Rachael T. Richardson

Polypyrrole-coated electrodes for the delivery of charge and neurotrophins to cochlear neurons.

Sensorineural hearing loss is associated with gradual degeneration of spiral ganglion neurons (SGNs), compromising hearing outcomes with cochlear implant use. Combination of neurotrophin delivery to the cochlea and electrical stimulation from a cochlear implant protects SGNs, prompting research into neurotrophin-eluting polymer electrode coatings. The electrically conducting polypyrrole/para-toluene sulfonate containing neurotrophin-3 (Ppy/pTS/NT3) was applied to 1.7 mm2 cochlear implant electrodes. Ppy/pTS/NT3-coated electrode arrays stored 2 ng NT3 and released 0.1 ng/day with electrical stimulation. Guinea pigs were implanted with Ppy/pTS or Ppy/pTS/NT3 electrode arrays two weeks after deafening via aminoglycosides. The electrodes of a subgroup of these guinea pigs were electrically stimulated for 8 h/day for 2 weeks. There was a loss of SGNs in the implanted cochleae of guinea pigs with Ppy/pTS-coated electrodes indicative of electrode insertion damage. However, guinea pigs implanted with electrically stimulated Ppy/pTS/NT3-coated electrodes had lower electrically-evoked auditory brainstem response thresholds and greater SGN densities in implanted cochleae compared to non-implanted cochleae and compared to animals implanted with Ppy/pTS-coated electrodes (p<0.05). Ppy/pTS/NT3 did not exacerbate fibrous tissue formation and did not affect electrode impedance. Drug-eluting conducting polymer coatings on cochlear implant electrodes present a clinically viable method to promote preservation of SGNs without adversely affecting the function of the cochlear implant.

Resprouting and survival of guinea pig cochlear neurons in response to the administration of the neurotrophins brain-derived neurotrophic factor and neurotrophin-3.

Degeneration of auditory neurons occurs after deafening and is associated with damage to the organ of Corti. The administration of neurotrophins can protect auditory neurons against degeneration if given shortly after deafening. However, it is not known whether the delayed administration of neurotrophins, when significant degeneration has already occurred, will provide similar protection. Furthermore, little is known about the effects of neurotrophins on the peripheral processes of the auditory neurons or whether these neurons can resprout. This study examined the morphological effects on auditory neurons following deafening and the administration of brain‐derived neurotrophic factor and neurotrophin‐3. Results showed that neurotrophins were effective in preventing death of auditory neurons if administered 5 days after deafening and were also effective in preventing the continued loss of neurons if the administration was delayed by 33 days. The peripheral processes of auditory neurons in cochleae that received neurotrophins were in greater number and had larger diameters compared with the untreated cochleae. Localized regions of resprouting peripheral processes were observed in deafened cochleae and were enhanced in response to neurotrophin treatment, occurring across wider regions of the cochlea. These findings have significant implications for an improvement in the performance of the cochlear implant and for future therapies to restore hearing to the deaf. J. Comp. Neurol. 487:147–165, 2005.

Conducting polymers, dual neurotrophins and pulsed electrical stimulation — Dramatic effects on neurite outgrowth

In this study the synergistic effect of delivering two neurotrophins simultaneously to encourage neuron survival and neurite elongation was explored. Neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) were incorporated into polypyrrole (PPy) during electrosynthesis and the amounts incorporated and released were determined using iodine-125 ((125)I) radio-labelled neurotrophins. Neurite outgrowth from cochlear neural explants grown on the conducting polymer was equivalent to that on tissue culture plastic but significantly improved with the incorporation of NT-3 and BDNF. Neurite outgrowth from explants grown on polymers containing both NT-3 and BDNF showed significant improvement over PPy doped only with NT-3, due to the synergistic effect of both neurotrophins. Neurite outgrowth was significantly improved when the polymer containing both neurotrophins was electrically stimulated. It is envisaged that when applied to the cochlear implant, these conducting and novel polymer films will provide a biocompatible substrate for storage and release of neurotrophins to help protect auditory neurons from degradation after sensorineural hearing loss and encourage neurite outgrowth towards the electrodes.

Effects of round window dexamethasone on residual hearing in a Guinea pig model of cochlear implantation.

