Cells in the BALF were counted, the cell BAY 87-2243? suspension was stained by Wright Giemsa, and two hundred cells were classified according to cell morphology using a light microscope. The results are expressed as the numbers of each type of cell population in one ml of BALF. Lung histopathology Lungs were infused via trachea with 1 ml of 10% neutral formalin. Sections of 5 um thickness were prepared and stained with hematoxylin eosin. To determine the severity of inflammatory cell infiltration, peribron chial eosinophil cell number was blindly counted and the severity was evaluated using a 5 point scoring sys tem described previously. Briefly, the scoring sys tem was 5 marked, 4 moderate, 3 medium, 2 mild, 1 minimal and 0 no eosinophil cells.

Lung and brain homogenates preparation The procedure of lung and brain homogenates prepara tion was used as described in details in our previous study. Briefly, after BALF, the lung artery was per fused with PBS to remove blood cells. Then the left lung and hemisphere were scissored into Inhibitors,Modulators,Libraries 1 mm1 mm1 mm cubes and homogenized in ice cold Hanks Inhibitors,Modulators,Libraries buf fer. Samples were diluted with methanol to precipitate proteins, and centrifuged at 3500g for 10 min at 4 C. The supernatant was diluted with ultra pure water to obtain a final methanol concentration of 25%, and extracted on a Sep Pak C18 column prewashed with 20 uL of ethanol and 20 uL of water. After 200 ng PGB2 was added as Inhibitors,Modulators,Libraries internal standard, samples were washed through the column with 0. 1% edetic acid, ultra pure water, 15% ethanol, petroleum ether and methanol in sequence.

The methanolic Inhibitors,Modulators,Libraries fraction was dried under argon and stored at 80 C, and the residual mixture was dissolved in methanol before RP HPLC assay. To mini mize absorption of LTB4, only tubes, vials and pipette tips made of polypropylene were used. All steps of the procedure were performed under 4 C. Measurement of LTB4 content in tissue homogenization using RP HPLC system RP HPLC was performed using a HP1100 separation module consisting of multiple solvent delivery systems, and equipped with UV detector, analytical pump, on line degasser, and column thermostat. Samples were separated by a Waters symmetry C18 reversed phase column which was protected by a Waters sentry C18 guard column. Absorbance of the column effluent was monitored using a dual wave length absorbance detector adjusted to 270 nm for LTB4.

Peak areas were calculated with a chromatogra phy manager program. The mobile phase for LTB4 was methanolwateracetic acid adjusted to pH 5. 6 with NH4OH. A flow Inhibitors,Modulators,Libraries rate of 1 mLmin at 35 C for LTB4 was used. Based on the peak areas, the LTB4 con centration of biological samples tested was estimated using the internal standard PGB2. Results are expressed as ng of LTB4 per g wet weight of lung or brain. Plasma ACTH and CORT assay Blood samples were collected from orbital vein selleck compound at two time points 30 min after LTB4 i. c. v.