Protective effect of lutein against acrolein-induced ototoxicity in rats.

BACKGROUND: Acrolein is a reactive aldehyde that forms during burning of wood and other fuels. It is also a product of lipid peroxidation (LPO) reactions and is present in cigarette smoke. Acrolein is known to cause oxidative stress and inflammatory nerve tissue damage. Lutein is a tetraterpenoid molecule with antioxidant and anti-inflammatory properties. There appear to be no studies on the effect of lutein on vestibulocochlear nerve damage induced by acrolein. The aim of this study was to investigate the effect of lutein on vestibulocochlear nerve damage induced by acrolein in rats using biochemical and histopathological methods. METHODS: The rats were divided into three groups (n = 6, for each group) a healthy control group (HG), an acrolein (ACR) group and a lutein and acrolein (LACR) group. In the LACR group, lutein was administered (1 mg/kg) via oral gavage. The ACR and HG groups received saline via oral gavage. Then, 1 h after the administration of lutein and saline, the LACR and ACR groups were treated with 3 mg/kg of acrolein via oral gavage. This procedure was repeated once a day for 30 days. RESULTS: The results of biochemical experiments showed that in the vestibulocochlear nerve tissues of the animals treated with acrolein, the levels of malondialdehyde, total oxidants, nuclear factor kappa b, tumor necrosis factor alpha and interleukin 1 beta significantly increased, whereas the levels of total glutathione and total antioxidants decreased as compared to those in the HG and LACR groups. In addition, severe histopathological damage was observed in vestibulocochlear nerve tissue of the acrolein group, whereas this damage was alleviated in the lutein group. CONCLUSION: Lutein protected vestibulocochlear nerve tissue from acrolein-associated oxidative and proinflammatory damage. This suggests that lutein might be useful in preventing or treating acrolein-induced ototoxicity.

Sensory Neuroblast Quiescence Depends on Vascular Cytoneme Contacts and Sensory Neuronal Differentiation Requires Initiation of Blood Flow.

In many organs, stem cell function depends on communication with their niche partners. Cranial sensory neurons develop in close proximity to blood vessels; however, whether vasculature is an integral component of their niches is yet unknown. Here, two separate roles for vasculature in cranial sensory neurogenesis in zebrafish are uncovered. The first involves precise spatiotemporal endothelial-neuroblast cytoneme contacts and Dll4-Notch signaling to restrain neuroblast proliferation. The second, instead, requires blood flow to trigger a transcriptional response that modifies neuroblast metabolic status and induces sensory neuron differentiation. In contrast, no role of sensory neurogenesis in vascular development is found, suggesting unidirectional signaling from vasculature to sensory neuroblasts. Altogether, we demonstrate that the cranial vasculature constitutes a niche component of the sensory ganglia that regulates the pace of their growth and differentiation dynamics.

Expression of MYOSIN VIIA in developing mouse cochleovestibular ganglion neurons.

Myosins make up a large super family of motor proteins responsible for actin-based motility in most eukaryotic cells. Myosin VIIA is essential for the development and function of sensory hair cells in the inner ear. The role of Myosin VIIA in the development of cochleovestibular ganglion (CVG) neurons in the mouse is largely unknown. Neurons of the CVG innervate sensory hair cells of the cochlea and vestibular organs to transmit hearing and balance information respectively to the brain. The aim of this study was to characterize the expression of MYOSIN VIIA in the CVG of mouse embryos. Spatiotemporal expression of MYOSIN VIIA was characterized in embryonic (E) mouse inner ear neurons from E9.5 to postnatal (P) day 0. At early stages, when otic neurons begin to delaminate to form the CVG, MYOSIN VIIA was co-expressed with TuJ1, ISLET1 and NEUROD in the otic epithelium and CVG. When CVG neurons were migrating and exiting mitosis, MYSOSIN VIIA was downregulated in a subset of neurons, which were NEUROD-negative and GATA3-positive. After segregation of the CVG, MYOSIN VIIA was observed in a subset of vestibular neurons marked by TUJ1 and absent in cochlear neurons, marked by GATA3. The differential expression of MYOSIN VIIA may indicate a role in inner ear neuron migration and specific labeling of vestibular neurons.

NLRP3 inflammasome activation in human vestibular schwannoma: Implications for tumor-induced hearing loss.

