Localized cell and drug delivery for auditory prostheses.

Localized cell and drug delivery to the cochlea and central auditory pathway can improve the safety and performance of implanted auditory prostheses (APs). While generally successful, these devices have a number of limitations and adverse effects including limited tonal and dynamic ranges, channel interactions, unwanted stimulation of non-auditory nerves, immune rejection, and infections including meningitis. Many of these limitations are associated with the tissue reactions to implanted auditory prosthetic devices and the gradual degeneration of the auditory system following deafness. Strategies to reduce the insertion trauma, degeneration of target neurons, fibrous and bony tissue encapsulation, and immune activation can improve the viability of tissue required for AP function as well as improve the resolution of stimulation for reduced channel interaction and improved place-pitch and level discrimination. Many pharmaceutical compounds have been identified that promote the viability of auditory tissue and prevent inflammation and infection. Cell delivery and gene therapy have provided promising results for treating hearing loss and reversing degeneration. Currently, many clinical and experimental methods can produce extremely localized and sustained drug delivery to address AP limitations. These methods provide better control over drug concentrations while eliminating the adverse effects of systemic delivery. Many of these drug delivery techniques can be integrated into modern auditory prosthetic devices to optimize the tissue response to the implanted device and reduce the risk of infection or rejection. Together, these methods and pharmaceutical agents can be used to optimize the tissue-device interface for improved AP safety and effectiveness.

Tonotopic distribution of short-term adaptation properties in the cochlear nerve of normal and acoustically overexposed chicks.

Cochlear nerve adaptation is thought to result, at least partially, from the depletion of neurotransmitter stores in hair cells. Recently, neurotransmitter vesicle pools have been identified in chick tall hair cells that might play a role in adaptation. In order to understand better the relationship between adaptation and neurotransmitter release dynamics, short-term adaptation was characterized by using peristimulus time histograms of single-unit activity in the chick cochlear nerve. The adaptation function resulting from 100-ms pure tone stimuli presented at the characteristic frequency, +20 dB relative to threshold, was well described as a single exponential decay process with an average time constant of 18.6+/-0.8 ms (mean+/-SEM). The number of spikes contributed by the adapting part of the response increased tonotopically for characteristic frequencies up to approximately 0.8 kHz. Comparison of the adaptation data with known physiological and anatomical hair cell properties suggests that depletion of the readily releasable pool is the basis of short-term adaptation in the chick. With this idea in mind, short-term adaptation was used as a proxy for assessing tall hair cell synaptic function following intense acoustic stimulation. After 48 h of exposure to an intense pure tone, the time constant of short-term adaptation was unaltered, whereas the number of spikes in the adapting component was increased at characteristic frequencies at and above the exposure frequency. These data suggest that the rate of readily releasable pool emptying is unaltered, but the neurotransmitter content of the pool is increased, by exposure to intense sound. The results imply that an increase in readily releasable pool size might be a compensatory mechanism ensuring the strength of the hair cell afferent synapse in the face of ongoing acoustic stress.

Cyclodextrins and Iatrogenic Hearing Loss: New Drugs with Significant Risk.

Cyclodextrins are a family of cyclic oligosaccharides with widespread usage in medicine, industry and basic sciences owing to their ability to solubilize and stabilize guest compounds. In medicine, cyclodextrins primarily act as a complexing vehicle and consequently serve as powerful drug delivery agents. Recently, uncomplexed cyclodextrins have emerged as potent therapeutic compounds in their own right, based on their ability to sequester and mobilize cellular lipids. In particular, 2-hydroxypropyl-ß-cyclodextrin (HPßCD) has garnered attention because of its cholesterol chelating properties, which appear to treat a rare neurodegenerative disorder and to promote atherosclerosis regression related to stroke and heart disease. Despite the potential health benefits, use of HPßCD has been linked to significant hearing loss in several species, including humans. Evidence in mice supports a rapid onset of hearing loss that is dose-dependent. Ototoxicity can occur following central or peripheral drug delivery, with either route resulting in the preferential loss of cochlear outer hair cells (OHCs) within hours of dosing. Inner hair cells and spiral ganglion cells are spared at doses that cause ~85% OHC loss; additionally, no other major organ systems appear adversely affected. Evidence from a first-to-human phase 1 clinical trial mirrors animal studies to a large extent, indicating rapid onset and involvement of OHCs. All patients in the trial experienced some permanent hearing loss, although a temporary loss of function can be observed acutely following drug delivery. The long-term impact of HPßCD use as a maintenance drug, and the mechanism(s) of ototoxicity, are unknown. ß-cyclodextrins preferentially target membrane cholesterol, but other lipid species and proteins may be directly or indirectly involved. Moreover, as cholesterol is ubiquitous in cell membranes, it remains unclear why OHCs are preferentially susceptible to HPßCD. It is possible that HPßCD acts upon several targets-for example, ion channels, tight junctions (TJ), membrane integrity, and bioenergetics-that collectively increase the sensitivity of OHCs over other cell types.

