Continuous Infusion Of Lipopolysaccharide Research Articles

Glycolytic reprogramming in macrophages and MSCs during inflammation.

Dysregulated inflammation is associated with many skeletal diseases and disorders, such as osteolysis, non-union of fractures, osteonecrosis, osteoarthritis and orthopaedic infections. We previously showed that continuous infusion of lipopolysaccharide (LPS) contaminated polyethylene particles (cPE) caused prolonged inflammation and impaired bone formation. However, the metabolic and bioenergetic processes associated with inflammation of bone are unknown. Mitochondria are highly dynamic organelles that modulate cell metabolism and orchestrate the inflammatory responses that involve both resident and recruited cells. Glycolytic reprogramming, the shift from oxidative phosphorylation (OXPHOS) to glycolysis causes inappropriate cell activation and function, resulting in dysfunctional cellular metabolism. We hypothesized that impaired immunoregulation and bone regeneration from inflammatory states are associated with glycolytic reprogramming and mitochondrial dysfunction in macrophages (Mφ) and mesenchymal stromal cells (MSCs). We used the Seahorse XF96 analyzer and real-time qPCR to study the bioenergetics of Mφ and MSCs exposed to cPE. To understand the oxygen consumption rate (OCR), we used Seahorse XF Cell Mito Stress Test Kit with Seahorse XF96 analyzer. Similarly, Seahorse XF Glycolytic Rate Assay Kit was used to detect the extracellular acidification rate (ECAR) and Seahorse XF Real-Time ATP Rate Assay kit was used to detect the real-time ATP production rates from OXPHOS and glycolysis. Real-time qPCR was performed to analyze the gene expression of key enzymes in glycolysis and mitochondrial biogenesis. We further detected the gene expression of proinflammatory cytokines in Mφ and genes related to cell differentiation in MSC during the challenge of cPE. Our results demonstrated that the oxidative phosphorylation of Mφ exposed to cPE was significantly decreased when compared with the control group. We found reduced basal, maximal and ATP-production coupled respiration rates, and decreased proton leak in Mφ during challenge with cPE. Meanwhile, Mφ showed increased basal glycolysis and proton efflux rates (PER) when exposed to cPE. The percentage (%) of PER from glycolysis was higher in Mφ exposed to cPE, indicating that the contribution of the glycolytic pathway to total extracellular acidification was elevated during the challenge of cPE. In line with the results of OCR and ECAR, we found Mφ during cPE challenge showed higher glycolytic ATP (glycoATP) production rates and lower mitochondrial ATP (mitoATP) production rates which is mainly from OXPHOS. Interestingly, MSCs showed enhanced glycolysis during challenge with cPE, but no significant changes in oxygen consumption rates (OCR). In accordance, seahorse assay of real-time ATP revealed glycoATP rates were elevated while mitoATP rates showed no significant differences in MSC during challenge with cPE. Furthermore, Mφ and MSCs exposed to cPE showed upregulated gene expression levels of glycolytic regulators and Mφ exposed to cPE expressed higher levels of pro-inflammatory cytokines. This study demonstrated the dysfunctional bioenergetic activity of bone marrow-derived Mφ and MSCs exposed to cPE, which could impair the immunoregulatory properties of cells in the bone niche. The underlying molecular defect related to disordered mitochondrial function could represent a potential therapeutic target during the resolution of inflammation.

Hyaluronic acid plasma levels during high versus low tidal volume ventilation in a porcine sepsis model.

BackgroundShedding of the endothelial glycocalyx can be observed regularly during sepsis. Moreover, sepsis may be associated with acute respiratory distress syndrome (ARDS), which requires lung protective ventilation with the two cornerstones of application of low tidal volume and positive end-expiratory pressure. This study investigated the effect of a lung protective ventilation on the integrity of the endothelial glycocalyx in comparison to a high tidal volume ventilation mode in a porcine model of sepsis-induced ARDS.MethodsAfter approval by the State and Institutional Animal Care Committee, 20 male pigs were anesthetized and received a continuous infusion of lipopolysaccharide to induce septic shock. The animals were randomly assigned to either low tidal volume ventilation, high tidal volume ventilation, or no-LPS-group groups and observed for 6 h. In addition to the gas exchange parameters and hematologic analyses, the serum hyaluronic acid concentrations were determined from central venous blood and from pre- and postpulmonary and pre- and postcerebral circulation. Post-mortem analysis included histopathological evaluation and determination of the pulmonary and cerebral wet-to-dry ratios.ResultsBoth sepsis groups developed ARDS within 6 h of the experiment and showed significantly increased serum levels of hyaluronic acid in comparison to the no-LPS-group. No significant differences in the hyaluronic acid concentrations were detected before and after pulmonary and cerebral circulation. There was also no significant difference in the serum hyaluronic acid concentrations between the two sepsis groups. Post-mortem analysis showed no significant difference between the two sepsis groups.ConclusionIn a porcine model of septic shock and ARDS, the serum hyaluronic acid levels were significantly elevated in both sepsis groups in comparison to the no-LPS-group. Intergroup comparison between lung protective ventilated and high tidal ventilated animals revealed no significant differences in the serum hyaluronic acid levels.

