Andreas Eckhard

Water channel proteins in the inner ear and their link to hearing impairment and deafness.

The inner ear is a fluid-filled sensory organ that transforms mechanical stimuli into the senses of hearing and balance. These neurosensory functions depend on the strict regulation of the volume of the two major extracellular fluid domains of the inner ear, the perilymph and the endolymph. Water channel proteins, or aquaporins (AQPs), are molecular candidates for the precise regulation of perilymph and endolymph volume. Eight AQP subtypes have been identified in the membranous labyrinth of the inner ear. Similar AQP subtypes are also expressed in the kidney, where they function in whole-body water regulation. In the inner ear, AQP subtypes are ubiquitously expressed in distinct cell types, suggesting that AQPs have an important physiological role in the volume regulation of perilymph and endolymph. Furthermore, disturbed AQP function may have pathophysiological relevance and may turn AQPs into therapeutic targets for the treatment of inner ear diseases. In this review, we present the currently available knowledge regarding the expression and function of AQPs in the inner ear. We give special consideration to AQP subtypes AQP2, AQP4 and AQP5, which have been studied most extensively. The potential functions of AQP2 and AQP5 in the resorption and secretion of endolymph and of AQP4 in the equilibration of cell volume are described. The pathophysiological implications of these AQP subtypes for inner ear diseases, that appear to involve impaired fluid regulation, such as Menières disease and Sjögrens syndrome, are discussed.

The subcellular distribution of aquaporin 5 in the cochlea reveals a water shunt at the perilymph-endolymph barrier.

Aquaporins are membrane water channel proteins that have also been identified in the cochlea. Auditory function critically depends on the homeostasis of the cochlear fluids perilymph and endolymph. In particular, the ion and water regulation of the endolymph is essential for sensory transduction. Within the cochlear duct the lateral wall epithelium has been proposed to secrete endolymph by an aquaporin-mediated flow of water across its epithelial tight junction barrier. This study identifies interspecies differences in the cellular distribution of aquaporin 5 (AQP5) in the cochlear lateral wall of mice, rats, gerbils and guinea pigs. In addition the cellular expression pattern of AQP5 is described in the human cochlea. Developmental changes in rats demonstrate longitudinal and radial gradients along the cochlear duct. During early postnatal development a pancochlear expression is detected. However a regression to the apical quadrant and limitation to outer sulcus cells (OSCs) is observed in the adult. This developmental loss of AQP5 expression in the basal cochlear segments coincides with a morphological loss of contact between OSCs and the endolymph. At the subcellular level, AQP5 exhibits polarized expression in the apical plasma membrane of the OSCs. Complementary, the basolateral membrane in the root processes of the OSCs exhibits AQP4 expression. This differential localization of AQP5 and AQP4 in the apical and basolateral membranes of the same epithelial cell type suggests a direct aquaporin-mediated transcellular water shunt between the perilymph and endolymph in the OSCs of the cochlear lateral wall. In the human cochlea these findings may have pathophysiological implications attributed to a dysfunctional water regulation by AQP5 such as endolymphatic hydrops (i.e. in Menieres disease) or sensorineural hearing loss (i.e. in Sjögrens syndrome).

All functional aquaporin-4 isoforms are expressed in the rat cochlea and contribute to the formation of orthogonal arrays of particles

The water channel aquaporin-4 (AQP4) is expressed in the cochlea and is essential for normal hearing. Unlike other AQPs, multiple isoforms of AQP4 have been reported in diverse tissues, three of which, M1, M23, and Mz, function as water channels. In addition, these protein isoforms are found in higher order complexes. Morphologically these higher order complexes correspond to orthogonal arrays of particles (OAPs) that are found in cell membranes by freeze fracture analysis. Using RT-PCR, quantitative PCR and blue-native PAGE immunoblots we identified all functional AQP4 isoforms -M1, M23, and Mz- and the formation of higher-order complexes in the organ of Corti of the rat. Complementary freeze-fracture studies revealed OAPs distributed in the lateral and basal membrane domains of the cochlear duct supporting cells, specifically Hensens cells and outer sulcus cells. The unique inter- and intracellular heterogeneity in size, density and shape of OAPs suggests exceptional physiological requirements for the maintenance of water homeostasis during auditory sensory transduction in the cochlea.