Vestibular schwannoma (VS) is the fourth most common intracranial tumor, arising from neoplastic Schwann cells of the vestibular nerve and often causing debilitating sensorineural hearing loss (SNHL) and tinnitus. Previous research suggests that the abnormal upregulation of inflammatory pathways plays a highly significant, though infrequently described role in VS pathobiology, and that VS-associated SNHL is due not only to mechanical compression of the auditory nerve but also to differences in the intrinsic biology of these tumors. We hypothesize that patients who present with poor hearing associated with VS experience a more robust inflammatory response to this tumor than VS patients who present with good hearing. To investigate this hypothesis, we conducted a comprehensive pathway analysis using gene expression data from the largest meta-analysis of vestibular schwannoma microarray data, comprising 80 tumors and 16 healthy peripheral nerves. We identified the NLRP3 inflammasome as a novel target worthy of further exploration in VS research and validated this finding at the gene and protein expression level in human VS tissue using qRT-PCR and immunohistochemistry. To date, NLRP3 inflammasome activation has not been reported in VS, and this finding may represent a new and potentially significant therapeutic avenue. Notably, after analysis of 30 VSs, we observe that overexpression of key components of the NLRP3 inflammasome is preferentially associated with tumors that produce increased hearing loss in VS patients. Therefore, therapeutic development for VS should include considerations for minimizing NLRP3-associated inflammation to best preserve hearing.

Spemann organizer gene Goosecoid promotes delamination of neuroblasts from the otic vesicle.

Neurons of the Statoacoustic Ganglion (SAG), which innervate the inner ear, originate as neuroblasts in the floor of the otic vesicle and subsequently delaminate and migrate toward the hindbrain before completing differentiation. In all vertebrates, locally expressed Fgf initiates SAG development by inducing expression of Neurogenin1 (Ngn1) in the floor of the otic vesicle. However, not all Ngn1-positive cells undergo delamination, nor has the mechanism controlling SAG delamination been elucidated. Here we report that Goosecoid (Gsc), best known for regulating cellular dynamics in the Spemann organizer, regulates delamination of neuroblasts in the otic vesicle. In zebrafish, Fgf coregulates expression of Gsc and Ngn1 in partially overlapping domains, with delamination occurring primarily in the zone of overlap. Loss of Gsc severely inhibits delamination, whereas overexpression of Gsc greatly increases delamination. Comisexpression of Ngn1 and Gsc induces ectopic delamination of some cells from the medial wall of the otic vesicle but with a low incidence, suggesting the action of a local inhibitor. The medial marker Pax2a is required to restrict the domain of gsc expression, and misexpression of Pax2a is sufficient to block delamination and fully suppress the effects of Gsc The opposing activities of Gsc and Pax2a correlate with repression or up-regulation, respectively, of E-cadherin (cdh1). These data resolve a genetic mechanism controlling delamination of otic neuroblasts. The data also elucidate a developmental role for Gsc consistent with a general function in promoting epithelial-to-mesenchymal transition (EMT).

The role of astrocyte mitochondria in differential regional susceptibility to environmental neurotoxicants: tools for understanding neurodegeneration.

In recent decades, there has been a significant expansion in our understanding of the role of astrocytes in neuroprotection, including spatial buffering of extracellular ions, secretion of metabolic coenzymes, and synaptic regulation. Astrocytic neuroprotective functions require energy, and therefore require a network of functional mitochondria. Disturbances to astrocytic mitochondrial homeostasis and their ability to produce ATP can negatively impact neural function. Perturbations in astrocyte mitochondrial function may accrue as the result of physiological aging processes or as a consequence of neurotoxicant exposure. Hydrophobic environmental neurotoxicants, such as 1,3-dinitrobenzene and α-chlorohydrin, cause regionally specific spongiform lesions mimicking energy deprivation syndromes. Astrocyte involvement includes mitochondrial damage that either precedes or is accompanied by neuronal damage. Similarly, environmental neurotoxicants that are implicated in the etiology of age-related neurodegenerative conditions cause regionally specific damage in the brain. Based on the regioselective nature of age-related neurodegenerative lesions, chemically induced models of regioselective lesions targeting astrocyte mitochondria can provide insight into age-related susceptibilities in astrocyte mitochondria. Most of the available research to date focuses on neuronal damage in cases of age-related neurodegeneration; however, there is a body of evidence that supports a central mechanistic role for astrocyte mitochondria in the expression of neural injury. Regional susceptibility to neuronal damage induced by aging by exposure to neurotoxicants may be a reflection of highly variable regional energy requirements. This review identifies region-specific vulnerabilities in astrocyte mitochondria in examples of exposure to neurotoxicants and in age-related neurodegeneration.