Moderate running exercise prevents excessive immune system activation

Benefits of exercise have been documented for many diseases with a chronic progression, including obesity, diabetes mellitus, cardiovascular diseases, neurodegenerative diseases, certain types of cancers, and overall mortality. Low-grade systemic inflammation is a key component of these pathologies and it has been demonstrated that can be prevented by performing regularly physical exercise. The aim of this study was to examine the effect of lipopolysaccharide (LPS)-induced inflammation on glucose and insulin tolerance, exercise performance, production of urinary neopterin and striatal neurotransmitters levels in adult male C57BL/6 mice. Increased blood glucose clearance and insulin sensitivity were observed after a single administration of glucose (2 g/kg, p.o.) or insulin (0.5 U/kg, i.p.). However, the repeated injection of LPS (0.33 mg/kg/day, i.p.) decreased glucose tolerance and increase urinary neopterin levels, pointing to systemic inflammation. In parallel to the urinary-increased neopterin, it was observed a significant reduction in the striatal dopamine levels and an increase in the serotonin/dopamine ratio. While a single LPS injection (0.33 mg/kg, i.p.) showed impaired performance in the incremental loading test (10 m/min, with 2 m/min increment every 3 min, at 9% grade), a moderate physical exercise protocol (treadmill for three weeks; 5 sessions/week; up to 50 min/day) prevented the exacerbation of immune system activation and preserved mitochondrial activity in skeletal muscle from mice with continuous LPS infusion (infusion pumps: 0.83 mg/kg/day, i.p.). In conclusion, the peripheral-induced inflammation elicited metabolic alterations that provoked impairment in striatal dopamine metabolism. The moderate exercise prevented the increase of urinary neopterin and preserved mitochondrial activity under LPS-induced inflammatory conditions.

Chronic maternal inflammation or high-fat-feeding programs offspring obesity in a sex-dependent manner.

The current world-wide obesity epidemic partially results from a vicious circle whereby maternal obesity during pregnancy predisposes the offspring for accelerated weight gain and development of metabolic syndrome. Here we investigate whether low-grade inflammation, characteristic of the obese state, provides a causal role for this disastrous fetal programming in mice. We exposed pregnant and lactating C57BL/6JBom female mice to either high-fat diet (HFD), or continuous infusion of lipopolysaccharide (LPS), a potent trigger of innate immunity, and studied offspring phenotypes. Both maternal LPS or HFD treatments rendered the offspring hyperphagic and inept of coping with a HFD challenge during adulthood, increasing their adiposity and weight gain. The metabolic effects were more pronounced in female offspring, while exposed male offspring mounted a larger inflammatory response to HFD at adulthood. This supports our hypothesis and highlights the programming potential of inflammation in obese pregnancies.

LPS infusion suppresses serum FGF21 levels in healthy adult volunteers.

ContextDuring the inflammatory acute phase response, plasma glucose and serum triglycerides are increased in humans. Fibroblast growth factor (FGF) 21 has plasma glucose and lipid-reducing actions, but its role in the acute inflammatory response in human is unknown.ObjectiveTo investigate circulating levels of FGF21 after lipopolysaccharide (LPS) infusion.DesignTwo randomized, single-blinded, placebo-controlled crossover trials were used.SettingThe studies were performed at a university hospital clinical research center.Patients and interventionsStudy 1 (LPS bolus): Eight young, healthy, lean males were investigated two times: (1) after isotonic saline injection and (2) after LPS injection (bolus of 1 ng/kg). Each study day lasted 4 h. Study 2 (continuous LPS infusion): Eight, healthy males were investigated two times: (1) during continuously isotonic saline infusion and (2) during continuous LPS infusion (0.06 ng/kg/h). Each study day lasted 4 h. Circulating FGF21 levels were quantified every second hour by an immunoassay.ResultsA LPS bolus resulted in a late suppression (t = 240 min) of serum FGF21 (P = 0.035). Continuous LPS infusion revealed no significant effects on FGF21 levels (P = 0.82).ConclusionsOur studies show that a bolus of LPS results in decreased FGF21 levels 4 h from exposure.

