Observation in inner ear of tree shrew using scanning electron microscope and the Atoh1 distribution in cochlea.

This study aimed to observe the ultrastructure on the surface of the inner ear of a normal tree shrew using scanning electron microscope (SEM). The specimens of cochlea, macula utriculi, macula sacculi, and crista ampullaris of the normal adult tree shrew were collected and observed by SEM. We used immunofluorescence for cochlear protein Atoh1 staining. We observed that cochlea of the tree shrew is centered on the cochlear axis, circling about 3.5 times from bottom to top of the cochlea. The organ of Corti is located between medial and lateral grooves, including inner and outer hair cells as well as supporting cells. Maculae staticae include macula of saccule and macula of utricle, and the surface of macula is covered with a large number of otoliths. We found a gelatinous layer below the otoliths, followed by the layer of the honeycomb structure. The hair cell cilia of macula and crista ampullaris include one kinocilium and more stereocilia. There is no obvious cross structure but numerous hair cell cilia on semicircular canal crista ampullaris. Immunofluorescence staining showed that protein Atoh1 is mainly distributed in the nucleus of the cochlea's inner and outer hair cells. The observation of the inner ear structure under SEM elucidate the fine surface morphological structure of the entire cochlea, the vestibular maculae staticae, and crista ampullaris, providing new insight into the structure and function of the inner ear of tree shrew. HIGHLIGHTS: This article is the first to describe the inner ear ultrastructure of a small primate tree shrew by scanning electron microscopy (SEM). Under an SEM, the phalangeal processes of Deiter cells in tree shrews were observed to be connected to the tip of a neighboring hair cell, which was different from that of Deiters' cells in guinea pigs, and this crossed one hair cell, and connected to the tip of the third hair cell. It was observed that the crista ampullaris of tree shrews were horseshoe-shaped, and similar to that of humans and monkeys, this had no obvious "cross-shaped hump" structure. Tree shrew's ABR threshold value curve conforms to the mammalian U-shaped curve, wave III is the main wave of ARB, its sensory frequency may be higher 8 kHz, and the characteristics of the stereocilia of tree shrew we have observed may be related to the perception of higher frequency hearing.

Targeted Allele Suppression Prevents Progressive Hearing Loss in the Mature Murine Model of Human TMC1 Deafness.

Hearing loss is the most common human sensory deficit. Its correction has been the goal of several gene-therapy based studies exploring a variety of interventions. Although these studies report varying degrees of success, all treatments have targeted developing inner ears in neonatal mice, a time point in the structural maturation of the cochlea prior to 26 weeks gestational age in humans. It is unclear whether cochlear gene therapy can salvage hearing in the mature organ of Corti. Herein, we report the first study to test gene therapy in an adult murine model of human deafness. Using a single intracochlear injection of an artificial microRNA carried in an AAV vector, we show that RNAi-mediated gene silencing can slow progression of hearing loss, improve inner hair cell survival, and prevent stereocilia bundle degeneration in the mature Beethoven mouse, a model of human TMC1 deafness. The ability to study gene therapy in mature murine ears constitutes a significant step toward its translation to human subjects.

A novel cerebellar commissure and other myelinated axons in the Purkinje cell layer of a pond turtle (Trachemys scripta elegans).

Parallel fibers in the molecular layer of the vertebrate cerebellum mediate slow spike conduction in the transverse plane. In contrast, electrophysiological recordings have indicated that rapid spike conduction exists between the lateral regions of the cerebellar cortex of the red-ear pond turtle (Trachemys scripta). The anatomical basis for this commissure is now examined in that species using neuronal tracing techniques. Fluorescently tagged dextrans and lipophilic carbocyanine dyes placed in one lateral edge of this nonfoliated cortex are transported across the midline of living brains in vitro and along the axonal membranes of fixed tissues, respectively. Surprisingly, the labeled commissural axons traversed the cortex within the Purkinje cell layer, and not in the white matter of the molecular layer or the white matter below the granule cell layer. Unlike thin parallel fibers that exhibit characteristic varicosities, this commissure is composed of smooth axons of large diameter that also extend beyond the cerebellar cortex via the cerebellar peduncles. Double labeling with myelin basic protein antibody demonstrated that these commissural axons are ensheathed with myelin. In contrast to this transverse pathway, an orthogonal myelinated tract was observed along the cerebellar midline. The connections of this transverse commissure with the lateral cerebellum, the vestibular nuclear complex, and the cochlear vestibular ganglia indicate that this commissure plays a role in bilateral vestibular connectivity.

