Investigation of Stent Prototypes for the Eustachian Tube in Human Donor Bodies.

Chronic otitis media is often connected to Eustachian tube dysfunction. As successful treatment cannot be guaranteed with the currently available options, the aim is to develop a stent for the Eustachian tube (ET). Over the course of this development, different prototypes were generated and tested in ex vivo experiments. Four different prototypes of an ET stent and one commercially available coronary stent were implanted in the ET of seven human donor bodies. The position of the stents was verified by cone beam CT. The implanted ETs were harvested, embedded in resin and ground at 200 µm steps. Resulting images of the single steps were used to generate 3D models. The 3D models were then evaluated regarding position of the stent in the ET, its diameters, amount of squeezing, orientation of the axes and other parameters. Virtual reconstruction of the implanted ET was successful in all cases and revealed one incorrect stent placement. The cross-section increased for all metal stents in direction from the isthmus towards the pharyngeal orifice of the ET. Depending on the individual design of the metal stents (open or closed design), the shape varied also between different positions along a single stent. In contrast, the cross-section area and shape remained constant along the polymeric prototype. With the current investigation, insight into the behavior of different prototypes of ET stents was gained, which can help in defining the specifications for the intended ET stent.

Refinement of systemic guinea pig deafening in hearing research: Sensorineural hearing loss induced by co-administration of kanamycin and furosemide via the leg veins.

Auditory disabilities have a large impact on the human population worldwide. Research into understanding and treating hearing disabilities has increased significantly in recent years. One of the most relevant animal species in this context is the guinea pig, which has to be deafened to study several of the hearing pathologies and develop novel therapies. Applying kanamycin subcutaneously and furosemide intravenously is a long-established method in hearing research, leading to permanent hearing loss without surgical intervention at the ear. The intravenous application of furosemide requires invasive surgery in the cervical area of the animals to expose the jugular vein, since a relatively large volume (1 ml per 500 g body weight) must be injected over a period of about 2.5 min. We have established a gentler alternative by applying the furosemide by puncture of the leg veins. For this, custom-made cannula-needle devices were built to allow the vein puncture and subsequent slow injection of the furosemide. This approach was tested in 11 guinea pigs through the foreleg via the cephalic antebrachial vein and through the hind leg via the saphenous vein. Frequency-specific hearing thresholds were measured before and after the procedure to verify normal hearing and successful deafening, respectively. The novel approach of systemic deafening was successfully implemented in 10 out of 11 animals. The Vena saphena was best suited to the application. Since the animals' condition, post leg vein application, was better in comparison to animals deafened by exposure of the Vena jugularis, the postulated refinement that reduced animal stress was deemed successful.

Dual Drug Delivery in Cochlear Implants: In Vivo Study of Dexamethasone Combined with Diclofenac or Immunophilin Inhibitor MM284 in Guinea Pigs.

Cochlear implants are well established to treat severe hearing impairments. Despite many different approaches to reduce the formation of connective tissue after electrode insertion and to keep electrical impedances low, results are not yet satisfying. Therefore, the aim of the current study was to combine the incorporation of 5% dexamethasone in the silicone body of the electrode array with an additional polymeric coating releasing diclofenac or the immunophilin inhibitor MM284, some anti-inflammatory substances not yet tested in the inner ear. Guinea pigs were implanted for four weeks and hearing thresholds were determined before implantation and after the observation time. Impedances were monitored over time and, finally, connective tissue and the survival of spiral ganglion neurons (SGNs) were quantified. Impedances increased in all groups to a similar extent but this increase was delayed in the groups with an additional release of diclofenac or MM284. Using Poly-L-lactide (PLLA)-coated electrodes, the damage caused during insertion was much higher than without the coating. Only in these groups, connective tissue could extend to the apex of the cochlea. Despite this, numbers of SGNs were only reduced in PLLA and PLLA plus diclofenac groups. Even though the polymeric coating was not flexible enough, MM284 seems to especially have potential for further evaluation in connection with cochlear implantation.

Effect of Immunophilin Inhibitors on Cochlear Fibroblasts and Spiral Ganglion Cells.

