β-defensin And Toll-like Receptor Correlates With The Pathogenesis Of Otitis Media With Effusion: An In Vivo Study In Two Rodent Models

Part one Expression ofβ-defensin mRNA in a rat model of otitis media with effusion induced by LPSObjective:To compare mRNA of rBD-1,rBD-2 in normal tubotympanum and inflammatory mucosa induced by lipopolysaccharide(LPS), which can be a foundation for further investigating the role of beta-defensin in the pathogenesis of otitis media with effusion.Methods:A total of 48 Sprague-Dawley rats (200-250g), free of middle ear disease, were used. The right bulla was exposed by neck incision and blunt separation and then 30μL LPS solution(lmg/ml) was injected in three groups(36 rats totally),30μl physiological saline was injected into the right bulla in another 12 rats(control group), the left bulla was intact to be normal group. Tubotympanum mucosa were obtained quickly after decapitation in 12 rats of control group and 36 rats inoculated by LPS at 1,3,7day(12 rats each time). Reverse transcription polymerase chain reaction (RT-PCR)was performed to detect the mRNA of rBD-1,rBD-2.Results:rBD-1mRNA and rBD-2mRNA were detected in normal rat tubotympanum and the expression of rBD-1 mRNA was more significant than rBD-2(P<0.01). rBD-2mRNA increased dramatically in the inflammatory mucosa (P<0.01) whereas rBD-1 mRNA did not augmented significantly. The up-regulation of rBD-2 gene was notable at 1,3 day group and declined to normal at 7 day group.Conclusion:rBD-1 may be part of defense system of normal rat middle ear, and the induced expression of rBD-2 is related to the clearance of invading pathogens. Part two Expression pattern of TLR-2,TLR-4 in a mouse model of otitis media with effusion induced by PAFObjective:To establish a mouse model of otitis media with effusion induced by PAF and evaluate the expression of TLR-2 and TLR-4 in tubotympanum mucosa of this model.Methods:50 BALB/c mouse(19-21g), free of middle ear disease, were used.5μL solution of PAF (1μg) injected into the bilateral middle ear transtympanically,5μl physiological saline was injected into the right bulla in another 10 mouse as a control, the left bulla was intact to be normal group. The tubotympanum mucosa was obtained quickly after decapitation in 8 mouse of control group and 32 mouse inoculated by PAF at 1,3,7,10day(8 mouse every time). Reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting and immunofluorescence (IF) staining were used to detect TLR-2 and TLR-4 in the tubotympanum.Results:Both TLR-2 and TLR-4 were detected in normal mouse tubotympanum mucosa and TLR-2 and TLR-4 increased in the inflammatory mucosa (P<0.01).WB and IF demonstrated that both TLR-2 and TLR-4 existed in normal mucosa and augmented in inflammatory mucosa(P<0.01).The green fluorescence of TLR-2 was significant in Eustachian tube mucosa and tympanic cavity mucosa, however, the red fluorescence of TLR-4 was significant in tympanic membrane, tympanic cavity mucosa and inflammatory cells in the middle ear effusion.Conclusion:This study demonstrated that the injection of PAF into the middle ear of mouse without any obstructed Eustachian tube can cause inflammation and effusion. This model can be used in further study to elucidate the interaction of PAF and TLR in the pathogenesis of OME.