Correlation between afferent rearrangements and behavioral deficits after local excitotoxic insult in the mammalian vestibule: a rat model of vertigo symptoms.

Damage to inner ear afferent terminals is believed to result in many auditory and vestibular dysfunctions. The sequence of afferent injuries and repair, as well as their correlation with vertigo symptoms, remains poorly documented. In particular, information on the changes that take place at the primary vestibular endings during the first hours following a selective insult is lacking. In the present study, we combined histological analysis with behavioral assessments of vestibular function in a rat model of unilateral vestibular excitotoxic insult. Excitotoxicity resulted in an immediate but transient alteration of the balance function that was resolved within a week. Concomitantly, vestibular primary afferents underwent a sequence of structural changes followed by spontaneous repair. Within the first two hours after the insult, a first phase of pronounced vestibular dysfunction coincided with extensive swelling of afferent terminals. In the next 24 h, a second phase of significant but incomplete reduction of the vestibular dysfunction was accompanied by a resorption of swollen terminals and fiber retraction. Eventually, within 1 week, a third phase of complete balance restoration occurred. The slow and progressive withdrawal of the balance dysfunction correlated with full reconstitution of nerve terminals. Competitive re-innervation by afferent and efferent terminals that mimicked developmental synaptogenesis resulted in full re-afferentation of the sensory epithelia. By deciphering the sequence of structural alterations that occur in the vestibule during selective excitotoxic impairment, this study offers new understanding of how a vestibular insult develops in the vestibule and how it governs the heterogeneity of vertigo symptoms.

A Novel ENU-Induced Mutation in Myo6 Causes Vestibular Dysfunction and Deafness.

Mouse N-ethyl-N-nitrosourea (ENU) mutagenesis has generated many useful animal models for human diseases. Here we describe the identification of a novel ENU-induced mouse mutant strain Turner (Tur) that displays circling and headtossing behavior and progressive hearing loss. Tur/Tur homozygous animals lack Preyer and righting reflexes and display severe headtossing and reaching response defect. We mapped the Tur mutation to a critical region of 11 cM on chromosome 9 that includes myosin VI. Direct sequence analysis revealed a c.820A>T substitution in exon 8 of the Myo6 gene that changes amino acid Asn200 to Ile (p.N200I) in the motor domain. Analysis of inner ear hair cells by immunohistochemistry, scanning electron microscopy and histology revealed degeneration of hair cells in the inner ear and structural malformation of the stereocilia in the cochlea of Turner homozygous mutant mice. Our data indicate that this novel mouse strain provides a useful model for future studies on the function of myosin VI in mammalian auditory and non-auditory systems and in human syndromes.

Vestibular dysfunction, altered macular structure and trait localization in A/J inbred mice.

A/J mice develop progressive hearing loss that begins before 1 month of age and is attributed to cochlear hair cell degeneration. Screening tests indicated that this strain also develops early onset vestibular dysfunction and has otoconial deficits. The purpose of this study was to characterize the vestibular dysfunction and macular structural pathology over the lifespan of A/J mice. Vestibular function was measured using linear vestibular evoked potentials (VsEPs). Macular structural pathology was evaluated using light microscopy, scanning electron microscopy, transmission electron microscopy, confocal microscopy and Western blotting. Individually, vestibular functional deficits in mice ranged from mild to profound. On average, A/J mice had significantly reduced vestibular sensitivity (elevated VsEP response thresholds and smaller amplitudes), whereas VsEP onset latency was prolonged compared to age-matched controls (C57BL/6). A limited age-related vestibular functional loss was also present. Structural analysis identified marked age-independent otoconial abnormalities in concert with some stereociliary bundle defects. Macular epithelia were incompletely covered by otoconial membranes with significantly reduced opacity and often contained abnormally large or giant otoconia as well as normal-appearing otoconia. Elevated expression of key otoconins (i.e., otoconin 90, otolin and keratin sulfate proteoglycan) ruled out the possibility of reduced levels contributing to otoconial dysgenesis. The phenotype of A/J was partially replicated in a consomic mouse strain (C57BL/6J-Chr 17(A/J)/NaJ), thus indicating that Chr 17(A/J) contained a trait locus for a new gene variant responsible to some extent for the A/J vestibular phenotype. Quantitative trait locus analysis identified additional epistatic influences associated with chromosomes 1, 4, 9 and X. Results indicate that the A/J phenotype represents a complex trait, and the A/J mouse strain presents a new model for the study of mechanisms underlying otoconial formation and maintenance.

