Charge-balanced biphasic electrical stimulation inhibits neurite extension of spiral ganglion neurons

Neuroscience Letters 624 (2016) 92–99

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Research paper

Charge-balanced biphasic electrical stimulation inhibits neurite extension of spiral ganglion neurons Na Shen a,b,d,1 , Qiong Liang a,d,1 , Yuehong Liu a,d , Bin Lai c , Wen Li e , Zhengmin Wang a,d,f,∗ , Shufeng Li a,d,f,∗ a

Department of Otolaryngology, Eye & ENT Hospital of Fudan University, Shanghai, China Department of Otolaryngology, Zhongshan Hospital of Fudan University, Shanghai, China c State Key Laboratory of Medical Neurobiology, Shanghai, China d NHFPC Key Laboratory of Hearing Medicine, Shanghai, China e Central laboratory, Eye & ENT Hospital of Fudan University, Shanghai, China f Shanghai Hearing Medical Center, Shanghai, China b

h i g h l i g h t s • • • •

We design a system of SGN dissociated culture under CI electrical stimulation. CI electrical stimulation inhibits SGN neurites extension. CI electrical stimulation reduces Schwann cell density in vitro. Inhibiting calcium influx through VDCCs reduces electrical stimulation’s effect.

a r t i c l e

i n f o

Article history: Received 1 February 2016 Received in revised form 17 April 2016 Accepted 28 April 2016 Available online 6 May 2016 Keywords: Spiral ganglion neuron Neurite extension Electrical stimulation Cochlear implant Voltage-dependent calcium channel Schwann cell

a b s t r a c t Intracochlear application of exogenous or transgenic neurotrophins, such as neurotrophin-3 (NT-3) and brain derived neurotrophic factor (BDNF), could promote the resprouting of spiral ganglion neuron (SGN) neurites in deafened animals. These resprouting neurites might reduce the gap between cochlear implant electrodes and their targeting SGNs, allowing for an improvement of spatial resolution of electrical stimulation. This study is to investigate the impact of electrical stimulation employed in CI on the extension of resprouting SGN neurites. We established an in vitro model including the devices delivering charge-balanced biphasic electrical stimulation, and spiral ganglion (SG) dissociated culture treated with BDNF and NT-3. After electrical stimulation with varying durations and intensities, we quantified neurite lengths and Schwann cell densities in SG cultures. Stimulations that were greater than 50 ␮A or longer than 8 h significantly decreased SG neurite length. Schwann cell density under 100 ␮A electrical stimulation for 48 h was significantly lower compared to that in non-stimulated group. These electrical stimulation-induced decreases of neurite extension and Schwann cell density were attenuated by various types of voltage-dependent calcium channel (VDCC) blockers, or completely prevented by their combination, cadmium or calcium-free medium. Our study suggested that charge-balanced biphasic electrical stimulation inhibited the extension of resprouting SGN neurites and decreased Schwann cell density in vitro. Calcium influx through multiple types of VDCCs was involved in the electrical stimulation-induced inhibition. © 2016 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

∗ Corresponding authors at: Department of Otolaryngology, Eye & ENT Hospital of Fudan University, 83 Fenyang Road, Shanghai, 200031, China. E-mail addresses: [email protected] (Z. Wang), [email protected] (S. Li). 1 Na Shen and Qiong Liang contributed equally to this work. http://dx.doi.org/10.1016/j.neulet.2016.04.069 0304-3940/© 2016 Elsevier Ireland Ltd. All rights reserved.

Cochlear implants (CIs) restore hearing perception by delivering electrical signals to spiral ganglion neurons (SGNs) through multiple channels. Most CI recipients experienced poor hearing in noise and music appreciation due to the insufficiency of independently functional channels [1,2]. Speech understanding abilities of CI recipients were also found to be strongly associated with the

