Figure 1.
Gentamicin causes cochlear hair cell loss in a dose-dependent manner.
A) Low magnification image of a postnatal 3-day-old (P3) mouse cochlea immunolabeled for myosin7a, a specific marker for inner and outer hair cells. B) Cochleae were isolated from P3 mice and cultured overnight. The following day, cultures were bathed in gentamicin (0–0.5 mM) for 1 hr at 37°C followed by a 48 hr recovery period in AG-free culture media. C) Schematic of aminoglycoside (AG, red) entry into hair cells via mechanotransduction channels (MET, green) located at the apical end of stereocilia, and proposed mechanism of MET channel closure preventing AG entry following tip-link (yellow) breakage. D–G) Representative images of the middle turn of cochleae treated with varying doses of gentamicin and labeled with anti-myosin7a. (D) Control. (E) 0.1 mM gentamicin (F) 0.25 mM gentamicin (G) 0.5 mM gentamicin. H) Myosin7a-positive hair cells per 400 µm middle turn were counted and 0.23 mM gentamicin was determined to cause a 50% hair cell loss, mostly among outer hair cells. Error bars = S.D., scale bars = 100 µm in A, 25 µm in D–G.
Figure 2.
Two-photon time lapse imaging of GTTR uptake into live Cdh23v2J transgenic mouse cochlear hair cells.
P3 cochleae of mouse litters from Cdh23v2J/+ breeding were isolated and cultured overnight before treatment with GTTR (3 µM×1 hr). A–C) GTTR rapidly entered into outer hair cells of wildtype and Cdh23v2J/+ cochleae, whereas GTTR did not enter hair cells of Cdh23v2J/v2J cochleae. Insets depict magnified views of outer hair cells of cochleae of each genotype after exposure to GTTR for 60 min. D) Quantification of fluorescence intensity (AU = arbitrary units) shows that the rate of GTTR uptake among outer hair cells from wildtype and heterozygous mice were comparable, while those from homozygous mice had no detectable drug uptake. Error bars = S.D., scale bar = 20 µm in A–C and 5 µm in insets.
Figure 3.
Cadherin 23 deficiency protects hair cells from gentamicin toxicity.
P3 cochleae of mouse litters from Cdh23v2J/+ breeding were cultured in control (A–C) or gentamicin-containing (0.25 mM) media (D–F). Cultured tissues were immunolabeled for myosin7a (green) and gentamicin (red). A–C) Untreated, cultured cochleae from wildtype, Cdh23v2J/+, and Cdh23v2J/v2J mice exhibited an organized array of hair cells and no gentamicin labeling. D–E) Wildtype and Cdh23v2J/+ littermates showed extensive hair cell loss and robust anti-gentamicin labeling following gentamicin treatment. F) Cdh23v2J/v2J cochleae exposed to gentamicin showed no hair cell loss or gentamicin labeling. G) Quantitative analyses show that hair cells from Cdh23v2J/v2J homozygous mice were significantly protected from gentamicin. * = p<0.01, ** = p<0.001, error bars = S.D., scale bar = 25 µm.
Figure 4.
Disruption of tip-links with BAPTA diminishes GTTR uptake.
A) P3 wildtype cochleae were cultured overnight, treated with BAPTA (5 mM), then exposed to GTTR (1 µM×1 hr). Control cultures were rinsed with BAPTA-free media and exposed to GTTR. All images were captured using identical microscope settings. B–D) In control cochleae, GTTR uptake into hair cells followed a basal-apical gradient, where hair cells in the basal turn were the most robustly labeled. E-G) BAPTA pre-treatment reduced GTTR uptake into hair cells throughout the cochlea. H-J) The fluorescence intensity of outer hair cells was quantified and normalized to the most intensely labeled cell among cultured organs at this time point. Histogram plots showing the distribution of normalized GTTR fluorescence intensity indicate that BAPTA exposure significantly reduced GTTR uptake (also see Table 1). A bimodal distribution was observed in the middle and basal turns. Scale bar = 25 µm in B–G and 5 µm in inset in D’.
Table 1.
Gentamicin-conjugated Texas Red uptake in hair cells after calcium chelation with BAPTA++.
Figure 5.
BAPTA pre-treatment reduces gentamicin toxicity in hair cells.
A) Cultured cochleae from P3 wildtype mice were treated with BAPTA (5 mM) before gentamicin exposure (0.5 mM×1 hr). After an additional 48 hr AG-free recovery period, tissues were fixed and immunolabeled for myosin7a (green). B–G) Untreated cochlear cultures and cochlear organs treated with BAPTA only did not show hair cell loss. H–J) Gentamicin-treated cultures showed degeneration and a disarrayed arrangement of hair cells. K–M) Pre-treatment with BAPTA led to improved hair cell survival in comparison to treatment with gentamicin alone. N) Quantitative analysis of myosin7a-positive hair cell counts show significantly more hair cells in BAPTA pre-treated organs than those exposed to gentamicin alone. Error bars = S.D., * = p<0.001, scale bar = 25 µm.
Figure 6.
BAPTA-treated hair cells gradually regain ability to take up GTTR.
A) Cochleae were cultured overnight, treated with BAPTA (5 mM), incubated in normocalcemic media for another 6 or 24 hr, and then exposed to GTTR (1 µM×1 hr). Control cochleae did not receive BAPTA treatment and were cultured for the same durations. B–D) At the 6 hr recovery time point, GTTR uptake was noted among hair cells from control tissues and followed a basal-apical gradient. E–G) GTTR uptake was diminished among BAPTA-treated organs. H–M) After another 24 hr in culture, BAPTA-treated hair cells and those from untreated, time-matched controls shared a similar degree and pattern of GTTR uptake. Scale bar = 25 µm.
Figure 7.
Histogram plots of hair cell GTTR fluorescence.
A–C) Six hours after BAPTA treatment, hair cells showed diminished GTTR uptake (green) in comparison to untreated controls. Bimodal distributions were observed in both BAPTA-treated and untreated organs. D–F) Twenty-four hours after BAPTA exposure, GTTR uptake in treated hair cells (green) was comparable to controls (red). D’–F’) GTTR uptake gradually increased with longer durations after calcium chelation treatment. Percentages indicate fluorescence intensities from the BAPTA-treated group in comparison to those from the same cochlear turn from untreated, time-matched controls. When one peak is present in the BAPTA group and two peaks in control, the peak with a larger area under the curve was chosen for comparison.
Figure 8.
Hair cells recovered from BAPTA treatment were susceptible to damage by gentamicin.
A) After BAPTA treatment, cochleae were incubated in BAPTA-free, normocalcemic media for 24 hr before exposure to gentamicin (0.5 mM×1 hr). Tissues were cultured for another 48 hr and then immunolabeled for myosin7a (green). B–G) Untreated cochlear cultures and cultures treated with BAPTA only did not show hair cell loss. H–M) Both cultures exposed to gentamicin alone or to gentamicin 24 hr after pre-treatment with BAPTA had extensive hair cell loss and disorganization. N) Quantitative analyses show that treatment with gentamicin alone or with gentamicin 24 hr after pre-treatment with BAPTA caused significant hair cell loss. Error bars = S.D., * = p<0.001, scale bar = 25 µm.