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

  • Marvin N. Peter
  • Athanasia WarneckeEmail author
  • Uta Reich
  • Heidi Olze
  • Agnieszka J. Szczepek
  • Thomas Lenarz
  • Gerrit Paasche
Original Article


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.


Electrical stimulation Spiral ganglion neurons Cochlear implant Safety limit Tissue damage Residual hearing 



The authors would like to thank Jasmin Bohlmann and Darja Werner for their excellent technical support.

Funding Information

Thus study was financed by the German Research Foundation (WA 2806/5-1 granted to Athanasia Warnecke).

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.


  1. Barclay M, Ryan AF, Housley GD (2011) Type I vs type II spiral ganglion neurons exhibit differential survival and neuritogenesis during cochlear development. Neural Dev 6:33. CrossRefGoogle Scholar
  2. Brummer SB, Turner MJ (1975) Electrical stimulation of the nervous system: the principle of safe charge injection with noble metal electrodes. Bioelectrochem Bioenerg 2:13–25. CrossRefGoogle Scholar
  3. Budni J, Molz S, Dal-Cim T, Martín-de-Saavedra M, Dolores E et al (2018) Folic acid protects against glutamate-induced excitotoxicity in hippocampal slices through a mechanism that implicates inhibition of GSK-3β and iNOS. Mol Neurobiol 55:1580–1589. CrossRefGoogle Scholar
  4. Burblies N, Schulze J, Schwarz H-C, Kranz K, Motz D, Vogt C, Lenarz T, Warnecke A, Behrens P (2016) Coatings of different carbon nanotubes on platinum electrodes for neuronal devices: preparation, cytocompatibility and interaction with spiral ganglion cells. PLoS One 11:e0158571. CrossRefGoogle Scholar
  5. Chiong CM, Burgess BJ, Nadol JB (1993) Postnatal maturation of human spiral ganglion cells: light and electron microscopic observations. Hear Res 67:211–219. CrossRefGoogle Scholar
  6. Coco A, Epp SB, Fallon JB, Xu J, Millard RE, Shepherd RK (2007) Does cochlear implantation and electrical stimulation affect residual hair cells and spiral ganglion neurons? Hear Res 225:60–70. CrossRefGoogle Scholar
  7. Cogan SF, Ludwig KA, Welle CG, Takmakov P (2016) Tissue damage thresholds during therapeutic electrical stimulation. J Neural Eng 13:021001. CrossRefGoogle Scholar
  8. Dabdoub A, Fritzsch B, Popper AN, Fay RR (2016) The primary auditory neurons of the mammalian cochlea, 52nd edn. Springer, Berlin Heidelberg, p 234CrossRefGoogle Scholar
  9. de Haro C, Mas R, Abadal G, Muñoz J, Perez-Murano F, Domı́nguez C (2002) Electrochemical platinum coatings for improving performance of implantable microelectrode arrays. Biomaterials 23:4515–4521. CrossRefGoogle Scholar
  10. Dziewas R, Stellato R, van der Tweel I, Walther E, Werner CJ, et al (2018) Pharyngeal electrical stimulation for early decannulation in tracheotomised patients with neurogenic dysphagia after stroke (PHAST-TRAC): a prospective, single-blinded, randomised trial. Lancet Neurol doi:, 17, 849, 859
  11. Gillespie LN, Clark GM, Bartlett PF, Marzella PL (2001) LIF is more potent than BDNF in promoting neurite outgrowth of mammalian auditory neurons in vitro. Neuroreport 12:275–279. CrossRefGoogle Scholar
  12. Habel B (2004) Elektrische Stimulation von Zellen und Geweben am besonderen Beispiel von Knochenzellen. Dissertaion, Humboldt-Universität zu BerlinGoogle Scholar
  13. Hartshorn DO, Miller JM, Altschuler RA (1991) Protective effect of electrical stimulation in the deafened Guinea pig cochlea. Otolaryngol Neck Surg 104:311–319. CrossRefGoogle Scholar
  14. Helbig S, Adel Y, Rader T, Stöver T, Baumann U (2016) Long-term hearing preservation outcomes after Cochlear implantation for electric-acoustic stimulation. Otol Neurotol 37:e353–e359. CrossRefGoogle Scholar
  15. Hudak EM, Kumsa DW, Martin HB, Mortimer JT (2017) Electron transfer processes occurring on platinum neural stimulating electrodes: calculated charge-storage capacities are inaccessible during applied stimulation. J Neural Eng 14:046012. CrossRefGoogle Scholar