Utricular afferents: morphology of peripheral terminals.

The utricle provides critical information about spatiotemporal properties of head movement. It comprises multiple subdivisions whose functional roles are poorly understood. We previously identified four subdivisions in turtle utricle, based on hair bundle structure and mechanics, otoconial membrane structure and hair bundle coupling, and immunoreactivity to calcium-binding proteins. Here we ask whether these macular subdivisions are innervated by distinctive populations of afferents to help us understand the role each subdivision plays in signaling head movements. We quantified the morphology of 173 afferents and identified six afferent classes, which differ in structure and macular locus. Calyceal and dimorphic afferents innervate one striolar band. Bouton afferents innervate a second striolar band; they have elongated terminals and the thickest processes and axons of all bouton units. Bouton afferents in lateral (LES) and medial (MES) extrastriolae have small-diameter axons but differ in collecting area, bouton number, and hair cell contacts (LES >> MES). A fourth, distinctive population of bouton afferents supplies the juxtastriola. These results, combined with our earlier findings on utricular hair cells and the otoconial membrane, suggest the hypotheses that MES and calyceal afferents encode head movement direction with high spatial resolution and that MES afferents are well suited to signal three-dimensional head orientation and striolar afferents to signal head movement onset.

The periductal channels of the endolymphatic duct, hydrodynamic implications.

OBJECTIVE: To describe the anatomy of a small network of channels surrounding the human endolymphatic duct. STUDY DESIGN: Archival temporal bone sections and a surgical specimen were studied using a variety of techniques. SETTING: Temporal bone laboratory of the House Research Institute. SUBJECTS AND METHODS: Archival temporal bone sections were examined by light microscopy, 3D reconstruction, and immunohistochemical labeling. A surgical specimen was examined using electron microscopy. Sections from temporal bones with blocked endolymphatic ducts or amputated sacs were examined for the manifestations of endolymphatic hydrops. RESULTS: Peri-endolymphatic duct channels were found to extend from the proximal cisternal part of the endolymphatic sac to the supporting tissue of the saccule and utricle. Tissue in the channels, as seen by conventional and electron microscopy, is continuous with and identical with the tissue surrounding the endolymphatic duct. Tissue in the channels labels with the S100 antibody similar to the spiral ligament and supporting tissue of the vestibular end organs and suggests a neural crest origin, as did the presence of melanocytes. Obstruction of the endolymphatic duct resulted in endolymphatic hydrops whereas amputation of the sac did not. CONCLUSION: Endolymph is probably absorbed in the endolymphatic duct. The peri-endolymphatic duct channels that extend from the proximal sac to the supporting tissue of the saccule label with the S100 antibody and contain melanocytes suggest a neural crest origin and involvement in fluid and potassium hydrodynamics similar to those described for the similarly staining spiral ligament of the cochlea.

Vestibulotoxic properties of potential metabolites of allylnitrile.

