This study was designed to examine the pattern and extent of cochlear lesions induced with a combination of a single dose of aminoglycoside, kanamycin followed by the loop diuretic, bumetanide. Mice that received a single injection of kanamycin at a dose of 1 mg/g showed no long-lasting systemic effects. Exceeding this dose, however, proved to be toxic, resulting in paralysis, altered breathing patterns, and subsequent death within 20 min (all six animals given 1.25 mg/g died). Following administration of 1 mg/g kanamycin alone, omitting the loop diuretic, no damage to the inner ear was evident at any time point examined; 24 h (n
3), 48 h (n
2), 7 days (n
2), and 14 days (n
2). Likewise, animals that received only the bumetanide showed no adverse effects or cochlear abnormalities at the time points examined [24 h (n
2); 48 h n
1; 7 days n
2]. In contrast, animals that received the combination of kanamycin followed by bumetanide showed an acute response as early as 18 h subsequent to the treatment.
In the cochlea, the kanamycin–bumetanide protocol resulted in virtually complete loss of OHC by 48 h postinjection (effects described in detail below). Phalloidin and DAPI staining of whole mount preparations, SEM, and thin sections revealed that the protocol was effective in 80% of injected mice (194 cochleae out of the 244 examined by these methods). In the remaining 20% of injected animals, no hair cell loss was evident. The cochleae of these animals appeared essentially unaffected. In some animals, during the initial subcutaneous injection of kanamycin, there was leakage of fluid from the injection site, and in others the injected bolus persisted for some time under the skin. It is likely that these animals received a lower systemic dose or there was slow uptake of the drug and this may account for the absence of damage in a proportion of injected animals. However, the animals in which this potentially inadequate dosing occurred were not systematically recorded, particularly in the earlier stages of the study, so whether the animals in which this happened were the only ones to show no effect after treatment is uncertain.
There was no obvious effect on the vestibular system. There was no evidence of hair loss in either utricular maculae or cristae, and no abnormal behavior was observed in any of the experimental mice in which loss of cochlear hair cells had occurred.
Effects on the organ of Corti
Pattern and extent of OHC loss
No damage, neither hair cell loss nor nuclear abnormalities, was detectable in phalloidin and DAPI-stained whole mounts from cochlea obtained at 6 h postinjection. At 18 h posttreatment, there was OHC loss in the basal coil. By 24 h, loss of all OHC in approximately the entire basal half of the cochlea was evident, and by 48 h, OHC loss had spread to the apical coil (Fig. ). At 24 h posttreatment, regions of OHC loss were separated from regions of no loss by a “transitional zone,” where scattered hair bundles existed amongst scars formed by the apical expansion of neighboring supporting cells to close the lesions resulting from OHC loss (Fig. B). Basal to this zone, there was total OHC loss (Fig. A), and apical to it, apparently intact OHC were present in all rows (Fig. C). By 48 h, the zone of ongoing death of OHC was confined to the apical end of the apical turn (Fig. F,H) with total OHC loss basally (Fig. D,E,G); often at this time, few OHC remained except at the extreme apex. At this stage, labeling with antibody to the calcium-binding protein calretinin, a preferential label of inner hair cells (IHC) and neurons (Fig. D), as well as SEM (Fig. G,H), revealed that all IHC were still present.
FIG. 1 Progression of OHC loss in the organ of Corti. A–C Twenty-four hours posttreatment. A Basal turn; B middle turn; C apical turn. Confocal images of whole mount preparations. Phalloidin-FITC (green) labels actin assemblies at the reticular lamina (more ...)
The presence of a “transitional” zone between regions of complete OHC loss and no loss afforded the opportunity to examine the region of ongoing hair cell death. Within this region, the majority of remaining OHC nuclei appeared apoptotic, with marginated and condensed chromatin as demonstrated with either propidium iodide or DAPI (Fig. A–C). Infrequently, a swollen nucleus was apparent amongst the apoptotic nuclei, suggesting that, in some instances, cells died via a necrotic pathway (Fig. A).