Manipulating gene expression in the mature inner ear.

It is possible to manipulate gene expression in cochlear tissue, but technical issues have made this challenging in the mature in vivo inner ear. Generally, the most common reasons for such manipulations involve basic science or therapeutic quests. Examples of experimental studies are those designed to elucidate the role of a specific gene or a gene expression cascade or to understand the function of a particular cell type. Therapeutic goals may include replacing a defective gene or enhancing tissue protection, repair, or regeneration. This review summarizes the main technical approaches that are viable options for in vivo manipulation of gene expression in the mature inner ear, as well as major research and clinical issues likely to benefit from such genetic manipulations.

The fate of outer hair cells after acoustic or ototoxic insults.

In epithelial sheets, clearance of dead cells may occur by one of several routes, including extrusion into the lumen, phagocytic clearance by invading lymphocytes, or phagocytosis by neighboring cells. The fate of dead cochlear outer hair cells is unclear. We investigated the fate of the "corpses" of dead outer hair cells in guinea pigs and mice following drug or noise exposure. We examined whole mounts and plastic sections of normal and lesioned organ of Corti for the presence of prestin, a protein unique to outer hair cells. Supporting cells, which are devoid of prestin in the normal ear, contained clumps of prestin in areas of hair cell loss. The data show that cochlear supporting cells surround the corpses and/or debris of degenerated outer hair cells, and suggest that outer hair cell remains are phagocytosed by supporting cells within the epithelium.

Postsynaptic TrkB-mediated signaling modulates excitatory and inhibitory neurotransmitter receptor clustering at hippocampal synapses.

Tyrosine receptor kinase B (TrkB)-mediated signaling modulates synaptic structure and strength in hippocampal and other neurons, but the underlying mechanisms are poorly understood. Full-length and truncated TrkB are diffusely distributed throughout the dendrites and soma of rat hippocampal neurons grown in vitro. Manipulation of TrkB-mediated signaling resulted in dramatic changes in the number and synaptic localization of postsynaptic NMDA receptor (NMDAR) and GABA(A) receptor (GABA(A)R) clusters. BDNF treatment resulted in an increase in the number of NMDAR and GABA(A)R clusters and increased the proportion of clusters apposed to presynaptic terminals. Downregulation of TrkB signaling resulted in a decrease in receptor cluster number and synaptic localization. Examination of the time course of the effects of BDNF on receptor clusters showed that the increase in GABA(A)R clusters preceded the increase in NMDAR clusters by at least 12 hr. Moreover, the TrkB-mediated effects on NMDAR clusters were dependent on GABA(A)R activation. Although TTX, APV, and CNQX treatment had no effect, blockade of GABA(A)Rs with bicuculline abolished the BDNF-mediated increase in NMDAR cluster number and synaptic localization. In contrast, application of exogenous GABA prevented the decrease in NMDAR clusters induced by BDNF scavenging. Together, these results suggest that TrkB-mediated signaling modulates the clustering of postsynaptic GABA(A)Rs and that receptor activity is required for a subsequent upregulation of NMDAR clusters. Therefore, TrkB-mediated effects on postsynaptic neurotransmitter clusters may be part of a mechanism that balances inhibitory and excitatory synaptic transmission in developing neural circuits.

