Figure 1.
ABR recordings reveal hypoacusis in AspalacZ/lacZ mice at 4 months.
Representative ABR waveforms from (A) Aspawt/wt and (B) AspalacZ/lacZ mice elicited by click stimulus. Note the substantial reduction of P4 and absence of P5 in the mutant waveform. Arrows indicate ABR thresholds. (C) ABR responses of AspalacZ/lacZ mice have higher thresholds for the click stimulus (29.0±1.0 db, n = 5) compared with Aspawt/wt controls (23.0±1.2 dB, n = 5; p = 0.027). ABR hearing threshold was not significantly different at 16 kHz (Aspawt/wt 13.0±1.2 dB; AspalacZ/lacZ 18.0±2.5 dB; p = 0.143) or at 24 kHz (Aspawt/wt 27.0±3.4 dB; AspalacZ/lacZ 27.0±2.0 dB; p = 0.782). (D) Analyses of peak latencies in response to click stimuli (30 dB above threshold) showed P1 were similar between AspalacZ/lacZ mice (1.62±0.03 ms, n = 5) and Aspawt/wt controls (1.59±0.03 ms, n = 5; p = 0.633), yet with significant differences for P2 (AspalacZ/lacZ, 2.49±0.07 ms; Aspawt/w, 2.25±0.04 ms; p<0.001) and P3 (AspalacZ/lacZ, 3.40±0.08 ms; Aspawt/w, 3.08±0.04 ms; p<0.001). Analysis of data was precluded for P4 and P5 due to the absence of the corresponding features in ABR waveforms from aspalacZ/lacZ animals. All data were analyzed by two-way ranked ANOVA and Holm-Sidak post-hoc comparison.
Figure 2.
Normal cochlear anatomy and outer hair cell function in AspalacZ/lacZ mice.
Representative transmitted light laser scanning microscopy images of midmodiolar sections of the cochleae of Aspawt/wt (A) and AspalacZ/lacZ mice (B) revealed normal gross anatomical organization of the cochlea including preserved organ of Corti (o/C), spiral ganglia (sg), Reissner's membrane (rm), scala vestibuli (SV), scala media (SM), scala tympany (ST), cochlear nerve (cn). (C, D) Close-up of β-III tubulin-expressing spiral ganglia neurons (sgn; red) showed no abnormalities. DAPI (blue) was used to label nuclei. (E, F) High power images of the organ of Corti, with the innervation of the hair cells (neurofilament immunofluorescence, red) overlaid on the transmitted light images. Inner hair cells (ihc) are appropriately innervated by spiral ganglion neurites. DAPI (blue) was used for counterstain. ohc, outer hair cells; Dc, Deiters' cells. (G) 2f1-f2 DPOAEs recorded at different primary tone frequencies showed normal thresholds, implying normal OHC electromotility and cochlear amplifier response in AspalacZ/lacZ mice compared with Aspawt/wt controls. Two-way ANOVA on ranked data and Holm-Sidak post-hoc comparison analyses showed no genotype differences for 8 kHz (AspalacZ/lacZ, 14.0±1.9; Aspawt/w, 13.0±1.2, p = 0.658), 16 kHz (AspalacZ/lacZ, 22.0±2.5; Aspawt/w, 22.0±2.5, p = 1.0), or 24 kHz (AspalacZ/lacZ, 40.0±6.1; Aspawt/w, 42.0±1.2, p = 0.556). n = 5; Bars: A–B, 100 µm; C–F, 20 µm.
Figure 3.
Schwann cells of the VIII cranial nerve express ASPA.
ASPA-immunoreactivity (red) was detected in cell bodies of Schwann cells in the VIII cranial nerve from Aspawt/wt animals (A) but was absent in AspalacZ/lacZ samples (B). The neurite staining detected by the neuronal marker β-III tubulin (green) appears similar in both control (C) and mutant (D). The Schwann cell density assessed by DAPI staining was also comparable between the control (E) and the mutant (F). Merged images for the control (G) and mutant nerve (H). Bars: 20 µm. Arrows indicate Schwann cell somata.
Figure 4.
Central histopathology of AspalacZ/lacZ mice.
(A–L) Representative overviews of coronal sections from Aspawt/w mice and AspalacZ/lacZ mice, stained with H&E (purple) and Luxol Fast Blue (blue) to visualize gross tissue integrity and myelination, respectively. Sections from forebrain (A–D), midbrain (E–H), and hindbrain (I–L) regions illustrate that vacuolization in AspalacZ/lacZ mice is moderate in the neocortex but prominent in posterior regions including the hippocampus, thalamus, cerebellar white matter, and dorsal brainstem. Widespread demyelination is observed by reduced intensity of the Luxol Fast Blue signal, particularly in white matter. Boxes in (I–L) indicate the brainstem region containing the cochlear nucleus and VIII cranial nerve. (I’–L’) Higher magnification of the areas containing the cochlear nucleus (CN). Arrowheads indicate myelination deficits in the VIII cranial nerve (K’–L’). Bars: A–L, 1 mm; I’–L’, 100 µm.
Figure 5.
Hypomyelination of central auditory structures in ASPA-deficient mice.
Luxol Fast Blue staining of brain sections from Aspawt/wt mice (A, C, E, G, I) and AspalacZ/lacZ mice (B, D, F, H, J) reveals demyelination in the brainstem (A–H) and midbrain (I, J) of the mutant. Hypomyelination is severe in white matter of the VIII cranial nerve adjacent to the cochlear nucleus (A, B), or axon tracts of the lateral lemniscus (G, H). Differences were less evident in inherently myelin-poor grey matter of the cochlear nucleus (C, D), superior olivary complex (E, F), and inferior colliculus (I, J). Note that Luxol Fast Blue-treated sections were counterstained with Cresyl Violet. VIII cranial nerve, VIII; cochlear nucleus, CN; superior olivary complex, SOC; lateral lemniscus, LL; inferior colliculus, CIC. Bars: 20 µm.