Targeted dorsal root regeneration towards spinal motoneurons by intramuscular Neurotrophin-3 gene therapy

• Grant number: 16/19954-2
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: November 01, 2016
• End date: June 30, 2017
• Field of knowledge: Biological Sciences - Morphology - Cytology and Cell Biology
• Principal Investigator: Alexandre Leite Rodrigues de Oliveira
• Grantee: Aline Barroso Spejo
• Supervisor: Lawrence Moon
• Host Institution: Instituto de Biologia (IB). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
• Institution abroad: King's College London, England
• Associated to the scholarship: 12/22750-9 - Use of fibrin sealant combined with mesenchymal stem cells for anterior funiculus injury repair: effects on inflammation, axonal regeneration and neuroprotection., BP.DR

Abstract

Dorsal root injury leads either to chronic pain or paresthesia, resulting in loss of function of the affected arm, even when motor roots remain intact, due to damage to proprioceptive fibers which lose contact with motoneurons. Regeneration through the dorsal root entry zone is difficult due to inhibitory molecules within the Central Nervous System, but possible in crushing injuries. In this sense, Neurotrophin-3 (NT-3) therapy seems to have a positive effect, promoting axon growth and synaptic plasticity in corticospinal tract and proprioceptive pathways, leading to sensorimotor function restoration. Thus, the present study pretends to evaluate the regeneration of sensory fibers into the spinal cord after C6-8 dorsal root crushing (DRC) and delayed intramuscular NT-3 gene therapy. Female Lister hooded rats will be divided in the following experimental groups (n=10 per group): (1) DRC + intramuscular injection of AAV1-CMV-NT3; (2) DRC + intramuscular injection of AAV1-CMV-GFP. During 8 to 12 weeks after injury animals functional recovery will be accessed weekly using the following behavioral tests: horizontal ladder test, gait analysis (DigiGait), automated in-cage single pellet reaching test and Hoffman reflex test. Cholera toxin subunit B (CTb) retrograde tracer will be applied to the ulnar nerve. At the end of the experiment, the spinal cords will be dissected out, allowing synaptic circuitry analysis by VGLUT1, CTb and VGAT immunostaining nearby motoneuron cell bodies and dorsal horn laminae.