Surgical Technique for Superior Cervical Ganglionectomy in a Murine Model

Summary

The present protocol describes a mouse model of the ablation of adrenergic innervation by identifying and resecting the superior cervical ganglion.

Abstract

Growing evidence suggests that the sympathetic nervous system plays an important role in cancer progression. Adrenergic innervation regulates salivary gland secretion, circadian rhythm, macular degeneration, immune function, and cardiac physiology. Murine surgical sympathectomy is a method for studying the effects of adrenergic innervation by allowing for complete, unilateral adrenergic ablation while avoiding the need for repeated pharmacologic intervention and the associated side effects. However, surgical sympathectomy in mice is technically challenging because of the small size of the superior cervical ganglion. This study describes a surgical technique for reliably identifying and resecting the superior cervical ganglion to ablate the sympathetic nervous system. The successful identification and removal of the ganglion are validated by imaging the fluorescent sympathetic ganglia using a transgenic mouse, identifying post-resection Horner's syndrome, staining for adrenergic markers in the resected ganglia, and observing diminished adrenergic immunofluorescence in the target organs following sympathectomy. This model enables future studies of cancer progression as well as other physiological processes regulated by the sympathetic nervous system.

Introduction

Multiple studies have reported that the nerves in the tumor microenvironment play an active role in supporting tumor progression. The ablation of adrenergic sympathetic nerves has been shown to impair tumor development and dissemination in prostate and gastric cancer in vivo^1,2,3, while the pharmacological blockade of adrenergic receptors inhibits tumor growth in head and neck cancer⁴. Sympathetic neural involvement has also been described in pancreatic, cervical, and basal cell carcinoma progression^5,6,7.

Within the sympathetic nervous system, the superior cervical ganglion (SCG) is the only ganglion of the sympathetic trunk that innervates the head. The SCG regulates various physiologic functions, such as salivary secretion and circadian rhythm, and directly innervates the cervical lymph nodes^8,9,10. The SCG has also been implicated in pathologic processes such as macular degeneration¹¹ and the progression of aortic dissection¹². Additionally, resection of the SCG has been reported to aggravate ischemia reperfusion-induced acute kidney injury¹³ and also alter the gut microbiota in rats¹⁴.

The complete ablation of the SCG in a mouse model would represent a valuable experimental technique to enable cancer and autonomic nervous system research. While many studies have utilized pharmacological adrenergic receptor blockade as an adrenergic ablation^{15,16,17,18,19,20}, surgical resection allows for complete, unilateral adrenergic ablation while avoiding the need for repeated pharmacologic intervention and the associated side effects^21,22,23.

Surgical resection of the SCG has been described in rats²⁴, and most reports studying the effect of superior cervical ganglionectomy (SCGx) have employed the rat model. Compared to the rat model, SCGx is technically more challenging in mice due to the small size of the SCG. However, mice are comparatively easier to handle, more cost-effective, and more amenable to genetic manipulation. Garcia et al. were one of the first to report SCGx in mice, and it was found to affect insulin release²⁵. More recently, Ziegler et al. described SCGx in mice based on the published technique described for rats^24,26. This and other articles describe a method in which the common carotid artery (CCA) is first identified and dissected, and the SCG is subsequently removed from the bifurcation of the CCA^21,22,27,28. In this article, a less invasive and safer technique is described in mice that avoids the dissection of the CCA, thereby minimizing the most serious complication of this procedure – bleeding from an injury to the CCA.

Protocol

The animal procedures described here were approved by the Institutional Animal Care and Use Committee at the Memorial Sloan Kettering Cancer Center. Eight-week-old male and female NSG mice were used here. The animals were obtained from a commercial source (see Table of Materials). The instruments are sterilized, the surgical working surface is disinfected, the animal's skin surface is disinfected, and the surgeon wears sterile gloves throughout the procedure.

1. Preparation of the mice and preoperative setup

1. On the day before surgery, anesthetize the mouse with 2% isoflurane in an induction chamber (3.75 in width x 9 in depth x 3.75 in height, see Table of Materials).
   ​NOTE: A surgical plane of anesthesia is usually achieved in 3-5 min, depending on the individual animal. Assess the adequacy of anesthesia by toe pinch, and increase the isoflurane percentage as appropriate.
   1. Shave the ventral aspect of the neck or use a chemical hair removal agent according to the manufacturer's instructions (see Table of Materials).
2. On the day of the surgery, anesthetize the mouse with 2% isoflurane in an induction chamber. Assess the adequacy of anesthesia by toe pinch, and increase the isoflurane percentage as appropriate.
3. Administer 2 mg/kg of meloxicam subcutaneously for preemptive systemic analgesia. Apply topical ophthalmic ointment (see Table of Materials) to prevent ocular injuries and dryness under anesthesia.
4. Place the mouse under a dissecting microscope on its dorsal side and provide thermal support. Maintain inhalational anesthesia with 2%-2.5% isoflurane using a precision vaporizer and nose cone. Gently secure both forelimbs with hypoallergenic tape (see Table of Materials).
5. Clean the shaved, ventral aspect of the neck with povidone-iodine, and then wipe with 70% alcohol. Repeat this process two more times. Ensure that the surgical site is free from any loose hair.
   NOTE: A pair of short curved forceps may also be used. Ensure to use a pair of fine or ophthalmic forceps to adequately work in this confined space. Additional pre-operative setup can be included as per the institutional guidelines. 

