[Reactions in the organ of Corti to electrical stimulation : StED technology for detecting changes]. / Reaktionen im Corti-Organ auf elektrische Stimulation : StED-Technologie zum Nachweis von Veränderungen.

Increasing numbers of cochlear implant patients have residual hearing. Despite surgical and pharmacological efforts to preserve residual hearing, a significant number of these patients suffer a late, unexplained loss of residual hearing. Surgical trauma can be excluded as the cause. To investigate this phenomenon and because cells in their native environment react differently to stimuli (such as electrical current) than isolated cells, whole-organ explants from cochleae may be a better model. For early detection of synaptic changes in the organ of Corti, a high-resolution microscopic technique such as stimulated emission depletion (StED) can be used. The aim of this study was establishment of a qualitative and quantitative technique to determinate changes in the organ of Corti and its synapses after electrical stimulation. Explanted organs of Corti from postnatal rats (P2-4) were cultured on a coverslip for 24 h and subsequently exposed to biphasic pulsed electrical stimulation (amplitude 0.44-2.0 mA, pulse width 400 µs, interpulse delay 120 µs, repetition 1 kHz) for another 24 h. For visualization, the cytoskeleton and the ribbon synapses were stained immunocytochemically. For an early detectable response to electrical stimulation, the number of synapses was quantified. Organs of Corti without electrical stimulation served as a reference. Initial research has shown that electrical stimulation can cause changes in ribbon synapses and that StED can detect these alterations. The herein established model could be of great importance for identification of molecular changes in the organ of Corti in response to electrical or other stimuli.

Intraluminal three-dimensional optical coherence tomography - a tool for imaging of the Eustachian tube?

OBJECTIVES: The cause of Eustachian tube dysfunction often remains unclear. Therefore, this study aimed to examine the feasibility and possible diagnostic use of optical coherence tomography in the Eustachian tube ex vivo. METHODS: Two female blackface sheep cadaver heads were examined bilaterally. Three conditions of the Eustachian tube were investigated: closed (resting position), actively opened and stented. The findings were compared (and correlated) with segmented histological cross-sections. RESULTS: Intraluminal placement of the Eustachian tube with the optical coherence tomography catheter was performed without difficulty. Regarding the limited infiltration depth of optical coherence tomography, tissues can be differentiated. The localisation of the stent was accurate as was the lumen. CONCLUSION: The application of optical coherence tomography in the Eustachian tube under these experimental conditions is considered to be a feasible, rapid and non-invasive diagnostic method, with possible diagnostic value for determining the luminal shape and superficial lining tissue of the Eustachian tube.

Biodegradable polymeric coatings on cochlear implant surfaces and their influence on spiral ganglion cell survival.

To improve the electrode-nerve interface of cochlear implants (CI), the role of poly(L-lactide) (PLLA) and poly(4-hydroxybutyrate) (P(4HB)) as potential coating matrices for CI was assessed both in vitro and in vivo in terms of degradation behavior and effects on spiral ganglion neurons, the main target of the electrical stimulation with a CI. Growth rates of fibroblasts on the polymers were investigated and a direct-contact test with freshly isolated spiral ganglion cells (SGC) was performed. In addition, the effects of the polymer degradation inside the inner ear were evaluated in vivo. The polymer degradation was assessed by use of scanning electron microscopy in combination with an energy-dispersive X-ray analysis. In vitro, no influence of the polymers was detected on fibroblasts' viability and on SGC survival rate. In vivo, SGC density was decreased only 6 months after implantation in the basal and middle turns of the cochlea in comparison to normal-hearing animals but not between implanted groups (coated or uncoated). The analysis of the electrode models showed that in vivo P(4HB) is characterized by a gradual degradation completed after 6 months; whereas, the PLLA coatings burst along their longitudinal axis but showed only little degradation within the same time frame. In conclusion, both polymers seem to justify further evaluation as possible coating for CI electrodes. Of the two options, due to its excellent coating adhesion/stability and optimal degradation behavior, P(4HB) may prove to be the more promising biodegradable polymer for designing a drug delivery system from the surface of CI electrodes.

Effects of metal ions on fibroblasts and spiral ganglion cells.

Degeneration of spiral ganglion cells (SGC) after deafness and fibrous tissue growth around the electrode carrier after cochlear implantation are two of the major challenges in current cochlear implant research. Metal ions are known to possess antimicrobial and antiproliferative potential. The use of metal ions could therefore provide a way to reduce tissue growth around the electrode array after cochlear implantation. Here, we report on in vitro experiments with different concentrations of metal salts with antiproliferative and toxic effects on fibroblasts, PC-12 cells, and freshly isolated spiral ganglion cells, the target cells for electrical stimulation by a cochlear implant. Standard cell lines (NIH/3T3 and L-929 fibroblasts and PC-12 cells) and freshly isolated SGC were incubated with concentrations of metal ions between 0.3 µmol/liter and 10 mmol/liter for 48 hr. Cell survival was investigated by neutral red uptake, CellQuantiBlue assay, or counting of stained surviving neurons. Silver ions exhibited distinct thresholds for proliferating and confluent cells. For zinc ions, the effective concentration was lower for fibroblasts than for PC-12 cells. SGC showed comparable thresholds for reduced cell survival not only for silver and zinc ions but also for copper(II) ions, indicating that these ions might be promising for reducing tissue growth on the surface of CI electrode arrays. These effects were also observed when combinations of two of these ions were investigated.

