Beta-caryophyllene Induces Axon Elongation In Motor Neurons In Vitro

Beta-caryophyllene Induces Axon Elongation In Motor Neurons In Vitro Authors: Riley Onoszko, BS; Erin Weber, MD PhD Affiliations: Indiana University, Indianapolis, IN, USA Abstract: PURPOSE: Patients with peripheral nerve injury to the upper extremity rarely regain all sensory and motor function. Many factors, including slow or inadequate axonal elongation, contribute to this poor recovery. Beta-caryophyllene (BCP) is an FDA-approved, natural phytochemical that binds to Trk receptors. Trk receptors, present in both motor and sensory neurons, are important for nerve development, function, and regeneration. Acting through Trk receptors, BCP has the potential to impact both motor and sensory nerve regeneration. BCP has been shown to positively impact neuropathic pain, but the effect on nerve regeneration after injury has not been studied. We sought to determine if BCP can enhance axonal elongation in peripheral motor neurons. METHODS: NSC-34 cells were subjected to low-serum differentiation medium (1:1 DMEM:F-12, 1% FBS, 1% nonessential amino acids, 0.5% Pen/Strep). NSC-34 cells are a hybrid cell line composed of mouse neuroblastoma cells and embryonic peripheral motor neurons and are commonly used to study peripheral motor neurons in vitro. Cells were then treated with a) 0.3 M cytosine arabinoside (CA, positive control), which further differentiates NSC-34 cells and promotes neuritogenesis, b) DMSO (negative control), c) 5, 10, or 15 M BCP, or d) CA and BCP. The cells were grown for 4-14 days, changing medium every 2 days. Cells were imaged daily with an Optika IM-7 microscope. Neurite length was measured with NeuronJ (ImageJ) software. RESULTS: CA treatment for 4 days produced an average neurite length of 216 um, compared to 111 um for the negative control. Treatment with 5, 10, and 15 uM BCP alone interestingly induced a differentiated mature neuronal phenotype, like CA, and produced intermediate neurite lengths of 140, 152, and 170 um, respectively. When treated with both CA and BCP, the average neurite lengths were synergistically increased to 217, 223, and 227 um. CONCLUSIONS: Beta-caryophyllene induces axonal elongation in motor neurons in a dose-dependent manner and synergizes with cytosine arabinoside to enhance neurite outgrowth in vitro.