- Email: [email protected]
Evidence for hair cell regeneration in the crista ampullaris of the lizard Podarcis sicula
Available online at www.sciencedirect.com R
Hearing Research 178 (2003) 79^88 www.elsevier.com/locate/heares
Evidence for hair cell regeneration in the crista ampullaris of the lizard Podarcis sicula Bice Avallone, Maria Porritiello, Daniela Esposito, Rosalia Mutone, Giuseppe Balsamo, Francesco Marmo Department of Genetics, General and Molecular Biology, University Federico II, via Mezzocannone 8, 80134 Naples, Italy Received 15 October 2002; accepted 17 January 2003
Abstract We studied hair cell regeneration in the crista ampullaris of the lizard Podarcis sicula both in untreated animals and at early and late time intervals following a single high dose of gentamicin. The study was carried out using the S-phase marker 5-bromo-2Pdeoxyuridine. Our ultrastructural and immunofluorescence studies showed that both apoptosis and hair cell regeneration happen in the lizard crista ampullaris in untreated animals, and that regenerative processes are greatly accelerated after treatment with the aminoglycoside antibiotic gentamicin. Our observations indicate that hair cell regeneration is strongly implicated in the repair of damaged sensory epithelium, and that new hair cells appear likely to arise from supporting cells. 1 2003 Elsevier Science B.V. All rights reserved. Key words: Regeneration; Lizard; Crista ampullaris; Bromodeoxyuridine ; Gentamicin
1. Introduction Hair cells (HCs), which are the sensory receptors for hearing, equilibrium and motion detection, located in the auditory, vestibular and lateral line organs, may be damaged by a number of agents including aminoglycoside antibiotics and severe over-stimulation (acoustic trauma). Postembryonic HC production occurs continually throughout life in the vestibular and auditory sensory epithelium in ¢shes and amphibians and in the vestibular end organs of birds (Popper and Hoxter, 1984; Corwin, 1985; J=rgensen and Mathiesen, 1988; Presson and Popper, 1990; Roberson et al., 1992; Stone
* Corresponding author. Tel.: +39 (81) 2535012 (o⁄ce), +39 (81) 2535006 (lab); Fax: +39 (81) 2535000. E-mail address: [email protected] (F. Marmo). Abbreviations: BrdU, 5-bromo-2P-deoxyuridine; DS, distal to septum; DW, distilled water; HC, hair cell; PBS, phosphatebu¡ered saline; PS, proximal to septum; SEM, scanning electron microscopy; TEM, transmission electron microscopy; TBS, Trisbu¡ered saline
et al., 1999). In the mature hearing organ of birds, the basilar papilla, HCs are produced after HC loss caused by exposure to noise (Cotanche, 1987; Corwin and Cotanche, 1988; Ryals and Rubel, 1988; Ryals and Westbrook, 1990), ototoxic drug treatment (Cruz et al., 1987; Lippe et al., 1991; Stone et al., 1999; Stone and Rubel, 2000) or laser ablation (Warchol and Corwin, 1996). According to Ryals and Westbrook (1990) there may be some very low level of HC production which is activated in the absence of trauma in adult quail cochlea. Analogous ongoing production of HCs during postembryonic life does not appear to occur in mammalian vestibular and auditory sensory epithelium. It has been demonstrated, however, that HCs reappeared in mammalian vestibular system after HC loss following treatment with aminoglycosides (Forge et al., 1993; Rubel et al., 1995; Tanyeri et al., 1995; Lopez et al., 1997; Zheng and Gao, 1997) but did not reappear in the organ of Corti, even after trauma (Sobkowicz et al., 1992). Several studies have sought to identify precursor cells for new HCs. These potential precursors include supporting cells, embryonic-like neuroepithelial cells, and cells immediately adjacent to the sensory epithelium
0378-5955 / 03 / $ ^ see front matter 1 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0378-5955(03)00040-6
HEARES 4664 1-4-03
80
B. Avallone et al. / Hearing Research 178 (2003) 79^88
(Corwin and Cotanche, 1988; Girod et al., 1989; Raphael, 1992; Weisleder and Rubel, 1993; Cotanche et al., 1994; Stone and Cotanche, 1994; Tsue et al., 1994; Bhave et al., 1995; Fekete et al., 1998; Stone et al., 1999). Few investigations have been carried out on the reptilian inner ear. A series of experiments with [3 H]thymidine injections in adders (Vipera berus) did not demonstrate any labelled nuclei in the sensory epithelium. Similar 3 H injections in two specimens of the turtle Pseudoemys elegans showed labelled supporting and sensorial cell nuclei (J=rgensen, 1991) which suggested cell turnover. The present work aims to study HC regeneration in the reptilian (Podarcis sicula ; Fig. 1) vestibular system, and if possible, to identify the precursor cells for new HCs. We studied the crista ampullaris (Fig. 2) of the lizard P. sicula both in normal animals and at early and late time intervals following a single high dose of gentamicin used to initiate the regenerative events (Janas et al., 1994). The resulting damage pattern was investigated by scanning electron microscopy (SEM). Transmission electron microscopy (TEM) studies were also undertaken to determine whether spontaneous apoptosis occurs, which would suggest a slow turnover of normal vestibular HCs. One S-phase-associated marker, 5-bromo-2P-deoxyuridine (BrdU), which is incorporated into the newly synthesised DNA strand (Gratzner, 1982), was used to study progression through S phase and mitosis. To detect BrdU-labelled cells a protocol for BrdU immuno£uorescence was followed.
USA) daily for 10 days. One lizard was sacri¢ced beginning at 24 h after the ¢rst BrdU injection, daily for 10 days. Then the samples were processed for immuno£uorescence. 2.3. Exclusive BrdU treatment Ten lizards were treated with BrdU only (100 mg/kg, in sterile physiological saline) daily for 10 days. The daily dose of BrdU was divided into three intraperitoneal injections given every 4 h. One lizard was sacri¢ced beginning at 24 h after BrdU injection, daily for 10 days. 2.4. Untreated group Six untreated lizards were used to verify the ototoxicity of gentamicin by SEM and to study the sensorial epithelium ultrastructure by TEM. The specimens were anaesthetised by deep exposure to fumes of ether and decapitated. 2.5. Positive control Intestines of lizards treated with BrdU only and gentamicin plus BrdU were removed and processed for immuno£uorescence the same as tissue from the crista ampullaris. 2.6. SEM
Thirty-seven adult specimens of P. sicula, weighing V7.00 g each, were collected near Naples, and maintained in the laboratory under natural conditions of light and temperature. The lizards were fed twice a week.
Heads were ¢xed in 2.5% glutaraldehyde in 0.1 M phosphate-bu¡ered saline (PBS) pH 7.4 for 3 h at 4‡C for SEM observation. After microdissection of the vestibular end organs the specimens were rinsed in PBS and post-¢xed in 1% OsO4 in the same bu¡er for 1 h at 4‡C. After several rinses in PBS, the specimens were subjected to serial dehydration steps followed by critical point drying. Specimens were mounted on aluminium stubs, coated with gold, and examined with a Cambridge Stereoscan 250 MK III Microscope.
2.1. Gentamicin treatment
2.7. TEM
One group (11 lizards) received a single subcutaneous injection of gentamicin sulphate (400 mg/kg; ScheringPlough, Italy). One lizard was sacri¢ced beginning from 4 h after gentamicin treatment and one lizard daily for 10 days. The samples were processed for SEM.
Heads were ¢xed in 2.5% glutaraldehyde in 0.1 M PBS pH 7.4 for 3 h at 4‡C for TEM observation. After microdissection of the vestibular end organs the specimens were rinsed in PBS and post-¢xed in 1% OsO4 in the same bu¡er for 1 h at 4‡C. Samples were rinsed several times in bu¡er, dehydrated, embedded in Epon 812 resin, and sectioned in a Super Nova Leica Ultratome. Ultrathin sections were placed on copper grids and counterstained with uranyl acetate and lead citrate. Specimens were observed and photographed in a Philips CM12 electron microscope.
