Pathological findings in the human auditory system following long-standing gentamicin ototoxicity

The clinical, audiovestibular and histopathological findings in a patient who suffered from a long-standing gentamicin-induced deafness are reported. In both temporal bones, the organ of Corti was completely absent, with only a few nerve fibres remaining in the apical part of the cochlea. Regenerative ingrowth of nerve fibers into the area of the degenerative organ of Corti was present apically in both ears. The stria vascularis exhibited considerable degeneration in all turns and loss of microvasculature was found in the basilar membrane. The spiral ganglion cells, the cochlear nerve and the central auditory pathways and nuclei appeared to be unaffected.     

Hearing loss and inner ear changes in a patient suffering from severe gentamicin ototoxicity

Summary The long-term histological effects of gentamicin ototoxicity could be studied in a human being in relation to the audiometric impairment. The possible sequence of degeneration of hair cells, supporting cells, nerve fibers, stria vascularis, spiral ganglion cells, and vascular supply is discussed.     

Experimental investigations on the influence of anemia on gentamicin ototoxicity

Summary The purpose of this study was to determine whether or not anemic patients have a higher risk of gentamicin ototoxicity. Using the laboratory rat as an animal model with auditory brain-stem response measurements and surface preparations of the organ of Corti, it could be shown that cochlear damage in anemic rats treated with gentamicin was significantly higher (P< 0.05) than in the nonanemic ones treated with the same dosages of antibiotic. Doses of gentamicin used were 50 mg/kg for 30 days and 40 mg/kg for another 75 days. Offprint requests to: P. Federspil     

Round window gentamicin absorption: an in vivo human model

OBJECTIVE/HYPOTHESIS: Using a novel human labyrinthine sampling model, in vivo gentamicin absorption through the round window can be measured. STUDY DESIGN: A prospective study. METHODS: Gentamicin was delivered either transtympanically (preoperative) or through a facial recess approach (intraoperative). The lateral semicircular canal and vestibule were opened, and by means of a microsyringe, labyrinthine fluid was aspirated. A sample of serum was also drawn. In all patients cerebrospinal fluid was also drawn. The samples were analyzed using a standard chemistry analyzer. RESULTS: Intratympanic gentamicin diffused through the round window membrane and achieved concentrations in the labyrinthine fluid ranging from 0 to 16 mg/L. Intratympanic gentamicin was absorbed into the systemic circulation in 4 of 11 patients with serum levels ranging from 0.3 to 0.4 mg/L. No gentamicin was detected in the cerebrospinal fluid. CONCLUSIONS: Intratympanic gentamicin diffuses rapidly through the round window membrane and achieves significant levels in the inner ear. Thus, this new model can be used to assess round window permeability to clinically relevant medications such as steroids and ototopical antibiotics.     

Spontaneous hair cell regeneration in the mouse utricle following gentamicin ototoxicity

Whereas most epithelial tissues turn-over and regenerate after a traumatic lesion, this restorative ability is diminished in the sensory epithelia of the inner ear; it is absent in the cochlea and exists only in a limited capacity in the vestibular epithelium. The extent of regeneration in vestibular hair cells has been characterized for several mammalian species including guinea pig, rat, and chinchilla, but not yet in mouse. As the fundamental model species for investigating hereditary disease, the mouse can be studied using a wide variety of genetic and molecular tools. To design a mouse model for vestibular hair cell regeneration research, an aminoglycoside-induced method of complete hair cell elimination was developed in our lab and applied to the murine utricle. Loss of utricular hair cells was observed using scanning electron microscopy, and corroborated by a loss of fluorescent signal in utricles from transgenic mice with GFP-positive hair cells. Regenerative capability was characterized at several time points up to six months following insult. Using scanning electron microscopy, we observed that as early as two weeks after insult, a few immature hair cells, demonstrating the characteristic immature morphology indicative of regeneration, could be seen in the utricle. As time progressed, larger numbers of immature hair cells could be seen along with some mature cells resembling surface morphology of type II hair cells. By six months post-lesion, numerous regenerated hair cells were present in the utricle, however, neither their number nor their appearance was normal. A BrdU assay suggested that at least some of the regeneration of mouse vestibular hair cells involved mitosis. Our results demonstrate that the vestibular sensory epithelium in mice can spontaneously regenerate, elucidate the time course of this process, and identify involvement of mitosis in some cases. These data establish a road map of the murine vestibular regenerative process, which can be used for elucidating the molecular events that govern this process.     