Effects of lipopolysaccharide-induced inflammation on initial lung fibrosis during open-lung mechanical ventilation in rats

This study aimed to assess the impact of pulmonary inflammation on early fibrotic response in rats challenged with increasing doses of lipopolysaccharide (LPS). Twenty-four rats were randomized and infused with three different increasing doses of continuous LPS infusion (n=8/group) while being ventilated with low tidal volumes and open-lung positive end-expiratory pressure. Another eight animals served as uninjured control group. Hemodynamics, gas exchange, respiratory system mechanics, lung histology, α-smooth muscle actin, plasma cytokines, and mRNA expression of cytokines and type I and III procollagen in lung tissue were assessed. We found impaired hemodynamics and gas exchange as well as higher histological lung injury scores and α-smooth muscle actin expressions in the medium LPS dose compared to control and the lower LPS dose. The highest LPS dose did not cause further aggravation of these findings. In all LPS groups type I and III procollagen decreased compared to controls and there was a negative correlation between type III procollagen-RNA expression and proinflammatory mediators.

Antagonism by salvianolic acid B of lipopolysaccharide-induced disseminated intravascular coagulation in rabbits.

The aim of the present study was to investigate the effects of salvianolic acid B on lipopolysaccharide (LPS)-induced disseminated intravascular coagulation (DIC) in rabbits. Continuous infusion of LPS was used to induce a DIC model in rabbits. Treatment with salvianolic acid B (1, 3 or 6 mg/kg) was started simultaneously with LPS infusion (0.5 mg/kg LPS in 60 mL saline; 10 mL/h over a period of 6 h) through the contralateral marginal ear vein. Activated partial thromboplastin time (APTT), prothrombin time (PT), platelet count and fibrinogen concentration were determined, as were plasma levels of fibrin-fibrinogen degradation products (FDP), alanine aminotransferase (ALT), blood urea nitrogen (BUN), protein C activity, antithrombin III (ATIII) and tumour necrosis factor (TNF)-α concentration. The gradual impairment of haemostatic parameters was induced by continuous infusion of LPS. There were marked increases in APTT, PT, BUN, ALT and plasma TNF-α and marked decreases in the platelet count, fibrinogen, FDP, protein C and ATIII. The intravenous administration of 1, 3 or 6 mg/kg salvianolic acid B attenuated the increases in APTT, PT, BUN, ALT and plasma TNF-α and the decreases in fibrinogen, platelet, FDP, protein C and ATIII induced by LPS infusion. These observations indicate that salvianolic acid B has an effect against LPS-induced DIC in rabbits.

Synergistic white matter protection with acute-on-chronic endotoxin and subsequent asphyxia in preterm fetal sheep

BackgroundPerinatal asphyxia and exposure to intrauterine infection are associated with impaired neurodevelopment in preterm infants. Acute exposure to non-injurious infection and/or inflammation can either protect or sensitize the brain to subsequent hypoxia-ischemia. However, the effects of subacute infection and/or inflammation are unclear. In this study we tested the hypothesis that acute-on-chronic exposure to lipopolysaccharide (LPS) would exacerbate white matter injury after subsequent asphyxia in preterm fetal sheep.MethodsFetal sheep at 0.7 gestational age received a continuous LPS infusion at 100 ng/kg for 24 hours, then 250 ng/kg/24 hours for 96 hours, plus 1 μg boluses of LPS at 48, 72, and 96 hours or the same volume of saline. Four hours after the last bolus, complete umbilical cord occlusion or sham occlusion was induced for 15 minutes. Sheep were sacrificed 10 days after the start of infusions.ResultsLPS exposure was associated with induction of microglia and astrocytes and loss of total and immature and mature oligodendrocytes (n = 9) compared to sham controls (n = 9). Umbilical cord occlusion with saline infusions was associated with induction of microglia, astrogliosis, and loss of immature and mature oligodendrocytes (n = 9). LPS exposure before asphyxia (n = 8) was associated with significantly reduced microglial activation and astrogliosis and improved numbers of immature and mature oligodendrocytes compared to either LPS exposure or asphyxia alone.ConclusionsContrary to our initial hypothesis, the combination of acute-on-chronic LPS with subsequent asphyxia reduced neuroinflammation and white matter injury compared with either intervention alone.