FIG. 2 OHC death. A Transitional zone at 24 h posttreatment. Confocal image of whole mount preparation labeled for actin (green), with nuclei labeled with propidium iodide (red). A single enlarged nucleus, suggesting necrosis (arrow), in an OHC (hair (more ...)
Apoptotic cells can be detected by terminal deoxynucleotidyl transferase (TdT)-mediated 2′-deoxyuridine 5′-triphosphate nick end labeling (TUNEL) in which the free 3′-OH termini of fragmented DNA are labeled with modified nucleotides. TUNEL-positive nuclei were located in the region of OHC loss at both 24 h posttreatment and 48 h (Fig. B). In all TUNEL-positive cells, the nuclei were pyknotic, with condensed chromatin. There were no TUNEL-positive nuclei in undamaged control tissue (not shown). To clarify whether hair cells died via a classical apoptotic pathway, immunohistochemistry was performed using an antibody to detect activated caspase-3, an effector of the caspase-dependent apoptotic pathway. This revealed cells positive for activated caspase-3 within the transitional zone (Fig. C). The nuclei of these cells were typical of apoptotic cells: pyknotic, with marginated or condensed chromatin. Phalloidin labeling revealed that hair bundles were retained on a number of cells positive for activated caspase-3, demonstrating that the hair cell death can already be underway prior to any obvious damage to stereocilia. No labeling for activated caspase-3 was found in areas of complete OHC loss, nor in undamaged control tissue (not shown).
In TEM of thin sections, regions of ongoing OHC death could be identified in the middle turn of the cochlea at 24 h and in the apical turn at 48 h posttreatment. In such regions, many OHC displayed morphological characteristics of apoptosis: nuclei with marginated and condensed chromatin, condensed cell cytoplasm, and distorted cell shape (Fig. D,E). OHC showing the morphological features of necrosis were not found in thin sections, but scattered debris apparently from ruptured OHC was free within the extracellular spaces of the organ of Corti at the locations of missing OHC (Fig. E,F). Often, such debris enclosed what appeared to be condensed chromatin – similar to an apoptotic nucleus – (Fig. F) at levels in the epithelium expected of an OHC nucleus. Confocal light microscopy of whole mount samples immunohistochemically labeled for prestin confirmed that such debris was derived from OHC. Within the spaces of the organ of Corti, irregular material, positively labeled for prestin, surrounded dense DAPI-labeled bodies indicative of condensed nuclear chromatin at the position of OHC nuclei (Fig. G). In the regions of the organ of Corti just basal to the area of ongoing hair cell degeneration where hair cell loss was complete, there was no debris, suggesting such cell fragments were cleared rapidly.
IHC death and survival
Total loss of OHC preceded any IHC loss, which was always significantly delayed in relation to loss of OHC, but it was inconsistent as to timing postinjection and variable in its occurrence. With the exception of the cochleae of one animal taken at 4 days postinjection, no IHC loss was apparent until at least 7 days posttreatment, and it was most frequently observed in samples taken at 2 and 4 weeks posttreatment. At both these times, whereas in some cochleae IHC loss appeared to be almost total along the entire length of the organ of Corti, in others it appeared to be ongoing, suggesting relatively recent initiation (Fig. A,B). In these latter examples, IHC loss could be seen generally to proceed base to apex, but sometimes, there was scattered loss of IHC, individual or groups of apparently intact IHC persisting within regions of otherwise absent IHC (Fig. A). However, loss of IHC did not always occur in cochleae in which there was total loss of OHC. In more than half of the cochleae from treated mice taken at 7 days posttreatment and later (32 out of 59) in which all OHC along the entire length of the organ of Corti had been lost, IHC persisted (Fig. C,D). Even in some animals taken at 3 months following injection in which there was total OHC lost, all IHC remained.
FIG. 3 Progression of damage in the organ of Corti 7 days to 3 months. A Seven days posttreatment. Confocal image of whole mount preparation (projection of sections through 18-μm depth of tissue) labeled for calretinin (red), with phalloidin-FITC (more ...)