N. Shen et al. / Neuroscience Letters 624 (2016) 92–99

degree of channel interactions [3,4]. The peripheral processes of spiral ganglion neurons usually degenerate gradually following the loss of hair cells, which produces a 250–1000 ␮m distance between the stimulating CI electrodes in the scala tympani and their targeting SGNs [5]. These distances result in non-specific electrical stimulation and poor spatial resolution in CI. Neurotrophin-3 (NT-3) and brain derived neurotrophic factor (BDNF) are critical to the development and function of spiral ganglia [6]. Introcochlear administration of BDNF and NT-3 could enhance the resprouting of SGN peripheral processes [7]. Adenoviralmediated expression of BDNF in the cochlea also induced SGN radial nerve fiber regrowth [8,9]. Recently, adeno-associated virus vectors with neurotrophin gene inserts, AAV.BDNF and AAV.Ntf3 were found to be able to induce considerable re-growth of peripheral auditory fibers in the basilar membrane area of deafened animals. The regrown SGN peripheral processes are supposed to be able to reduce the gap between CI electrodes and their targeting SGNs. As a potential supplementary therapy to CI recipients, delivering neurotrophins are supposed to be accompanied with electrical stimulation of CIs. However, the impact of electrical stimulation on the neurotrophin-supported extension of regrown SGN neurites remains unclear. Cochlear implants stimulate SGNs by chargebalanced biphasic pulses designed to prevent the damage to the stimulated tissues and the metal electrodes. The depolarization of SGN membrane caused by electrical stimulation results in calcium influx through multiple types of voltage-dependent calcium channels (VDCCs). It has been shown that excessive calcium influx caused by membranous depolarization could lead to SGN injury [10] and inhibition of SGN neurite extension [11]. In this study, we established a system consisting of spiral ganglia (SG) dissociated culture treated with BDNF and NT-3, and chamber slides connected with the devices delivering charge-balanced biphasic pulses. Using this system, we investigated the impact of charge-balanced biphasic electrical stimulation on the extension of resprouting SGN neurites and Schwann cell density, as well as the role of calcium influx through VDCCs in it. 2. Materials and methods 2.1. Spiral ganglion dissociated culture The following procedures using Sprague-Dawley rat pups were approved by the Ethics Review Board of Eye & ENT Hospital of Fudan University. The rat pups used in our study were from Shanghai SLAC Laboratory Animal Co. They were feed by their mother rats kept in “Specific Pathogen Free” (SPF) environment. One cage only kept one mother rat and her pups. The pups were delivered to our lab in a box for delivering SPF animals on the day of cell culture. Dissociated spiral ganglion culture was prepared as described previously [12]. Briefly, the Sprague-Dawley pups of 4–6 postnatal days were deeply anesthetized by being placed on ice for 20 min and then euthanized. The spiral ganglia were harvested and dissociated with 0.125% trypsin and 0.1% collagenase (Sigma-Aldrich, St. Louis, MO, USA) for 30 min at 37 ◦ C and gentle trituration. The culture was maintained in high glucose Dulbecco’s Modified Eagle’s Medium (DMEM, Life technologies, 11965) with N2 supplement (Life Technologies), 10% fetal bovine serum, 10 ␮g/ml insulin (Sigma-Aldrich, St. Louis, MO, I6634), 50 ng/ml NT-3 (Sigma-Aldrich, N1905) and 50 ng/ml BDNF (Sigma-Aldrich, B3795) in a 37 ◦ C humidified incubator with 5% CO2.

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Fig. 1. Schematics of spiral ganglia (SG) dissociated explant culture with chargebalanced biphasic electrical stimulation. (A) Eight-well chamber slides were used in SG dissociated culture or cochlear explant culture. Four holes adjacent to the floor were made on two opposite walls of each chamber to introduce two platinumiridium wires at the two opposite borders. The wires were connected to chargebalanced biphasic pulse generators. (B) The biphasic pluses used for the electrical stimulation had 50 ␮A or 100 ␮A amplitude, 65 ␮s pulse width, 8 ␮s open-circuit interphase gap, 4862 ␮s short-circuit phase, and 200 Hz frequency.

were made on two opposing walls near the floor of the chamber, two platinum-iridium wires were placed on two opposite sides of each chamber. Silicon glue was used to seal the holes and secure the wires. The wires were then connected to adjustable charge-balanced biphasic pulse generators with 12 parallel channels (Listent Medical Tech. Co., Ltd.). The charge-balanced biphasic pulses used for electrical stimulation held an amplitude of 50 ␮A or 100 ␮A, with a 65-␮s pulse width, 8-␮s open-circuit interphase gap, and 4.862-ms short-circuit phase at a frequency of 200 Hz (Fig. 1). 2.3. Manipulation of calcium influx Six hours after the plating of SG dissociated cultures, various VDCC blockers were added into culture medium. The VDCC blockers included L-type channel blocker Verapamil (VPL, 10 ␮M; V4629, Sigma-Aldrich), N-type channel blocker ␻-Conotoxin GVIA (GVIA, 1 ␮M; C9915, Sigma-Aldrich), P/Q-type channel blocker ␻-Agatoxin IVA (IVA, 1 ␮M; A6719, Sigma-Aldrich) and nonselective VDCC blocker cadmium (10 ␮M; Sigma-Aldrich). For the calcium-free environment, the medium was completely replaced by calcium-free DMEM (Life Technologies, 21068) with 1 mM EDTA, N2, BDNF, NT3 and insulin. 2.4. Immunocytochemistry