2. Materials and methods
2.2. Gentamicin plus BrdU treatment Ten lizards, beginning at 24 h after gentamicin injection (400 mg/kg), received a subcutaneous injection of BrdU (100 mg/kg, in saline ; Sigma, St. Louis, MO,
HEARES 4664 1-4-03
B. Avallone et al. / Hearing Research 178 (2003) 79^88
81
2.8. Indirect immuno£uorescence Heads were ¢xed in 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M cacodylate bu¡er pH 7.3 for 2.5 h at room temperature for immuno£uorescence observations. After several rinses in the same bu¡er, vestibular end organs were dissected and treated with 0.2 M ammonium chloride in cacodylate bu¡er for 1 h at room temperature. Specimens were dehydrated with ethanol and propylene oxide and embedded in Epon. Serial sections of specimens were cut at 2 Wm thickness, mounted on slides coated with poly-L-lysine (0.01%) and stained with toluidine blue. The plastic was removed from sections by immersing the slides in a su⁄cient amount of a solution of 2.4 M KOH in propylene oxide :methyl alcohol (2:1) for 6 min. Slides were rinsed with methyl alcohol and washed thoroughly ¢rst in water for 10 min and then in distilled water (DW) for 5 min. Slides were treated with 2 N HCl in PBS for 20 min at 37‡C to denature DNA and rinsed in DW. Subsequently, the sections were incubated in: (1) protease K (4 Wg/ml; Sigma) in 0.01 M Tris^HCl pH 7.4 for 8 min at room temperature and rinsed in 0.1 M PBS and 0.5% bovine serum albumin (BSA; Sigma); (2) rabbit serum (Sigma) 1:20 in 0.1 M Tris-bu¡ered saline (TBS), 0.25% Tween 20 and 0.5% BSA for 1 h at 37‡C ; (3) anti-BrdU monoclonal antibody (Sigma) 1:1000 in 0.1 M TBS, 0.25% Tween 20 and 0.5% BSA overnight at room temperature and rinsed in the same bu¡er; (4) rabbit anti-mouse IgG FITC conjugated (Sigma) 1:100 in the same bu¡er for 1 h at 37‡C and ¢nally rinsed several times in the same bu¡er. Preimmune sera, instead of speci¢c antisera, were used in the control section; each step was carried out in a moist chamber. Finally sections were mounted in glycerol and £uorescence observations were carried out with a Zeiss Axioskop microscope using the KS300 software (Kontron) to acquire microscope images and ProPalette 8000 for digital colour ¢lm recorder. 2.9. Statistical analysis One hundred sections, from each crista ampullaris of lizard, were used for quantitative analysis. Statistical analysis was performed with a single factorial analysis of variance (ANOVA). A P value less than 0.01 was considered to be signi¢cant.
Fig. 1. P. sicula. Fig. 2. Light micrograph of crista ampullaris of lizard. Sensory epithelium (se), transitional cell zone (TCz) and dark cell zone (DCz). Bar = 10 Wm. Fig. 3. SEM of crista ampullaris of P. sicula. Bar = 10 Wm.
3. Results 2.10. Use of animals 3.1. SEM observations Samples were captured with authorisation of 1/06/ 2000, No. SCN/2D/2000/RB of the Ministero dell’Ambiente.
The crista sensory area (Fig. 3) of untreated specimens shows HCs surrounded by supporting cells with
HEARES 4664 1-4-03
82
B. Avallone et al. / Hearing Research 178 (2003) 79^88
HEARES 4664 1-4-03
B. Avallone et al. / Hearing Research 178 (2003) 79^88
83
Fig. 5. TEM of crista ampullaris. (A) Bottle-shaped HC I enclosed in a nerve calyx. Bar = 1.7 Wm. (B) Apoptotic HC I with dark cytoplasmic matrix and a condensed nucleus. Bar = 1.9 Wm. (C) Apoptotic HC I with dark cytoplasm and dense nucleus enclosed in a nerve calyx together with a light sensory cell. Bar = 1.1 Wm.
small microvilli projecting from the luminal surfaces (Fig. 4A,B). In the crista sensory area, two zones characterised by di¡erent HC ciliary bundles are observed : a central zone, proximal to septum (PS) (Fig. 4A), with cells showing both long kinocilium and long stereocilia,
decreasing in length, and a peripheral zone, distal to septum (DS) (Fig. 4B) with cells showing a long kinocilium and short stereocilia compact bundles with a gradual increase in length. The region of the crista included between the two
6 Fig. 4. SEM of crista ampullaris. (A) Lizard, untreated. Zone proximal to septum cruciatum (PS): cells with both a long kinocilium and long stereocilia, decreasing in length. Bar = 3.8 Wm. (B) Lizard, untreated. Zone distal to septum cruciatum (DS): cells with a long kinocilium and short stereocilia compact bundles, which gradually increase in length. Bar = 4 Wm. (C) Lizard, untreated. Smooth central portion (asterisk) of the crista ampullaris, included between the two protrusions of the septum cruciatum, with small polygonal cells. Bar = 4.4 Wm. (D) Lizard, untreated. Septum cruciatum: sparse tufts of microvilli of ‘microvillous’ cells (arrows) are observed. Bar = 4.7 Wm; (Inset) Higher magni¢cation of microvilli tufts. Bar = 2.1 Wm. (E) PS zone of the crista 4 h after gentamicin injection. Damaged sensory cells with fused stereocilia (arrows). Bar = 5 Wm. (F) DS zone of the crista 4 h after gentamicin injection. Supporting cells lacking small microvilli and sensory cells with su¡ering and fused stereocilia (arrows) are observed. Bar = 5 Wm. (G) PS zone of the crista 1 day after gentamicin injection. Sensory cells with short (arrow) or lacking hairs (asterisk) are observed. Bar = 5.4 Wm. (H) DS zone of the crista 1 day after gentamicin injection. Extrusion of cellular contents into the fused bundles. Bar = 5.4 Wm. (I) PS zone of the crista 10 days after gentamicin injection. Sensory cells with mature-appearing hairs and some HCs with immature stereocilia bundles (arrows). Bar = 5 Wm. (L) DS zone of the crista 10 days after gentamicin injection. Sensory cells regain normal appearance. Bar = 5 Wm.
HEARES 4664 1-4-03
84
B. Avallone et al. / Hearing Research 178 (2003) 79^88
At 1 day after gentamicin injection, there is a widespread loss of hairs of the sensory cells (Fig. 4G), and extrusion of both entire HCs and cellular contents into the fused bundle (Fig. 4H). At 10 days after gentamicin injection, the sensorial area shows an apparently normal morphology: very few cells with hair loss are present so that some HCs are evident with immature hair bundles and signi¢cant numbers of HCs with mature hair bundles (Fig. 4I,L). 3.2. TEM observations
Fig. 6. TEM of crista ampullaris. (A) Cylindrical HC II, innervated by terminal buttons. Bar = 2 Wm. (B) Apoptotic HC II with dark cytoplasmic matrix and a condensed nucleus. Bar = 2.2 Wm.
protrusions of the septum cruciatum is smooth (Fig. 4C), lacking stereo- or kinocilia, with small polygonal cells. In the septum region, the size of these cells increases, and sparse tufts of microvilli appear among them (Fig. 4D). The HCs already appear damaged 4 h after gentamicin injection and the stereocilia seem fused in both the DS and PS zones of the crista (Fig. 4E,F).