Development of a drug delivery device: using the femtosecond laser to modify cochlear implant electrodes.

Animal experiments suggest that pharmacological intervention could possibly enhance cochlear implant performance. One of the key aspects is therefore a drug delivery device for the human inner ear. The objective of this study was to investigate the possibility of using the femtosecond laser for modifying a cochlear implant electrode for the purpose of drug delivery to the cochlea. Using silicone sheets, the best parameters for creating defined channels at calculated diameters were investigated using a femtosecond laser. The results were transferred to a cochlear implant electrode array (Nucleus Contour). The capability of delivering substances through the drilled openings was tested in vitro. By variation of the output of the laser, spot distance, repetition rate, number of cycles and introducing several focus planes, it was possible to drill holes with nearly vertical walls in the silicone sheets. Transferring these data to the cochlear implant electrode resulted in prototypes for drug delivery with various openings along the array. The use of the femtosecond laser allows rapid modification and adaptation of designs to experimental prototypes of cochlear implant electrodes for the purpose of drug delivery to the inner ear.

Technical report: modification of a cochlear implant electrode for drug delivery to the inner ear.

OBJECTIVE: To investigate the possibility of modifying a cochlear implant electrode for the purpose of drug delivery to the cochlea. BACKGROUND: Animal experiments suggest that local therapy of the inner ear could be a promising new approach to the interventional treatment of inner ear disease, and that pharmacologic intervention could possibly enhance cochlear implant performance. One of the key aspects is the deployment of a means of drug delivery to the human inner ear. METHODS: The tip of the Contour electrode array was cut to open the lumen of the array, and a connecting piece was developed to connect the electrode to a pump. The feasibility of using the array for drug delivery was tested using both an Alzet mini-osmotic pump and a mechanical pump. The connection was tested for its stability in terms of leakage and resistance to tractive forces. The system was also applied to temporal bones to evaluate its applicability to the human cochlea. RESULTS: The modified Contour electrode is easy to handle in temporal bones and can be used to simulate drug delivery to the inner ear. The connection to the pump was sealed for all tested pump rates and resisted tractive forces up to 50 N. CONCLUSIONS: The described modified electrode could provide a safe and easy-to-handle means of combining electrical stimulation with the beneficial effects of a local drug therapy applied to the inner ear.

Mitochondria of retinal Müller (glial) cells: the effects of aging and of application of free radical scavengers.

Age-related changes of mitochondria were studied in Müller (retinal glial) cells from guinea pigs fed with or without externally applied Ginkgo biloba extract EGb 761, an established radical scavenger. When Müller cell mitochondria from aged animals were compared with those from young adults, they displayed (1) a diminished number of well-defined cristae at the ultrastructural level, (2) a reduced membrane potential, as revealed by fluorimetry using the voltage-sensitive dye tetramethyl rhodamine methylester, and (3) a slightly reduced index of vitality assayed by tetrazolium salt colorimetry. Müller cell mitochondria were also studied in aged guinea pigs which had been fed daily by EGb 761 during the last 2 months before they were sacrificed. Such mitochondria displayed (1) many well-defined cristae at the ultrastructural level, and, compared with mitochondria from untreated aged animals, (2) a significantly enhanced membrane potential and (3) a significantly enhanced index of vitality. No age- or drug-related changes were observed in the mitochondrial content of GABA transaminase, as revealed by immunocytochemistry/densitometry. These results suggest that many but not all structural and functional parameters of aging Müller cell mitochondria are impaired by accumulating oxidative damage, and that externally applied radical scavengers may protect the organelles from the damaging actions of free radicals. As it has been shown earlier that EGb 761 treatment enhances the intrinsic glutathione content of aged guinea pig Müller cells, the protective radical-scavenging effect of the drug may be mediated both directly and indirectly.

Association of lysozyme with phospholipid vesicles is accompanied by membrane surface dehydration.

