Endotoxin In Rats Research Articles

Expression of NADPH-diaphorase in nucleus tractus solitarius after peripheral injection of E. coli endotoxin in rats

1. The possibility that peripheral exogenous pyrogens can activate brainstem nuclei by abdominal viscera afferents was studied using the NADPH-diaphorase method in E. coli lipopolysaccharide treated rats with and without ibuprofen pre-treatment. 2. NADPH-d staining revealed: (i) a significant increase in the number of NADPH-d labeled neurons in the subpostremal area of the nucleus tractus solitarius lipopolysaccharide treated rats compared with control animals ( p<0.05), and (ii) an almost similar number of NADPH-d positive neurons in both control and ibuprofen pre-treated rats ( p=0.091). 3. These data indicate that peripheral administration of an exogenous pyrogen stimulates the synthesis of nitric oxide in the nucleus tractus solitarius, and are consistent with the hypothesis of a direct neural link between the periphery and the hypothalamus.

Cloning and Characterization of Novel Mouse and Human Secretory Phospholipase A2s

Mammalian secretory phospholipase A(2)s (sPLA(2)s) are classified into several groups according to molecular structure and the localization of intramolecular disulfide bridges. Among them, group IIA sPLA(2) has been thought to be one of the key enzymes in the pathogenesis of inflammatory diseases owing to its augmented expression under various inflammatory conditions. However, in a number of inbred mouse strains, the group IIA sPLA(2) gene is naturally disrupted by a frameshift mutation. Here, we report the cloning of a cDNA encoding a novel sPLA(2) expressed in the spleen of group IIA sPLA(2)-deficient mouse. We also cloned its human homolog and mapped its gene location on chromosome 1p36.12 near the loci of group IIA and V sPLA(2) genes. The human mature sPLA(2) protein consists of 125 amino acids (M(r) = 14,500) preceded by a 20-residue prepeptide and is most similar to group IIA sPLA(2) with respect to the number and positions of cysteine residues as well as overall identity (48%). Based on these structural properties, the novel sPLA(2) should be categorized into group II, called group IID to follow the already identified IIA to IIC sPLA(2)s. When the cDNA was expressed in COS-7 cells, PLA(2) activity preferentially accumulated in the culture medium. It is maximally active at neutral to alkaline pH and with 2 mM Ca(2+). In assays with individual substrates, L-alpha-1-palmitoyl-2-linoleoyl phosphatidylethanolamine was more efficiently hydrolyzed than the other phospholipids examined. An RNA blot hybridized with the cDNA exhibited two transcripts (2.0 and 1.0 kb) in human spleen, thymus, and colon. The expression of a novel sPLA(2) mRNA was elevated in the thymus after treatment with endotoxin in rats as well as in group IIA sPLA(2)-deficient mice, suggesting its functional role in the progression of the inflammatory process.

Persistence and in vivo effects of Yersinia enterocolitica 0:3 endotoxin in rats

In vivo effects of Yersinia enterocolitica 0:3 lipopolysaccharide (prepared from bacteria grown at 25°C and 37°C) were investigated after intraperitoneal (i.p.) and intraarticular (i.a.) injection in rats during 30 days of examination. The persistence of endotoxin in the peritoneal and the synovial cavities was demonstrated by the immunofluorescence technique. Peritoneal and synovial exudative cell infiltration, as well as changes in some parameters (glycolytic and acid phosphatase activity, and killing ability of peritoneal cells; lactate dehydrogenase concentration in synovial fluid) were studied. The results indicated that endotoxin could persist longer in the synovial than in the peritoneal cavity.

Persistence and in vivo effects of Yersinia enterocolitica 0:3 endotoxin in rats.

In vivo effects of Yersinia enterocolitica 0:3 lipopolysaccharide (prepared from bacteria grown at 25 degrees C and 37 degrees C) were investigated after intraperitoneal (i.p.) and intraarticular (i.a.) injection in rats during 30 days of examination. The persistence of endotoxin in the peritoneal and the synovial cavities was demonstrated by the immunofluorescence technique. Peritoneal and synovial exudative cell infiltration, as well as changes in some parameters (glycolytic and acid phosphatase activity, and killing ability of peritoneal cells; lactate dehydrogenase concentration in synovial fluid) were studied. The results indicated that endotoxin could persist longer in the synovial than in the peritoneal cavity.