TEM observations of crista sensory epithelium of untreated specimen of P. sicula show that HCs are located at the summit of the crista in the luminal part of the sensory epithelium and they never extend to the basal membrane. Two major types of HC populations (HC I and HC II according to Wersa«ll, 1956) are found: HC I, bottle-shaped and enclosed in a nerve chalice, and HC II, cylindrical and innervated by terminal buttons. HCs are surrounded by supporting cells in which the cytoplasm shows extensive arrays of parallel microtubules extending lengthwise (Figs. 5A and 6A). Occasionally, a third type of sensory cells, i.e. dark HCs, was observed in both the DS and PS zones of the crista. These cells show the morphology of apoptotic cells. We consider these dark cells to be sensory cells not only because of the presence of a cuticular plate, but also because these cells are sometimes enclosed in a nerve calyx together with a light sensory cell. Furthermore, these cells present rare synaptic buttons, both a¡erent and e¡erent. These cells show an increase in electronic density of both the nucleus and cytoplasm, accompanied by a clump of nuclear chromatin lying against the nuclear membrane. However, mitochondria appear to be intact and endoplasmic reticulum dilated (Figs. 5B,C and 6B). 3.3. BrdU immuno£uorescence In the animals treated with BrdU only (Fig. 8), at 2 days very few labelled supporting cell nuclei are found (Fig. 7A). In the following days, the number of labelled supporting cell nuclei progressively increased and, only
C Fig. 7. Fluorescence micrograph of crista ampullaris. (A) Crista ampullaris of sample treated with BrdU only, for 2 days. Very few BrdU-labelled supporting cell nuclei (arrows) are observed. Bar = 24 Wm. (B) Crista ampullaris of a sample treated with BrdU only, for 5 days. Numerous supporting cells and few sensory cell (arrows) BrdU-labelled nuclei are observed. Bar = 22.2 Wm. (C) Crista ampullaris of a sample treated with BrdU only, for 10 days. Some labelled sensory cell nuclei (arrows) are observed. Bar = 22.6 Wm. (D) Crista ampullaris of a sample treated with BrdU only, for 2 days. Numerous labelled nuclei of ‘microvillous’ dark cells are observed. Bar = 22.2 Wm. (E) Crista ampullaris of a sample treated with BrdU for 3 days, after gentamicin injection. Numerous supporting cells and some BrdU-labelled sensory cells (arrows) are observed. Bar = 22.2 Wm. (F) Crista ampullaris of a sample treated with BrdU for 5 days after gentamicin injection. A cluster of BrdU-labelled nuclei of sensory cells (arrow) is observed. Bar = 30.4 Wm. (G) Crista ampullaris of a sample treated with BrdU for 10 days, after gentamicin injection. Numerous £uorescent HC I cell nuclei surrounded by supporting positive cells are observed. Bar = 26.6 Wm. (H) Portion of crista ampullaris of a sample treated with BrdU for 10 days, after gentamicin injection. BrdU-labelled nuclei of ‘microvillous’ dark cells are observed. Bar = 22.2 Wm.
HEARES 4664 1-4-03
B. Avallone et al. / Hearing Research 178 (2003) 79^88
85
D
A
E
B
F
C
G
HEARES 4664 1-4-03
H
86
B. Avallone et al. / Hearing Research 178 (2003) 79^88
supporting cell 60
hair cell
supporting cell
60
50
50 Positive cell
40 positive cell
hair cell
70
30 20
40 30 20
10
8
10
0 2d
5d
9
10d
0 2d
5d
10d
Fig. 8. Mean number of labelled hair and supporting cell nuclei, W S.D., after 2, 5 and 10 days, in animals treated with BrdU only. Signi¢cance of di¡erences was evaluated at P 6 0.01 (ANOVA). Fig. 9. Mean number of labelled hair and supporting cell nuclei, W S.D., after 2 days, 5 days and 10 days, in animals treated with Gentamicin plus BrdU. Signi¢cance of di¡erences has been evaluated at P 6 0.01 (ANOVA).
in the samples treated with BrdU for 5 days, some BrdU-labelled nuclei of the sensory cells were detected (Fig. 7B). Their number increased in the samples treated with BrdU for 10 days (Fig. 7C). From the ¢rst day of treatment, numerous labelled nuclei of ‘microvillous’ (according to Hamilton, 1965) dark cells were always evident (Fig. 7D). In the epithelium of the cristae of animals treated with BrdU after gentamicin injection, already 1 day after BrdU treatment we observed chie£y BrdU-labelled nuclei of supporting cells. At 3 days, £uorescence was present not only in the majority of nuclei of the supporting cells (i.e. in the supporting cell layer of epithelium) and in the nuclei of the ‘microvillous’ dark cells but also in some sensory cell nuclei (i.e. in the HC layer of epithelium) (Fig. 7E). The number of labelled sensory cell nuclei increased proportionally to the duration of the treatment. The animals treated with BrdU for 5 days after gentamicin showed a greater number of labelled sensory cell nuclei, often clustered in some zones of the crista (Fig. 7F). After 10 days of treatment a large number of £uorescent HCs surrounded by positive supporting cells was observed (Fig. 7G). Intense and wide positivity was present in the septum ‘microvillous’ dark cells (Fig. 7H). Control gut sections both in the gentamicin plus BrdU and in the BrdU-only-treated animals always showed numerous epithelium cells with BrdU-labelled nuclei. All negative controls showed no £uorescence. 3.4. Statistical analysis Fig. 8 shows the mean number of labelled hair and supporting cell nuclei, W S.D., after 2, 5 and 10 days, in animals treated with BrdU only. Fig. 9 shows the mean
number of labelled hair and supporting cell nuclei, W S.D., after 2, 5 and 10 days, in animals treated with gentamicin plus BrdU. Signi¢cance of di¡erences was evaluated at P 6 0.01 (ANOVA).
4. Discussion Our SEM results on the crista of P. sicula treated with gentamicin show that this aminoglycoside antibiotic induces damage primarily to HCs. SEM observations indicate that this damage is precocious : 4 h after gentamicin injection, sensory cells with fused stereocilia are visible in the crista. Sensorial epithelium repairs progressively and, 10 days after the injection, di¡erentiated HCs and cells with small hair bundles (and therefore probably immature) are evident in this area. Besides, the immuno£uorescence studies show that regeneration phenomena are present and rather di¡use. In fact, 1 day after gentamicin injection numerous labelled supporting cell nuclei are evident and, starting from 3 days, sensory cell nuclei also are BrdU-labelled. In the following days, the number of labelled supporting cell nuclei progressively increases. Immuno£uorescence studies and SEM observations therefore indicate that regeneration processes are strongly implicated in the damaged sensory epithelium repair. Incorporation of BrdU at ¢rst in the supporting cell nuclei and only after some days in the HC nuclei (both in gentamicin plus BrdU and in BrdU only animals) leads us to hypothesise that sensory cells di¡erentiate from supporting cells, in agreement with suggestions from several previous studies on regeneration in the cochlea of chicken (Corwin and Cotanche, 1988; Raphael, 1992; Stone and Cotanche, 1994; Bhave et al.,
HEARES 4664 1-4-03
B. Avallone et al. / Hearing Research 178 (2003) 79^88
1995; Fekete et al., 1998; Stone et al., 1999), in the vestibular organs of chicks (Weisleder and Rubel, 1992, 1993) and in the lateral line organs of amphibians (Corwin, 1986; Corwin et al., 1989; Balak et al., 1990; Jones, 1991; Jones and Corwin, 1993). The earliest cells labelled with the S phase marker resemble supporting cells, and all our studies reveal no labelled HCs immediately following the onset of ototoxic treatment. The gradual increase of BrdU-positive cells in the crista sensory epithelium may also be attributable to the fact that supporting cells may go through more than one cell division cycle after HC degeneration (Jones and Corwin, 1993; Stone and Cotanche, 1994). Moreover, TEM observations, showing that di¡erentiated HCs undergo apoptosis during normal life, may indirectly indicate the presence of proliferation phenomena and cell di¡erentiation connected to spontaneous HC turnover in reptilian vestibular systems. This was already demonstrated in ¢shes (Corwin, 1981, 1983; Popper and Hoxter, 1984, 1990) which continually produce cells replacing apoptotic dying cells, as well as in other groups of vertebrates (amphibians, avians) (Corwin, 1985; J=rgensen and Mathiesen, 1988; Roberson et al., 1992; Weisleder and Rubel, 1993; Stone et al., 1999; Goodyear et al., 1999) in the absence of acoustic trauma or treatment with aminoglycoside antibiotics. In summary, our observations suggest that in the lizard P. sicula new HCs do not arise from pre-existing HCs. Most likely, labelled supporting cells divide and produce daughter cells with labelled DNA which become HCs and regenerate the epithelium. This process is limited or slow in normal conditions but is greatly ampli¢ed by treatment with the aminoglycoside gentamicin.
