Characterizing human vestibular sensory epithelia for experimental studies: new hair bundles on old tissue and implications for therapeutic interventions in ageing

Abstract

Balance disequilibrium is a significant contributor to falls in the elderly. The most common cause of balance dysfunction is loss of sensory cells from the vestibular sensory epithelia of the inner ear. However, inaccessibility of inner ear tissue in humans severely restricts possibilities for experimental manipulation to develop therapies to ameliorate this loss. We provide a structural and functional analysis of human vestibular sensory epithelia harvested at trans-labyrinthine surgery. We demonstrate the viability of the tissue and labeling with specific markers of hair cell function and of ion homeostasis in the epithelium. Samples obtained from the oldest patients revealed a significant loss of hair cells across the tissue surface, but we found immature hair bundles present in epithelia harvested from patients >60 years of age. These results suggest that the environment of the human vestibular sensory epithelium could be responsive to stimulation of developmental pathways to enhance hair cell regeneration, as has been demonstrated successfully in the vestibular organs of adult mice.

Keywords: Aging pathologies; Hair cells; Human vestibular; Stereocilia; Supporting cells; Utricle.

Supplementary Fig. 1

Mouse utricular maculae. (A) A 22-month-old CBA/Ca mouse. Stereocilia of hair bundles in orderly arrays. Hair cells missing over a large area where supporting cells have closed the lesions. (B) A 24-month-old CBA/Ca mouse. Longitudinal section of hair cell with granules (arrow) of an appearance consistent with lipofuscin. (C) A 24-month-old CBA/Ca mouse. The macula is covered with hair cells, but there are many regions where hair cells are missing (asterisks) throughout the tissue. (D–F) Immature bundles. (D) Immature hair bundle in early postnatal (P4) C57Bl/6 mouse. Stereocilia are of equal height and cross-linked. (E, F) Immature-appearing hair bundles in old mice: 24-month-old CBA/Ca. (G–I) Stereocilia anomalies in old (24 months) CBA/Ca mice. (G) Giant, fused stereocilia (arrow). (H) Multiple fusions and swellings along shaft and at tip. (I) Fusion and enlarged swelling (arrow). Scale bars: (A) 10 μm; (B) 1 μm; (C) 10 μm; (D–I) 1 μm.

Supplementary Fig. 2

Junctional actin assemblies in mouse. (A) Rapidly frozen (by high pressure freezing), freeze-substituted tissue shows electron-dense structures in contact with the lateral membrane in the region of the adherens junction are composed of microfilaments. (B) Explanted utricular macula from a 22-month-old CBA/Ca mouse, maintained in culture for 19 days after exposure to gentamycin. All hair cells lost. Phalloidin labeling of whole mount preparation shows actin bands in the junctional region of supporting cells to be relatively thin, but more prominent than in the equivalent junctional complexes of the cells in the surrounding nonsensory epithelium. (C–E) Thin sections of utricle from a 22-month-old CBA/Ca mouse maintained in culture for 6 days after exposure to gentamicin; hair cells lost. (C) The adherens junctional region between supporting cells is simple; there is little evidence large actin assemblies. (D, E) Electron-dense structures with the appearance of junctional actin assemblies are displaced to the basal region of the cells (arrows) and detached from the plasma membrane (E). Scale bars: (A) 0.5 μm; (B) 10 μm; (C) 5 μm; (D, E) 2 μm.

Fig. 1

(A–C) FM1-43 uptake. (A) Utricular macula. (B) Crista. Labeling across the entire tissue surface. (C) Utricular macula. Dye is confined to cells with the shapes and pattern of distribution of hair cells. Inset shows cells that take up the dye have the shape expected of hair cells. (D) Hair cells labeled for myosin VIIa (green) are distributed across the epithelium (filamentous actin labeled with phalloidin-TRITC [red]). (E) Hair bundles (arrowheads) and hair cell bodies labeled for calretinin. Phalloidin labeling of filamentous actin (red) in hair bundles (arrowheads) and in wide bands at the junctions between supporting cells, but in some regions from which hair cells have been lost and the lesions closed by expansion supporting cells, the junctional actin bands are thinner (arrows). (F) Phalloidin labeling reveals actin “rods” through the bodies of some hair cells (arrows) in fresh-fixed tissue. Scale bars: (A, B) 20 μm; (C) 10 μm; inset (10) μm; (D) 20 μm; (E, F) 10 μm.