This study addressed the hypothesis that epoxidation of the double bond in allylnitrile mediates its vestibular toxicity, directly or after subsequent metabolism by epoxide hydrolases. The potential metabolites 3,4-epoxybutyronitrile and 3,4-dihydroxybutyronitrile were synthesized and characterized. In aqueous solutions containing sodium or potassium ions, 3,4-epoxybutyronitrile rearranged to 4-hydroxybut-2-enenitrile, and this compound was also isolated for study. Male adult Long-Evans rats were exposed to allylnitrile or 3,4-epoxybutyronitrile by bilateral transtympanic injection, and vestibular toxicity was assessed using a behavioral test battery and scanning electron microscopy (SEM) observation of the sensory epithelia. Overt vestibular toxicity was caused by 3,4-epoxybutyronitrile at 0.125 mmol/ear and by allylnitrile in some animals at 0.25 mmol/ear. Additional rats were exposed by unilateral transtympanic injection. In these studies, behavioral evidences and SEM observations demonstrated unilateral vestibular toxicity after 0.125 mmol of 3,4-epoxybutyronitrile and bilateral vestibular toxicity after 0.50 mmol of allylnitrile. However, 0.25 mmol of allylnitrile did not cause vestibular toxicity. Unilateral administration of 0.50 mmol of 3,4-dihydroxybutyronitrile or 4-hydroxybut-2-enenitrile caused no vestibular toxicity. The four compounds were also evaluated in the mouse utricle explant culture model. In 8-h exposure experiments, hair cells completely disappeared after 3,4-epoxybutyronitrile at concentrations of 325 or 450µM but not at concentrations of 150µM or lower. In contrast, no difference from controls was recorded in utricles exposed to 450µM or 1.5mM of allylnitrile, 3,4-dihydroxybutyronitrile, or 4-hydroxybut-2-enenitrile. Taken together, the present data support the hypothesis that 3,4-epoxybutyronitrile is the active metabolite of allylnitrile for vestibular toxicity.

Reduced systemic toxicity and preserved vestibular toxicity following co-treatment with nitriles and CYP2E1 inhibitors: a mouse model for hair cell loss.

Several nitriles, including allylnitrile and cis-crotononitrile, have been shown to be ototoxic and cause hair cell degeneration in the auditory and vestibular sensory epithelia of mice. However, these nitriles can also be lethal due in large part to the microsomal metabolic release of cyanide, which is mostly dependent on the activity of the 2E1 isoform of the cytochrome P450 (CYP2E1). In this study, we co-administered mice with a nitrile and, to reduce their lethal effects, a selective CYP2E1 inhibitor: diallylsulfide (DAS) or trans-1,2-dichloroethylene (TDCE). Both in female 129S1/SvImJ (129S1) mice co-treated with DAS and cis-crotononitrile and in male RjOrl:Swiss/CD-1 (Swiss) mice co-treated with TDCE and allylnitrile, the nitrile caused a dose-dependent loss of vestibular function, as assessed by a specific behavioral test battery, and of hair cells, as assessed by hair bundle counts using scanning electron microscopy. In the experiments, the CYP2E1 inhibitors provided significant protection against the lethal effects of the nitriles and did not diminish the vestibular toxicity as assessed by behavioral effects in comparison to animals receiving no inhibitor. Additional experiments using a single dose of allylnitrile demonstrated that TDCE does not cause hair cell loss on its own and does not modify the vestibular toxicity of the nitrile in either male or female 129S1 mice. In all the experiments, high vestibular dysfunction scores in the behavioral test battery predicted extensive to complete loss of hair cells in the utricles. This provides a means of selecting animals for subsequent studies of vestibular hair cell regeneration or replacement.

Characterization of a novel ENU-generated myosin VI mutant mouse strain with congenital deafness and vestibular dysfunction.

Myosin VI (Myo6) is known to play an important role in the mammalian auditory and vestibular systems. We have identified a novel N-ethyl-N-nitrosourea mutagenised mouse strain, charlie, carrying an intronic Myo6 splice site mutation. This mutation (IVS5+5G > A) results in skipping of exon 5, and is predicted to cause a frameshift and premature termination of the protein. We detected essentially no Myo6 transcript in tissue from charlie homozygous mutant mice (Myo6(chl/chl)). Myo6(chl/chl) mice exhibit vestibular dysfunction and profound hearing impairment when first tested at four weeks of age. Analysis of vestibular and cochlear hair cells by scanning electron microscopy and immunohistochemistry revealed highly disorganised hair bundles with irregular orientation and kinocilium position at postnatal stage P2-P3. Within a few weeks, the majority of hair cell stereocilia are missing, or fused and elongated, and degeneration of the sensory epithelium occurs. This novel mouse strain will be an important resource in elucidating the role myosin VI plays in the mammalian auditory system, as well as its non-auditory functions.

Retinoic acid deficiency impairs the vestibular function.