INTRODUCTION: Loss of hair cells and degeneration of spiral ganglion neurons (SGN) lead to severe hearing loss or deafness. The successful use of a cochlear implant (CI) depends among other factors on the number of surviving SGN. Postoperative formation of fibrous tissue around the electrode array causes an increase in electrical impedances at the stimulating contacts. The use of immunophilin inhibitors may reduce the inflammatory processes without suppressing the immune response. Here, we report on in vitro experiments with different concentrations of immunophilin inhibitors MM284 and compound V20 regarding a possible application of these substances in the inner ear. METHODS: Standard cell lines (NIH/3T3 fibroblasts), freshly isolated SGN, and fibroblasts from neonatal rat cochleae (p3-5) were incubated with different concentrations of immunophilin inhibitors for 48 h. Metabolic activity of fibroblasts was investigated by MTT assay and cell survival by counting of immunochemically stained neurons and compared to controls. RESULTS: MM284 did not affect SGN numbers and neurite growth at concentrations of 4 × 10-5 mol/L and below, whereas V20 had no effect at 8 × 10-6 mol/L and below. Metabolic activity of fibroblasts was unchanged at these concentrations. CONCLUSION: Especially MM284 might be considered as a possible candidate for application within the cochlea.

Impedance development after implantation of hybrid-L24 cochlear implant electrodes.

OBJECTIVE: Shorter and thinner electrodes were developed for preserving residual hearing after cochlear implantation by minimising trauma. As trauma is regarded as one of the causes of fibrous tissue formation after implantation, and increase in impedance is considered to be connected to fibrous tissue formation, the aim of the current study was to evaluate impedance development after implantation of Hybrid-L electrodes. DESIGN: Impedance values were retrospectively collected from our clinical database and evaluated for all active contacts and basal, middle and apical contacts separately for up to 10 years. STUDY SAMPLES: All 137 adult patients received a Hybrid-L electrode and had to be implanted for at least 1 year. RESULTS: On average impedances increased to 13 kOhm before first fitting and dropped to 5-7 kOhm under electrical stimulation with lower values measured on apical contacts. Mean values remained stable over years, but variability increased. Values before first fitting were independent of age at implantation whereas lower values were found later in patients of higher age at implantation. CONCLUSION: Despite smaller contacts, impedance values after start of electrical stimulation were comparable to published values of Contour electrodes. This might suggest less tissue growth with the Hybrid-L electrode array.

Intravascular Ultrasonography (IVUS)-A Tool for Imaging the Eustachian Tube?

The Eustachian tube (ET) has a key role in the pathogenesis of otitis media. Until now, there has been a lack of meaningful imaging methods to investigate the ET and its surrounding tissue. The aim of the current study was to investigate the possibilities of imaging the ET using Intravascular Ultrasonography (IVUS). ETs from sheep were scanned ex vivo and in vivo with different IVUS probes. In addition to native ETs, water was also used to improve coupling. Scans were subsequently compared with histological sections and a 3D model of the ET. In addition, ETs with a stenosis induced by a hyaluronic acid depot, after stent insertion, and during lower jaw movement were examined. The IVUS catheter was inserted into the ET lumen without any problems or injuries in all cases. The surrounding structures of the ET were identified in the ultrasound image. In addition, a change in size of the ET lumen due to movement was observed, and the position of the stent and the depot of hyaluronic acid could be examined. With the use of IVUS, a non-invasive possibility to examine the ET over its course with the adjacent structures as well as after different treatments is presented.

Development of an In Vivo Model for Eustachian Tube Dysfunction.

Otitis media is often connected to Eustachian tube dysfunction (ETD). Until now, there was no large animal model available for the examination of new treatment methods such as stents for the Eustachian tube (ET). Thus, the aim of the study was to develop a method to reproducibly induce ETD by injection of fillers and without permanent closure of the ET. Tools for safe injection of hyaluronic acid (HA) in the surrounding of the ET were developed. In ex vivo experiments, HA mixed with Imeron® was injected close to the nasopharyngeal orifice of the ET of blackface sheep. The established depot was visualized using cone beam computer tomography and magnetic resonance imaging, and stents could be placed into the ET. A reliable position of the HA depot was achieved. This method was transferred to in vivo, and middle ear ventilation was investigated by tympanometry. ETD was achieved with amounts of 2.5 mL HA or higher. None of the animals showed any sign of discomfort or complications. The induced ETD lasted for 3 to 13 (maximum observation period) weeks and was also combined with middle ear effusion. A model of ETD based on injection of HA next to the ET was successfully established and is now available to test novel treatment options for ET functionality.