Granulocyte colony-stimulating factor modulates the pulmonary host response to endotoxin in the absence and presence of acute ethanol intoxication.

Alcohol impairs neutrophil function and predisposes the host to infectious complications. Granulocyte colony-stimulating factor (G-CSF) increases both the number and functional activities of neutrophils. This study investigated the effects of G-CSF on the pulmonary response to endotoxin in rats with or without acute ethanol intoxication. Acute ethanol intoxication inhibited tumor necrosis factor (TNF)-alpha and macrophage inflammatory protein (MIP)-2 production in the lung and suppressed the recruitment of neutrophils into the lung. Ethanol also inhibited CD11b/c expression on recruited neutrophils and suppressed the phagocytic activity of circulating neutrophils. G-CSF pretreatment up-regulated CD11b/c expression on circulating polymorphonuclear leukocytes, augmented the recruitment of neutrophils into the lung, and enhanced the phagocytic activity of circulating and recruited neutrophils in both the absence and presence of acute ethanol intoxication. G-CSF inhibited MIP-2 but not TNF-alpha production in the lung. These data suggest that G-CSF may be useful in the prevention or treatment of infections in persons immunocompromised by alcohol.

Chemotactic gradients predict neutrophilic alveolitis in endotoxin-treated rats.

We hypothesized that the intensity of neutrophilic alveolitis is related to establishing a gradient of neutrophil attractant chemokines across the alveolar-capillary barrier. In these experiments, a positive chemokine gradient toward the alveoli was induced by intratracheal instillation of endotoxin in rats (IT LPS). Alteration of the chemotactic gradient was induced by combining IT LPS (0.1 mg/kg) with an intraperitoneal injection of endotoxin (IP LPS, 6.0 mg/kg). Bronchoalveolar lavage (BAL) and peripheral blood cell counts and differentials, and lavage and serum CXC chemokines were measured 4 h after LPS treatment. Compared with IT LPS treatment alone, IP + IT LPS resulted in a 30-fold reduction in neutrophil (PMN) count in BAL and a decreased percentage of PMNs in lavage (from 82 to 24%, p < 0.01). Total lung myeloperoxidase activity, a reflection of total PMN burden, was increased in all three treatment groups compared with the control group, but differences were not apparent between treatment groups. For the rat CXC chemokines MIP-2 and CINC, high concentrations were detected in BAL from both IT and IP + IT LPS groups; however, significantly higher concentrations were found in the sera of rats treated with IP + IT LPS compared with IT LPS alone. The calculated chemokine BAL-serum gradients were significantly higher for both MIP-2 and CINC in the IT LPS group than in the IT + IP LPS or IP LPS group, and correlated with neutrophil influx into the alveolar spaces 4 h after LPS treatment. In addition, the BAL-serum MIP-2 gradient was found to be increased 24 h after IP LPS, which is the time point of peak neutrophilic alveolitis. In summary, these data show that local chemokine gradients predict the intensity of neutrophilic alveolitis after treatment with endotoxin. Interventions to limit neutrophilic alveolitis could either be targeted to block local lung chemokine production or, paradoxically, to increase systemic production of chemokines.

SUPPRESSION OF RENAL INFLAMMATION WITH VITAMINS A AND E IN ASCENDING PYELONEPHRITIS IN RATS

We evaluated the effects of vitamin A and E supplementation alone or in combination with non-steroidal anti-inflammatory drugs (NSAIDs) on the development of inflammation in an animal model of ascending pyelonephritis. Ascending pyelonephritis was induced in adult rats by surgical bladder inoculation with P-pili-forming Escherichia coli. Treatment of pyelonephritic rats was initiated at 72 hours post-infection. Treatment groups included no treatment, or a five day regimen of antibiotic only, antibiotic plus vitamins A and E, or antibiotic, vitamins and either of two NSAIDs. Kidneys were harvested at six weeks post-infection and assessed for histopathologic inflammation. Antibiotic treatment of pyelonephritic rats with vitamins A and E alone or in combination with NSAIDs resulted in significantly less kidney inflammation, as compared with untreated rats or rats treated with antibiotic alone. There was no significant difference in inflammation between animals treated with vitamins alone or vitamins plus NSAIDs. Antibiotic therapy and diet supplementation with vitamins A and E can significantly reduce the inflammation associated with ascending pyelonephritis, suggesting a potential use in the medical management of reflux nephropathy in children.

