In contrast to results following systemic administration, following local application of gentamicin the H&E-stained sections showed that there was often obvious degeneration of the organ of Corti, the spiral ligament, the spiral limbus, and spiral ganglion cells. Initial experiments were done with the gentamicin for injection formulation. With this formulation there were cases with degeneration so severe that there were few surviving cell classes remaining to analyze. For this reason the formulation of the drug was changed. In cases employing the drug dissolved in artificial perilymph, there was greater variability in severity of the pathology observed following drug administration. In some of these cases there was widespread disruption of cochlear structures (the most severe is shown in ), but in four cases (survival times of 1 week, 1 month, 3 months, and 6 months) there was no cell loss apparent in H&E sections. In contrast, all cases with the gentamicin for injection formulation and survival times of 4 days or greater showed overt cochlear pathology. In keeping with previous studies, OHCs were obviously vulnerable to gentamicin toxicity, but this vulnerability was equal to or closely matched by that of Deiters' cells. Usually degeneration of the organ of Corti was most severe in the lower basal turn, with less severe effects extending apically for variable distances. Cases with the least degeneration had missing OHCs and Deiters' cells (). In such cases the height of the organ of Corti was also reduced, indicating that pillar cells, although still present, were structurally abnormal (). In some cases with missing OHCs, some Deiters' cells survived but were dysmorphic and their nuclei were near the basilar membrane ().
Figure 4 H&E staining of cochleas treated with local application of gentamicin. The pair of images at the top are from the upper basal turn and show a gentamicin-treated ear (B) and the corresponding image of the contralateral control ear (A) in a case (more ...)
In several cases with minimal cell loss in the organ of Corti, there was mild endolymphatic hydrops (), which indicated that nonsensory cells had been affected by the drug treatment. In the case illustrated in , hydrops was perhaps some cell loss in the spiral ligament (area of the white arrowhead), but clear instances of spiral ligament cell loss were not apparent in every case where there was hydrops. However, cell loss is the extreme condition of toxicity and it may be necessary to seek evidence of more subtle forms of cellular dysfunction that can account for hydropic conditions. Mild hydrops was present in two cases with 4 day survival and one case with 7 day survival. Longer-term survival cases with hydrops also showed fibrosis of scala vestibuli ().
A common form of degeneration was the complete loss of the organ of Corti and its replacement by a squamous epithelium (). This condition was accompanied by degeneration of either the stria vascularis (), and/or the spiral ligament (), and/or the spiral limbus (). There appeared to be no obvious relationships between the susceptibilities of cell loss in these different structures. The most severe form of degeneration included reduction of the membranous labyrinth to a squamous epithelium, accompanied by a complete fibrosis of the scala vestibuli (). Fibrosis of the scala tympani invariably included some degree of degeneration of the spiral limbus (). The most severe case of degeneration also included new bone formation in the spiral ligament and scala vestibuli, as well as on the middle ear surface of the otic capsule (). In animals with greater than 1 week survival times and a totally absent organ of Corti, there was usually a clear reduction in the thickness of the adjacent stria vascularis (e.g., ). In some cases the cross-sectional area of the stria was reduced in some cochlear turns and edematous or degenerated in other turns (e.g., ). Spiral ganglion cell loss appeared in parts of the cochlea where there was a totally degenerated organ of Corti. In contrast to other cell losses, ganglion cell loss was not usually apparent until survival times of one month or greater.
Application of gentamicin on the round window resulted in such high levels of the drug within the perilymph that it was difficult to differentially stain cells in the cochlea within 6 h post administration due to diffuse staining of all tissue. Consequently, the early time course of drug uptake could not be documented as precisely as in the systemically treated animals. As staining of extracellular tissue became reduced with increasing post administration time, diffuse staining of most cells was discernible. The cell types that were found to be positively stained for gentamicin following local administration were, by and large, the same cell types that were positive following systemic administration. A striking feature of locally treated cases was a marked basal to apical gradient in apparent drug uptake which corresponded to the drug application at the base of the cochlea. This gradient of staining matched the gradient of cellular degeneration which was most extreme basally.
There was variability between animals with respect to the degree of staining for gentamicin and in the extent of tissue that was stained. There was a strong correlation of the intensity of staining for gentamicin and the degree of cellular degeneration in individual cases. In cases of obvious cochlear degeneration, some cell types, such as root cells, which had stained only mildly in cases with no apparent degeneration, were intensely stained. The same relation was found in vestibular organs. In cases with missing vestibular cells, remaining nearby cells were far more intensely stained for gentamicin than corresponding cells in cases where there was no degeneration. Animals receiving the commercial solution of the drug usually had more intense immunostaining for gentamicin and more profound cell losses. There was no immunostaining for gentamicin and no obvious morphological changes in the animal which received the vehicle only.
There was considerable variability between animals in which sensory organs stained darkly for the drug when it was applied locally. Usually both the cochlea and vestibular organs were stained, but in some instances not all vestibular organs were stained, although there was no consistent pattern regarding which organs were stained. Usually when a given organ was heavily stained, perivascular tissue within the nearby bone was also stained.