Purification of Fibroblasts From the Spiral Ganglion.

Using cultures of freshly isolated spiral ganglion cells (SGC) is common to investigate the effect of substances on spiral ganglion neurons (SGN) in vitro. As these cultures contain more cell types than just neurons, and it might be beneficial to have cochlear fibroblasts available to further investigate approaches to reduce the growth of fibrous tissue around the electrode array after cochlear implantation, we aimed at the purification of fibroblasts from the spiral ganglion in the current study. Subcultivation of the primary SGC culture removed the neurons from the culture and increased the fibroblast to glial cell ratio in the preparations, which was revealed by staining for vimentin, the S100B-protein, and the 200-kD neurofilament. We performed direct immunolabeling for the Thy1-glycoprotein and the p75NGFR-enabled fluorescence-based cell sorting. This procedure resulted in a cell culture of cochlear fibroblasts with a purity of more than 99%. The received fibroblasts can be subcultivated for up to 10 passages before proliferation rates drop. Additionally, 80% of the cells survived the first attempt of cryopreservation and exhibited a fibroblast-specific morphology. Using the described approach provides a purified preparation of cochlear fibroblasts, which can now be used in vitro for further investigations.

PLLA Coating of Active Implants for Dual Drug Release.

Cochlear implants, like other active implants, rely on precise and effective electrical stimulation of the target tissue but become encapsulated by different amounts of fibrous tissue. The current study aimed at the development of a dual drug release from a PLLA coating and from the bulk material to address short-term and long-lasting release of anti-inflammatory drugs. Inner-ear cytocompatibility of drugs was studied in vitro. A PLLA coating (containing diclofenac) of medical-grade silicone (containing 5% dexamethasone) was developed and release profiles were determined. The influence of different coating thicknesses (2.5, 5 and 10 µm) and loadings (10% and 20% diclofenac) on impedances of electrical contacts were measured with and without pulsatile electrical stimulation. Diclofenac can be applied to the inner ear at concentrations of or below 4 × 10-5 mol/L. Release of dexamethasone from the silicone is diminished by surface coating but not blocked. Addition of 20% diclofenac enhances the dexamethasone release again. All PLLA coatings serve as insulator. This can be overcome by using removable masking on the contacts during the coating process. Dual drug release with different kinetics can be realized by adding drug-loaded coatings to drug-loaded silicone arrays without compromising electrical stimulation.

Functional aspects of the Eustachian tube by means of 3D-modeling.

The extent of dysfunction of the Eustachian tube (ET) is relevant in understanding the pathogenesis of secondary otological diseases such as acute or chronic otitis media. The underlying mechanism of ET dysfunction remains poorly understood except for an apparent genesis such as a nasopharyngeal tumor or cleft palate. To better describe the ET, its functional anatomy, and the biomechanical valve mechanism and subsequent development of diagnostic and interventional tools, a three-dimensional model based on thin-layer histology was created from an ET in this study. Blackface sheep was chosen as a donor. The 3-D model was generated by the coherent alignment of the sections. It was then compared with the cone-beam computed tomography dataset of the complete embedded specimen taken before slicing. The model shows the topographic relation of the individual components, such as the bone and cartilage, the muscles and connective tissue, as well as the lining epithelium with the lumen. It indicates a limited spiraling rotation of the cartilaginous tube over its length and relevant positional relationships of the tensor and levator veli palatine muscles.

Optical absorbance of the tympanic membrane in rat and human samples.

Chronic infections are often connected to biofilm formation. In presence of implants, this can lead to loss of the implant. Systemic or local application of drugs is relatively ineffective in case of biofilm formation. One technique to provide antibacterial properties on demand is the antibacterial photodynamic therapy (aPDT). Using this technique, these properties can be "switched on" by light illumination. In the middle ear with the semitransparent tympanic membrane, it might be possible in future to activate the antibacterial effect without opening the membrane. Therefore, we investigated the optical absorbance spectra of the tympanic membrane. Optical absorbance spectra were measured in ex vivo preparations from neonatal and adult rats with the membrane still being attached to the surrounding bony ring and four human samples. After performing area scans, the spot with the lowest absorbance being surrounded by a ring like structure with higher absorbance was chosen as region of interest for scanning wavelengths between 300 and 900 nm. Absorbance is generally higher at lower wavelengths with a local absorbance maximum at 420 nm and a weak second maximum with two neighbouring peaks at 540 / 580 nm and is significantly higher in adult rats compared to neonatal rats where about 10% of light was transmitted. The human samples show similar characteristics with a little higher absorbance. For activation of aPDT through the tympanic membrane, larger wavelengths are more promising. Whether the amount of light transmitted through the membrane would be sufficient to induce aPDT remains to be tested in further experiments.

