Inner and outer hair cells were damaged in an ototoxicity mouse model

Since aminoglycosides are important as a first and second agent in the treatment of many serious infections28,30, an aminoglycoside-induced ototoxicity model was generated by the application of kanamycin and furosemide in this study. Before 5-aza treatment, screening of IHC and OHC damage was performed to confirm degeneration of hair cells and determine the time for 5-aza treatment. The numbers of IHCs and OHCs were screened throughout the cochleae from 3 days to 14 days after deafening. Mouse cochlear samples of 3, 7, 10 and 14 days post deafening were collected throughout the cochleae and received immunofluorescence using hair cell specific anti-Myosin VIIa antibodies (Fig. 1a). As being consistent with other studies31,32, OHCs seemed to be more sensitive to ototoxicity, and were completely lost approximately 3–7 days post deafening, whereas IHCs were entirely damaged around 10–14 days after deafening (Fig. 1b). Because cell counting data demonstrated that over 90% OHCs had been damaged 3 days post deafening and also because we focused on regeneration of OHCs in this study, 5-aza was injected into mice 3 days post deafening. After determining the time for 5-aza injection, the timeline of the experimental procedures was designed and shown in Fig. 1c.

Figure 1 Loss of hair cell (HC) in chemically-damaged mature mouse inner ears. (a) Cochlear sections show HCs in normal hearing mice (arrows), whereas HCs are lost following chemical damage (arrowheads). (b) Quantitative study shows HC survival following chemical damage. In normal mouse cochlea, there are one row of IHCs and three rows of OHCs. Thus, the percentage of IHC survival is calculated by (number of IHCs/1) × 100%. The percentage of OHC survival is calculated by (number of OHCs/3) × 100%]. Scale bar: 50 µm. (c) The diagram shows the experimental design. The day of mice being deafened was set as experimental day 0. 5-aza or saline was injected into the mouse cochlea 3 days post deafening. Mice were followed up for 1–6 weeks. During one-week, two-week, four-week and six-week post-surgery, one group of animals were sacrificed, and their cochleae were cryosectioned to evaluate HCs. New HCs characterization and 5-aza concentration study were performed on animals two-week after 5-aza treatment. Full size image

Auditory hair cells were regenerated by Dnmt1 inhibition

To understand whether the Dnmt1 inhibitor-5-aza stimulates the regeneration of OHCs, 5-aza or saline (control solution used to dissolve 5-aza) was injected into the left inner ear of chemically-deafened wildtype mature mice via the round window (Fig. 1c). Animals were maintained for two weeks after treatment to observe potential new hair cell regeneration (Fig. 1c). Double labeling of Myosin VIIa and F-actin was used to evaluate the presence of hair cells (HCs). The right cochleae (non-treated side) of the 5-aza treatment group were also kept and immunostained with Myosin VIIa to ensure the death of OHCs (data not shown). The cochlear cryosection of normal mice showed one IHC and three OHCs, double labeled by Myosin VIIa and F-actin (arrow and arrowheads in Fig. 2a). In the saline-treated group, the hair cell protein Myosin VIIa or F-actin immunostaining was not observed in the organ of Corti two weeks after saline injection (arrow and arrowheads in Fig. 2a). The deafened mouse cochlear section was also absent of Myosin VIIa or F-actin immunostaining (arrow and arrowheads in Fig. 2a). However, in the 5-aza group cells in the organ of Corti expressed both Myosin VIIa and F-actin (arrow and arrowheads in Fig. 2a). Basilar membrane surface preparation showed the number of Myosin VIIa-expressing HCs in the 5-aza group was greater than the saline-treated group, in which HCs were totally absent (arrow and arrowheads in Fig. 2b).