Time-Dependent Compensatory Responses to Chronic Neuroinflammation in Hippocampus and Brainstem: The Potential Role of Glutamate Neurotransmission

Chronic neuroinflammation is characteristic of neurodegenerative diseases and is present during very early stages, yet significant pathology and behavioral deficits do not manifest until advanced age. We investigated the consequences of experimentally-induced chronic neuroinflammation within the hippocampus and brainstem of young (4 mo) F-344 rats. Lipopolysaccharide (LPS) was infused continuously into the IVth ventricle for 2, 4 or 8 weeks. The number of MHC II immunoreactive microglia in the brain continued to increase throughout the infusion period. In contrast, performance in the Morris water maze was impaired after 4 weeks but recovered by 8 weeks. Likewise, a transient loss of tyrosine hydroxylase immunoreactivity in the substantia nigra and locus coeruleus was observed after 2 weeks, but returned to control levels by 4 weeks of continuous LPS infusion. These data suggest that direct activation of microglia is sufficient to drive, but not sustain, spatial memory impairment and a decrease in tyrosine hydroxylase production in young rats. Our previous studies suggest that chronic neuroinflammation elevates extracellular glutamate and that this elevation underlies the spatial memory impairment. In the current study, increased levels of GLT1 and SNAP25 in the hippocampus corresponded with the resolution of performance deficit. Increased expression of SNAP25 is consistent with reduced glutamate release from axonal terminals while increased GLT1 is consistent with enhanced clearance of extracellular glutamate. These data demonstrate the capacity of the brain to compensate for the presence of chronic neuroinflammation, despite continued activation of microglia, through changes in the regulation of the glutamatergic system.

Systemic Administration of Enamel Matrix Derivative to Lipopolysaccharide-Challenged Pigs: Effects on the Inflammatory Response

Periodontitis is the primary clinical indication for enamel matrix derivative (EMD). Recent investigations, showing that EMD inhibits the production of tumor necrosis factor-alpha (TNF-alpha) when added to human whole blood, indicate a novel role for EMD as a modulator of systemic inflammation. In the present study, we investigated the systemic effects of EMD in lipopolysaccharide (LPS)-challenged pigs. In a preparatory study, seven pigs received a prophylactic EMD bolus injection (5 mg/kg), followed by a continuous infusion (50 mg/kg/min). Thirty minutes later, a continuous infusion of LPS (1.7 mcg/kg/h) was started. An additional 12 pigs were randomized into two groups. Six of these animals were given the same treatment, except that EMD was administered 30 min after LPS. The remainder served as controls. The groups were compared according to organ injury and function, hemodynamics, and systemic markers of inflammation. Prophylactic administration of EMD triggered transient hemodynamic instability in two of seven pigs. In the randomized pigs, no or only nonspecific changes were observed in biopsies from vital organs, independent of treatment. Enamel matrix derivative did not modify systemic TNF-alpha, interleukin (IL)-1 beta, or IL-6 concentrations. In the formulation and dosages used, EMD did not modulate the inflammatory response. No true allergic or immunotoxic reactions were seen. To be usable for systemic application, a new formulation should be developed, or the active part of the protein(s) should be identified and produced in a soluble form designed for infusion. The potential of EMD as a systemic immune modulator is still unsettled.

