The Controversial Role of Folic Acid on Diabetic Auditory Neuropathy

PurposeDiabetic auditory neuropathy(DAN) is a common complication of diabetes that seriously affects the quality of life in patients. In this study, we investigate the role of folic acid in the treatment of DAN in an experimental rat model.MethodsThirty-two Sprague-Dawley rats were equally divided into 4 groups: group 1, normal; group 2, diabetic rats; group 3and 4, rats treated with folic acid (40 and 80 mg/kg, respectively). The tools we used in this study to investigate the effect of folic acid on DAN were auditory brain stem response, stereology methodfor estimation ofthe volume and number ofspiral ganglion,volume of stria vascularis, and spiral ligament, and measurement of homocysteine (HCY), malondealdehyde(MDA) and superoxide dismutase.ResultsOur study showed that folic acid treatment was not significantly effective in improving structural and functional disorders in DAN, despite its effect in reducing HCY and MDA as oxidative biomarkers.ConclusionFolic acid is not effective in relieving morphological and functional disorders in DAN.

Cochlear Fibrocyte and Osteoblast Lineages Expressing Type 2 Deiodinase Identified with a Dio2CreERt2 Allele

Type 2 deiodinase (Dio2) amplifies levels of 3,5,3′-L-triiodothyronine (T3), the active form of thyroid hormone, and is essential for cochlear maturation and auditory development. However, cellular routes for endocrine signaling in the compartmentalized, anatomically complex cochlea are little understood. Dio2 generates T3 from thyroxine (T4), a more abundant thyroid hormone precursor in the circulation, and is dramatically induced in the cochlea before the onset of hearing. The evidence implies that specific Dio2-expressing cell types critically mediate T3 signaling but these cell types are poorly defined because Dio2 is expressed transiently at low levels. Here, using a Dio2CreERt2 knockin that activates a fluorescent reporter, we define Dio2-expressing cochlear cell types at high resolution in male or female mice. Dio2-positive cells were detected in vascularized supporting tissues but not in avascular internal epithelia, indicating segregation of T3-generating and T3-responding tissues. In the spiral ligament and spiral limbus, Dio2-positive fibrocytes clustered around vascular networks that convey T4 into cochlear tissues. In the otic capsule, Dio2-positive osteoblasts localized at cartilage surfaces as the bony labyrinth matures. We corroborated the identities of Dio2-positive lineages by RNA-sequencing of individual cells. The results suggest a previously unrecognized role for fibrocytes in mediating hormonal signaling. We discuss a model whereby fibrocytes mediate paracrine-like control of T3 signaling to the organ of Corti and epithelial target tissues.

Disruption of Glutamate Release and Uptake-Related Protein Expression After Noise-Induced Synaptopathy in the Cochlea

High-intensity noise can cause permanent hearing loss; however, short-duration medium-intensity noise only induces a temporary threshold shift (TTS) and damages synapses formed by inner hair cells (IHCs) and spiral ganglion nerves. Synaptopathy is generally thought to be caused by glutamate excitotoxicity. In this study, we investigated the expression levels of vesicle transporter protein 3 (Vglut3), responsible for the release of glutamate; glutamate/aspartate transporter protein (GLAST), responsible for the uptake of glutamate; and Na+/K+-ATPase α1 coupled with GLAST, in the process of synaptopathy in the cochlea. The results of the auditory brainstem response (ABR) and CtBP2 immunofluorescence revealed that synaptopathy was induced on day 30 after 100 dB SPL noise exposure in C57BL/6J mice. We found that GLAST and Na+/K+-ATPase α1 were co-localized in the cochlea, mainly in the stria vascularis, spiral ligament, and spiral ganglion cells. Furthermore, Vglut3, GLAST, and Na+/K+-ATPase α1 expression were disrupted after noise exposure. These results indicate that disruption of glutamate release and uptake-related protein expression may exacerbate the occurrence of synaptopathy.

Targeted Deletion of Loxl3 by Col2a1-Cre Leads to Progressive Hearing Loss

Collagens are major constituents of the extracellular matrix (ECM) that play an essential role in the structure of the inner ear and provide elasticity and rigidity when the signals of sound are received and transformed into electrical signals. LOXL3 is a member of the lysyl oxidase (LOX) family that are copper-dependent amine oxidases, generating covalent cross-links to stabilize polymeric elastin and collagen fibers in the ECM. Biallelic missense variant of LOXL3 was found in Stickler syndrome with mild conductive hearing loss. However, available information regarding the specific roles of LOXL3 in auditory function is limited. In this study, we showed that the Col2a1-Cre-mediated ablation of Loxl3 in the inner ear can cause progressive hearing loss, degeneration of hair cells and secondary degeneration of spiral ganglion neurons. The abnormal distribution of type II collagen in the spiral ligament and increased inflammatory responses were also found in Col2a1–Loxl3–/– mice. Amino oxidase activity exerts an effect on collagen; thus, Loxl3 deficiency was expected to result in the instability of collagen in the spiral ligament and the basilar membrane, which may interfere with the mechanical properties of the organ of Corti and induce the inflammatory responses that are responsible for the hearing loss. Overall, our findings suggest that Loxl3 may play an essential role in maintaining hearing function.