2.2. Chamber slide culture with electrical stimulation Eight-well chamber slides (Nalge Nunc International) were used in spiral ganglion (SG) dissociated culture. After four holes

The cultures were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.2% Triton X-100, 10% donkey serum in PBS for 1 h. For immunostaining, the cultures were incubated

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Fig. 2. Electrical stimulation inhibited neurite extension in SG dissociated cultures. (A–I) Representative images of SGN neurites in SG cultures under electrical stimulation with various intensities and durations. (J) Electrical stimulation of 8 h/100 ␮A, 24 h/50 ␮A, 24 h/100 ␮A, 48 h/50 ␮A and 48 h/100 ␮A, all significantly decreased SG neurite length compared to those with same culture durations but no electrical stimulation, while 8 h/50 ␮A stimulation did not decrease neurite length. *p < 0.05, Kruskal-Wallis one-way ANOVA on ranks followed by Dunn’s method. n value in this columns are the number of neurites scored in each condition. Data represent means ± SEM.

sequentially with primary and secondary antibodies diluted in PBS with 10% donkey serum, primary antibodies at 4 ◦ C overnight and secondary antibodies for 1 h at room temperature. Primary antibodies used were as follows: anti-NF200 (1:400; N0142, SigmaAldrich) to label the SGNs and their peripheral axons, anti-S100 (1: 400; SAB4500474, Sigma-Aldrich) to label the Schwann cells. Secondary antibodies were conjugated with Alexa Fluor-488 or Alexa Fluor-546, (1: 800; Life Sciences).

MetaMorph software (Molecular Devices) with an inverted fluorescence microscope (ZEISS AXIOVERT 40CFL). Neurite length was measured in ImageJ software (NIH). Neurite length was defined as the distance from the soma to the end of the longest neurite. Schwann cell densities were determined as the numbers of Schwann cells in each 10× field by using the measurement tool in ImageJ. 2.6. Statistical analysis

2.5. Measurement of neurite length and Schwann cell density Digital images of anti-NF200 immunofluorescence in dissociated SG cultures were acquired by scan-slide function of

Each condition had four wells in one experiment. All experiments have been repeated three times. Statistical analysis was performed by GraphPad Prism 5.01 (GraphPad Software, Inc.; CA,

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Fig. 3. Electrical stimulation decreased Schwann cell densities in SG dissociated cultures. (A–C) Representative images of Schwann cells in cultures without electrical stimulation (A), under 48 h/50 ␮A stimulation (B) and under 48 h/100 ␮A stimulation. (D) Schwann cell density in SG cultures under 48 h/100 ␮A electrical stimulation was lower than that in cultures under no electrical stimulation or 48 h/50 ␮Astimulation. *p < 0.05, Kruskal-Wallis one-way ANOVA on ranks followed by Dunn’s method. n value in this columns are the number of 10 × fields scored in each condition. Data represent means ± SEM.

USA). Significances of differences among various conditions were compared by unpaired t-test or One-way ANOVA Kruskal-Wallis test on ranks followed by Dunn’s method. 3. Results 3.1. Electrical stimulation inhibited neurite extension in SG dissociated cultures After 8-h electrical stimulation with charge-balanced biphasic pulses, SG neurite length with 50 ␮A stimulation was still comparable to that without electrical stimulation (P = 0.3086), while neurite length under 100 ␮A stimulation was significantly shorter compared to that without electrical stimulation (P = 0.0003). Twenty-four-hour or forty-eight-hour stimulation with the amplitude of either 50 ␮A or 100 ␮A all significantly inhibited neurite extension (P < 0.05) (Fig. 2). There was no significant difference among the numbers of SGNs in all conditions (P > 0.05). 3.2. Electrical stimulation decreased Schwann cell densities in SG dissociated cultures After 48-h stimulation, the mean cell densities of Schwann cells in the control group, 50 ␮A group and 100 ␮A group were 4749 ± 608.0, 3600 ± 290.3 and 2756 ± 229.7 each 10× field, respectively. There was no statistical differences among the cell densities of 50 ␮A group and that of the control group (p = 0.0965),