Figure 2 5-aza injection stimulates the generation of new HCs in a deafened mouse model. (a) Cochlear and organ of Corti (OC) images show expression of the hair cell protein Myosin VIIa (Myo7a) in the normal hearing mouse (arrow for IHC and arrowheads for OHCs), but not in saline-treated deafened mouse cochlea (arrow and arrowhead indicate IHC and OHC area respectively, without expression of HC protein Myosin VIIa). Two weeks post 5-aza injection into deafened mouse cochleae, cells express Myosin VIIa in the OC (arrows for IHCs and arrowheads for OHCs), suggesting new HC generation. Scale bar: 50 µm in cochlear section overview; 20 µm in OC highlight. (b) Basilar membrane surface preparations were obtained from mice two weeks after 5-aza or saline injection. Apical turn basilar membrane surface preparation shows Myosin VIIa-expressing HCs in normal hearing inner ear, but not in saline-treated deafened mouse inner ear. However, new HCs are found in 5-aza-treated deafened mouse inner ear. Arrows indicate the IHC area, whereas arrowheads suggest the OHC area. Scale bar: 20 µm. (c) Quantification of HCs in different cochlear turns in basilar membrane surface preparation two weeks after 5-aza or saline injection into deafened mice. One-way ANOVA was performed to compare the difference among the three groups, which showed a significant difference in all three cochlear turns (P < 0.01). Full size image

In the quantitative assay using whole mount study of counting HCs per 100 µm length of the basilar membrane, apex, middle and basal turns were separately quantified. In the apex turn of basilar membrane, 50 ± 6, 2 ± 3 and 28 ± 7 HCs per 100 µm were found in the normal hearing, saline- and 5-aza-treated groups respectively (Fig. 2c). In the middle turn, 55 ± 5, 2 ± 2 and 37 ± 9 HCs per 100 µm were found in the normal hearing, saline- and 5-aza-treated groups respectively (Fig. 2c). In the basal turn, 50 ± 5, 4 ± 2 and 14 ± 5 HCs per 100 µm were found in the normal hearing, saline- and 5-aza-treated groups respectively (Fig. 2c). One-way ANOVA was performed to compare the difference among the three groups. All three turns showed significant difference among the normal hearing, saline- and 5-aza-treated groups (Supplemental data Table 1). Within the 5-aza-treated group, there are significant differences among the apex, middle and basal turns (Supplemental data Table 1). Thus, it is concluded from the quantification of basilar membrane preparation that compared with the saline-treated group, there is a significant number of regenerated HCs in the 5-aza-treated group. In three cochlear turns, the middle turn showed best results of hair cell regeneration compared with apex and basal turns.

Further immunofluorescence study showed that regenerated OHCs expressed other hair cell proteins including Myosin VI and Pou4f3 (Fig. 3). These cells simultaneously expressed several hair cell specific proteins in multiple labeling immunofluorescence (Fig. 3), indicating that these cells are likely HC-like cells. These data suggest that new HCs may be observed in the deafened mouse following 5-aza treatment.

Figure 3 Newly-generated HCs express multiple hair cell proteins. New hair cell generation is observed two weeks following injection of 5-aza into the deafened mouse inner ear. These cells simultaneously express multiple hair cell specific proteins Myosin VIIa, Myosin VI and Pou4f3. Myosin VIIa and Myosin VI are found to co-express in the new HC-like cells (arrows). Myosin VIIa and Pou4f3 co-labeling is found in some cells (arrows), whereas some cells only express either Myosin VIIa (arrowheads) or Pou4f3 (double arrow). Scale bar: 50 μm in cochlear section overview; 20 μm in organ of Corti (OC) highlight. Full size image

Determine the 5-aza concentration that is optimal for promoting new HC generation

It was shown in our previous in vitro study that 40 µM was suitable for in vitro application of 5-aza while higher concentration caused cell death24. To determine the 5-aza concentration optimal for new HC generation in vivo, 0.4, 1, 4 and 40 mM 5-aza were injected into deafened mouse inner ears. New HCs were found in 12.5%, 37.5%, 75% and 25% of animals in these four groups two weeks post injection respectively (Fig. 4a,b). The chi-square statistic of the four-group comparison was 7.4667 and P = 0.058421, which was statistically insignificant. In a two-group comparison, the chi-square statistics of 0.4 vs 4 mM and 4 vs 40 mM groups were 6.3492 (P = 0.011743) and 4.0 (P = 0.0455) respectively, whereas the other two-group comparisons were statistically insignificant (P > 0.05; Supplemental data Table 2). In a quantification study to evaluate the average number of new HCs in each concentration, one-way ANOVA and Tukey post hoc test showed that there was no significant difference among the four concentration groups (P = 0.7415). Although not statistically significant, 4 mM group show a higher percentage of animals bearing new HCs and more HCs in cochlear sections (Fig. 4b, n = 8 mice in each group). Thus, 4 mM may be the concentration optimal for HC generation and was selected for the following studies.