The fine structure of lipofuscin in the human inner ear

Summary Lipofuscin inclusions in the human membranous labyrinth were studied by electron microscopy. Lipofuscin is morphologically an irregularly shaped, membrane-bound inclusion consisting of an electron-dense structure. The most common component was a fine, granular, osmiophilic substance which was always associated with a homogenous, spherical structure resembling a lipid droplet. The combination of these two components was frequently observed in the human inner ear. Distended inclusions containing lipofuscin components were also observed within the supporting cells, saccular, utricular and ampullar wall, the epithelial cells of the transitional zone and in the dark cells. Lipofuscin is closely associated with lysosome and is known to accumulate in the tissue as a result of aging. The high lysosomal activity possibly may result in lipofuscin formation in the human inner ears. Also some other unknown metablic conditions may provide the deposits of lipofuscin.I wish to express my thanks to Professor Nobuo Ueda of the Biochemistry Department for his advice and to Mr. Dominic W. Hughes for English editing of this paper.     

Distribution of Gentamicin in the Guinea Pig Inner Ear after Local or Systemic Application

Uptake and retention of gentamicin by cells in the guinea pig inner ear after a single peritoneal injection or local application on the round window were investigated using immunocytochemistry to localize the drug. The cells that accumulated the drug under the two conditions were the same, but staining for the drug was more intense and was often accompanied by widespread cochlear degeneration following local application. Soon after drug administration by either route, there was diffuse staining for the drug throughout all tissue within the labyrinth, including bone. At later times when distinct cell staining became evident, virtually all cell types were found to be positive, with several cell types staining more darkly for the drug than hair cells, indicating that hair cells were not the most avid in accumulating gentamicin. The infracuticular portion of auditory and vestibular hair cells as well as type III fibrocytes of the spiral ligament were positively stained in almost all cases and these sites were found to be positive for as long as six months post administration. In animals with loss of the organ of Corti, there was unusually intense staining for gentamicin in root cells of the spiral ligament, in marginal cells of the stria vascularis, and in cells of the spiral limbus. Dark staining of surviving cells in cases with overt tissue destruction suggests that variability in the extent of damage caused by the drug was determined more by the degree of its local uptake than by differences in animals' capacities to metabolize the drug systemically. The present results show that gentamicin may damage or destroy all cochlear cells following a single round window application. The findings broaden the scope of our knowledge of cochlear gentamicin uptake and damage and have implications for treatment of patients with vestibular disorders by infusion of aminoglycosides into the middle ear, as well as implications for prospects of rehabilitating patients that have been deafened by aminoglycosides.     

Distribution of gentamicin by immunofluorescence in the guinea pig inner ear

Summary We studied the distribution of gentamicin in the inner ear, brain and kidney of the guinea pig following intraperitoneal administration or perfusion of gentamicin through the perilymphatic space. The resulting histopathologcial changes were examined by immunofluorescence using antigentamicin antiserum. After perfusion of gentamicin through the perilymphatic space, specific fluorescence was found in the cochlea, and was especially prominent in the outer hair cells, basilar membrane and basilar crest. Although no fluorescence was observed in the cochlea following intraperitoneal administration of high doses of gentamicin, type I hair cells in the vestibule were seen to be selectively stained with the antibody. Furthermore, some of the vestibular ganglion cells, Purkinje cells and unidentified nuclei in the brain stem were also stained. In particular, fine granules showing relatively intense fluorescence were recognized in the cytoplasm of the stained cells. In the cortex of kidney, only proximal tubular cells were stained with intense fluorescence. Our results suggest that the aminoglycoside antibiotics have two sites of action: one is the cell membrane of the sensory hair cells and the other is the cytoplasm.This study was supported in part by a grant from the Ministry of Education, Science and Culture of Japan, and by a Research Grant for Specific Diseases from the Ministry of Health and Welfare to the Acute Profound Deafness Research Committee of Japan     

