On the pathomechanism of cochlear dysfunction in experimental perilymph fistulas

The mechanism leading to hearing impairment in perilymph fistulas was investigated in guinea pigs with perforated round window membranes (RWM) by analyzing alterations of inner ear fluid pressure, changes of auditory function following manipulations to get presumed air bubbles out of the cochlea (“positional audiometry”), and temporal bone sections. The instantaneous loss of normal positive inner ear fluid pressure after RWM perforation had no immediate effect on auditory function. Inner ear pressure was restored 4 days following RWM perforation. “Positional audiometry” was negative in guinea pigs with perforated RWM. All ears in which auditory thresholds had increased had additional iatrogenic lesions at the spiral lamina. Fistulas in the RWM per se do not affect auditory thresholds. The question of the surgical repair of fistulas was not directly addressed; it only can be concluded that there are no direct sequelae of an isolated fistula which induce auditory impairment and which could be prevented by surgical repair of the fistula.     

Round window membrane permeability to human serum albumin in antigen-induced otitis media

The round window membrane (RWM) permeability to human serum albumin (HSA) was investigated in both normal chinchillas and chinchillas sensitized with HSA. The effect of a corticosteroid agent (triamcinolone) on the RWM permeability was also analyzed. It was found that HSA could not be detected in either the perilymph or the cerebrospinal fluid of normal chinchillas within 1 hour after instillation into the middle ear bulla. Perilymph levels of HSA peaked 24 hours after instillation. In antigen-induced otitis media, the HSA level in the perilymph at 12 hours after challenge was significantly higher than that in normal chinchillas (P less than .01), but it did not increase with the development of otitis media. A significant difference of HSA level in the perilymph of animals with and without steroid treatment was noted (P less than .025 at 24 hours and P less than .05 at 48 hours). It is concluded that little HSA passes through the normal RWM in a short time and that the RWM permeability to HSA increases in the early stage (about 12 hours) of antigen-induced otitis media. Corticosteroids can affect the RWM permeability to HSA by reducing the level of antigen-induced otitis media.     

Changes of the permeability of round window membrane in otitis media

The effects of endotoxin, exotoxin, and otitis media on the permeability of the round window membrane (RWM) in chinchillas was investigated by detecting tetraethylammonium chloride, applied to the RWM, using a potassium-selective microelectrode in the scala tympani. The RWM, 48 hours following the application of endotoxin or exotoxin, became significantly more permeable to tetraethylammonium chloride than the normal RWM. Two weeks after the obstruction of the eustachian tube, the permeability of the RWM was decreased. These results suggest that bacterial toxins and the consequential migration of chemical inflammatory mediators act as promotive factors of RWM permeability, and that a pathologic thickness of the RWM and the presence of effusion induced by the obstruction of the eustachian tube acts as an inhibitory factor. In the clinical role of RWM permeability in human otitis media, these two factors must be taken into consideration.     

An immunocytochemical study of Matrixmetalloproteinase 1 in cholesteatoma and deep meatal skin

Aim: To investigate the hypothesis that bacterial toxins produced in the middle ear during acute otitis media (AOM), travel via the round window membrane (RWM) to cause cochlear damage and hence sensorineural hearing loss (SNHL). Methods: We investigated toxins produced by the two most common pathogens isolated from middle ear effusions in children. These were Pneumolysin, a product of S. pneumoniae and a cytoplasmic extract of Haemophilus influenza. Both electrophysiological measurements and morphological investigations were made after these toxins were placed on the RWM of guinea pigs. Control experiments were done using artificial perilymph as a substitute for the toxins. In each experiment one of the cochleas served as an internal control. Results: Losses in both the Cochlear Microphonics (CM) and Compound Action Potential (CAP) were observed indicating impaired hair cell function resulting in SNHL. Morphological studies showed hair cell destruction. Examination of the RWM exposed to pneumolysin showed disruption of this three-layered structure. Conclusions: The RWM is a probable portal for access to the inner ear by substances produced during AOM. Sensorineural hearing loss could result from the type of damage observed. More experiments are needed to verify this hypothesis and to determine the extent of damage that can be caused by these substances, especially with regard to the position of the damaged hair cells. Most of the damage observed occurred in the basal turn. Careful audiological examination (especially at high frequencies) of children who suffer with AOM might reveal a population of children with sensorineural hearing loss which might otherwise go undetected.     

