The total number of cells was counted from a standard area in the vicinity of the end of the microcatheter, a standard area at a location approximately 100 m from the catheter, a standard area at a location approximate 200 m from the catheter, and a standard area at a location approximate 500 m from the catheter, using SPOT computer software calibrated to the appropriate magnification. The ratio of TUNEL positive cells to the total number of cells counted was determined, and this ratio was multiplied by 100. This was defined as the percentage of TUNEL positive cells. The percentages AZD1480 of TUNEL positive cells in the six locations were compared using one way analysis of variance followed by Tukey Kramer,s t test post hoc to determine statistical differences. A P value of 0.05 was considered significant. Statview 5.0 was used for statistical analysis. PCNA staining. A Zymed PCNA staining kit was used as a marker for cell proliferation, because proliferating cell nuclear antigen levels are elevated in actively proliferating cells during the S, G2, andMphases of cell mitosis.
Tissue preparation was performed as outlined above for TUNEL, and staining and visualization were done according to the manufacturer,s instructions. Briefly, the slides were processed through 3 H2O2 for 10 min to quench endogenous peroxidase activity. Sections were then blocked using the kit blocking solution and incubated with biotinylated mouse anti PCNA Ab for 60 Flavopiridol min at room temperature, followed by streptavidinperoxidase incubation for 10 min and incubation for 2 min with the DAB chromogen solution. All specimens were lightly counterstained with methyl green. The percentage of PCNA labeled cells was determined using the same methodology as that for the percentage of TUNEL positive cells. Cytotoxicity assay.
To assess the level of cytotoxicity of the dosage of SP600125 used in the mini osmotic pump, the bullae of six male albino Hartley guinea pigs weighing 300 to 350 g were dissected, divided, and cultured in the manner described above. The explants were cultured with the JNK inhibitor SP600125 or with vehicle only. All explants were maintained in culture for 3 days. The explants were then exposed for 2 h to a 0.1 protease solution dissolved in PBS. Enzymatically dissociated cells were harvested, centrifuged at 1,000 g for 5 min, and resuspended in Dulbecco,s modified Eagle,s medium. Aliquots of cell suspension were mixed with 0.4 trypan blue solution and placed on a hemocytometer. Data 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.
RESULTS Levels of total JNK and pJNK. Figure 1A illustrates the presence of total JNK and pJNK in the control ME mucosae and in the infected ME mucosae at various time points, as assessed by Western blotting. A pJNK signal was not detected in the control ME mucosae. pJNK1 was first observed 6 h after bacterial inoculation, was maximal between 24 h and 72 h, and returned to undetectable levels by 7 days. pJNK2 was first observed at 1 h and was also maximal from 24 h to 72 h after bacterial inoculation. pJNK2 decreased at 5 to 7 days but remained well above the control level. Figure 1B shows band density ratios of pJNK to total JNK. The ratio of pJNK1 to total JNK1 showed a high degree of phosphorylation at 6 h, a maximum between 24 h and 72 h, and a decline from 5 to 7 days. The ratio of pJNK2 to total JNK2 was maximal between 6 h and 48 h and moderate from 72 h to 7 days.