Key inflammatory cytokines and ion homeostasis genes evaluated by PCR array.

<p>Key inflammatory cytokines and ion homeostasis genes evaluated by PCR array.</p

Comparison of treatments on cytokine gene expression.

<p>A comparison was made of untreated controls, <i>H flu</i> only mice, and the two steroid treatments to determine how effective the steroids were is returning homeostasis genes back to a normal level. Claudin 4 was upregulated by <i>H flu</i> and its levels were not reduced by the steroids. The remaining genes were significantly up or down regulated by the <i>H flu</i> treatment, and steroid treatments returned expression of these genes back to normal levels.</p

Histologic comparison of the murine middle ear 24 hours after inoculation with <i>H flu</i> before (A) and after (B) glucocorticoid treatment.

<p><b>A:</b> Note the extensive fluid (arrows) in the middle ear (ME) space, particularly against the round window membrane (left arrow) adjacent to the inner ear (IE) scala tympani <b>B:</b> The murine middle ear 24 hours after inoculation with <i>H flu</i> and treatment with transtympanic prednisolone three hours later. The middle ear is nearly clear of fluid except for a small amount against the round window membrane.</p

Cytokine gene expression for the control animals that received no treatment three hours and six hours after transtympanic inoculation with <i>H flu</i>.

<p>This reflects fold change in gene expression seven and ten hours, respectively, after inoculation compared to animals receiving no bacteria. All inflammatory cytokine genes were significantly upregulated with the exception of IL-10 and TNFα. By ten hours, most were still elevated, although the trend was for their gradual decline in the overexpression seen at the earlier time point.</p

Ion homeostasis gene expression for the control animals that received no treatment three hours and six hours after transtympanic inoculation with <i>H flu</i>.

<p>This reflects fold change in gene expression seven and ten hours after inoculation compared to animals receiving no bacteria. Although there were some genes that were expressed at higher than normal levels (claudin 4, gap junction a1, aquaporin 1), the majority of homeostasis genes in the middle ear were suppressed in expression. Many other genes were reduced to 10–30% of normal expression (Clcnka, claudin 14, aquaporins 2 and 3, most postassiumchannels, and ENac channels Scnn1b and Scnn1g), but these did not reach statistical significance due to variability. In general, the expression levels seen in the three hour controls were similar to those in the six hour controls.</p

Impact of steroid treatment on ion homeostasis gene expression.

<p>Mice received either prednisolone (Pred) or aldosterone (Aldo) three hours after after transtympanic inoculation with <i>H flu</i> and harvested four hours later (total seven hours post inoculation). With the exception of Gja1, Aqp1, and Tmprss3, most of the genes controlling ion and fluid balances were upregulated by the steroids.</p

Impact of steroids on cytokine gene expression.

<p>Mice received either prednisolone (Pred) or aldosterone (Aldo) three hours after after transtympanic inoculation with <i>H flu</i> and harvested four hours later (total seven hours post inoculation). Expression of several of the cytokine genes was reduced compared to <i>H flu</i> only mice shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119228#pone.0119228.g001" target="_blank">Fig. 1</a>. In general, both Pred and Aldo were similar in their suppression of the genes impacted by the bacteria. Although a few genes showed significant effects with Pred and not Aldo (IL-6, Cxcl2, TNFα), the differences in effects of the two steroids were minimal.</p