As described above, IHC death was considerably delayed in comparison with OHC death, and in more than half the cochleae examined from 2 weeks onwards in which all OHC were lost, IHC were still present. In regions of ongoing IHC loss, some nuclei were TUNEL-positive (Fig. A), suggesting apoptotic cell death. Thin sections through regions of ongoing IHC death revealed a variety of features associated with degeneration in different cells. In many IHC, nuclear chromatin was condensed and marginated, the cell cytoplasm was condensed and cell shape distorted, although mitochondria appeared largely normal (Fig. B). These cells with morphology consistent with apoptosis often possessed seemingly intact hair bundles. A few IHC displayed ruptured plasma membrane and loss of cytoplasmic contents, features consistent with necrosis (Fig. C). In such cells, stereocilia were often fused, and extensively fused stereocilia upon IHC were evident in SEM (Fig. D). Other IHC, whose shape and nuclei appeared normal, had an unusually electron dense cytoplasm, suggesting condensation of cytoplasmic contents, but there were also swellings around the base of the IHC at the sites of the afferent nerve terminals (Fig. E). Many IHC with this morphology seemed to be resorbing their stereocilia; the microfilament bundles were enclosed beneath the apical plasma membrane (Fig. F). In SEM, IHC with no or only vestigial stereocilia were apparent amongst hair cells with normal hair bundles and close to the sites of missing IHC (Fig. G). The apical surface of the membrane of these cells appeared smooth, with no indications of the sites of the stereocilia. The apical cytoplasm of cells with internalized stereocilia contained numerous vesicles, often double-membraned, enclosing cellular material (Fig. H,I). These structures are reminiscent of autophagic vesicles in various stages of the autophagic progression (Eskelinen 2005
FIG. 4 IHC loss. A TUNEL in whole mount preparation at 7 days posttreatment. TUNEL, green; actin, red; nuclei with DAPI, blue. In a region of scattered IHC loss, one IHC nucleus is TUNEL-positive. Scale bar, 10 μm. B Thin section 4 weeks (more ...)
At least some IHC appeared to fragment just below the level of the cuticular plate with the apical fragment released into the endolymphatic space. SEM revealed globular structures, reminiscent of cellular material, at the apical surface at the sites of IHC (Fig. J). Apical fragments of IHC, consisting of stereocilia, the cuticular plate, and some cytoplasmic material containing apparently intact mitochondria, were sometimes found lying atop the reticular lamina above the locations of IHC, in the endolymphatic space below the tectorial membrane (Fig. K).
FM1-43 labeling of hair cells
One reason for the persistence of IHC when OHC die might be that they do not take up aminoglycoside as readily as OHC. To examine this issue, we investigated the uptake of the fluorescent lipophilic dye FM1-43 into hair cells in the mature organ of Corti. FM1-43 is of similar size to aminoglycoside (MW FM1-43 452, kanamycin 484) and, like aminoglycosides, FM1-43 is a cation. In sensory epithelia of the inner ear, FM1-43 preferentially labels hair cells. It has been suggested that the dye enters hair cells via the transduction channel at the tip of the stereocilia (Gale et al. 2001
; Meyers et al. 2003
), and because aminoglycosides can bind to the transduction channel and FM1-43 can inhibit the aminoglycoside-induced injury to hair cells in organotypic culture of the early postnatal organ of Corti, it has been argued that FM1-43 and aminoglycosides enter hair cells by the same route (Gale et al. 2001
). Consequently, FM1-43 may act as a nontoxic “mimic” for aminoglycosides.
When the isolated, intact, but opened, mature cochleae were exposed to FM1-43 for 10 s, dye labeling was present in both IHC and OHC (Fig. A). Labeling in IHC was at least as intense if not greater than that in OHC. This suggested that the dye may be able to enter IHC and OHC equally readily in the mature cochlea.
FIG. 5 FM1-43 uptake. A In vitro isolated cochlea exposed for 10 s at 37°C; lower apical cochlear coil. FM1-43 has labeled only hair cells and has labeled both IHC and OHC. B Organ of Corti 6 h after coadministration of FM1-43 and bumetanide (more ...)
