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Dispersed equine vascular endothelial cells grown in tissue culture, and freshly isolated neutrophils were used to determine direct effects of endotoxin on cyclooxygenase and lipoxygenase products. Endothelial cells (10(7)/ml) or neutrophils (2 X 10(6)/ml) were incubated with (a) buffer, (b) endotoxin (10 micrograms/ml), (c) endotoxin + flunixin meglumine (10 micrograms/ml), or (d) calcium ionophore, A23187 (10 micrograms/ml). Thromboxane (TxB2), prostacyclin (6-keto-PGF1 alpha), and leukotriene C4 (LTC4) were determined in the incubation fluid by radioimmunoassay. Thromboxane and prostacyclin levels increased in endothelial cells incubated with endotoxin. Treatment with flunixin meglumine prevented the endotoxin-induced release of these cyclooxygenase products to levels below those observed in control cells. Leukotriene production was increased in endothelial cells incubated with endotoxin plus flunixin meglumine. Endotoxin as well as endotoxin plus flunixin meglumine increased the production of prostacyclin and LTC4 by freshly isolated neutrophils. Cells exposed to endotoxin plus flunixin meglumine produced more LTC4 than cells exposed to endotoxin. The data revealed that endotoxin has a direct effect on arachidonic acid metabolism in endothelial cells and neutrophils. Flunixin meglumine reduced the level of cyclooxygenase products but increased the level of lipoxygenase products. Therefore, the well-established beneficial effects of cyclooxygenase inhibitors during endotoxemia may be improved even more if they are used in conjunction with lipoxygenase inhibitors or a combined cyclooxygenase-lipoxygenase inhibitor.

A study was made of flunixin meglumine, an analgesic agent with antiinflammatory and antiprostaglandin activity, for the management of endotoxin-induced cardiovascular derangements. Three groups of 5 ponies each were used: controls--group 1; given endotoxin but not treated--group 2; and given endotoxin and treated with flunixin meglumine--group 3. Shock was induced in anesthetized ponies with IV injection of Escherichia coli endotoxin. Hemodynamic changes were monitored, and regional blood flow was determined at 4 different times, using microspheres labeled with 1 of 4 nuclides. There were extensive vasodilation and decreased blood return to the heart of group 2 ponies, as indicated by decreased mean arterial blood pressure and central venous pressure and by increased heart rate and cardiac output. Blood flow, as determined by radioactive microspheres, to gastrointestinal regions, skeletal muscle, and skin was increased and that to the CNS was decreased. Treatment with flunixin meglumine (group 3 ponies) exerted selective microvascular actions which helped to reverse endotoxin-induced changes. This included the maintenance of mean arterial blood pressure and the enhanced perfusion of vital organs (eg, brain and heart) by preventing extensive vasodilation in the gastrointestinal tract.

An in vitro system of cultured equine endothelial cells was evaluated as a model for endotoxin (ET) exposure in the horse. Primary cell lines from pulmonary vessels and aortas were cultured from tissues of 6 horses. Effects of ET alone with and without serum and in combination with the cyclo-oxygenase inhibitor flunixin meglumine and isolated equine neutrophils were evaluated by transmission electron microscopy. Cells plus serum were incubated with 10, 25, 50, or 100 micrograms of ET/ml of incubation medium for 1, 3, 8, or 24 hours. Cells without serum were cultured for 1 and 3 hours. Flunixin meglumine was used at a concentration of 20 micrograms/ml. Cells also were incubated in the presence of 1,000, 5,000, or 20,000 neutrophils/ml plus ET and in the presence of a combination of ET and flunixin meglumine for 1 or 3 hours. Endotoxin alone did not cause cell damage, and the only evidence of an effect was an increased number of secondary lysosomes at incubation hour 8. At incubation hour 24, cells appeared normal. Endotoxin plus neutrophils caused cells to become round and detach from the growth substrate. Cell pathologic changes included swollen and distorted mitochondria and cytoplasmic vacuolization. Response to the ET plus neutrophil combination was variable and ranged from 5% to 50% of the cells being affected. The variability appeared to have some correlation with cell age, as well as individual preparation of neutrophils.