Epidural Analgesia Prevents Endotoxin-Induced Gut Mucosal Injury in Rabbits

In the present study, we evaluated the effect of epidural analgesia on the alterations of gut barrier function elicited by endotoxin in rabbits. After the placement of an epidural catheter, 28 male rabbits were randomized into either 0.5% lidocaine (group E) or saline (group C) group. The solutions (0.4 mL/kg) were epidurally injected, followed by continuous infusion (0.1 mL . kg(-1) . h(-1)) throughout the study period. Under a continuous infusion of lipopolysaccharide (15 microg . kg(-1) . h(-1)), mean arterial blood pressure, intramucosal pH, and plasma thrombomodulin concentrations were measured. At 4 h, mean arterial blood pressure was lower (P < 0.05), intramucosal pH was higher (P < 0.01), and the progression of hemodilution more profound (P < 0.05) in group E versus group C, whereas plasma thrombomodulin levels were increased to a similar extent between the groups. With less wet-to-dry weight ratio of ileum, histopathological injury scores of gut mucosa were significantly less in group E versus group C (P < 0.01). In a separate series of experiments (n = 10 each group), mucosal permeability in group E was significantly less compared with group C (P < 0.05). Collectively, these studies showed that despite a significant decrease of perfusion pressure and arterial oxygen content, epidural analgesia minimized endotoxin-induced functional and structural injury of gut mucosa possibly through endothelium-independent mechanisms.

Regional and temporal changes in cardiovascular responses to norepinephrine and vasopressin during continuous infusion of lipopolysaccharide in conscious rats

Reduced pressor responsiveness to norepinephrine (NE) in sepsis is well documented but the associated regional haemodynamic changes are less well characterized, and there are varying reports of changes in haemodynamic responses to arginine vasopressin (AVP). We compared changes in regional haemodynamic responsiveness to AVP and NE during a 24 h continuous infusion of lipopolysaccharide (LPS) in conscious rats. Conscious, male Sprague-Dawley rats were infused with saline (0.4 ml h(-1)) or LPS (150 micro g kg(-1) h(-1)). Renal, mesenteric, and hindquarter haemodynamic responses to 3 min infusions of AVP (0.25, 0.625, and 1.25 ng kg(-1) min(-1)) or NE (75, 250, and 750 ng kg(-1) min(-1)) were assessed 2, 6, and 24 h after the onset of LPS or saline. Two and six hours after the onset of LPS, all haemodynamic effects of NE were markedly reduced, but by 24 h, there was some recovery in the vasoconstrictor actions of NE although the pressor and bradycardic effects were still depressed. Two hours after the onset of LPS, the cardiovascular effects of AVP were depressed but there was some recovery in vascular responsiveness at 6 h. By 24 h, only the mesenteric vasoconstrictor effect of AVP was consistently reduced. During low dose LPS infusion, there are differential changes in haemodynamic responsiveness to AVP and NE, which show different temporal and regional profiles of recovery with time. Furthermore, reduced pressor responsiveness to NE is not necessarily accompanied by a reduced capacity of vessels for vasoconstriction.

Pentoxifylline reduces acute lung injury in chronic endotoxemia

Background. Pentoxifylline (PTX) attenuates end-organ injury in models of sepsis and hemorrhage. PTX is thought to act by inhibiting phosphodiesterase, thus increasing cAMP and decreasing tumor necrosis factor-α (TNF-α) synthesis. The effects of PTX on neutrophil and endothelial cell adhesion molecules and, ultimately, organ injury in a chronic endotoxemia model have not been studied. We hypothesized that continuous infusion of PTX reduces acute lung injury (ALI) caused by chronic lipopolysaccharide (LPS) exposure. Materials and methods. Male Sprague-Dawley rats were given continuous infusion of LPS, PTX + LPS combined, or saline (sham) by implantable pumps. Neutrophil CD11b expression, lung histopathology, lung intercellular adhesion molecule-1 (ICAM-1) expression assessed by immune staining, serum TNF-α, serum interleukin-6 (IL-6), and bronchoalveolar lavage (BAL) IL-8 were evaluated at different time points. Lung injury was graded in a blinded fashion from 0 (normal) to 4 (severe) for interstitial inflammation, neutrophil infiltration, congestion, and edema. Total lung injury score (TLIS) was calculated by adding listed categories. White cell count in the peripheral blood and in the BAL was also performed. Results. Animals treated with PTX + LPS showed a significant reduction in lung injury score, a marked decrease in ICAM-1 expression, and a significant decrease in IL-8 levels in the BAL and serum IL-6 levels when compared with LPS-treated animals. Conclusions. Continuous infusion of PTX reduces ALI caused by chronic endotoxemia. The effect seems to be a result of decreased expression of endothelial and epithelial ICAM-1 and modulation of proinflammatory cytokine synthesis.