Differential roles for EphA and EphB signaling in segregation and patterning of central vestibulocochlear nerve projections.

Auditory and vestibular afferents enter the brainstem through the VIIIth cranial nerve and find targets in distinct brain regions. We previously reported that the axon guidance molecules EphA4 and EphB2 have largely complementary expression patterns in the developing avian VIIIth nerve. Here, we tested whether inhibition of Eph signaling alters central targeting of VIIIth nerve axons. We first identified the central compartments through which auditory and vestibular axons travel. We then manipulated Eph-ephrin signaling using pharmacological inhibition of Eph receptors and in ovo electroporation to misexpress EphA4 and EphB2. Anterograde labeling of auditory afferents showed that inhibition of Eph signaling did not misroute axons to non-auditory target regions. Similarly, we did not find vestibular axons within auditory projection regions. However, we found that pharmacologic inhibition of Eph receptors reduced the volume of the vestibular projection compartment. Inhibition of EphB signaling alone did not affect auditory or vestibular central projection volumes, but it significantly increased the area of the auditory sensory epithelium. Misexpression of EphA4 and EphB2 in VIIIth nerve axons resulted in a significant shift of dorsoventral spacing between the axon tracts, suggesting a cell-autonomous role for the partitioning of projection areas along this axis. Cochlear ganglion volumes did not differ among treatment groups, indicating the changes seen were not due to a gain or loss of cochlear ganglion cells. These results suggest that Eph-ephrin signaling does not specify auditory versus vestibular targets but rather contributes to formation of boundaries for patterning of inner ear projections in the hindbrain.

Limited inner ear morphogenesis and neurosensory development are possible in the absence of GATA3.

Haploinsufficiency of Gata3 causes hypoparathyroidism, deafness and renal dysplasia (HDR) syndrome in mice and humans. Gata3 null mutation leads to early lethality around embryonic day (E)11.5, but catecholamine precursor administration can rescue Gata3 null mutants to E16.5. At E11.5, GATA3 deficiency results in the development of an empty otocyst with an endolymphatic duct. However, using rescued mice we found that some morphogenesis and neurosensory development is possible in the ear without Gata3. Extending previous studies, we find that at E16.5, Gata3 mutant inner ears can undergo partial morphogenesis and develop an endolymphatic duct, a utricular and saccular recess, and a shortened cochlear duct. In addition to the obvious morphogenic aberrations, these studies demonstrate that a subset of neurons develop and connect a fragmented sensory patch of MYO7A-positive hair cells to the vestibular nuclei of the brainstem. In situ hybridization studies reveal altered expression of several transcription factors relevant to ear development and we hypothesize that this may relate to the observed dysmorphia and restricted neurosensory development. While a cochlear duct can form, there is no concurrent cochlear neurosensory development, observations consistent with specific hearing defects encountered by HDR patients and mice with Gata3-associated expression alterations. Gata3 null mutant phenocopies the otic maldevelopment (cochlear duct formation in the absence of neurosensory development) seen in Foxg1cre mediated conditional deletion of microRNA processing enzyme, Dicer1. Finally, while GATA3 is expressed in the developing vestibulo-cochlear efferent (VCE) neurons, and its absence in the null mutants disrupts VCE projections to the ear, loss of GATA3 does not affect VCE progenitor cell migration.

Receptors for leptin in the otic labyrinth and the cochlear-vestibular nerve of guinea pig are modified in hormone-induced anorexia.

