Effect of Neurotrophins and Depolarization on Survival of Spiral Ganglion Neurons in Dissociation Cell Culture

Kim, Myung Soon; Chung, Won Ho; Cho, Yang Sun; Shin, Dong Bong; Bang, Eun Guk; Lyoo, Sung Hee; Hong, Sung Hwa

Abstract

BACKGROUND AND OBJECTIVES: Several neurotrophic factors have been shown to play an essential trophic role in the development, maintenance and regulation of neuronal function. Specific neurotrophins are currently used in clinical trials for the treatment of some neurodegenerative diseases. The purposes of this experiment were twofold. Firstly, we aimed to determine the trophic effects of BDNF, NT-3, and 25 mM K+ on auditory neurons in dissociated cultures of early postnatal spiral ganglia. Secondly, we tried to collect pure neural cells after dissociating the spiral ganglions using the immunomagnetic sorting method with one of neuronal surface antigens.
MATERIALS AND METHODS
Dissociated spiral ganglion cell cultures were pre-pared from cochleae of Sprague Dawley rats of 5-6 days old, and maintained in a neurobasal medium with modified N2 supplements. BDNF (50 ng/ml), NT-3 (50 ng/ml), and 25 mM K+ were added to the cultures, respectively. These cells were grown during the time course (24hr, 48hr, 72hr, 98hr) and stained with NF-200 to identify survival of spiral ganglion neurons. Immunomagnetic cell sorting for separation of spiral ganglion neurons in dissociated cells was carried out using the MiniMACS Separating System. Magnetically separated cells were analysed by flow cytometry.
RESULTS
Survival of the auditory neurons in the dissociated cells was significantly increased by addition of BDNF, NT-3, and 25K. The effect of 25 mM K+ on neuronal survival showed the highest in the experimental conditions. BDNF dramatically increased the neurite length compared with those under other conditions. After immunomagnetic sorting in dissociated cultures, spiral ganglion neurons were shown to contain 50% of the fluorescently labeled positive cells.
CONCLUSIONS
Neurotrophins (BDNF, NT-3) and depolarization by 25 mM K+ were essential trophic factors for postnatal auditory neurons and BDNF stimulated neuritogenesis in cultured spiral ganglion neurons. The immunomagnetic cell sorting method is not appropriate for collecting pure neural cells from the dissociated cells of spiral ganglia (50% purity).