However, it is possible that the isolation of the cochlea, opening it, and the exposure to in vitro conditions lead to abnormal, sustained opening of the transduction channels in both OHC and IHC and/or the dye is not efficiently washed away and uptake occurs through several routes, including endocyototic activity (although the use of low temperatures for postexposure washing in buffer maintained on ice was included to restrict endocytotic activity). Thus, the entry of FM1-43 into hair cells in vivo and the effects of coadministration of bumetanide upon uptake were examined. Two hours after administration either with or without coadministration of bumetanide, no dye uptake was evident in the organ of Corti. At 6 h after systemic injection of FM1-43 in conjunction with bumetanide, both IHC and OHC in the lower apical coil were labeled (Fig. B). At 24 h, IHC and OHC were both labeled, IHC seemingly more intensely than OHC (Fig. C). In the cochleae of mice that received only FM1-43, but not bumetanide, there was very little labeling of the hair cells at 6 h, and the intensity of labeling at 24 h (Fig. D) appeared less than in the cochleae of animals coadministered the diuretic, but again, both IHC and OHC were labeled. In utricular maculae from mice coadministered FM1-43 and bumetanide, the intensity of FM1-43 labeling in hair cells was markedly lower than that in organs of Corti of the same set of animals (Fig. E) and very little different from labeling of utricular hair cells in mice that received only FM1-43. However, dye labeling was present and quite intense in the capillaries underlying the utricular macula, demonstrating that FM1-43 had reached the vestibular organs (Fig. E).
Supporting cell rearrangement following OHC loss
The loss of OHC was accompanied by closure of the lesions by expansion of supporting cells into the area once occupied by the hair cell. Neither in SEM preparations (Fig. A,B) nor in thin sections (Fig. C) was there evidence of obvious lesions breaching through the reticular lamina as hair cells died, suggesting that, despite the rapid and extensive loss of OHC, lesion repair still occurred efficiently and in a manner similar to that described previously for OHC loss following chronic gentamicin treatment in guinea pigs (Forge 1985
). At the apical surface of the reticular lamina at the site from which an OHC was missing, there was a depression, the shape of a hair cell apex, that was closed at its base, indicating supporting cells had already closed the lesion (Fig. A). Other stages in ongoing repair captured at the time of tissue fixation also revealed the closure of the lesion as hair cell remnants separated, and the absence of open wounds (Fig. B). In thin sections (Fig. C), the site at the reticular lamina of a missing hair cell was identifiable by a depression in the surface, sometimes with some cellular debris within it, and immediately below it, the lesion was closed by a junction between adjacent supporting cells with the electron density of a tight junction, indicating the sealing of the lesion as the hair cell died.
FIG. 6 Supporting cell reorganization. A In a region of ongoing OHC loss, a depression in the reticular lamina at the site of a lost hair cell (arrow) is closed at the bottom. Scale bar, 5 μm. B Apical fragments of OHC are being lost from the (more ...)
There was considerable structural reorganization amongst the supporting cells during and following lesion closure. Normally, the phalangeal processes of Deiters’ cells rise at an angle from the cell body region, enclosing one OHC body to contact the apices of OHC at least one OHC distant crossing over another OHC body on the way (Fig. D). In the repaired organ of Corti, the Deiters’ cell phalangeal processes rose straight up from the cell body without angulation, suggesting movements of the heads of the supporting cells at the level of the reticular lamina (Fig. E,F). Some Deiters’ cells were rounded as the reorganization proceeded and the cell body enclosing the nucleus was at a more apical (luminal) position than normal (Fig. F,G); although, whether these cells lost contact with the basilar membrane was not confirmed. Despite these morphological changes, Deiters’ cell bodies retained close contact with their neighbors (Fig. G). The repaired epithelium was narrower, with the outermost edge of the epithelium closer to the pillar cells than in undamaged tissue. The epithelial surface was buckled so that an inward furrow was created along the length of the epithelial surface with the Hensen’s closely approaching the pillar cells (Fig. H,I). However, although there was rearrangement of Deiters’ cells, they retained most of their obvious structural specializations, including prominent, organized microtubule bundles in the cell body region and at the base of the cell in contact with the basilar membrane (Fig. G). Other than the expansion of the head of the outer pillar cell to close lesions where OHC in the first row were lost (Fig. A,B), and retraction of the head of some inner pillar cells (Figs. C and I), there was little alteration of the phalangeal processes or cell bodies of the pillar cells. The tunnel of Corti remained widely open (Fig. B,D), flanked by the phalangeal processes both of the outer and the inner pillar cells, which remained erect, supported by their bundles of microtubules, which remained undisturbed (Fig. J). Overall, there was little structural evidence of any significant de- or redifferentiation of supporting cells even after quite prolonged periods following loss of OHC.
