Surgical Lumbar Sympathectomy in Mice

Summary

This manuscript presents a protocol for surgically removing the postganglionic lumbar sympathetic neurons from a mouse. This procedure will facilitate a multitude of studies aimed at investigating the role of sympathetic innervation in distal tissue targets.

Abstract

Peripheral nerve injuries are common, and full functional recovery after injury is achieved in only 10% of patients. The sympathetic nervous system plays many critical roles in maintaining bodily homeostasis, but it has rarely been studied in the context of peripheral nerve injury. The extent of postganglionic sympathetic neuronal functions in distal targets in the periphery is currently unclear. To better explore the role of sympathetic innervation of peripheral targets, a surgical "knock-out" model provides an alternative approach. Although this can be achieved chemically, chemical destruction of postganglionic sympathetic neurons can be nonspecific and dose-dependent. The use of a surgical lumbar sympathectomy in mice, once thought to be "virtually not practicable" in small animals, allows for specific targeting of postganglionic sympathetic neurons that innervate the hind limbs. This manuscript describes how to surgically remove the L2-L5 lumbar sympathetic ganglia from a mouse as a survival surgery, which reliably decreases the hind paw sweat response and the number of sympathetic axons in the sciatic nerve.

Introduction

Peripheral nerve injuries (PNIs) can lead to motor, sensory, and sympathetic deficits in distal tissue targets that rarely fully functionally recover¹. PNI research has often focused on the motor and sensory regeneration; however, nearly one-quarter of the rat sciatic nerve consists of unmyelinated sympathetic axons². The role of sympathetic innervation in the peripheral tissues, nevertheless, is not fully understood³. The sympathetic nervous system plays a major role in maintaining bodily homeostasis, participating in immune regulation, thermoregulation, vascular tone, mitochondrial bio....

Protocol

All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Emory University (under the IACUC protocol number PROTO202100177). Four adult female wild-type C57BL/6J mice, aged 14 weeks and weighing between 16-21 g, were used in this study. The details of the reagents and equipment used here are listed in the Table of Materials.

1. Presurgical preparation

1. Autoclave the surgical tools: 1 pair of sharp scissors, 2 fine-tipped tweezers, 1 needle driver.
2. Warm a heating pad to 37 °C and place it under the surgical tabletop.
3. Spray the surgical are....

Results

This protocol describes the surgical removal of postganglionic lumbar sympathetic neurons from a mouse. Two mice received lumbar sympathectomies, and two mice served as controls. To achieve a successful surgical lumbar sympathectomy, adequate visualization of at least the L2 and L3 bilateral lumbar sympathetic ganglia must be achieved, as seen in Figure 1. Removal of the L4 and L5 ganglia would achieve complete sympathetic denervation of the lower body; however, visualization of the lower ga.......

Discussion

The lumbar sympathetic ganglia are very small structures located behind many critical abdominal organs and large vessels. Therefore, this procedure requires significant precision and accuracy. Much of the difficulty lies in identifying the sympathetic ganglia intraoperatively. It is suggested that the learner first be able to identify the ganglia in a mouse cadaver prior to attempting this procedure in a live mouse. Troubleshooting will often need to occur when identifying the sympathetic ganglia after the diversion of t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
5-0 absorable suture	CP Medical	421A
5-0 nylon suture	Med-Vet International	MV-661
70% ethanol	Sigma-Aldrich	E7023-4L
Anesthesia Induction Chamber	Kent Scientific VetFlo	VetFlo-0530XS
Anesthesia Vaporizer	Kent Scientific VetFlo	13-005-202
Betadine	HealthyPets	BET16OZ
C57BL/6J mice	Jackson Laboratory	#000664
Chicken anti-neurofilament-heavy	Abcam	ab72996
Cryostat	Leica	CM1850
Data Analysis Software	Prism
Fine-tipped tweezers	World Precision Instruments	500233
Fluorescent microscope	Nikon	Ti-E
Goat anti-chicken 488	Invitrogen	A32931
Goat anti-rabbit 647	Invitrogen	A21245
Heating pad	Braintree Scientific	39DP
Image Analysis Software	Fiji
Imaging Software	Nikon	NIS-Elements
Isoflurane	Med-Vet International	RXISO-250
Meloxicam	Med-Vet International	RXMELOXIDYL32
Needle driver	Roboz Surgical Store	RS-7894
Normal Goat Serum	Abcam	ab7481
Ophthalmic ointment	Refresh	Refresh P.M.
Phox2bCre:tdTomato mutant mice	Jackson Laboratory	#016223, #007914
Pilocarpine hydrochloride	Sigma-Aldrich	P6503
Rabbit anti-tyrosine hydroxylase	Abcam	ab112
Small straight scissors	Fine Science Tools	14084-09
Sterile cotton swabs 2x2	Dynarex	3252
Sterile cotton tipped applicators	Dynarex	4301
Sterile drape	Med-Vet International	DR4042
Sterile saline solution	Med-Vet International	1070988-BX
ThCre:mTmG mutant mice	Mutant Mouse Resource and Research Centers	strain #017262-UCD	Jackson Laboratory, strain #007576
ThCre:tdTomato mutant mice	European Mouse Mutant Archive	strain #00254	Jackson Laboratory, strain #007914