TNF-a but not IL-1a: ANIMALS AND METHODS
Reagents: All chemicals were obtained from Sigma Chemical Co (St Louis, Missouri). D-GalN was obtained from Sigma Chemical Co. PGE1 (alprostadil) was purchased from The Upjohn Co (Kalamazoo, Michigan). Antigen and polyclonal antibody obtained from Genzyme Diagnostics (Cambridge, Massachusetts) were used for quantification of TNF-a and IL-1a by ELISA. Nitrate reductase and lactate dehydrogenase (LDH) were obtained from Boehringer Mannheim (Mannheim, Germany). Animals and treatments: Male Wistar rats (175 to 225 g) were housed in a climate-controlled (21°C) room under a 12 h light-dark cycle and were given tap water and standard laboratory rat chow ad libitum. Animal care standards and experimental protocols were according to the Guide for the Care and Use of Laboratory Animals. Rats were divided into four groups of 30 according to treatment: control, D-GalN, PGEj, and PGE^+D-GalN. Each group was further divided into another six groups according to the time point of the kinetics (0, 1, 2.5, 5, 10 and 15 mins) in relation to the last treatment. All operations were performed under general (pentobarbital) anesthesia. The abdomen was entered through a midline incision to observe clearly the hepatic lobules and portal vein. To restrain and accelerate liver response, treatments (in a volume of 500 pL) were infused as a bolus through the cannulated portal vein instead of intraperitoneally. Control and PGE1-treated animals received the corresponding volume of 0.9% sodium chloride or PGEj (250 pg/kg) dissolved in 20% ethanol. D-GalN was also dissolved in 0.9% sodium chloride, and the dose (200 mg/kg) administered was adjusted according to the mode of administration compared with higher doses used in intraperitoneal administration. In the PGEj+D-GalN group, PGE1 was infused 10 mins before D-GalN infusion.
The dose of PGEj was in the range used in several experimental studies and the total infusion dose used during clinical trials. To check the effect of 20% ethanol as a solvent of PGEj, an equal amount of ethanol to that used in the PGEj-treated groups was infused to another group of rats 10 mins before infusion of the volume of 0.9% sodium chloride. Serum was obtained from blood collected after puncturing the suprahepatic vein. Alanine aminotransferase (ALT) was assayed immediately, but TNF-a, IL-1 a and nitrite/nitrate (NOx) were measured afterwards in frozen serum samples. No differences in ALT, TNF-a, IL-1 a and NOx concentrations were observed between the control group and the group treated with 20% ethanol in the time intervals studied. ALT quantification in rat serum: ALT was assayed with a commercial kit obtained from Merck (Darmstadt, Germany) based on a routine method. TNF-a quantification in rat serum: TNF-a was measured in serum by competitive ELISA. The standards used were calibrated against the National Institute for Biological Standards and Control (NIBSC) reference standard. Wells of ELISA plates were coated with 200 pL of TNF-a (2.5 ng/mL) for 1 h at 37°C. Afterwards, wells were blocked with a solution containing 2% bovine serum albumin in 10 mM phosphate buffer solution comprising 0.05% Tween in phosphate buffered saline (PBS-T) pH 7.4. At the same time, samples and polyclonal antibodies (32.5 ng/mL) were incubated for 2 h at 37°C, transferred to wells and incubated for 2 h at37°C. Afterwards, wells were washed with PBS-T and incubated with biotinylated secondary antibody (125 pg/mL) for 1 h at 37°C. Furthermore, wells were washed with tris buffer (100 mM) pH 7.6 and incubated with commercial streptavidin-alkaline phosphatase (MD, Granada, Spain) solution for 30 mins at 37°C. Finally, the plates were washed and incubated with the alkaline phosphatase substrate (Sigma 104, Sigma Chemical Co) and read after 1 h with the 405 nm filter in a titrated Organon Teknika 510 ELISA reader (Organon Teknika, Turnhout, Belgium).
IL-1 a quantification in rat serum: IL-1 a was measured in serum by competitive ELISA. The standards used were calibrated against the NIBSC reference standard. Wells of ELISA plates were coated with 200 pL of IL-1-a (5 ng/mL) for 1 h at 37°C. Afterwards, wells were blocked with a solution containing 2% bovine serum albumin in PBS-T pH 7.4. At the same time, samples and polyclonal antibodies (2 ng/mL) were incubated for 2 h at37°C, transferred to wells and incubated for 2 h at37°C. Afterwards, wells were washed with PBS-T and incubated with biotinylated secondary antibody (17.5 ng/mL) for 1 h at 37°C. Furthermore, wells were washed with tris buffer (100 mM) pH 7.6 and incubated with commercial streptavidin-alkaline phosphatase (MD, Granada, Spain) solution for 30 mins at 37°C. Finally, the plates were washed and incubated with the alkaline phosphatase substrate (Sigma 104) and read after 1 h with the 405 nm filter in a titrated Organon Teknika 510 ELISA reader. NOx quantification in rat serum: NOx measurements were used as an index of nitric oxide production. Nitrate was reduced to nitrite after incubation of samples with nitrate reductase (0.2 U/mL), flavin adenine dinucleotide (5 mM) and reduced nicotine adenine dinucleotide phosphate (50 mM) for 20 mins at 37°C. The reaction was stopped by adding sodium pyruvate (10 mM) and LDH (24 mg/mL) for 5 mins at 37°C, and precipitated with 1.4% zinc sulphate. Total nitrite was reacted with Griess reagent (1% sulpha- nilamide, 2.5% phosphoric acid, 0.1% N-naphthyl-ethyl- ene-diamine) for 10 mins at 37°C and read with the 540 nm filter in a titrated Organon Teknika 510 ELISA reader. Statistical analysis: Results are expressed as means ± standard errors. Data were evaluated by one-way ANOVA, with four groups per variable. Because the homogeneity of variances assessed by the Bartlett test was sufficient, groups were subjected to the multiple comparison least significant differences test. Statistical significance was set at P<0.05.
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