Efficiency of a transtympanic approach to the round window membrane using a microendoscope

There has been increasing interest in cochlear drug delivery through the round window membrane (RWM). However, placing drugs on the RWM is difficult because of anatomical barriers. We examined the efficacy of a microendoscope for a transtympanic approach to the RWM. We evaluated the visibility of the RWM using four approaches: transtympanic microendoscopic, transtympanic microscopic, transmastoid microendoscopic, and transmastoid microscopic in ten human temporal bones. For the transtympanic approach, we made a fenestration (2 × 1 mm) in the postero-inferior quadrant of the tympanic membrane. For the transmastoid approach, conventional posterior hypotympanotomy was performed. The transtympanic microendoscopic approach enabled visualization of the RWM in all specimens, whereas the transtympanic microscopic approach only permitted visualization in three specimens. Through the transmastoid approach, the RWM was visible in all specimens using either a microendoscope or a microscope. The transtympanic microendoscopic approach can be utilized for cochlear drug delivery through the RWM.     

Effect of round window membrane application of nitric oxide on hearing and nitric oxide concentration in perilymph

Nitric oxide (NO), a free radical, has been found to be important in the development of middle ear effusions. However, the effect of NO in the middle ear effusion on cochlear function and on perilymph concentrations of NO has not been reported. We placed S-nitroso-N-acetylpenicillamine (SNAP), a NO donor compound, on the round window membrane (RWM) of adult chinchillas. Auditory brainstem response (ABR) thresholds were measured before and after the placement of SNAP on the RWM and hourly for 8 h after SNAP placement. Samples of perilymph were collected 2 h after application of SNAP and were assayed for total nitrate and nitrite, the end products of NO. Experimental ears demonstrated significant ABR threshold elevations after 5 h and elevated nitrate/nitrite in the perilymph. These findings suggest that NO present in the middle ear passes through the RWM into the inner ear and can cause significant hearing loss.     

Experimental defects of the round window membrane]

Previous and current studies of experimentally induced fistulas in the round window membrane (RWM) are reviewed. After puncture (or "micro-perforation") of the RWM leaking perilymph was not been observed and auditory evoked potentials have remained unaltered. In contrast, most publications have reported that gross incisions ("macro-perforations") of the RWM have resulted in the deterioration of auditory evoked potentials, with leaking perilymph and/or cerebrospinal fluid and/or with entrance of air bubbles into the scala tympani depending on the site of the perforation. Spontaneous healing was then observed within 4 to 8 days and was associated with normalization of the auditory evoked potentials. In a very few experimental studies pathophysiological mechanisms were simulated that may be involved clinically in patients with RWM fistulas, i.e. implosive rupture of the RWM due to an acute increase in middle ear pressure or explosive rupture of the RWM that may occur in patients trying to use forced Valsalva maneuvers to equalize decreased middle ear pressures due to blocked Eustachian tubes.     

Characterization of the Sheep Round Window Membrane

Intratympanic injection is a clinically used approach to locally deliver therapeutic molecules to the inner ear. Drug diffusion, at least in part, is presumed to occur through the round window membrane (RWM), one of the two openings to the inner ear. Previous studies in human temporal bones have identified a three-layered structure of the RWM with a thickness of 70–100 μm. This is considerably thicker than the RWM in rodents, which are mostly used to model RWM permeability and assess drug uptake. The sheep has been suggested as a large animal model for inner ear research given the similarities in structure and frequency range for hearing. Here, we report the structure of the sheep RWM. The RWM is anchored within the round window niche (average vertical diameter of 2.1 ± 0.3 mm and horizontal diameter of 2.3 ± 0.4 mm) and has a curvature that leans towards the scala tympani. The centre of the RWM is the thinnest (55–71 μm), with increasing thickness towards the edges (< 171 μm), where the RWM forms tight attachments to the surrounding bony niche. The layered RWM structure, including an outer epithelial layer, middle connective tissue and inner epithelial layer, was identified with cellular features such as wavy fibre bundles, melanocytes and blood vessels. An attached “meshwork structure” which extends over the cochlear aqueduct was seen, as in humans. The striking anatomical similarities between sheep and human RWM suggest that sheep may be evaluated as a more appropriate system to predict RWM permeability and drug delivery in humans than rodent models.     

