Mice were placed on a low-thiamine diet (2 vs. 22 mg/kg in normal chow) for 11, 19, or 26 days. By 11 days, cochlear neural output was significantly reduced in transporter-null mice, as seen in the threshold shifts for auditory brainstem responses (ABRs), especially for frequencies < 22 kHz (Fig. ). There was little further threshold shift after 19 or 26 days on low thiamine. Wild-type mice on the same low-thiamine diet showed no threshold elevation, even after 26 days (Fig. ). Shifts in distortion product otoacoustic emissions (DPOAE) thresholds were also larger in transporter-null mice than in wild-types (Fig. ). However, in the transporter-null mice the DPOAE shifts were smaller than the shifts in ABR thresholds (p
= 0.003, F
= 86.634, for group differences by two-way ANOVA). Compare, for example, the ABR threshold shifts of ~50 dB at 11.3 and 16 kHz in the transporter null mice after 26 days on low-thiamine to the DPOAE shifts measured at the same time, which were only 10–20 dB. This mismatch between ABR and DPOAE threshold shifts suggests that a significant component of the cochlear dysfunction in the null mice arises at, or “downstream” of, the inner hair cell (e.g., Liberman et al. 1997
Fig. 1 Threshold shifts for wild-type and transporter-null mice placed, at age 45 days, on a low-thiamine diet for 11, 19, or 26 days, as indicated in the key. Until age 45 days, all animals in this cohort were maintained on a normal (more ...)
To assess effects on the auditory nerve fibers per se, we measured wave 1 latencies from the ABRs in Figure . When latencies are plotted in dB re ABR threshold, there are no significant differences between wild-type and transporter-null animals at 11 days after thiamine restriction, a time when ABR thresholds are already elevated by as much as 40 dB (Fig. ).
Fig. 2 Latencies for ABR wave 1 are unchanged in transporter-null mice, even when thresholds are elevated by 30–40 dB. Data are for 22.6-kHz tone pips from the cohort of animals shown in Figure : for transporter nulls, data are from (more ...)
When another group of transporter-null and wild-type mice were placed on a low-thiamine diet with 3 mg/kg thiamine (rather then 2 mg/kg used for first cohort) no significant threshold differences were observed, by either ABR or DPOAE measures, even after 70 days on the diet (data not shown). Similarly, when a third group of transporter-null mice was maintained on a diet with normal thiamine levels, no threshold shifts were seen (Fig. ; differences between wild-type and transporter-null thresholds were not significant: p = 0.5, F = 0.505, two-way ANOVA).
Fig. 3 Transporter-null mice maintained on a diet with normal thiamine levels showed no threshold shifts. (A) ABR data from transporter-null animals plotted as threshold shifts; (B) ABR data replotted as absolute thresholds and compared to the wild-type data (more ...)
A final cohort of wild-type and transporter null animals was placed on 2 mg/kg thiamine at age 45 days and then returned to a normal thiamine diet after 19 days, to see if the threshold shifts could be “rescued.” As shown in Figure , the initial threshold shift in the transporter null animals seen after 19 days on low thiamine was, on average, 20 dB at the lower frequencies. There was a slight improvement in average thresholds over the next 5 weeks; however, thresholds did not return to normal levels. Variability was high in this group, because one of the null animals showed minimal threshold shift at 19 days (differences between “initial” and “rescued” thresholds were not significant: p = 0.089, F = 6.193, two-way ANOVA).
Fig. 4 Partial recovery in a rescue experiment in which transporter-null mice were maintained on a low-thiamine diet for 19 days (initial shift), and then switched back to normal diet for 35 days. For the first 45 days of life, these (more ...)
Cochleas were plastic-embedded to allow the high-resolution evaluation of all cochlear structures at the light microscopic level. The most striking histopathology in the transporter-null mice maintained on low thiamine was selective loss of inner hair cells (Fig. ). The overall architecture of the organ of Corti remained remarkably normal; thus inspection with low-power objectives might fail to reveal the pathology. In the high-power view (insets), the nucleus and stereocilia of a normal inner hair cell are clearly visible in the wild-type (filled arrows) and clearly absent in the transporter null ear. Supporting cells in the inner hair cell area are present in both cases (unfilled arrows point to their nuclei). There was no sign of spiral ganglion cell loss, nor was there an obvious loss of peripheral axons in the osseous spiral lamina; however, the longest postdiet-onset survival was only 36 days. The stria vascularis, spiral ligament, and spiral limbus all looked normal in transporter-null mice.
Fig. 5 When transporter-null mice were maintained on a low-thiamine diet until ABR thresholds began to shift, their cochleas showed selective loss of inner hair cells. (A, C) Place-matched images from the upper basal turn of a transporter-null and a wild-type (more ...)
Cytocochleograms prepared from mice in the cohort in Figure reveal a regular progression of hair cell loss with increasing duration of dietary thiamine restriction (Fig. ). Hair cell loss is minimal in wild-type animals (Fig. A) and in transporter null mice sacrificed after 11 days on low thiamine (Fig. B), although ABR shifts were visible at this time in the latter group. By 19 days on low thiamine, the transporter-null mice show loss restricted to the inner hair cells in the apical half of the cochlea (Fig. C). By 26 days, inner hair cell loss was virtually complete throughout a large midcochlear region; however, the basal 1/3 of the cochlea showed no cell loss (Fig. D). In the animals maintained on the diet for 36 days, inner hair cell loss had spread slightly toward the basal cochlear extreme; however, there was more extensive loss of outer hair cells (OHC) as well (Fig. E).
Fig. 6 Patterns of hair cell loss in representative mice from the cohort in Figure : i.e., animals maintained on a low-thiamine diet for variable periods (see header to each panel). (A) Data from a wild-type ear, (B–E) data from four transporter-null (more ...)
The time progression of hair cell loss is explicitly plotted in Figure , in which the fractional survival of inner or outer hair cells is averaged over the apical 2/3 of the cochlea. The data suggest that loss of inner hair cells dramatically increases in transporter-null mice between 19 and 26 days after the onset of low-thiamine diet, and that OHC loss progresses at a significantly slower rate.
Fig. 7 Loss of inner and outer hair cells in wild-type and transporter null ears, as a function of days on low-thiamine diet. All animals were placed on the diet at 45 days of age after initial maintenance on a normal diet. Each point represents data (more ...)