References Balak, K.J., Corwin, J.T., Jones, J.E., 1990. Regenerated hair cells can originate from supporting cell progeny: evidence from photoxicity and laser ablation experiments in the lateral line system. J. Neurosci. 10, 2502^2512. Bhave, S.A., Stone, J.S., Rubel, E.W., Coltrera, M.D., 1995. Cell cycle progression in gentamicin-damaged avian cochleas. J. Neurosci. 15, 4618^4628. Corwin, J.T., 1981. Postembryonic production and ageing of inner ear hair cells in sharks. J. Comp. Neurol. 201, 541^553. Corwin, J.T., 1983. Postembryonic growth of the macula neglecta auditory detector in the ray, Raja clavata. Continual increases in hair cell number neural convergence and physiological sensitivity. J. Comp. Neurol. 217, 345^356. Corwin, J.T., 1985. Perpetual production of hair cells and maturational changes in hair cell ultrastructure accompany postembryonic growth in amphibian inner ear. Proc. Natl. Acad. Sci. USA 82, 3911^3915. Corwin, J.T., 1986. Regeneration and self-repair in hair cell epithelia:
87
experimental evaluation of capacities and limitations. In: Ruben, R.J., Van De Water, T.R., Rubel, E.W. (Eds.), The Biology of Change in Otolaryngology. Elsevier, New York. Corwin, J.T., Cotanche, D.A., 1988. Regeneration of sensory hair cells after acoustic trauma. Science 240, 1772^1774. Corwin, J.T., Balak, K.J., Borden, P.C., 1989. Cellular events underlying the regenerative replacement of lateral line sensory epithelia in amphibians. In: Combs, S., Gorner, P., Munz, H. (Eds.), The Mechanosensory Lateral Line: Neurobiology and Evolution. Springer, New York, pp. 161^183. Cotanche, D.A., 1987. Regeneration of hair cell stereociliary bundles in the chick cochlea following severe acoustic trauma. Hear. Res. 30, 181^196. Cotanche, D.A., Lee, K.H., Stone, J.S., Picard, D.A., 1994. Hair cell regeneration in the bird cochlea following noise damage or ototoxic drug damage. Anat. Embryol. (Berl.) 189, 1^18. Cruz, R.M., Lambert, P.R., Rubel, E.W., 1987. Light microscopic evidence of hair cell regeneration after gentamicin toxicity in chick cochlea. Arch. Otolaryngol. Head Neck Surg. 113, 1058^1062. Fekete, D.M., Muthukumar, S., Karagogeos, D., 1998. Hair cell and supporting cells share a common progenitor in the avian inner ear. J. Neurosci. 18, 7811^7821. Forge, A., Li, L., Corwin, J.T., Nevil, G., 1993. Ultrastructural evidence for hair cell proliferation in the mammalian inner ear. Science 259, 1616^1619. Girod, D.A., Duckert, L.G., Rubel, E.W., 1989. Possible precursors of regenerated hair cells in the avian cochlea following acoustic trauma. Hear. Res. 42, 175^194. Goodyear, R.J., Gates, R., Lukashkin, A.N., Richardson, G.P., 1999. Hair-cell numbers continue to increase in the utricular macula of the early post-hatch chick. J. Neurocytol. 28, 851^861. Gratzner, H.G., 1982. Monoclonal antibody to 5-bromo-and 5-iododeoxyuridine: a new reagent for detection of DNA replication. Science 218, 474. Hamilton, D.W., 1965. Microvillous cells in ampullae of the lizard inner ear. J. Morphol. 116, 339^354. Janas, J.D., Cotanche, D.A., Rubel, E.W., 1994. Avian cochlear hair cell regeneration: a quantitative analysis of damage and recovery from single high dose gentamicin injection. Assoc. Res. Otolaryngol. 17, 66A. Jones, J.E., 1991. Regeneration in the Lateral Line: Identities of Progenitor Cells and the Action of Leukocytes. PhD Thesis, University of Virginia, Charlottesville, VA. Jones, J.E., Corwin, J.T., 1993. Replacement of lateral line sensory organs during tail regeneration in salamanders: identi¢cation of progenitor cells and analysis of leukocyte activity. J. Neurosci. 13, 1022^1034. J=rgensen, J.M., 1991. Regeneration of lateral line and inner ear vestibular cells. Ciba Foundation Symposium 160, Wiley, Chichester, pp. 151^170. J=rgensen, J.M., Mathiesen, C., 1988. The avian inner ear. Continuous production of hair cells in vestibular sensory organs, but not in the auditory papilla. Naturwissenschaften 75, 319^320. Lippe, W.R., Westbrook, E.W., Ryals, B.M., 1991. Hair cell regeneration in the chicken cochlea following aminoglycoside toxicity. Hear. Res. 56, 203^210. Lopez, I., Honrubia, V., Lee, S.C., Schoeman, G., Beykirch, K., 1997. Quanti¢cation of the process of hair cell loss and recovery in the chinchilla crista ampullaris after gentamicin treatment. Int. J. Dev. Neurosci. 15, 447^461. Popper, A.N., Hoxter, B., 1984. Growth of ¢sh ear, I: quantitative analysis of hair cell and ganglion cell proliferation. Hear. Res. 15, 133^142. Popper, A.N., Hoxter, B., 1990. Growth of a ¢sh ear. II. Location of newly proliferated sensory hair cells in the saccular epithelium of Astronotus ocellatus. Hear. Res. 45, 3340.
HEARES 4664 1-4-03
88
B. Avallone et al. / Hearing Research 178 (2003) 79^88
Presson, J.C., Popper, A.N., 1990. Possible precursors to new hair cells, support cells, and Schwann cells in the ear of a post-embryonic ¢sh. Hear. Res. 46, 9^22. Raphael, Y., 1992. Evidence for supporting cell mitosis in response to acoustic trauma in the avian inner ear. J. Neurocytol. 21, 663^671. Roberson, D.F., Weisleder, P., Bohrer, P.S., Rubel, E.W., 1992. Ongoing production of sensory cells in the vestibular epithelium of the chick. Hear. Res. 57, 166^174. Rubel, E., Dew, L.A., Roberson, D.W., 1995. Mammalian vestibular hair cell regeneration. Science 267, 701^703. Ryals, B.M., Rubel, E.W., 1988. Hair cell regeneration after acoustic trauma in adult Coturnix quail. Science 240, 1774^1776. Ryals, B.M., Westbrook, E.W., 1990. Hair cell regeneration in senescent quail. Hear. Res. 50, 87^96. Sobkowicz, H., August, B.K., Slapnick, S.M., 1992. Epithelia repair following mechanical injury of the developing organ of Corti in culture: an electron microscopic and autoradiographic study. Exp. Neurol. 115, 44^49. Stone, J.S., Cotanche, D.A., 1994. Identi¢cation of the timing of S phase and the patterns cell proliferation during hair cell regeneration in the chick cochlea. J. Comp. Neurol. 331, 97^110. Stone, J.S., Rubel, E.W., 2000. Temporal, spatial, and morphologic features of hair cell regeneration in the avian basilar papilla. J. Comp. Neurol. 417, 1^16. Stone, J.S., Choi, Y.S., Woolley, S.M., Yamashita, H., Rubel, E.W., 1999. Progenitor cell cycling during hair cell regeneration in the
vestibular and auditory epithelia of the chick. J. Neurocytol. 28, 863^876. Tanyeri, H., Lopez, I., Honrubia, V., 1995. Histological evidence for hair cell regeneration after ototoxic cell destruction with local application of gentamicin in the chinchilla crista ampullaris. Hear. Res. 89, 194^202. Tsue, T.T., Watling, D.L., Weislader, P., Coltrera, M.D., Rubel, E.W., 1994. Identi¢cation of hair cell progenitors and intermitotic migration of their nuclei in the normal and regenerating avian inner ear. J. Neurosci. 14, 140^152. Warchol, M.E., Corwin, J.T., 1996. Regenerative proliferation in organ cultures of the avian cochlea: Identi¢cation of the initial progenitors and determination of the latency of the proliferative response. J. Neurosci. 16, 5466^5477. Weisleder, P., Rubel, E.W., 1992. Hair cell regeneration in the avian vestibular epithelium. Exp. Neurol. 115, 2^6. Weisleder, P., Rubel, E.W., 1993. Hair cell regeneration after streptomycin toxicity in the avian vestibular epithelium. J. Comp. Neurol. 931, 97^110. Wersa«ll, J., 1956. Studies on the structure and innervation of the sensory epithelium of the cristae ampullares in the guinea pig. Acta Otolaryngol. 126 (Suppl.), 1^85. Zheng, J.L., Gao, W.Q., 1997. Analysis of rat vestibular hair cell development and regeneration using calretinin as an early marker. J. Neurosci. 17, 8270^8282.
