Recovery of hair cell function after damage induced by gentamicin in organ culture of rat vestibular maculae

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Recovery of hair cell function after damage induced by gentamicin in organ culture of rat vestibular maculae Akiko Taura a , Ken Kojima a , Juichi Ito a , Harunori Ohmori b,⁎ a

Department of Otolaryngology-Head and Neck Surgery, Japan Department of Physiology, Graduate School of Medicine, Kyoto University, Kyoto, Japan

b

A R T I C LE I N FO

AB S T R A C T

Article history:

Here, we report the functional and morphological evidence of hair cell recovery after

Accepted 17 April 2006

damages induced by gentamicin (GM) in cultured explants of rat vestibular maculae. We

Available online 9 June 2006

evaluated mechano-electrical transduction (MET) function in hair cells, by measuring Ca2+ responses in the explants with fura-2 when hair bundles were stimulated. After the MET

Keywords:

testing, hair bundles were observed in high resolution by scanning electron microscopy, or

Vestibular hair cell

by fluorescence microscopy after staining with phalloidin-FITC (fluorescent isothiocyanate).

Mechano-electrical transduction

In the control culture, the number of hair bundles on the explants gradually decreased, and

(MET)

the percentage of explants showing Ca2+ responses decreased and disappeared after 17 days

Steroid hormone

in culture. Following GM (1–2 mM) treatment, most of the hair bundles were eliminated initially, but the hair bundles gradually increased in number during culture. Short hair bundle-like structures emerged in the areas where hair bundles had been completely lost. Consistent with the morphological observations, Ca2+ responses disappeared after GM treatment, and they gradually recovered to a peak 13–17 days after treatment and were even induced at 17 days or more in culture. Furthermore, cells accumulated FM1–43, a dye permeable through the MET channel, when Ca2+ responses recovered after GM treatment. Application of steroid hormone increased the percentage of explants showing MET activity, and enhanced the recovery of MET after GM treatment. We investigated Ki-67 immunoreactivity to detect cell proliferation and TUNEL staining to detect apoptotic cell death. Ki-67 immunoreactivity was negative after GM treatment, however TUNEL staining was positive and the positivity was GM dose dependent. Therefore, this functional recovery of transduction activity was not owing to the proliferation of hair cells but was likely the self-repair of the hair bundle. © 2006 Elsevier B.V. All rights reserved.

1.

Introduction

The major cause of sensorineural hearing loss and vestibular disorders is the loss of hair cells (Nadol, 1993); damage to the stereocilial bundle of hair cells is sufficient to cause hearing deficits (Liberman and Dodds, 1984; Morton, 1991). Functional

recovery from hearing loss or vestibular disorders is extremely rare in mammals after damage by loud noise or ototoxic drugs (Stone et al., 1998). In contrast, the recovery of inner ear function is observed in non-mammalian vertebrates. In avians, hair cells are known to recover when they are lost after acoustic trauma or treatment with aminoglycoside

⁎ Corresponding author. Fax: +81 753 4349. E-mail address: [email protected] (H. Ohmori). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.04.090

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damage in mammals, there still remains a possibility that these functional recoveries in vivo are made by some central compensation, and direct evidence is needed to show the functional recovery of the hair cells themselves. In this study, we established an organ culture of the vestibular sensory epithelium of postnatal rats, and investigated the recovery of hair cells after damage induced by

Fig. 1 – Mechanical stimulation of hair bundles. Hair bundles were stimulated mechanically by water flow ejected from a pipette. (A) A control, before stimulation. ROI was indicated by a square. At least 5–10 hair bundles were included within the ROI. Inset in panel a shows an ejected fluid marked by dye. (B) Hair bundles were displaced away from the pipette by a positive pressure. (C) Hair bundles were displaced toward the pipette by the release of a positive pressure. The pipette tip diameter was 50 μm. Scale bar in panel A indicates 10 μm for panels A, B and C, and 100 μm for Aa.

antibiotics (Corwin and Cotanche, 1988; Cotanche et al., 1987; Cruz et al., 1987; Jones and Nelson, 1992; Ryals and Rubel, 1988). Recently, in mammals, morphological evidence has been demonstrated for the self-repair (Zheng et al., 1999) and the renewal (Berggren et al., 2003) of hair cells. The regeneration of hair cells was also proposed morphologically in the vestibular epithelia (Forge et al., 1993), where the capabilities have been demonstrated, of making new hair cells by the overexpression of Math1 (Zheng and Gao, 2000). Furthermore, a recovery of vestibular function, nystagmus, in mammals was reported in vivo (Meza et al., 1992). Although these data should support the idea that hair cells can recover after

Fig. 2 – Frame number and % hair bundle along the day in vitro (DIV). (A) Phalloidin-FITC-stained images for the control (Aa–d) and after GM treatment (Ae–h). Culture days are indicated in the panel. Ab, hair bundles and reticular lamina of 0 DIV are shown with a larger magnification, and three frames are indicated by a dotted line. The some frames had a hair bundle (arrows) and we identified these frames as a hair cell. Scale bar is 10 μm for panels Aa, c–h, and 5 μm for Ab. (B) Mean number of frames was calculated every 4 days in vitro. There were no significant differences in the number of frames among three different conditions of culture (P > 0.05, ANOVA). DIV is the same as the abscissa in C. (C) Dependence of the percentage of a frame which had a hair bundle (% hair bundle) to total frame numbers on DIV. * indicates a significant difference (P < 0.05, t test). Plots in panels B and C are mean ± SEM. Each measure was from 3 to 6 explants at each DIV. GM was applied for 2 days as indicated by the bar and the arrow on the abscissa.

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gentamicin (GM) both functionally and morphologically. The function of hair cells was monitored by Ca2+ measurement using fura-2 and by accumulation of FM1-43, which is known to reflect the activity of mechano-electrical transducer (MET) channels (Gale et al., 2001). Our investigation further concluded that a hair cell proliferation is not likely participated in this functional recovery of transduction activity. Nevertheless, we believe that the experiments described here might be a useful strategy in the investigation to identify the underlying mechanisms of the functional recovery of hair cells (Fig. 1).

2.

Results

2.1.

Percentage of cells having a hair bundle during culture

We measured the percentage of cells having a hair bundle in phalloidin-stained explants as a function of days in vitro (Fig.

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2). As illustrated in Fig. 2A (Aa, Ab; 0DIV), the cell boundaries within the reticular lamina were phalloidin-positive, and some of these delimited polygonal areas had a hair bundle (Fig. 2Ab, arrows). We called this delimited area a frame, and measured the fraction of frames, which had a hair bundle (% hair bundle). The % hair bundle was 23% in the control even during the first week in culture (Fig. 2C), indicating that these frames reflect not only the hair cell boundary but also the boundary of supporting cells constructing the sensory epithelium. During the first week in culture, there was no statistical difference in % hair bundle among different days in culture (P > 0.05, ANOVA). After 1 week in culture, the % hair bundle decreased with the progress of days in vitro (DIV) (Figs. 2Ac, Ad and C). These tendencies were the same when explants were cultured with retinoic acid (1 × 10−7/10−8 mM, Sigma) or Epidermal Growth Factor (100 ng/ml, Sigma) (data not shown). We could not find the reason for the damage of hair cells after 1 week in culture.

