Fig 1.
Progressive hearing loss in Enpp1asj/asj mice.
ABR threshold means are shown for Enpp1asj/asj mice and littermate control mice, tested at the ages of 3 (n = 6 mutant ears/8 control ears), 6 (n = 10/10), 8 (n = 6/6), 12 (n = 22/16), 18 (n = 8/6), 26 (n = 10/8), and 30 (n = 12/14) weeks. Starting from 6 weeks of age, the mutant mice exhibit significantly higher mean ABR threshold values at all the stimulus frequencies tested (click, 8 kHz, 16 kHz, 32 kHz) compared to those of the littermate controls. Thresholds of mutant mice continue to increase with age and by 18 weeks most of the Enpp1asj/asj mice are profoundly hearing impaired. The increase in 32 kHz ABR thresholds of the control mice is due to the B6 background, a strain known to exhibit age-related hearing loss starting at high frequencies. Error bars indicate standard errors of the mean.
Table 1.
ABR thresholds of asj/asj mutant mice
Table 2.
ABR thresholds of +/+ or +/asj control mice
Fig 2.
Otitis media in Enpp1asj/asj mice.
A, B: Representative images of pathological changes in the middle ears of Enpp1asj mutant mice (B) compared with controls (A) at the age of 5 months. The middle ear cavity (MEC) of Enpp1asj mutant mice is filled with effusions (black asterisk), fibroblastic and amorphous tissue masses (long arrows), and inflammatory cells (arrow head). Ectopic mineralization of the otic capsule is also evident in mutant mice (open arrow in B). Control mice show a clear middle ear cavity without fibroblastic proliferation (A). C, D: The thickness of the middle ear epithelium (double headed arrows) is greater in Enpp1asj mutant mice (D) than controls (C). E, F: The stapedial artery wall (arrows) is thicker in Enpp1asj mutant mice (F) than controls (E). G, H: Representative images of discharge in mutant mice (H) and littermate controls (G) at the age of 5 months. The external ear cavity of the mutant mice is filled with discharge (red asterisk), while control mice have a clear external ear canal and easily observable tympanic membrane (black asterisk). Scale bars: A, B = 200μm, C, D = 50μm, E, F = 80 μm.
Fig 3.
Development of otitis media in Enpp1asj/asj mice.
A-D: 6–12 week time course of middle ear pathology of Enpp1asj mutant mice (B, C, D) compared with a 12-week-old control (A). At 6 weeks of age (B), an aqueous effusion (black asterisk) and a slightly thickened epithelium (arrow) are observed in the middle ear cavity of the Enpp1asj mutant mice. At 8 weeks (C), the middle ear cavity of the Enpp1asj mutant mouse is filled with an aqueous effusion (black asterisk) and contains amorphous tissue masses (green asterisk). The middle ear epithelium is much thickened (arrow), and the otic capsule exhibits regions of ectopic mineralization (arrowhead) with adjacent fibroblastic proliferation. At 12 weeks (D), the middle ear cavity of mutant mice is filled with pus-like secretions (black asterisk), a thickened epithelium (arrows), and an amorphous tissue mass (green asterisk). E, F: Eustachian tube morphology of mutant (F) and control (E) mice at 12 weeks of age. In mutant mice, an amorphous tissue mass (green asterisk) is present near the orifice of the Eustachian tube (empty arrow), which may impede Eustachian tube function and lead to an accumulation of effusion in the middle ear cavity. G, H: Cochlear morphology and round window membranes of mutant (H) and control (G) mice. Cochlear morphology is grossly normal in Enpp1asj mutant mice compared with controls; however, the round window membrane (black arrow) of mutant mice is thicker. MEC: middle ear cavity. Scale bars: A, B, C, D, E, F = 200μm, G, H = 100μm.
Table 3.
Histological assessment of the middle ears of the Enpp1 mutant and control mice.
MEE: Middle Ear Effusion; EH: Epithelial Hyperplasia; IC: Inflammatory Cells; ETO: Eustahian Tube Opening; EM: Ectopic Mineralization; CGC: Cluster of Goblet Cells.
Fig 4.
Increased density of goblet cells in Enpp1asj/asj mice.
Mayer's mucincarmine method was used to visualize goblet cells (stained red) in the epithelia lining the Eustachian tube (A, B) and middle ear cavity (C, D) of mutant and control mice. A, B: Few goblet cells are seen in the epithelia lining the Eustachian tube of littermate control mice (A, empty arrow points to Eustachian tube, insert shows higher magnification of the Eustachian tube epithelia). By contrast, goblet cells are present at high density in the epithelium lining the Eustachian tube of Enpp1asj mutant mice (B, empty arrow points to Eustachian tube, magnified inset shows goblet cells, marked by arrows, in the Eustachian tube epithelia). C, D: More goblet cells are present in the epithelia in the middle ear cavity (MEC) of the asj mutant mouse (arrows in D) than in the control (C). Scale bars: A, B = 200 μm, C, D = 50 μm, A and B inserts = 20 μm.
Fig 5.
Scanning electron micrographs of epithelium lining the middle ear cavity.
Compared with littermate controls (A), the middle ear epithelium in 1-month-old Enpp1asj mutant mice (B) shows an increased number of goblet cells (red asterisks). Scattered goblet cells (green asterisks) and cilia are seen in the mucociliary epithelia of control mice at 6-months of age (C); however, a layer of mucin obscures most cilia and goblet cells (arrows) in age-matched Enpp1asj mutant mice (D). Scale bars = 25μm.
Fig 6.
Calcification of middle ear structures (or tympanosclerosis) in Enpp1asj/asj mice.
In the control mice (A), the outer ear canal is clear without any discharge. The tympanic membrane appears to be transparent and malleus (blue asterisk) is clearly visible. In the age matched mutant mice (B), surrounding bone must be removed to expose the tympanic membrane, most of which is completely covered by discharge. White patches (inside black dashed lines) were observed on the tympanic membrane of the mutant mice (B). The ossicle bones of the age matched control and mutant mice appear to have similar morphology (C-D), but malleus and incus are fused in the mutant mice (D, red arrow). M: Malleus; I: Incus; S: Stapes. Alizarin red staining reveals extensive mineralization in the stapedial artery wall (green asterisk) in Enpp1as/asjj mice (F), but not in the control mice (E).