A rapid, novel model of culturing cranial nerve X-derived motoneurons for screening trophic factor outgrowth response.
OBJECTIVE
After cranial nerve X (CN X) injury, vocal fold paralysis treatments currently face a myriad of obstacles in achieving non-synkinetic, functional reinnervation. Of particular therapeutic interest is the targeted administration of locally expressed biological neurotrophic factors (NFs). To date, a method to culture mature CN X motoneurons for NF responsiveness screening has not been described.


METHODS
We herein present a novel method for establishing mature murine CN X motoneuron cultures, and use the model to test CN X motoneuron outgrowth response to individual and paired ascending concentrations of selected neurotrophic factors [glial cell-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), and ciliary neurotrophic factor (CNTF)].


RESULTS
Findings demonstrated low concentration (5 ng/ml) CNTF to have the greatest positive effect on motoneuron outgrowth, beyond that of both indivual NF and paired NF combinations, based on total neurite outgrowth [mean total neurite outgrowth = 445.7±84.45 μm in the (5 ng/ml) CNTF group versus 179.7±13.63 μm in saline controls (P<0.01)]. Paired treatments with CNTF/GDNF, and CNTF/BDNF promoted motoneuron branching at a variety of concentrations beyond saline controls, and paired GDNF/BDNF had inhibitory effects on motoneuron branching.


CONCLUSIONS
Our described in vitro model of establishing mature CN X cultures allowed rapid screening for responsiveness to therapeutic NFs at a variety of concentrations and combinations. While the model ultimately may be used to investigate the molecular mechanisms of CN X motoneuron regeneration, the current study identified CNTF as a promising therapeutic candidate for the promotion of CN X outgrowth.
Department of Otolaryngology–Head and Neck Surgery, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
Bryan R
BR
McRae
Matthew
M
Shew
Geoffrey P
GP
Aaron
Khadijeh
K
Bijangi-Vishehsaraei
Stacey L
SL
Halum
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D005455:Fluorescent Antibody Technique; D009046:Motor Neurons / Q000166:cytology* / Q000187:drug effects; D009414:Nerve Growth Factors / Q000378:metabolism* / Q000494:pharmacology; D016501:Neurites / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors; D014630:Vagus Nerve / Q000166:cytology* / Q000187:drug effects
D009414:Nerve Growth Factors