Effects of a new C5a receptor antagonist on C5a- and endotoxin-induced neutropenia in the rat.

A new C5a receptor antagonist, the cyclic peptide Phe-[Orn-Pro-D-cyclohexylalanine-Trp-Arg], (F-[OPdChaWR]), was tested for its ability to antagonize the neutropenic effects of both C5a and endotoxin in rats. Human recombinant C5a (2 microg kg(-1) i.v.) caused rapid neutropenia, characterized by an 83% decrease in circulating polymorphonuclear leukocytes (PMNs) at 5 min. Administration of F-[OPdChaWR] (0.3-3 mg kg(-1) i.v.), did not affect the levels of circulating PMNs but, when given 10 min prior to C5a, it inhibited the C5a-induced neutropenia by up to 70%. Administration of E. Coli lipopolysaccharide (LPS, 1 mg kg(-1) i.v.) also caused neutropenia with an 88% decrease in circulating PMNs after 30 min. When rats were pretreated with F-[OPdChaWR] (0.3 - 10 mg kg(-1) i.v.) 10 min prior to LPS, there was a dose-dependent antagonism of the neutropenia caused by LPS, with up to 69% reversal of neutropenia observed 30 min after LPS administration. These findings suggest that C5a receptor antagonists may have therapeutic potential in the many diseases known to involve either endotoxin or C5a.

P–13 - The role of nitric oxide on decreasing the hepatic drug metabolizing enzyme activity by bacterial endotoxin in rats

P–13 - The role of nitric oxide on decreasing the hepatic drug metabolizing enzyme activity by bacterial endotoxin in rats

PBN amplifies the production of the anti-inflammatory cytokine interleukin-10 in a rat endotoxin shock model: [formula omitted], H. Sang, G. L. Wallis, C. A. Stewart, Nai-ying Zheng, Guoying Bing, Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA

PBN amplifies the production of the anti-inflammatory cytokine interleukin-10 in a rat endotoxin shock model: [formula omitted], H. Sang, G. L. Wallis, C. A. Stewart, Nai-ying Zheng, Guoying Bing, Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA

O.07 The ratio of N-6 to N-3 fatty acids in parenteral nutrition influences the bactericidal defence of the splanchnic region in an endotoxin rat model

O.07 The ratio of N-6 to N-3 fatty acids in parenteral nutrition influences the bactericidal defence of the splanchnic region in an endotoxin rat model

Chylomicrons alter the hepatic distribution and cellular response to endotoxin in rats.

Chylomicrons (CM) can bind endotoxin (lipopolysaccharide [LPS]), forming CM-LPS complexes, and protect against endotoxic shock and death in rodent models of gram-negative sepsis. The liver appears to play a central role in this process, as demonstrated by the increased uptake of LPS by this organ. We examined the effect of CM on the uptake and cellular response to injected 125I-LPS by hepatocytes and hepatic nonparenchymal cells. Whereas CM increased the uptake of LPS by both hepatocytes and Kupffer cells, the increase was proportionately greater in hepatocytes than Kupffer cells. Importantly, CM-LPS complexes inhibited inducible nitric oxide synthase (iNOS) mRNA expression and NO production in Kupffer cells and endothelial cells, reducing mRNA levels by 45% to 50% as compared with LPS alone. CM-bound LPS also reduced NO production by hepatocytes in response to cytokine stimulation. Lastly, CM-LPS complexes yielded a concentration-dependent inhibition of LPS-induced tumor necrosis factor alpha (TNF-alpha) production by Kupffer cells in vitro. These data indicate that the mechanism by which CM protect against endotoxicity may involve an increased uptake of LPS by hepatocytes. Moreover, uptake of CM-bound LPS by liver cells attenuates the capacity of these cells to respond to proinflammatory stimulation. These results highlight important anti-inflammatory properties of CM.