The pharmacokinetic profiles of dexamethasone and methylprednisolone concentration in perilymph and plasma following systemic and local administration

CONCLUSION: Both methylprednisolone (MTH) and dexamethasone (DEX) could successfully and effectively penetrate the round window membrane (RWM) into perilymph. RWM topical application and otocyst infusion with MTH and DEX result in high perilymph drug concentrations and low plasma levels. An intratympanic administration schedule for DEX or MTH could be carried out twice daily. OBJECTIVE: To explore the pharmacokinetics of DEX and MTH in the inner ear fluids and plasma following systemic and local administration. MATERIALS AND METHODS: Three routes of administration of steroids were used in guinea pigs: intracardial injection, otocyst infusion and RWM topical application by granule gelfoam soaked with steroids. Samples of blood or perilymph of the scala tympani were collected at 1-6 h after administration. High-performance liquid chromatography was used to assay concentrations of steroids. RESULTS: Both the topical application and infusion administration resulted in a significantly higher concentration of steroids in perilymph than intracardial injection. The level of steroids in the perilymph reached a peak at 1-2 h after dosing, and this was maintained at a relatively high level for several hours. The intracardial injection with steroids yielded very low perilymph levels at all sampling times after administration.     

Pharmacokinetics of caroverine in the inner ear and its effects on cochlear function after systemic and local administrations in Guinea pigs

Caroverine, an N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist, has been shown to protect the inner ear from excitotoxicity and to be effective in the treatment of cochlear synaptic tinnitus. Local administration of caroverine on the round window membrane (RWM) could be a more effective means of administration to avoid systemic side/adverse effects. The present study investigates the pharmacokinetics of caroverine in the perilymph, cerebrospinal fluid (CSF) and plasma following intravenous and local applications at different dosages. High-performance liquid chromatography was used to determine the drug concentrations. Our results show much higher caroverine concentrations in the perilymph with lower concentrations in CSF and plasma following local applications compared with systemic administration. Auditory brainstem responses were measured to evaluate the changes in auditory function. The effects on hearing were transient and fully reversible 24 h after local caroverine applications. The findings suggest that local application of caroverine on the RWM for the treatment of excitotoxicity-related inner ear diseases, such as tinnitus and noise-induced hearing loss, might be both safe and more efficacious while avoiding high blood and CSF caroverine levels seen with systemic administration.     

Efficacy of gene transfer through the round window membrane: an in vitro model

The viral vector-transgene soaked gelatin-sponge method has been shown to be successful in mediating transgene expression across an intact round window membrane (RWM) in mouse in vivo. However, there are many confounding factors which make it difficult to evaluate the role of the RWM in gene transfer. We have created an in vitro model to test the feasibility of gene delivery through an intact RWM. The round window including the bony niche of a CD1 mouse was removed under an operating microscope and fixed with adhesive on the base of a petri dish through which a hole had been drilled. Toluidine blue was injected into the niche containing a hyaluronic acid ester sponge against the round window membrane. The niche was closed with a fascia. A plastic tube containing PBS was fixed on the opposite side, from where the samples were collected at different time points. The concentration of toluidine blue was evaluated spectrophotometrically. An adenoviral vector containing green fluorescent protein (GFP) marker gene was injected into the niche. Samples were collected from the opposite side at different time points. The presence of the vector was studied with GFP PCR. We also modulated the permeability of the RWM by treating it with clinically applicable detergents, histamine or silver nitrate. Silver nitrate and trichloracetic acid caused destruction of the surface epithelium of the RWM as shown by light microscopy. Both toluidine blue and adenoviral vectors passed through the RWM in a time-dependent fashion. RWM cells expressed GFP after Ad-GFP treatment. The permeability of the RWM was decreased after treatment with different detergents, histamine or silver nitrate. RWM offers an atraumatic route to the inner ear. Compared with more invasive gene delivery methods, this technique represents a safer and clinically more viable route of cochlear gene delivery.     

