Figure 4 shows the typical microscopic appearances of the healthy guinea pig ME mucosa, bacterially inoculated ME mucosa, and bacterially inoculated ME mucosa treated with vehicle or SP600125 in vivo. Inhibition of mucosal hyperplasia by SP600125 was observed in a restricted region around the microcatheter end. Figure 5 shows a quantitative analysis of the effects of subepithelial delivery of vehicle or SP600125 on mucosal hyperplasia in bacterially inoculated guinea pig MEs in vivo. Continuous delivery of SP600125 ARRY-142886 to the subepithelial compartment locally inhibited epithelial and stromal hyperplasia, decreasing the thickness by 19.4 and 18.0 , respectively. In contrast, delivery of vehicle had no effect on mucosal hyperplasia. Inhibition of cell proliferation and increase in apoptosis by SP600125 in vivo. Figure 6 shows the typical microscopic appearance of TUNEL staining and PCNA immunohistochemistry in bacterially inoculated ME mucosae treated with SP600125 or vehicle in vivo.
An increase in TUNEL positive cells and a decrease in PCNA immunoreactivity by SP600125 were observed in a restricted region around the microcatheter end. Decreased cell proliferation was also observed in a region 0 to 300 m adjacent Agomelatine to the catheter. Apoptosis was increased only very near the catheter and involved primarily polymorphonuclear cells. In contrast, delivery of vehicle had no effect on TUNEL staining or PCNA immunoreactivity. Figure 7 shows a quantitative analysis of the effects of subepithelial delivery of SP600125 on mucosal hyperplasia in bacterially inoculated guinea pig MEs in vivo. Continuous delivery of SP600125 to the subepithelial compartment locally increased the percentage of TUNEL positive cells only in the immediate catheter area, not in areas 100 m, 200 m, and 500 m from the catheter.
In contrast, continuous delivery of SP600125 to the subepithelial compartment locally decreased the percentage of PCNA labeled cells in the catheter area, and also at 100 m and 200 m, compared with that 500 m from the catheter. There was no significant difference in either variable at any location when vehicle alone was delivered. Cytotoxicity assay for 1 mM SP600125. We observed no evidence of mucosal cell cytotoxicity by 1 mM SP600125 in the region at the end of the microcatheter supplied by the miniosmotic pump. In mucosal explants, the percentage of viable cells detected by trypan blue exclusion 72 h after 1 mM SP600125 treatment was 89.6 2.1 . In the explants maintained in medium alone, the percentage of viable cells was 88.
8 1.8 . OM is a common disease and the leading reason for prescribing antibiotics during childhood. Mucosal hyperplasia is a major feature of OM and can contribute to its symptoms and to irreversible ME disease. It is therefore of interest to determine how mucosal hyperplasia is regulated in the ME. To our knowledge, this study is the first to provide evidence that ME mucosal hyperplasia involves signaling by the JNK MAPK cascade. Our Western blotting and cytochemical data indicate that inactive JNK is present in the healthy ME mucosa and that JNK activation occurs during OM. Moreover, the time course of JNK activation is well related to the kinetics of mucosal hyperplasia. As previously reported, the rat ME mucosa is maximally thickened by 4 days after NTHI inoculation, and it recovers substantially by day 8.