Effects of in vivo lipopolysaccharide infusion on vasoconstrictor function of rat isolated mesentery, kidney, and aorta.

Continuous infusion of lipopolysaccharide (LPS) into conscious rats elicits regionally selective cardiovascular disturbances. The aim of the present study was to assess contractile function in different vascular preparations (renal, mesenteric, and thoracic aorta) taken from rats infused with LPS for 2 or 24 h. Sustained responses to continuous infusion of methoxamine but not to KCl were reduced in the aorta (at 2 and 24 h LPS) and mesentery (at 24 h LPS) but not in the renal vascular bed. In contrast, transient responses to bolus doses of methoxamine were unchanged in the mesentery. In Ca2+-imaging experiments with fura-2, challenge with a single concentration of methoxamine (10 microM, which showed an impaired contractile response at 24 h LPS) induced a rise in intracellular Ca2+ in the mesenteric artery that was not different from the control. Furthermore, in the aorta, the contractile response to caffeine was attenuated only in the 2 h LPS group. These results show that there is regional heterogeneity in in vitro vascular responsiveness in preparations taken from LPS-infused rats. Thus, in mesenteric beds and aortae, but not renal beds, there is hypocontractility to methoxamine that is not due to a generalized inability of the smooth muscle to contract, which is evident with sustained but not transient application of agonist (mesentery) and which, in late endotoxemia (24 h LPS), does not appear to involve abnormalities in Ca2+ mobilization or entry.

Vasoconstrictor responsiveness of tail arteries from endotoxaemic rats

Continuous infusion of lipopolysaccharide in conscious rats mimics some aspects of cardiovascular dysfunction in septic shock. In the present study, contractile responsiveness of tail arteries taken from rats infused with lipopolysaccharide was investigated. Contractile responses to α,β-methylene ATP and potassium chloride, but not to methoxamine, were greater after 24 h lipopolysaccharide infusion than in 2-h saline, 24-h saline and 2-h lipopolysaccharide groups. N G-nitro- l-arginine methyl ester augmented contractions to α,β-methylene ATP and methoxamine in the 2-h saline, 24-h saline and 2-h lipopolysaccharide groups, but had no significant effect in the 24-h lipopolysaccharide group. Endothelium-independent vasorelaxant responses to sodium nitroprusside were greater in the 24-h lipopolysaccharide group compared to the other three groups. Relaxations to acetylcholine were not significantly different. In vitro incubation in medium containing lipopolysaccharide for 24 h had no significant effect on contractile responses of tail arteries compared to controls incubated in medium alone. These data indicate a possible impaired nitric oxide and/or endothelial function in tail arteries isolated from rats 24 h after lipopolysaccharide infusion. As hypercontractility was not evoked following in vitro incubation with lipopolysaccharide, the involvement of in vivo neurohumoral factors/mechanisms in the pathology of these changes is implicated.

S-Methylisothiourea sulfate improves renal, but not hepatic dysfunction in canine endotoxic shock model.

Excess production of nitric oxide (NO) by inducible NO synthase (iNOS) has been implicated in the pathophysiology of septic shock. This study was designed to see whether S-methylisothiourea sulfate (SMT), a selective inhibitor for iNOS, prevents cardiovascular changes and multiple organ damage in the canine endotoxic shock model. Prospective, comparable, experimental study. Laboratory at a university hospital. Twenty male mongrel dogs were studied under pentobarbital anesthesia. Dogs were divided into three groups: bacterial lipopolysaccharide (LPS) group (n = 7) receiving continuous infusion of LPS (2 mg/kg/h for 1 h); LPS plus SMT group (n = 7) receiving LPS and SMT (1 mg/kg, bolus i. v., followed by continuous infusion of 1 mg/kg/h for 1 h); and vehicle plus SMT group (n = 6). Hemodynamics, blood gas parameters, and urine output were measured during 6 h observation periods. Serum levels of lactate, transaminases, and bilirubin were measured at baseline, 1 and 6 h. Creatinine and free water clearance, urine sodium excretion and fractional excretion of sodium were calculated. LPS caused a profound hypotension associated with decreases in cardiac output and oxygen delivery, lactic acidosis, renal and liver dysfunction, and thrombocytopenia. SMT prevented the LPS-induced hypotension and renal dysfunction, whereas it did not affect the LPS-induced decreases in cardiac output or oxygen delivery, hyperlactatemia, liver dysfunction, or thrombocytopenia. SMT alone had no appreciable effects on hemodynamics, blood gases, liver or renal functions. These findings show that SMT improves renal, but not hepatic dysfunction, in dogs with endotoxic shock, suggesting that iNOS-derived NO plays differential roles in sepsis-associated multiple organ dysfunction.