To study electric acoustic stimulation, we have developed a model of guinea pig cochlear implantation via a cochleostomy. Thirty minutes prior to implantation, a hyaluronic acid/carboxymethylcellulose bead, loaded with either dexamethasone or normal saline, was placed upon the round window membrane. Animals that did not receive beads acted as controls. Pure-tone auditory brainstem response thresholds were estimated before and after electrode insertion, and 1 and 4 weeks later. Selected cochlear histology was performed. Results: Dexamethasone could be detected in the cochlea for 24 h after cochlear implantation. Thresholds were elevated across frequencies in all animals immediately after surgery. These thresholds recovered completely at and below 2 kHz, and partially at higher frequencies by 1 week after implantation. At 32 kHz, but not the lower frequencies, the presence of dexamethasone had a significant protective effect upon hearing, which increased in magnitude over time. The protection was greatest in difficult implantations where an intractable resistance to electrode insertion was met. There was a persistent foreign body reaction at the site of implantation of saline-treated implanted ears but not in the dexamethasone-treated implanted ears. Conclusion: Short-term preoperative delivery of dexamethasone through the round window can protect residual hearing during cochlear implantation, especially during technically difficult surgery.

Effects of Localized Neurotrophin Gene Expression on Spiral Ganglion Neuron Resprouting in the Deafened Cochlea

A cochlear implant may be used to electrically stimulate spiral ganglion neurons (SGNs) in people with severe sensorineural hearing loss (SNHL). However, these neurons progressively degenerate after SNHL due to loss of neurotrophins normally supplied by sensory hair cells (HCs). Experimentally, exogenous neurotrophin administration prevents SGN degeneration but can also result in abnormal resprouting of their peripheral fibers. This study aimed to create a target-derived neurotrophin source to increase neuron survival and redirect fiber resprouting following SNHL. Adenoviral (Ad) vectors expressing green fluorescent protein (GFP) alone or in combination with brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT3) were injected into the cochlear scala tympani or scala media of guinea-pigs (GPs) deafened via aminoglycosides for 1 week. After 3 weeks, cochleae were examined for gene expression, neuron survival, and the projection of peripheral fibers in response to gene expression. Injection of vectors into the scala media resulted in more localized gene expression than scala tympani injection with gene expression consistently observed within the partially degenerated organ of Corti. There was also greater neuron survival and evidence of localized fiber responses to neurotrophin-expressing cells within the organ of Corti from scala media injections (P < 0.05), a first step in promoting organized resprouting of auditory peripheral fibers via gene therapy.

Effect of the dopant anion in polypyrrole on nerve growth and release of a neurotrophic protein

The dopant anion in polypyrrole plays a critical role in determining the physical and chemical properties of these conducting polymers. Here we demonstrate an additional effect on the ability to incorporate and release a neurotrophic protein - neurotrophin-3. The multi-faceted role of the dopant is critical in ensuring optimal performance of polypyrroles in their use as platforms for nerve growth. In this paper, the effect of changing the co-dopant used in electrochemical polypyrrole synthesis on the compatibility with primary auditory nerve tissue is considered and compared to some of the physical properties of the films. Significant differences in the controlled-release properties of the films were also observed. The ability of the polymers to enhance nerve growth and survival in vitro with neurotrophin-3 release was also studied, which is a function of both compatibility with the neural tissue and the ability of the polymer to release sufficient neurotrophic protein to affect cell growth. A small synthetic dopant, para-toluene sulphonate, was found to perform favourably in both aspects and ultimately proved to be the most suitable material for the application at hand, which is the delivery of neurotrophins for inner-ear therapies.

Factors influencing the efficacy of round window dexamethasone protection of residual hearing post-cochlear implant surgery

AIM To protect hearing in an experimental model of cochlear implantation by the application of dexamethasone to the round window prior to surgery. The present study examined the dosage and timing relationships required to optimise the hearing protection. METHODS Dexamethasone or saline (control) was absorbed into a pledget of the carboxymethylcellulose and hyaluronic acid and applied to the round window of the guinea pig prior to cochlear implantation. The treatment groups were 2% w/v dexamethasone for 30, 60 and 120min; 20% dexamethasone applied for 30min. Auditory sensitivity was determined pre-operatively, and at 1 week after surgery, with pure-tone auditory brainstem response audiometry (2-32kHz). Cochlear implantation was performed via a cochleostomy drilled into the basal turn of the cochlea, into which a miniature cochlear implant dummy electrode was inserted using soft-surgery techniques. RESULTS ABR thresholds were elevated after cochlear implantation, maximally at 32kHz and to a lesser extent at lower frequencies. Thresholds were less elevated after dexamethasone treatment, and the hearing protection improved when 2% dexamethasone was applied to the round window for longer periods of time prior to implantation. The time that dexamethasone need be applied to achieve hearing protection could be reduced by increasing the concentration of steroid, with a 20% application for 30min achieving similar levels of protection to a 60min application of 2% dexamethasone. CONCLUSIONS Hearing protection is improved by increasing the time that dexamethasone is applied to the round window prior to cochlear implantation, and the waiting time can be reduced by increasing the steroid concentration. These results suggest that the diffusion dexamethasone through the cochlea is the prime determinant of the extent of hearing protection.