while the cell density of 100 ␮A group was significantly lower compared to that of non-stimulated group (p = 0.0040) (Fig. 3). 3.3. VDCC blockers attenuated electrical stimulation-induced inhibition of SG neurite extension To investigate the role of calcium influx through VDCCs in electrical stimulation-induced inhibition of neurite extension, we treated SG culture under 48 h electrical stimulation by various VDCC blockers, i.e. 10 ␮M VPL, 1 ␮M GVIA, 1 ␮M IVA or their combination. With the application of VPL, GIVA, IVA or their combination, neurite lengths under either 50 ␮A or 100 ␮A stimulation were all significantly longer than those without the treatment of VDCC blockers (P < 0.05). Meanwhile, SGN neurite length without electrical stimulation was not changed by the application of VDCC blockers (P > 0.05). Neurite length with a single VDCC blocker was still significantly shorter than that without electrical stimulation (P < 0.05). Notably, with the combination of VDCC blockers, neurite length under electrical stimulation was comparable to that without stimulation (P > 0.05) (Fig. 4). 3.4. VDCC blockers attenuated electrical stimulation-induced decrease of Schwann cell density Similar to the effect on neurite extension, Schwann cell density under 48 h/100 ␮A electrical stimulation was higher with the application of VDCC blocker combination (P < 0.05 compared to normal

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Fig. 4. Inhibiting calcium influx through VDCCs attenuated electrical stimulation-induced inhibition of SG neurite extension. (A–C) Neurite lengths in SG cultures with the application of 1 ␮m IVA (A), 1 ␮M GVIA (B), 10 ␮M VPL (C) or their combination (COM; D) in SG cultures under 48 h/50 ␮A or 48 h/100 ␮A stimulation were all longer than those in stimulated cultures without these VDCC blockers. The neurite length in stimulated cultures with single VDCC blocker was still shorter than that in non-stimulated cultures, while neurite length in stimulated cultures was comparable to that in non-stimulated cultures. *p < 0.05, Kruskal-Wallis one-way ANOVA on ranks followed by Dunn’s method. n value in this columns are the number of neurites scored in each condition. Data represent means ± SEM. (E–J).Representative images of SGN neurites in differently stimulated cultures with or without COM (48 h stimulation).

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medium), and was comparable to that without electrical stimulation (P > 0.05) (Fig. 5). 3.5. Reduced calcium influx attenuated electrical stimulation-induced inhibition of neurite extension and decrease of Schwann cell density To further investigate the role of calcium influx in the electrical stimulation-induced inhibition of neurite extension, we treated SG dissociated culture that underwent 48 h/100 ␮A electrical stimulation with the application of cadmium, a non-selective VDCC blocker, and calcium-free culture medium. With cadmium-treated or calcium-free medium, SG neurite length under electrical stimulation was significantly longer than that in normal medium (P < 0.05) and was comparable to that without electrical stimulation (P > 0.05) (Fig. 4). Meanwhile, Schwann cell density under 48 h/100 ␮A electrical stimulation was higher in both cadmiumcontaining medium and calcium-free medium than that in normal medium (P < 0.05) and was comparable to that without electrical stimulation (P > 0.05) (Fig. 6). 4. Discussion In the present study, we used SG dissociated culture to investigate the extension of resprouting SGN neurites under CI-using electrical stimulations. Our results suggested that charge-balanced biphasic electrical stimulation inhibited SG neurite extension and decreased Schwann cell via calcium influx through multiple types of VDCCs. A close position of electrode array to auditory peripheral fibers of could significantly increase input-output function of electrically evoked auditory brainstem responses (EABRs) [13]. BDNF could promote neurite extension in dissociated SG culture [14]. Intracochlear application of BDNF alone or the combination of BDNF and NT-3 resulted in higher density of myelinated radial nerve fibers and sprouting of fibers into the scala tympani [7,15–17]. Similarly, transgenic BDNF or BDNF in epithelial or mesothelial cells induced robust regrowth of nerve fibers towards the cells that secrete the neurotrophin [8,18]. The resprouting SG neurites are likely to reduce the distance between CI electrodes and their targeting SGNs, allowing an improved electrode-neuron interface. Take together, the application exogenous or transgenic NTs provides

Fig. 5. Inhibiting calcium influx through VDCCs attenuated electrical stimulationinduced decrease of Schwann cell density. (A–D). Representative images of Schwann cells in SG cultures without electrical stimulation and COM (A), under 48 h/100 ␮A stimulation and without COM (B), without electrical stimulation and but with COM (C), under 48 h/100 ␮A stimulation and with COM. (E) In 48 h/100 ␮A-stimulated SG cultures, Schwann cell density with COM treatment was significantly higher than that without COM treatment. *p < 0.05 compared with all other groups, KruskalWallis one-way ANOVA on ranks followed by Dunn’s method. n value in this columns are the number of 10× fields scored in each condition. Data represent means ± SEM.

