Fate of mammalian cochlear hair cells and stereocilia after loss of the stereocilia

Abstract

Cochlear hair cells transduce mechanical stimuli into electrical activity. The site of hair cell transduction is the hair bundle, an array of stereocilia with different height arranged in a staircase. Tip links connect the apex of each stereocilium to the side of its taller neighbor. The hair bundle and tip links of hair cells are susceptible to acoustic trauma and ototoxic drugs. It has been shown that hair cells in lower vertebrates and in the mammalian vestibular system may survive bundle loss and undergo self-repair of the stereocilia. Our goals were to determine whether cochlear hair cells could survive the trauma and whether the tip link and/or the hair bundle could be regenerated. We simulated the acoustic trauma-induced tip link damage or stereociliary loss by disrupting tip links or ablating the hair bundles in the cultured organ of Corti from neonatal gerbils. Hair-cell fate and stereociliary morphology and function were examined using confocal and scanning electron microscopies and electrophysiology. Most bundleless hair cells survived and developed for approximately 2 weeks. However, no spontaneous hair-bundle regeneration was observed. When tip links were ruptured, repair of tip links and restoration of mechanotransduction were observed in <24 h. Our study suggests that the dynamic nature of the hair cell's transduction apparatus is retained despite the fact that regeneration of the hair bundle is lost in mammalian cochlear hair cells.

Figure 1.

Images of the tissue culture of neonatal organ of Corti and procedures to remove the hair bundles. A, Survey microphotograph of a 1-d-old culture of basilar membrane—organ of Corti prepared from the basal turn of a neonatal gerbil cochlea. The culture shown in this picture was explanted on the gridded coverslip. B, Three glass fibers with diameter of ∼5–6 μm were inserted underneath the culture to mark the area where the hair bundles were removed and the area used for control. C, The hair bundles observed under high magnification before the bundles were ablated. The bundles in the first row of OHCs were in focus. A suction pipette was positioned nearby. D, The hair bundles in first row OHCs were removed by the suction pipette. The bundles of the second row of OHCs were damaged. The focus in D was slightly readjusted to show the bundleless reticular lamina in the first row of OHCs and the damaged hair bundles in the second row of OHCs. Scale bars: B, 50 μm; C, D, 10 μm.

Figure 2.

Confocal images of prestin expression in the OHCs. A, One day after the hair bundles were removed. No prestin immunoactivity was seen at this age. B, C, Six and eleven days after the bundles were removed. Prestin was expressed at 6 PID and strongly expressed at 11 PID. Scale bar: 10 μm for A–C. D, E, Prestin expression in the preparations isolated from the developing gerbils at 7 (D) and 12 (E) d after birth. Both were from middle turns. F, G, Hair bundles and prestin expression at 1 PID. The hair bundle was labeled with rhodamine-phalloidin. Three rows of “V”-shaped OHC bundles and one row of IHC bundles could clearly be seen in the control areas. The hair bundles were no longer present in the area (middle of the image) where the hair bundles were removed. No prestin immunoactivity was seen at this age. G, Hair bundles and prestin expression at 11 PID. The hair cell region expanded significantly at this age. Prestin immunoactivity could be seen in the OHCs with and without the hair bundles.

Figure 3.

NLC and whole-cell currents recorded from bundleless OHCs and IHCs. A, NLC measured from two representative OHCs 4 and 11 d after their hair bundles were ablated. The cells were held at 0 mV and two-sinusoidal voltage protocols were used to probe voltage-dependent NLC. NLC was plotted in black thin lines while the Boltzmann function used to fit the response was in heavy black lines. B, Whole-cell currents measured from two representative IHCs in cultures 4 and 11 d after their hair bundles were removed. The cells were held at −70 mV. The membrane potential varied from −120 to 80 mV in 20 mV steps.

Figure 4.

SEM pictures of cultured organ of Corti and hair cell stereocilia. A, Surface view of the reticular lamina at 7 PID. The area where the bundle was removed is marked by double-head arrows. The tissue was from the middle turn. No regrowth or elongation of the truncated stereocilia was seen. B, Apical surface of a bundleless OHC under high magnification. The rootlets of three rows of truncated stereocilia were clearly visible. A white arrow indicates one of the rootlets. A kinocilium is still present in the apical surface. C, A bundleless IHC. Some microvilli together with a kinocilium are also seen. B and C are both from the damaged area from A. D, Surface view of the reticular lamina at 13 PID. No bundle repair was observed. The tissue was from basal turn. E, F, High-magnification images of the apical surface of an OHC (E) and IHC (F). Arrows mark the rootlets of stereocilia. E and F are from the damaged area shown in D. Scale bars in A and D represent 10 μm, while the bars in the rest of the images represent 1 μm.

Figure 5.

SEM picture of hair cell stereocilia. A, Stereocilia of a 2-d-old OHC in culture. Kinocilium and abundant microvilli are clearly visible at this age. B, Stereocilia of a 14-d-old OHC in culture. The kinocilium disappears and the number of microvilli is significantly reduced at this age. C, Apical surface of an OHC at 7 PID. The kinocilium remains while no elongation of the stereocilia is seen. D, Apical surface of an OHC at 7 PID. Half of the stereocilia bundle is undamaged and the kinocilium is also present. No repair is seen on the truncated stereocilia.

Figure 6.

SEM picture of hair cell stereocilia and the MET currents measured from cultured hair cells. All cultures were prepared from the cochlear apical turn from 8-d-old gerbils for all experiments. A, SEM of stereociliary bundles after cultured for 24 h. At this age, the kinocilium in some hair cells already disappeared (indicated by a black arrow) and in other hair cells, the kinocilium was still present (marked by a white arrow). B, Newly regenerated tip links 24 h after being cut. Tip links could clearly be seen in the apex of the innermost (shortest) stereocilia to the side of their taller neighbor. Two black arrows mark the two examples of the newly regenerated tip link. C, F, MET current recorded 10 min after BAPTA treatment. E, F, MET current recorded from control IHC and OHC after 24 h culture. D, G, MET current returned in the BAPTA-treated preparations after 24 h culture. The cells were held at −70 mV. The bundle was vibrated in response to fluid jet stimulation at 100 Hz. Three presentations were averaged for each trial. E, H, MET currents measured from cultured hair cells whose tip links were not cut (control group). I, J, Adaptation of MET currents measured from control (I) and BAPTA-treated (J) OHCs after 24 h culturing. The hair bundle was deflected by a rigid fiber which was driven by piezoelectric actuator. Three presentations were averaged for each trial.