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.

Avaliação audiológica em crianças com baixo nível de exposição cumulativa ao chumbo

OBJETIVO: verificar a ocorrência de perda auditiva sensorioneural em crianças com baixo nível de exposição cumulativa ao chumbo. MÉTODO: 156 crianças intoxicadas por chumbo, 94 do sexo masculino e 62 do sexo feminino, na faixa etária entre 18 meses a 14 anos e 5 meses, foram submetidas a análise longitudinal do nível de Plumbemia em sangue, bem como audiometria tonal liminar e emissões otoacústicas evocadas por estímulo transiente. RESULTADOS: a população pesquisada apresentou um valor médio de Plumbemia estimada de 12,2±5,7mg/dL (faixa entre 2,4-33mg/dL); todas as crianças apresentaram resposta normal na audiometria tonal liminar em 20 dBNA nas frequências testadas, 0,5; 1; 2 e 4 kHz, para ambas as orelhas; as emissões otoacústicas evocadas por estímulo transiente estiveram presentes para todas as frequências bilateralmente, nas 79 crianças pesquisadas. CONCLUSÃO: não foi constatada perda auditiva sensorioneural em crianças com histórico de baixo nível de exposição cumulativa por chumbo, assim como não foi encontrada lesão de células ciliadas externas na cóclea, mesmo que subclínicas.

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.

Inner-Hair Cells Parameterized-Hardware Implementation for Personalized Auditory Nerve Stimulation

In this paper the hardware implementation of an inner hair cell model is presented. Main features of the design are the use of Meddis’ transduction structure and the methodology for Design with Reusability. Which allows future migration to new hardware and design refinements for speech processing and custom-made hearing aids

Visualization of α9 acetylcholine receptor expression in hair cells of transgenic mice containing a modified bacterial artificial chromosome

The α9 acetylcholine receptor (α9 AChR) is specifically expressed in hair cells of the inner ear and is believed to be involved in synaptic transmission between efferent nerves and hair cells. Using a recently developed method, we modified a bacterial artificial chromosome containing the mouse α9 AChR gene with a reporter gene encoding green fluorescent protein (GFP) to generate transgenic mice. GFP expression in transgenic mice recapitulated the known temporal and spatial expression of α9 AChR. However, we observed previously unidentified dynamic changes in α9 AChR expression in cochlear and vestibular sensory epithelia during neonatal development. In the cochlea, inner hair cells persistently expressed high levels of α9 AChR in both the apical and middle turns, whereas both outer and inner hair cells displayed dynamic changes of α9 AChR expression in the basal turn. In the utricle, we observed high levels of α9 AChR expression in the striolar region during early neonatal development and high levels of α9 AChR in the extrastriolar region in adult mice. Further, simultaneous visualization of efferent innervation and α9 AChR expression showed that dynamic expression of α9 AChR in developing hair cells was independent of efferent contacts. We propose that α9 AChR expression in developing auditory and vestibular sensory epithelia correlates with maturation of hair cells and is hair-cell autonomous.

Reciprocal electromechanical properties of rat prestin: The motor molecule from rat outer hair cells

Cochlear outer hair cells (OHCs) are responsible for the exquisite sensitivity, dynamic range, and frequency-resolving capacity of the mammalian hearing organ. These unique cells respond to an electrical stimulus with a cycle-by-cycle change in cell length that is mediated by molecular motors in the cells' basolateral membrane. Recent work identified prestin, a protein with similarity to pendrin-related anion transporters, as the OHC motor molecule. Here we show that heterologously expressed prestin from rat OHCs (rprestin) exhibits reciprocal electromechanical properties as known for the OHC motor protein. Upon electrical stimulation in the microchamber configuration, rprestin generates mechanical force with constant amplitude and phase up to a stimulus frequency of at least 20 kHz. Mechanical stimulation of rprestin in excised outside-out patches shifts the voltage dependence of the nonlinear capacitance characterizing the electrical properties of the molecule. The results indicate that rprestin is a molecular motor that displays reciprocal electromechanical properties over the entire frequency range relevant for mammalian hearing.