Triamcinolone acetonide protects auditory hair cells from 4-hydroxy-2,3-nonenal (HNE) ototoxicity in vitro

Abstract

Conclusion. Triamcinolone acetonide crystalline suspension (e.g. Volon A®) was not ototoxic to the auditory hair cells present within organ of Corti explants and protected them from an ototoxic molecule, i.e. 4-hydroxy-2,3-nonenal (HNE), that is produced within the organ of Corti as a result of oxidative stress-induced damage. Objectives. To test the corticosteroid, triamcinolone acetonide, for ototoxicity and otoprotective capacity in organ of Corti explants. Materials and methods. Organ of Corti explants excised from 4-day-old rats were the test system, HNE was the ototoxin challenge. Hair cell integrity counts were performed with fluorescent microscopy on fixed explants stained with FITC-labeled phalloidin. Statistical significance was set atp <0.05. Results. Triamcinolone acetonide did not affect hair cell integrity in the organ of Corti explants and it provided a high level of protection of hair cells against the ototoxic effects of a damaging level of HNE as determined by hair cell density counts.     

Structural abnormalities in the stria vascularis following chronic gentamicin treatment

Freeze-fracture and thin-sectioning have been used to examine the stria vascularis of albino guinea pigs chronically treated with gentamicin. Immediately following the end of treatment, most marginal cells showed lipid bodies in the cell body region and freeze-fracture revealed alterations to the marginal cell plasma membrane. Intermediate cells also showed peculiarities including a dilation of the rough endoplasmic reticulum. A co-incidence was noted in the location in the cochlea in which effects in the stria and in outer hair cells occurred. At 4 weeks post-treatment, the stria was significantly thinner than normal and appeared less structurally complex. A minority of marginal cells degenerated. Some morphological features associated with degeneration resembled those of apoptosis, a process of controlled, cellular self-destruction. There were also indications of turnover of gap-junctions throughout the post-treatment period examined. The results indicate significant ototoxic effects of gentamicin occur in the stria and that changes to plasma membranes are one of the initial alterations.     

A new immunohistochemical method for the detection of gentamicin in inner ear fluid compartments

A new method was developed for frozen section detection of antigens that natively occur in the cochlear peri- and endolymph. A combination of immunohistochemistry and immunoblot assay enabled topological and quantitative detection of small and hydrophilic molecules (such as the aminoglycoside antibiotics) in frozen sections of the inner ear compartments (scala tympani, scala vestibuli and cochlear duct). A selective localization is possible in the peri- and endolymphatic region of each coil of the cochlea. During sectioning of the cochlea, a small piece of a nitrocellulose membrane is placed to the surface of the intersection and briefly warmed. The sections are cut, simultaneously attached to a nitrocellulose membrane on which the aminoglycoside antibiotics remain adsorbed without any fixation procedure. Using this method, immunoincubation to detect gentamicin was performed in a way usually done in western blot analysis. Results with two different enzyme reactions with the enzyme conjugated to a second antibody (i.e., dye as substrate and the chemiluminescence detection system) are presented and compared. This histoimmunoblot assay provides a general non-radioactive and sensitive immunohistochemical tool for the localization of compounds occurring in extracellular body fluid compartments. For inner ear research this method now enables the investigation of the penetration and distribution of therapeutics in peri-and endolymphatic sites and can even be applied to separately quantifying concentrations of a substance in different coils of the same cochlear section.     