Perilymphatic fistula in the guinea pig

Clinical reports on perilymphatic fistulae (PLF) of the round window membrane (RWM) have shown different degrees of hearing loss. However, the hearing can also be entirely unaffected. Experimentally induced PLF of the RWM in animals showed results similar to those of the clinical reports, although some researchers have concluded that perforation of the RWM per se does not necessarily induce significant hearing loss. The purpose of the present study was to clarify if, and how, PLF of RWM in the guinea pig actually affects the auditory action potentials. During 1 h of observation following perforation of the RWM, the immediate and continuous effect of the PLF was evaluated. In more than 50% of the animals, different amounts of threshold shift were obtained. In another group of guinea pigs an attempt was made to clarify the findings by reducing the cerebrospinal fluid pressure before perforating the RWM.     

Otic preparations altered permeability and thickness of the round window membrane of the chinchilla

The effects of topical otic preparations (Cortisporin, Coly-Mycin, Aristocort, and Bestron) upon the permeability of the round window membrane (RWM) in chinchillas were investigated. Using K(+)-selective microelectrodes, the concentration of tetraethylammonium (TEA) ions was measured. Changes in the thickness of the RWM were measured using light microscopy. The RWM permeability was reduced significantly in Cortisporin- and Coly-Mycin-treated ears. Moreover, these two drugs resulted in a marked thickening of the RWM. In contrast, Aristocort or Bestron resulted in no alteration of the RWM permeability.     

Evaluation of round window accessibility to cochlear implant insertion

Emphasis on hearing preservation has led to recognition of the round window membrane (RWM) as a portal for the cochlear implant electrode array. The St Thomas’ Hospital (STH) classification was devised to evaluate the accessibility of RWM electrode insertion. The objectives of this study were: (1) to prospectively evaluate the STH classification in selecting the appropriate cochlear insertion route in “RWM-intentioned” cases, and (2) to ascertain if RWM accessibility differs from adults to children. This was a prospective cohort study of consecutive patients (adult and paediatric) undergoing cochlear implantation at a specialist auditory implant centre. Visibility of the RWM was graded according to the STH classification after an “optimal” posterior tympanotomy had been performed and any overhang of the bony round window niche removed without breaching the RWM. Most adult (89 %) and paediatric (78 %) cases had more than 50 % of the RWM exposed (Types I and IIa). Cases having less than 50 % of RWM exposed (Types IIb) or none exposed (Type III) were twice as common in children (p = 0.004). 96 % of Type I and 63 % of Type IIa cases underwent RWM insertion through a membranous cochleostomy. 71 % of Type IIb necessitated an extended membranous cochleostomy. All Type III cases required a conventional bony cochleostomy. When more than 50 % of RWM was visible (Types I and IIa), 88 % underwent a membranous cochleostomy. For “RWM-intentioned” cases, the STH classification can be a sensible method of relating RWM visibility/accessibility to the optimal route for insertion. This study also demonstrates that RWM insertion is more challenging in children.     

Effects of Intratympanic Injection of Isosorbide on the Vestibular Function of Animal Models of Endolymphatic Hydrops

