Encapsulated cell device approach for combined electrical stimulation and neurotrophic treatment of the deaf cochlea.

Profound hearing impairment can be overcome by electrical stimulation (ES) of spiral ganglion neurons (SGNs) via a cochlear implant (CI). Thus, SGN survival is critical for CI efficacy. Application of glial cell line-derived neurotrophic factor (GDNF) has been shown to reduce SGN degeneration following deafness. We tested a novel method for local, continuous GDNF-delivery in combination with ES via a CI. The encapsulated cell (EC) device contained a human ARPE-19 cell-line, genetically engineered for secretion of GDNF. In vitro, GDNF delivery was stable during ES delivered via a CI. In the chronic in vivo part, cats were systemically deafened and unilaterally implanted into the scala tympani with a CI and an EC device, which they wore for six months. The implantation of control devices (same cell-line not producing GDNF) had no negative effect on SGN survival. GDNF application without ES led to an unexpected reduction in SGN survival, however, the combination of GDNF with initial, short-term ES resulted in a significant protection of SGNs. A tight fibrous tissue formation in the scala tympani of the GDNF-only group is thought to be responsible for the increased SGN degeneration, due to mechanisms related to an aggravated foreign body response. Furthermore, the fibrotic encapsulation of the EC device led to cell death or cessation of GDNF release within the EC device during the six months in vivo. In both in vitro and in vivo, fibrosis was reduced by CI stimulation, enabling the neuroprotective effect of the combined treatment. Thus, fibrous tissue growth limits treatment possibilities with an EC device. For a stable and successful long-term neurotrophic treatment of the SGN via EC devices in human CI users, it would be necessary to make changes in the treatment approach (provision of anti-inflammatories), the EC device surface (reduced cell adhesion) and the ES (initiation prior to fibrosis formation).

Optimal electrode length to match patient specific cochlear anatomy.

Cochlear implantation (CI) has reached over years of practicing high standards of surgical outcomes. Even patients with significant residual hearing are nowadays benefiting from a cochlear implant. However, the speech perception still depends to great extent on the adequate pitch match between the frequency components delivered by an electrode array and individual cochlear tonotopic map. Compression, deletion or shift of frequency components can be tolerated by patients only to some extent. Furthermore, low frequency information delivered to the cochlear apex is particularly important for spatial hearing. It is therefore important to use the electrode array of an appropriate length for each individual cochlea. The large variability in the anatomy makes this task difficult as a single design does not fit all cochlear shapes. Fortunately, preoperative CT imaging, routinely taken in most of ENT clinics, can be exploited also for the prediction of the cochlear duct length (CDL). It turns out that a single radiological measurement, the diameter of the basal turn, is highly correlated with CDL and its measurement can be used for the informed selection of the most suitable electrode array length from the available array portfolio for each CI patient.

Mammalian prestin is a weak Cl⁻/HCO3⁻ electrogenic antiporter.

The lateral membrane of mammalian cochlear outer hair cells contains prestin, a protein which can act as a fast voltage-driven actuator responsible for electromotility and enhanced sensitivity to sound. The protein belongs to the SLC26 family of transporters whose members are characterised as able to exchange halides for SO(4)(2-) or HCO(3)(-) yet previous analyses of mammalian prestin have suggested that such exchange functions were minimal. Here anion transport is investigated both in guinea-pig outer hair cells (OHCs) and in an expression system where we employ a sensitive intracellular pH (pH(i)) probe, pHluorin, to report HCO(3)(-) transport and to monitor the small pH(i) changes observable in the cells. In the presence of extracellular HCO(3)(-), pH(i) recovered from an acid load 4 times faster in prestin-transfected cells. The acceleration required a chloride gradient established by reducing extracellular chloride to 2 mm. Similar results were also shown using BCECF as an alternative pH(i) sensor, but with recovery only found in those cells expressing prestin. Simultaneous electrophysiological recording of the transfected cells during bicarbonate exposure produced a shift in the reversal potential to more negative potentials, consistent with electrogenic transport. These data therefore suggest that prestin can act as a weak Cl(-)/HCO(3)(-) antiporter and it is proposed that, in addition to participating in wide band cochlear sound amplification, prestin may also be involved in the slow time scale (>10 s) phenomena where changes in cell stiffness and internal pressure have been implicated. The results show the importance of considering the effects of the endogenous bicarbonate buffering system in evaluating the function of prestin in cochlear outer hair cells.

A homology model of the pore region of HCN channels.

HCN channels are activated by membrane hyperpolarization and regulated by cyclic nucleotides, such as cyclic adenosine-mono-phosphate (cAMP). Here we present structural models of the pore region of these channels obtained by using homology modeling and validated against spatial constraints derived from electrophysiological experiments. For the construction of the models we make two major assumptions, justified by electrophysiological observations: i), in the closed state, the topology of the inner pore of HCN channels is similar to that of K(+) channels. In particular, the orientation of the S5 and S6 helices of HCN channels is very similar to that of the corresponding helices of the K(+) KcsA and K(+) KirBac1.1 channels. Thus, we use as templates the x-ray structure of these K(+) channels. ii), In the open state, the S6 helix is bent further than it is in the closed state, as suggested (but not proven) by experimental data. For this reason, the template of the open conformation is the x-ray structure of the MthK channel. The structural models of the closed state turn out to be consistent with all the available electrophysiological data. The model of the open state turned out to be consistent with all the available electrophysiological data in the filter region, including additional experimental data performed in this work. However, it required the introduction of an appropriate, experimentally derived constraint for the S6 helix. Our modeling provides a structural framework for understanding several functional properties of HCN channels: i), the cysteine ring at the inner mouth of the pore may act as a sensor of the intracellular oxidizing/reducing conditions; ii), the bending amplitude of the S6 helix upon gating appears to be significantly smaller than that found in MthK channels; iii), the reduced ionic selectivity of HCN channels, relative to that of K(+) channels, may be caused, at least in part, by the larger flexibility of the inner pore of HCN channels.

BiaCore analysis of leptin-leptin receptor interaction: evidence for 1:1 stoichiometry.

Leptin is a hormonal protein involved in energy homeostatis that acts to inhibit food intake, to stimulate energy expenditure, and to influence insulin secretion, lipolysis, and sugar transport. Its action is mediated by a specific receptor whose activation is highly controversial. As a member of the cytokine receptor superfamily, it has been predicted to be activated by ligand-induced dimerization. However, recent evidence has indicated that this receptor exists as a dimer in both ligand-free and ligand-bound states. Here, the BiaCore has been used to measure the kinetics and stoichiometry of the interaction between the leptin and its receptor. Human or mouse receptor chimeras comprising two receptor extracellular domains fused to the Fc region of IgG(1) were captured on to the sensor via protein G. Kinetic data fitted to the simplest 1/1 model. The observed stoichiometry at ligand saturation was 1:1. Analyzing the binding mode and the reaction stoichiometry allowed us to conclude that the leptin receptor dimerization is not induced by ligand binding. This contradicts the common paradigm of cytokine receptor activation. Furthermore, data demonstrated a high-affinity interaction. The KD was 0.23+/-0.08 nM, with ka = (1.9 +/- 0.4) x 10(6) M(-1)s(-1) and kd = (4.4 +/- 0.6) x 10(-4) s(-1) for human leptin with its cognate receptor. Similar results were observed for the affinity of different species of leptin binding to mouse leptin receptor.