HEARES 4664 1-4-03
Hearing Research 178 (2003) 79^88 www.elsevier.com/locate/heares
Evidence for hair cell regeneration in the crista ampullaris of the lizard Podarcis sicula Bice Avallone, Maria Porritiello, Daniela Esposito, Rosalia Mutone, Giuseppe Balsamo, Francesco Marmo Department of Genetics, General and Molecular Biology, University Federico II, via Mezzocannone 8, 80134 Naples, Italy Received 15 October 2002; accepted 17 January 2003
Abstract We studied hair cell regeneration in the crista ampullaris of the lizard Podarcis sicula both in untreated animals and at early and late time intervals following a single high dose of gentamicin. The study was carried out using the S-phase marker 5-bromo-2Pdeoxyuridine. Our ultrastructural and immunofluorescence studies showed that both apoptosis and hair cell regeneration happen in the lizard crista ampullaris in untreated animals, and that regenerative processes are greatly accelerated after treatment with the aminoglycoside antibiotic gentamicin. Our observations indicate that hair cell regeneration is strongly implicated in the repair of damaged sensory epithelium, and that new hair cells appear likely to arise from supporting cells. 1 2003 Elsevier Science B.V. All rights reserved. Key words: Regeneration; Lizard; Crista ampullaris; Bromodeoxyuridine ; Gentamicin
1. Introduction Hair cells (HCs), which are the sensory receptors for hearing, equilibrium and motion detection, located in the auditory, vestibular and lateral line organs, may be damaged by a number of agents including aminoglycoside antibiotics and severe over-stimulation (acoustic trauma). Postembryonic HC production occurs continually throughout life in the vestibular and auditory sensory epithelium in ¢shes and amphibians and in the vestibular end organs of birds (Popper and Hoxter, 1984; Corwin, 1985; J=rgensen and Mathiesen, 1988; Presson and Popper, 1990; Roberson et al., 1992; Stone
* Corresponding author. Tel.: +39 (81) 2535012 (o⁄ce), +39 (81) 2535006 (lab); Fax: +39 (81) 2535000. E-mail address: [email protected] (F. Marmo). Abbreviations: BrdU, 5-bromo-2P-deoxyuridine; DS, distal to septum; DW, distilled water; HC, hair cell; PBS, phosphatebu¡ered saline; PS, proximal to septum; SEM, scanning electron microscopy; TEM, transmission electron microscopy; TBS, Trisbu¡ered saline
et al., 1999). In the mature hearing organ of birds, the basilar papilla, HCs are produced after HC loss caused by exposure to noise (Cotanche, 1987; Corwin and Cotanche, 1988; Ryals and Rubel, 1988; Ryals and Westbrook, 1990), ototoxic drug treatment (Cruz et al., 1987; Lippe et al., 1991; Stone et al., 1999; Stone and Rubel, 2000) or laser ablation (Warchol and Corwin, 1996). According to Ryals and Westbrook (1990) there may be some very low level of HC production which is activated in the absence of trauma in adult quail cochlea. Analogous ongoing production of HCs during postembryonic life does not appear to occur in mammalian vestibular and auditory sensory epithelium. It has been demonstrated, however, that HCs reappeared in mammalian vestibular system after HC loss following treatment with aminoglycosides (Forge et al., 1993; Rubel et al., 1995; Tanyeri et al., 1995; Lopez et al., 1997; Zheng and Gao, 1997) but did not reappear in the organ of Corti, even after trauma (Sobkowicz et al., 1992). Several studies have sought to identify precursor cells for new HCs. These potential precursors include supporting cells, embryonic-like neuroepithelial cells, and cells immediately adjacent to the sensory epithelium
0378-5955 / 03 / $ ^ see front matter 1 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0378-5955(03)00040-6
HEARES 4664 1-4-03
80
B. Avallone et al. / Hearing Research 178 (2003) 79^88
(Corwin and Cotanche, 1988; Girod et al., 1989; Raphael, 1992; Weisleder and Rubel, 1993; Cotanche et al., 1994; Stone and Cotanche, 1994; Tsue et al., 1994; Bhave et al., 1995; Fekete et al., 1998; Stone et al., 1999). Few investigations have been carried out on the reptilian inner ear. A series of experiments with [3 H]thymidine injections in adders (Vipera berus) did not demonstrate any labelled nuclei in the sensory epithelium. Similar 3 H injections in two specimens of the turtle Pseudoemys elegans showed labelled supporting and sensorial cell nuclei (J=rgensen, 1991) which suggested cell turnover. The present work aims to study HC regeneration in the reptilian (Podarcis sicula ; Fig. 1) vestibular system, and if possible, to identify the precursor cells for new HCs. We studied the crista ampullaris (Fig. 2) of the lizard P. sicula both in normal animals and at early and late time intervals following a single high dose of gentamicin used to initiate the regenerative events (Janas et al., 1994). The resulting damage pattern was investigated by scanning electron microscopy (SEM). Transmission electron microscopy (TEM) studies were also undertaken to determine whether spontaneous apoptosis occurs, which would suggest a slow turnover of normal vestibular HCs. One S-phase-associated marker, 5-bromo-2P-deoxyuridine (BrdU), which is incorporated into the newly synthesised DNA strand (Gratzner, 1982), was used to study progression through S phase and mitosis. To detect BrdU-labelled cells a protocol for BrdU immuno£uorescence was followed.
USA) daily for 10 days. One lizard was sacri¢ced beginning at 24 h after the ¢rst BrdU injection, daily for 10 days. Then the samples were processed for immuno£uorescence. 2.3. Exclusive BrdU treatment Ten lizards were treated with BrdU only (100 mg/kg, in sterile physiological saline) daily for 10 days. The daily dose of BrdU was divided into three intraperitoneal injections given every 4 h. One lizard was sacri¢ced beginning at 24 h after BrdU injection, daily for 10 days. 2.4. Untreated group Six untreated lizards were used to verify the ototoxicity of gentamicin by SEM and to study the sensorial epithelium ultrastructure by TEM. The specimens were anaesthetised by deep exposure to fumes of ether and decapitated. 2.5. Positive control Intestines of lizards treated with BrdU only and gentamicin plus BrdU were removed and processed for immuno£uorescence the same as tissue from the crista ampullaris. 2.6. SEM
Thirty-seven adult specimens of P. sicula, weighing V7.00 g each, were collected near Naples, and maintained in the laboratory under natural conditions of light and temperature. The lizards were fed twice a week.
Heads were ¢xed in 2.5% glutaraldehyde in 0.1 M phosphate-bu¡ered saline (PBS) pH 7.4 for 3 h at 4‡C for SEM observation. After microdissection of the vestibular end organs the specimens were rinsed in PBS and post-¢xed in 1% OsO4 in the same bu¡er for 1 h at 4‡C. After several rinses in PBS, the specimens were subjected to serial dehydration steps followed by critical point drying. Specimens were mounted on aluminium stubs, coated with gold, and examined with a Cambridge Stereoscan 250 MK III Microscope.
2.1. Gentamicin treatment
2.7. TEM
One group (11 lizards) received a single subcutaneous injection of gentamicin sulphate (400 mg/kg; ScheringPlough, Italy). One lizard was sacri¢ced beginning from 4 h after gentamicin treatment and one lizard daily for 10 days. The samples were processed for SEM.
Heads were ¢xed in 2.5% glutaraldehyde in 0.1 M PBS pH 7.4 for 3 h at 4‡C for TEM observation. After microdissection of the vestibular end organs the specimens were rinsed in PBS and post-¢xed in 1% OsO4 in the same bu¡er for 1 h at 4‡C. Samples were rinsed several times in bu¡er, dehydrated, embedded in Epon 812 resin, and sectioned in a Super Nova Leica Ultratome. Ultrathin sections were placed on copper grids and counterstained with uranyl acetate and lead citrate. Specimens were observed and photographed in a Philips CM12 electron microscope.