Neurotrophic Factors and Neural Prostheses: Potential Clinical Applications Based Upon Findings in the Auditory System

Spiral ganglion neurons (SGNs) are the target cells of the cochlear implant, a neural prosthesis designed to provide important auditory cues to severely or profoundly deaf patients. The ongoing degeneration of SGNs that occurs following a sensorineural hearing loss is, therefore, considered a limiting factor in cochlear implant efficacy. We review neurobiological techniques aimed at preventing SGN degeneration using exogenous delivery of neurotrophic factors. Application of these proteins prevents SGN degeneration and can enhance neurite outgrowth. Furthermore, chronic electrical stimulation of SGNs increases neurotrophic factor-induced survival and is correlated with functional benefits. The application of neurotrophic factors has the potential to enhance the benefits that patients can derive from cochlear implants; moreover, these techniques may be relevant for use with neural prostheses in other neurological conditions

Promoting neurite outgrowth from spiral ganglion neuron explants using polypyrrole/BDNF‐coated electrodes

Release of neurotrophin-3 (NT3) and brain-derived neurotrophic factor (BDNF) from hair cells in the cochlea is essential for the survival of spiral ganglion neurons (SGNs). Loss of hair cells associated with a sensorineural hearing loss therefore results in degeneration of SGNs, potentially reducing the performance of a cochlear implant. Exogenous replacement of either or both neurotrophins protects SGNs from degeneration after deafness. We previously incorporated NT3 into the conducting polymer polypyrrole (Ppy) synthesized with para-toluene sulfonate (pTS) to investigate whether Ppy/pTS/NT3-coated cochlear implant electrodes could provide both neurotrophic support and electrical stimulation for SGNs. Enhanced and controlled release of NT3 was achieved when Ppy/pTS/NT3-coated electrodes were subjected to electrical stimulation. Here we describe the release dynamics and biological properties of Ppy/pTS with incorporated BDNF. Release studies demonstrated slow passive diffusion of BDNF from Ppy/pTS/BDNF, with electrical stimulation significantly enhancing BDNF release over 7 days. A 3-day SGN explant assay found that neurite outgrowth from explants was 12.3-fold greater when polymers contained BDNF (p < 0.001), although electrical stimulation did not increase neurite outgrowth further. The versatility of Ppy to store and release neurotrophins, conduct electrical charge, and act as a substrate for nerve-electrode interactions is discussed for specialized applications such as cochlear implants.

Delivery of neurotrophin-3 to the cochlea using alginate beads.

Objective: The aim of this study was to design a novel cochlear neurotrophin (NT) delivery system for the rescue of auditory neurons after ototoxicity-induced deafening. Background: NT-3 is a trophic growth factor that promotes the survival of the auditory nerve and may have a potential therapeutic role in slowing neuron loss in progressive deafness, especially as an adjunct to the current cochlear implant. Beads made from alginate are biodegradable, slow release substances that can be placed at the round window or inside the cochlea. This study investigated the loading properties, release kinetics, and implantation potential of alginate beads loaded with NT-3. Methods: Alginate beads were prepared using an ionic gelation technique and postloaded with NT-3. Release of NT-3 was measured using enzyme-linked immunosorbent assay over 5 days. Alginate beads were implanted into deafened guinea pigs for 28 days, after which survival of auditory neurons was assessed. Results: Enzyme-linked immunosorbent assay studies demonstrated a 98% to 99% loading of NT-3 with a slow, partial release over 5 days in Ringers solution. Furthermore, the addition of heparin to the delivery system modulated NT-3 release to a steadier pattern. Implantation of alginate-heparin beads in guinea pig cochleae produced minimal local tissue reaction. NT-3 loaded beads implanted at both the round window and within the scala tympani of the basal turn provided auditory neurons significant protection from degradation and apoptosis compared with unloaded beads or untreated cochleae. Conclusions: This study demonstrates alginate beads to be a safe, biodegradable and effective delivery system for NT-3 to the cochlea.