A novel model of spontaneous otitis media with effusion (OME) in the Oxgr1 knock-out mouse

Abstract

Objective: A novel mouse model with a specific genetic mutation in a G protein coupled receptor (GPCR) encoded by the Oxgr1 gene results in a predisposition to spontaneous otitis media with effusion. As a primary component of interest in OME, mucin expression was examined in this model to assess expression as compared to wild type animals and suitability as a murine model of OME.

Method: Mutant (Oxgr1(-/-)) and wild-type (Oxgr1(+/+)) mice between ages of 2 and 5 months were examined by otoscopy and auditory brainstem response (ABR). Histology changes in the middle ear were evaluated. Expression of mucin genes in the middle ear epithelium was determined using RT-PCR and quantitative PCR.

Result: Otoscopic exam showed signs of inflammation in 82% of mutant mice. Significant elevated ABR thresholds were detected in mutant mice indicating hearing loss. Histology analysis of the middle ears demonstrated the presence of inflammatory cells, changes in the mucosal epithelium, and middle ear fluid. RT PCR using universal primers for bacterial 18s rRNA suggested the absence of bacteria in the middle ear. The knockout mice demonstrated expression of Muc1, Muc2, Muc3, Muc4, Muc5AC, Muc5B, Muc9, Muc10, Muc13, Muc15, Muc16, Muc18, Muc19 and Muc20. There was a trend of increase in Muc5B and Muc19 expression in the middle ear of the knockout mice compared to that of wild-type. There was no significant change in the level of Muc2, and Muc5AC was expressed at a level below the detection limit of quantification.

Conclusion: Development of a murine model with genetic defect has several attractive features. The rate of OME in these animals is high at 82%. It is clear that this OME is related to histopathologic changes in the middle ear epithelium of these knock-out mice. Induction of mucus effusion is evident though the viation in dysregulation of GFM does exist in this non-challenge study condition. The underlying cause of these differences between individual animal requires further investigation. Given this, the Oxgr1(-/-) model is likely to be an ideal model to examine mucin regulation in MEE and potentially develop novel GPCR-specific targeted interventions to regulate these processes.

Figure 1

Genotype verification in litter mates by PCR amplification of tail genomic DNA. (A) Specific wild type allele primers generated a 294bp fragment in the homozygote wild type (oxgr1^+/+) and heterozygotes (oxgr1^+/−) animals. Specific mutant allele primers amplified a 378bp fragment of the selection cassette that generated homozygotes null (oxgr1^−/−) via homologous recombination. (B) Restriction analysis of the amplified fragments from wild type and mutant alleles. The amplified wild type fragment yielded a 152 and 142bp in SmaI digestion, and a 150 and 144bp in XmaI digestion. The amplified mutant allele subjected to BamHI digestion resulted in 177 and 201bp, while HindIII reduced the fragment to 168 and 210bp. M; 50–1000bp DNA ladder, U; uncut, S; SmaI, X; XmaI, B; BamHI, and H; HindIII.

Figure 2

Comparison of auditory brainstem thresholds in affected ears of oxgr1^−/−, as indicated by otoscopic exam, and wild type mice. Error bar indicated standard error of mean. Two Way Repeated Measures Analysis of Variance showed a significant hearing loss (p<0.001).

Figure 3

Histology of the middle ear from oxgr1⁻/⁻ mice compared to wild type mice. (A,D) H&E staining of middle ears from wild type (WT) mice revealed no significant in inflammatory or chronic reactive changes. In A, notice the intact ciliated epithelium overlaying delicate lamina propria abutting bone. In D, notice the normally scant, wispy eosinophillic luminal material (open white arrow) that has survived histology processing and the lightly mineralized ossicular bone (black asterisk). The tympanic membrane (TM) is thin and delicate. (B, C, E) H&E staining of middle ear from mutant mice demonstrate evidence of chronic tissue injury and host response. In B and C, notice clusters of large foamy macrophages (long black arrows) adherent to injured ciliated epithelium and set within abundant densely eosinophillic fluid (C and E, open white arrow). Focal deposits of golden brown hemosiderin are present within the macrophages and subepithelial stroma (B, see small green arrows in enlarged inset). The submucosa (B) is fibrotic. Ossicular bone (black asterisks) with the middle ear cavity (MEC) is much more heavily mineralized in the mutant mice (E) than in the wild type control (D).

Figure 4

Conventional RT PCR identified mucin genes expressed in middle ear mucosa of wild type (w) and mutant (m) mice. This expression pattern was 100% correlated to the mouse and human middle ear epithelium culture models.

Figure 5

Relative quantification of gel forming mucin genes in the temporal bone of oxgr1^−/− mutant demonstrated comparable level of muc2. The mRNA of muc5AC was observed at lower level than the detection limit with cycler thresholds higher than 40 on both wild type and mutant. A trend of increasing in muc5B and muc19 level was observed in mutant mice.