Combining Cell-Based Therapies and Neural Prostheses to Promote Neural Survival
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- Wise, A.K., Fallon, J.B., Neil, A.J. et al. Neurotherapeutics (2011) 8: 774. doi:10.1007/s13311-011-0070-0
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Cochlear implants provide partial restoration of hearing for profoundly deaf patients by electrically stimulating spiral ganglion neurons (SGNs); however, these neurons gradually degenerate following the onset of deafness. Although the exogenous application of neurotrophins (NTs) can prevent SGN loss, current techniques to administer NTs for long periods of time have limited clinical applicability. We have used encapsulated choroid plexus cells (NTCells; Living Cell Technologies, Auckland, New Zealand) to provide NTs in a clinically viable manner that can be combined with a cochlear implant. Neonatal cats were deafened and unilaterally implanted with NTCells and a cochlear implant. Animals received chronic electrical stimulation (ES) alone, NTs alone, or combined NTs and ES (ES + NT) for a period of as much as 8 months. The opposite ear served as a deafened unimplanted control. Chronic ES alone did not result in increased survival of SGNs or their peripheral processes. NT treatment alone resulted in greater SGN survival restricted to the upper basal cochlear region and an increased density of SGN peripheral processes. Importantly, chronic ES in combination with NTs provided significant SGN survival throughout a wider extent of the cochlea, in addition to an increased peripheral process density. Re-sprouting peripheral processes were observed in the scala media and scala tympani, raising the possibility of direct contact between peripheral processes and a cochlear implant electrode array. We conclude that cell-based therapy is clinically viable and effective in promoting SGN survival for extended durations of cochlear implant use. These findings have important implications for the safe delivery of therapeutic drugs to the cochlea.
KeywordsCell-based therapyCochlear implantationNeurotrophinNerve protectionElectrical stimulation
In 2005, the World Health Organization estimated that 278 million people were living with a significant hearing impairment. The most common form of impairment is a sensorineural hearing loss (SNHL), which occurs when the sensory hair cells of the cochlea are damaged or absent. For patients with a severely profound SNHL, the only clinical treatment is a cochlear implant, which is a neural prosthesis designed to electrically stimulate spiral ganglion neurons (SGNs) to partially restore hearing to deaf patients. However, SNHL also results in the gradual degeneration of SGNs, the target cells of the cochlear implant [1–3]. SGN degeneration is characterized by retraction of the peripheral processes of the SGNs and ultimately leads to degeneration of the SGN soma and central process [4, 5]. It is believed that a loss of endogenous neurotrophic support normally provided by the sensory hair cells [6, 7] is the primary cause of SGN death, a factor likely to limit the effectiveness of cochlear implants. Therefore, research focusing on strategies to prevent loss of the SGNs and their peripheral processes following deafness should lead to improvements in cochlear implant performance .
Administration of neuroprotective agents (e.g., the neurotrophins [NTs], brain derived neurotrophic factor, and NT3) in profoundly deafened cochleae has been shown to prevent SGN loss [9–16]. However, the survival promoting effects were short lived once the NT delivery had ceased [17, 18], suggesting that ongoing delivery is required for lasting effects. Combining NT delivery with chronic intracochlear electrical stimulation (ES) from a cochlear implant can enhance SGN survival , even after the withdrawal of the exogenous NT supply , indicating the potential synergies between treatment with NTs and chronic ES.
In most studies to date, mini-osmotic pumps have been used to deliver NTs over relatively short time periods (weeks) to the cochleae of deaf rodents [8, 9, 11–15, 17, 18]. The use of mini-osmotic pumps in rodent deafness models, in which SGNs degenerate much more rapidly than in other species, such as the cat (in months ) and human (in years ), has limited the examination of NT therapies to relatively short timeframes. Furthermore, the limited reservoir and significant risk of infection associated with mini-pumps reduces their potential clinical application . Consequently, the promise of NT therapy to rescue SGNs after SNHL has yet to be realized, and effective and safe drug administration strategies for application in the inner ear remains elusive. The use of cell-based therapies provides a clinically viable NT delivery technique that is less susceptible to adverse side effects than pump-based delivery methods [21–23]. In the present study, choroid plexus cells (NTCells, Living Cell Technologies, Auckland, New Zealand), which produce and release a cocktail of NTs , were encapsulated in biocompatible alginate and implanted into the neonatal-deafened cat cochlea in combination with a cochlear implant electrode array. The results showed that treatment with cell-based NT therapy for an extended duration (as much as 8 months) provided protection of SGNs and their peripheral processes from deafness-induced changes, and also that survival effects were enhanced by concurrent chronic ES via a cochlear implant.
