The Effect of Two Experimental Treatments (R13 & Compound 11) on Sensory Axon Regeneration After Peripheral Nerve Injury Open Access

Abstract

Recovery from peripheral nerve injuries (PNIs) is poor because axon regeneration is slow and inefficient. Experimental therapies that increase signaling of neuronal brain-derived neurotrophic factor (BDNF) through its TrkB receptor enhance axon regeneration, but treatments using recombinant human BDNF, or even the BDNF mimetic, 7,8-dihydroxyflavone (7,8-DHF), are not feasible because of their short biological half-life. A prodrug, R13, is metabolized in the liver and releases 7,8-DHF gradually. A target of BDNF-TrkB activation is the inhibition of asparagine endopeptidase (AEP), a lysosomal protease that can be inhibited by Compound 11 (CP11). The goals of this project were to compare the effects of treatments with R13 or CP11 on the regeneration of sensory axons following PNI and to evaluate the proportions of different classes of sensory (dorsal root ganglion, DRG) neurons that successfully reinnervate muscle targets following either treatment. We hypothesized that both treatments would result in enhanced regeneration of sensory axons, but the proportions of neurons expressing proteins associated with different classes of DRG neurons would not be changed. Following sciatic nerve transection and repair, C57BL/6J mice were treated for two weeks with either R13, CP11, or a control vehicle. Four weeks after injury, different fluorescent retrograde tracers were injected into the gastrocnemius (GAST) and tibialis anterior (TA) muscles to mark newly regenerated DRG neurons that had reinnervated these muscles. Using immunofluorescence, labeled DRG neurons expressing TrpV1, IB4, TH, VGLUT1, or multiple proteins were counted. Treatments with R13 or CP11 resulted in muscle reinnervation by twice as many DRG neurons as vehicle-treated controls, but neurons expressing proteins associated with different classes of DRG neurons were in roughly the same proportions. Both treatments are promising drug-based approaches to enhancing axon regeneration and function, and potentially, quality of life, for individuals living with PNI.

Table of Contents

Introduction.......................................................................................... 1

Methods................................................................................................ 7

           Animal surgeries........................................................................... 7

           Immunostaining for antibodies in regenerated sensory neurons....... 8

           Imaging...................................................................................... 10

Results................................................................................................. 11

           R13 and CP11 administration enhances sensory axon regeneration.......................................... 11

           Six different phenotypes of sensory DRG neurons found in GAST and TA muscles..................... 12

Imaging of DRG neurons following TrpV1 and IB4 staining............................................................... 13

           TrpV1...................................................................................... 13

           IB4.......................................................................................... 14

           TrpV1 & IB4............................................................................. 15

Imaging of DRG neurons following TH and VGLUT1 staining............................................................ 16

           TH........................................................................................... 17

           VGLUT1................................................................................... 18

           TH & VGLUT1.......................................................................... 19

Discussion........................................................................................ 21

Bibliography..................................................................................... 28

About this Honors Thesis

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School	Emory College
Department	Neuroscience and Behavioral Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Cell Biology, Neuroscience
Keyword	sciatic nerve peripheral nerve
Committee Chair / Thesis Advisor	English, Arthur, Emory University
Committee Members	Ward, Jill, Emory University Hue, Gillian, Emory University

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