Figure 4 Generation of new hair cells by 5-aza is concentration dependent. (a) Deafened mice received 0.4, 1, 4 and 40 mM 5-aza injection and maintained for 2 weeks after injection. New Myosin VIIa-expressing HCs are observed in deafened mouse inner ear in 5-aza treated inner ear (arrows for the IHC area and arrowheads for the OHC area). Flask-like shape cells are observed in the IHC area (arrows), whereas columnar-shaped cells are found in the OHC area (arrowheads). Scale bar: 50 µm in cochlear section overview; 20 µm in organ of Corti (OC) highlight. (b) Quantification of the number of new OHCs in cochlear section in each 5-aza concentration group. Full size image

New HCs were survived for at least 6 weeks

After determination of the optimal concentration of 5-aza, the survival time of new HCs was investigated by an additional set of animal experiment. Animals were deafened using the previous methods, and 4 mM 5-aza was applied to the deafened mice, which were kept for 1, 2, 4 and 6 weeks. New HCs were found in 37.5%, 75%, 50% and 62.5% of animals 1, 2, 4 and 6 weeks post injection respectively (Fig. 5, n = 8 mice in each group), suggesting that newly-generated HCs were able to survive for at least six weeks following 5-aza treatment.

Figure 5 Survival of new HCs following 5-aza injection into deafened mouse inner ear. Following 4 mM 5-aza injection into the deafened mouse inner ear, new hair cell survival is seen for at least 6 weeks post 5-aza injection. Scale bar: 50 µm in cochlear section overview; 20 µm in organ of Corti highlight. Full size image

Normal hearing mice were not affected by 5-aza

To explore the effect of 5-aza in normal hearing mice, 4 mM 5-aza was injected into the inner ear of young adult normal hearing mice (without deafening treatment). Two weeks after 5-aza injection, mice were euthanized. To be consistent with the majority of data in Figs 1–5, cochleae were continuously sectioned and labeled with Myosin VIIa to observe if there was any difference in the number of HCs between deafened and normal mice. All cochlear sections were collected for evaluation. There were one IHC and three OHCs in the section of normal mice with 5-aza treatment (arrows and arrowheads in Fig. 6), without significant changes in the number of HCs. These data indicate that 5-aza may not affect the number of HCs in normal hearing mice.

Figure 6 Normal hearing mice treated by 5-aza injection. After injection of 4 mM 5-aza into the cochleae of normal mice, the mice were followed up for two weeks. No changes of HCs are observed in cochlear sections. There are one row of IHC (arrowheads) and three rows of OHCs (arrows) in each cochlear section. Scale bar: 50 µm. Full size image

Initial investigation of the mechanisms of new HCs generated from 5-aza treatment

In experiments studying whether new HCs are generated via cell cycle reentry, Ki67 was used to label the sections of 5-aza-treated group two weeks after 5-aza injection. Ki67 immunostaining signal was not observed in the cochlear sections of 4 mM 5-aza treated mice (Fig. 7a, positive controls shown in Supplemental Data Fig. S1). Although negative Ki67 immunostaining cannot completely exclude the possibility of cell proliferation, it at least suggests that cell reentry was not identified at the observation time points or after hair cell differentiation. The origin of new HCs remains unclear, which may deserve an independent study.

