Immunohistochemical staining of TLR₄ in trachea and effects of sevoflurane on TLR₄ protein and mRNA expression after LPS challenge <i>in vivo</i>.

<p>(<b>A</b>) Representative micrographs of TLR₄ in trachea of mice anesthetized with phenobarbital or sevoflurane in absence or presence of LPS challenge. The presence of TLR₄ was observed only in the smooth muscle cell layer, whose cell membrane and cytoplasm stained tan as detected by horseradish peroxidase–labeled antibodies. Scale bars = 50 μm. (<b>B</b>) Histogram for quantification of TLR₄ in smooth muscle cell layer. The mean optical density values (MOD) were calculated after normalizing against PN. (<b>C</b>) Representative western blot and quantitative analysis of TLR₄ isolated from smooth muscle cell layer after LPS challenge. (<b>D</b>) Quantitative real-time PCR of TLR₄ mRNA expressions in isolated smooth muscle cell layer. (<b>E</b>) Representative western blot and quantitative protein analysis of NF-κB in nuclear extracts from smooth muscle cell layer after LPS challenge. (<b>F</b>) Quantitative real-time PCR of NF-κB mRNA expressions in smooth muscle cell layer after LPS challenge. Sevoflurane prevented significant increases in protein and mRNA expressions of TLR₄ and NF-κB in isolated smooth muscle cell layer from mice after LPS challenge. The relative integral density values (IDVs) were calculated after normalizing against GAPDH in each sample and presented as relative protein expression units. Data are presented as means ± SEM (n = 8 per group). *<i>P</i> < 0.05 versus PN; #<i>P</i> < 0.05, versus PL.