Metabolic syndromic inner ear pathology is a recognized condition in clinical practice but the possible causes remain controversial. We have previously reported that chronically-implanted estrogen implants in guinea pig results in hyperprolactinemia and hearing loss together with otic bone dysmorphology. The animals also present with anorexia. The hormone leptin has major roles in the regulation of satiety as well as bone metabolism and so we hypothesized that leptin might contribute to pathology of the otic labyrinth. We employed immunohistochemistry to investigate leptin receptor (ObR) expression. In control animals, ObR immunolabeling was not detected in the bone of the otic capsule but immunolabeling was observed in the cochlear-vestibular nerve. The labeling was associated with the astrocytic glial dome area, which marks the transition between central and peripheral parts of the nerve. In estrogen-treated animals, positive-ObR immunolabeling was observed in osteoblasts in new bone of the otic capsule and the ObR labeling was reduced in the cochlear-vestibular nerve compared to controls. The data provide evidence that leptin may target the labyrinth - affecting the bone and the nerve - and so could contribute to ongoing protection of the inner ear. Leptin disturbance might contribute to metabolic syndromes involving the audiovestibular system.

Localization of vesicular glutamate transporters in the peripheral vestibular system of rat.

OBJECTIVE: To examine the vesicular glutamate transporters (VGluTs: VGluT1-VGluT3) in the peripheral vestibular system. METHODS: The vestibular structures, including Scarpa's ganglion (vestibular ganglion, VG), maculae of utricle and saccule, and ampullary cristae, from normal Sprague-Dawley rats were processed immunohistochemically for VGluTs, by avidin-biotinylated peroxidase complex method, with 3-3'-diaminobenzidine (DAB) as chromogen. RESULTS: (1) VGluT1 was localized to partial neurons of VG and to the putative primary afferent fibers innervating vestibular end-organs. (2) Intense VGluT3 immunoreactivity was detected in large number of sensory epithelia cells, and weak labeling of VGluT3-positive afferent fibers was in the maculae and ampullary cristae. (3) No or very weak VGluT2 immunoreactivity was observed in the VG and acoustic maculae. CONCLUSION: These results provide the morphological support that glutamate exists in the peripheral vestibular system, and it may play an important role in the centripetal vestibular transmission.

Differential brain-derived neurotrophic factor and transforming growth factor-beta expression in the rat cochlea following deafness.

Brain derived neurotrophic factor (BDNF) and transforming growth factor-beta (TGFbeta) subtypes have demonstrated their importance in cochlear functions. The aim of this study was to determine gene and protein expression patterns of BDNF, TGFbeta1/2 and their main receptors trkB and TGFbetaR1/R2 in the auditory nerve and inferior colliculus of normal hearing and deafened rats by reverse-transcriptase polymerase chain reaction and immunohistochemistry. Deafening was performed by cochlear injection of neomycin (10%). Significant gene expression changes were not found in the inferior colliculus after deafness; however, in the auditory nerve, BDNF, TGFbeta1 and TGFbetaR1 mRNA in particular were upregulated. Additionally, BDNF protein and the cytokines were found to be expressed in the auditory nerve after hair cell loss. These data indicate the importance of BDNF and TGFbeta1 as endogenous survival factors.

Episodic blockade of cranial nerve VIII provokes asymmetric changes in lobule X of the rat.

Although debilitating syndromes like Ménière's disease are in part characterized by recurrent or episodic vestibular disturbance the study of episodic vestibular disruption has only recently been possible with the introduction of a new model utilizing tetrodotoxin (TTX). In the present study, serial unilateral transtympanic administration of TTX produced behavioral symptoms indicative of transient vestibular disruption and novel patterns of Fos activity in the brainstem and cerebellum. Following two or three serial injections of TTX and a final survival time of 2 h, Fos immunocytochemistry revealed a distinct pattern of labeling in the brainstem that differed temporally from that observed following a single unilateral TTX injection. Specifically there was protracted expression of Fos in the beta subdivision of the inferior olive (IO) on the side ipsilateral to TTX treatment. In the cerebellum, the hallmark of episodic vestibular blockade was an asymmetric pattern of Fos labeling that involved all three layers of the cortex. In particular, there was prominent Fos labeling of Purkinje cells in the contra-TTX half of lobule X. In view of the fact that Fos labeling is not found in Purkinje cells following a single transient event or following peripheral vestibular ablation, it is suggested that Fos expression in Purkinje cells is a unique feature of episodic vestibular disruption and may represent a novel plastic response by a select population of Purkinje cells to episodic functional deafferentation.