Twenty-four cats were used in these experiments. All animal procedures were carried out with the approval of the Royal Victorian Eye and Ear Hospital Animal Research and Ethics Committee and conformed to the Code of Practice of the National Health and Medical Research Council of Australia.
Deafening and Implant Surgery
Experimental groups, the number of animals used, and the duration of treatment (± SEM)
Mean duration of treatment
195 ± 2.8 days
234 ± 5.3 days
ES + NT
217 ± 8.7 days
Cells were harvested by Living Cell Technologies from the choroid plexus of neonatal pigs, as previously described . The pigs (2–6 kg) were sourced from a pathogen-free and isolated colony on the Auckland Islands (New Zealand). The choroid plexus was removed, finely dissected, digested with collagenase and allowed to settle before the supernatant was removed and filtered. The preparation was adjusted to approximately 5000 clusters/mL in culture medium before being encapsulated in an alginate membrane using good manufacturing practice techniques previously described . The alginate membrane is biocompatible and semi-permeable, permitting the transfer of secreted NTs, gases, nutrients, and waste products across the membrane and isolating the implanted cells from the immune system of the host .
Intracochlear Electrode Array and Chronic Electrical Stimulation
The intracochlear electrode assembly contained 8 intracochlear platinum electrodes on a silicone carrier and 1 extracochlear platinum ball return electrode. The intracochlear electrodes were 0.3-mm wide with an inter-electrode separation of 0.45 mm. A percutaneous lead wire connected the electrode array to a clinical cochlear implant (CI 24, Cochlear Ltd, Sydney, Australia) and speech processor (ESPrit 3G, Cochlear Ltd, Sydney, Australia) as described previously . Chronic environmentally-derived ES was delivered as monopolar charge-balanced biphasic current pulses (8 μs phase, 25 μs gap) at a rate of 400 pps/electrode, consistent with clinical configurations. The ES was provided by clinical speech processors and stimulators, and therefore delivered ES identical to that which a cochlear implant patient would receive in the same sound environment. These sounds included vocalizations of the animals and self-generated sounds (e.g., playing with noisy toys) that were shown to drive the processors and thus deliver ES at perceptible levels. This is 1 of a handful of studies to use such clinical stimulation in an animal model, and the first to combine clinical ES with clinically viable NT delivery. Stimulation commenced 2 weeks after surgery and the speech processors were programmed to map the full dynamic range of the acoustic environment to electrical stimulus levels from 3 dB below to 6 dB above the electrically-evoked auditory brainstem response (EABR) thresholds. Standard electrophysiological techniques (as described for ABRs) were used to measure EABRs and thresholds were defined as the current level that elicited a peak-to-trough response amplitude of at least 0.2 mV at a latency of 2 to 3 ms . Animals received ES in a clinically relevant manner >16 h/day, 7 days/week for a period of as much as 8 months. The electrical stimulus did not distress the animals as indicated by an absence of stress behavior, such as withdrawal, aggression, appetite, or weight loss and the continuance of normal behavior, such as hunting and inquisitiveness to environmental stimulants and participation in normal play with other cats. Electrode functioning was assessed by daily monitoring of electrode impedances and monthly measurements of EABR thresholds. Stimulation parameters were adjusted in accordance with any changes observed to EABR thresholds, to ensure that ES was delivered at levels sufficient to activate the auditory pathway.
Histology, Immunohistology, and SGN Quantification
At the conclusion of the treatment period, the animal was euthanized and intracardially perfused with 4% paraformaldehyde. The cochleae were removed, locally perfused, and postfixed in the same fixative overnight at 4°C. The cochleae were decalcified in a solution containing 10% (wt./vol.) ethylenediamine tetra-acetic acid in 0.1 mol/L phosphate buffer and then cryopreserved in 30% sucrose solution. The cochleae were infiltrated with O.C.T.™ (Tissue-Tek, Torrance, CA) and snap frozen before being sectioned on a cryostat at 12 μm and mounted onto Superfrost-Plus slides (Menzel-Gläser, Braunschweig, Germany) . A representative series of sections of the entire cochlea was stained with hematoxylin and eosin.