  16. Jurawitz M-C, Büchner A, Harpel T, Schüssler M, Majdani O, Lesinski-Schiedat A, Lenarz T (2014) Hearing preservation outcomes with different Cochlear implant electrodes: nucleus® HybridTM-L24 and nucleus FreedomTM CI422. Audiol Neurotol 19:293–309. CrossRefGoogle Scholar
  17. Kiang N, Rho J, Northrop C, Liberman M, Ryugo D (1982) Hair-cell innervation by spiral ganglion cells in adult cats. Science 217:175–177. CrossRefGoogle Scholar
  18. Kopelovich JC, Reiss LAJ, Etler CP, Xu L, Bertroche JT, Gantz BJ, Hansen MR (2015) Hearing loss after activation of hearing preservation Cochlear implants might be related to afferent Cochlear innervation injury. Otol Neurotol 36:1035–1044. CrossRefGoogle Scholar
  19. Kujawa SG (2006) Acceleration of age-related hearing loss by early noise exposure: evidence of a misspent youth. J Neurosci 26:2115–2123. CrossRefGoogle Scholar
  20. Kujawa SG, Liberman MC (2009) Adding insult to injury: cochlear nerve degeneration after “temporary” noise-induced hearing loss. J Neurosci 29:14077–14085. CrossRefGoogle Scholar
  21. Leake PA, Hradek GT, Rebscher SJ, Snyder RL (1991) Chronic intracochlear electrical stimulation induces selective survival of spiral ganglion neurons in neonatally deafened cats. Hear Res 54:251–271. CrossRefGoogle Scholar
  22. Liberatore F, Bucci D, Mascio G, Madonna M, Di Pietro P et al (2017) Permissive role for mGlu1 metabotropic glutamate receptors in excitotoxic retinal degeneration. Neuroscience 363:142–149. CrossRefGoogle Scholar
  23. Liu W, Edin F, Atturo F, Rieger G, Löwenheim H, Senn P, Blumer M, Schrott-Fischer A, Rask-Andersen H, Glueckert R (2015) The pre- and post-somatic segments of the human type I spiral ganglion neurons - structural and functional considerations related to cochlear implantation. Neuroscience 284:470–482. CrossRefGoogle Scholar
  24. Liu M, Yin C, Jia Z, Li K, Zhang Z, Zhao Y, Gong X, Liu X, Li P, Fan Y (2018) Protective effect of moderate exogenous electric field stimulation on activating Netrin-1/DCC expression against mechanical stretch-induced injury in spinal cord neurons. Neurotox Res 34:285–294. CrossRefGoogle Scholar
  25. Lobarinas E, Salvi R, Ding D (2016) Selective inner hair cell dysfunction in chinchillas impairs hearing-in-noise in the absence of outer hair cell loss. JARO JARO - J Assoc Res Otolaryngol 17:89–101. Google Scholar
  26. Long Y, Wei H, Li J, Yao G, Yu B, et al (2018) Effective Wound Healing Enabled by Discrete Alternative Electric Fields from Wearable Nanogenerators. ACS Nano acsnano.8b07038. doi:
  27. Lousteau RJ (1987) Increased spiral ganglion cell survival in electrically stimulated, deafned guinea pig cochleae. Laryngoscope 97:836–842. CrossRefGoogle Scholar
  28. Merrill DR, Bikson M, Jefferys JGR (2005) Electrical stimulation of excitable tissue: design of efficacious and safe protocols. J Neurosci Methods 141:171–198. CrossRefGoogle Scholar
  29. Mitchell A, Miller JM, Finger PA, Heller JW, Raphael Y, Altschuler RA (1997) Effects of chronic high-rate electrical stimulation on the cochlea and eighth nerve in the deafened guinea pig. Hear Res 105:30–43. CrossRefGoogle Scholar
  30. Nayagam B, Muniak M, Ryugo D (2011) The spiral ganglion: connecting the peripheral and central auditory systems. Hear Res 278:2–10. CrossRefGoogle Scholar
  31. Olney JW, Ho O, Rhee V (1971) Cytotoxic effects of acidic and sulphur containing amino acids on the infant mouse central nervous system. Exp Brain Res 14:61–76. CrossRefGoogle Scholar
  32. Puel J-L, Ruel J, DʼAldin CG, Pujol R (1998) Excitotoxicity and repair of cochlear synapses after noise-trauma induced hearing loss. Neuroreport 9:2109–2114. CrossRefGoogle Scholar
  33. Reich U, Warnecke A, Szczepek AJ, Mazurek B, Olze H (2015) Establishment of an experimental system to study the influence of electrical field on cochlear structures. Neurosci Lett 599:38–42. CrossRefGoogle Scholar
