Mouse Sciatic Nerve Transection/Resuture Procedure for Studying Nerve Repair and Functional Recovery

Summary

This protocol describes a sciatic nerve transection and resuture model in mice to study peripheral nerve regeneration using adult dorsal root ganglion neurons. It enables in vivo analysis of axon regrowth, muscle reinnervation, and functional recovery following nerve injury.

Abstract

Nerve repair is crucial for restoring function following axonal injury. However, mature neurons in the central nervous system (CNS) have a limited ability to regenerate their axons after injury due to diminished intrinsic growth capacity and a hostile environment. In contrast, neurons in the peripheral nervous system (PNS) exhibit robust regenerative potential. Among them, adult dorsal root ganglion (DRG) neurons are particularly well-known for their ability to regenerate effectively following peripheral nerve injuries, making them an ideal model for studying the cellular and molecular mechanisms underlying nerve repair. Here, a modified microsuture technique, termed "loop-before-transection," is described for generating a sciatic nerve transection and resuture model in mice, followed by analysis of axon regeneration. Behavioral assessments and muscle reinnervation are also demonstrated to evaluate functional recovery. After sciatic nerve transection and resuture, animals initially exhibit behavioral deficits, but full recovery is typically observed by 30 days post-injury. Applying drugs that promote axonal fusion at the injury site significantly accelerates functional recovery. This model system offers a powerful tool for investigating the mechanisms governing mammalian nerve repair in vivo.

Introduction

Axonal injury is a common and severe consequence of brain and spinal cord damage, often caused by traumatic injuries, neurotoxins, or neurological disorders that disrupt neural connectivity and impair axonal regeneration^1,2. Not allneurons possess an intrinsic capacity for regeneration, which is crucial for the repair of severed axons.The rate of axonal regeneration is influenced by multiple factors, including the neuron's intrinsic growth potential, protein synthesis efficiency, cytoskeletal dynamics, clearance of myelin debris, and the availability of growth factors^3,

Protocol

All animal procedures were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) at the University of Texas Health Science Center at San Antonio (Protocol Number: 20200082AR). Adult male and female FVB mice (8-10 weeks old, 20-25 g) were used for the JoVE experimental procedures described here. C57BL/6 mice (8-10 weeks old, 20-25 g) were used only in preliminary practice surgeries conducted prior to the formal experiments to optimize handling and suturing precision. These practice sessions confirmed that the "loop-before-transection" procedure produced comparable outcomes in both strains; therefore, all formal d....

Results

The previous PEG-fusion protocol¹⁸ was modified to maintain consistent surgical results and minimize nerve damage caused by suturing²⁵. By placing the suture to penetrate through the nerve before transection, damage caused by pinching with tweezers or pricking with a needle after transection was minimized. To test the effect of tying a suture penetrating a nerve on its function, the suture was passed through an intact nerve twice and knotted (Figure 2A<.......

Discussion

Nerve regeneration studies using nerve transection models are widely performed on the mouse sciatic nerve^31,39. However, due to its small size and soft texture, the mouse sciatic nerve is difficult to handle gently with surgical tools, and the tissue is prone to tearing during suturing. The microsuture process, often involving excessive manipulation, can lead to nerve deformation and trauma. Even when unnecessary instrument stimulation is minimized, excessive mov.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
#55 Forceps	Dumostar-Biology	11295-51	One in each hand. You will need two to grasp a 10-0 nylon suture.
10-0 nylon suture	DemeTECH	NL761000,4F0P
4-0 nylon suture	Matrix Wizard	0197
Anti-beta-tubulin	R&D Systems	MAB1195
Anti-NF-200	Millipore Sigma	N4142
Austerlitz size 000			For Pinprick test
CF488 conjugated-alpha-bungarotoxin	Biotium	00005
Dissecting scissors	Kent Scientific
Fine suturing scissors	Kent Scientific
Iris forceps	Kent Scientific
K&H Small Animal Heated Pad	K&H pet products
ML162	Millipore Sigma	SML2561
Mouse surgical kit	Kent Scientific
Needle holder	Kent Scientific
Polyethylene glycol (PEG)	Millipore Sigma	P5413
SCG10	Novus Biologicals	NBP1-49461
SomnoSuite	Kent Scientific		For isoflurane anesthesia
Splitter forcep			Used to expose the sciatic nerve.