Immunohistochemistry was performed on cochlear sections to identify the mitochondrial content of the NTCells within the implanted capsules and to analyze the SGN peripheral processes within the osseous spiral lamina (OSL). Standard immunohistochemistry procedures were used, whereby sections were washed and blocked before incubation overnight with the primary antibody. After another wash, sections were incubated in the secondary antibody (Alexa Fluro, Molecular Probes Eugene, OR) and mounted with Vectashield mounting media containing DAPI (Vector Laboratories, CA, USA). Antibodies for cytochrome C oxidase (complex IV, subunit Va; Mitosciences, Eugene, OR) were used on sections containing encapsulated NTCells to detect intracellular mitochondria and obtain evidence of metabolic activity. Sections were imaged on a Zeiss Axioplan microscope (Carl Zeiss, Jena, Germany).
Antibodies to neurofilament-200 (NF-200; Chemicon International, Australia) were used to examine the effects of treatment on the radially projecting peripheral processes of the SGNs. Sections were imaged on a confocal microscope (LSM 510 META, Carl Zeiss, Jena, Germany) and confocal stacks containing up to 10 images were collected. A projected image of the stack was used for analysis. The inbuilt “moment” threshold filter in Image J software (National Institutes of Health, USA) was used to threshold the image, and a region within the OSL containing the peripheral processes was selected for analysis. The density of NF-200 labelled pixels was determined for each section and averages across the 4 sections (76-μm apart) were calculated for each cochlea.
SGNs were quantified in mid-modiolar sections using a Zeiss microscope by a single observer blinded to the experimental cohorts. SGNs were identified within Rosenthal’s canal and counted within the lower basal, upper basal, lower middle, upper middle, and apical cochlear regions (Fig. 1). Only SGNs exhibiting a clear nucleus and nucleoli were counted . The area of Rosenthal’s canal was measured using Image J software and the density of the SGNs was determined. SGN density data for each cochlear region were averaged from 5 sections that were spaced 76-μm apart, ensuring that no SGN was counted more than once.
Cochlear Response to Implantation
The tissue response of the cochlea to the presence of the electrode array and the capsules was determined to assess the biocompatibility of the NT therapy. This was done by measuring the percentage of the area of the scala tympani occupied by an inflammatory response (fibrous tissue and new bone). Tissue response was quantified in hematoxylin and eosin-stained sections at 4 cochlear locations within the basal turn (Fig. 1) using a Zeiss Axioplan microscope, which was then analyzed using Image J.
Statistical comparisons of SGN density and peripheral process density were made by comparing the treated cochlea (left) with the unimplanted contralateral control cochlea (right) via a paired t test or a repeated measures (RM) analysis of variance (ANOVA) using p < 0.05 level of significance. Post hoc comparisons were made using the Holm Sidak method.
Deafness-Induced Degeneration of SGNs
SGN Survival following ES Treatment
SGN Survival following NT Treatment
SGN Survival following Combined ES and NT Treatment
Summary of SGN survival effects for each treatment condition across cochlear region
ES + NT
Peripheral Process Density
The peripheral process density was analyzed for all treatment groups in the basal cochlear region. Examples are shown for peripheral processes in an ES + NT treated cochlea (Fig. 6b) that exhibited a greater density of peripheral processes compared to the untreated control cochlea (Fig. 6c). There was a significant main effect of treatment on the density of peripheral processes (RM ANOVA, p < 0.001). Post hoc analysis indicated that there was a significantly greater density of processes in ES + NT treated cochlea (Holm-Sidak, p < 0.001) and in NT-treated cochleae (p < 0.001) compared to their respective contralateral control cochleae. There was no difference in the density of peripheral processes in the ES-treated cochleae compared to the control. The density of processes in normal cochleae was significantly greater than all treatment groups (1-way ANOVA, p < 0.001).
Peripheral Process Re-Sprouting
Tissue Response to Implantation
This study has shown that cell-based delivery of NTs promoted the survival of SGNs and their peripheral processes following long-term deafness, and the extent of survival was enhanced when combined with cochlear implant use. Implantation of encapsulated NTCells was compatible with cochlear implant use in terms of surgical access, tissue biocompatibility, and chronic neural excitation, indicating that this therapy is both effective and clinically viable. A major advantage of the present study is the use of a long-term neonatal deafness model with chronic ES delivered by clinical devices, a model of early cochlear implantation for deaf infants.