2. Dissection

1. Make a 1.5 cm midline skin incision on the ventral aspect of the neck using small scissors from approximately 2 mm below the chin to 2 mm above the sternal notch.
2. Retract the edges of the skin laterally with forceps to expose the underlying fascia and submandibular salivary glands. Separate the skin from the underlying fascia by inserting pointed scissors under the skin on each side and spreading. Pull down the submandibular glands caudally with forceps to reveal the underlying muscles.
3. Locate the junction of the posterior belly of the digastric muscle and omohyoid muscle (Figure 1A, black circle). The anterior jugular vein is seen running longitudinally and lateral to the omohyoid muscle.
   NOTE: The omohyoid muscle covers the trachea longitudinally, while the digastric muscle lies transversely at the cranial aspect of the trachea (Figure 1C).
   1. Insert the tip of 45° angled forceps at this junction, lateral to the anterior jugular vein, to pierce and spread an opening in the overlying deep cervical fascia.
4. Keep this window created in step 2.3.1 open with the 45° angled forceps. Expand this opening wider by performing spreading maneuvers with a pair of curved forceps in the other hand.

3. Identification and resection of the ganglion

1. Locate the superior cervical ganglion (SCG) on the lateral wall of the revealed space. It appears as a round, pearly tissue.
   NOTE: If the SCG is not identified, the tissues in this space need to be examined more laterally and superiorly. The SCG may be easily confused with fat, which is often present in this region. Fat has a slightly yellow tinge, while in contrast, the SCG appears pearl white.
2. While maintaining the opening with forceps with the other hand, gently grasp the SCG with forceps, and pull it out of the opening to bring it into better view.
3. Once the SCG is in view, grasp the lateral base of the SCG, where it is still attached to the surrounding tissues. Using the other hand, slowly and gently retract the SCG in a ventral and caudal direction.
   1. Retract the SCG multiple times to gradually avulse the ganglion little by little. Keep the ganglion intact during this maneuver to ensure no residual ganglion remnants are left behind.
      NOTE: Pull the ganglion gently, as bleeding may occur during this step. If minor bleeding occurs, use oxidized regenerated cellulose or a small strip of sterile gauze to hold pressure over the opening for 30 s to 1 min. Then, slowly lift the gauze and reassess. Repeat the process of holding pressure over the opening as necessary until the bleeding has stopped.
4. Slowly release the other forceps holding the base of the ganglion. Check for bleeding by looking for blood pooling.
   NOTE: Slight oozing at this time is normal. Monitor and ensure there is no persistent or significant bleeding before closing and finishing the procedure. If this occurs, hold pressure over the opening, as described in step 3.3.1.
5. Move the salivary glands back into their normal anatomic positions. Approximate and close the skin using simple interrupted 5-0 nylon sutures (see Table of Materials).
6. Place the mouse in a clean cage by itself to allow for full recovery from the anesthesia.
   NOTE: It may take 5-15 min for the mouse to awaken fully from the anesthesia. Do not leave the mouse unattended until it has regained sufficient consciousness to maintain sternal recumbency. Do not place the mouse in a cage with other mice until it has fully recovered. Assess the mouse for postsurgical recovery at least once every 24 h for 72 h.

Results

This protocol describes the surgical removal of the SCG in a mouse model. Figure 2 illustrates the anatomical landmarks, including the CCA, the anterior jugular vein, and the SCG. With dissection (Figure 2A), the right anterior jugular vein can be seen coursing alongside the lateral border of the trachea. As it is located deeper than the anterior jugular vein, the left CCA and its bifurcation into the internal carotid artery (ICA) and external carotid artery (EC...

Discussion

This protocol describes a mouse model for the surgical unilateral ablation of SCG input. This technique allows for studying the effects of adrenergic innervation in various settings. In addition, the resected sympathetic ganglion can also be grown in 3D matrigel culture for in vitro experiments³⁰.

Studies involving SCGx have mostly been performed in rats, as their larger anatomy allows for easier anatomic visualization and dissection. While SCGx in mice has bee...

Materials

Name	Company	Catalog Number	Comments
Anti-Tyrosine Hydroxylase Antibody	EMD Millipore	AB152
Artificial Tears Lubricant Ophthalmic Ointment	Akorn	59399-162-35
Curity 2 x 2 Inch Gauze Sponge 8 Ply, Sterile	Covidien	1806
Derf Needle Holder	Thomas Scientific	1177K00
Dissecting Microscope
Dumont #5/45 Forceps	Fine Science Tools	11251-35
Dumont #7b Forceps	Fine Science Tools	11270-20
ETHILON Nylon Suture	Ethicon	698H
Fine Scissors - ToughCut	Fine Science Tools	14058-09
Hypoallergenic Surgical Tape	3M Blenderm	70200419342
Induction Chamber, 2 Liter	VetEquip	941444
Isoflurane	Baxter	1001936060
Nair	Church & Dwight Co., Inc	40002957	chemical hair removing agent
NORADRENALINE RESEARCH ELISA	Labor Diagnostika Nord (Rocky Mountain Diagnostics)	BA E-5200
NSG Mouse	Jackson Laboratory	JAX:005557
Povidone-Iodine Swabstick	PDI	S41350
Webcol Alcohol Preps	Covidien	5110