Increased Plasma P-Selectin Induced by Intravenous Administration of Endotoxin in Rats

We developed a competitive enzyme immunoassay for detection of rat P-selectin and examined temporal changes of plasma P-selectin levels in an endotoxin-induced injury model in rats. Soluble P-selectin was detected in rat plasma after intravenous administration of lipopolysaccharide (LPS), and increased to a maximum level of five-fold over baseline after 24 hours. Plasma P-selectin was partially purified by gel-filtration and was identified as a 122 kDa band by Western blotting. Using the ELISA system we developed, monitoring of plasma P-selectin has become possible in the rat.

Are the capsaicin-sensitive structures of ventral medulla involved in the temperature response to endotoxin in rats?

In chronic experiments on rats pretreated with bilateral microinjection of 25 nl 1% capsaicin to the caudal ventrolateral medulla under ketamine-xylazine-acepromazine anesthesia, an enhancement of the temperature response to intraperitoneal application of 3 μg/kg E. coli lipopolysaccharide as compared to animals who received vehicle to the caudal ventrolateral medulla was found. This is indicative of the involvement of the capsaicin-sensitive bulbar structures in thermoregulatory processes during endotoxemia.

Soluble glucan protects against endotoxin shock in the rat: the role of the scavenger receptor.

Soluble carboxymethyl-b-1,3-glucan (CMG), a possible ligand for scavenger receptors, has macrophage-activating action but lacks the granulomatose inflammatory side effect: it is a promising immunomodulator that may mitigate the severity of sepsis. This motivated us to study in rats the effect of CMG (25 mg/kg), injected into the tail vein at 48 and 24 h prior to the administration of 5 mg/kg Escherichia coli 0127.B8 endotoxin on survival, hemodynamic condition, and, in vitro, on the chemiluminescence of PMNs and macrophages, and on macrophagal tumor necrosis factor (TNF) production. Acetylated low density lipoprotein (AcLDL) clearance in vivo and in vitro binding to macrophages was used to study scavenger receptor function. In the nonpretreated group 9 of 10 rats died during the first 24 h after endotoxin, but all CMG-pretreated rats survived. CMG-pretreatment prevented severe decreases in cardiac output and blood pressure after endotoxin. Chemiluminescence of macrophages and PMNs from CMG-pretreated rats was about two times less (p < .05) than that from nonpretreated ones; the endotoxin induced TNF production by macrophages also decreased. Pretreatment with CMG increased, but coinjection of CMG and AcLDL decreased the AcLDL clearance, while coinjection of endotoxin and AcLDL decreased the survival rate. In vitro AcLDL uptake by macrophages decreased after coinjection with CMG. Our results thus showed that CMG was protective in rat endotoxin shock, which seemed partly connected with enhancement of endotoxin clearance through scavenger receptors and to decreased TNF production.

Superoxide–Mediated Glomerulopathy in the Endotoxin–Treated Pregnant Rat

In the present study the role of superoxide in the glomerular damage in the low–dose endotoxin–infused pregnant rats was investigated. On day 14 of pregnancy, 12 rats were infused for 1 h with 1.0 μg/kg bw endotoxin via a permanent jugular vein cannula. Of these rats, 6 were treated with SOD both prior to endotoxin infusion (7,000 U/kg) and 30 min (7,000 U/kg) and 4 h (14,000 U/kg) after the start of the infusion (SOD rats). The other 6 rats received no SOD treatment (endotoxin rats). Control pregnant rats were infused for 1 h with saline (saline rats; n = 6). Urinary albumin was measured on days 15 and 19 of pregnancy. On day 21, rats were sacrificed and kidney specimens were snap–frozen. Cryostat kidney sections were stained for fibrinogen, ecto–ATP diphosphohydrolase (e–ATPase) activity, polymorphonuclear cells, monocytes and various adhesion molecules on the endothelium and the leukocytes. SOD treatment appeared to significantly prevent the increased urinary albumin excretion and the decrease of glomerular e–ATPase activity which were observed in endotoxin–treated rats. This effect of SOD treatment after endotoxin infusion was associated with a significant inhibition of glomerular monocyte influx and a significant inhibition of adhesion molecule expression (glomerular ICAM–1 and VCAM–1 and leukocyte LFA–1 and VLA–4).The present data suggest that in the endotoxin–infused pregnant rat, production of superoxide in the first few hours after the infusion plays a role in the induction of glomerular damage, leading to albuminuria and diminished e–ATPase expression during the following days.

