Persistência do efeito otoprotetor: qual a duração da otoproteção à amicacina?

Há comprovação de que o fenômeno de resistência ocorre quando a dose não lesiva da amicacina protege as células ciliadas contra a ototoxicidade da própria amicacina. OBJETIVO: O objetivo deste trabalho é verificar se o fenômeno de resistência é temporalmente persistente. MÉTODO: Estudo experimental com 14 cobaias albinas (Cavia porcellus) divididas em três grupos. Avaliação da função auditiva por emissões otoacústicas por produto de distorção (EOAPD): na pré-exposição à amicacina, no 15º dia de aplicação da dose não lesiva, no final da aplicação da dose lesiva e antes da decapitação. RESULTADOS: O Grupo A (controle) apresentou função auditiva e padrão histológico normais. No Grupo B (amicacina 20mg/kg/dia intramuscular por 30 dias e dose lesiva (400 mg/kg/dia) por 12 dias) e no Grupo C (mesmo esquema do grupo B, porém mantidos por 60 dias e sacrificados), as OEA-PD confirmaram função auditiva normal no período pré-exposição e manutenção do padrão após dose não lesiva, porém, houve perda importante da função auditiva após término do período de aplicação da dose lesiva. CONCLUSÃO: Não houve manutenção do fenômeno da autodefesa estendida por um período de 30 a 60 dias após a aplicação de doses lesivas de amicacina.

Il coniglio come modello sperimentale per lo studio degli effetti neurologici indotti da Cobalto

Sono stati studiati gli effetti tossici dell’esposizione cronica a cobalto e cromo. In passato, questa tossicità, che colpiva lavoratori esposti per ragioni occupazionali, è stata un problema molto sentito. Tuttavia, recenti pubblicazioni hanno descritto una specifica tossicità mediata da elevati livelli di cobalto e cromo, anche in pazienti portatori di protesi metalliche, quali gli impianti d’anca. Anche se sintomi clinici tra cui, cecità, sordità e neuropatia periferica, suggeriscono uno specifico neurotropismo, ancora poco è conosciuto delle basi neuropatologiche di questo processo ed oltretutto non ne è ancora stata apportata un’evidenza sperimentale. In questo progetto di ricerca, quindi, si è voluto approfondire il meccanismo patogenetico da cui scaturiscono tali sintomi neurologici, utilizzando come modello sperimentale il coniglio. Conigli New Zealand White sono stati trattati con dosi endovenose ripetute di cobalto e cromo, inoculati singolarmente od in associazione tra loro. Nessuna evidente alterazione clinica o patologica è stata associata alla somministrazione di solo cromo, nonostante gli elevati livelli in sangue e tessuti, mentre i trattati con cobalto-cromo o solo cobalto hanno mostrato segni clinici gravanti sul sistema vestibolo-cocleare; il cobalto, quindi, è stato identificato come il maggiore elemento scatenante neurotossicità. Inoltre all’esame istopatologico gli animali hanno mostrato severa deplezione delle cellule gangliari retiniche e cocleari, assieme a danno al nervo ottico e perdita di cellule sensitive capellute dell’orecchio. È risultato infine evidente che la gravità delle alterazioni è stata correlata al dosaggio ed al tempo di esposizione; dati questi che confermano, quindi, le precedenti osservazioni fatte su pazienti umani esposti a rilascio abnorme di cobalto e cromo da usura di protesi d’anca. È stato ipotizzato che il cobalto agisca sui mitocondri provocando l’incremento di produzione di specie reattive dell’ossigeno e il rilascio di fattori proapoptotici, causando sulle cellule neuronali un danno proporzionale al loro fabbisogno energetico e grado di mielinizzazione.

[Stem-cell-based approaches for treating inner ear diseases]

The capacity of stem cells to regenerate lost tissue cells has gained recognition among physicians. Despite the successful use of blood stem cells for treating blood cancers, other stem cell types have not yet been widely introduced into clinical practice. Therapy options involving stem cells for inner ear diseases consequently have not been implemented. Nonetheless, several reports have recently been published describing the generation of morphologically and immunologically distinctive inner ear cell types-such as hair cells, supporting cells, and spiral ganglion neurons-from stem cells. Although promising, all of these studies still lack functional results regarding hearing restoration or vestibular function.