Fig. 3 – A hair bundle emerged at the delimited location after GM treatment. Application of 2 mM GM (2 GM) eliminated most of hair bundles. A pieces of coverslip delimited the location where repeatedly observed; by DIC optics in panels A, A′, B, B′ and by SEM in panels C, D. Locations of two pieces of coverslip are indicated in panels A, B, and C by broken lines. (A) DIC image 3 days after GM treatment. No hair bundles were found. (B) DIC image 17 days after GM treatment. (A′, B′) Different focal plane images to show the delimiting coverslips. Several bud-like structures emerged; one of them is indicated by an arrow. Note the arrow in panels A and B indicates the corresponding site. (C) SEM image (low magnification). A small white mass (arrow) was observed in the area indicated by the arrow in panel B. (D) SEM image (high magnification) showing the area pointed by the arrow and delimited by a square in panel C. Although small, a hair bundle-like structure was observed. Scale bars are 10 μm for A, A′, B, B′, C, and 1 μm for panel D.

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After GM treatment for 2 days, hair bundles disappeared almost completely and the % hair bundle became very close to 0 (Figs. 2Ae, Ag and C; GM application was indicated by a bar below abscissa in Fig. 2C). The % hair bundle did not stay at the bottom, however, it gradually increased after GM treatment (Figs. 2Af and Ah) and peaked at about 11 DIV after 1 mM GM treatment (1 GM), and at about 15 DIV after 2 mM GM treatment (2 GM; Fig. 2C). The difference in % hair bundle was significant at days (near 11 DIV) when % hair bundle peaked after 1 GM (between the control and 1 GM, P < 0.05, t test); however, there was no significant difference between the control and 2 GM even at the days when % hair bundle peaked. Fig. 2B shows the number of frame as a function of DIV. The number of frame decreased a little immediately after GM administration (around 10 DIV), however the number of frames per explant did not significantly change during cultures, irrespective of the culture conditions; the control, and after 1 or 2 mM GM treatment (Fig. 2B, mean 6600 ± 320, n = 201 explants; P > 0.05, ANOVA).

2.2.

Regrowth of hair bundles

The delayed peak of % hair bundle after GM treatments (Fig. 2C) strongly indicates a regrowth of hair bundles. We tried to see the exact regrowth of hair bundles in a particular area where a complete loss of hair bundles was confirmed by DIC optics (Fig. 3). Hair bundles of explants were first eliminated by the treatment of 2 mM GM, then one area was marked by placing several pieces of broken coverslip (Figs. 3A and A′). This area was repeatedly observed by DIC optics (see Experimental procedures). Fig. 3A shows the area of the explant at 5 DIV where hair bundles were eliminated. Two pieces of broken coverslip were placed to demarcate this area (glass 1 and glass 2). Although hair bundles were eliminated, the structure of reticular lamina remained (Fig. 3A). After 2 weeks in culture, several small stump-like structures were observed (an arrow indicates one of them in Fig. 3B). After fixation, fortunately two pieces of broken coverslip remained on the explant and delimited the area, which was observed by SEM (Fig. 3C). At higher magnification (Fig. 3D), the SEM image showed a small and short, but stereocilial bundle-like structure in this location. We expected to find that the form of reticular lamina to be constant throughout the culture, but it changed substantially during these 2 weeks, and the pieces of coverslip became the only landmarks to identify the area (Figs. 3A′ and B′). The observation of regrowth of hair bundles in such demarcated area occurred only once during the 50 trials. However, in 15 cases out of 50 explants, we observed the emergence of hair bundle-like structures in nearby areas, but not in the particularly delimited, repeatedly observed areas. Thirty-five cases failed in the regrowth of hair bundles.

2.3. Ca2+ responses induced by the mechanical stimulation of hair bundles Fig. 4A shows fura-2 fluorescence changes recorded in the explant at 0 DIV when the fluid puff stimulus was applied. F340 and f380 showed reciprocal changes during mechanical

Fig. 4 – Ca2+ responses. MET of explants was monitored as Ca2+ signals using the fluorescence indicator fura-2. (A) Fluorescence responses measured in the control culture, 0 DIV. Ratio (f340/f380), f340 and f380 from the top to the bottom (arbitrary units). Note that the fluorescence intensity of f340 and f380 was changed in a reciprocal manner when mechanical stimuli were applied to the hair bundles. Timing of mechanical stimuli is indicated by a stitched bar on the abscissa (A–C). B, C, f340/f380. (B) 3 days after 2 mM GM (2GM) treatment, and mechanical stimulation did not generate any fluorescence responses. (C) 17 days after 2 GM treatment, and small fluorescence responses were induced repeatedly by mechanical stimuli. stimulation (Fig. 4A two middle traces), and the ratio (f340/ f380) showed a large increase (Fig. 4A top trace) repetitively when 4 stimuli were applied (timing of stimuli is marked by stitched bars on the abscissa in Figs. 4A–C). These fluorescence changes indicate an increase of [Ca2+]i (Grynkiewicz et al., 1985). These fluorescence responses disappeared completely when 1 mM GM was applied in the bathing medium for 5 min, and were reversed almost completely by washing (data not shown). GM is known to block the transduction of hair cells reversibly (Kroese et al., 1989). These fluorescence responses also disappeared when the L-type Ca2+ channel blocker nicardipine (10 μM) was bath applied. However, nicardipine did not block the hair cell accumulation of FM1–43 by mechanical stimulation, even after the incubation of explant for 30 min (personal communication of Dr. Matsumoto, see also Fig. 6). Therefore, the fura-2 fluorescence responses observed here were likely generated through the activation of L-type Ca2+ channels by membrane depolarization induced