Despite considerable reorganization of the sensory epithelium in the OHC region, in many cases, IHC persisted (Figs. C and H). They remained even when there was buckling of the reticular lamina caused by the inward movement of the Hensen’s cells (Fig. H). Conversely, there was IHC loss when pillar cells and the arch of Corti were intact (Figs. B and J). The death of IHC, when it occurred, therefore, did not appear to be directly related to the reorganization of the epithelium occasioned by death of OHC and the subsequent structural rearrangements amongst the supporting cells.
Effects on the SV
In TEM sections from control animals (Fig. A), and from those which had received the drugs but showed no effect upon the hair cells (“undamaged, injected” animals), no obvious abnormalities of the stria were detected. Administration of a single dose of bumetanide is known to cause extensive edema in the stria by 1 h following injection, but this rapidly resolves and no continuing abnormalities are evident by 24 h posttreatment (Santi and Duvall 1979
). There was no edema in the SV of any animal taken at 24 h posttreatment in which there was hair cell loss (Fig. B), and in these mice, the stria appeared no different from that of controls or undamaged, injected animals. Likewise, there were no obvious abnormalities in the striae of any animal taken at 2–4 days posttreatment in which there was hair cell loss. Measurements of the thickness of the striae in these animals (basal coil; mean 35.01 μm, range 24.6–45.8 μm; apical coil: mean 29.6 μm; range 21.1–37.0 μm) showed no significant difference from that of controls (basal coil: mean 34.1 μm, range 28–42 μm; apical coil: mean 31.1 μm; range 28.7–34.8 μm) (Fig. ). Striae of two untreated older mice, one aged 12 months the other 2 years old, were also examined. There was no detectable hair cell loss at 12 months, and at 2 years, there was a very low level of scattered hair cell loss, and the mean strial width in these animals (12 months, 32.2 μm; 2 years, 31.3 μm) was within the normal range (Fig. C). There were, however, indications of cellular degeneration in the stria from the 2-year-old animal (Fig. C), most noticeably the presence of cells within the body of the stria containing clusters of electron dense inclusions and vesicles filled with amorphous electron dense material.
FIG. 7 SV atrophy. A Normal SV from an untreated mouse. MC, marginal cell; IC, intermediate cell; BC, basal cell; cap, capillary; sp lig, spiral ligament. B Twenty-four hours after kanamycin–bumetanide treatment. SV looks normal. C SV from untreated (more ...)
In contrast, in animals in which there was hair cell loss taken at 7 days posttreatment and at subsequent time points, there was a significant decrease in strial thickness (Figs. D,E; ). One-way ANOVA statistics revealed a significant decrease for both apical and basal coils at 2 weeks posttreatment in comparison with controls and a further significant decrease between 4 weeks posttreatment and 8 weeks and later times. This would indicate a progressive reduction in strial thickness continuing for some time concomitant with the reorganization of the sensory epithelium following hair cell loss. In some animals at the later time points, (2–3 months) the strial thickness was only 10–12 μm and the basal cell layer was still intact, intermediate cells were difficult to distinguish, and marginal cells were severely reduced (Fig. E).
