AZ 960 was collected and the residual volume assessed to ensure that normal agent delivery had occurred

The microcatheter was secured to the surrounding bone of the bulla. The osmotic pump was placed in the subcutaneous pocket of the back. The incision was then closed. A 28 1 2 gauge syringe needle was then used to inject 105 CFU ml NTHI through the left tympanic membrane. After the animals were sacrificed, the osmotic pumps were collected and the residual volume assessed to ensure that normal agent delivery had occurred. Histology. Animals were AZ 960 sacrificed 5 days after surgery. Following decapitation under deep anesthesia with a combination of ketamine, xylazine, and acepromazine intramuscularly, the temporal bones were immediately removed and the bullae were perfused with 4 paraformaldehyde in phosphate buffer for 6 h. The bullae were decalcified in 8 EDTA for 2 weeks, dehydrated in graded ethanol, and embedded in paraffin.
Sections were cut, stained with hematoxylin and eosin, and evaluated under a KU-0063794 light microscope. The ME mucosa was examined, and any temporal bones in which the microcatheter was not in its proper placement in the subepithelial compartment were discarded. Each specimen was photographed with an RT digital color camera. For both the SP600125 and vehicle groups, seven ME mucosae were evaluated. Mucosal thickness was calculated at the end of the microcatheter and at a location approximately 500 m from the catheter, using SPOT computer software calibrated to the appropriate magnification. The thickness data from the two locations were compared using the Wilcoxon signed rank test. A P value of 0.05 was considered significant. Statview 5.0 was used for statistical analysis. Detection of apoptosis.
Terminal deoxynucleotidyltransferase mediated dUTP biotin nick end labeling was performed with a TACS TdT DAB kit, following the manufacturer,s instructions. Three guinea pigs were sacrificed 72 h after surgery and bacterial inoculation. Following decapitation under deep anesthesia with a combination of ketamine , xylazine, and acepromazine intramuscularly, the temporal bones were immediately removed and the bullae were perfused with 4 paraformaldehyde in phosphate buffer for 1 h. The bullae were decalcified in 8 EDTA for 2 weeks, embedded in an optimal cutting temperature compound, and sectioned with a cryostat. Sections were digested with proteinase K at a concentration of 20 g ml for 15 min. Endogenous peroxidase activity was quenched with 3 H2O2 for 5 min. The slides were immersed in terminal deoxynucleotidyltransferase buffer.
TdT, 1 mM Mn2, and biotinylated deoxynucleoside triphosphates in TdT buffer were added to cover the sections and incubated in a humidity chamber at 37 for 60 min. The slides were washed with PBS and incubated with streptavidin horseradish peroxidase for 10 min. After being rinsed with PBS, the slides were immersed in DAB solution for 2 min. All specimens were lightly counterstained with methyl green. The ME mucosa was examined, and any temporal bones in which the microcatheter was not in its proper placement in the subepithelial compartment were discarded. Each specimen was photographed with an RT digital color camera.