2. Materials and methods
2.2. Gentamicin plus BrdU treatment Ten lizards, beginning at 24 h after gentamicin injection (400 mg/kg), received a subcutaneous injection of BrdU (100 mg/kg, in saline ; Sigma, St. Louis, MO,
HEARES 4664 1-4-03
B. Avallone et al. / Hearing Research 178 (2003) 79^88
81
2.8. Indirect immuno£uorescence Heads were ¢xed in 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M cacodylate bu¡er pH 7.3 for 2.5 h at room temperature for immuno£uorescence observations. After several rinses in the same bu¡er, vestibular end organs were dissected and treated with 0.2 M ammonium chloride in cacodylate bu¡er for 1 h at room temperature. Specimens were dehydrated with ethanol and propylene oxide and embedded in Epon. Serial sections of specimens were cut at 2 Wm thickness, mounted on slides coated with poly-L-lysine (0.01%) and stained with toluidine blue. The plastic was removed from sections by immersing the slides in a su⁄cient amount of a solution of 2.4 M KOH in propylene oxide :methyl alcohol (2:1) for 6 min. Slides were rinsed with methyl alcohol and washed thoroughly ¢rst in water for 10 min and then in distilled water (DW) for 5 min. Slides were treated with 2 N HCl in PBS for 20 min at 37‡C to denature DNA and rinsed in DW. Subsequently, the sections were incubated in: (1) protease K (4 Wg/ml; Sigma) in 0.01 M Tris^HCl pH 7.4 for 8 min at room temperature and rinsed in 0.1 M PBS and 0.5% bovine serum albumin (BSA; Sigma); (2) rabbit serum (Sigma) 1:20 in 0.1 M Tris-bu¡ered saline (TBS), 0.25% Tween 20 and 0.5% BSA for 1 h at 37‡C ; (3) anti-BrdU monoclonal antibody (Sigma) 1:1000 in 0.1 M TBS, 0.25% Tween 20 and 0.5% BSA overnight at room temperature and rinsed in the same bu¡er; (4) rabbit anti-mouse IgG FITC conjugated (Sigma) 1:100 in the same bu¡er for 1 h at 37‡C and ¢nally rinsed several times in the same bu¡er. Preimmune sera, instead of speci¢c antisera, were used in the control section; each step was carried out in a moist chamber. Finally sections were mounted in glycerol and £uorescence observations were carried out with a Zeiss Axioskop microscope using the KS300 software (Kontron) to acquire microscope images and ProPalette 8000 for digital colour ¢lm recorder. 2.9. Statistical analysis One hundred sections, from each crista ampullaris of lizard, were used for quantitative analysis. Statistical analysis was performed with a single factorial analysis of variance (ANOVA). A P value less than 0.01 was considered to be signi¢cant.
Fig. 1. P. sicula. Fig. 2. Light micrograph of crista ampullaris of lizard. Sensory epithelium (se), transitional cell zone (TCz) and dark cell zone (DCz). Bar = 10 Wm. Fig. 3. SEM of crista ampullaris of P. sicula. Bar = 10 Wm.
3. Results 2.10. Use of animals 3.1. SEM observations Samples were captured with authorisation of 1/06/ 2000, No. SCN/2D/2000/RB of the Ministero dell’Ambiente.
The crista sensory area (Fig. 3) of untreated specimens shows HCs surrounded by supporting cells with
HEARES 4664 1-4-03
82
B. Avallone et al. / Hearing Research 178 (2003) 79^88
HEARES 4664 1-4-03
B. Avallone et al. / Hearing Research 178 (2003) 79^88
83
Fig. 5. TEM of crista ampullaris. (A) Bottle-shaped HC I enclosed in a nerve calyx. Bar = 1.7 Wm. (B) Apoptotic HC I with dark cytoplasmic matrix and a condensed nucleus. Bar = 1.9 Wm. (C) Apoptotic HC I with dark cytoplasm and dense nucleus enclosed in a nerve calyx together with a light sensory cell. Bar = 1.1 Wm.
small microvilli projecting from the luminal surfaces (Fig. 4A,B). In the crista sensory area, two zones characterised by di¡erent HC ciliary bundles are observed : a central zone, proximal to septum (PS) (Fig. 4A), with cells showing both long kinocilium and long stereocilia,
decreasing in length, and a peripheral zone, distal to septum (DS) (Fig. 4B) with cells showing a long kinocilium and short stereocilia compact bundles with a gradual increase in length. The region of the crista included between the two
6 Fig. 4. SEM of crista ampullaris. (A) Lizard, untreated. Zone proximal to septum cruciatum (PS): cells with both a long kinocilium and long stereocilia, decreasing in length. Bar = 3.8 Wm. (B) Lizard, untreated. Zone distal to septum cruciatum (DS): cells with a long kinocilium and short stereocilia compact bundles, which gradually increase in length. Bar = 4 Wm. (C) Lizard, untreated. Smooth central portion (asterisk) of the crista ampullaris, included between the two protrusions of the septum cruciatum, with small polygonal cells. Bar = 4.4 Wm. (D) Lizard, untreated. Septum cruciatum: sparse tufts of microvilli of ‘microvillous’ cells (arrows) are observed. Bar = 4.7 Wm; (Inset) Higher magni¢cation of microvilli tufts. Bar = 2.1 Wm. (E) PS zone of the crista 4 h after gentamicin injection. Damaged sensory cells with fused stereocilia (arrows). Bar = 5 Wm. (F) DS zone of the crista 4 h after gentamicin injection. Supporting cells lacking small microvilli and sensory cells with su¡ering and fused stereocilia (arrows) are observed. Bar = 5 Wm. (G) PS zone of the crista 1 day after gentamicin injection. Sensory cells with short (arrow) or lacking hairs (asterisk) are observed. Bar = 5.4 Wm. (H) DS zone of the crista 1 day after gentamicin injection. Extrusion of cellular contents into the fused bundles. Bar = 5.4 Wm. (I) PS zone of the crista 10 days after gentamicin injection. Sensory cells with mature-appearing hairs and some HCs with immature stereocilia bundles (arrows). Bar = 5 Wm. (L) DS zone of the crista 10 days after gentamicin injection. Sensory cells regain normal appearance. Bar = 5 Wm.
HEARES 4664 1-4-03
84
B. Avallone et al. / Hearing Research 178 (2003) 79^88
At 1 day after gentamicin injection, there is a widespread loss of hairs of the sensory cells (Fig. 4G), and extrusion of both entire HCs and cellular contents into the fused bundle (Fig. 4H). At 10 days after gentamicin injection, the sensorial area shows an apparently normal morphology: very few cells with hair loss are present so that some HCs are evident with immature hair bundles and signi¢cant numbers of HCs with mature hair bundles (Fig. 4I,L). 3.2. TEM observations
Fig. 6. TEM of crista ampullaris. (A) Cylindrical HC II, innervated by terminal buttons. Bar = 2 Wm. (B) Apoptotic HC II with dark cytoplasmic matrix and a condensed nucleus. Bar = 2.2 Wm.
protrusions of the septum cruciatum is smooth (Fig. 4C), lacking stereo- or kinocilia, with small polygonal cells. In the septum region, the size of these cells increases, and sparse tufts of microvilli appear among them (Fig. 4D). The HCs already appear damaged 4 h after gentamicin injection and the stereocilia seem fused in both the DS and PS zones of the crista (Fig. 4E,F).