Fig. 6. Reduced calcium influx by cadmium or calcium free culture medium attenuated the inhibition of neurite extension and the decrease of Schwann cell density. (A) In cultures under 48 h/100 ␮A stimulation, neurite lengths with the treatment of cadmium or calcium-free culture medium were longer than those without these treatments. (B) Schwann cell densities with the treatment of cadmium or calcium-free culture medium were higher than those without these treatments. *p < 0.05 compared with all other groups in the same figure, Kruskal-Wallis one-way ANOVA on ranks followed by Dunn’s method.

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promising methods to improve the clinical outcomes of CIs. However, while the effect of electrical stimulation on SGN survivals have been thoroughly investigated, the impact of electrical stimulation on the extension of resprouting neurites is unknown. In our study, dissociated SG cultures treated with BDNF and NT3 were used to investigate the extension of resprouting neurites under electrical stimulation. Unlike animal models usually used in the investigations of electrical stimulation effect on spiral ganglion neurons, SG neurites in the present system were all resprouting ones with the treatment of BDNF and NT-3. Compared to animal models, cell culture models can provide uniform electrical fields and cell population, allowing molecular research demanding a large amount of samples. The present system is modified from our system reported previously [19]. Other reports demonstrated two instruments used for the investigation of electrical stimulation on cultured SGNs or cochlear tissues. However, it is not the chargebalanced biphasic pulses that were used for electrical stimulation [20,21]. Here we used charge-balanced biphasic pulses used in CIs to prevent the toxicity from DC current. Current intensities used in cochlear implant are up to 1.75 mA. In our study, we used relatively low current intensities of 50 ␮A and 100 ␮A to reduce the possibility of damage from residual direct current. Our results did not support a positive effect of charge-balanced biphasic electrical stimulation on the extension of resprouting SGN neurites. In concordance with our results, it was reported that membrane depolarization accomplished by raising extracellular K+ inhibited SG neurite extension via activation of multiple types of VDCCs [11]. It was also shown that charge-balanced biphasic electrical stimulation could turn the SGN growth cones away from the electrodes [19]. The investigations on the trophic effect of chronic intracochlear electrical stimulation alone on SGNs did not deliver consistent results (as reviewed by Landry et al.) [22]. Meanwhile, it has been reported that neurotrophin-mediated SGN survival could not be enhanced by chronic electrical stimulation [16,17,22] though other early reports supported the existence of this enhancement [23]. Inconsistent with our results, animal studies have suggested that electrical stimulation-, neurotrophin- and electrical stimulation/neurotrophin -treated animals had significant longer SG neurites compared to the untreated control group [16]. However, NT and electrical stimulation treatment were applied at 2–3 weeks after deafening in this study. At this time, even after subsequent 28 days without electrical stimulation and neurotrophin treatment, some original SG peripheral fibers were still kept. It is possible that some of these long neurites might be the original neurites rescued by electrical stimulation or neurotrophin treatment, but not be the resprouting ones. In our study, we employed dissociated SG cultures in which SG neurites were all resprouting ones. The difference between cultured neurons from neonatal animals and mature neurons may also contribute to this diversity. Calcium is a well-known critical mediator of neurite growth. It has been shown that calcium influx from extracellular sources plays an important role in BDNF-induced axon outgrowth and steering [24]. Cultured SGNs express the subunits for L-type, N-type, and P/Q type VGCCs [11]. Continuous electrical stimulation on SGNs might activate VGCCs and cause excessive calcium influx, which could induce the death of SGNs [10]. Calcium influx through VGCCs also could activate calpains to inhibit neurite extension of cultured SGNs [11]. In our study, blocking calcium influx through multiple types of VDCCs by the combination of VDCC blockers, using non-selective VDCC blocker cadmium or removal of extracellular calcium appeared to completely abolish the electrical stimulationinduced inhibition of neurite growth. This suggested that calcium influx through multiple types of VDCCs were involved in the suppression of neurite extension induced by electrical stimulation. However, further investigation is necessary to clarify how calcium influx caused by electrical stimulation inhibits the extension of