Fig. 2

(A–G) Hair bundles in recently excised utricular maculae. (A) Adjacent bundles of differing height. (B–D) Different bundle “types.” In (B) the stereocilia, especially the shorter ones are generally thicker than those in (C), but there is variability in the width of stereocilia of similar height within individual bundles. In (D), the tallest stereocilia are markedly longer than the shorter ones. Although there is height gradation across the bundles, there is variability in the differential lengths of adjacent stereocilia. (E–G) Stereocilia cross-links. (E) Tip-links (arrows) and top connectors (arrowheads). (F) Top connectors (arrowheads) and shaft connectors (arrows). (G) Ankle links (arrows). (H, I) Almost complete loss of hair cells in utricular maculae from elderly people. (H) An 80-year-old woman; the surface consists almost entirely of a pavement of supporting cell surfaces which are covered with short microvilli. (I) A 65-year-old man. A single hair bundle (arrow) is evident across the field. Scale bars: (A–D) 1 μm; (E) 0.1 μm; (F) 0.5 μm; (G) 0.25 μm; (H, I) 10 μm.

Fig. 3

Thin sections of hair cells in utricular macula. (A, B) Striated bodies (arrows) associated with cuticular plate: type 1 (A) and type 2 (B) hair cell. (C, D) Innervation. (C) Thin afferent calyx (arrow) surrounding body of type 1 hair cell. (D) Base of type 2 hair cell showing 2 bouton nerve endings (asterisks). (E) Two synaptic ribbons (arrows) opposite a single afferent nerve bouton. Arrowheads in (A–C) indicate lipofuscin granules. Scale bars: (A–D) 1 μm; (E) 0.2 μm.

Fig. 4

Bundles with immature characteristics in utricular maculae. (A) Bundle with short stereocilia of equal height (arrow) among mature-appearing bundles in tissue from a 60-year-old woman. (B) Stereocilia of approximately equal height cover almost the entire apical surface of the cell, are extensively cross-linked and surround the thicker kinocilium (arrow); from a 65-year-old man. (C) Cluster of short stereocilia occupying entire cell surface are extensively cross-linked; from a 65-year-old man. (D) Cluster of elongated “microvilli” with eccentrically positioned thicker projection, reminiscent of a kinocilium (arrow); from 85-year-old woman. Scale bars: (A) 5 μm; (B–D) 1 μm.

Fig. 5

Stereociliary pathologies. (A–G) Scanning electron microscopy. (A) Stereocilia of varying width along length (arrowhead) and kinocilium fused with stereocilium (arrow). (B) Individual stereocilia vary in width along their length. (C) Stereocilia show swellings along the shaft (arrow). (D) Fused stereocilia and various swellings at distal ends. (E) Fused, elongated, and swollen stereocilia. (F) Single fused “giant” “stereocilium” on an individual hair cell. (G) Stereocilia apparently internalized at the apical surface of the hair cell (arrow), which shows numerous vesicle openings (arrowheads) around the periphery of the of the luminal surface. (H–M) Thin sections. (H) Swollen apical tip of a stereocilium. (I) Swelling along the shaft of the stereocilium in which cytoplasmic-like material is enclosed by the stereociliary membrane and a dense rod-like structure (arrowed) is located at the periphery of the stereociliary filament bundle. (J) Stereocilium showing a thinning along its shaft and a dense rod structure within the shaft. (K) Stereociliary fusion of 1 stereocilium at the site of a swelling along the shaft of another (arrowhead). (L) Hair cell apical membrane in the region between the points of insertion of stereocilium is detached from the cuticular plate and is rising between and fusing with adjacent stereocilia. (M) Fused stereocilia (arrowhead) and fusion between stereocilia and kinocilium (arrow). There seem to be 2 ciliary axonemes within the limiting membrane of the kinocilium. Scale bars: (A–G) 1 μm; (H, I) 0.2 μm; (J, K) 0.5 μm; (L, M) 0.2 μm.

