of decreases and recovery of hepatic arterial, portal venous and superior mesenteric arterial blood flows (electromagnetic flow probes) and splanchnic O2 deliveries. Unrelated to perfusion pressure and O2 delivery, there were early and sustained decreases in ileal mucosal surface partial pressure of oxygen (PO2) (multiwire PO2 electrode) and pH (tonometry). This was not reflected by ileal serosal surface PO2, O2 uptake and arteriomesenteric venous pH and partial pressure of carbon dioxide (PCO2) gradients. There was little evidence of concomitant hepatic dysoxia as evaluated by surface PO2.ConclusionsThe study demonstrates early and sustained regional (mucosa) intestinal hypoxia with little evidence of simultaneous hepatic dysoxia during initial endotoxaemia.

Elevated serum neopterin level: its relation to endotoxaemia and sepsis in patients with major burns.

The present study was conducted to determine the relationship between levels of neopterin and endotoxin in the circulation, and whether the neopterin level was related to the development of severe sepsis after extensive burns. This prospective study included 35 patients with burn size greater than 30% (30-98%), and 22 healthy volunteers who served as a comparison group. Neopterin levels increased in most patients on day 3 post-burn, but they were not significantly correlated with the extent of the burn surface (P > 0 center dot 05). A high serum neopterin level was found in patients with sepsis (n = 15), and a marked elevation persisted throughout the observation period. The difference between septic and non-septic patients (n = 20) became significant on 14 and 28 days post-burn. Although the presence of early endotoxaemia did not influence the alterations in serum neopterin, patients with endotoxaemia had much higher neopterin values than those who showed no endotoxaemia from the second week onward (P < 0 center dot 05-0 center dot 01). In addition, circulating endotoxin and neopterin levels were positively correlated in patients who developed endotoxaemia on day 14 (r = 0 center dot 368, P < 0 center dot 05) and day 21 (r = 0 center dot 439, P < 0 center dot 01) after major burns. These results suggest that thermal injury can lead to an elevation of serum neopterin independent of the burn surface area. The initial increase in the neopterin level may be a part of the acute-phase response to tissue injury itself, whereas the endotoxin release in the circulation may be responsible for the continuous induction of neopterin during the late stage. In addition, the presence of a constant high neopterin level is associated with a critical event in the development of severe burn sepsis.

Sequential changes in the splanchnic circulation during continuous endotoxin infusion in sedated sheep: evidence for a selective increase of hepatic artery blood flow and loss of the hepatic arterial buffer response

On-line recording of the sequential changes in systemic, pulmonary, mesenteric, hepatic and renal circulations during onset of endotoxaemia and at 24 h of established hyperdynamic sepsis were evaluated in seven chronically instrumented and sedated sheep receiving a continuous intravenous infusion of Escherichia coli endotoxin (20 ng min-1 kg-1). A transient and significant (P < 0.05) pulmonary arterial vaso-constriction was noted after 13 +/- 4 min, and was followed immediately by a simultaneous significant decrease of coeliac trunk, superior mesenteric artery, and portal vein blood flow to below 50% of baseline values. The superior mesenteric artery and portal vein blood flows partially recovered pre-endotoxin levels to 69 and 75% of baseline, respectively, after 70 min of endotoxin infusion. In contrast, the coeliac trunk blood flow remained reduced for a more prolonged period of time, but then completely recovered baseline values at 100 min. The response of the hepatic artery was biphasic, and consisted of a transient (5-10 min) vasoconstriction at 40 min followed by transitory increase of hepatic artery blood flow reaching a maximum of 921% of baseline values at 102 min. Contrasting with the early changes observed in mesenteric vascular resistances mostly unrelated to systemic haemodynamics, the response of the renal vasculature appeared to be more dependent on changes of renal perfusion pressure. A follow-up at 24 h revealed that the continuous intravenous infusion of endotoxin reproduced some of the most characteristic features of human sepsis with increased cardiac output and decreased vascular resistances of all vascular beds. We conclude that hepatic artery blood flow is selectively and considerably increased in early endotoxaemia in sheep independently of changes in portal vein blood flow, suggesting a disregulation of the physiologic hepatic arterial buffer response, most probably secondary to an increased liver oxygen demand required for phagocytosis, transport, and digestion of the the sudden overload of bacterial endotoxins.