Damage and functional recovery of the Atlantic cod (Gadus morhua) inner ear hair cells following local injection of gentamicin

This study addresses the ultrastructural and functional damage and subsequent recovery of the inner ear in the Atlantic cod following intrasaccular gentamicin injection. Inner ear damage was assessed using SEM and measurements of AEP following 250-Hz pure-tone stimuli. Data from gentamicin-treated fish were compared with control (no injection) and sham (injection of saline) fish. Control fish had normal response thresholds associated with well-developed hair cell bundles in their macula sacculi. Sham fish had higher response thresholds compared with control fish during the first week post-intervention, but response thresholds were subsequently normal. Treated fish displayed significant inner ear damage associated with an increased average AEP threshold on the third day following treatment. Thereafter, inner ear tissue displayed signs of progressive regeneration until it was comparable to controls from the 14th day. Response thresholds were similar to those of control fish from the 17th day following treatment. These observations suggest that the macula sacculi of Atlantic cod can regenerate towards a near-complete functional and ultrastructural recovery within 17–21 days following ototoxic gentamicin treatment.     

Epigenetic Influences on Sensory Regeneration: Histone Deacetylases Regulate Supporting Cell Proliferation in the Avian Utricle

The sensory hair cells of the cochlea and vestibular organs are essential for normal hearing and balance function. The mammalian ear possesses a very limited ability to regenerate hair cells and their loss can lead to permanent sensory impairment. In contrast, hair cells in the avian ear are quickly regenerated after acoustic trauma or ototoxic injury. The very different regenerative abilities of the avian vs. mammalian ear can be attributed to differences in injury-evoked expression of genes that either promote or inhibit the production of new hair cells. Gene expression is regulated both by the binding of cis-regulatory molecules to promoter regions as well as through structural modifications of chromatin (e.g., methylation and acetylation). This study examined effects of histone deacetylases (HDACs), whose main function is to modify histone acetylation, on the regulation of regenerative proliferation in the chick utricle. Cultures of regenerating utricles and dissociated cells from the utricular sensory epithelia were treated with the HDAC inhibitors valproic acid, trichostatin A, sodium butyrate, and MS-275. All of these molecules prevent the enzymatic removal of acetyl groups from histones, thus maintaining nuclear chromatin in a “relaxed” (open) configuration. Treatment with all inhibitors resulted in comparable decreases in supporting cell proliferation. We also observed that treatment with the HDAC1-, 2-, and 3-specific inhibitor MS-275 was sufficient to reduce proliferation and that two class I HDACs—HDAC1 and HDAC2—were expressed in the sensory epithelium of the utricle. These results suggest that inhibition of specific type I HDACs is sufficient to prevent cell cycle entry in supporting cells. Notably, treatment with HDAC inhibitors did not affect the differentiation of replacement hair cells. We conclude that histone deacetylation is a positive regulator of regenerative proliferation but is not critical for avian hair cell differentiation.     

Ultrastructural correlates of selective outer hair cell destruction following kanamycin intoxication in the chinchilla

Kanamycin ototoxicity, combined with behavioral audiometry to evaluate threshold shifts, was used to destroy outer hair cells (OHCs) in the basal cochlea of the chincilla while leaving the inner hair cell (IHC) population largely intact. After survival times of four weeks to one year, transmission electron microscopy was employed to determine the condition of surviving hair cells and neural elements. Throughout the region of OHC loss, IHCs and their innervation were normal in appearance if their adjacent supporting cells were undamaged. When IHC supporting cells, specifically the inner pillar cells, were damaged or absent, damage to IHCs was commonly obsereved. Such supporting cell-related damage included extrusion of the cuticular plate from the surface of the reticular lamina, encapsulation and/or fusion of stereocilla, and gross distortion of hair cell shape. When the outer supporting cells of the organ of Corti were undamaged following OHC loss, outer spiral fibers were found to have survived in near-normal numbers in the region from 0.5–1.0 mm basalmost surviving OHC, but suffered progressive attrition toward the basal end of the cochlea. It is concluded that kanamycin-induced OHC loss can occur without concommitant IHC damage or outer spinal fiber loss.     