Dio2-CreErt2 Knockin Model to Identify T3-Generating Cells That Express Type 2 Deiodinase in Tissues

Background: Thyroid hormone promotes many developmental and homeostatic functions. Apart from adequate circulating levels, the concentration of the active hormone T3 within tissues may be amplified by type 2 deiodinase (Dio2) by conversion from the precursor T4. Dio2 is critical in auditory development, bone maturation, brain function and control of the hypothalamic-pituitary-thyroid axis. Despite its crucial role, an obstacle to studying Dio2 is that the protein has a short half-life, is at low levels and is often transiently expressed, making it difficult to identify Dio2 in tissues at cellular resolution. Methods: We derived a Dio2-CreERt2 knockin mouse that expresses tamoxifen-dependent Cre recombinase from the endogenous Dio2 gene. When crossed onto Ai6 reporter mice, following tamoxifen treatment, Dio2-CreERt2 expression is detected as fluorescent signal in specific cells in brain regions, pituitary, and other tissues. We showed previously that Dio2 is essential for hearing with rising expression levels in the cochlea prior to onset of hearing. The Dio2-CreERt2 model identified positive cell types in the cochlear spiral ligament, septal divisions and modiolus around the sensory epithelium. Dio2-positive fibrocytes were adjacent to and extended projections around blood capillary networks, the source of T4 supply. Transcriptome analysis of isolated positive cells revealed bone lineage-related origins for many of these cells. Conclusion: The Dio2-CreERt2 model detects Dio2 expression sensitively at cellular resolution. In the cochlea, Dio2-positive cell types reside in vascularized support tissues, suggesting combined endocrine and paracrine-like control of the T3 supply. Analysis of cell origins suggests novel interactions between endocrine and skeletal systems in promoting T3 action required for hearing.

Lack of collagen α6(IV) chain in mice does not cause severe-to-profound hearing loss or cochlear malformation, a distinct phenotype from nonsyndromic hearing loss with COL4A6 missense mutation

Congenital hearing loss affects 1 in every 1000 births, with genetic mutations contributing to more than 50% of all cases. X-linked nonsyndromic hereditary hearing loss is associated with six loci (DFNX1-6) and five genes. Recently, the missense mutation (c.1771G>A, p.Gly591Ser) in COL4A6, encoding the basement membrane (BM) collagen α6(IV) chain, was shown to be associated with X-linked congenital nonsyndromic hearing loss with cochlear malformation. However, the mechanism by which the COL4A6 mutation impacts hereditary hearing loss has not yet been elucidated. Herein, we investigated Col4a6 knockout (KO) effects on hearing function and cochlear formation in mice. Immunohistochemistry showed that the collagen α6(IV) chain was distributed throughout the mouse cochlea within subepithelial BMs underlying the interdental cells, inner sulcus cells, basilar membrane, outer sulcus cells, root cells, Reissner’s membrane, and perivascular BMs in the spiral limbus, spiral ligament, and stria vascularis. However, the click-evoked auditory brainstem response analysis did not show significant changes in the hearing threshold of Col4a6 KO mice compared with wild-type (WT) mice with the same genetic background. In addition, the cochlear structures of Col4a6 KO mice did not exhibit morphological alterations, according to the results of high-resolution micro-computed tomography and histology. Hence, loss of Col4a6 gene expression in mice showed normal click ABR thresholds and normal cochlear formation, which differs from humans with the COL4A6 missense mutation c.1771G>A, p.Gly591Ser. Therefore, the deleterious effects in the auditory system caused by the missense mutation in COL4A6 are likely due to the dominant-negative effects of the α6(IV) chain and/or α5α6α5(IV) heterotrimer with an aberrant structure that would not occur in cases with loss of gene expression.

Mast Cells in the Auditory Periphery of Rodents

Mast cells (MCs) are densely granulated cells of myeloid origin and are a part of immune and neuroimmune systems. MCs have been detected in the endolymphatic sac of the inner ear and are suggested to regulate allergic hydrops. However, their existence in the cochlea has never been documented. In this work, we show that MCs are present in the cochleae of C57BL/6 mice and Wistar rats, where they localize in the modiolus, spiral ligament, and stria vascularis. The identity of MCs was confirmed in cochlear cryosections and flat preparations using avidin and antibodies against c-Kit/CD117, chymase, tryptase, and FcεRIα. The number of MCs decreased significantly during postnatal development, resulting in only a few MCs present in the flat preparation of the cochlea of a rat. In addition, exposure to 40 µM cisplatin for 24 h led to a significant reduction in cochlear MCs. The presence of MCs in the cochlea may shed new light on postnatal maturation of the auditory periphery and possible involvement in the ototoxicity of cisplatin. Presented data extend the current knowledge about the physiology and pathology of the auditory periphery. Future functional studies should expand and translate this new basic knowledge to clinics.

Type 1 Diabetes Induces Hearing Loss: Functional and Histological Findings in An Akita Mouse Model

The relationship between type 1 diabetes and hearing loss is not well known, although based on many pathological studies, type 2 diabetes induced hearing loss is associated with microcirculation problems in the inner ear. The purpose of this study was to investigate the correlation between type 1 diabetes and hearing loss through hearing function and immunohistochemical analyses using type 1 diabetic Akita or wild-type (WT) mice. The Akita mice had a significant increase in hearing thresholds, blood glucose, and insulin tolerance compared to WT mice. Histological analysis showed that the loss of cells and damage to mitochondria in the spiral ganglion neurons of Akita mice were significantly increased compared to WT. Also, the stria vascularis showed decreased thickness, loss of intermediate cells, and disturbance in blood capillary shape in the Akita mice. Moreover, a reduction in type I, II, and IV fibrocytes and Na+/K+-ATPase α1 expression in spiral ligament was also observed. Cleaved caspase-3 expression was highly expressed in spiral ganglion neurons. In conclusion, hearing loss in type 1 diabetes is caused not only by ion imbalance and blood flow disorders of cochlear endolymph, but through the degenerative nervous system via apoptosis-mediated cell death.