Active hair-bundle movements can amplify a hair cell’s response to oscillatory mechanical stimuli

To enhance their mechanical sensitivity and frequency selectivity, hair cells amplify the mechanical stimuli to which they respond. Although cell-body contractions of outer hair cells are thought to mediate the active process in the mammalian cochlea, vertebrates without outer hair cells display highly sensitive, sharply tuned hearing and spontaneous otoacoustic emissions. In these animals the amplifier must reside elsewhere. We report physiological evidence that amplification can stem from active movement of the hair bundle, the hair cell’s mechanosensitive organelle. We performed experiments on hair cells from the sacculus of the bullfrog. Using a two-compartment recording chamber that permits exposure of the hair cell’s apical and basolateral surfaces to different solutions, we examined active hair-bundle motion in circumstances similar to those in vivo. When the apical surface was bathed in artificial endolymph, many hair bundles exhibited spontaneous oscillations of amplitudes as great as 50 nm and frequencies in the range 5 to 40 Hz. We stimulated hair bundles with a flexible glass probe and recorded their mechanical responses with a photometric system. When the stimulus frequency lay within a band enclosing a hair cell’s frequency of spontaneous oscillation, mechanical stimuli as small as ±5 nm entrained the hair-bundle oscillations. For small stimuli, the bundle movement was larger than the stimulus. Because the energy dissipated by viscous drag exceeded the work provided by the stimulus probe, the hair bundles powered their motion and therefore amplified it.

Thyroid hormone receptor β-dependent expression of a potassium conductance in inner hair cells at the onset of hearing

To elucidate the role of thyroid hormone receptors (TRs) α1 and β in the development of hearing, cochlear functions have been investigated in mice lacking TRα1 or TRβ. TRs are ligand-dependent transcription factors expressed in the developing organ of Corti, and loss of TRβ is known to impair hearing in mice and in humans. Here, TRα1-deficient (TRα1−/−) mice are shown to display a normal auditory-evoked brainstem response, indicating that only TRβ, and not TRα1, is essential for hearing. Because cochlear morphology was normal in TRβ−/− mice, we postulated that TRβ regulates functional rather than morphological development of the cochlea. At the onset of hearing, inner hair cells (IHCs) in wild-type mice express a fast-activating potassium conductance, IK,f, that transforms the immature IHC from a regenerative, spiking pacemaker to a high-frequency signal transmitter. Expression of IK,f was significantly retarded in TRβ−/− mice, whereas the development of the endocochlear potential and other cochlear functions, including mechanoelectrical transduction in hair cells, progressed normally. TRα1−/− mice expressed IK,f normally, in accord with their normal auditory-evoked brainstem response. These results establish that the physiological differentiation of IHCs depends on a TRβ-mediated pathway. When defective, this may contribute to deafness in congenital thyroid diseases.

The ionic basis of frequency selectivity in hair cells of the bullfrog's sacculus

Hair cells from the bull frog's sacculus, a vestibular organ responding to substrate-borne vibration, possess electrically resonant membrane properties which maximize the sensitivity of each cell to a particular frequency of mechanical input. The electrical resonance of these cells and its underlying ionic basis were studied by applying gigohm-seal recording techniques to solitary hair cells enzymatically dissociated from the sacculus. The contribution of electrical resonance to frequency selectivity was assessed from microelectrode recordings from hair cells in an excised preparation of the sacculus.

Electrical resonance in the hair cell is demonstrated by damped membrane-potential oscillations in response to extrinsic current pulses applied through the recording pipette. This response is analyzed as that of a damped harmonic oscillator. Oscillation frequency rises with membrane depolarization, from 80-160 Hz at resting potential to asymptotic values of 200-250 Hz. The sharpness of electrical tuning, denoted by the electrical quality factor, Q_e, is a bell-shaped function of membrane voltage, reaching a maximum value around eight at a membrane potential slightly positive to the resting potential.

In whole cells, three time-variant ionic currents are activated at voltages more positive than -60 to -50 mV; these are identified as a voltage-dependent, non-inactivating Ca current (I_ca), a voltage-dependent, transient K current (I_a), and a Ca-dependent K current (I_c). The C channel is identified in excised, inside-out membrane patches on the basis of its large conductance (130-200 pS), its selective permeability to Kover Na or Cl, and its activation by internal Ca ions and membrane depolarization. Analysis of open- and closed-lifetime distributions suggests that the C channel can assume at least two open and three closed kinetic states.