Do Impedance Changes Correlate With a Delayed Hearing Loss After Hybrid L24 Implantation?

OBJECTIVES: Preservation of residual hearing is one of the main goals in present cochlear implantation surgery. Especially for this purpose, smaller and softer electrode carriers were developed that are to be inserted through the round window membrane to minimize trauma. By using these electrodes and insertion technique, residual hearing can be preserved in a large number of patients. Unfortunately, some of these patients with initially preserved residual hearing after cochlear implantation lose it later on. The reason for this is unknown but it is speculated about a correlation with an increase in impedance, since increased impedance values are linked to intracochlear inflammation and tissue reaction. Our hypothesis for this study design was that an increase in impedance predicts changes in residual hearing under clinical conditions. DESIGN: Data of all adult patients (N = 122) receiving a Hybrid-L24 cochlear implant at our center between 2005 and early 2015 were retrospectively evaluated. Impedance values in Common Ground mode as measured during clinical routine and referring audiological test data (audiometric thresholds under headphones) were collected. Changes between consecutive measurements were calculated for impedance values and hearing thresholds for each patient. Correlations between changes in impedances and acoustic hearing thresholds were calculated. Average values were compared as well as patients with largest impedance changes within the observation period were evaluated separately. RESULTS: Group mean values of impedances were between 5 and 7 kΩ and stable over time with higher values on basal electrode contacts compared with apical contacts. Average hearing thresholds at the time of initial fitting were between 40 to 50 dB (250 Hz) and 90 dB (1 kHz) with a loss of about 10 dB compared with preoperative values. Correlation between impedance changes and threshold changes was found, but too inconsistently to imply a true relationship. When evaluating the 20 patients with the largest impedance changes during the observation period (all >1 kΩ from one appointment to the next one), some patients were found where hearing loss is timely connected and highly correlated with an unusual impedance change. But large impedance changes were also observed without affecting hearing thresholds and hearing loss was found without impedance change. CONCLUSIONS: Changes in impedance as measured during clinical routine cannot be taken as an indicator for a late acoustic hearing loss.

Consecutive Treatment with Brain-Derived Neurotrophic Factor and Electrical Stimulation Has a Protective Effect on Primary Auditory Neurons.

Degeneration of neurons, such as the inner ear spiral ganglion neurons (SGN), may be decelerated or even stopped by neurotrophic factor treatment, such as brain-derived neurotrophic factor (BDNF), as well as electrical stimulation (ES). In a clinical setting, drug treatment of the SGN could start directly during implantation of a cochlear implant, whereas electrical stimulation begins days to weeks later. The present study was conducted to determine the effects of consecutive BDNF and ES treatments on SGN density and electrical responsiveness. An electrode drug delivery device was implanted in guinea pigs 3 weeks after deafening and five experimental groups were established: two groups received intracochlear infusion of artificial perilymph (AP) or BDNF; two groups were treated with AP respectively BDNF in addition to ES (AP + ES, BDNF + ES); and one group received BDNF from the day of implantation until day 34 followed by ES (BDNF ⇨ ES). Electrically evoked auditory brainstem responses were recorded. After one month of treatment, the tissue was harvested and the SGN density was assessed. The results show that consecutive treatment with BDNF and ES was as successful as the simultaneous combined treatment in terms of enhanced SGN density compared to the untreated contralateral side but not in regard to the numbers of protected cells.

Differential Effects of Low- and High-Dose Dexamethasone on Electrically Induced Damage of the Cultured Organ of Corti.