Investigation of the ototoxicity of gadoteridol (ProHance) and gadodiamide (Omniscan) in mice

Conclusion: In the mouse, when a tympanic perforation is present, gadoteridol does not seem to cause ototoxicity. Gadodiamide may cause mild ototoxicity other than toxicity to the outer hair cells of the cochlea.

Objectives: Endolymphatic hydrops have been visualized through intra-tympanic injection of gadolinium-based contrast agents (GBCAs) and three-dimensional fluid-attenuated inversion recovery (3-D FLAIR) magnetic resonance imaging. However, reports on the safety of GBCAs are limited. This study aimed to assess ototoxicity of gadoteridol and gadodiamide.

Method: In a prospective, randomized, controlled trial, myringotomies in the left ear were performed in 20 male C57 BL/6 mice. After testing the baseline auditory brainstem response (ABR) (range?=?8–32?kHz), the test solution (gadoteridol, gadodiamide, saline, or cisplatin) was injected into the left ear. ABR testing was repeated 14 days after test solution application. In morphological experiments, images of post-mortem surface preparations were assessed for cochlear hair cell status.

Results: At 14 days following gadoteridol application, there was no significant change in ABR thresholds at 8, 16, or 32?kHz. Gadodiamide application caused a significant change in the ABR threshold at 8?kHz. Apparent cochlear hair cell loss was not observed in the surface preparation after gadoteridol or gadodiamide application.     

Aquaporin-2 in the human endolymphatic sac

Objective To detect and localize aquaporin-2 (AQP-2), a water channel regulated by the antidiuretic hormone, in human endolymphatic sac.

Material and Methods Human endolymphatic sacs were sampled during removal of vestibular schwannomas via a translabyrinthine approach. Samples were immediately fixed in 10% formalin (24 h) and embedded in paraffin; in situ hybridization and immunohistochemistry were performed with an AQP-2-specific probe and a polyclonal antibody.

Results Both AQP-2 mRNA and protein were detected in the epithelium of the endolymphatic sac. AQP-2 immunostaining was mainly cytoplasmic, suggesting that most AQP-2 was located in intracellular pools.

Conclusion In the endolymphatic sac, AQP-2 probably participates in the homeostasis of endolymph; the possibility of reducing the volume of endolymph by inhibiting its expression and membranous insertion using an antidiuretic hormone inhibitor represents a new therapeutic approach for the treatment of Ménière's disease.     

The presence of laminin in the fetal human inner ear

Summary The expression of laminin was analyzed in the human fetal inner ear using immunohistochemical methods. In the 11-week-old human fetus, the presence of laminin was found in the basement membrane of the immature cochlea, endolymphatic sac and vestibular end organs. The reaction of the basement membrane of the endolymphatic sac was strong in the 15-week-old human fetus. A laminin reaction was seen in the cochlea, Reissner's membrane, epithelial cells of the limbus spiralis, the basilar membrane and the stria vascularis. In particular, the capillaries and basement membrane of the stria vascularis were strongly positive. These results suggest that laminin may be an essential component in the development of the inner ear and may possibly be related to filtration of the endolymph.     

Expression of epidermal growth factor, epidermal growth factor receptor and transforming growth factor-alpha in the human fetal inner ear

The expression of epidermal growth factor (EGF), EGF receptor and transforming growth factor (TGF)-alpha was analyzed in the human fetal inner ear using immuno-histochemical methods. EGF receptor was observed only in 9.5-week-old fetal vestibular epithelia. In 14- and 16-week-old fetuses, EGF receptor could not be detected. TGF-alpha was observed strongly in the 9- and 11-week-old vestibular epithelia, whereas only trace amounts were detectable in the 14- and 16-week-old vestibular epithelia. These findings suggest that EGF and TGF-alpha probably have a mitogenic effect in the sensory epithelia of the fetal inner ear, especially at early stages of development.