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by MET of hair cells (Ohmori, 1984, 1985). The ratio responses were small even in the control, probably because of the neutralizing effects of background fluorescence (see Experimental procedures); the peak ratio was 1.87 ± 0.05 (n = 4 explants) when the basal level was 1.68 ± 0.09 (n = 4 explants). After exposure to 2 mM GM (between 1 and 2 DIV), Ca2+ responses were not observed when explants were examined 3 days later (5 DIV) (Fig. 4B). However, on 20 DIV (17 days after GM treatment), small ratio responses were observed in some explants (Fig. 4C). These ratio responses were small, but were repeatedly generated during a test. The ratio responses after exposure to 1 GM were maximum at 11–12 DIV (ΔR = 0.018 ± 0.003, n = 10 explants) and those after 2 GM were at 15–16 DIV (ΔR = 0.017 ± 0.005, n = 5 explants). ΔR indicates the difference between the peak and the base fluorescence ratios; the base fluorescence ratios were not different throughout all DIV between the control and after treatments with GM (Table 1, P > 0.05, ANOVA). Because the ratio responses were small, particularly after GM treatment, and the size varied extensively from explant to explant, we scored the MET as the percentage of explants showing MET-positive responses (definitions in Experimental procedures) rather than the absolute size or the average size of Ca2+ responses (Fig. 5). Fig. 5 shows the fraction of MET-positive explants as a function of DIV. In the control, the % of MET-positive explants gradually decreased with the progress of DIV and followed a time course almost parallel with that of the % hair bundle (Fig. 2C), and no MET was observed after 17 DIV (Fig. 5, filled squares). After GM treatment, MET was not observed in any explants for the next few days; however, the percentage of MET-positive explants gradually increased after 5 DIV and peaked at 13–17 DIV (Fig. 5, filled circles for 1 GM, and filled triangles for 2 GM). The Ca2+ responses were even generated at 27 DIV. After that, the percentage of MET-positive explants gradually decreased as

Fig. 5 – A fraction of MET-positive explants as a function of days in culture. Criteria to score the explant as MET-positive are described in Experimental procedures. Control without GM treatment (filled squares, n = 5–11 explants at each measure), after 1 mM GM (1GM) treatment (filled circles, n = 5–14 explants), and after 2 mM GM (2GM) treatment (filled triangles, n = 5–18 explants). Note that even after the DIV when control explants lost MET activity, a significant fraction of explants generated MET after GM treatment. Differences between the control and after GM treatment at the peak of MET positive explants (13 DIV for 1 GM, and 17 DIV for 2 GM) were both statistically significant (*P < 0.05, Fisher's exact probability test).

control. The maximum % of MET-positive explants in 1 GM was larger than that in 2 GM (50% in 1 GM vs. 30% in 2 GM) and emerged earlier (13 DIV in 1 GM vs. 17 DIV in 2 GM, Fig. 5). Incidences of MET-positive explants after 1 GM and 2 GM were statistically significant over the control when compared around the peaks of MET-positive DIV: at 13 DIV in 1 GM and 17 DIV in 2 GM (P < 0.05, Fisher's exact probability test).

Table 1 – Effect of DEX on the MET and the hair bundle Control

1GM

2GM

MET 1. Base level of fluorescence ratio (f340/f380) at 17–20DIV a DEX (−) 1.58 ± 0.15 (11) DEX (+) 1.97 ± 0.24 (14) 2. Ca2+ responses (ΔR) at 17–20 DIV a DEX (−) n.r. (12) DEX (+) 0.01 ± 0.01 (8)

1.46 ± 0.07 (16) 1.67 ± 0.13 (11)

1.39 ± 0.07 (25) 1.53 ± 0.06 (18)

0.02 ± 0.002 (8) 0.02 ± 0.01 (5)

0.02 ± 0.003 (5) 0.02 ± 0.01 (7)

Number of hair bundle (/10,000 μm2) at 17–20 DIV a DEX (−) 18.3 ± 2.0 (6) DEX (+) 47.3 ± 9.5 b (6)

23.9 ± 4.9 (5) 11.6 ± 3.3 (5)

10.8 ± 2.3 (6) 7.9 ± 1.8 (5)

Integrity of hair bundle (%) at 19–20 DIV c DEX (−) 6.7 ± 2.5 (4) DEX (+) 46.2 ± 6.7 b (4)

10.5 ± 1.6 (4) 43.5 ± 10.4 b (7)

6.5 ± 3.6 (4) 21.0 ± 2.6 b (5)

n.r., no response. DIV, day in vitro. Values are presented as means ± SEM. Numbers in parenthesis indicate the number of explants. a Base levels are the averages of base fluorescence ratio (f340/f380). Ca2+ responses were the difference of f340/f380 between the stimulus and the base. b Statistical significant difference (P < 0.05, t test) against DEX(−). c Integrity of hair bundle is defined in the text.

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FM1–43 accumulation in the cultured hair cells

In the control, FM1–43 accumulation was observed in many cells in response to mechanical stimulation, and these fluorescent cells matched the cells having a hair bundle by overlaying (Fig. 6A, overlaying of DIC image and fluorescence image). Three days after 2 GM treatment, there were neither FM1–43 positive cells nor hair bundles (Fig. 6B). However, after 17 days of 2 GM treatment, short hair bundle-like structures were observed and the fluorescence spots of FM1– 43 were overlaid (Fig. 6C, two arrows). These results strongly indicate that these small hair bundles have MET activity. After 17–18 days of GM treatment, the accumulation of FM1– 43 was observed in 60% of explants (12 out of 20 explants); 7 out of 10 explants after 1GM and 5 out of 10 explants after 2 GM. Fourteen explants among these 20 explants were tested FM1–43 accumulation after the test using fura-2. Six out of 14 explants were positive for both fura-2 response and FM1– 43 accumulation. And other 5 explants were negative for both fura-2 and FM1–43. Another 3 explants showed different results between fura-2 and FM1–43. Therefore, among these 14 explants tested for both fura-2 and FM1– 43, 11 explants showed the parallel results (80%). This indicates a strong correlation between fura-2 and FM1–43 accumulation.

2.5.

Effects of a steroid hormone

Steroid hormones are clinically used in the treatment of hearing loss in the acute phase (Stolroos and Albers, 1996). The treatment is occasionally effective. We tested and evaluated

MET using the same Ca2+ imaging protocol as control. When DEX (1 μM) was included in the culture medium, a large fraction of explants demonstrated MET even at much later days in culture (Fig. 7A, filled squares for DEX and open squares for control). The control is the same as in Fig. 5. On 5 DIV, 97% of explants showed MET when DEX was added, while less than 75% did in the control. Twenty-seven percent (7 out of 26) of explants generated MET, well after the day (17 DIV) when MET disappeared in the control (Fig. 7A). These differences during 5–17 DIV were statistically significant (P < 0.05, Fisher's exact probability test). Furthermore, DEX enhanced MET activity after GM treatment (Fig. 7B). The fraction of explants having MET at 21 DIV almost doubled in DEX after 1GM (from 27% to 66%), and tripled after 2 GM (from 14% to 56%). These differences between use and non-use of DEX were significant; statistical evaluations were made at 21 DIV for 1GM and 19–23 DIV for 2 GM (P < 0.05, Fisher's exact probability test). The basal [Ca2+]i was slightly higher after DEX application, but the Ca2+ responses (ΔR) were not different (Table 1). Furthermore, the enhanced activity of MET by DEX was confirmed by FM1–43 accumulation when measured at 20 DIV after the loss of hair bundles by 1 GM treatment (Fig. 6D1–4). A large number of cells with a hair bundle were labeled with FM1–43; in 8 out of 11 explants maintained in culture with DEX (1 μM) after GM treatments. We have tested several other agents but they were not effective, neither functionally nor morphologically; brain-derived neurotrophic factor (BDNF) (25 or 50 ng/ml, Sigma), z-VAD-FMK a general caspase inhibitor (100 μM, R&D Systems, Inc.), leupeptin (1 mM, Sigma) and DMSO (1%, Wako, Japan).