  34. Rosso IM, Crowley DJ, Silveri MM, Rauch SL, Jensen JE (2017) Hippocampus glutamate and N-acetyl aspartate markers of excitotoxic neuronal compromise in posttraumatic stress disorder. Neuropsychopharmacology 42:1698–1705. CrossRefGoogle Scholar
  35. Roth JA, Salvi R (2016) Ototoxicity of divalent metals. Neurotox Res 30:268–282CrossRefGoogle Scholar
  36. Roxo RS, Xavier VB, Miorin LA, Magalhães AO, dos Santos Sens YA et al (2016) Impact of neuromuscular electrical stimulation on functional capacity of patients with chronic kidney disease on hemodialysis. J Bras Nefrol.
  37. Santa Maria PL, Domville-Lewis C, Sucher CM, Chester-Browne R, Atlas MD (2013) Hearing preservation surgery for Cochlear implantation—hearing and quality of life after 2 years. Otol Neurotol 34:526–531. CrossRefGoogle Scholar
  38. Scheper V, Paasche G, Miller J, Warnecke A, Berkingali N et al (2009) Effects of delayed treatment with combined GDNF and continuous electrical stimulation on spiral ganglion cell survival in deafened guinea pigs. J Neurosci Res 87:1389–1399. CrossRefGoogle Scholar
  39. Schwieger J, Warnecke A, Lenarz T, Esser K-H, Scheper V (2015) Neuronal survival, morphology and outgrowth of spiral ganglion neurons using a defined growth factor combination. PLoS One 10:e0133680. CrossRefGoogle Scholar
  40. Schwieger J, Esser K-H, Lenarz T, Scheper V (2016) Establishment of a long-term spiral ganglion neuron culture with reduced glial cell number: effects of AraC on cell composition and neurons. J Neurosci Methods 268:106–116. CrossRefGoogle Scholar
  41. Shannon RV (1992) A model of safe levels for electrical stimulation. IEEE Trans Biomed Eng 39:424–426. CrossRefGoogle Scholar
  42. Shea GKH, Tsui AYP, Chan YS, Shum DKY (2010) Bone marrow-derived Schwann cells achieve fate commitment – a prerequisite for remyelination therapy. Exp Neurol 224:448–458. CrossRefGoogle Scholar
  43. Shen N, Liang Q, Liu Y, Lai B, Li W, Wang Z, Li S (2016) Charge-balanced biphasic electrical stimulation inhibits neurite extension of spiral ganglion neurons. Neurosci Lett 624:92–99. CrossRefGoogle Scholar
  44. Skarzynski H, van de Heyning P, Agrawal S, Arauz SL, Atlas M, Baumgartner W, Caversaccio M, de Bodt M, Gavilan J, Godey B, Green K, Gstoettner W, Hagen R, Han DM, Kameswaran M, Karltorp E, Kompis M, Kuzovkov V, Lassaletta L, Levevre F, Li Y, Manikoth M, Martin J, Mlynski R, Mueller J, O'Driscoll M, Parnes L, Prentiss S, Pulibalathingal S, Raine CH, Rajan G, Rajeswaran R, Rivas JA, Rivas A, Skarzynski PH, Sprinzl G, Staecker H, Stephan K, Usami S, Yanov Y, Zernotti ME, Zimmermann K, Lorens A, Mertens G (2013) Towards a consensus on a hearing preservation classification system. Acta Otolaryngol 133:3–13. CrossRefGoogle Scholar
  45. Stalmann U (2015) Altersabhängige Degeneration und Lärmempfindlichkeit des Corti-Organs bei tauben Otof-Knockout-Mäusen. Dissertation, Georg-August-Universität zu GöttingenGoogle Scholar
  46. Sucher NJ, Lipton SA, Dreyer EB (1997) Molecular basis of glutamate toxicity in retinal ganglion cells. Vis Res 37:3483–3493. CrossRefGoogle Scholar
  47. Warnecke A, Sasse S, Wenzel GI, Hoffmann A, Gross G, Paasche G, Scheper V, Reich U, Esser KH, Lenarz T, Stöver T, Wissel K (2012) Stable release of BDNF from the fibroblast cell line NIH3T3 grown on silicone elastomers enhances survival of spiral ganglion cells in vitro and in vivo. Hear Res 289:86–97. CrossRefGoogle Scholar
  48. Whitlon D, Grover M, Tristano J, Williams T, Coulson MT (2007) Culture conditions determine the prevalence of bipolar and monopolar neurons in cultures of dissociated spiral ganglion. Neurosci 146:833–840. CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Otorhinolaryngology, Head and Neck SurgeryHannover Medical SchoolHannoverGermany
  2. 2.Cluster of Excellence “Hearing4all” of the German Research FoundationOldenburgGermany
  3. 3.Department of Otorhinolaryngology, Head and Neck SurgeryBerlin Institute of Health, Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu BerlinBerlinGermany

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