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 vivo1,2,3, 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 progression5,6,7.
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 nodes8,9,10. 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 ablation15,16,17,18,19,20, surgical resection allows for complete, unilateral adrenergic ablation while avoiding the need for repeated pharmacologic intervention and the associated side effects21,22,23.
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 rats24,26. 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 CCA21,22,27,28. 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.
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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
- 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.- Shave the ventral aspect of the neck or use a chemical hair removal agent according to the manufacturer's instructions (see Table of Materials).
- 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.
- 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.
- 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).
- 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
- 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.
- 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.
- 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).- 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.
- 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
- 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. - 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.
- 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.
- 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.
- 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.
- 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. - 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).
- 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.
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Representative 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 (ECA) are only faintly seen lateral to the vein. When examining this in NSG.B6-P0TdTomato transgenic mouse (a P0-Cre TdTomato mouse where the Schwann cells are fluorescent red, unpublished work) with red fluorescent neurons under a fluorescent microscope, the fluorescent vagus nerve can be seen coursing laterally to the CCA, and the fluorescent SCG can be seen at the bifurcation of the CCA, lateral to the anterior jugular vein (Figure 2B).
After the resection of the SCG in a normal mouse and a transgenic mouse, the resected tissue was confirmed by its red fluorescence compared to the non-fluorescent SCG control (Figure 3A) and immunofluorescent staining for tyrosine hydroxylase (TH), a marker for adrenergic nerves13,29 (Figure 3B).
If the procedure is performed correctly, the mouse develops ipsilateral Horner's syndrome immediately after surgery upon regaining full consciousness24. Ptosis, the drooping of the eyelid, was observed, which is a sign of Horner's syndrome (Figure 4B).
The submandibular salivary gland is one of the tissues innervated by the SCG. To validate successful SCGx, immunofluorescence staining for TH was performed on the right submandibular salivary gland following right SCGx and confirmed the successful ablation of the adrenergic signaling with absent TH nerve staining (right side of the dotted line, Figure 5A). In contrast, the left control submandibular gland (no SCGx) maintained its adrenergic input and intact TH nerve staining (left side of the dotted line, Figure 5A). These findings were confirmed by quantification (Figure 5B). ELISA quantification of norepinephrine13,30,31 in these tissues further confirmed a significant reduction in norepinephrine expression in the submandibular gland on the side of SCGx in contrast to the control sham surgery side (Figure 6). The quantification for both was analyzed by an unpaired, two-tailed Student's t-test.
Figure 1: The left anterior jugular vein serves as an anatomical landmark. (A) The left anterior jugular vein (blue arrow) can be seen coursing longitudinally and alongside the lateral edge of the omohyoid muscle. When piercing the deep cervical fascia between the angle of the posterior belly of the digastric and omohyoid muscles, the piercing should also be lateral to the anterior jugular vein (black circle). (B) When the deep cervical fascia is slightly stretched, the vagus nerve (white arrow) and the common carotid artery with its bifurcation (red arrow) can also be seen. (C) Stylized illustration of (B). Scale bar = 100 µm. Abbreviation: M = muscle. Please click here to view a larger version of this figure.
Figure 2: The SCG and its relation to anatomical landmarks in a transgenic mouse with fluorescent neurons. (A) Dissection in a transgenic mouse with red fluorescent neurons, illustrating the right common carotid artery (red arrow pointing to its bifurcation) and the anterior jugular vein. The common carotid artery bifurcates into the external carotid artery (ECA) and internal carotid artery (ICA). The yellow arrow points to the vagus nerve running laterally to the common carotid artery. The distance between the common carotid artery and the vagus nerve appears wider here as the mouse's head is turned to capture all the structures in this image. (B) The same dissection examined with fluorescent imaging. The vagus nerve (yellow arrow) has red fluorescence and is again seen running laterally to the common carotid artery (red arrow pointing to its bifurcation). The fluorescent SCG is located at the bifurcation of the carotid artery (yellow arrowhead). The anterior jugular vein (blue arrow) runs medial to the common carotid artery. The deep cervical fascia overlying these structures can be seen with its glistening reflection. Scale bar = 1 mm. Please click here to view a larger version of this figure.
Figure 3: Microscopic images of the resected ganglion. (A) Resected superior cervical ganglia under fluorescent microscopy. The left shows the SCG resected from a normal mouse, serving as a non-fluorescent control. The right shows a fluorescent SCG resected from a transgenic mouse with red fluorescent neurons. Scale bar = 500 µm. (B) Immunofluorescent staining for tyrosine hydroxylase (TH), a marker for adrenergic nerves, in the resected red fluorescent ganglion (P0). Scale bar = 100 µm. Please click here to view a larger version of this figure.
Figure 4: Development of Horner's syndrome following SCGx. (A) A normal mouse before SCGx. (B) The development of ptosis (black arrow), the drooping of the eyelid, following ipsilateral SCGx, which is a sign of Horner's syndrome. Please click here to view a larger version of this figure.