Fig. 6

Utricular maculae maintained in organotypic culture. (A) Seven days in vitro. Confocal projection image of apical surface of whole mount preparation. Hair cells, labeled for myosin VIIa (green), are distributed throughout the epithelium. Phalloidin labeling of filamentous actin delineates the junctional regions around the necks of supporting cells. Where some hair cells have been lost, the heads of supporting cells have expanded to close the lesions producing characteristic scar formations where the junctional actin bands are thinner than elsewhere (arrows). (B) Nine days in vitro. Confocal image of single optical section with orthogonal projections. Both flask-shaped type 1 and more cylindrical type 2 hair cells are retained. (C) Twenty-one days in vitro. Scanning electron microscopy shows hair bundles with long stereocilia, but some evidence of fusion after this period of ex corporeal maintenance. Scale bars: (A) 10 μm; (B, C) 5 μm.

Fig. 7

Hair cells survive without hair bundles. (A–D) Utricular macula fixed immediately after harvesting. (A) Hair cells devoid of bundles, many with impressions of the insertion points of stereocilia at the apical surface, are scattered across the epithelium. (B) Scanning electron microscopy (SEM) of the surface of an individual hair cell. Short stumps of the stereocilia and of the kinocilium are covered over; their internal structure is not exposed. (C) Thin section of the apical end of a type 2 hair cell shows continuous plasma membrane mounded over the stereociliary rootlets descending into the cuticular plate (arrow). The cytoplasm below the cuticular plate shows no evidence of degenerative changes. (D) In a type 1 hair cell, the apical plasma membrane is continuous over the cuticular plate with no indication of disruption at the sites of the stereociliary rootlets. There is no indication of cellular degeneration in the cytoplasm below the cuticular plate. The arrow points to large mitochondria in the afferent nerve calyx. (E–H) Four days in vitro. (F–H) Images collected by backscatter detection in the SEM of sections of the same sample examined by conventional SEM shown in panel. Hair cells without bundles persist in the epithelium (E). The hair cells show no obvious evidence of degeneration (F). At the apical ends of the cells ([G], type 2 hair cell; [H], type 1), the cuticular plate with persisting stereociliary rootlets (arrow in [G]) is covered by a continuous plasma membrane, and striated bodies are retained (arrow [H]). Scale bars: (A) 10 μm; (B) 1 μm; (C) 0.5 μm; (D) 1 μm; (E) 10 μm; (F–H) 1 μm.

Fig. 8

Utricular maculae after incubation with 2 mmol/L gentamicin for 48 hours. (A, B) Two days after the end of gentamycin exposure. (A) Few intact hair cells remain, but myosin VIIa–labeled debris and some pyknotic nuclei (DAPI, blue) are present at the level of hair cell nuclei. (B) Most remaining, apparently intact hair cells (positive labeling for myosin VIIa) contain an actin cable running through the length of the cell. (C) Seven days after gentamicin exposure. Phalloidin labeling of f-actin at the cell junctions, shows numerous “scar” formations in the positions once occupied by hair cells where supporting cells have closed the lesions repair in which the junctional actin bands are relatively thin. (D) Four days after gentamicin exposure. Thin section shows no hair cells are present and supporting cells, with nuclei predominantly positioned at the basal aspect of the epithelium, have repaired efficiently the epithelium. The arrow indicates a relatively simple adherens junctional assembly. (E) Twenty-one days after gentamicin exposure. Scanning electron microscopy reveals that the apical surface of the epithelium is composed of a pavement of supporting cell apices, with no indication of the presence of remaining hair cells or of immature hair bundles. Scale bars: (A–C) 10 μm; (D) 5 μm; (E) 10 μm.