Fig. 6 – FM1–43 accumulation in hair cells. After exposing explants to FM1–43 for 10 s with mechanical stimulation, fluorescence-positive spots emerged. The FM1–43-positive fluorescence image (red) was overlaid with the DIC image. (A) Control (0 DIV). FM1–43 positive spots were superimposed on hair cells; identified by the presence of a hair bundle. B, 3 days after 2 GM. No hair bundles and no fluorescence-positive spots were found. C, 17 days after 2 GM. Two short hair bundle-like structures (arrows) overlaid the fluorescence spots of FM1–43. The hair bundle indicated by the left arrow appeared separated from the FM1–43 spot in this focus; however, it was matched with the fluorescence spot by focusing downward. D1–4, FM1–43 accumulation in explants cultured 17 days with DEX after 1 GM. Scale bar is 10 μm.

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gradually lost during cultures, and at 19 DIV, the number of stereocilia in a bundle looked reduced (Figs. 8A and B). The stereocilia were disarranged and fanned out, and the integrity as a bundle was completely lost (Fig. 8B). MET generally disappeared in these explants (Fig. 5). MET was sensitive to the integrity of hair bundles, and once the stereocilial bundle was disarranged, MET was lost (Rastel et al., 1993). With DEX, the hair bundle appeared more normally integrated both in the control (Figs. 8C and D) and after 1GM (Figs. 8E and F) even at this DIV (19 DIV). After DEX treatment, the integrity was significantly greater than in the control (P < 0.05 ANOVA, Table 1). This is in good agreement with the percentage of METpositive explants, which was almost zero in the control but was 26% in DEX at 19 DIV (Fig. 7A). Well-integrated tall and short hair bundles were observed after 1GM treatment when incubated with DEX (Fig. 8F). At 19 DIV after 1 GM treatment, small hair bundle-like structures were observed sometimes even without DEX treatment (in 11 explants out of 58, Figs. 8G and H).

2.7.

Fig. 7 – DEX prolonged the days having MET activity in the control, and enhanced MET after GM treatment. (A) Incubation with DEX prolonged the culture days having MET activities in the control explants, and enhanced the fraction of MET-positive explants during the first week in culture (filled squares). The fraction of MET-positive explants was significantly larger at 5–17 DIV with DEX over the control (*P < 0.05 Fisher's exact probability test, n = 5–11 explants at each DIV for both control and DEX). MET was observed even after 27 DIV with DEX. The control data is the same as in Fig. 5. (B) After GM, the incubation with DEX increased the fraction of MET-positive explants. 1 mM GM (1GM) (circles), and 2 mM GM (2GM) (triangles). However, the total time course of the MET positivity was not different. Differences of MET-positive explants between incubation with (filled symbols) and without DEX (open symbols) after GM treatment were statistically significant when compared at 21 DIV in 1 GM and at 19–23 DIV (largest at 21 DIV) in 2 GM (*P < 0.05 Fisher's exact probability test, n = 5–18 explants at each DIV).

2.6.

Integrity of the hair bundle

DEX prolonged the days in vitro when MET could be observed in the control culture, and enhanced the proportion of MET positive explants after GM treatment (Fig. 7). However, paradoxically, the number of hair bundle was not significantly improved by DEX application when compared at 17–20 DIV in groups of GM treatment (Table 1). A large fraction of explants produced MET in the control culture until 7 DIV (Fig. 5), and hair bundle morphology was relatively normal (Fig. 2Ac). The integrity of hair bundles was

The viability of FM1–43 positive cells during culture

After loading the cells with FM1–43 on the day of explantation (0 DIV), the number of FM1–43 positive cells gradually decreased with the progress of days in culture (Fig. 9). We could find about 70 FM1–43 positive cells over 10,000 μm2 in 3– 5 DIV, and this corresponds to 24% of the frame (Fig. 9D). These numbers are approximately equal to the total number of hair cells and the % hair bundle in these DIV. The number of FM1– 43 positive cells decreased significantly at 9 DIV and after (Fig. 9D). However, with DEX, the rate of decrease was reduced (Fig. 9D, filled squares). This effect of DEX was statistically significant when evaluated at 9 and 15 DIV (P < 0.05, t test), and was not different whether explants were of utricle or saccule origin. There was a good correlation in DEX effects studied by fura-2 and FM1–43 (Figs. 7A and 9D; correlation coefficient of 0.944, P < 0.0001, Spearman's rank correlation). After GM treatment, FM1–43 signals appeared granular and apparently scattered over number of adjacent cells. However, there were some cells, which had a short hair bundle and were FM1–43 positive (Fig. 9C). These cells may indicate a reemergence of a hair bundle in hair cells after GM treatment.

2.8. Apoptotic cell death but absence of cell proliferation after GM treatment We tested TUNEL positivity at 5 DIV where the % hair bundle was still minimum after GM treatment. The percentage of TUNEL positive cells was increased with GM dose (Fig. 10). Fig. 10B shows % of TUNEL positive cells both in the hair cell layer (P < 0.05 ANOVA; n = 3–4 explants), and in the supporting cell layers (0% in 1 GM, 0.9 ± 0.8% in 2 GM and 7.6 ± 3.0% in 3 GM; statistically not significant). Incubation with DEX apparently reduced the cell death in the hair cell layer, however this decrease was not statistically significant (P = 0.1 in 1 GM and P = 0.3 in 2 GM, Fig. 10B). Effects of DEX in the supporting cell layer were not clear, partly because of the absence of TUNEL positive cells detected in this layer. With immuno-histochemistry of Ki-67 (Fig. 11), we tested cell proliferation in the phase of re-growth of hair bundle after

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Fig. 8 – DEX maintained the shape of hair bundles. The shape of hair bundles was observed by a fluorescence microscope after phalloidin-FITC staining and by SEM, at 19 DIV in the control and after GM treatment. (A, C, E, G) phalloidin-FITC images. (B, D, F, H) SEM images. (A and B) the control cultures. The number of hair bundles and the stereocilia within a bundle was extremely reduced (A). The remaining stereocilia were disarrayed (B–D) with DEX in the control culture, a large number of hair bundles was left (C). Each hair bundle was composed of well-integrated stereocilia (D–F) after 1 GM treatment with DEX. Several phalloidinpositive small hair bundle-like structures were observed in E. SEM showed both tall and short hair bundles, and they were composed of well-integrated stereocilia (F–H) after 1 GM treatment without DEX. Phalloidin-positive small hair bundle-like structures were found in G. These short stereocilia like-structures formed a bundle, and appeared like a hair bundle, H. Scale bar is 10 μm in panels A, C, E, G, 1 μm in panels B, D, F, H.