Figure 5: Immunofluorescence and corresponding H&E staining for the adrenergic marker in the target tissue after SCGx versus sham surgery. (A) Left, immunofluorescence staining for tyrosine hydroxylase (TH) in the submandibular salivary gland following SCGx or sham surgery. Right, the corresponding H&E staining of the same tissue. Scale bar = 200 µm. (B) Quantification of the TH staining. Data represent mean ± SEM. Statistical analysis by unpaired, two-tailed Student's t-test, p < 0.0001. Please click here to view a larger version of this figure.
Figure 6: ELISA quantification of norepinephrine in the salivary gland after SCGx versus sham surgery. Data represent mean ± SEM. Statistical analysis by unpaired, two-tailed Student's t-test, p < 0.0001. Please click here to view a larger version of this figure.
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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 been described before by Ziegler et al.26 and briefly reported in other studies21,22,27,28, the technique was based on that used in rats, in which the CCA is exposed and dissected prior to the resection of the SCG. In contrast to the rat model, the CCA in mice is smaller and thinner, rendering the dissection more difficult and, therefore, more prone to the serious complication of major bleeding from the CCA. Additionally, the exposure of the CCA requires more extensive manipulation, including the displacement of the sternocleidomastoid muscle, as well as the dissection and lateral rotation of the salivary gland26. In contrast, the present method uses the anterior jugular vein instead of the CCA as the anatomical landmark. In comparison to the CCA, the anterior jugular vein is located more superficially and extends further cranially (Figure 2A). This offers a few advantages. First, this landmark is more readily seen without the dissection and displacement of the salivary gland and sternocleidomastoid muscle, making the surgery less invasive; this protocol, therefore, only requires the salivary to be pulled down slightly (step 2.2). The minimal dissection also shortens the surgery time and the duration of anesthesia for the animal. Furthermore, by avoiding extensive dissection of the CCA, this minimizes the chances of injuring the CCA, which can lead to major and fatal bleeding in serious cases. Manipulation of the CCA is inevitable, as the SCG is located at the bifurcation of the CCA, but by approaching this region posteromedially via a piercing next to the anterior jugular vein rather than opening the fascia directly overlying the CCA, this protocol minimizes the contact with and, therefore, the risk of injury to this major artery.
Two major challenges are faced when performing this surgery. The first is the successful identification of the SCG, especially given the very small size of the anatomical landmarks and the ganglion itself in mouse models. The careful dissection and identification of the landmarks are, therefore, essential. In step 2.3, angled forceps must be inserted to pierce the deep cervical fascia at the angle of the posterior belly of the digastric and omohyoid muscles. During this step, the anterior jugular vein is usually seen running alongside the lateral edge of the omohyoid muscle and should be kept medial to the insertion point (Figure 1); this is an important landmark and will help entry into the correct space to find the SCG. If the SCG is not seen in the lateral region of this space, the tissues need to be explored more laterally and superiorly. During this dissection, the carotid sheath is visualized lateral to the field of view to avoid bleeding of the surrounding tissues and to help identify the SCG medial to this structure.
The second major challenge for this procedure is managing the risk of bleeding. There are multiple critical vascular structures adjacent to the SCG, including the CCA, the external carotid artery, and the internal jugular vein. In our experience, if bleeding occurs, it is encountered intraoperatively rather than postoperatively. Bleeding might be encountered during the step of unclamping the forceps in step 3.4. Injury to the vessels is most likely to occur when trying to peel and gently avulse the ganglion from the surrounding vessels and tissues. Active bleeding might not be seen immediately because a pair of forceps is clamped near the vessels in that region. Therefore, bleeding may be identified once the forceps are released, and it is important to inspect the area carefully after the ganglion is removed. In the rare instance of exsanguination due to a tear in a major vessel, holding pressure over the area is futile because of the rapid rate of bleeding. In this situation, the surgery must be terminated, and the mouse must be euthanized.
Given the challenges of SCG identification and possible bleeding complications, it is recommended to first practice SCG dissection and removal on cadaveric mice to become familiar with the anatomy prior to performing the experimental survival surgery.
This method may also be affected by the surgeon's handedness. The procedure is easier to perform on the same side as the surgeon's dominant hand. For example, when performing SCGx on the right side of the mouse, the surgeon's left hand would be used to grasp the base of the ganglion, and the right hand would be used to peel out the ganglion, thus meaning the surgery would require more finesse with the right hand. If bilateral SCGx is to be performed, it may be more time-consuming and require more training to perform on the surgeon's non-dominant side.
This surgical technique of SCGx in a murine model enables future experimental studies examining the effects of the sympathetic nervous system in both oncologic and physiologic settings. The mouse model has multiple advantages over other in vivo models, including the low cost, the ease of handling, and the amenability to genetic manipulation, thus enabling more powerful experimental models to be created.
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Disclosures
The authors have nothing to disclose.
Acknowledgments
Q. W. was supported by NIH T32CA009685. R. J. W. was supported by NIH R01CA219534. The Memorial Sloan Kettering Cancer Center Core Facilities were supported by NIH P30CA008748.
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 |
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