Neural network and "ganglion" formations in vitro: a video microscopy and scanning electron microscopy study on adult cultured spiral ganglion cells.
OBJECTIVE
To analyze if adult-dissociated spiral ganglion cells may be propagated in vitro for later use in transplantation models to form integrated neural networks.


BACKGROUND
Hearing loss is often associated with primary or secondary spiral ganglion cell degeneration. New strategies for cell repair and tissue engineering warrants further elucidation of the regenerative capacity of the auditory nerve.


METHODS
We used in vitro/in video microscopy in combination with immunocytochemistry and field emission scanning electron microscopy to analyze neural development and network formation from dissociated adult guinea pig spiral ganglion cells. Cells were cultured in serum-free medium and in the presence of brain-derived neurotrophic factor, neurotrophin 3, and glia cell line-derived neurotrophic factor for up to 8 weeks.


RESULTS
Time-lapse video microscopy and scanning electron microscopy exposed the propagation of auditory neurons and the role of neural growth cones in axon locomotion, fasciculation, and nuclear migration, often ensuing in cell congregation (ganglion-like formations) during network formation. Axons were sometimes ensheathed by adjoining S-100/glia fibrillary acidic protein-expressing cells. A few expanding neurons were nestin positive and sometimes incorporated the markers of proliferating cells Ki67 and 5'-bromo-2-deoxyuridine. Neurons expressed the markers and transcription factors for neural development neurogenin 1, neurogenic differentiation factor 1, Brn3a, and GATA binding protein 3, as well as the neural markers beta-III tubulin, NeuN, and neurofilament 160 during this process.


CONCLUSIONS
This method of culturing and expanding spiral ganglion neurons in vitro may be useful in further studies of cell transplantation models aiming to restore the injured inner ear.
Boström
Marja
M
Department of Otolaryngology, Uppsala University Hospital, Uppsala University, Uppsala, Sweden.
Anderson
Malin
M
Lindholm
Dan
D
Park
Kyoung-Ho
KH
Schrott-Fischer
Annelies
A
Pfaller
Kristian
K
Glueckert
Rudolf
R
Rask-Andersen
Helge
H
D000818:Animals; D000963:Antimetabolites; D001369:Axons / Q000502:physiology; D019208:Brain-Derived Neurotrophic Factor / Q000494:pharmacology; D001973:Bromodeoxyuridine; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival; D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique; D005819:Genetic Markers; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000494:pharmacology; D020439:Growth Cones / Q000187:drug effects; D006168:Guinea Pigs; D007150:Immunohistochemistry; D008855:Microscopy, Electron, Scanning; D008858:Microscopy, Phase-Contrast; D018715:Microscopy, Video; D009414:Nerve Growth Factors / Q000494:pharmacology; D009415:Nerve Net / Q000166:cytology / Q000187:drug effects / Q000254:growth & development*; D009419:Nerve Tissue Proteins / Q000096:biosynthesis; D020933:Neurotrophin 3 / Q000494:pharmacology; D013136:Spiral Ganglion / Q000166:cytology / Q000187:drug effects / Q000254:growth & development*
D000963:Antimetabolites; D019208:Brain-Derived Neurotrophic Factor; D005819:Genetic Markers; D051100:Glial Cell Line-Derived Neurotrophic Factor; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D020933:Neurotrophin 3; D001973:Bromodeoxyuridine