Estrogen receptor subtype beta2 is involved in neuromast development in zebrafish (Danio rerio) larvae

Estrogens are known to play a role in both reproductive and non-reproductive functions in mammals. Estrogens and their receptors are involved in the development of the central nervous system (brain development, neuronal survival and differentiation) as well as in the development of the peripheral nervous system (sensory-motor behaviors). In order to decipher possible functions of estrogens in early development of the zebrafish sensory system, we investigated the role of estrogen receptor beta(2) (ERbeta(2)) by using a morpholino (MO) approach blocking erbeta(2) RNA translation. We further investigated the development of lateral line organs by cell-specific labeling, which revealed a disrupted development of neuromasts in morphants. The supporting cells developed and migrated normally. Sensory hair cells, however, were absent in morphants' neuromasts. Microarray analysis and subsequent in situ hybridizations indicated an aberrant activation of the Notch signaling pathway in ERbeta(2) morphants. We conclude that signaling via ERbeta(2) is essential for hair cell development and may involve an interaction with the Notch signaling pathway during cell fate decision in the neuromast maturation process.

Zebrafish (Danio rerio) neuromast: promising biological endpoint linking developmental and toxicological studies

Aquatic toxicology is facing the challenge to assess the impact of complex mixtures of compounds on diverse biological endpoints. So far, ecotoxicology focuses mainly on apical endpoints such as growth, lethality and reproduction, but does not consider sublethal toxic effects that may indirectly cause ecological effects. One such sublethal effect is toxicant-induced impairment of neurosensory functions which will affect important behavioural traits of exposed organisms. Here, we critically review the mechanosensory lateral line (LL) system of zebrafish as a model to screen for chemical effects on neurosensory function of fish in particular and vertebrates in general. The LL system consists of so-called neuromasts, composed of centrally located sensory hair cells, and surrounding supporting cells. The function of neuromasts is the detection of water movements that is essential for the fish's ability to detect prey, to escape predator, to socially interact or to show rheotactic behaviour. Recent advances in the study of these organs provided researchers with a broad area of molecular tools for easy and rapid detection of neuromasts dysfunction and/or disturbed development. Further, genes involved in neuromasts differentiation have been identified using auditory/mechanosensory mutants and morphants. A number of environmental toxicants including metals and pharmaceuticals have been shown to affect neuromasts development and/or function. The use of the LL organ for toxicological studies offers the advantage to integrate the available profound knowledge on developmental biology of the neuromasts with the study of chemical toxicity. This combination may provide a powerful tool in environmental risk assessment.

Integrity and regeneration of mechanotransduction machinery regulate aminoglycoside entry and sensory cell death

Sound perception requires functional hair cell mechanotransduction (MET) machinery, including the MET channels and tip-link proteins. Prior work showed that uptake of ototoxic aminoglycosides (AG) into hair cells requires functional MET channels. In this study, we examined whether tip-link proteins, including Cadherin 23 (Cdh23), regulate AG entry into hair cells. Using time-lapse microscopy on cochlear explants, we found rapid uptake of gentamicin-conjugated Texas Red (GTTR) into hair cells from three-day-old Cdh23(+/+) and Cdh23(v2J/+) mice, but failed to detect GTTR uptake in Cdh23(v2J/v2J) hair cells. Pre-treatment of wildtype cochleae with the calcium chelator 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA) to disrupt tip-links also effectively reduced GTTR uptake into hair cells. Both Cdh23(v2J/v2J) and BAPTA-treated hair cells were protected from degeneration caused by gentamicin. Six hours after BAPTA treatment, GTTR uptake remained reduced in comparison to controls; by 24 hours, drug uptake was comparable between untreated and BAPTA-treated hair cells, which again became susceptible to cell death induced by gentamicin. Together, these results provide genetic and pharmacologic evidence that tip-links are required for AG uptake and toxicity in hair cells. Because tip-links can spontaneously regenerate, their temporary breakage offers a limited time window when hair cells are protected from AG toxicity.

5-HT and GABA modulate intrinsic excitability of type I interneurons in Hermissenda.