resprouting SGN neurites. Due to the complexity of in vivo environments, animal experiments are definitely required to investigate the effect of VDCC blockers on neurite extension under electrical stimulation. Furthermore, we used a classic SGN cultures from newborn rat pups in our study. Some of the neurites might be from stem cells which might grow in a different way from the neurites from adult SGNs. This study also showed that increasing duration and strength of electrical stimulation (48 h/100 ␮A) had a negative impact on the density of Schwann cells, which could be diminished by blocking calcium influx through VDCCs. This suggested that electrical stimulation decreased Schwann cell densities in SG cultures via calcium influx through multiple types of VDCCs. In dissociated SG cultures, Schwann cells could secrete BDNF and NT-3, which support SGN survival and neurite growth [25–28]. In our study, NT3 and BDNF were supplemented in culture medium, which could compensate for the decrease of neurotrophic factors from Schwann cells. In addition, only 48 h/100 ␮A stimulation induced a decrease of Schwann cell density whereas neurite length changed at as low as 8 h/100 ␮A stimulation. This suggested that the effect of electrical stimulation on SG neurite extension precedes its effect on Schwann cell density. 5. Conclusion In this study, we introduced a reliable system for investigating the impact of charge-balanced biphasic electrical stimulation on the extension of resprouting SG neurites. Our results suggested that charge-balanced biphasic electrical stimulation used in CIs inhibited SGN neurite extension and decreased Schwann cell density via calcium influx through multiple types of VDCCs. Acknowledgements This work was supported by National Natural Science Foundation of China (NSFC, No. 81171482), the research special fund for public welfare industry of health (201202001) and Fund of the Science and Technology Commission of Shanghai Municipality (12DZ2251700). References [1] G.N. Esquia Medina, S. Borel, Y. Nguyen, E. Ambert-Dahan, E. Ferrary, O. Sterkers, A.B. Grayeli, Is electrode-modiolus distance a prognostic factor for hearing performances after cochlear implant surgery? Audiol. Neurootol. 18 (2013) 406–413. [2] R. Filipo, P. Mancini, V. Panebianco, M. Viccaro, E. Covelli, V. Vergari, R. Passariello, Assessment of intracochlear electrode position and correlation with behavioural thresholds in CII and 90 K cochlear implants, Acta. Otolaryngol. 128 (2008) 291–296. [3] G.S. Stickney, P.C. Loizou, L.N. Mishra, P.F. Assmann, R.V. Shannon, J.M. Opie, Effects of electrode design and configuration on channel interactions, Hear. Res. 211 (2006) 33–45. [4] J.J. Hanekom, R.V. Shannon, Gap detection as a measure of electrode interaction in cochlear implants, J. Acoust. Soc. Am. 104 (1998) 2372–2384. [5] F. Hassepass, S. Bulla, W. Maier, R. Laszig, S. Arndt, R. Beck, L. Traser, A. Aschendorff, The new mid-scala electrode array: a radiologic and histologic study in human temporal bones, Otol. Neurotol. 35 (2014) 1415–1420. [6] U. Pirvola, J. Ylikoski, J. Palgi, E. Lehtonen, U. Arumae, M. Saarma, Brain-derived neurotrophic factor and neurotrophin 3 mRNAs in the peripheral target fields of developing inner ear ganglia, Proc. Natl. Acad. Sci. U. S. A. 89 (1992) 9915–9919. [7] A.K. Wise, R. Richardson, J. Hardman, G. Clark, S. O’Leary, Resprouting and survival of guinea pig cochlear neurons in response to the administration of the neurotrophins brain-derived neurotrophic factor and neurotrophin-3, J. Comp. Neurol. 487 (2005) 147–165. [8] S.B. Shibata, S.R. Cortez, L.A. Beyer, J.A. Wiler, A. Di Polo, B.E. Pfingst, Y. Raphael, Transgenic BDNF induces nerve fiber regrowth into the auditory epithelium in deaf cochleae, Exp. Neurol. 223 (2010) 464–472. [9] A.K. Wise, C.R. Hume, B.O. Flynn, Y.S. Jeelall, C.L. Suhr, B.E. Sgro, S.J. O’Leary, R.K. Shepherd, R.T. Richardson, Effects of localized neurotrophin gene expression on spiral ganglion neuron resprouting in the deafened cochlea, Mol. Ther. 18 (2010) 1111–1122.

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