Effects of Chronic ES on SGN Survival
There was no significant effect of chronic ES alone on SGN survival, a finding consistent with previous studies in the cat [27, 31, 32] and guinea pig [8, 33, 34], although this is not a uniform finding [25, 35–41]. Differences in the methodology (e.g., the duration and severity of deafness, the age at deafness onset, electrode design and position, duration, and mode of chronic ES) complicate direct comparisons between these experiments . However, a lack of ES-induced survival effects on SGNs reported here is supported by evidence from human temporal bones following cochlear implant use [43–47], although clinical studies lack the control offered by animal experiments.
Effects of NT Treatment on SGN Survival
The choroid plexus produces and secretes a wide range of NTs and other cell survival factors at physiological levels (pg/mL) [24, 48]. Significant SGN survival for the NT treatment group was observed in the upper basal cochlear region only (35% greater SGN density compared to the contralateral deafened untreated cochleae), providing evidence that the NTs released by the NTCells were protective for SGNs in this cochlear region. In addition to the production of NTs, the choroid plexus also produces anti-oxidants that can prevent oxidative stress in neurons by destroying oxygen free radicals in addition to limiting their production. The expression levels of anti-oxidants are in the top 5 to 10% of all genes expressed , and could represent another mechanism, whereby encapsulated NTCells exerted their protective effects on SGNs. Immunohistochemistry on the cat cochlea (Fig. 8) indicated that the NTCells were positively labeled with DAPI and cytochrome C oxidase, providing evidence that they were alive and metabolically active at the conclusion of a treatment period of at least 6 months. In a previous study, the viability of implanted capsules containing NTCells was assessed following implantation in the striatum of rats. After 4 months of implantation, NTCell viability was the same as pre-implant levels, but decreased to 67.5% following 6 months of implantation . Therefore, it is likely that there was a reduction in NT production and release during the timeframe of the current experiments.
Capsules containing NTCells were observed primarily in the lower and upper basal cochlear regions (Figs. 5a and 8b), and infrequently in more apical cochlear regions (middle turn) (Fig. 9a). The region-specific survival effect on SGNs in the NT treatment group is likely to reflect local differences in the effective concentration of NTs available to the SGNs, and there are a number of factors that may contribute to the finding. Unlike the use of mini-osmotic pumps, whereby relatively high levels of NTs were delivered (for more detail see Ernfors et al. , Staecker et al. , Miller et al. , Wise , McGuinness and Shepherd , Glueckert et al. , and Agterberg et al. ), NTCells produce and release more physiological levels of NTs and therefore physical barriers (e.g., fibrous tissue, or simply the distance between the capsules and the target neurons) are far more critical. In addition, the relatively large area of the lower basal turn of the scala tympani (~5.0 mm2) compared to the upper basal turn (~0.5 mm2)  meant that the capsules containing NTCells were located relatively further away from the SGNs located within Rosenthal’s canal, potentially reducing the local concentration of NTs in this region.
Effects of Combined ES and NT Treatment on SGN Survival
Combined ES + NT treatment resulted in significantly greater SGN density throughout a large extent of the cochlea (i.e., in the lower basal [44%], upper basal [68%], and lower middle [63%] regions). Previous studies have shown that chronic ES can potentiate the pro-survival effects of growth factors such as NTs [8, 41]. ES may lead to an up-regulation of NT receptors [50–52] or increased intracellular trafficking of NT receptors that amplify the neuronal responsiveness to limited amounts of NTs in vivo . ES also enhances the internalization of NT receptor complexes  (a process that is critical for neuronal responses to target-derived NTs ), and may play an important role in the enhanced SGN survival seen with the combined treatment. Another possibility is an ES-induced increase in the production and secretion of neuroprotective factors by the NTCells themselves. Finally, the regional specific effects of combined ES + NT treatment (no enhanced SGN survival in the upper middle turn) may also relate to the SGN population that was electrically activated throughout the treatment period. A previous study has shown enhanced SGN survival in cochlear regions likely to be electrically activated by chronic ES following long-term NT delivery .
Peripheral Process Responses
There was significantly greater density of peripheral processes within the OSL in cochleae that received NT treatment than in the untreated control cochleae, with the largest average density observed in the combined ES + NT treatment group. The increased process density is likely to be a combination of NT-induced protection of the peripheral processes and enhanced process re-sprouting and/or branching. Indeed, it was noticeable that the projection profiles of processes within the OSL of treated cochleae were different to the normal radial projection profile that typically exhibits punctate neurofilament staining in the process cross sections. The staining of processes in treated cochleae was less punctate, indicative of an increased ectopic projection of the processes within the OSL in these cochleae.