Organ of Corti
Changes in the pattern of staining for gentamicin in the organ of Corti in locally treated animals followed the same sequence that occurred in systemically treated animals. That is, initially staining of OHCs was punctate and located in cytoplasm (). More diffuse staining, often with a reticular appearance, was present at 12 h and beyond (). Gradually the cytoplasmic staining diminished with increasing survival times except for that in the infracuticular portions of the OHCs (). Apical staining of OHCs was present in animals with survival times as long as 6 months (). Staining present at the longest survival time was predominantly in the basal turn. In animals in which the organ of Corti was collapsed and replaced by cuboidal epithelium, the remaining cells were immunoreactive for gentamicin at survival times of 1 month or greater (). In contrast, when only a squamous epithelium remained, it showed little or no staining for gentamicin (not illustrated).
Figure 5 Immunostaining for gentamicin in the cochlea following local application. Numbers on the photos indicate survival periods. A. The cytosol of OHCs shows intense immunoreaction for gentamicin. The arrow indicates heavily stained radial nerve fibers. B. (more ...)
Ganglion cells were darkly stained with punctate reaction product at post injection times of 4 days and less. At longer post injection times, use of the standard dilution of the antibody (1:40,000), which resulted in intense staining of type III fibrocytes, produced little or no staining of ganglion cells.
Mesenchymal cells beneath the basilar membrane (arrowheads in ) were immunoreactive at 6 h post administration. This staining was present throughout the length of the basilar membrane, but only cells in basal turn remained stained for as long as 6 months (). Staining of the mesenchymal cells was present regardless of whether the overlying organ of Corti was degenerated (). Nearby mesenchymal cells lining the scala tympani were often intensely stained.
There was distinct staining in the basal cells and intermediate cells of the stria vascularis at 6 h post administration (, arrowhead), but no staining of these cells remained beyond 1 week survival (). In ears with a badly damaged organ of Corti, there was granular staining of the marginal cells of the stria vascularis (arrowhead in ). The spiral ligament was diffusely stained at 6–24 h post administration (not illustrated). Diffuse staining initially obscured individual cells, but distinct staining of type I fibrocytes, which was present chiefly in the basal half of the cochlea, was obvious at 4 days post administration (, region of the asterisk). This staining was more pronounced than in systemically treated animals but was diffuse instead of punctate as in most other cells. Staining of type I fibrocytes was considerably diminished at 7 days survival (). Staining of type II fibrocytes was predominantly in the basal turn. This staining was punctate and intense. Many stained type II fibrocytes were present at survival times up to 1 week. Thereafter, the number of stained type II fibrocytes declined and only a few were present at 1 month post administration (small arrows in ). Staining of type III fibrocytes (large arrows in ) was predominantly in the basal turn. These cells were the most avid cochlear cells in accumulating locally applied gentamicin. In several cases where there was very little staining of the cochlea, type III fibrocytes were the only cochlear cells that were stained. Unlike OHCs, which avidly accumulated the drug and were highly susceptible to degeneration, type III fibrocytes were clearly the most robust in the presence of high levels of accumulated gentamicin. They stained intensely soon following administration and remained so for as long as 6 months (). Despite this avid accumulation of the drug, in the most severely degenerated cases, these cells were the only remaining identifiable cell type within the cochlea.
External sulcus cells and root cells within the spiral ligament were stained predominantly in the basal and second turns, but sometimes in the third and apical turns (small arrows in ). Staining of external sulcus cells was always less intense than that of root cells, which extend into the ligament from the external sulcus cells. In contrast to systemically injected cases where root cells were seldom darkly stained, in locally treated animals, root processes were often darkly and diffusely stained (small arrows in ). Such staining occurred in regions adjacent to sites of degenerated or totally missing organ of Corti and was present in cases with as long as 3 months survival.
The spiral limbus was darkly and diffusely stained in all turns at short survival times. At 4 days post administration the extracellular diffuse staining was sufficiently eliminated to show that both fibrocytes (arrow in ) and interdental cells were stained (). Dark staining was present in interdental cells (arrowheads in ) longer than in fibrocytes. Punctate staining of both cell types was present as long as 6 months in all cochlear turns. Irregular diffuse staining also remained in the limbus for as long as 6 months.
The patterns of immunostaining in vestibular organs were similar to those seen in systemically treated animals. There was diffuse immunostaining in the subepithelial connective tissue of all of the vestibular organs at 6 h post administration (asterisks in ). This staining gradually diminished with increasing survival time. In contrast, staining of hair cells, which was clearly evident at 24 h, became increasingly distinct. Staining of hair cells was present in the cytosol and the stereocilia of some animals with 24 h (not shown) and 4 day survival times (). In animals with longer survival times, staining of hair cells was restricted to the region beneath the cuticular plate and was present for as long as 6 months post administration (). In comparison to staining intensities of hair cells, there was weak staining in vestibular ganglion cells in animals with less than 4 days survival (not illustrated).
Figure 6 Immunostaining for gentamicin in the vestibular system following local application. Numbers on the photos indicate survival periods. Each row shows the progression of staining with increasing survival time for a given tissue. A, D, G. Hair cells and fibrocytes (more ...)