Characterization of the Interaction of Primary Cells from the Rat Inner Ear with Polymer Films As Coatings for Cochlear Implant Electrode Surface

Summary

Here, we present an immunocytochemical and electron microscopical protocol that enables qualitative and quantitative characterization of the interaction of the primary spiral ganglion neurons and other cell types within ultrathin polymer films.

Abstract

The spiral ganglion (SG) cells prepared from the inner ear of postnatal rats represent one of the key cell culture models in hearing research. Numerous projects in hearing research aim at improving nerve-electrode-interactions by inhibiting the formation of connective tissue following cochlear implant insertion, i.e., by coating the carrier material with ultrathin polymer films for selective cell attachment. Here, we established scanning electron microscopy (SEM) and immunocytochemical (ICC) staining to enable the characterization of the interactions of fibroblasts, glial cells, and spiral ganglion neurons (SGN) growing on polymers, i.e., poly(N,N-dimethylacrylamide) (PDMAA), poly(2-ethyloxazoline) (PEtOx), and poly([2-methacryloyloxy)ethyl]trimethylammoniumchloride) (PMTA). For this purpose the primary cells dissociated from the SG of postnatal rats were cultivated for 48 h on the polymer films. ICC was used to demonstrate the preferences of cell adhesion on the polymer coatings. It could be shown that glial cells and SGN mainly adhered on PMTA monolayers forming long processes, but not on PDMAA and PEtOx films. Also, SEM imaging showed that only PMTA enabled SG neuron survival and neurite outgrowth. In conclusion, the ability of the SGN to survive and to form neurites was associated with glial cell adhesion on different coatings.

Introduction

Numerous projects in hearing research aim at improving nerve-electrode-interactions by inhibiting the formation of connective tissue following cochlear implant insertion by either application of drugs like dexamethasone^1,2,3,4, coating the carrier material with ultrathin polymer films^5,6,7,8, or nano structuring of the carrier materials for selective cell attachment^9,

Protocol

Neonatal Spraque-Dawley rats (P3-5, n = 18 per experiment, n = 5 independent experiments) were used for SG dissection in accordance with the institutional guidelines for animal welfare of Hannover Medical School following the standards described by the German "Law on protecting animals" (Tierschutzgesetz).

1. Dissection of the Cochlear SG from Neonatal Rats ^13,14

1. Sacrifice the postnatal rats, which should not be old.......

Discussion

This study represents for the first time the differential interactions of SGN, glial cells, and fibroblasts on varying polymer films. Staining of the specific intermediary filaments and the neurotrophic growth factor receptor enabled not only a strong distinction between the cell types and their morphology following adhesion, but also the quantitative determination of the interesting cell types. Hereby, as described in Hadler et al.⁷, the growth of fibroblasts was clearly scaled down on t.......