Previous studies have shown that NTs delivered to the deafened cochlea via a mini-osmotic pump can promote re-sprouting of the peripheral processes in the guinea pig cochlea [12, 14]. In the present study, re-sprouting peripheral processes were observed in all treatment conditions, including cochleae that received ES only. Processes were observed within the scala media, projecting onto the basilar membrane and also coursing on the inner sulcus and up to the tectorial membrane. Importantly, re-sprouting processes were observed within the scala tympani of implanted cochleae and were incorporated into the fibrous tissue matrix associated with the tissue response to the intracochlear electrode within the scala tympani. Extensive re-sprouting, particularly if processes project longitudinally along the electrode array as reported in this study (Fig. 7b), may act to reduce the specificity of electrical stimulation delivered by a multi-channel cochlear implant, a factor that may contribute to the variability in implant performance observed between cochlear implant recipients; this is currently under investigation.
Tissue Response to Treatment
As with the implantation of any foreign body, there is an immune response from the cochlea to the presence of an electrode array. In the current study, cochlear implantation was associated with a loose fibrotic response, a tissue capsule surrounding the electrode array, and in some instances small areas of new bone growth that was typically restricted to the scala tympani of the basal turn. The extent of the tissue response and new bone growth to cochlear implantation was consistent to a previous report in which a similar electrode array (without capsules) was chronically implanted into the basal turn of the cat . The presence of a tissue response in the present study was likely to contribute to the increase in electrode impedance that was observed during the treatment period. Importantly, there was no significant difference in the tissue response for cochleae that were implanted with empty capsules and those that received NTCells, indicating that the factors released did not promote osteoneogenesis or cause significant increases in the tissue response. Indeed, capsules were often associated with very few inflammatory cells (Figs. 5, 8, and 9), indicating that the capsule membrane provided a robust immunological barrier to the porcine NTCells. Therefore, the role of the choroid plexus in the production of NTs and other factors (at physiologically relevant levels) makes it an attractive option for SGN protection that is safe and compatible with the cochlear environment.
One methodological issue warrants discussion. Higher SGN densities were observed in the contralateral (unimplanted) cochleae of the ES group compared to the NT and ES + NT group. A factor likely contributing to this result is the shorter duration of deafness in the ES group (Table 1). All statistical analysis used “within animal” comparisons, taking advantage of the symmetrical degeneration following systemic neomycin administration , and therefore eliminating the influence of any differences in neural survival between animals.
Clinical Implications and Future Directions
It is commonly believed that a robust population of SGNs is beneficial for cochlear implant performance. However, to date it has not been possible to show a positive correlation between cochlear implant performance and SGN density [45, 46, 57, 58]. The conductive nature of the cochlear fluids leads to relatively low spatial precision of SGN activation by cochlear ES meaning that higher SGN densities may not offer improvements in resolution with contemporary devices. New electrode designs that can benefit from biological interventions aimed at protecting the SGNs and regenerating their peripheral processes coupled with new stimulation strategies aimed at delivering more focused ES are likely to lead to improvements in cochlear implant performance in the future [59, 60]. Moreover, the development of high resolution electrode arrays for direct implantation into the auditory nerve  would rely on very good neural survival. Finally, future interventions that combine drug delivery and new electrode designs may be able to take advantage of the re-sprouting capacity of SGN peripheral processes to achieve a more direct interface between the electrode and the nerve to provide high resolution electrical stimulation.
This study has shown that cell-based delivery of therapeutic agents can be effectively combined with a cochlear implant, and therefore it is likely to be a viable strategy of protecting neural populations when combined with other neural prostheses, such as deep brain or retinal stimulation. Neuroprotective agents produced and released via cell-based techniques may reduce or prevent changes at the nerve-electrode interface, such as cell loss and scar tissue formation, which can reduce the efficacy of therapeutic electrical stimulation.
The authors would like to acknowledge the contributions of Jin Xu, Helen Feng, Maria Clarke, Prudence Nielsen, Anette Fransson, Mohannad Fallatah and Jacqueline Andrew. Funding was provided by the National Institutes of Health (HHS-N-263-2007-00053-C), The Garnett Passe and Rodney Williams Memorial Foundation, and the National Health and Medical Research Council. The authors would like to acknowledge the support from the State Government of Victoria’s Operational Infrastructure Program.
A full conflict of interest disclosure is available in the electronic supplementary material for this article.