The retinaldehyde dehydrogenase 3 (Raldh3) gene encodes a major retinoic acid synthesizing enzyme and is highly expressed in the inner ear during embryogenesis. We found that mice deficient in Raldh3 bear severe impairment in vestibular functions. These mutant mice exhibited spontaneous circling/tilted behaviors and performed poorly in several vestibular-motor function tests. In addition, video-oculography revealed a complete loss of the maculo-ocular reflex and a significant reduction in the horizontal angular vestibulo-ocular reflex, indicating that detection of both linear acceleration and angular rotation were compromised in the mutants. Consistent with these behavioral and functional deficiencies, morphological anomalies, characterized by a smaller vestibular organ with thinner semicircular canals and a significant reduction in the number of otoconia in the saccule and the utricle, were consistently observed in the Raldh3 mutants. The loss of otoconia in the mutants may be attributed, at least in part, to significantly reduced expression of Otop1, which encodes a protein known to be involved in calcium regulation in the otolithic organs. Our data thus reveal a previously unrecognized role of Raldh3 in structural and functional development of the vestibular end organs.

Localization of aquaporins 1, 2, and 3 and vasopressin type 2 receptor in the mouse inner ear.

CONCLUSION: It is suggested that aquaporins (AQPs) 1, 2, and 3, and vasopressin type 2 receptors (V2Rs) in the fluid transporting cells, such as stria vascularis, vestibular dark and transitional cells, and endolymphatic sac epithelial cells, have an important role in fluid transport in the inner ear, while those in the sensory and ganglion cells may play a functional role in the sensory cell transduction system. OBJECTIVE: To analyze expression of AQP1, AQP2, and AQP3 as well as V2Rs in the normal mouse inner ear. METHODS: CBA/J mice were used in this study. Localization of AQP1, AQP2, AQP3, and V2Rs in the inner ear, i.e. cochlea, vestibular end organs, and endolymphatic sac, was investigated by immunohistochemistry. RESULTS: The results show that AQP1, AQP2, AQP3, and V2Rs are abundantly distributed in many inner ear structures, i.e. stria vascularis, inner and outer hair cells, spiral ganglion cells, vestibular sensory and ganglion cells, vestibular dark and transitional cells, and the endolymphatic sac.

Morphology and innervation of the vestibular lagena in pigeons.

The morphological characteristics of the pigeon lagena were examined using histology, scanning electron microscopy, and biotinylated dextran amine (BDA) neural tracers. The lagena epithelium was observed to lie partially in a parasagittal plane, but was also U-shaped with orthogonal (lateral) directed tips. Hair cell planar polarities were oriented away from a central reversal line that ran nearly the length of the epithelium. Similar to the vertebrate utricle and saccule, three afferent classes were observed based upon their terminal innervation pattern, which include calyx, dimorph, and bouton fibers. Calyx and dimorph afferents innervated the striola region of the lagena, whereas bouton afferents innervated the extrastriola and a small region of the central striola known as the type II band. Calyx units had large calyceal terminal structures that innervated only type I hair cells. Dimorph afferents innervated both type I and II hair cells, with calyx and bouton terminals. Bouton afferents had the largest most complex innervation patterns and the greatest terminal areas contacting many hair cells.

Severe vestibular dysfunction and altered vestibular innervation in mice lacking prosaposin.

Prosaposin, a precursor of four glycoprotein activators (saposin A, B, C and D) for lysosomal hydrolases, has previously been shown to be important for normal adult cochlear innervation and the maintenance of normal hearing. In these studies, we now investigate prosaposin in normal vestibular epithelium and the functional impairment of balance caused by prosaposin ablation. In normal mice, prosaposin is localized to all 3 vestibular end-organs (ampullae, saccule, and utricle) and Scarpa's ganglion by RT-PCR, Western blot analysis and immunofluorescence. Ablation of prosaposin function caused severe vestibular dysfunction on a battery of behavioral tasks. Histologically, the KO mice demonstrated an exuberant cellular proliferation below the vestibular hair cells with disruption of the supporting cells. Electron microscopy further demonstrated inclusion bodies and cellular proliferation disturbing the normal neuroepithelial structure of the vestibular end-organs. Lastly, immunofluorescence (neurofilament 200 and synaptophysin) staining suggests that this cellular proliferation corresponds to afferent and efferent neuronal overgrowth. These data suggest that prosaposin plays a role not only in the maintenance of normal hearing but also an important role in the neuronal maturation processes of the vestibular sensory epithelium and the maintenance of normal vestibular system function.

Roles of the espin actin-bundling proteins in the morphogenesis and stabilization of hair cell stereocilia revealed in CBA/CaJ congenic jerker mice.