GM treatment. Ki-67 was generally negative in the vestibular explants, irrespective of the GM treatment or the control. Even during the phase when % hair bundle increased after GM treatment (6, 10 and 13 DIV), only a small percentage of cells were found Ki-67 positive (less than 1%). DAPI positivity

indicates a presence of many cells in this area. The otocyst of embryonic rat (E13) and the surrounding structures were proliferating and Ki-67 positive (Fig. 11A bottom). Administration of DEX did not affect the situation, and the small base percentages persisted (Fig. 11B). Because Ki-67 is positive in

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the occasion of cell proliferation (Schluter et al., 1993), this may indicate that GM treatment of the explants or the incubation with DEX does not induce proliferation of hair cells.

3.

Discussion

Recovery of hair cells after damage is a well-known phenomenon in avians, not only morphologically but also functionally (Matsui et al., 2000; Ryals and Rubel, 1988). In mammals, although some morphological evidence is available pointing to the recovery of hair cells (Forge et al., 1993; Sobkowicz et al., 1996; Warchol et al., 1993; Zheng et al., 1999), functional recovery was not confirmed yet; it is not clear whether those re-emerged hair bundles have functional MET channels or not. In the present study, we confirmed that the recovered hair

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cells have the MET activity and found that steroid hormones facilitate the hair cell recovery. GM treatment caused a loss of hair bundles that was followed by an increase in the % hair bundles, probably consistent with the increase in the number of hair cells having the hair bundle (Fig. 2), and this increase paralleled the increase in the proportion of MET-positive explants (Fig. 5), and the increase was facilitated by the incubation with a steroid hormone (Fig. 7). However, we could not find any evidence to support this functional recovery of transduction activity being accompanied with proliferation of hair cells (Fig. 11).

3.1. Acute block of the transducer and chronic effects on the hair bundle by gentamicin GM blocks reversibly the MET channel with IC50 of 7.6 μM (Lambert et al., 1997). We used higher concentration of GM (1– 2 mM) for 2 days to damage hair cells (see (Forge and Schacht, 2000)); this was in contrast to the acute block of MET channel, since MET was blocked reversibly by the shorter exposure time of 5 min (1 GM). GM induces the loss of hair bundles or the death of hair cells due to chronic toxicity. The mechanism of chronic toxicity is sill unclear; GM-induced formation of free radicals was proposed as being critical (Forge and Schacht, 2000). Aminoglycoside is reported to eliminate hair cells in a doseand exposure time-dependent manner (Kotecha and Richardson, 1994; Matsui et al., 2000). Because of the slower time course and the smaller extent of recovery after 2 GM than after 1 GM, both in % hair bundle and in MET activity (Figs. 2 and 5), more loss of hair cells have likely occurred after 2 GM treatment. This is consistent with the increased percentage of TUNEL positive cells with GM dose (Fig. 10). GM concentrations we adopted (1 mM and 2 mM) might be high compared with preceding papers (Gale et al., 2002). However, Zheng et al. used 1 mM GM to damage hair cells (Zheng and Gao, 1997), and reported that 54% hair cells survived after the treatment (Zheng et al., 1999). They used a medium containing 15% serum. In the preliminary experiment, we

Fig. 9 – Long-term viability of FM1–43 accumulated cells in cultured explants. A viability of hair cells during a long time culture was evaluated by counting the number of cells accumulated with FM1–43. Hair cells were loaded with FM1–43 on the first day of culture, and the fluorescence was measured on the 3rd, 5th, 9th, and 15th DIV. (A) FM1–43 positive cells in the explant under the control culture, at 15 DIV. (B) Explant with DEX incubation, at 15 DIV. (C1,2) 1 mM GM treated explants at 15 DIV. Some of FM1–43 positive cells had a small hair bundle (arrow). (D) Dependence of % frame and the number of FM1–43 positive cell on DIV. Open squares are for the control, and filled squares with DEX. The % of FM1– 43 positive cells was 24% immediately after the loading. Bars indicate mean ± SEM. Each measure was from 4 to 5 explants at each DIV. The scale bar on the left shows the % of the frame which is FM1–43 positive. The scale on the right shows the number of FM1–43 positive cells countered in 10,000 μm2 area. Scale bars indicate 50 μm in panels A, B and 10 μm in panel C. *Indicates a significant difference (P < 0.05, t test).

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Fig. 10 – TUNEL staining after GM treatment. (A) TUNEL staining; fluorescence image for DAPI and merged image, for the control, 1 mM GM (1GM) and 2 mM GM (2GM) evaluated on 5 DIV. HC: hair cell layer, SC: supporting cell layer. Scale bar 50 μm. (B) The percentage of TUNEL-positive cells in the hair cell layer (open box) and in the supporting cell layer (filled box) (*P < 0.05 ANOVA; n = 88–155 cells, sampled from 4 to 6 explants). Bars indicate mean ± SEM.

could not eliminate hair bundles completely with 0.5 mM GM. As we conducted recovery experiments partly on the imaging ground, it was necessary to eliminate almost all the hair bundles. A high concentration of GM we used was partly because of a culture condition where a high concentration of serum was included to maintain a long-term culture. Therefore, the concentration of GM we used might be high, but was necessary and could be still in an appropriate range.

3.2.

Functional recovery of hair cells

In the preceding papers, functional recoveries were evaluated mainly through in vivo experiments in mammals, where the auditory brain stem response was used to examine cochlear function (Wang et al., 2002), or vestibulo-ocular reflexes (Kopke et al., 2001), swimming behavior (Meza et al., 1996)

and nystagmus (Meza et al., 1992) were examined to evaluate vestibular function. However, in the evaluation of vestibular function in vivo, a possible involvement of some central compensation mechanism should be excluded, but seems difficult. In the present in vitro study, we confirmed the reemergence of MET activity to begin almost immediately after GM treatment (Fig. 7B): Some explants had immature hair bundle-like structures after some days in vitro (Figs. 8G and H), and transducer activities of these structures were confirmed by FM1–43 accumulation (Fig. 6C). These observations indicate a functional recovery of hair cells. FM1–43 accumulation by hair cells is supposed to be mediated by a mechano-electrical transduction dependent process. Zebrafish larval hair cells whose transduction is abolished, such as mar mutants or nompC morphants, or zebrafish hair cells exposed to blockers of the transduction channel, do not accumulate FM1–43 (Seiler and Nicolson, 1999;

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Fig. 11 – Immuno-reactivity to Ki-67. (A) Fluorescence image of Ki-67 and DAPI was made at 10 DIV. HC: hair cell layer, SC: supporting cell layer. The bottom images were from the otocyst and the surrounding area of embryonic rat E13. HB: hind brain, OC: otocyst, →: abdominal portion of central nervous tissue. Scale bar 50 μm. (B) The percentage of Ki-67-positive cells in the hair cell layer and in the supporting cell layer (n = 125–225 cells, sampled from 5 explants). Differences in these percentages were not significant, irrespective of GM treatment or incubation with DEX. Similar distributions of Ki-67 positive cells were found at 6 DIV or at 13 DIV. Bars indicate mean ± SEM.