Peripheral administration of novel anti-inflammatories can attenuate the effects of chronic inflammation within the CNS

In the present study we investigated whether nitroflurbiprofen (NFP) or nitro-aspirin can reduce the inflammatory response induced by continuous infusion of lipopolysaccharide (LPS) into the fourth ventricular space of the rat's brain for 30 days. The chronic LPS infusion produced an extensive inflammation that was particularly evident in the hippocampus, subiculum and entorhinal and piriform cortices. Daily peripheral administration of NFP dose-dependently, and significantly, attenuated the brain inflammation as indicated by the decreased density and reactive state of microglial cells. Daily peripheral administration of nitro-aspirin also attenuated the brain inflammation, but to a much lesser degree than NFP. The results demonstrated that nonsteroidal anti-inflammatory drugs (NSAIDs) could reduce brain inflammation and that NFP is an effective anti-inflammatory agent.

Lack of effect of TNF antibodies on the cardiovascular sequelae of lipopolysaccharide infusion in conscious rats

1. The aims of the present study were to determine the profile of tumour necrosis factor (TNF) release, and the effect of monoclonal antibodies to TNF, on the changes in regional haemodynamics and the responses to vasodilator and vasoconstrictor challenges, during a continuous 24 h low dose infusion of lipopolysaccharide (LPS) in conscious rats. 2. Male Long Evans rats were chronically instrumented for measurement of regional haemodynamics (renal, superior mesenteric and hindquarters) and were challenged with 3 min infusions of acetylcholine (22 nmol min-1), methoxamine (120 nmol min-1), salbutamol (0.83 nmol min-1) and bradykinin (14.4 nmol min-1). The rats were given either saline, or the TNF antibodies, TN3g1 or TN3g2a, 1 h before the start of a continuous infusion of LPS (150 micrograms kg-1 h-1) and were subsequently re-tested with the vasodilator and vasoconstrictor challenges 2, 6 and 24 h after the start of the LPS infusion. 3. Prior to infusion of LPS, TNF was not detectable in the plasma. During continuous infusion of LPS there was a transient elevation in plasma TNF levels, reaching a maximum (approximately 2300 pg ml-1) after approximately 1 h, and returning to undetectable levels after approximately 3 h of LPS infusion. 4. In the saline pretreated group, after 1-2 h of LPS infusion, there was a small hypotension and a marked renal vasodilation; 6 h after the start of LPS infusion there was a minor elevation in MAP above control levels, renal vasodilatation was maintained and a hindquarters vasoconstriction occurred; after 24 h of LPS infusion, there was a hypotension and renal and hindquarters vasodilatation. There were significant reductions in the tachycardic and renal vasodilator responses and an enhanced depressor response to acetylcholine after 24 h of LPS infusion. LPS infusion also caused a generalized hyporesponsiveness to the cardiovascular effects of methoxamine and salbutamol. The major changes in response to bradykinin were reduced tachycardic and enhanced depressor responses throughout the LPS infusion, a biphasic decrease and increase in renal conductance and enhanced hindquarters vasodilatation at 24 h. 5. Pretreatment with either TN3g1 or TN3g2a antibodies had no major effects on the changes in resting haemodynamics, or on the changes in response to methoxamine, salbutamol or bradykinin challenges during LPS infusion. However, the tachycardic responses to acetylcholine were generally preserved, and its hypotensive effect after 24 h of LPS infusion was not enhanced after TN3g1 or TN3g2a pretreatment. Thus, despite substantial, but transient, elevation of plasma TNF levels during continuous LPS infusion, it appears that the majority of cardiovascular changes were dependent on factors other than plasma TNF.