Figure 7 Initial mechanistic study of new HCs regeneration. (a) No Ki67 staining is observed in cochlear sections of mice two weeks after receiving 5-aza injection (Ki67 positive control in Supplemental Data Fig. S1). Scale bar: 50 µm in cochlear section overview; 20 µm in organ of Corti (OC) highlight. (b) Relative Dnmt1 mRNA expression levels were measured in the saline- and 5-aza-injected deafened mice two weeks after injection. The results show decreased Dnmt1 mRNA expression in the 5-aza group compared to the saline group. (c) Relative mRNA expression of Notch and Wnt signaling genes in saline- and 5-aza-treated deafened mice two weeks after injection. It is observed that expression of Notch signaling genes Hes1 and Hey1 as well as Wnt signaling genes Fzd2 and Gsk3b are significantly downregulated, whereas Ctnnb1 was not affected. Scale bar: 50 µm. Full size image

Since 5-aza has been shown as a DNA methylation inhibitor by irreversibly binding to methyltrasferase22,23, it is logical to evaluate the expression of genes encoding DNA methyltransferase to understand whether the DNA methylation level is affected by 5-aza treatment. Dnmt1, which encodes DNA methyltransferase 1, was investigated in this study. Quantitative PCR was used to measure the level of Dnmt1 mRNA in the control and 5-aza treated groups. The results show that compared to the control group, 5-aza treated mice exhibited significantly decreased Dnmt1 expression (Fig. 7b). These data indicate possible DNA demethylation in the 5-aza group after 5-aza injection.

To evaluate whether Notch and Wnt signalings are involved in new HC generation, quantitative PCR was performed to determine the expression of Notch and Wnt signaling genes. It was observed that the expression of Notch signaling genes Hes1 and Hey1 as well as Wnt signaling genes Gsk3b and Fzd2 was significantly reduced in the 5-aza group compared to the saline group12,33 (Fig. 7c). Down-regulation of Notch-related genes is consistent with the previous report10. However, reduced expression of Wnt signaling genes does not seem to be in lines with the data from the neonatal mice11,12,13, which deserves independent studies in the future.

Evaluate the hair bundle of new HCs and initiate a protein-based functional assay

Comparing to the hair cell bundles of normal animals (Fig. 2), the bundles of newly-generated HCs seem abnormal and planar cell polarity seems to be lost. These observations raised our interests to investigate the hair bundle of new HCs. To further evaluate if hair cell bundles of new HCs are normal, F-actin labeling was performed in the surface preparations of normal, saline- and 5-aza-treated mice. The morphology of newly-regenerated HCs is shown in Fig. 8a. The hair bundle F-actin labeling in the OHC area of saline-treated mice was totally absent (arrows in Fig. 8a), indicating lack of hair bundle/OHCs in the saline group. In the 5-aza treated animals, abnormal hair cell bundles were observed (arrowheads in Fig. 8a) compared to the HCs in the normal animals. In addition, the planar cell polarity was lost in 5-aza treated mice with disorganized hair cell bundles. These abnormal hair cell bundles may indicate hair cell dysfunction. Notably, the F-actin labeling was missing in some of the OHC area, which also suggested impaired function of new HCs. Taken together, the abnormality of hair cell bundle F-actin labeling may indicate compromised functionality of new HCs.

Figure 8 Hair bundle and presynaptic protein study of new HCs. (a) Basilar membrane preparation of mouse cochleae 2 weeks following 5-aza and saline treatment. Untreated normal hearing mice serve as a control (arrowheads showing W-shape of OHC hair bundles). Fluorescent phalloidin was used to label F-actin of HCs in the surface preparation. OHCs were absent in the saline group (arrows). Compared with the untreated group, the 5-aza group showed disorganized hair bundles (arrowheads). Scale bar: 10 µm. (b) Basilar membrane preparation of mouse cochleae 2 weeks following 5-aza and saline treatment. CtBP2 was used to label presynaptic vesicles of HCs in surface preparation. CtBP2 puncta are observed in untreated normal control and 5-aza groups (arrows), whereas they were not found in the saline group. Scale bar: 10 µm. Full size image

To initiate the functional evaluation of new HCs, CtBP2 immunostaining was used to examine presynaptic vesicles. CtBP2 immunostaining showed that CtBP2 puncta were observed in the HCs of 5-aza treated mice and normal mice (arrows in Fig. 8b), whereas CtBP2 puncta expression was not detected in the saline-treated group (Fig. 8b). The positive CtBP2 staining suggests the possibility of presynaptic function of regenerated HCs.