Sidi et al., 2003). Although an endocytotic entry was reported in guinea pig inner HCs (Griesinger et al., 2002), other studies suggest that FM1–43 likely passes through the transduction channel (Gale et al., 2001; Meyers et al., 2003).

3.3.

Effects of a steroid hormone (DEX)

Steroid hormones are used frequently in the clinical therapy against inner ear damage. In the present study, DEX kept the

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hair cells alive longer (Fig. 9), maintained the integrity of hair bundles (Fig. 8) and improved the MET activity (Fig. 7). Several mechanisms may contribute to these DEX effects. First, DEX has anti-oxidative effects (Nagura et al., 1999) and can prevent aminoglycoside ototoxicity (Himeno et al., 2002). Second, DEX stabilizes the lysosomal membrane and inhibits the release of inflammatory enzymes (Hinz and Hirschelmann, 2000). Third, DEX stabilizes the actin filament (Castellino et al., 1992), and induces the actin polymerization through the increase of cAMP hydrolysis (Koukouritaki et al., 1996). Fourth, DEX protects against apoptosis by decreasing the tumor suppressor p53 (Maya et al., 2001), increasing the antiapoptotic protein Bcl-xL, and inhibiting the apoptosisinducing factor (AIF) (Wada et al., 2005). In our study, DEX apparently decreased the percentage of apoptotic hair cells after GM treatment (Fig. 10) and enhanced the viability of FM1–43 positive cells (Fig. 9). Still further investigations are required to understand how DEX works in details; however, this study demonstrated that DEX inhibits apoptosis of hair cells. This may have some potentially important clinical consequences.

3.4. Re-emergence of hair bundles is likely by some natural turnover mechanisms By repeated observations with DIC optics, we have confirmed a re-emergence of a hair bundle-like structure in the delimited area where hair bundles had completely disappeared (Fig. 3). The morphology of a new hair bundle was similar to the immature hair bundle of the inner ear observed at E13.5–14 of mice during development (Denman and Forge, 1999; Mbiene et al., 1988), and to those undergoing regeneration (Forge et al., 1998). There are currently three proposed mechanisms that could contribute to the hair cell recovery: (1) some supporting cells may proliferate and become new hair cells, (2) some nonproliferative supporting cells may convert its phenotype into hair cells (Forge et al., 1998), and (3) some partially damaged hair cells may be repaired (Zheng et al., 1999). In this study, we could not find evidence to support cell proliferation after GM treatment, neither in the hair cell layer nor in the supporting cell layer (Fig. 11); although GM induced apoptosis (Fig. 10). We may not be able to eliminate the possibility of conversion of supporting cells to hair cells. However, a finding of a short hair bundle-like structure on a GM treated cell (Fig. 9C), which had FM1–43 accumulation within the cell since the first day of culture, might suggest a possibility of self-repair of the hair bundle. This re-emergence of hair bundles is proposed to be due to self-repair of the damaged hair bundle, because the number of cells in proliferation and in apoptosis are small and cannot match the number of recovered hair cells in the rat vestibular organ (Zheng et al., 1999). The repair of hair bundles may be triggered by the loss of hair bundle and cytoplasmic structure in the apical part of cells, such as cuticular plate (Corwin and Warchol, 1991; Gale et al., 2002). Some constitutive renewal of actin bundles of the stereocilium is reported (Schneider et al., 2002). The natural turnover of hair bundles might also be in progress during the culture period (Forge et al., 1993;

Lambert et al., 1997; Rubel et al., 1995). These may indicate a possibility that the re-emergence of hair bundles we observed utilized the same mechanism underlying the natural turnover process. Although the mechanism of functional recovery of hair cells after damages induced by GM in mammals is not yet clear, our approach using physiological measurements from cultured hair cell epithelia provides a promising strategy for future investigation.

4.

Experimental procedures

4.1.

Organ cultures of vestibular maculae

Wistar rat pups (P1–3) were deeply anesthetized with diethyl ether and killed by decapitation, and the vestibular organ was dissected out. Briefly, the temporal bone was removed and transferred into a Petri dish containing Hank's balanced salt solution (HBSS, GIBCO, Grand Island, NY, USA). The capsule covering the vestibule was opened with a pair of fine needles under a dissecting stereomicroscope (SZX9, Olympus, Tokyo, Japan). The utricular and saccular maculae were then quickly dissected out with a pair of fine forceps and the otoconial membrane was removed with a fine needle and forceps. The vestibular explants were then transferred onto a millicell membrane (Millicell CM, 30 mm, Millipore, USA), facing the hair cell layer top (Zine and De Ribaupierre, 1998). Four to 6 explants were placed on each membrane. The explants were maintained in a Falcon dish containing the culture medium, composed of 35% HBSS supplemented with L-glutamine (18.6 mM, Sigma-Aldrich, Steinheim, Germany) and glucose (108 mM, Sigma), 25% minimum essential medium (MEM, GIBCO) buffered with HEPES (5 mM, Sigma) enriched with 40% horse serum (Sigma) (Rastel et al., 1993). The osmolarity of this medium was about 330 mOsm/l. The millicell membrane was pretreated with penicillin G (50 U/ml, Sigma) in MEM, exposed to UV illumination 30 min and rinsed with MEM before use. The cultures were maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO2. The total culture medium was replaced every 2 days. The cultures were denoted as 0 day in vitro (DIV) on the day of explantation. The experimental protocol adopted in this paper was approved by the Animal Research Committee, Graduate School of Medicine, Kyoto University.

4.2.

Administration of GM and steroid hormone

After 24 h culture, hair cells were damaged by incubation with three different dosages of GM for 2 days (0.5 mM, 1 mM or 2 mM). Then, the explants were rinsed with GM-free control medium three times and the cultures were continued. When incubated with a lower concentration of GM (0.5 mM), some hair bundles were remained and hair cells still showed MET activities. So, the following experiments were made by applying 1–2 mM GM to damage hair cells. In some experiments, dexamethasone (DEX, 1 μM, Sigma) was added when GM was introduced to the culture. DEX was present for the next 2 weeks after rinsing GM. In some control experiments, DEX was applied without GM treatment.

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4.3.