ObjectivesThe aims of this study were to investigate the effects of intratympanic injections of isosorbide on vestibular function in animal models of endolymphatic hydrops and to find a new treatment option for the acute onset of vertigo in Ménière disease (MD).MethodsSeventy male guinea pigs received intratympanic injection of isosorbide (IT-ISB). The animals were divided into three study groups: control, a chronic hydrops model, and an acute hydrops model. Intracochlear drug concentrations were measured using high-performance liquid chromatography. Vestibular function was analyzed using an animal rotator test with bidirectional sinusoidal harmonic acceleration before and after IT-ISB administration. Histological changes were also investigated.ResultsISB successfully permeated the perilymph through the round window membrane (RWM) at all three concentrations (25%, 50%, and 100%). In the chronic hydrops model, while IT-ISB histologically induced a reduction of endolymphatic hydrops, vestibular function was unchanged. In the acute hydrops model, no endolymphatic hydrops was histologically observed, and vestibular symmetry was also preserved after IT-ISB.ConclusionISB passed through the RWM into the perilymphatic space even at lower concentrations. IT-ISB histologically reduced hydrops in the chronic model and preserved symmetrical vestibular function in the acute model. IT-ISB could be a treatment candidate for acute attacks of vertigo in MD.     

Cochlear microdialysis for quantification of dexamethasone and fluorescein entry into scala tympani during round window administration

Before new drugs for the treatment of inner ear disorders can be studied in controlled clinical trials, it is important that their pharmacokinetics be established in inner ear fluids. Microdialysis allows drug levels to be measured in perilymph without the volume disturbances and potential cerebrospinal fluid contamination associated with fluid sampling. The aims of this study were to show: (i) that despite low recovery rates from miniature dialysis probes, significant amounts of drug are removed from small fluid compartments, (ii) that dialysis sampling artifacts can be accounted for using computer simulations and (iii) that microdialysis allows quantification of the entry rates through the round window membrane (RWM) into scala tympani (ST). Initial experiments used microdialysis probes in small compartments in vitro containing sodium fluorescein. Stable concentrations were observed in large compartments (1000 microl) but significant concentration declines were observed in smaller compartments (100, 10 and 5.6 microl) comparable to the size of the inner ear. Computer simulations of these experiments closely approximated the experimental data. In in vivo experiments, sodium fluorescein 10 mg/ml and dexamethasone-dihydrogen-phosphate disodium salt 8 mg/ml were simultaneously applied to the RWM of guinea pigs. Perilymph concentration in the basal turn of ST was monitored using microdialysis. The fluorescein concentration reached after 200 min application (585+/-527 microg/ml) was approximately twice that of dexamethasone phosphate (291+/-369 microg/ml). Substantial variation in concentrations was found between animals by approximately a factor of 34 for fluorescein and at least 41 for dexamethasone phosphate. This is, to a large extent, thought to be the result of the RWM permeability varying in different animals. It was not caused by substance analysis variations, because two different analytic methods were used and the concentration ratio between the two substances remained nearly constant across the experiments and because differences were apparent for the repeated samples obtained in each animal. Interpretation of the results using computer simulations allowed RWM permeability to be quantified. It also demonstrated, however, that cochlear clearance values could not be reliably obtained with microdialysis because of the significant contribution of dialysis to clearance. The observed interanimal variation, e.g., in RWM permeability, is likely to be clinically relevant to the local application of drugs in patients.     

Estimation of perilymph concentration of agents applied to the round window membrane by microdialysis

OBJECTIVE: The round window membrane (RWM) is known to be permeable to various biological substances. Application of biological substances to the RWM has been shown to affect inner ear fluid composition and damage hair cells, resulting in functional changes RWM instillation of gentamicin, a preferentially vestibulotoxic aminoglycoside, is used as a therapeutic treatment for patients with intractable vertigo and is gaining acceptance as a chemical vestibular ablation agent, despite considerable variations in the incidence and severity of hearing loss associated with gentamicin. Clearly, the susceptibility of vestibular and auditory hair cells to the ototoxic effects of gentamicin is not well understood. The aim of this study was to understand the kinetics of urea and methylene blue instilled into the inner ear space through the RWM and to establish a method for determining the optimal dosage for the treatment of inner ear disorders. MATERIAL AND METHODS: We used inner ear microdialysis to quantify changes in perilymph concentration of low molecular weight agents applied to the RWM in a chinchilla model. RESULTS: Preliminary results after placement of a microdialysis probe and application of a low molecular weight marker (urea) to the RWM were extrapolated from a time versus concentration plot from dialysates sampled over a 3-min interval using modifications of standard microdialysis equations for estimation of in vivo recovery. Our data suggest that inner ear microdialysis can be used to measure the pharmacokinetics of a low molecular weight agent within the perilymphatic space without the need for repeated direct sampling. CONCLUSION: Inner ear microdialysis may be a useful method for establishing a therapeutic dosage for ototoxic agents used in the treatment of inner ear disorders.     

