Rapid Screening Protocol: In Vivo Peripheral Nerve Injury Model for Rapid Screening of Potential Neurotrophic Compounds
Hilton M Kaplan, MBBCh FCSSA PhD1; Wei Chang, PhD1; Derek J Woloszyn, BS1,2; Matthew Richtmyer, BS1; Nickolas Rupertus, BS1; Joachim Kohn, PhD FBSE3; (1)Rutgers University, Piscataway, NJ, (2)Boston University School of Medicine, Boston, MA, (3)Rutgers - The State University of New Jersey, Piscataway, NJ
INTRODUCTION: Peripheral nerve injuries result in limited recovery, especially over large gaps. Nerve guidance conduits (NGCs) are an attractive option, but prove largely insufficient on their own. Creating a conducive micro-environment by incorporating bioactive moieties to aide speed and accuracy of regeneration, requires lengthy processes (screening, optimizing delivery, in vivo testing), which can take a year or longer. We describe a rapid "pump screening" model that delivers test compounds reliably over 4 weeks, without requiring any special formulation development. At 6 weeks, quantifiable histological outcomes are evaluated, dramatically reducing: study duration (6 vs. 16-24 weeks), animals (by ³75%), and costs (by ±68%).
MATERIALS & METHODS:
MODEL: A 1 cm rat sciatic nerve injury is repaired with a tyrosine-derived polycarbonate NGC (1.5 mm ID x 12 mm), with a drug-delivery cannula affixed from a 2 ml Alzet® osmotic pump (DURECT, Cupertino, CA) implanted subcutaneously on the back. The pump delivers drug throughout the NGC volume over every 20 hour period for 4 weeks. At 6 weeks samples are assessed histologically.
GROUPS: There are 10 Experimental groups (test compounds that demonstrated promise in vitro, and remain efficacious for 4 weeks at 37 OC); and 4 Control groups: (i) Validation controls using hydrophilic (fluorescein, n=2) and hydrophobic (Nile red, n=2) compounds; (ii) Positive control (BDNF, n=6); (iii) Negative control (No drug, n=6); (iv) Contralateral control (Uninjured nerve, n=6).
HISTOLOGY: Tissues are stained with toluidine blue and osmium tetroxide. Cross-sections (XS) of proximal nerve (3 mm from NGC) and conduit (3 mm into proximal NGC), and longitudinal sections (LS) of the remaining NGC, are assessed for 6 quantifiable parameters: XS = axon count, density, diameter, g-ratio, nerve area; LS = propagation rate.
STATISTICS: Experimental groups are compared to each control group (Tukey test), and to each other (ANOVA), absolutely and for % change from proximal nerve into conduit.
RESULTS: Validation controls demonstrated both hydrophilic and lipophilic compounds were consistently delivered throughout NGCs. Analyses of experimental groups are being completed and will be presented. Currently, one uric acid precursor has demonstrated significant retrograde protection and enhanced sprouting vs. controls (p<0.05).
CONCLUSIONS: We have developed an in vivo model to rapidly screen potentially neurotrophic factors. Major advantages are significant savings in time (avoids months-years formulating slow-release drug delivery platforms for each compound; 6 vs. 16-24 week in vivo studies), animals (6 per group vs. 6 per timepoint = 75+% less); and costs (±68% savings).
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