Microtubule-associated protein 2 (MAP2) expression during synaptic plasticity in the guinea pig cochlea.

The expression of different isoforms of microtubule-associated proteins 2 (MAP2), including the low molecular weight form MAP2c present mainly in developing neurons, was investigated in the primary auditory neurons after alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) perfusion in the guinea pig cochlea. MAP2 expression appeared to be tightly regulated in the repairing neurons. Neurite regrowth seems to involve the MAP2c isoform. In cochlear neurons, mechanisms involved in the period of development might be reactivated after excitotoxic injury in the mature cochlea.

Distribution of Fos labeling in the inferior olive following transient blockade of the VIIIth cranial nerve.

The sodium channel blocker, tetrodotoxin (TTX), is an effective tool for blockade of action potentials in neurons. Unilateral transtympanic administration of 3 mM TTX produced behavioral symptoms paralleling those previously reported following unilateral vestibular ablation. Behavioral symptoms were evident as early as 15 min post-TTX. Fos immunocytochemistry revealed an initial bilateral distribution of Fos in the inferior olive (IO) followed by an almost exclusively unilateral distribution of Fos. By 1 h, Fos was predominantly localized in subdivisions of the IO contralateral to TTX treatment. Fos labeling in the IO was most pronounced at 2- and 6-h survival times and was localized in the contralateral IOA, IOB, IOC, IOBe, and IOK subdivisions and bilaterally in the IOM and IODM. Other regions of the brainstem including the vestibular nuclei, prepositus hypoglossi, dorsal paragigantocellular reticular nucleus, nucleus of the tractus solitarius and locus coeruleus also exhibited altered patterns of Fos labeling following TTX. The finding that Fos activity in the IO is initially bilateral and then rapidly becomes unilateral has not been reported for the traditional vestibular ablation models and may be unique to the TTX model. In addition, since altered Fos activity is readily detected in the IO at time-points prior to detectable changes in Fos in the central vestibular complex it is possible that the IO is particularly sensitive to events precipitated by unilateral vestibular disturbance.

Glial cell line-derived neurotrophic factor and chronic electrical stimulation prevent VIII cranial nerve degeneration following denervation.

As with other cranial nerves and many CNS neurons, primary auditory neurons degenerate as a consequence of loss of input from their target cells, the inner hair cells (IHCs). Electrical stimulation (ES) of spiral ganglion cells (SGCs) has been shown to enhance their survival. Glial cell line-derived neurotrophic factor (GDNF) has also been shown to increase survival of SGCs following IHC loss. In this study, the combined effects of the GDNF transgene delivered by adenoviral vectors (Ad-GDNF) and ES were tested on SGCs after first eliminating the IHCs. Animal groups received Ad-GDNF or ES or both. Ad-GDNF was inoculated into the cochlea of guinea pigs after deafening, to overexpress human GDNF. ES-treated animals were implanted with a cochlear implant electrode and chronically stimulated. A third group of animals received both Ad-GDNF and ES (GDNF/ES). Electrically evoked auditory brainstem responses were recorded from ES-treated animals at the start and end of the stimulation period. Animals were sacrificed 43 days after deafening and their ears prepared for evaluation of IHC survival and SGC counts. Treated ears exhibited significantly greater SGC survival than nontreated ears. The GDNF/ES combination provided significantly better preservation of SGC density than either treatment alone. Insofar as ES parameters were optimized for maximal protection (saturated effect), the further augmentation of the protection by GDNF suggests that the mechanisms of GDNF- and ES-mediated SGC protection are, at least in part, independent. We suggest that GDNF/ES combined treatment in cochlear implant recipients will improve auditory perception. These findings may have implications for the prevention and treatment of other neurodegenerative processes. .

Zinc inhibition of group I mGluR-mediated calcium homeostasis in auditory neurons.

