doi:10.1016/j.heares.2007.09.009 
Copyright © 2007 Elsevier B.V. All rights reserved.
Research paper
Gentamicin ototoxicity in the saccule of the lizard Podarcis Sicula induces hair cell recovery and regeneration
Bice Avallonea, 
, 
, Umberto Fasciob, Giuseppe Balsamoa and Francesco Marmoa
aDepartment of Biological Science, Section of Genetics and Molecular Biology, University of Naples “Federico II”, via Mezzocannone 8, 80134 Naples, Italy
bCIMA, University of Milan, 20133 Milan, Italy
Received 20 June 2007;
revised 6 September 2007;
accepted 14 September 2007.
Available online 1 October 2007.
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Abstract
There is little information available on the susceptibility of reptilian saccule hair cells to ototoxin-induced sensory damage. In this study, we report morphological evidence of hair cell recovery and regeneration after damage induced by gentamicin in the saccule of a lizard. We perform morphological analysis using scanning electron microscopy and confocal laser scanning microscopy with actin and calbindin as markers for hair cells and tubulin as a marker for supporting cells. The data were consistent: gentamicin induced damage in the hair cells, and the damage increased with increasing duration of treatment. Initially, the saccule appeared unhealthy. Subsequently, the sensory hair cells became compromised, with fused stereovilli, followed by widespread loss of hair cell bundles from the hair cells. Finally, numerous hair cells were lost. Morphologically, the saccule appeared normal 28 days after gentamicin treatment. Using a mitogenic marker, we tested whether or not there is hair cell regeneration following administration of gentamicin. We found evidence of bromodeoxyuridine incorporation first in supporting cell nuclei and subsequently in hair cell nuclei. This indicates that a process of sensory epithelium repair and hair cell regeneration occurred, in both extrastriolar and striolar regions, and that the recovery was due to both the proliferation of supporting cells and, as seems likely, self-repair of hair cell bundles.
Keywords: Aminoglycoside antibiotics; Calbindin; BrdU; Inner ear; Recovery
Abbreviations: BrdU, 5-bromo-2′-deoxyuridine; CaB, calbindin-D-28K; CaM, calmodulin; CBPs, calcium-binding proteins; CLSM, confocal laser scanning microscope; DABCO, diazabicyclo-octane; DW, distilled water; FITC, fluorescein isothiocyanate; Gm, gentamicin; PBS, buffer phosphate saline; SEM, scanning electron microscope; TBS, Tris buffer saline; TRITC, tetramethylrhodamine isothiocyanate
Fig. 1. SEM (A) saccule sensory epithelium of untreated lizards: hair cells with an enlarged, bulb-like kinocilium (arrow) and stereovilli in an organ pipe configuration (arrowhead); (B) (1Gm/4 h): the hair cells appear compromised (arrows); (C) (2Gm/4 h): hair cells appear damaged and the stereovilli seem to be fused; (D) (3Gm/4 h): several hair cell bundles have disappeared from the saccule (arrow), and many hair cells are abnormal, with swellings or blebs on the apical surfaced (arrowheads); (E, F) (3Gm/3 d): widespread loss of hair bundles from the hair cells in both extrastriolar (E) and striolar (F) regions is evident (arrows), apical blebs are present (arrowheads).
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Fig. 2. SEM (A) (3Gm/8 d): in extrastriolar region numerous hair cells are missing; note the supporting cells invading the region of pre-existing hair cells to produce a “scar” (asterisk). A few remaining mature hair cell bundles and some mature hair cell bundles undergoing degeneration are evident (arrow); some hair cells that had lost their hair bundles retain cuticular plates (arrowheads); (B) (3Gm/8 d): in striolar region the epithelium appears like a mosaic of supporting cells forming a “scar”; (C) (3Gm/18 d): in extrastriolar region small hair cell bundles show features of immaturity with stereovilli of almost equal height surrounding a much longer kinocilium (arrows); (D) (3Gm/18 d): in striolar region hair cell showing small hair cell bundles and a longer kinocilium, which are therefore probably immature, are present (arrow); (E, F) (3Gm/28 d): sensory epithelium has regained an apparently normal morphology comprising hair cells with an enlarged, bulb-like kinocilium and stereovilli that are graduated in length.