The major cause of the strial thinning appeared predominantly to be a reduction in the basal infoldings of marginal cells (Fig. F,G), first apparent in samples taken at 7 days posttreatment, and degeneration of marginal cells themselves (Fig. A–G), which was more pronounced in samples taken at 14 days postinjection and subsequently. In damaged cochlea, the extensive basal infoldings were absent from many marginal cells (Fig. F) or reduced to thin, apposing stacks of short processes (Fig. G), and the interdigitations with intermediate cells were retracted such that ramifying, thin processes of the intermediate cells were revealed (Fig. F). What were presumably marginal cells, judging from their location at the endolymphatic surface but which possessed no obvious basal infoldings and were almost cuboidal or rounded in shape, exhibited a reduced electron density in comparison with more normal neighboring marginal cells (Fig. A,B). Some cells with this feature (Fig. A) were filled with long, almost parallel, closely packed filamentous structures, ca. 20–25 nm in width, reminiscent of microtubules (Fig. C,D). These ran throughout the cell, from the apical cell body region (Fig. C) to the basal extremities (Fig. D). Transverse sections (Fig. D) confirmed the microtubule-like character of these structures. At their apical surfaces, some cells exhibited unusual numbers of small microvilli and cell width along the apical plasma appeared reduced concomitant with apparent enchroachment of lateral expansions of the immediately neighboring marginal cells (Fig. A,B), such that the affected cell seemed to be becoming enclosed apically and withdrawing into the epithelium. Enclosed within the body of the stria, there were also degenerating cells (Fig. E), which the size of their still intact mitochondria suggested were marginal cells. Nuclei in some marginal cells with abnormal features were fragmented with condensed chromatin (Fig. A,E) suggestive of apoptosis. Within the stria at a level similar to that of intermediate cells, there were often approximately rounded cells with normal appearing nuclei that contained electron dense inclusions and filled membrane-bound vesicles (Fig. F,G), precisely similar to the features indicating cellular degeneration in the stria of the aged 2-year-old mouse (Fig. C). Also found in the body of the stria were concentric whorls of membrane enclosing what appeared to be degenerating cellular material (Fig. H,I). These latter abnormalities – cells with inclusions and concentric membrane whorls – while occasionally present at 7 days posttreatment, were common in SV at the later time points.
FIG. 9 A Marginal cell at 7 days posttreatment has much lighter cytoplasm than normal marginal cells, as evident in neighbors. The apex shows numerous small microvilli. Neighboring, more normal-appearing marginal cells appear to be extending processes (more ...)
Amikacin or gentamicin with bumetanide
Mice tolerated amikacin as equally well as kanamycin. All animals survived injections with doses of amikacin at 1 mg/g (n
6). When amikacin was administered with bumetanide, complete loss of OHC was evident, but IHC survived for at least 14 days (n
3). As others have noted (Wu et al. 2001), gentamicin was not well tolerated. Single injections of doses greater than 0.3 mg/g caused rapid death of the animals (n
6). Gentamicin at 0.25 mg/g, when given in combination with bumetanide, had no damaging effect on the cochlea (n
3). However, some animals survived injection of gentamicin at 0.3 mg/g followed by bumetanide. In these animals, complete loss of all OHC but persistence of IHC was also evident at 7 (n
3) and 14 days posttreatment (n
2). These observations indicate that the relative resistance of IHC following kanamycin–bumetanide treatment is not drug-specific.
Age effects on OHC loss
In animals at 18–21 days of age, the organ of Corti is structurally mature and the animals can hear. Animals of this age were used partly to keep injection volumes as small as possible, partly because it has been suggested that animals of this age are more sensitive to the aminoglycoside–diuretic combination than older animals (Prieve and Yanz 1984
). To test the age-dependence of hair cell loss, animals aged 25, 30, and 35 days (n
3 in each case) were treated and their cochleae examined 7 days after treatment. Complete loss of OHC from base to apex was observed in the 25- and 29-day-old animals, but in two out of the three animals at 35 days, OHC remained in the apical coil, although there was still scattered loss in this region too.