Mechanical stimulation of hair bundles

MET of hair cells was tested every 2 days in separate groups of cultures throughout the culture period. The explants were fixed immediately after MET testing, and hair bundles were investigated morphologically. When MET was tested, explants on the cut-out millicell membrane were mechanically stabilized on the glass floor of a recording chamber using a U shaped platinum wire with a grid of parallel nylon strings. The recording chamber was continuously perfused with artificial perilymphatic solution (concentrations in mM: 125 NaCl, 2.5 KCl, 26 NaHCO3, 1.25 NaH2PO4, 17 glucose, 2 CaCl2, 1 MgCl2, pH 7.3). Hair bundles were mechanically stimulated by a gently applied fluid flow from a nearbyplaced pipette with a diameter of 50 μm at the tip (Fig. 1). The fluid flow was controlled to fan out over a wide area by a handmade piezoelectric pump using a layered piezo-electric element (Fig. 1A, inset), by applying a driving signal of 1 Hz sinusoidal waveform between 0 and 100 V. Mechanical stimulation was applied to the explants for approximately 20 s, and was repeated several times at approximately 100-s intervals. Because of the wide diameter of the pipette tip and fanning out of the flow some 60 degrees radial from the pipette tip, at least 5–10 hair cell hair bundles were stimulated simultaneously in the square region of interest (ROI) covering an area approximately of 30 × 50 μm (Fig. 1). Because the stimulus flow was radiating, each hair bundle was not always displaced towards its optimal orientation (Fig. 1B) (Flock, 1965).

4.4.

Fura-2 fluorometry and evaluation of MET activity

MET activity is the most representative of the hair cell function. We tried first to measure directly the MET current from hair cells by the whole cell patch recording technique, but the experiment was difficult and the current could not be recorded with confidence throughout all DIV. Therefore, MET activity of hair cells was evaluated indirectly as Ca2+ responses by loading fura-2 onto the explants, or by the accumulation of FM1–43 as described below. Explants were incubated with fura-2AM (Molecular Probes, Junction City, OR, USA) 30 min at 37 °C at a final concentration of 5 μM (10 mM fura-2AM stock solution in DMSO mixed with an equal volume of F-127, a non-ionic detergent; 25% weight/ volume in DMSO, Molecular Probe). The final concentration of DMSO was 0.1%. After the incubation, the explants were rinsed twice with MEM and transferred to a recording chamber with a glass bottom, on a microscope stage (BX50WI, Olympus, Tokyo, Japan). Fura-2 fluorescence (measured through a long-pass filter longer than 510 nm) was excited alternately at 340 nm (f340) and 380 nm (f380) wavelengths, and was monitored with a time resolution of 1.12 s. Fluorescence images were visualized using an ×60 water immersion objective and were captured with a CCD camera through an image intensifier unit. Ratio images (f340/f380) were calculated using an image processing system (Aquacosmos HiSCa, Hamamatsu Photonics, Hamamatsu, Japan). The fluorescence ratio was calculated from a ROI where hair bundles were stimulated (Fig. 1A), and simultaneously from another ROI where hair bundles were not stimulated, as a control from the

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same explant. All experiments were performed at room temperature (22–25 °C). Both temporal and spatial resolutions were limited in these experiments, because of the neutralization effects of the background fluorescence created by a number of non-responsive cells; only 5–10 hair cells were stimulated within a ROI. We therefore did not calculate the absolute concentration of intracellular Ca2+, rather the Ca2+ responses were indicated as the ratio of f340/f380 of a ROI. We calculated the temporal average during mechanical stimulation, and the response was scored either positive or negative for each explant by the following criteria: The explant was scored MET-positive when the ratio (f340/f380) change during stimulation was at least three times larger than the standard deviation of the ratio noise level before stimulation (SD = 0.002–0.008 in Fig. 4). An explant was scored MET-negative when no positive ratio response was observed even by the test performed on more than 10 ROIs randomly placed on the explant (Fig. 4B). The percentage of MET positive explants were plotted against DIV (Figs. 5 and 7), after taking moving averages among nearby three DIV. Plots on the first and the last DIV were the average between the second and 1 day before the last DIV, respectively.

4.5.

FM1–43 accumulation of hair cells

A lipophilic dye, FM1–43 (Molecular Probes, Inc), has been shown to enter hair cells through transducer channels (Gale et al., 2001), suggesting a possibility of detecting hair cells with active transducer channels as the accumulation of FM1–43 (5 μM FM1–43 in artificial perilymphatic solution, prepared from 10 mM stock solution in DMSO; Fig. 6). FM1–43 accumulation within the cell was observed with a fluorescence microscope (excitation 510–550 nm, emission 600 nm and long pass) using either ×10, ×60 or ×100 water-immersion lenses. The MET activity of a hair cell was tested as the dye accumulation, by applying mechanical stimulation for 10 s as was done in fura-2 fluorometry (Fig. 6). Five seconds stimulation might be sufficient to detect active MET for normal hair cells (Si et al., 2003). But we examined recovering hair cells, therefore a longer time stimulation of 10 s was adopted. Gale et al. also stimulated for 10 s (Gale et al., 2001). Without mechanical stimulation, dye accumulation did not occur. Immediately after the mechanical stimulus, the explant was washed 30 min with artificial perilymphatic solution. Fluorescence and bright field DIC images were then captured using a cooled color 3 CCD camera (Fig. 6, C7780, Hamamatsu Photonics, Hamamatsu, Japan), and were overlaid. In some experiments, FM1–43 imaging was made on the same explants after Ca2+ imaging with fura-2. The long-term viability of cells that retained FM1–43 was evaluated by counting the number of cells (Fig. 9). In these experiments, explants were loaded with FM1–43 on the first day of culture by pipetting for 1 min in the phosphate-buffered saline (PBS; pH 7.4) containing 5 μM FM1–43. A long stimulus time was adopted here; because (1) we presumed the efficacy of mechanical stimulation was low because the pipetting of solution was not necessarily directed to hair bundles, and (2) we intended to load the cell with the dye at the maximum concentration possible, to make it certain the dye presence within the cell during a long-term culture. After rinsing the

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dye, the explants were cultured as usual. On the 3rd, 5th, 9th, and 15th DIV, the explants were observed under a fluorescence microscope without fixation. The number of FM1–43 positive cells per 10,000 μm2 was counted. Approximately this area corresponds to 300 frames (frame is defined below) and contained about 70 hair cells. These numbers of FM1–43 positive cells per 10,000 μm2 were converted to % frame of FM1–43 positive cells (Fig. 9D), in order to facilitate comparison with % hair bundle (Fig. 2). Cells were counted FM1–43 positive when the average fluorescence intensity of the cell was more than 1.5 times greater than the background.

vented by sterilizing the ×100 water-immersion objective with 70% ethanol and by subsequent exposure to UV illumination 30 min. SEM observation was made after fixation; 2% glutaraldehyde 2 h at 4 °C, followed by rinsing three times with 0.1 M phosphate buffer (PB) 20 min each, and postfixation with 1% OsO4 40 min at room temperature. They were then dehydrated in a graded series of alcohol (50% ∼ absolute), dried using the freeze and dry method in tbutyl-alcohol (Nakalai tesque, Kyoto, Japan) and gold spattered at a pressure 5 × 10−2 Torr.