Lysozyme is a globular protein which is known to bind to negatively charged phospholipid vesicles. In order to study the relationship between binding of the protein and the subsequent destabilization of the phospholipid vesicles a set of experiments was performed using phospholipid monolayers and vesicles. Using microelectrophoresis the binding of lysozyme to phospholipid vesicles made of PS was determined. At low ionic strength and mild acidic pH of the solution lysozyme reduced the magnitude of the negative zeta potential of PS vesicles at lower concentrations compared to neutral pH and high ionic strength. In contrast, the bound fraction of lysozyme to PS vesicles was nearly constant at acidic and neutral pH. At low pH, the binding of lysozyme was accompanied by a strong aggregation of the vesicles. Lysozyme binding to PS vesicles is accompanied by its penetration into the PL monolayer. This was measured by surface tension and film balance measurements at low pH and low ionic strength. The interaction of lysozyme with negatively charged vesicles lead to a decrease of the vesicle surface hydration as measured by the shift of the emission peak of the fluorescent probe DPE. The binding of bis-ANS increased at low pH after addition of lysozyme to the vesicles. This indicates that more hydrophobic patches of the lysozyme-PS complex are exposed at low pH. At low pH the binding process of lysozyme to PS vesicles was followed by an extensive intermixing of phospholipids between the aggregated vesicles, accompanied by a massive leakage of the aqueous content of vesicles.

Investigation of phospholipid area compression induced by calcium-mediated dextran sulfate interaction.

The association of anionic polyelectrolytes such as dextran sulfate (DS) to zwitterionic phospholipid surfaces via Ca(2+) bridges results in a perturbation of lipid packing at physiologically relevant Ca(2+) concentrations. Lipid area compression was investigated in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) multilamellar bilayer dispersions by (2)H-NMR and in monolayer studies. Binding of DS to DMPC surfaces via Ca(2+) results in denser lipid packing, as indicated by higher lipid chain order. DMPC order parameters are homogeneously increased throughout the lipid bilayer. Higher order translates into more extended hydrocarbon chains and decreased average lipid area per molecule. Area compression is reported as a function of DS concentration and molecular weight. Altering the NaCl and Ca(2+) concentrations modified electrostatic interactions between DS and phospholipid. A maximal area reduction of DeltaA = 2.7 A(2) per DMPC molecule is observed. The lipid main-phase transition temperature increases upon formation of DMPC/Ca(2+)/DS-complexes. Lipid area compression after addition of DS and Ca(2+) to the subphase was also observed in monolayer experiments. A decrease in surface tension of up to 3.5 mN/m at constant molecular area was observed. DS binds to the lipid headgroups by formation of Ca(2+) bridges without penetrating the hydrophobic region. We suggest that area compression is the result of an attractive electrostatic interaction between neighboring lipid molecules induced by high local Ca(2+) concentration due to the presence of DS. X-ray diffraction experiments demonstrate that DS binding to apposing bilayers reduces bilayer separation. We speculate that DS binding alters the phase state of low-density lipoproteins that associate with polyelectrolytes of the arterial connective tissue in the early stages of arteriosclerosis.

The glutathione content of retinal Müller (glial) cells: the effects of aging and of application of free-radical scavengers.

The dependence of intracellular glutathione (GSH), an important radical scavenger, on aging with or without externally applied Ginkgo biloba extract EGb 761, another established radical scavenger, was studied in guinea pig M¿ller (retinal glial) cells by using the fluorescent dye monochlorobimane. The GSH content of freshly dissociated cells from untreated aged animals was significantly lower than that of young controls; most of this reduction was prevented by application of EGb 761. Culturing the cells in amino-acid-free caused a loss of up to 50% of the initial GSH content. When the culture medium contained 100 microM glutamate and 100 microM cystine, ongoing GSH synthesis counteracted the loss of GSH. The rates of net GSH synthesis were equal for the two groups of aged animals but significantly higher for cells from young controls. It is concluded that externally applied radical scavengers may enhance the protective glutathione 'reserve' of M¿ller cells in cases of neuronal degeneration.

Distribution of mitochondria within Müller cells--I. Correlation with retinal vascularization in different mammalian species.

The distribution of mitochondria within retinal glial (Müller) cells and neurons was studied by electron microscopy, by confocal microscopy of a mitochondrial dye and by immunocytochemical demonstration of the mitochondrial enzyme GABA transaminase (GABA-T). We studied sections and enzymatically dissociated cells from adult vascularized (human, pig and rat) and avascular or pseudangiotic (guinea-pig and rabbit) mammalian retinae. The following main observations were made. (1) Müller cells in adult euangiotic (totally vascularized) retinae contain mitochondria throughout their length. (2) Müller cells from the periphery of avascular retinae display mitochondria only within the sclerad-most end of Müller cell processes. (3) Müller cells from the vascularized retinal rim around the optic nerve head in guinea-pigs contain mitochondria throughout their length. (4) Müller cells from the peripapillar myelinated region ('medullary rays') of the pseudangiotic rabbit retina contain mitochondria up to their soma. In living dissociated Müller cells from guinea-pig retina, there was no indication of low intracellular pH where the mitochondria were clustered. These data support the hypothesis that Müller cells display mitochondria only at locations of their cytoplasm where the local O2 pressure (pO2) exceeds a certain threshold. In contrast, retinal ganglion cells of guinea-pig and rabbit retinae display many mitochondria although the local pO2 in the inner (vitread) retinal layers has been reported to be extremely low. It is probable that the alignment of mitochondria and the expression of mitochondrial enzymes are regulated by different mechanisms in various types of retinal neurons and glial cells.