Evidence that rapid vesicle replenishment of the synaptic ribbon mediates recovery from short-term adaptation at the hair cell afferent synapse.

We have employed both in vitro patch clamp recordings of hair cell synaptic vesicle fusion and in vivo single unit recording of cochlear nerve activity to study, at the same synapse, the time course, control, and physiological significance of readily releasable pool dynamics. Exocytosis of the readily releasable pool was fast, saturating in less than 50 ms, and recovery was also rapid, regaining 95% of its initial amplitude following a 200-ms period of repolarization. Longer depolarizations (greater than 250 ms) yielded a second, slower kinetic component of exocytosis. Both the second component of exocytosis and recovery of the readily releasable pool were blocked by the slow calcium buffer, EGTA. Sound-evoked afferent synaptic activity adapted and recovered with similar time courses as readily releasable pool exhaustion and recovery. Comparison of readily releasable pool amplitude, capture distances of calcium buffers, and number of vesicles tethered to the synaptic ribbon suggested that readily releasable pool dynamics reflect the depletion of release-ready vesicles tethered to the synaptic ribbon and the reloading of the ribbon with vesicles from the cytoplasm. Thus, we submit that rapid recovery of the cochlear hair cell afferent fiber synapse from short-term adaptation depends on the timely replenishment of the synaptic ribbon with vesicles from a cytoplasmic pool. This apparent rapid reloading of the synaptic ribbon with vesicles underscores important functional differences between synaptic ribbons in the auditory and visual systems.

Hearing loss and hair cell death in mice given the cholesterol-chelating agent hydroxypropyl-ß-cyclodextrin.

Cyclodextrins are sugar compounds that are increasingly finding medicinal uses due to their ability to complex with hydrophobic molecules. One cyclodextrin in particular, 2-hydroxypropyl-ß-cyclodextrin (HPßCD), is used as a carrier to solubilize lipophilic drugs and is itself being considered as a therapeutic agent for treatment of Niemann-Pick Type C disease, due to its ability to mobilize cholesterol. Results from toxicological studies suggest that HPßCD is generally safe, but a recent study has found that it causes hearing loss in cats. Whether the hearing loss occurred via death of cochlear hair cells, rendering it permanent, was unexplored. In the present study, we examined peripheral auditory function and cochlear histology in mice after subcutaneous injection of HPßCD to test for hearing loss and correlate any observed auditory deficits with histological findings. On average, auditory brainstem response thresholds were elevated at 4, 16, and 32 kHz in mice one week after treatment with 8,000 mg/kg. In severely affected mice all outer hair cells were missing in the basal half of the cochlea. In many cases, surviving hair cells in the cochlear apex exhibited abnormal punctate distribution of the motor protein prestin, suggesting long term changes to membrane composition and integrity. Mice given a lower dose of 4,000 mg/kg exhibited hearing loss only after repeated doses, but these threshold shifts were temporary. Therefore, cyclodextrin-induced hearing loss was complex, involving cell death and other more subtle influences on cochlear physiology.

Temperature insensitivity of short-term adaptation in single-units of the chick cochlear nerve.

Short-term adaptation in acoustically stimulated chick cochlear nerve fibers has recently been shown to have similar kinetics as the readily-releasable vesicle pool in patch-clamped chick hair cells, suggesting that short-term adaptation depends on the dynamics of hair cell exocytosis. Our understanding of the relationship between these two phenomena has been hampered by differences in the temperatures at which the two types of data have been collected. In this report, the effect of temperature on short-term adaptation was studied in single-units of the chick cochlear nerve. Compared to units recorded at 38-41 degrees C, spontaneous and evoked firing rates were markedly decreased when the temperature was lowered to 28-32 degrees C, but the rate of short-term adaptation during 100 ms tone bursts was relatively unchanged, with a temperature Q(10), of approximately 1.2. The continued similarity of the adaptation time-constant of cooled units to vesicle depletion kinetics in chick hair cells measured at room temperature suggests that comparison of in vitro hair cell exocytosis and in vivo cochlear nerve firing properties may not be confounded by temperature differences between the two approaches.