TEM observations of crista sensory epithelium of untreated specimen of P. sicula show that HCs are located at the summit of the crista in the luminal part of the sensory epithelium and they never extend to the basal membrane. Two major types of HC populations (HC I and HC II according to Wersa«ll, 1956) are found: HC I, bottle-shaped and enclosed in a nerve chalice, and HC II, cylindrical and innervated by terminal buttons. HCs are surrounded by supporting cells in which the cytoplasm shows extensive arrays of parallel microtubules extending lengthwise (Figs. 5A and 6A). Occasionally, a third type of sensory cells, i.e. dark HCs, was observed in both the DS and PS zones of the crista. These cells show the morphology of apoptotic cells. We consider these dark cells to be sensory cells not only because of the presence of a cuticular plate, but also because these cells are sometimes enclosed in a nerve calyx together with a light sensory cell. Furthermore, these cells present rare synaptic buttons, both a¡erent and e¡erent. These cells show an increase in electronic density of both the nucleus and cytoplasm, accompanied by a clump of nuclear chromatin lying against the nuclear membrane. However, mitochondria appear to be intact and endoplasmic reticulum dilated (Figs. 5B,C and 6B). 3.3. BrdU immuno£uorescence In the animals treated with BrdU only (Fig. 8), at 2 days very few labelled supporting cell nuclei are found (Fig. 7A). In the following days, the number of labelled supporting cell nuclei progressively increased and, only
C Fig. 7. Fluorescence micrograph of crista ampullaris. (A) Crista ampullaris of sample treated with BrdU only, for 2 days. Very few BrdU-labelled supporting cell nuclei (arrows) are observed. Bar = 24 Wm. (B) Crista ampullaris of a sample treated with BrdU only, for 5 days. Numerous supporting cells and few sensory cell (arrows) BrdU-labelled nuclei are observed. Bar = 22.2 Wm. (C) Crista ampullaris of a sample treated with BrdU only, for 10 days. Some labelled sensory cell nuclei (arrows) are observed. Bar = 22.6 Wm. (D) Crista ampullaris of a sample treated with BrdU only, for 2 days. Numerous labelled nuclei of ‘microvillous’ dark cells are observed. Bar = 22.2 Wm. (E) Crista ampullaris of a sample treated with BrdU for 3 days, after gentamicin injection. Numerous supporting cells and some BrdU-labelled sensory cells (arrows) are observed. Bar = 22.2 Wm. (F) Crista ampullaris of a sample treated with BrdU for 5 days after gentamicin injection. A cluster of BrdU-labelled nuclei of sensory cells (arrow) is observed. Bar = 30.4 Wm. (G) Crista ampullaris of a sample treated with BrdU for 10 days, after gentamicin injection. Numerous £uorescent HC I cell nuclei surrounded by supporting positive cells are observed. Bar = 26.6 Wm. (H) Portion of crista ampullaris of a sample treated with BrdU for 10 days, after gentamicin injection. BrdU-labelled nuclei of ‘microvillous’ dark cells are observed. Bar = 22.2 Wm.
HEARES 4664 1-4-03
B. Avallone et al. / Hearing Research 178 (2003) 79^88
85
D
A
E
B
F
C
G
HEARES 4664 1-4-03
H
86
B. Avallone et al. / Hearing Research 178 (2003) 79^88
supporting cell 60
hair cell
supporting cell
60
50
50 Positive cell
40 positive cell
hair cell
70
30 20
40 30 20
10
8
10
0 2d
5d
9
10d
0 2d
5d
10d
Fig. 8. Mean number of labelled hair and supporting cell nuclei, W S.D., after 2, 5 and 10 days, in animals treated with BrdU only. Signi¢cance of di¡erences was evaluated at P 6 0.01 (ANOVA). Fig. 9. Mean number of labelled hair and supporting cell nuclei, W S.D., after 2 days, 5 days and 10 days, in animals treated with Gentamicin plus BrdU. Signi¢cance of di¡erences has been evaluated at P 6 0.01 (ANOVA).
in the samples treated with BrdU for 5 days, some BrdU-labelled nuclei of the sensory cells were detected (Fig. 7B). Their number increased in the samples treated with BrdU for 10 days (Fig. 7C). From the ¢rst day of treatment, numerous labelled nuclei of ‘microvillous’ (according to Hamilton, 1965) dark cells were always evident (Fig. 7D). In the epithelium of the cristae of animals treated with BrdU after gentamicin injection, already 1 day after BrdU treatment we observed chie£y BrdU-labelled nuclei of supporting cells. At 3 days, £uorescence was present not only in the majority of nuclei of the supporting cells (i.e. in the supporting cell layer of epithelium) and in the nuclei of the ‘microvillous’ dark cells but also in some sensory cell nuclei (i.e. in the HC layer of epithelium) (Fig. 7E). The number of labelled sensory cell nuclei increased proportionally to the duration of the treatment. The animals treated with BrdU for 5 days after gentamicin showed a greater number of labelled sensory cell nuclei, often clustered in some zones of the crista (Fig. 7F). After 10 days of treatment a large number of £uorescent HCs surrounded by positive supporting cells was observed (Fig. 7G). Intense and wide positivity was present in the septum ‘microvillous’ dark cells (Fig. 7H). Control gut sections both in the gentamicin plus BrdU and in the BrdU-only-treated animals always showed numerous epithelium cells with BrdU-labelled nuclei. All negative controls showed no £uorescence. 3.4. Statistical analysis Fig. 8 shows the mean number of labelled hair and supporting cell nuclei, W S.D., after 2, 5 and 10 days, in animals treated with BrdU only. Fig. 9 shows the mean
number of labelled hair and supporting cell nuclei, W S.D., after 2, 5 and 10 days, in animals treated with gentamicin plus BrdU. Signi¢cance of di¡erences was evaluated at P 6 0.01 (ANOVA).
4. Discussion Our SEM results on the crista of P. sicula treated with gentamicin show that this aminoglycoside antibiotic induces damage primarily to HCs. SEM observations indicate that this damage is precocious : 4 h after gentamicin injection, sensory cells with fused stereocilia are visible in the crista. Sensorial epithelium repairs progressively and, 10 days after the injection, di¡erentiated HCs and cells with small hair bundles (and therefore probably immature) are evident in this area. Besides, the immuno£uorescence studies show that regeneration phenomena are present and rather di¡use. In fact, 1 day after gentamicin injection numerous labelled supporting cell nuclei are evident and, starting from 3 days, sensory cell nuclei also are BrdU-labelled. In the following days, the number of labelled supporting cell nuclei progressively increases. Immuno£uorescence studies and SEM observations therefore indicate that regeneration processes are strongly implicated in the damaged sensory epithelium repair. Incorporation of BrdU at ¢rst in the supporting cell nuclei and only after some days in the HC nuclei (both in gentamicin plus BrdU and in BrdU only animals) leads us to hypothesise that sensory cells di¡erentiate from supporting cells, in agreement with suggestions from several previous studies on regeneration in the cochlea of chicken (Corwin and Cotanche, 1988; Raphael, 1992; Stone and Cotanche, 1994; Bhave et al.,
HEARES 4664 1-4-03
B. Avallone et al. / Hearing Research 178 (2003) 79^88
1995; Fekete et al., 1998; Stone et al., 1999), in the vestibular organs of chicks (Weisleder and Rubel, 1992, 1993) and in the lateral line organs of amphibians (Corwin, 1986; Corwin et al., 1989; Balak et al., 1990; Jones, 1991; Jones and Corwin, 1993). The earliest cells labelled with the S phase marker resemble supporting cells, and all our studies reveal no labelled HCs immediately following the onset of ototoxic treatment. The gradual increase of BrdU-positive cells in the crista sensory epithelium may also be attributable to the fact that supporting cells may go through more than one cell division cycle after HC degeneration (Jones and Corwin, 1993; Stone and Cotanche, 1994). Moreover, TEM observations, showing that di¡erentiated HCs undergo apoptosis during normal life, may indirectly indicate the presence of proliferation phenomena and cell di¡erentiation connected to spontaneous HC turnover in reptilian vestibular systems. This was already demonstrated in ¢shes (Corwin, 1981, 1983; Popper and Hoxter, 1984, 1990) which continually produce cells replacing apoptotic dying cells, as well as in other groups of vertebrates (amphibians, avians) (Corwin, 1985; J=rgensen and Mathiesen, 1988; Roberson et al., 1992; Weisleder and Rubel, 1993; Stone et al., 1999; Goodyear et al., 1999) in the absence of acoustic trauma or treatment with aminoglycoside antibiotics. In summary, our observations suggest that in the lizard P. sicula new HCs do not arise from pre-existing HCs. Most likely, labelled supporting cells divide and produce daughter cells with labelled DNA which become HCs and regenerate the epithelium. This process is limited or slow in normal conditions but is greatly ampli¢ed by treatment with the aminoglycoside gentamicin.
