Abstract
This study investigated micro-patterned, high-density complementary metal–oxide–semiconductor (CMOS) electrode array to be used as biologically permissive environment for organization, guidance and electrical stimulation of spiral ganglion neurons (SGN). SGNs extracted and isolated from cochleae of P5-P7 rat pups and adult guinea pigs were cultured 1, 4 and 7 days in vitro on glass coverslips (control) and CMOS electrode array. The cultures were analyzed visually and immunohistochemically for SGN presence, outgrowth, neurite alignment, neurite length, neurite asymmetry as well as the contact of a neuronal soma and neurites with the micro-electrodes. Our findings indicate that topographical environment of CMOS chip with micro-patterned pillars enhanced growth, survival, morphology, neural orientation and alignment of SGNs in vitro compared to control. Smaller spacing (0.8–1.6 µm) between protruding pillars on CMOS led SGNs to develop structured and guided neurites oriented along three topographical axes separated by 60°. We found morphological basis for positioning of the micro-electrodes on the chip that was appropriate for direct contact of SGNs with them. This configuration allowed CMOS electrode array to electrically stimulate the SGN whose responses were observed with live Fluo 4 calcium imaging.
Highlights
The cochlear implant (CI) is currently the prevailing neuro-prosthetic treatment for partial restoration of hearing in deaf people
In each complementary metal–oxide–semiconductor (CMOS) sample, we identified neurons with bipolar morphology to be analyzed for neurite width asymmetry with the following procedure: first, we submitted scanning electron microscopy (SEM) images consisting of neurons with clear bipolar morphology into ImageJ
Survival rates of spiral ganglion neurons (SGN) grown on the flat CMOS group (Flat) surfaces of the chip and glass coverslips were similar (9.8 ± 3.4% for control vs. 11.5 ± 5.4% for Flat areas after 4 days in vitro (DIV)) and were significantly lower, almost halved, compared to SGNs grown on the narrow pillar CMOS group (Narrow) and wide pillar CMOS group (Wide) areas of the chip (22.8 ± 6.1% for Narrow areas and 19.7 ± 6.9% for Wide areas after 4 DIV)
Summary
The cochlear implant (CI) is currently the prevailing neuro-prosthetic treatment for partial restoration of hearing in deaf people. In vivo studies have demonstrated that electrical stimulation from CI electrodes promotes survival of SGN36,37 Another important issue, investigated in this study, is whether neuronal morphology is affected by the position of CMOS micro-electrodes’ embedded by CMOS among the pillars. For successful electrical stimulation it is crucial to investigate electrophysiologically relevant morphological specializations of SGN cultured in vitro in this active model and to assess whether its topography can serve as favorable environment for the organization and guidance of SGNs. Adult-derived SGNs from guinea pigs were cultured on these CMOS substrates and were compared with SGN cultures from neonatal (P5-P7) rat pups in order to assess the effect of micro-patterned surfaces of CMOS chips on neuronal presence, survival, morphology and alignment as well as the effect of age on sensitivity to topography. CMOS electrode array was used to investigate electrical stimulation of the SGN
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