Zinc is widely distributed in the central nervous system (CNS), it functions normally as a synaptic modulator, and it contributes to neuronal death under pathologic conditions. Zinc colocalizes with glutamate in excitatory synapses, and the presence of zinc is well characterized in the synapses of the auditory system. Since chick cochlear nucleus neurons depend upon synaptic activation of metabotropic glutamate receptors (mGluRs) for maintenance and survival, the goal of this study was to determine (1) if zinc is released from the eighth nerve calyces onto nucleus magnocellularis (NM) neurons in the chick cochlear nucleus, and, if so, (2) what effect it has on group I mGluR-mediated calcium homeostasis of these neurons. Using in vitro slices and a fluorescent dye relatively specific to vesicularized zinc, we show that zinc is indeed localized to the presynaptic calyces and is released upon nerve stimulation or KCl depolarization. Experiments employing fura-2 calcium imaging show that zinc inhibits group I mGluR release of calcium from internal stores of NM neurons and disrupts activity-dependent calcium homeostasis in a manner identical to the mGluR5-specific antagonist 2-methyl-6-(phenylethynyl)pyridine. The mGluR1-specific antagonist 7-hydroxyiminocyclopropan-[b]chromen-la-carboxylic acid ethyl ester did not affect release of calcium from stores by the nonspecific mGluR agonist aminocyclopentane dicarboxylic acid, nor did it affect activity-dependent calcium homeostasis. We conclude that zinc is present in and released from the glutamatergic eighth nerve calcyes. The presence of zinc inhibits mGluR5, a major component of calcium homeostasis of NM neurons, and plays a modulatory role in the activity-dependent, mGluR-mediated calcium homeostasis of auditory neurons.

Could antioxidant therapy reduce the incidence of deafness following bacterial meningitis?

Sensorineural hearing loss following acute bacterial meningitis could be caused by hydroxyl radicals generated by the inflammatory response. Obstruction of cerebrospinal fluid circulation through the tela choroidae of the choroid plexuses, with subsequent rupture of the tela choroidae, would expose the auditory nerve to selective radical damage. Acute administration of lipophilic antioxidants might provide the auditory nerve with increased protection.

RT-PCR analysis of mRNA expression of natriuretic peptide family and their receptors in rat inner ear.

To assess the possible physiological role of the atrial natriuretic peptide (ANP) family, we investigated the expression of mRNA of ANP, brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and their receptors in rat inner ear using the reverse transcription-polymerase chain reaction method. ANP and CNP message bands were detected in the inner ear, but the BNP message band was not. Amplification products of the expected sizes of ANP-A, ANP-B and ANP-C receptors were detected in the inner ear. These results suggest that natriuretic peptide family may influence the function of the inner ear through the ANP-A, ANP-B, and ANP-C receptors.

ARIA is heavily expressed in rat peripheral auditory and vestibular ganglia.

The ARIA (acetylcholine receptor inducing activity) polypeptide is a member of the neuregulin gene family. It was originally purified on the basis of its ability to induce skeletal muscle nicotinic acetylcholine receptors (nAChRs). ARIA mRNA is expressed in ventral horn motor neurons and brain cholinergic neurons. We report here that ARIA mRNA is heavily expressed in the embryonic, developing, and adult peripheral auditory and vestibular ganglia, the spiral ganglion and Scarpa's ganglion. Neither ganglion is cholinergic, but both express mRNAs for nicotinic and muscarinic receptors. The expression of ARIA in these ganglia may be related to the regulation of cholinergic receptors or a more general role for ARIA in growth and development.

Expression of neurite outgrowth factor and gicerin during inner ear development and hair cell regeneration in the chick.

Several cell adhesion molecules are expressed in the developing inner ear. The present study focused on gicerin, a novel member of the immunoglobulin superfamily, in an attempt to improve our understanding of the development and regeneration of chick inner ear. Gicerin is known to homophilically interact with itself and to bind to neurite outgrowth factor (NOF). The data collected herein show that gicerin is highly expressed in auditory epithelium and acoustic ganglion during early embryogenesis. The immunoreactivity of gicerin in the auditory epithelium decreases more rapidly than that in the acoustic ganglion as the mature hair cells become distinguishable. At the post-hatch stage, the expression of gicerin is not observed. In contrast, NOF was expressed on the basement membranes around the auditory epithelium, and in the acoustic ganglion during development and after birth, but not in the auditory epithelium. Following noise damage, gicerin is transiently re-expressed on the damage receptor epithelium when active cell proliferation is observed in the epithelium. This positive reaction immediately disappears as immature short hair cells appear. These results suggest that gicerin may be associated with cell proliferation in the auditory epithelium, and play a role in neurite extension of the acoustic ganglion cells in conjunction with NOF.