References Balak, K.J., Corwin, J.T., Jones, J.E., 1990. Regenerated hair cells can originate from supporting cell progeny: evidence from photoxicity and laser ablation experiments in the lateral line system. J. Neurosci. 10, 2502^2512. Bhave, S.A., Stone, J.S., Rubel, E.W., Coltrera, M.D., 1995. Cell cycle progression in gentamicin-damaged avian cochleas. J. Neurosci. 15, 4618^4628. Corwin, J.T., 1981. Postembryonic production and ageing of inner ear hair cells in sharks. J. Comp. Neurol. 201, 541^553. Corwin, J.T., 1983. Postembryonic growth of the macula neglecta auditory detector in the ray, Raja clavata. Continual increases in hair cell number neural convergence and physiological sensitivity. J. Comp. Neurol. 217, 345^356. Corwin, J.T., 1985. Perpetual production of hair cells and maturational changes in hair cell ultrastructure accompany postembryonic growth in amphibian inner ear. Proc. Natl. Acad. Sci. USA 82, 3911^3915. Corwin, J.T., 1986. Regeneration and self-repair in hair cell epithelia:
87
experimental evaluation of capacities and limitations. In: Ruben, R.J., Van De Water, T.R., Rubel, E.W. (Eds.), The Biology of Change in Otolaryngology. Elsevier, New York. Corwin, J.T., Cotanche, D.A., 1988. Regeneration of sensory hair cells after acoustic trauma. Science 240, 1772^1774. Corwin, J.T., Balak, K.J., Borden, P.C., 1989. Cellular events underlying the regenerative replacement of lateral line sensory epithelia in amphibians. In: Combs, S., Gorner, P., Munz, H. (Eds.), The Mechanosensory Lateral Line: Neurobiology and Evolution. Springer, New York, pp. 161^183. Cotanche, D.A., 1987. Regeneration of hair cell stereociliary bundles in the chick cochlea following severe acoustic trauma. Hear. Res. 30, 181^196. Cotanche, D.A., Lee, K.H., Stone, J.S., Picard, D.A., 1994. Hair cell regeneration in the bird cochlea following noise damage or ototoxic drug damage. Anat. Embryol. (Berl.) 189, 1^18. Cruz, R.M., Lambert, P.R., Rubel, E.W., 1987. Light microscopic evidence of hair cell regeneration after gentamicin toxicity in chick cochlea. Arch. Otolaryngol. Head Neck Surg. 113, 1058^1062. Fekete, D.M., Muthukumar, S., Karagogeos, D., 1998. Hair cell and supporting cells share a common progenitor in the avian inner ear. J. Neurosci. 18, 7811^7821. Forge, A., Li, L., Corwin, J.T., Nevil, G., 1993. Ultrastructural evidence for hair cell proliferation in the mammalian inner ear. Science 259, 1616^1619. Girod, D.A., Duckert, L.G., Rubel, E.W., 1989. Possible precursors of regenerated hair cells in the avian cochlea following acoustic trauma. Hear. Res. 42, 175^194. Goodyear, R.J., Gates, R., Lukashkin, A.N., Richardson, G.P., 1999. Hair-cell numbers continue to increase in the utricular macula of the early post-hatch chick. J. Neurocytol. 28, 851^861. Gratzner, H.G., 1982. Monoclonal antibody to 5-bromo-and 5-iododeoxyuridine: a new reagent for detection of DNA replication. Science 218, 474. Hamilton, D.W., 1965. Microvillous cells in ampullae of the lizard inner ear. J. Morphol. 116, 339^354. Janas, J.D., Cotanche, D.A., Rubel, E.W., 1994. Avian cochlear hair cell regeneration: a quantitative analysis of damage and recovery from single high dose gentamicin injection. Assoc. Res. Otolaryngol. 17, 66A. Jones, J.E., 1991. Regeneration in the Lateral Line: Identities of Progenitor Cells and the Action of Leukocytes. PhD Thesis, University of Virginia, Charlottesville, VA. Jones, J.E., Corwin, J.T., 1993. Replacement of lateral line sensory organs during tail regeneration in salamanders: identi¢cation of progenitor cells and analysis of leukocyte activity. J. Neurosci. 13, 1022^1034. J=rgensen, J.M., 1991. Regeneration of lateral line and inner ear vestibular cells. Ciba Foundation Symposium 160, Wiley, Chichester, pp. 151^170. J=rgensen, J.M., Mathiesen, C., 1988. The avian inner ear. Continuous production of hair cells in vestibular sensory organs, but not in the auditory papilla. Naturwissenschaften 75, 319^320. Lippe, W.R., Westbrook, E.W., Ryals, B.M., 1991. Hair cell regeneration in the chicken cochlea following aminoglycoside toxicity. Hear. Res. 56, 203^210. Lopez, I., Honrubia, V., Lee, S.C., Schoeman, G., Beykirch, K., 1997. Quanti¢cation of the process of hair cell loss and recovery in the chinchilla crista ampullaris after gentamicin treatment. Int. J. Dev. Neurosci. 15, 447^461. Popper, A.N., Hoxter, B., 1984. Growth of ¢sh ear, I: quantitative analysis of hair cell and ganglion cell proliferation. Hear. Res. 15, 133^142. Popper, A.N., Hoxter, B., 1990. Growth of a ¢sh ear. II. Location of newly proliferated sensory hair cells in the saccular epithelium of Astronotus ocellatus. Hear. Res. 45, 3340.
HEARES 4664 1-4-03
88
B. Avallone et al. / Hearing Research 178 (2003) 79^88
Presson, J.C., Popper, A.N., 1990. Possible precursors to new hair cells, support cells, and Schwann cells in the ear of a post-embryonic ¢sh. Hear. Res. 46, 9^22. Raphael, Y., 1992. Evidence for supporting cell mitosis in response to acoustic trauma in the avian inner ear. J. Neurocytol. 21, 663^671. Roberson, D.F., Weisleder, P., Bohrer, P.S., Rubel, E.W., 1992. Ongoing production of sensory cells in the vestibular epithelium of the chick. Hear. Res. 57, 166^174. Rubel, E., Dew, L.A., Roberson, D.W., 1995. Mammalian vestibular hair cell regeneration. Science 267, 701^703. Ryals, B.M., Rubel, E.W., 1988. Hair cell regeneration after acoustic trauma in adult Coturnix quail. Science 240, 1774^1776. Ryals, B.M., Westbrook, E.W., 1990. Hair cell regeneration in senescent quail. Hear. Res. 50, 87^96. Sobkowicz, H., August, B.K., Slapnick, S.M., 1992. Epithelia repair following mechanical injury of the developing organ of Corti in culture: an electron microscopic and autoradiographic study. Exp. Neurol. 115, 44^49. Stone, J.S., Cotanche, D.A., 1994. Identi¢cation of the timing of S phase and the patterns cell proliferation during hair cell regeneration in the chick cochlea. J. Comp. Neurol. 331, 97^110. Stone, J.S., Rubel, E.W., 2000. Temporal, spatial, and morphologic features of hair cell regeneration in the avian basilar papilla. J. Comp. Neurol. 417, 1^16. Stone, J.S., Choi, Y.S., Woolley, S.M., Yamashita, H., Rubel, E.W., 1999. Progenitor cell cycling during hair cell regeneration in the
vestibular and auditory epithelia of the chick. J. Neurocytol. 28, 863^876. Tanyeri, H., Lopez, I., Honrubia, V., 1995. Histological evidence for hair cell regeneration after ototoxic cell destruction with local application of gentamicin in the chinchilla crista ampullaris. Hear. Res. 89, 194^202. Tsue, T.T., Watling, D.L., Weislader, P., Coltrera, M.D., Rubel, E.W., 1994. Identi¢cation of hair cell progenitors and intermitotic migration of their nuclei in the normal and regenerating avian inner ear. J. Neurosci. 14, 140^152. Warchol, M.E., Corwin, J.T., 1996. Regenerative proliferation in organ cultures of the avian cochlea: Identi¢cation of the initial progenitors and determination of the latency of the proliferative response. J. Neurosci. 16, 5466^5477. Weisleder, P., Rubel, E.W., 1992. Hair cell regeneration in the avian vestibular epithelium. Exp. Neurol. 115, 2^6. Weisleder, P., Rubel, E.W., 1993. Hair cell regeneration after streptomycin toxicity in the avian vestibular epithelium. J. Comp. Neurol. 931, 97^110. Wersa«ll, J., 1956. Studies on the structure and innervation of the sensory epithelium of the cristae ampullares in the guinea pig. Acta Otolaryngol. 126 (Suppl.), 1^85. Zheng, J.L., Gao, W.Q., 1997. Analysis of rat vestibular hair cell development and regeneration using calretinin as an early marker. J. Neurosci. 17, 8270^8282.
HEARES 4664 1-4-03