Spiral Form of the Human Cochlea Results from Spatial Constraints.

The human inner ear has an intricate spiral shape often compared to shells of mollusks, particularly to the nautilus shell. It has inspired many functional hearing theories. The reasons for this complex geometry remain unresolved. We digitized 138 human cochleae at microscopic resolution and observed an astonishing interindividual variability in the shape. A 3D analytical cochlear model was developed that fits the analyzed data with high precision. The cochlear geometry neither matched a proposed function, namely sound focusing similar to a whispering gallery, nor did it have the form of a nautilus. Instead, the innate cochlear blueprint and its actual ontogenetic variants were determined by spatial constraints and resulted from an efficient packing of the cochlear duct within the petrous bone. The analytical model predicts well the individual 3D cochlear geometry from few clinical measures and represents a clinical tool for an individualized approach to neurosensory restoration with cochlear implants.

Involvement of calpain in 4-hydroxynonenal-induced disruption of gap junction-mediated intercellular communication among fibrocytes in primary cultures derived from the cochlear spiral ligament.

The endocochlear potential in the inner ear is essential for hearing ability, and maintained by various K(+) transport apparatuses including Na(+), K(+)-ATPase and gap junction-mediated intercellular communication (GJ-IC) in the lateral wall structures of the cochlea. Noise-induced hearing loss is known at least in part due to disruption of GJ-IC resulting from an oxidative stress-induced decrease in connexins (Cxs) level in the lateral wall structures. The purpose of this study was to investigate, using primary cultures of fibrocytes from the cochlear spiral ligament of mice, the mechanism underlying GJ-IC disruption induced by 4-hydroxynonenal (4-HNE), which is formed as a mediator of oxidative stress. An exposure to 4-HNE produced the following events: i.e., an increase in 4-HNE-adducted proteins; a decrease in the protein levels of Cx43, ß-catenin, and Cx43/ß-catenin complex along with intracellular translocation of this complex from the cell membrane to the cytoplasm; enhanced calpain-dependent degradation of endogenous α-fodrin; and disruption of GJ-IC. The 4-HNE-induced decrease in these protein levels and disruption of GJ-IC were most completely abolished by the calpain inhibitor PD150606. Taken together, our data suggest that 4-HNE disrupted GJ-IC through calpain-mediated degradation of Cx43 and ß-catenin in primary cultures of fibrocytes derived from the cochlear spiral ligament.

NKCCs in the fibrocytes of the spiral ligament are silent on the unidirectional K⁺ transport that controls the electrochemical properties in the mammalian cochlea.

Unidirectional K(+) transport across the lateral cochlear wall contributes to the endocochlear potential (EP) of +80 mV in the endolymph, a property essential for hearing. The wall comprises two epithelial layers, the syncytium and the marginal cells. The basolateral surface of the former and the apical membranes of the latter face the perilymph and the endolymph, respectively. Intrastrial space (IS), an extracellular compartment between the two layers, exhibits low [K(+)] and a potential similar to the EP. This IS potential (ISP) dominates the EP and represents a K(+) diffusion potential elicited by a large K(+) gradient across the syncytial apical surface. The K(+) gradient depends on the unidirectional K(+) transport driven by Na(+),K(+)-ATPases on the basolateral surface of each layer and the concomitant Na(+),K(+),2Cl(-)-cotransporters (NKCCs) in the marginal cell layer. The NKCCs coexpressed with the Na(+),K(+)-ATPases in the syncytial layer also seem to participate in the K(+) transport. To test this hypothesis, we examined the electrochemical properties of the lateral wall with electrodes measuring [K(+)] and potential. Blocking NKCCs by perilymphatic perfusion of bumetanide suppressed the ISP. Unexpectedly and unlike the inhibition of the syncytial Na(+),K(+)-ATPases, the perfusion barely altered the electrochemical properties of the syncytium but markedly augmented [K(+)] of the IS. Consequently, the K(+) gradient decreased and the ISP declined. These observations resembled those when the marginal cells' Na(+),K(+)-ATPases or NKCCs were blocked with vascularly applied inhibitors. It is plausible that NKCCs in the marginal cells are affected by the perilymphatically perfused bumetanide, and these transporters, but not those in the syncytium, mediate the unidirectional K(+) transport.