Materials

Name	Company	Catalog Number	Comments
Animals
Sprague-Dawley rats	Charles River, Sulzfeld, Germany		Inbreeding strain, ordered and provided by the Laboratory Animal Sciences, Medical School Hannover, Germany
Laboratory equipment
Transmission light microscope Leica MZ-6	Leica, Germany		Preparation of the cochlear structures
Inverse microscope: Keyence BZ 9000 Biorevo	Keyence International, Mechelen, Belgium		Fluorescence microscopy
SEM 505	Philips, NL		Scanning electron microscopy
CPD 030	Balzers Union, Balzers FL		Critical point drying
Polaron E5400	Watford Hertfordshire, England		High resolution sputter coater
Laboratory tools
Standard Pattern Forceps, curved/12 cm	Fine Science Tools, Heidelberg, Germany	11001-12	Dissection of the spiral ganglia from postnatal rats
Adson-Brown Forceps, Shark Teeth	Fine Science Tools, Heidelberg, Germany	11627-12	Dissection of the spiral ganglia from postnatal rats
Forceps Dumont #3c, straight/11 cm	Fine Science Tools, Heidelberg, Germany	11231-20	Dissection of the spiral ganglia from postnatal rats
Forceps Dumont Medical #5, straight	Fine Science Tools, Heidelberg, Germany	11255-20	Dissection of the spiral ganglia from postnatal rats
Scissor, pointed-pointed, straight/11 cm	Wirtschaftsgenossenschaft deutscher Tierärzte (WDT) eG, Garbsen, Germany	27040	Dissection of the spiral ganglia from postnatal rats
Plastic and glass material
48well-microtiter plate	Nunc/thermo Scientific	150787
Coverslips Menzel-Gläser 24 x 60 mm	VWR International GmbH, Darmstadt, Germany	631-0973
Buffers, enzymes, proteins, chemicals, cell culture supplements
Acetone	Mallinckrodt Baker R.V., Griesheim, Germany	9002-02
Argon	Linde Gas, Pullach, Germany	Argon 5.0
Bovine serum albumine	Sigma-Aldrich, St.Louis, USA	A3294	Lyophilized powder, initial fraction by heat shock, fraction V.
DNase I	Roche, Basel, Switzerland	11284932001
Fetal bovine serum (FBS)	Biochrom, Berlin, Germany	S0415
Glucose 40 % concentrate			Our lab is receiving glucose by the hospital pharmacy.
Glutardialdehyde	Polysciences, Warrington, PA, USA	01909-10
Hank’s balanced salt solution (HBSS)	Invitrogen/Fisher Scientific, Waltham, MA, USA	14170-070	Ca2+/ Mg2+-free
HEPES	Invitrogen/Fisher Scientific, Waltham, MA, USA	15630-056	1 M
Insulin from bovine pancreas	Sigma-Aldrich, St.Louis, USA	I0516
Leit-C	Plano, Wetzlar, Germany	G3300	fluid carbon in xylol
N2-Supplement	Invitrogen/Fisher Scientific, Waltham, MA, USA	17502-048	100x
Natural Mouse Laminin	Invitrogen/Fisher Scientific, Waltham, MA, USA	23017-015
Panserin 401 neuro medium	PAN Biotech, Aidenbach, Germany	P04-710401	Neuro medium for cultivation neuronal cells
Phosphate buffered saline (PBS) tablets	Invitrogen/Fisher Scientific, Waltham, MA, USA	18912-014	Ca2+/ Mg2+-free
Penicillin G Sodium salt	Sigma-Aldrich, St.Louis, USA	PENNA-10MU
Poly-DL-Ornithin hydrobromid	Sigma-Aldrich, St.Louis, USA	P8638
Prolong anti-fade Gold with DAPI	Invitrogen/Fisher Scientific, Waltham, MA, USA	P36941	DAPI containing mounting gel
Sodium cacodylate	Sigma-Aldrich, St.Louis, USA	C4945-10G
Triton-X 100	Sigma-Aldrich, St.Louis, USA	T8787
Trypan Blue	Sigma-Aldrich, St.Louis, USA	T 8154
Trypsin	Biochrom, Berlin, Germany	L2123	1:250 in PBS, Ca2+/ Mg2+-free
Primary antibodies
Neurofilament 200kD, mouse, monoclonal	Novocastra, Newcastle upon Tyne, UK	NCL-NF200
p75, rabbit, polyclonal	Abcam, Cambridge, UK	38335
Vimentin clone V9, mouse, monoclonal	Dako GmbH, Jena, Germany	M0725
Vimentin, chicken, polyclonal	Abcam, Cambridge, UK	ab24525
Secondary antibodies
Goat anti-chicken, conjugated with Texas Red	Santa Cruz Biotech., Dallas, TX, USA	sc2994
Goat anti-mouse, conjugated with New dylight 488	Jackson-Immunoresearch Laboratories Inc., West Grove, PA, USA	115-485-008
Goat anti-rabbit, conjugated with Alexa Fluor 594	Jackson-Immunoresearch Laboratories Inc., West Grove, PA, USA	111-515-144