Hearing and vestibular function depend on mechanosensory staircase collections of hair cell stereocilia, which are produced from microvillus-like precursors as their parallel actin bundle scaffolds increase in diameter and elongate or shorten. Hair cell stereocilia contain multiple classes of actin-bundling protein, but little is known about what each class contributes. To investigate the roles of the espin class of actin-bundling protein, we used a genetic approach that benefited from a judicious selection of mouse background strain and an examination of the effects of heterozygosity. A congenic jerker mouse line was prepared by repeated backcrossing into the inbred CBA/CaJ strain, which is known for excellent hearing and minimal age-related hearing loss. We compared stereocilia in wild-type CBA/CaJ mice, jerker homozygotes that lack espin proteins owing to a frameshift mutation in the espin gene, and jerker heterozygotes that contain reduced espin levels. The lack of espins radically impaired stereociliary morphogenesis, resulting in stereocilia that were abnormally thin and short, with reduced differential elongation to form a staircase. Mean stereociliary diameter did not increase beyond ∼0.10-0.14 µm, making stereocilia ∼30%-60% thinner than wild type and suggesting that they contained ∼50%-85% fewer actin filaments. These characteristics indicate a requirement for espins in the appositional growth and differential elongation of the stereociliary parallel actin bundle and fit the known biological activities of espins in vitro and in transfected cells. The stereocilia of jerker heterozygotes showed a transient proximal-distal tapering suggestive of haploinsufficiency and a slowing of morphogenesis that revealed previously unrecognized assembly steps and intermediates. The lack of espins also led to a region-dependent degeneration of stereocilia involving shortening and collapse. We conclude that the espin actin-bundling proteins are required for the assembly and stabilization of the stereociliary parallel actin bundle.

Organophosphate-related ototoxicity: Description of the vestibulocochlear system ultrastructural aspects of guinea pigs.

UNLABELLED: Organophosphate toxic agents are used in agriculture and are currently part of the group of toxic agents which can lead to hearing loss, in which we have solvents, metals and asphyxiation agents. AIM: To analyze the acute ototoxic action of a group of organophosphate agents in the vestibulo-cochlear system. This is a prospective experimental study. MATERIALS AND METHODS: We used male albino guinea pigs, broken down into three groups, to which we provided distilled water (group 1 - control), agrotoxic agent - 0.3 mg/Kg/day (group 2), agrotoxic - 3 mg/Kg/day (group 3), during 7 seven consecutive days. The most used agrotoxic agent was Tamaron BR (metamidophos). The anatomical evaluation of the cochlea, saccule and utricle was carried out by means of electronic scanning microscopy after the use of the agrotoxic agent. RESULTS: The guinea pigs submitted to the organophosphate presented cochlear morphological alterations with lesions on the three turns analyzed, as well as cilia alterations in the saccule and utricle, intensified according to the agent dosage. CONCLUSION: The morphological alterations seen in the hair cells exposed to daily doses of organophosphate promote evidences of an acute deleterious effect of agrotoxic agents on the vestibulo-cochlear system.

Ototoxicidade por organofosforados: descrição dos aspectos ultraestruturais do sistema vestibulococlear de cobaias / Organophosphate-related ototoxicity: Description of the vestibulocochlear system ultrastructural aspects of guinea pigs

Os agrotóxicos organofosforados são amplamente utilizados na agricultura, e atualmente fazem parte do grupo de agentes químicos que podem levar à perda auditiva, no qual já estavam incluídos os solventes, os metais e os asfixiantes. OBJETIVO: Analisar a ação ototóxica aguda de um agrotóxico do grupo dos organofosforados na citoarquitetura do sistema vestibulococlear. Trata-se de um estudo experimental prospectivo. MATERIAL E MÉTODO: Foram utilizadas cobaias albinas machos, divididas em três grupos, nos quais se administrou água destilada (grupo 1 - controle), agrotóxico - 0,3mg/Kg/dia (grupo 2), agrotóxico - 3 mg/Kg/dia (grupo 3), durante sete dias consecutivos. O agrotóxico utilizado foi Tamaron BR (metamidofós). A avaliação anatômica da cóclea, sáculo e utrículo foi realizada através da microscopia eletrônica de varredura, após o período de aplicação do agrotóxico. RESULTADOS: As cobaias submetidas ao organofosforado apresentaram alterações morfológicas cocleares, com lesões nas três espiras analisadas, bem como alterações ciliares de sáculo e utrículo, intensificadas de acordo com a dosagem recebida do agente. CONCLUSÃO: As alterações morfológicas observadas nas células ciliadas nos grupos expostos a doses diárias de organofosforado promovem evidências de um efeito agudo degradante dos agrotóxicos no sistema vestibulococlear.

