Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Summary

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

Abstract

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

Introduction

Achieving axon regeneration after nervous system injury is a challenging task¹. To study the failure of axon regeneration in the central nervous system (CNS), researchers have used a plethora of nerve injury models. As regions of the CNS differ, it is important to use an anatomically appropriate model to study axon regeneration. By using the appropriate model, researchers can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration, as opposed to a "one-for-all" treatment strategy.

In spinal cord injury, for exa....

Protocol

All the following animal procedures were conducted in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986. If unfamiliar with these procedures, please check with local/national regulations and seek veterinary advice before starting the protocol.

1. Choosing a Suitable Strain of Animals

NOTE: A dorsal root crush injury results in the loss of sensation and paw deafferentation. Common adverse effects of forepaw deafferentation can include over-grooming, self-mutilation, and paw autotomy.

1. Obtain animals for the procedure.
   NOTE: For rats, the more active strains, such as Lister-Hooded and Wistar,....

Results

As a representation, a transverse spinal cord section with the attached DRG is presented to show the effectiveness of this protocol in transducing DRG neurons and tracing sensory axons in the spinal cord four weeks after injecting a control virus, AAV5-GFP, directly into the C7 DRG without a dorsal root crush injury (Figure 1A). Axons in both the dorsal column and the dorsal horn of the spinal cord express GFP (Figure 1B), as well as the cell bodies and a.......

Discussion

In this article, we present a step-by-step guide to perform a DRG injection and dorsal root crush injury in the lower cervical spinal cord of an adult rat. As this is an extremely invasive and delicate surgery, we strongly recommend that all potential users obtain sufficient training and practice before advancing to live animal surgery. The users should be familiar not only with spinal cord anatomy, but also with the surrounding muscle tissues, vertebral bone structure, and vasculature. Ideally, a competent user should b.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fast Green FCF dye	Sigma-Aldrich	F7258	For visualizing colorless solution. Recommended concentration: 1%
Cholera Toxin B subunit	List Biological Laboratories	104	For anterograde axonal tracing. Recommended concentration: 1%
IsoFlo	Zoetis	115095	Inhalation anesthetic (active ingredient: isoflurane)
Baytril 2.5% injectable	Bayer	05032756093017	Antibiotic (active ingredient: enrofloxacin). Manufacturer's recommended dosage: 10 mg/kg
Carprieve 5.0% w/v	Norbrook	02000/4229	Analgesic (active ingredient: carprofen). Manufacturer's recommended dosage: 4 mg/kg
Lacri-Lube	Allergan	PL 00426/0041	Eye ointment
Olsen-Hegar Needle Holder	Fine Science Tools	FST 12502-12
Friedman Pearson Rongeur Curved 0.7 mm Cup	Fine Science Tools	FST 16121-14
Bonn Micro Forceps	Fine Science Tools	FST 11083-07	For performing dorsal root crush injury
Tissue Separating Scissors	Fine Science Tools	FST 14072-10
Fine Scissors	Fine Science Tools	FST 14058-11
Micro-Adson Forceps	Fine Science Tools	FST 11018-12
Goldstein Retractor	Fine Science Tools	FST 17003-03
Vannas Spring Scissors (straight)	Fine Science Tools	FST 15018-10
SURGIFOAM Absorbable Gelatin Sponge	Ethicon	1972	For bleeding control
Microliter Syringe RN701 (10 μL)	Hamilton	80330
Custom-made Removable Needle (for DRG injection)	Hamilton	7803-05	33 gauge, 38 mm, point style 3
Custom-made Removable Needle (for CTB injection)	Hamilton	7803-05	33 gauge, 10 mm, point style 3
UltraMicroPump with SYS-Micro4 Controller	World Precision Instruments	UMP3-1