The sensory neurons (photoreceptors) in the visual system of Hermissenda are one site of plasticity produced by Pavlovian conditioning. A second site of plasticity produced by conditioning is the type I interneurons in the cerebropleural ganglia. Both photoreceptors and statocyst hair cells of the graviceptive system form monosynaptic connections with identified type I interneurons. Two proposed neurotransmitters in the graviceptive system, serotonin (5-HT) and gamma-aminobutyric acid (GABA), have been shown to modify synaptic strength and intrinsic neuronal excitability in identified photoreceptors. However, the potential role of 5-HT and GABA in plasticity of type I interneurons has not been investigated. Here we show that 5-HT increased the peak amplitude of light-evoked complex excitatory postsynaptic potentials (EPSPs), enhanced intrinsic excitability, and increased spike activity of identified type I(e(A)) interneurons. In contrast, 5-HT decreased spike activity and intrinsic excitability of type I(e(B)) interneurons. The classification of two categories of type I(e) interneurons was also supported by the observation that 5-HT produced opposite effects on whole cell steady-state outward currents in type I(e) interneurons. Serotonin produced a reduction in the amplitude of light-evoked complex inhibitory PSPs (IPSPs), increased spontaneous spike activity, decreased intrinsic excitability, and depolarized the resting membrane potential of identified type I(i) interneurons. In contrast to the effects of 5-HT, GABA produced inhibition in both types of I(e) interneurons and type I(i) interneurons. These results show that 5-HT and GABA can modulate the intrinsic excitability of type I interneurons independent of the presynaptic effects of the same transmitters on excitability and synaptic efficacy of photoreceptors.

The pre- and post-somatic segments of the human type I spiral ganglion neurons--structural and functional considerations related to cochlear implantation.

Human auditory nerve afferents consist of two separate systems; one is represented by the large type I cells innervating the inner hair cells and the other one by the small type II cells innervating the outer hair cells. Type I spiral ganglion neurons (SGNs) constitute 96% of the afferent nerve population and, in contrast to other mammals, their soma and pre- and post-somatic segments are unmyelinated. Type II nerve soma and fibers are unmyelinated. Histopathology and clinical experience imply that human SGNs can persist electrically excitable without dendrites, thus lacking connection to the organ of Corti. The biological background to this phenomenon remains elusive. We analyzed the pre- and post-somatic segments of the type I human SGNs using immunohistochemistry and transmission electron microscopy (TEM) in normal and pathological conditions. These segments were found surrounded by non-myelinated Schwann cells (NMSCs) showing strong intracellular expression of laminin-β2/collagen IV. These cells also bordered the perikaryal entry zone and disclosed surface rugosities outlined by a folded basement membrane (BM) expressing laminin-β2 and collagen IV. It is presumed that human large SGNs are demarcated by three cell categories: (a) myelinated Schwann cells, (b) NMSCs and (c) satellite glial cells (SGCs). Their BMs express laminin-β2/collagen IV and reaches the BM of the sensory epithelium at the habenula perforata. We speculate that the NMSCs protect SGNs from further degeneration following dendrite loss. It may give further explanation why SGNs can persist as electrically excitable monopolar cells even after long-time deafness, a blessing for the deaf treated with cochlear implantation.

Response profiles of murine spiral ganglion neurons on multi-electrode arrays.

OBJECTIVE Cochlear implants (CIs) have become the gold standard treatment for deafness. These neuroprosthetic devices feature a linear electrode array, surgically inserted into the cochlea, and function by directly stimulating the auditory neurons located within the spiral ganglion, bypassing lost or not-functioning hair cells. Despite their success, some limitations still remain, including poor frequency resolution and high-energy consumption. In both cases, the anatomical gap between the electrode array and the spiral ganglion neurons (SGNs) is believed to be an important limiting factor. The final goal of the study is to characterize response profiles of SGNs growing in intimate contact with an electrode array, in view of designing novel CI devices and stimulation protocols, featuring a gapless interface with auditory neurons. APPROACH We have characterized SGN responses to extracellular stimulation using multi-electrode arrays (MEAs). This setup allows, in our view, to optimize in vitro many of the limiting interface aspects between CIs and SGNs. MAIN RESULTS Early postnatal mouse SGN explants were analyzed after 6-18 days in culture. Different stimulation protocols were compared with the aim to lower the stimulation threshold and the energy needed to elicit a response. In the best case, a four-fold reduction of the energy was obtained by lengthening the biphasic stimulus from 40 μs to 160 μs. Similarly, quasi monophasic pulses were more effective than biphasic pulses and the insertion of an interphase gap moderately improved efficiency. Finally, the stimulation with an external electrode mounted on a micromanipulator showed that the energy needed to elicit a response could be reduced by a factor of five with decreasing its distance from 40 μm to 0 μm from the auditory neurons. SIGNIFICANCE This study is the first to show electrical activity of SGNs on MEAs. Our findings may help to improve stimulation by and to reduce energy consumption of CIs and thereby contribute to the development of fully implantable devices with better auditory resolution in the future.