Fig. 3. CLSM: anti-actin (green) labels the cytoplasm of hair cells, anti-tubulin (red) labels the cytoplasm of supporting cells; DAPI (blue) stains the nuclei. (A) Saccule sensory epithelium of untreated lizards: anti-actin (green) labels the cytoplasm of hair cells (arrows); anti-tubulin (red) labels the cytoplasm of supporting cells (arrowheads); (B) (3Gm/4 h): hair cells appear damaged (arrows); (C) (3Gm/3 d): widespread loss of the sensory cells’ hair bundles and damage is evident (arrows), just a few intact hair cells bodies are present; (D) (3Gm/8 d): many hair cells are lost (arrowheads), some hair cell debris are evident in the luminal space above the epithelium (arrows), but no intact hair cell bodies are evident; (E) (3Gm/18 d): large supporting cells invade the region previously occupied by hair cells (arrowheads); note a few newly formed hair cells with small tufts of stereovilli (arrows); (F) (3Gm/28 d): sensory epithelium shows a normal appearance.
Fig. 4. CLSM: anti-calbindin (green) labels the cytoplasm of hair cells; DAPI (blue) stains the nuclei. (A) Saccule sensory epithelium of untreated lizards: anti-calbindin (green) strongly labels the cytoplasm of hair cells (arrows), but not the supporting cells’ cytoplasm (arrowheads); (B) (3Gm/4 h): hair cells appear damaged; (C) (3Gm/3 d): widespread loss of hair bundles of the hair cells and damage is evident (arrows), just a few intact hair cells bodies are present; (D) (3Gm/8 d): many hair cells are lost and cellular contents are present in the lumen (arrow), but no intact hair cell bodies are evident; (E) (3Gm/18 d): note a few newly formed hair cells with small tufts of stereovilli (arrows); (F) (3Gm/28 d): sensory epithelium shows a normal appearance.
Fig. 5. Mean number of hair cells in the saccule, for each group of treated lizards (3Gm/4 h, 3Gm/3 d, 3Gm/8 d, 3Gm/18 d, 3Gm/28 d) using calbindin as marker, expressed as percentage of the mean number of hair cells in the respective control samples (untreated), ±SD. The significance of differences was P < 0.01 (ANOVA).
Fig. 6. Immunofluorescence of saccule treated with Gm + BrdU (A–E), and with BrdU only (F). (A) 2Gm + 2BrdU/4 h: a few BrdU-labeled nuclei of supporting cells (i.e. in the supporting cell layer of epithelium) are already present; (B) 3Gm + 6BrdU/3 d: incorporation of BrdU in hair cells nuclei (i.e. in the HC layer of epithelium) appears in a few nuclei; (C) 3Gm + 11BrdU/8 d: some BrdU-labeled nuclei of hair cell are detected; (D) 3Gm + 21BrdU/18 d: BrdU-labeled nuclei of hair cells increase; (E) 3Gm + 31BrdU/28 d: incorporation of BrdU in hair cell nuclei appear increased significantly; (F) 3BrdU/4 h: lizards treated with BrdU only show evidence of BrdU incorporation in some hair cell nuclei; (G) control intestinal sections in the Gm plus BrdU showed BrdU incorporation evidence; (H) control intestinal sections in the BrdU-only-treated lizards showed some epithelium cells with BrdU-labeled nuclei.
Fig. 7. Mean number of labelled hair and supporting cell nuclei, ±SD, for each group of the Gm + BrdU-treated lizards. The significance of difference was P < 0.01 (ANOVA).
Table 1.
Animals used for each group and gentamicin and BrdU treatment
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