Endoscopic Transcanal Retrocochlear Approach to the Internal Auditory Canal with Cochlear Preservation Pilot Cadaveric Study

Contemporary operative approaches to the internal auditory canal (IAC) require the creation of large surgical portals for visualization with associated morbidity, including hearing loss, vestibular dysfunction, facial nerve injury, and skull base defects that increase the risk of cerebrospinal fluid leak. Transcanal approaches to the IAC have been possible only via a transcochlear technique. To preserve cochlear function, we describe a novel endoscopic transcanal infracochlear approach to the IAC in cadaveric temporal bones. Navigation fiducials were secured on fresh cadaveric heads, and real-time computed tomography imaging was used for surgical guidance. With a combination of curved instruments and rigid angled endoscopy, a transcanal hypotympanotomy and subcochlear tunnel were created with superior extension to access the IAC. Postprocedure imaging and temporal bone dissection confirmed access to the IAC without injury to the cochlea or neighboring neurovascular structures.

Regulation of the perilymphatic–endolymphatic water shunt in the cochlea by membrane translocation of aquaporin-5

Volume homeostasis of the cochlear endolymph depends on radial and longitudinal endolymph movements (LEMs). LEMs measured in vivo have been exclusively recognized under physiologically challenging conditions, such as experimentally induced alterations of perilymph osmolarity or endolymph volume. The regulatory mechanisms that adjust LEMs to the physiological requirements of endolymph volume homeostasis remain unknown. Here, we describe the formation of an aquaporin (AQP)-based “water shunt” during the postnatal development of the mouse cochlea and its regulation by different triggers. The final complementary expression pattern of AQP5 (apical membrane) and AQP4 (basolateral membrane) in outer sulcus cells (OSCs) of the cochlear apex is acquired at the onset of hearing function (postnatal day (p)8–p12). In vitro, hyperosmolar perfusion of the perilymphatic fluid spaces or the administration of the muscarinic agonist pilocarpine in cochlear explants (p14) induced the translocation of AQP5 channel proteins into the apical membranes of OSCs. AQP5 membrane translocation was blocked by the muscarinic antagonist atropine. The muscarinic M3 acetylcholine (ACh) receptor (M3R) was identified in murine OSCs via mRNA expression, immunolabeling, and in vitro binding studies using an M3R-specific fluorescent ligand. Finally, the water shunt elements AQP4, AQP5, and M3R were also demonstrated in OSCs of the human cochlea. The regulation of the AQP4/AQP5 water shunt in OSCs of the cochlear apex provides a molecular basis for regulated endolymphatic volume homeostasis. Moreover, its dysregulation or disruption may have pathophysiologic implications for clinical conditions related to endolymphatic hydrops, such as Ménière’s disease.

Methyl methacrylate embedding to study the morphology and immunohistochemistry of adult guinea pig and mouse cochleae.

BACKGROUND Histological analysis of the cochlea is required to understand the physiological and pathological processes in the inner ear. In the past, many embedding techniques have been tested in the cochlea to find an optimal protocol that gives both good morphological and immunohistochemical results. Resins provide high quality cochlear morphology with reduced immunogenicity due to the higher polymerization temperature. NEW METHOD We used Technovit 9100 New(®), a low temperature embedding system based on methyl methacrylate, on adult guinea pig and mouse cochleae to evaluate preservation of the morphology and maintenance of the antigenicity. RESULTS Conventional toluidine blue staining, as well as immunohistochemical staining with a set of commonly used antibodies, showed highly preserved morphology and immunogenicity of decalcified adult guinea pig and mouse cochleae. COMPARISON WITH EXISTING METHOD(S) We demonstrate both, well-preserved morphology and preservation of antigenicity, superior to other embedding techniques. CONCLUSIONS Our results showed that the Technovit 9100 New(®) embedding system provided highly preserved morphology and immunogenicity with our protocol in adult guinea pig and mouse cochleae.