Organophosphate toxic agents are used in agriculture and are currently part of the group of toxic agents which can lead to hearing loss, in which we have solvents, metals and asphyxiation agents. AIM: to analyze the acute ototoxic action of a group of organophosphate agents in the vestibulo-cochlear system. This is a prospective experimental study. MATERIALS AND METHODS: we used male albino guinea pigs, broken down into three groups, to which we provided distilled water (group 1 - control), agrotoxic agent - 0.3mg/Kg/day (group 2), agrotoxic - 3 mg/Kg/day (group 3), during 7 seven consecutive days. The most used agrotoxic agent was Tamaron BR (metamidophos). The anatomical evaluation of the cochlea, saccule and utricle was carried out by means of electronic scanning microscopy after the use of the agrotoxic agent. RESULTS: the guinea pigs submitted to the organophosphate presented cochlear morphological alterations with lesions on the three turns analyzed, as well as cilia alterations in the saccule and utricle, intensified according to the agent dosage. CONCLUSION: the morphological alterations seen in the hair cells exposed to daily doses of organophosphate promote evidences of an acute deleterious effect of agrotoxic agents on the vestibulo-cochlear system.

Vestibular morphology in the German Waltzing guinea pig.

OBJECTIVE: The German waltzing guinea pig is a special strain of animal with a recessively inherited inner ear defect, resulting in deafness and a severe vestibular dysfunction. The hearing loss in the cochlea of the German strain is a result of a collapse of the Reissner membrane and the absence of scala media. The vestibular organ has not yet been described. MATERIALS AND METHODS: German waltzing guinea pigs (homozygote and heterozygote) of different ages ranging from embryologic age 25 days to adulthood were investigated. The living animals were tested with four different vestibular tests, and the fetuses were controlled according to breeding. The morphology of the vestibular parts (ampulla, saccule, and utricle) was observed by using the light and transmission electron microscopy. RESULTS: Collapse of the membranous labyrinth was found already at embryologic age 50 days and progressed over time. Vestibular dysfunction was noted already from birth. CONCLUSIONS: Vestibular atelectasis has been shown to have the same morphology as the reported vestibular dysfunction in the German waltzing guinea pig. Owing to this similarity, this animal can be a good model for vestibular research.

A rapid approach to ultrastructural evaluation and DNA analysis of the vestibular labyrinth and ganglion in dogs and cats.

The vestibular labyrinth is the organ for sensation of equilibrium. It is part of the inner ear and located in the caudodorsal aspect of the temporal bone which makes it very difficult to access. This study evaluated a preparation technique in cats and dogs for morphological and DNA analysis. The study included 44 temporal bones of 14 cats and 11 dogs collected within 48h after death. Preparation was performed after peri-/endolymphatic injection of Fast-Green-FCF through the fenestra vestibuli to visualize the membranous labyrinth. The vestibular nerve, including its ganglion, and the vestibular labyrinth were exposed by drilling and cracking of the petrous temporal bone along the meatus acusticus internus. The posterior ampulla was collected for histology and transmission electron microscopy whereas the vestibular nerve, the utriculus, sacculus, and the lateral and anterior ampullae were harvested for subsequent DNA analysis. Histology and electron microscopy showed well-preserved cells. A total DNA amount of 4753+/-1502ng in cats and 5865+/-2911ng in dogs was retrieved from the ganglion, and 2390+/-561ng in cats and 2544+/-1277ng in dogs, respectively, from membranous vestibular organs. Polymerase chain reaction of a 229 base pair product of the Gapdh-gene proved for presence of amplifiable DNA. Taken together, mechanical bone removal after Fast-Green-FCF injection allows for reliable gross, microscopic and ultrastructural examination of the feline and canine vestibular labyrinth, and it does not interfere with DNA analysis via PCR. This technique is feasible for multimodal investigation of the vestibular labyrinth retrieved from individual necropsy cases.