Fine control of drug delivery for cochlear implant applications

Cochlear implants are neuroprostheses that are inserted into the inner ear to directly electrically stimulate the auditory nerve, thus replacing lost cochlear receptors, the hair cells. The reduction of the gap between electrodes and nerve cells will contribute to technological solutions simultaneously increasing the frequency resolution, the sound quality and the amplification of the signal. Recent findings indicate that neurotrophins (NTs) such as brain derived neurotrophic factor (BDNF) stimulate the neurite outgrowth of auditory nerve cells by activating Trk receptors on the cellular surface (1–3). Furthermore, small-size TrkB receptor agonists such as di-hydroxyflavone (DHF) are now available, which activate the TrkB receptor with similar efficiency as BDNF, but are much more stable (4). Experimentally, such molecules are currently used to attract nerve cells towards, for example, the electrodes of cochlear implants. This paper analyses the scenarios of low dose aspects of controlled release of small-size Trk receptor agonists from the coated CI electrode array into the inner ear. The control must first ensure a sufficient dose for the onset of neurite growth. Secondly, a gradient in concentration needs to be maintained to allow directive growth of neurites through the perilymph-filled gap towards the electrodes of the implant. We used fluorescein as a test molecule for its molecular size similarity to DHF and investigated two different transport mechanisms of drug dispensing, which both have the potential to fulfil controlled low-throughput drug-deliverable requirements. The first is based on the release of aqueous fluorescein into water through well-defined 60-μm size holes arrays in a membrane by pure osmosis. The release was both simulated using the software COMSOL and observed experimentally. In the second approach, solid fluorescein crystals were encapsulated in a thin layer of parylene (PPX), hence creating random nanometer-sized pinholes. In this approach, the release occurred due to subsequent water diffusion through the pinholes, dissolution of the fluorescein and then release by out-diffusion. Surprisingly, the release rate of solid fluorescein through the nanoscopic scale holes was found to be in the same order of magnitude as for liquid fluorescein release through microscopic holes.

Comparing in vitro, in situ, and in vivo experimental data in a three-dimensional model of mammalian cochlear mechanics

Normal mammalian hearing is refined by amplification of the motion of the cochlear partition. This partition, comprising the organ of Corti sandwiched between the basilar and tectorial membranes, contains the outer hair cells that are thought to drive this amplification process. Force generation by outer hair cells has been studied extensively in vitro and in situ, but, to understand cochlear amplification fully, it is necessary to characterize the role played by each of the components of the cochlear partition in vivo. Observations of cochlear partition motion in vivo are severely restricted by its inaccessibility and sensitivity to surgical trauma, so, for the present study, a computer model has been used to simulate the operation of the cochlea under different experimental conditions. In this model, which uniquely retains much of the three-dimensional complexity of the real cochlea, the motions of the basilar and tectorial membranes are fundamentally different during in situ- and in vivo-like conditions. Furthermore, enhanced outer hair cell force generation in vitro leads paradoxically to a decrease in the gain of the cochlear amplifier during sound stimulation to the model in vivo. These results suggest that it is not possible to extrapolate directly from experimental observations made in vitro and in situ to the normal operation of the intact organ in vivo.

Gene disruption of p27Kip1 allows cell proliferation in the postnatal and adult organ of Corti

Hearing loss is most often the result of hair-cell degeneration due to genetic abnormalities or ototoxic and traumatic insults. In the postembryonic and adult mammalian auditory sensory epithelium, the organ of Corti, no hair-cell regeneration has ever been observed. However, nonmammalian hair-cell epithelia are capable of regenerating sensory hair cells as a consequence of nonsensory supporting-cell proliferation. The supporting cells of the organ of Corti are highly specialized, terminally differentiated cell types that apparently are incapable of proliferation. At the molecular level terminally differentiated cells have been shown to express high levels of cell-cycle inhibitors, in particular, cyclin-dependent kinase inhibitors [Parker, S. B., et al. (1995) Science 267, 1024–1027], which are thought to be responsible for preventing these cells from reentering the cell cycle. Here we report that the cyclin-dependent kinase inhibitor p27Kip1 is selectively expressed in the supporting-cell population of the organ of Corti. Effects of p27Kip1-gene disruption include ongoing cell proliferation in postnatal and adult mouse organ of Corti at time points well after mitosis normally has ceased during embryonic development. This suggests that release from p27Kip1-induced cell-cycle arrest is sufficient to allow supporting-cell proliferation to occur. This finding may provide an important pathway for inducing hair-cell regeneration in the mammalian hearing organ.

