Surgical Lumbar Sympathectomy in Mice

Published: July 05, 2024

doi:

¹Medical Scientist Training Program,Emory University School of Medicine, ²Neuroscience Graduate Program, Laney Graduate School,Emory University, ³Department of Cell Biology,Emory University School of Medicine

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 biogenesis, and more⁴^,⁵^,⁶^,⁷^,⁸^,⁹^,¹⁰^,¹¹. When sympathetic innervation at the neuromuscular junction is lost, persistent muscle weakness and synaptic instability are observed despite the maintenance of motoneuron innervation¹². This sympathetic regulation of synaptic transmission at the neuromuscular junction has been shown to decline with aging¹³^,¹⁴, which contributes to sarcopenia, defined as the age-dependent reduction in muscle mass, force, and power¹⁵. A better understanding of the role of sympathetic innervation of peripheral tissues is necessary for the development of therapies that will optimize functional outcomes for patients with PNIs and other forms of sympathetic dysfunction.

A sympathectomy is a powerful experimental tool that will allow for investigations of the role of sympathetic innervation in distal target tissues. Specifically, removal of the L2-L5 level sympathetic ganglia removes a majority of the sympathetic innervation to the lower limbs, which is especially useful for investigators interested in the sciatic nerve.

This protocol details the removal of L2-L5 level postganglionic sympathetic neurons from a mouse as a survival surgery. This procedure requires rodent microsurgical skills and familiarity with mouse anatomy, and when performed effectively, does not cause any visible phenotypic differences. A surgical lumbar sympathectomy has been used in rodent research, more so in rats than in mice¹⁶^,¹⁷^,¹⁸^,¹⁹^,²⁰^,²¹; however, a detailed protocol describing the protocol does not currently exist. Previous studies utilizing the lumbar sympathectomy have primarily focused on the role of sympathetic innervation in the pain response, which is generally attenuated by sympathectomy in various nerve injury models. Fewer studies have used this technique in mice²², likely due to the smaller size of anatomic landmarks, as the use of surgical sympathectomy was believed to be "virtually not practicable" in small animals²³^,²⁴. Localized sympathectomies in the form of microsympathectomies have also been utilized in rodent models, also mostly in the context of pain behaviors²⁵^,²⁶^,²⁷. The microsympathectomy, in contrast to the total lumbar sympathectomy, utilizes a dorsal approach through which a segment of the gray ramus to a specific spinal nerve is disconnected and removed, allowing for a very targeted sympathectomy that will avoid wider spread side effects.

Because mouse models are critical for many studies requiring genetic manipulation, this procedure will have versatile applications beyond the breadth of peripheral nerve injuries as well. Using a transabdominal approach, the lumbar sympathetic ganglia can be reliably visualized and resected from the mouse with no apparent adverse effects. Although protocols for the chemical destruction of postganglionic sympathetic neurons are available, such as the use of 6-hydroxydopamine (6-OHDA)²³^,²⁴, this surgical procedure allows for anatomically specific targeting of the postganglionic lumbar sympathetic ganglia. The use of a surgical sympathectomy also avoids the nonspecific and dose-dependent concerns related to pharmacological methods²⁸^,²⁹.

The use of chemical sympathectomies via administration of 6-OHDA was described in 1967 as a simple way to achieve selective destruction of adrenergic nerve endings since surgical sympathectomies in small animals were not favored²³^,²⁴. 6-OHDA is a catecholaminergic neurotoxin that is endogenously formed in patients with Parkinson's disease, and its toxicity is derived from its ability to form free radicals and inhibit the electron transport chain in mitochondria³⁰^,³¹. Through norepinephrine uptake-1 transport mechanisms, 6-OHDA is able to accumulate within noradrenergic neurons, such as postganglionic sympathetic neurons²⁸. Eventually, the neuron is destroyed by 6-OHDA; however, terminals in the peripheral nervous system do regenerate, with the restoration of functional activity even when the amine levels are still reduced. Different dosage thresholds are also present for different organs in response to 6-OHDA, and higher doses of 6-OHDA have been shown to exhibit more nonspecific effects, extending its neurotoxic consequences to non-catecholamine-containing neurons and even non-neuronal cells. Aside from noradrenergic neurons, dopaminergic neurons are affected by 6-OHDA as well²⁹, making the chemical sympathectomy ultimately less specific to postganglionic sympathetic neurons than the surgical sympathectomy.

Therefore, a surgical lumbar sympathectomy enables the targeted ablation of the sympathetic innervation to the lower limbs, which can be combined with a variety of experimental techniques and genetic manipulations in the mouse to study how the sympathetic nervous system contributes to various injury and disease states.

Protocol

All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Emory University (under the IACUC protocol number PROTO201700371). 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 Autoclave the surgical tools: 1 pair of sharp scissors, 2 fine-t…

Representative 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…

Declarações

The authors have nothing to disclose.

Acknowledgements

This work was supported by the NIH National Institute of Neurological Disorders and Stroke under award number K01NS124912 and in part by a developmental grant from the NIH-funded Emory Specialized Center of Research Excellence in Sex Differences U54AG062334 and the Medical Scientist Training Program of Emory University School of Medicine. Thank you to David Kim, postbaccalaureate, for sectioning sciatic nerves and to HaoMin SiMa, research specialist, for 3D printing a phone mount for our stereo microscope that allowed for the filming of the video.

Materials

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
Eye lubricant	Refresh	Refresh P.M.
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
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 2×2	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

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