18, p

18, p. higher concentration of (2.5 108 CFU). Further studies revealed that only animals receiving indomethacin prior to displayed elevated plasma and liver cytokines at early time points (0.5 and 1 h), suggesting prostaglandins control early inflammatory cytokine production. Overall, these data indicate prostaglandins mainly mediate the early rise in plasma corticosterone, while inflammatory cytokines contribute to maintaining levels of corticosterone at later on time points. (0111:B4; ATCC 15746; American Type Tradition Collection) was rehydrated and produced over night in 40 ml of brain-heart infusion broth (DIFCO Laboratories) at 37C at 5% CO2. Bacterial cultures were then aliquoted into 1 ml brain-heart infusion broth with 10% glycerol and freezing at ?20C. All studies used bacteria from these stock cultures. One day time prior to experimentation, stock cultures were thawed and cultured over night in 40 ml of brain-heart infusion broth at 37C TMPRSS2 and 5% CO2. Bacteria were quantified by extrapolating from previously identified growth curves. Cultures were centrifuged for 20 min at 3,000 AB05831 rpm, and supernatants were discarded. Bacteria were resuspended in sterile 0.9% saline at a concentration of 1 1 108 CFU/ml for and and (2.5 107 CFU for and and 2.5 108 CFU for and and and tail vein blood was collected at designated time points. Rats were softly wrapped inside a towel and lightly secured having a Velcro strap prior to tail blood collection. A lateral tail vein was nicked in the posterior end of the tail using a scalpel knife, and the tail was stroked from anterior to posterior to facilitate the movement of blood. Approximately 200C300 l of blood was collected in an Eppendorf snap-cap tube within 60C90 s of eliminating the animal from your cage. Blood was centrifuged for 10 min at 10,000 rpm, and plasma was collected and stored at ?80C until time of assay. Cells Collection For and = 6C8/group) were injected intraperitoneally with either saline or (2.5 107 CFU) and euthanized by decapitation 0.5, 1, 2, 4, or 24 h later. One or two saline-control animals were euthanized at each time and offered like a 0-h time point. Trunk blood was collected for AB05831 measurement of cytokines, ACTH, corticosterone, and PGE2. A small specimen of liver was quickly harvested, and cytokines was measured by ELISA. Study 2. A baseline sample of tail vein blood was collected prior to drug and administration. Animals (= 6/group) were injected intraperitoneally with either vehicle or indomethacin (5 mg/kg) 30 min prior to intraperitoneal administration (2.5 107 CFU). Tail vein blood was then collected at 1, 2, and 4 h after challenge for measurement of corticosterone. PGE2 was also measured in blood samples to verify blockade of PGE2 formation by indomethacin. Study 3. A baseline sample of tail vein blood was collected prior to drug and administration. Animals (6C9/group) were injected intraperitoneally with either saline or IL-6 antibody (4.2 g/kg) 30 min prior to intraperitoneal administration (2.5 107 CFU). Tail vein blood was collected at 1, 2, and 4 h AB05831 after challenge for measurement of corticosterone. Study 4. A baseline sample of tail vein blood was collected prior to drug and AB05831 administration. Animals (6C9/group) were injected with either DMSO or indomethacin 30 min prior to intraperitoneal challenge (2.5 108 CFU). Tail vein blood was collected at 1, 2, and 4 h after challenge for measurement of corticosterone. Study 5. Animals (6C9/group) were injected with either AB05831 DMSO or indomethacin 30 min prior to intraperitoneal saline or (2.5 108 CFU) administration. Animals were killed by decapitation at 0.5 and 1 h, and trunk blood was collected for measurement of cytokines, corticosterone, and PGE2 by ELISA or enzyme immunoassay. A small specimen of liver was quickly harvested for measurement of cells cytokines. Statistical Analysis Data from on numerous mediators of HPA axis activation, were analyzed using a 1 6 ANOVA across time (0, 0.5, 1, 2, 4, and 24 h). In and 0.05 was used in all instances as a measure of significance to reject the null hypothesis. RESULTS Study 1: Time Course of Mediators Involved.