A rapid, novel model of culturing cranial nerve X-derived motoneurons for screening trophic factor outgrowth response

Objectives: 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), brainderived 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 mm 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. Discussion: 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.