An increased number of patients with residual hearing are undergoing cochlear implantation. A subset of these experience delayed hearing loss post-implantation, and the aetiology of this loss is not well understood. Our previous studies suggest that electrical stimulation can induce damage to hair cells in organ of Corti (OC) organotypic cultures. Dexamethasone has the potential to protect residual hearing due to its multiple effects on cells and tissue (e.g., anti-inflammatory, free radical scavenger). We therefore hypothesized that dexamethasone treatment could prevent electrical stimulation induced changes in the OC. Organ of Corti explants from neonatal rats (P2-4) were cultured for 24 h with two different concentrations of dexamethasone. Thereafter, OC were subjected to a charge-balanced biphasic pulsed electrical stimulation (0.44-2 mA) for a further 24 h. Unstimulated dexamethasone-treated OC served as controls. Outcome analysis included immunohistochemical labelling of ribbon synapses, histochemical analysis of free reactive oxygen species and morphological analysis of stereocilia bundles. Overall, the protective effects of dexamethasone on electrically induced damage in cochlear explants were moderate. High-dose dexamethasone protected bundle integrity at higher current levels. Low-dose dexamethasone tended to increase ribbon density in the apical region.

Dimensions and position of the Eustachian tube in Humans.

Eustachian tube (ET) dysfunction is one of the causes for chronic otitis media. To develop new therapies such as stents to facilitate middle ear ventilation, a better knowledge on dimensions and positions of the ET in individual patients is necessary. Cone beam CT scans of 143 patients were retrospectively investigated. Parameters such as lengths of the ET and its cartilaginous and bony parts, diameters, angles as well as distance of the ostium from the nasal conchae were determined and evaluated for side, gender and age specific differences. The average length of the cartilaginous and bony tubes was smaller in women than men. The average deviation from the horizontal plane was 1.7° larger on the left side (35.4°) compared to the right side (33.7°). Tools to manipulate the ET or to insert stents into the ET should cover angles from at least 42° to 64°. The distance of the pharyngeal orifices from the conchae nasalis inferior increased with age, becoming most prominent above 70 years of age. This investigation provides necessary information to develop stents for human application and tools for safe positioning of the stents.

Correction to: Influence of In Vitro Electrical Stimulation on Survival of Spiral Ganglion Neurons.

The first multiplication sign (.) for unit µC cm¯2·phase¯1 was not placed, which is part of the author's correction. Furthermore, the unit appears anywhere in the article.

Influence of In Vitro Electrical Stimulation on Survival of Spiral Ganglion Neurons.

Patients scheduled for cochlear implantation often retain residual hearing in the low frequencies. Unfortunately, some patients lose their residual hearing following implantation and the reasons for this are not well understood. Evidence suggests that electrotoxicity could be one of the factors responsible for this late adverse effect. Therefore, the aim of this study was to investigate the survival of spiral ganglion neurons (SGN) subjected to in vitro electrical stimulation (ES). A stimulation setup was developed to provide defined electrical fields at given points of the chamber. SGN isolated from Sprague Dawley rats (P3-4) were dissociated and cultured in the chamber for 24 h prior to biphasic, pulsed electrical field exposure for another 24 h. The current varied in the range of 0 to 2 mA and the pulse width from 10 to 400 µs. Neurite growth and survival were evaluated with respect to the charge density at the position of the cells. Non-exposed SGN cultures served as control. Charge densities below 2.2 µC·cm-2·phase-1 appeared to have no effect on SGN survival and neurite outgrowth. Charge densities above 4.9 µC·cm-2·phase-1 were detrimental to almost all cells in culture. After fitting results to a sigmoidal dose response curve, a LD50 of 2.9 µC·cm-2·phase-1 was calculated. This screening regarding survival and outgrowth of SGN provides parameters that could be used to further investigate the effect of ES on SGN and to develop possible protection strategies, which could potentially rescue residual hearing in the implanted patients.

Stenting the Eustachian tube to treat chronic otitis media - a feasibility study in sheep.

