Fig. 2

Neuronal density in OHCs at different timepoints of B1 deprivation. OHCs challenged with B1 deprivation lost approximately 71% of neurons from the 5th day on. Surprisingly, between the 7th and 14th day of culture without B1, a pulse of neurogenesis followed by a new massive loss of neurons was observed. A Left Y axis shows the neuronal density means (NeuN+/mm²) of the CT (controls) (n = 12) and TD (thiamine deficiency) (n = 12). Groups were compared with the bidirectional analysis of variance (ANOVA) test followed by the Tukey’s multiple comparisons test. Data were expressed as mean ± standard deviation (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001). Statistical differences represented by the red horizontal bars and asterisks refer to the variation in the TD group between the different culture time points. Gray vertical bars and asterisks refer to the intergroup differences (CT versus TD) at the same timepoint. Right Y axis shows the OHC area means of the CT (n = 12) and TD (n = 12). Groups were compared with the bidirectional ANOVA test followed by the Tukey's multiple comparisons test. B BrdU assay identify proliferating cells. The graph shows post-mitotic neurons density (BrdU+ neurons/mm²) between days 7 and 10 of B1 deprivation. N sample: CT (n = 3) and TD (n = 4). Two-tailed T test was used (*P ≤ 0.05) and data were expressed as mean ± standard deviation. C Confocal microscopy images (×60) of OHCs show mature neurons (NeuN+) (in red), cell nuclei (DAPI) (in blue), and BrdU (in green). In detail, a digital zoom (×8) of typical BrdU staining. CT controls, TD thiamine deficiency (scale bar = 50 μm)