Neuronal erythropoietin overexpression is protective against kanamycin-induced hearing loss in mice

Aminoglycosides have detrimental effects on the hair cells of the inner ear, yet these agents indisputably are one of the cornerstones in antibiotic therapy. Hence, there is a demand for strategies to prevent aminoglycoside-induced ototoxicity, which are not available today. In vitro data suggests that the pleiotropic growth factor erythropoietin (EPO) is neuroprotective against aminoglycoside-induced hair cell loss. Here, we use a mouse model with EPO-overexpression in neuronal tissue to evaluate whether EPO could also in vivo protect from aminoglycoside-induced hearing loss. Auditory brainstem response (ABR) thresholds were measured in 12-weeks-old mice before and after treatment with kanamycin for 15 days, which resulted in both C57BL/6 and EPO-transgenic animals in a high-frequency hearing loss. However, ABR threshold shifts in EPO-transgenic mice were significantly lower than in C57BL/6 mice (mean difference in ABR threshold shift 13.6 dB at 32 kHz, 95% CI 3.8-23.4 dB, p = 0.003). Correspondingly, quantification of hair cells and spiral ganglion neurons by immunofluorescence revealed that EPO-transgenic mice had a significantly lower hair cell and spiral ganglion neuron loss than C57BL/6 mice. In conclusion, neuronal overexpression of EPO is protective against aminoglycoside-induce hearing loss, which is in accordance with its known neuroprotective effects in other organs, such as the eye or the brain.

[Water regulation in the cochlea : Do molecular water channels facilitate potassium-dependent sound transduction?].

ZusammenfassungHintergrundDie Schalltransduktion in der Cochlea ist von der Zirkulation von Kaliumionen (K+) zwischen der Endolymphe und der Perilymphe entlang so genannter „K+-Recyclingrouten“ abhängig. Diese K+-Flüsse erzeugen hohe ionale und osmotische Gradienten, welche die Erregbarkeit der Haarsinneszellen sowie die zelluläre Integrität des gesamten cochleären Epithels gefährden. Molekulare Wasserkanäle – Aquaporine (AQP) – werden in allen cochleären Stützzellen entlang der K+-Recyclingrouten exprimiert; ihre Bedeutung für die osmotische Äquilibration der Zellen des Ductus cochlearis ist nicht bekannt.MethodenDiffusive und osmotische Wasserpermeabilitäten der Reissner-Membran, des Corti-Organs sowie des gesamten Epithels des Ductus cochlearis wurden bestimmt. Die Expression des Kaliumkanals Kir4.1 und des Wasserkanals AQP4 im Ductus cochlearis wurde immunhistochemisch untersucht.ErgebnisseDie ermittelten Wasserpermeabilitätswerte weisen auf durch AQP beschleunigte Wasserflüsse im Epithel des Ductus cochlearis hin. Immunhistochemisch zeigt sich eine Kolokalisation von Kir4.1 und AQP4 in distinkten Membrandomänen der Stützzellen entlang der K+-Recyclingrouten.SchlussfolgerungDiese Ergebnisse legen einen durch AQP mediierten schnellen Wasseraustausch zwischen der Endolymphe, den Zellen des Ductus cochlearis und der Perilymphe nahe. Die subzelluläre Kolokalisation von Kir4.1 und AQP4 in epithelialen Stützzellen spricht für eine enge funktionelle Kopplung von Kalium- und Wasserflüssen in der Cochlea. Dies bietet eine Erklärung für die bei Menschen mit Mutationen im AQP4-Gen beobachtete Schwerhörigkeit.AbstractBackgroundSound transduction in the cochlea critically depends on the circulation of potassium ions (K+) along so-called “K+ recycling routes” between the endolymph and perilymph. These K+ currents generate high ionic and osmotic gradients, which potentially impair the excitability of sensory hair cells and threaten cell survival in the entire cochlear duct. Molecular water channels—aquaporins (AQP)—are expressed in all cochlear supporting cells along the K+ recycling routes; however, their significance for osmotic equilibration in cochlear duct cells is unknown.MethodsThe diffusive and osmotic water permeabilies of Reissner’s membrane, the organ of Corti and the entire cochlear duct epithelium were determined. Expression of the potassium channel Kir4.1 and the water channel AQP4 in the cochlear duct was investigated by immunohistochemistry.ResultsThe calculated water permeability values indicate the extent of AQP-facilitated water flux across the cochlear duct epithelium. Immunohistochemically, Kir4.1 and AQP4 were found to colocalize in distinct membrane domains of supporting cells along the K+-recycling routes.ConclusionThese observations suggest the presence of a rapid AQP-mediated water exchange between the endolymph, the cells of the cochlear duct and the perilymph. The subcellular colocalization of Kir4.1 and AQP4 in epithelial supporting cells indicates functional coupling of potassium and water flow in the cochlea. Finally, this offers an explanation for the hearing impairment observed in individuals with mutations in the AQP4 gene.