The Notch ligand Jagged1 is required for inner ear sensory development

Within the mammalian inner ear there are six separate sensory regions that subserve the functions of hearing and balance, although how these sensory regions become specified remains unknown. Each sensory region is populated by two cell types, the mechanosensory hair cell and the supporting cell, which are arranged in a mosaic in which each hair cell is surrounded by supporting cells. The proposed mechanism for creating the sensory mosaic is lateral inhibition mediated by the Notch signaling pathway. However, one of the Notch ligands, Jagged1 (Jag1), does not show an expression pattern wholly consistent with a role in lateral inhibition, as it marks the sensory patches from very early in their development—presumably long before cells make their final fate decisions. It has been proposed that Jag1 has a role in specifying sensory versus nonsensory epithelium within the ear [Adam, J., Myat, A., Roux, I. L., Eddison, M., Henrique, D., Ish-Horowicz, D. & Lewis, J. (1998) Development (Cambridge, U.K.) 125, 4645–4654]. Here we provide experimental evidence that Notch signaling may be involved in specifying sensory regions by showing that a dominant mouse mutant headturner (Htu) contains a missense mutation in the Jag1 gene and displays missing posterior and sometimes anterior ampullae, structures that house the sensory cristae. Htu/+ mutants also demonstrate a significant reduction in the numbers of outer hair cells in the organ of Corti. Because lateral inhibition mediated by Notch predicts that disruptions in this pathway would lead to an increase in hair cells, we believe these data indicate an earlier role for Notch within the inner ear.

Notch signaling in the development of the inner ear: Lessons from Drosophila

The sensory patches in the ear of a vertebrate can be compared with the mechanosensory bristles of a fly. This comparison has led to the discovery that lateral inhibition mediated by the Notch cell–cell signaling pathway, first characterized in Drosophila and crucial for bristle development, also has a key role in controlling the pattern of sensory hair cells and supporting cells in the ear. We review the arguments for considering the sensory patches of the vertebrate ear and bristles of the insect to be homologous structures, evolved from a common ancestral mechanosensory organ, and we examine more closely the role of Notch signaling in each system. Using viral vectors to misexpress components of the Notch pathway in the chick ear, we show that a simple lateral-inhibition model based on feedback regulation of the Notch ligand Delta is inadequate for the ear just as it is for the fly bristle. The Notch ligand Serrate1, expressed in supporting cells in the ear, is regulated by lateral induction, not lateral inhibition; commitment to become a hair cell is not simply controlled by levels of expression of the Notch ligands Delta1, Serrate1, and Serrate2 in the neighbors of the nascent hair cell; and at least one factor, Numb, capable of blocking reception of lateral inhibition is concentrated in hair cells. These findings reinforce the parallels between the vertebrate ear and the fly bristle and show how study of the insect system can help us understand the vertebrate.

Cochlear mechanisms from a phylogenetic viewpoint

The hearing organ of the inner ear was the last of the paired sense organs of amniotes to undergo formative evolution. As a mechanical sensory organ, the inner-ear hearing organ's function depends highly on its physical structure. Comparative studies suggest that the hearing organ of the earliest amniote vertebrates was small and simple, but possessed hair cells with a cochlear amplifier mechanism, electrical frequency tuning, and incipient micromechanical tuning. The separation of the different groups of amniotes from the stem reptiles occurred relatively early, with the ancestors of the mammals branching off first, approximately 320 million years ago. The evolution of the hearing organ in the three major lines of the descendents of the stem reptiles (e.g., mammals, birds-crocodiles, and lizards-snakes) thus occurred independently over long periods of time. Dramatic and parallel improvements in the middle ear initiated papillar elongation in all lineages, accompanied by increased numbers of sensory cells with enhanced micromechanical tuning and group-specific hair-cell specializations that resulted in unique morphological configurations. This review aims not only to compare structure and function across classification boundaries (the comparative approach), but also to assess how and to what extent fundamental mechanisms were influenced by selection pressures in times past (the phylogenetic viewpoint).