Since the aim in the study was to find out the bactericidal activity as will likely be determined by viable cell count and not the survival, the endpoint was selected to be 6 hours just after the initiation of therapy. Mice re ceiving mixture therapy received 0. 1 mL of AMP, promptly followed by 0. 1 mL of AZM. These dosing intervals had been selected so as to simulate the in vivo effi cacy of quick term high dose remedy from the drugs in humans. Untreated SP infected animals had been regarded as as manage and received identical volume of isotonic saline. MPO activity as a marker of neutrophil infiltration Myeloperoxidase enzyme activity was analyzed as index of neutrophil infiltration inside the lung tissue, be trigger it’s closely associated with all the quantity of neutrophil present in the tissue.
Blood free of charge lung homogenates was homogenized and centrifuged at 3000 ? g for 30 minutes at four C. MPO activity was estimated against a standard curve selleck chemicals produced with commercially obtainable MPO, by techniques previously described. Lung vascular permeability The Evans blue permeability assay was applied to quantify lung capillary permeability. Evans blue avidly binds to serum albumin and may therefore be made use of as a tracer for transcapillary flux of macromolecules. Evans blue was injected in a tail vein 30 min before the sacrifice. Lungs had been homogenized in two ml of potassium phosphate buffer. Evans blue was extracted by incubating samples in 4 ml of formamide at 60 C for 24 h, followed by centrifugation at five,000 ? g for 30 min. The concentration of Evans blue was esti mated by dual wavelength spectropho tometry, which permitted correction of optical densities for contaminating heme pigments.
As a result, the following formula was made use of Cytokine levels in lung For cytokine measure ments, lung homogenates were lysed in lysis buffer pH 7. four consisting of 300 mM NaCl L, 15 mM TRIS L, 2 mM MgCl2 L, two mM Triton X one hundred L, 20 ng pepstatin A mL, 20 ng leupeptin mL, and 20 ng aprotinin mL, and were centrifuged at 1500 selleckchem ? g for 15 min at four C, the supernatant was frozen at 20 C, till cytokine measurement by ELISA as per producers protocol. Sample preparation for cytokine measurement from serum Blood samples have been transferred into micro centrifuge tubes and permitted to clot at 4 C followed by centrifuga tion at 3000 ? g for 5 min at four C. The supernatant pale yellow colored serum was pipette out carefully together with the support of micropipettes into fresh micro centrifuge tubes, labeled and used for cytokine evaluation.
Serum from dif ferent groups had been normalized to the protein content material by Bradford process just before the assay and levels of cytokines have been determined by Sandwich ELISA based on the suppliers in struction in a Bio Rad ELISA Reader. Expression of Cox two in lung tissue Expression of cyclooxegenase 2 in lung tissues was determined by immunoblotting by techniques described elsewhere.