Twelve male neonatal calves (39 to 50 kg) were allotted to 3 groups of 4 calves each. All calves were anesthetized with halothane, and then Escherichia coli endotoxin was given intravenously (3 times) and intraperitoneally (3 times) during a 6-hour period. Group-1 calves were untreated, group-2 calves were pretreated with a low dose of flunixin meglumine (1.1 mg/kg of body weight), and group-3 calves were pretreated with a high dose of flunixin meglumine (4.4 mg/kg). In calves of group 1, the mean systemic arterial blood pressure (MABP) and cardiac output (CO) decreased, but pulmonary arterial pressure increased after the initial intravenous and intraperitoneal injections of endotoxin. In calves of this group, these changes were accompanied by increased plasma thromboxane B2 (TxB2) concentration. During this period, increased plasma TxB2 concentration or hemodynamic changes were not detected in calves of groups 2 and 3. Only calves of group 1 had altered hemodynamics early in the experiment; however, after 6 hours, calves of all 3 groups had similarly decreased CO and MABP. In calves of the untreated group, plasma 6-keto-prostaglandin (PG)F1 alpha concentration increased steadily from the beginning of the experiment until 3 hours later. The CO and MABP were low at the time when serum 6-keto-PGF1 alpha concentration was high; however, these 2 measurements also were low in treated calves who did not have correspondingly high plasma 6-keto-PGF1 alpha concentration. Regional blood flow analysis did not reveal correlations between prostanoid concentrations and altered blood flow to selected tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma concentrations of the stable hydrolysis products of thromboxane (Tx) A2, TxB2, and prostacyclin (6-keto-prostaglandin F1 alpha; PGF1 alpha) were evaluated in 22 neonatal calves given endotoxin (0.5 microgram/kg) by slow IV infusion over a 5-hour period. The effect of pretreatment with bovine antiserum to a mutant of Escherichia coli O111:B4 (J-5) on plasma concentrations of these eicosanoids was determined. Plasma concentrations of TxB2 and 6-keto-PGF1 alpha were increased in response to endotoxin infusion. The mean plasma concentrations of TxB2 peaked 1 hour after endotoxin infusion began and was significantly increased from base line through 8 hours. Mean plasma 6-keto-PGF1 alpha concentration increased more slowly and was increased from base line only at 2 hours after endotoxin infusion began. Administration of bovine J-5 antiserum had no significant effect on endotoxin-induced thromboxane and prostacyclin production.

Ponies with electromagnetic blood flow transducers implanted around the main pulmonary and left main coronary arteries, were used to evaluate effects of chronic sublethal endotoxin on cardiac output (CO), stroke volume, and left coronary blood flow (LCBF). Plasma thromboxane (TX), as indicated by TXB2, prostacyclin as indicated by 6-keto-prostaglandin (PG) F1 alpha, and hematologic and blood chemical values also were evaluated. Over 24 hours, 2 groups of ponies were given progressively increasing IV and intraperitoneal doses of Escherichia coli lipopolysaccharide (LPS) at 0, 6, 12, and 18 hours. Group 1 was not treated and group 2 was treated with flunixin meglumine, before each LPS insult. Initial LPS inoculation in group 1 led to 10-fold increases in TXB2 and 6-keto-PGF1 alpha values by 30 and 90 minutes, respectively. These eicosanoid values returned to base line by 6 hours after each insult. Although repeated LPS injections stimulated recurring high plasma concentrations of 6-keto-PGF1 alpha, TXB2 production became less with each successive LPS insult. Cardiac output decreased to 55% to 60% of base-line values in association with increased 6-keto-PGF1 alpha values. Left coronary blood flow could not be evaluated accurately. Severe lactic acidosis developed in group 1. Group-2 ponies remained clinically normal, indicating protection of cardiovascular function and peripheral perfusion with flunixin meglumine. Seemingly, flunixin meglumine helped to maintain acceptable cardiovascular function and tissue perfusion during endotoxemia. Flunixin meglumine given to healthy ponies had no effect on cardiovascular function.(ABSTRACT TRUNCATED AT 250 WORDS)