Fig. 9

Supporting cell characteristics. (A) Labeling for Sox-9 (red) almost exclusively confined to nuclei (counterstained with DAPI, blue) at the level of those of supporting cells. Nuclei in hair cells (labeled for myosin VIIa, green) do not usually label for Sox-9, but an occasional nucleus in a myosin VIIa–labeled cell showed weak labeling for Sox-9. (Actin labeling in magenta.) (B) Labeling for l-glutamate/l-aspartate transporter (GLAST) delineates lateral membranes of supporting cells, extending down to the level of the basement membrane. (C, D) Labeling for connexins. Both CX26 (red in [C]) and CX30 (red in [D]) are present in large plaques around the cell body regions of supporting cells. Smaller labeled puncta are also present in the region close to the junctional complexes at the luminal end of supporting cells (arrows). Scale bars: (A–D) 10 μm.

Fig. 10

Dye transfer in viable slices of utricular maculae of a young adult (P30) mouse (A, B), and a 65-year-old human (C, D). Individual supporting cells are injected with neurobiotin (red) and fluorescein-dextran (green). Dextran is confined to the injected cell but neurobiotin spreads to all supporting cells in the slice. There is no spread to hair cells. In the human tissue, there are relatively few hair cells. Supporting cells have enlarged to occupy the spaces from which hair cells have been lost, but neurobiotin distribution shows maintenance of extensive coupling between supporting cells, and that the coupling is confined to the sensory epithelium. Data is typical of 4–5 maculae per species. Scale bars: 10 μm.

Fig. 11

Remodeling of actin assemblies at adherens junctional complexes. (A–C) Normal morphology of junctional complexes in human utricular maculae. (A) Large, electron-dense actin structures (1 denoted by asterisk) in the region of the adherens junction contact the plasma membrane in scalloped fashion at both sides of the junction. The basal limits of the tight junctions are indicated by the arrowheads; the electron densities at the plasma membrane, which indicate the adherens junction itself, are indicated by arrows. The actin assemblies often, but not always contact the plasma membrane at the site of the adherens junction. (B) Section parallel to the long axis of the supporting cell shows the actin assembly extends quite deeply into the cells (asterisk). The arrow indicates where there is no actin assembly to appose that contacting the lateral plasma membrane in the adjacent cell. (C) Cross-section (almost parallel to the apical surface) shows the width of the actin assemblies (arrows indicate the limits at 1 intercellular junction in each of the apposed cells). (D, E) Displaced actin assemblies. In (D), arrows indicate actin assemblies in apposed cells displaced basally. The inset shows that in the lower indicated structure each actin assembly contacts the plasma membrane in the respective apposed cells. In (E), structures with an appearance identical to that of the junctional actin assemblies (asterisks) are contained within the cytoplasm of the cell body region of the supporting cell. The inset shows these structures are composed of closely packed filaments of a size consistent with that of f-actin. (F–H) Junctions in tissue after gentamicin-induced hair cell loss. (F) Seven days after the end of incubation with gentamicin. Longitudinal section shows relatively small electron densities contacting plasma membrane in the adherens junction region (arrows). An electron-dense structure similar to the junctional actin assembly is located in the center of the cell detached from the plasma membrane. (G) Four days after gentamicin exposure. Cross-section show the actin band associated with the adherens junction is thinner that in normal tissue as in panel (C). (H) Confocal image of phalloidin-labeled whole mount of tissue 19 days after gentamicin exposure reveals actin bands in the junctional region of supporting tissue to be thinner than those in “normal” tissue (e.g., Fig. 1F), although still denser and more prominent than the actin associated with the adherens junctions between cells in the nonsensory epithelium. Scale bars: (A) 0.5 μm; (B) 1 μm; (C) 2 μm; (D) 5 μm, inset 0.5 μm; (E) 2 μm, inset 0.5 μm; (F) 1 μm; (G) 2 μm; (H) 10 μm.