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Developing an In-Vitro System to Test Enhanced Neurite Outgrowth by Electrical Stimulation of a Conductive Polymer
Chandan G. Reddy, MD; Huan Wang, MD, PhD; M. Brett Runge, PhD; Michael J. Yaszemski; Robert J. Spinner; Anthony J. Windebank
Mayo Clinic, Rochester, MN, USA
Introduction: Slow rates of peripheral nerve regeneration limit functional recovery of distal targets. Electrical stimulation has been demonstrated to consistently stimulate neurite outgrowth, both in-vitro and in-vivo. However, there remains considerable variability in the parameters of electrical stimulation delivered, with variations in total duration, frequency, amount of current, and number of treatments. Previous work has demonstrated that the biocompatible conductive polymer, polycaprolactone fumarate-polypyrrole doped with naphthalene-2-sulfonic acid sodium salt anion (PCLF-PPyNSA) is favorable for nerve regeneration. The goal of the present work is to develop a consistent in-vitro assay for assessing the effectiveness of various parameters of electrical stimulation on rat embryonic dorsal root ganglia (DRG) explants cultured on PCLF-PPyNSA substrate.
Method: DRG were extracted from E15 rat embryos and cultured on PCLF-PPyNSA discs, which had been inserted into 24 well culture plates. All polymer discs had been presterilized in 70% ethanol, coated with a thin layer of type I collagen, and subsequently incubated for 24 hours in growth medium supplemented with NGF at 5ng/mL. Electrical stimulation was performed using platinum wires inserted through presterilized silicon tubes to make electrical contact with the conductive polymer. DRG were allowed to attach for 12 hours prior to initiation of 1 hour of electrical stimulation at either 10uA or 100uA current intensity at 20Hz DC. DRG were fixed at 72h, stained with beta-tubulin and DAPI, and imaged using an inverted confocal microscope. For each DRG, maximal neurite length was measured using NIH Image J software. Overall size and mean-intensity of the fluorescent beta-tubulin antibody of the DRG culture was measured using KS-400 software.
Results: Several iterations were required to develop culture parameters that led to consistent DRG growth in the control condition. The final protocol entailed collagen coated PCLF-PPyNSA with one stimulation for one hour duration after 12 hours of DRG incubation, using 20 Hz DC. At 72 hours, electrically stimulated DRG showed consistently longer neurite growth than non-stimulated DRG (900 μm +/- 122 vs. 356 μm +/- 185, p <0.001). Overall size of DRG cultures was larger in the stimulated group than the non-stimulated group (1.63 mm2 vs. 0.68 mm2, p<0.001), though mean intensity was unchanged.
Conclusion: Preliminary results demonstrated electrical stimulation consistently enhances neurite outgrowth of DRG explants cultured on an electrically conductive polymer. Further work is now necessary to characterize the optimal electrical stimulation parameters that will benefit the length, density and directionality of neurite outgrowth.
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