A study was conducted to determine whether body fluids undergo a net shift from one compartment to another during endotoxin-induced shock in the pony, and whether flunixin meglumine alters these endotoxin-induced changes in the volumes of body fluid compartments. Total blood, RBC, and plasma volumes were determined, using 51Cr-labeled RBC and PCV that were corrected for trapped plasma. Total body water was measured by distribution of 3HOH. Arterial blood pressure was measured directly, using a blood pressure transducer. Treatment (flunixin meglumine, 1.1 mg/kg of body weight) was given to 6 of the 12 ponies 1 minute before an IV injection of Escherichia coli endotoxin (100 micrograms/kg of body weight, LD100). The PCV and RBC volume increased in both groups; however, the hemoconcentration was less in flunixin meglumine-treated ponies. In nontreated ponies, total blood volume and plasma volume decreased significantly during the first hour after endotoxin administration. In treated ponies, total blood volume did not vary significantly, and plasma volume decreased only slightly. In both groups, the increase in PCV was apparently due to splenic contraction, which increased the number of circulating RBC. Hemoconcentration was further increased in nontreated ponies by the loss of plasma into the interstitial space. Flunixin meglumine reduced plasma loss, minimized hemoconcentration, and maintained normal blood volume. Total body water remained constant in treated and nontreated ponies.

Endotoxic shock was induced in 5 ponies by intraperitoneal injections of 20, 40, 60, 80, and 80 micrograms of Escherichia coli endotoxin (LPS)/kg of body weight at 0, 6, 12, 18, and 24 hours, respectively. At 24 hours, the ponies also were given 20 micrograms of LPS/kg via catheter in the left ventricle of the heart. A 2nd group of 4 ponies was given 1.1 mg of flunixin meglumine (FM)/kg, IV, at 6, 12, 18, and 24 hours just before the corresponding LPS injection. Two hours after the 24-hour LPS injection, the ponies in both groups were anesthetized, the lungs were perfused with fixative, and portions of the pulmonary arteries and veins and right and left ventricles were prepared for scanning and transmission electron microscopy. In ponies that were given only LPS, some areas of pulmonary vascular endothelium appeared normal when compared with untreated controls, but other areas had disoriented endothelial cells or had varying amounts of sloughing, which ranged from focal areas of a few cells to large areas of denuded endothelium. Ponies treated with FM before LPS had less severe and less extensive endothelial cell damage. In both groups, leukocytes were attached to areas of the vessel wall; endothelial cell damage was greater in these regions. Administration of FM before LPS administration attenuated the LPS-induced endothelial cell damage.

To evaluate the effect of certain drugs on hematologic changes, blood chemical values, and survival in endotoxin shock, anesthetized ponies were given (IV) endotoxin (Escherichia coli O55:B5) and then treated as follows: Group A ponies--given a saline infusion at 5 minutes and at 3 hours after they were given endotoxin; group B ponies--given flunixin meglumine at 5 minutes and at 3, 6, 9, and 24 hours after they were given endotoxin; group C ponies--treated with dexamethasone; and group D ponies--treated with prednisolone at 5 minutes and at 3, 9, and 24 hours after they were given endotoxin. Anesthesia was maintained for 4 hours, after which time the ponies were allowed to recover. Throughout the experiment, samples of blood were collected for blood gas, hematologic, and blood chemical values. The endotoxin effects were seen in the 4 groups: lactic acidosis, prolonged coagulation times, leukopenia, hemoconcentration, and elevated blood chemical values. Although none of the treatments prevented the effects of endotoxin, changes were less severe and survival times were longer in ponies treated with flunixin meglumine.