BACKGROUND: Untreated chronic otitis media severely impairs quality of life in affected individuals. Local destruction of the middle ear and subsequent loss of hearing are common sequelae, and currently available treatments provide limited relief. Therefore, the objectives of this study were to evaluate the feasibility of the insertion of a coronary stent from the nasopharynx into the Eustachian tube in-vivo in sheep and to make an initial assessment of its positional stability, tolerance by the animal, and possible tissue reactions. METHODS: Bilateral implantation of bare metal cobalt-chrome coronary stents of two sizes was performed endoscopically in three healthy blackface sheep using a nasopharyngeal approach. The postoperative observation period was three months. RESULTS: Stent implantation into the Eustachian tube was feasible with no intra- or post-operative complications. Health status of the sheep was unaffected. All stents preserved their cylindrical shape. All shorter stents remained in position and ventilated the middle ear even when partially filled with secretion or tissue. One of the long stents became dislocated toward the nasopharynx. Both of the others remained fixed at the isthmus but appeared to be blocked by tissue or secretion. Tissue overgrowth on top of the struts of all stents resulted in closure of the tissue-lumen interface. CONCLUSION: Stenting of the Eustachian tube was successfully transferred from cadaver studies to an in-vivo application without complications. The stent was well tolerated, the middle ears were ventilated, and clearance of the auditory tube appeared possible. For fixation, it seems to be sufficient to place it only in the cartilaginous part of the Eustachian tube.

Local inner ear application of dexamethasone in cochlear implant models is safe for auditory neurons and increases the neuroprotective effect of chronic electrical stimulation.

Dexamethasone (DEX) can reduce fibrous tissue growth as well as loss of residual hearing which may occur after cochlear implantation. Little is known about the effect of local inner ear DEX treatment on the spiral ganglion neurons (SGN), which are the target of the electrical stimulation with a cochlear implant (CI). Three different clinically relevant strategies of DEX-delivery into the inner ear were used. DEX was either eluted from the electrode carriers' silicone, released from a reservoir by passive diffusion, or actively applied using a pump based system. The effect of the locally applied DEX on SGN density, size and function was evaluated. DEX did not affect the SGN density compared to the relevant control groups. Simultaneously applied with chronic electrical stimulation (ES), DEX increased the neuroprotective effect of ES in the basal region and the hearing threshold tended to decrease. The EABR thresholds did not correlate with the relevant SGN density. When correlating the SGN number with fibrosis, no dependency was observed. DEX concentrations as applied in these animal models are safe for inner ear delivery in terms of their effect on SGN density. Additionally, DEX tends to improve the neuroprotective effect of chronic electrical stimulation by increasing the number of surviving neurons. This is an important finding in regard to clinical applications of DEX for local treatment of the inner ear in view of cochlear implantation and other applications.

Effect of hyperbaric oxygen on BDNF-release and neuroprotection: Investigations with human mesenchymal stem cells and genetically modified NIH3T3 fibroblasts as putative cell therapeutics.

Hyperbaric oxygen therapy (HBOT) is a noninvasive widely applied treatment that increases the oxygen pressure in tissues. In cochlear implant (CI) research, intracochlear application of neurotrophic factors (NTFs) is able to improve survival of spiral ganglion neurons (SGN) after deafness. Cell-based delivery of NTFs such as brain-derived neurotrophic factor (BDNF) may be realized by cell-coating of the surface of the CI electrode. Human mesenchymal stem cells (MSC) secrete a variety of different neurotrophic factors and may be used for the development of a biohybrid electrode in order to release endogenously-derived neuroprotective factors for the protection of residual SGN and for a guided outgrowth of dendrites in the direction of the CI electrode. HBOT could be used to influence cell behaviour after transplantation to the inner ear. The aim of this study was to investigate the effect of HBOT on the proliferation, BDNF-release and secretion of neuroprotective factors. Thus, model cells (an immortalized fibroblast cell line (NIH3T3)-native and genetically modified) and MSCs were repeatedly (3 x - 10 x) exposed to 100% oxygen at different pressures. The effects of HBO on cell proliferation were investigated in relation to normoxic and normobaric conditions (NOR). Moreover, the neuroprotective and neuroregenerative effects of HBO-treated cells were analysed by cultivation of SGN in conditioned medium. Both, the genetically modified NIH3T3/BDNF and native NIH3T3 fibroblasts, showed a highly significant increased proliferation after five days of HBOT in comparison to normoxic controls. By contrast, the number of MSCs was decreased in MSCs treated with 2.0 bar of HBO. Treating SGN cultures with supernatants of fibroblasts and MSCs significantly increased the survival rate of SGN. HBO treatment did not influence (increase / reduce) this effect. Secretome analysis showed that HBO treatment altered the protein expression pattern in MSCs.