Substituted 2-iminopiperidines as inhibitors of human nitric oxide synthase isoforms.

A series of analogues of 2-iminopiperidine have been prepared and shown to be potent inhibitors of the human nitric oxide synthase (NOS) isoforms. Methyl substitutions on the 4-position (3) or 4- and 6-positions (8) afforded the most potent analogues. These compounds exhibited IC50 values of 0.1 and 0.08 microM, respectively, for hiNOS inhibition. Substitution with cyclohexylmethyl at the 6-position (13) afforded an inhibitor that showed the best selectivity for hiNOS versus heNOS (heNOS IC50/hiNOS IC50 = 64). Following oral administration, inhibitors were found to decrease serum nitrite/nitrate levels in an in vivo rat endotoxin assay. This series of 2-iminopiperidines were prepared via the described synthetic methodologies. The effect of ring substitutions on potency and selectivity for this class of cyclic amidines as NOS inhibitors is described.

Selective inhibition of cyclooxygenase-2 by NS-398 in endotoxin shock rats in vivo.

The role of cyclooxygenase (COX)-2 was examined using a rat endotoxin shock model and the potency and selectivity of NS-398, a COX-2 selective inhibitor in vitro, for COX-2 activity was examined in vivo. Male Wistar rats (weighing 140-180 g) were used. Lipopolysaccharide (LPS, 1 mg/kg, i.v.) was administered to rats (LPS-treated rats) and expression of COX-1 mRNA and COX-2 mRNA in the aorta and peripheral blood leukocytes was examined by RT-PCR. COX activity was assessed by measuring the plasma 6-keto prostaglandin (PG) F1 alpha, PGE2 and thromboxane (TX)B2 30s after administration of arachidonic acid (AA, 3 mg/kg, i.v.), NS-398 (0.3-100 mg/kg, p.o.) or indomethacin (0.3-3 mg/kg, p.o.) was administered 1 h before the AA injection. COX-2 mRNA was detectable in the aorta and peripheral blood leukocytes at least from 3 to 9 h after the LPS injection but not in non-LPS-treated rats. Plasma 6-keto PGF1 alpha, PGE2 and TXB2 levels after AA injection into LPS-treated rats were significantly enhanced compared to findings in non-LPS-treated rats. NS-398 showed significant inhibition of the increase in PGs in LPS-treated rats, the ED50 values being 0.35 mg/kg for 6-keto PGF1 alpha, 1.5 mg/kg for PGE2 and < 0.3 mg/kg for TXB2. NS-398 even at 100 mg/kg did not significantly suppress the increased PGs levels in non-LPS-treated rats. In contrast, indomethacin significantly inhibited plasma PGs levels after AA injection into LPS-treated rats and non-LPS-treated rats. The ED50 values in LPS-treated rats, determined by 6-keto PGF1 alpha, PGE2 and TXB2 production, were 1.0, 1.3 and 2.3 mg/kg and those in non-LPS-treated rats were 0.42, 0.24 and 0.93 mg/kg, respectively. In a rat endotoxin shock model, expression of COX-2 plays a role in an increase in COX activity. NS-398 showed preferential inhibitory effects on COX-2 activity in vivo. This approach is useful to directly analyze the inhibitory activity of NSAIDs for COX-1 and COX-2 in vivo.

O.57 Omega-3 fatty acids enriched fat emulsions modulate splanchnic blood flow and microbial translocation in a low dose endotoxin rat model

O.57 Omega-3 fatty acids enriched fat emulsions modulate splanchnic blood flow and microbial translocation in a low dose endotoxin rat model

P.67 Improvement of gut blood flow by enteral nutritionsupplemented with glutamine, omega-3 fatty acids and antioxidants in an endotoxin rat model

P.67 Improvement of gut blood flow by enteral nutritionsupplemented with glutamine, omega-3 fatty acids and antioxidants in an endotoxin rat model