Contractility in type III cochlear fibrocytes is dependent on non-muscle myosin II and intercellular gap junctional coupling.

The cochlear spiral ligament is a connective tissue that plays diverse roles in normal hearing. Spiral ligament fibrocytes are classified into functional sub-types that are proposed to carry out specialized roles in fluid homeostasis, the mediation of inflammatory responses to trauma, and the fine tuning of cochlear mechanics. We derived a secondary sub-culture from guinea pig spiral ligament, in which the cells expressed protein markers of type III or "tension" fibrocytes, including non-muscle myosin II (nmII), α-smooth muscle actin (αsma), vimentin, connexin43 (cx43), and aquaporin-1. The cells formed extensive stress fibers containing αsma, which were also associated intimately with nmII expression, and the cells displayed the mechanically contractile phenotype predicted by earlier modeling studies. cx43 immunofluorescence was evident within intercellular plaques, and the cells were coupled via dye-permeable gap junctions. Coupling was blocked by meclofenamic acid (MFA), an inhibitor of cx43-containing channels. The contraction of collagen lattice gels mediated by the cells could be prevented reversibly by blebbistatin, an inhibitor of nmII function. MFA also reduced the gel contraction, suggesting that intercellular coupling modulates contractility. The results demonstrate that these cells can impart nmII-dependent contractile force on a collagenous substrate, and support the hypothesis that type III fibrocytes regulate tension in the spiral ligament-basilar membrane complex, thereby determining auditory sensitivity.

Lactate dilates cochlear capillaries via type V fibrocyte-vessel coupling signaled by nNOS.

Transduction of sound in the inner ear demands tight control over delivery of oxygen and glucose. However, the mechanisms underlying the control of regional blood flow are not yet fully understood. In this study, we report a novel local control mechanism that regulates cochlear blood flow to the stria vascularis, a high energy-consuming region of the inner ear. We found that extracellular lactate had a vasodilatory effect on the capillaries of the spiral ligament under both in vitro and in vivo conditions. The lactate, acting through monocarboxylate transporter 1 (MCT1), initiated neuronal nitric oxide (NO) synthase (nNOS) and catalyzed production of NO for the vasodilation. Blocking MCT1 with the MCT blocker, α-cyano-4-hydroxycinnamate (CHC), or a suppressing NO production with either the nonspecific inhibitor of NO synthase, N(G)-nitro-L-arginine methyl ester (L-NAME), or either of two selective nNOS inhibitors, 3-bromo-7-nitroindazole or (4S)-N-(4-amino-5[aminoethyl]aminopentyl)-N'-nitroguanidine (TFA), totally abolished the lactate-induced vasodilation. Pretreatment with the selective endothelial NO synthase inhibitor, L-N(5)-(1-iminoethyl)ornithine (L-NIO), eliminated the inhibition of lactate-induced vessel dilation. With immunohistochemical labeling, we found the expression of MCT1 and nNOS in capillary-coupled type V fibrocytes. The data suggest that type V fibrocytes are the source of the lactate-induced NO. Cochlear microvessel tone, regulated by lactate, is mediated by an NO-signaled coupling of fibrocytes and capillaries.

Visualization and contractile activity of cochlear pericytes in the capillaries of the spiral ligament.

Pericytes, mural cells located on microvessels, are considered to play an important role in the formation of the vasculature and the regulation of local blood flow in some organs. Little is known about the physiology of cochlear pericytes. In order to investigate the function of cochlear pericytes, we developed a method to visualize cochlear pericytes using diaminofluorescein-2 diacetate (DAF-2DA) and intravital fluorescence microscopy. This method can permit the study of the effect of vasoactive agents on pericytes under the in vivo and normal physiological condition. The specificity of the labeling method was verified by the immunofluorescence labeling of pericyte maker proteins such as desmin, neural proteoglycan (NG2), and thymocyte differentiation antigen 1 (Thy-1). Superfused K(+) and Ca(2+) to the cochlear lateral wall resulted in localized constriction of capillaries at pericyte locations both in vivo and in vitro, while there was no obvious change in cochlear capillary diameters with application of the adrenergic neurotransmitter noradrenaline. The method could be an effective way to visualize cochlear pericytes and microvessels and study lateral wall vascular physiology. Moreover, we demonstrate for the first time that cochlear pericytes have contractility, which may be important for regulation of cochlear blood flow.