Exposing hair cells to external solutions that inhibit the Ca or C conductances degrades the electrical resonance properties measured under current-clamp conditions, while blocking the A conductance has no significant effect, providing evidence that only the Ca and C conductances participate in the resonance mechanism. To test the sufficiency of these two conductances to account for electrical resonance, a mathematical model is developed that describes I_ca, I_c, and intracellular Ca concentration during voltage-clamp steps. I_ca activation is approximated by a third-order Hodgkin-Huxley kinetic scheme. Ca entering the cell is assumed to be confined to a small submembrane compartment which contains an excess of Ca buffer; Ca leaves this space with first-order kinetics. The Ca- and voltage-dependent activation of C channels is described by a five-state kinetic scheme suggested by the results of single-channel observations. Parameter values in the model are adjusted to fit the waveforms of I_ca and I_c evoked by a series of voltage-clamp steps in a single cell. Having been thus constrained, the model correctly predicts the character of voltage oscillations produced by current-clamp steps, including the dependencies of oscillation frequency and Q_e on membrane voltage. The model shows quantitatively how the Ca and C conductances interact, via changes in intracellular Ca concentration, to produce electrical resonance in a vertebrate hair cell.

A comparison of the haemagglutinating and enzymic activities of Bacteroides fragilis whole cells and outer membrane vesicles

The haemagglutinating and enzymic activities of the obligately anaerobic pathogenic bacterium Bacteroides fragilis were examined. Outer membrane vesicles are released from the surface of B. fragilis. They can be detected by electron microscopy in ultrathin sections and bacterial suspensions after negative staining. Electron microscopy and immunogold labelling with a MAb specific for surface polysaccharide of B. fragilis confirmed that the vesicles carried outer membrane associated epitopes. The haemagglutinating activity of whole cells from populations of B. fragilis strains NCTC9343, BE3 and LS66 enriched by Percoll density gradient centrifugation for a large capsule (LC), electron dense layer (EDL); non-capsulate by light microscopy) and outer membrane vesicles (OMV) which had been purified by centrifugation from EDL-enriched populations were compared using human and horse erythrocytes. The enzymic activity of OMV, LC- and EDL-enriched populations, as detected by the API ZYM kit, was compared for strains NCTC 9343 and BE3. Purified OMV from the strains examined exhibited both haemagglutinating and enzymatic activity. Haemagglutination by the EDL-enriched population was sensitive to treatment with sodium periodate. The LC-enriched population haemagglutinated only after ultrasonic removal of the capsule. This indicates that the LC masks a haemagglutinin. The results suggest a potential role for OMV in the virulence of B. fragilis.

From embryonic stem cells to sensory hair cells : a programming approach

Tese de doutoramento, Ciências Biomédicas (Biologia do Desenvolvimento), Universidade de Lisboa, Faculdade de Medicina, 2014

The selectivity of the hair cell’s mechanoelectrical-transduction channel promotes Ca2+ flux at low Ca2+ concentrations

The mechanoelectrical-transduction channel of the hair cell is permeable to both monovalent and divalent cations. Because Ca2+ entering through the transduction channel serves as a feedback signal in the adaptation process that sets the channel’s open probability, an understanding of adaptation requires estimation of the magnitude of Ca2+ influx. To determine the Ca2+ current through the transduction channel, we measured extracellular receptor currents with transepithelial voltage-clamp recordings while the apical surface of a saccular macula was bathed with solutions containing various concentrations of K+, Na+, or Ca2+. For modest concentrations of a single permeant cation, Ca2+ carried much more receptor current than did either K+ or Na+. For higher cation concentrations, however, the flux of Na+ or K+ through the transduction channel exceeded that of Ca2+. For mixtures of Ca2+ and monovalent cations, the receptor current displayed an anomalous mole-fraction effect, which indicates that ions interact while traversing the channel’s pore. These results demonstrate not only that the hair cell’s transduction channel is selective for Ca2+ over monovalent cations but also that Ca2+ carries substantial current even at low Ca2+ concentrations. At physiological cation concentrations, Ca2+ flux through transduction channels can change the local Ca2+ concentration in stereocilia in a range relevant for the control of adaptation.