4.8. 4.6. Phalloidin staining of the hair bundle and the cell boundary (frame) The actin filaments of the hair bundles were stained with phalloidin after fixation (Figs. 2 and 8). Explants were fixed with 4% paraformaldehyde at 4 °C overnight and washed 15 min with PBS three times. The preparations were incubated with 0.5% Triton X-100 (Sigma) 20 min and then with 1% BSA (Sigma). Hair bundles were then stained with fluorescent isothiocyanate (FITC)-labeled phalloidin solution (0.5 μg/ml in PBS, Sigma) 45 min in the dark and washed three times with PBS 15 min each. Explants were mounted on a slide glass using Vectashield mounting media (Vector). The phalloidin-FITCstained explants were observed under a fluorescence microscope equipped with a digital image capture system (460– 490 nm excitation and emission 510 nm and long pass, Nikon, Tokyo, Japan). Phalloidin-FITC stained the hair bundles and the submembranous actin matrix, which visualized the cell boundary as a polygonal frame (Fig. 2Ab, indicated by a dotted line). The polygonal frame is the edges cell adhesion, and is the mixed boundaries of hair cells and supporting cells constituting the sensory epithelium. We defined the area enclosed by the phalloidin-positive cell boundary as a frame. The number of frames per explant was calculated from the average number of frames within ROIs and the areal ratio of these ROIs to the total area of an explant (237,500 ± 21,868 μm2 n = 110, Fig. 2B); averaging was made on 3 ROIs randomly set on an explant. Each ROI covered approximately the area 60 × 60 μm2 and its perimeter was on the edge of frames and contained about 40–50 frames. The total number of frames (Fig. 2B) and the percentage of frames which had a hair bundle (% hair bundle, Fig. 2C) were plotted as a function of DIV; they were plotted on odd DIV after taking an average between the next even DIV (Fig. 2C).

4.7. Observation of the hair bundle re-emergence by differential interference contrast (DIC) optics and scanning electron microscope (SEM) After the elimination of hair bundles by exposing the explants to GM, the process of hair-bundle re-emergence was observed by DIC optics with an ×100 water-immersion objective. Small pieces of cracked cover glass were placed on the explants to mark the site of observation (Figs. 3A and A′). The same site was observed by DIC several times for the next 2–3 weeks; then the explants were fixed and were observed using a scanning electron microscope (SEM S-430, HITACHI, Tokyo, Japan). Bacterial contamination was pre-

Integrity of hair bundle

A percentage of cells having a well-integrated hair bundle was counted using SEM (Fig. 8, Table 1). The integrity of stereocilial array was evaluated as being positive or negative according to the following 3 criteria: (1) presence of more than a few stereocilia in a bundle, 2) absence of stereocilial fusion appearance within the bundle, and (3) absence of fanning out of stereocilia from the bundle. When all three criteria were satisfied, the hair bundle was classified as integrity-positive, and the percentage of integrity-positive hair bundles in each explant was calculated from more than 20 hair bundles.

4.9. TUNEL staining for detecting apoptosis and immuno-histochemistry of Ki-67 for detecting cell proliferation 4.9.1.

TUNEL staining

Apoptotic cell death was evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method (Fig. 10), using an Apoptag Fluorescein Direct In situ Apoptosis Detection Kit (Intergen, Purchase, NY, USA). The explants were sectioned 20 μm by a cryostat, then TUNEL staining was made according to the supplier's instruction. In brief, the specimens were incubated with 0.5% Triton-X in PBS (PBS-Triton) 20 min at room temperature, then incubated with terminal-deoxynucleotidyl-transferase (TdT) and fluorescein nucleotide in a humid atmosphere at 37 °C 1 h.

4.9.2.

Immuno-histochemistry of Ki-67

In order to investigate the underlying mechanism of hair bundle recovery, we examined cell proliferation activities which were detected by Ki-67 immuno-histochemistry (Fig. 11). Ki-67 can detect the granular component within the nucleus during proliferating stage, and is used to indicate cell proliferation. Thin sections of explants were incubated with 0.2% PBSTriton 30 min at room temperature, then immersed in 1% bovine serum albumin (BSA) in PBS-Triton to block nonspecific immunoglobulin binding. The samples were incubated at 4 °C overnight with anti-Ki-67 rabbit monoclonal antibody solution (1:500; Lab Vision, Fremont, CA, USA) diluted with 1% BSA in PBS-Triton. After two washes in PBS-Triton, the sections were incubated with Alexa Fluor 594-conjugated antirabbit IgG (1:500; Molecular Probes) diluted with 1% BSA in PBSTriton 6 h at 4 °C. As a control for Ki-67 immuno-histochemistry, the otocyst of embryonic rat (E13) was processed similarly, showing a large number of proliferating cells (Fig. 11A bottom).

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4.9.3.

DAPI staining

At the end of immuno-staining procedures, specimens were stained with 4′,6-diamidino-2-phenylindole (DAPI; 0.1μg/ml, Molecular Probes, Eugene, OR, USA) 30 min at room temperature to demonstrate the nuclear chromatin. TUNEL or Ki-67 immunoreactive images were made under a fluorescence microscope, together with DAPI images (ECLIPSE E600, Nikon, Tokyo, Japan). Percentages of TUNEL positive cells were counted on 5 DIV, and Ki-67 immunoreactive cells on 6, 10 and 13 DIV over the DAPI-positive cells (Figs. 10 and 11). Supporting cell layer and hair cell layer were separately counted; the supporting cell layer was condensed with the nuclei (DAPI) and the hair cell layer was located on top of the supporting cell layer. Some sections were stained with phalloidin-FITC in order to confirm the hair cell layer.

4.10.

Statistical analysis

Statistically significant differences were evaluated using ANOVA, t test or Fisher's exact probability test, and the significance was evaluated at the level of P < 0.05. Spearman's rank correlation method was used to evaluate the correlation coefficient. Means and standard errors are indicated with the number of explants.

Acknowledgments We acknowledge Professor E.W. Rubel for critical comments and valuable suggestions on the manuscript, Drs. T.M. Ishii and H. Kuba and T. Nakagawa and T. Kita for discussions and Professor T. Kaneko and Ms. K. Okamoto for assistance in scanning electron microscope studies, and Dr. Matsumoto of Department of Otolaryngology-Head and Neck Surgery, Graduate School of Medicine, Kyoto University for the personal communication. This paper was supported by a grant-in-aid of priority area (12053233) to H.O., and supported in part by Establishment of International COE for Integration of Transplantation Therapy and Regenerative Medicine (COE program of the Ministry of Education, Culture, Sports, Science and Technology, Japan). REFERENCES

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