Cardiac and regional haemodynamics, inducible nitric oxide synthase (NOS) activity, and the effects of NOS inhibitors in conscious, endotoxaemic rats

1. A reproducible model of the hyperdynamic circulatory sequelae of endotoxaemia in conscious, chronically-instrumented Long Evans rats, was achieved with a continuous infusion of lipopolysaccharide (LPS, 150 micro g kg(-1) h(-1)) for 32 h. Over the first 2 h of LPS infusion, there was a transient hypotension and tachycardia, accompanied by a marked increase in renal flow and vascular conductance, although there were reductions in cardiac and stroke index. Between 4-8 after the start of LPS infusion, there was slight hypotension and tachycardia, and a transient rise in mesenteric flow and conductance, but reductions in the hindquarters vascular bed; the hyperaemic vasodilatation in the renal vascular bed was maintained. At this stage, all cardiac haemodynamic variables were not different from baseline. At this stage, cardiac and stroke index were substantially elevated, in association with marked increases in peak aortic flow, dF/dtmax and total peripheral conductance; these changes were well-maintained over the following 8 h of LPS infusion. 2. By 2 h after the start of LPS infusion, only lung inducible nitric oxide synthase (iNOS) activity was increased, but at 6 h there were significant increases in iNOS activity in lung, liver, spleen, heart and aorta. (43.3 +/- 7.8, 28.8 +/- 3.3, 50.8 +/- 7.2, 3.04 +/- 0.29, 3.76 +/- 0.94 pmol min(-1) mg(-1) protein, respectively). However, by 24 h after the start of LPS infusion, iNOS activity was not elevated significantly in any tissue examined, and kidney iNOS activity did not change significantly during LPS infusion. Plasma nitrite/nitrate levels were increased after 2 h infusion of LPS (from 6.07 +/- 1.23 to 29.44 +/- 7.08 micromol l(-1)), and further by 6 h (228.10 +/- 29.20 micromol l(-1)), but were less 24 h after onset of LPS infusion (74.96 +/- 11.34 micromol l(-1)). Hence, the progressive hypotension, increasing cardiac function and developing hyperaemic vasodilatation in renal and hindquarters vascular beds between 8-24 h after the onset of LPS infusion, occurred when tissue iNOS activity and plasma nitrite/nitrate levels were falling. 3. Pretreatment with NG-monomethyl-L-arginine (L-NMMA, 30 mg kg(-1) bolus, 30 mg kg(-1) h(-1) infusion) 1 h before LPS infusion did not prevent the early hypotension, but abolished the initial renal vasodilatation and the later (6-8 h) fall in mean arterial pressure (MAP), and the additional renal vasodilatation.

Nitric oxide mediates lipopolysaccharide-induced alteration of mitochondrial function in cultured hepatocytes and isolated perfused liver

The influence of endogenous nitric oxide, which is generated by stimulation with lipopolysaccharide, on the mitochondrial energization of rat hepatocytes was investigated in vitro and ex vivo. Using a fluorescence microscope equipped with a silicon intensifier target camera, we visualized fluorescence of rhodamine-123, a mitochondrial energization-sensitive fluorescence probe, in individual hepatocytes and measured the fluorescence intensity with a digital imaging processor. Although addition of Kupffer cells or lipopolysaccharide in a range of 0.1 to 1.0 microgram/ml caused no significant alteration in the fluorescence in hepatocytes, Kupffer cells plus 1.0 microgram/ml lipopolysaccharide reduced fluorescence intensity in the cocultured hepatocytes. The alteration of rhodamine-123 fluorescence in the hepatocytes induced by lipopolysaccharide-activated Kupffer cells was significantly inhibited by the addition of NG-monomethyl-L-arginine, a selective inhibitor of nitric oxide synthesis. The transportal infusion of lipopolysaccharide also decreased rhodamine-123 fluorescence in perfused rat liver. The decrease was significantly enhanced in the pericentral regions. Autofluorescence of NADH was elicited by continuous infusion of lipopolysaccharide; this reaction was also enhanced in the pericentral regions. We showed the main site of uptake of infused lipopolysaccharide in the hepatic lobule to be in the periportal regions with fluorescein isothiocyanate-labeled lipopolysaccharide. Our results indicate that the inhibition of mitochondrial energization occurs mainly in pericentral regions, which are distant from the lipopolysaccharide uptake site. The continuous administration of NG-monomethyl-L-arginine significantly attenuated the lipopolysaccharide-induced decrease in rhodamine-123 fluorescence and increase of the NADH contents of the hepatic lobule.(ABSTRACT TRUNCATED AT 250 WORDS)