The pharmacokinetic profiles of dexamethasone and methylprednisolone concentration in perilymph and plasma following systemic and local administration

Conclusion. Both methylprednisolone (MTH) and dexamethasone (DEX) could successfully and effectively penetrate the round window membrane (RWM) into perilymph. RWM topical application and otocyst infusion with MTH and DEX result in high perilymph drug concentrations and low plasma levels. An intratympanic administration schedule for DEX or MTH could be carried out twice daily. Objective. To explore the pharmacokinetics of DEX and MTH in the inner ear fluids and plasma following systemic and local administration. Materials and methods. Three routes of administration of steroids were used in guinea pigs: intracardial injection, otocyst infusion and RWM topical application by granule gelfoam soaked with steroids. Samples of blood or perilymph of the scala tympani were collected at 1–6 h after administration. High-performance liquid chromatography was used to assay concentrations of steroids. Results. Both the topical application and infusion administration resulted in a significantly higher concentration of steroids in perilymph than intracardial injection. The level of steroids in the perilymph reached a peak at 1–2 h after dosing, and this was maintained at a relatively high level for several hours. The intracardial injection with steroids yielded very low perilymph levels at all sampling times after administration.     

Measurement of Basilar Membrane Motion During Round Window Stimulation in Guinea Pigs

Driving the cochlea in reverse via the round window membrane (RWM) is an alternative treatment option for the hearing rehabilitation of a nonfunctional or malformed middle ear. However, cochlear stimulation from the RWM side is not a normal sound transmission pathway. The basilar membrane (BM) motion elicited by mechanical stimulation of the RWM is unknown. In this study, the BM movement at the basal turn was investigated in both reverse via RWM drive and acoustic stimulation in the ear canal or forward drive in postmortem isolated temporal bone preparations of guinea pigs. During reverse drive, a magnet-coil was coupled on RWM, and the BM vibration at the basal turn and the movement of the incus tip were measured with laser Doppler vibrometry. During forward drive, the vibration of the incus tip induced by sound pressure in the ear canal resulted in BM vibration and the BM movement at the same location as that in the reverse stimulation was measured. The displacement ratio of the BM to RWM in reverse drive and the ratio of the BM to incus in forward drive were compared. The results demonstrated that the BM response measured in both situations was similar in nature between forward and reverse drives. This study provides new knowledge for an understanding of BM movement induced by reverse drive via the RWM stimulation.     

Hyaluronan applied to lesioned round window membrane is free from cochlear ototoxicity

Hyaluronan (HYA) in 1% solution was instilled into the round window (RW) niche of rats (n = 6) prior to perforating the round window membrane (RWM). Cochlear functioning and structure were then monitored by recording auditory brainstem responses (ABRs) at 2-31.5 kHz and by scanning electron microscopy. Perforation of the RWM alone (n = 6) resulted in immediate loss of ABR thresholds between 6 and 31.5 kHz in 2 of 6 animals. Similar results were obtained after instilling HYA into the RW niche and subsequent RWM perforation (n = 6). After 2 months, ABR thresholds were recorded at all frequencies in the HYA-treated animals, whereas in 2 of the controls no ABR thresholds could be elicited at 20 and 31.5 kHz. However, in both treatment groups the mean ABR thresholds and mean latencies for wave II at the ABR threshold returned to the pre-surgical (normal) range after 2 months. With respect to the cochlear morphology the results in both treatment groups were also alike including minor structural changes in hair cell stereociliae but no loss of hair cells. It is concluded that HYA, when instilled into the middle ear with the inner ear opened, is free from cochlear otoxicity.