Name: surgical technique for superior cervical ganglionectomy in a murine model
Uploaded: 2022-12-02T13:30:00
Description: Watch this Scientific Journal Video about Surgical Technique for Superior Cervical Ganglionectomy in a Murine Model at JoVE.com

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.

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&#39;s skin surface is disinfected, and the surgeon wears sterile gloves throughout the procedure.
1. Prep...

<ol>
	<li>Magnon, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autonomic+nerve+development+contributes+to+prostate+cancer+progression.">Autonomic nerve development contributes to prostate cancer progression.</a> Science. 341 (6142), 1236361 (2013).</li><li>Zhao, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Denervation+suppresses+gastric+tumorigenesis.">Denervation suppresses gastric tumorigenesis.</a> Science Translational Medicine. 6 (250), 115 (2014).</li><li>Zahalka, A. 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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...

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 &#8211; bleeding from an injury to the CCA.

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			<td>Dissecting Microscope</td>
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			<td>Fine Science Tools</td>
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			<td>Hypoallergenic Surgical Tape</td>
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			<td>Induction Chamber, 2 Liter</td>
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			<td>Isoflurane</td>
			<td>Baxter</td>
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			<td>Church &#38; Dwight Co., Inc</td>
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			<td>NSG Mouse</td>
			<td>Jackson Laboratory</td>
			<td>JAX:005557</td>
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			<td>Povidone-Iodine Swabstick</td>
			<td>PDI</td>
			<td>S41350</td>
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			<td>Webcol Alcohol Preps</td>
			<td>Covidien</td>
			<td>5110</td>
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surgical technique for superior cervical ganglionectomy in a murine model

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.

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...

Watch this Scientific Journal Video about Surgical Technique for Superior Cervical Ganglionectomy in a Murine Model at JoVE.com

Surgical Technique for Superior Cervical Ganglionectomy in a Murine Model

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

Growing evidence suggests that the sympathetic nervous system plays an important role in cancer progression. Adrenergic innervation regulates salivary ...

The sympathetic nervous system plays an important role in cancer progression and other diseases. Surgical sympathectomy is a valuable method for studying these processes by inducing complete unilateral adrenergic ablation. This technique avoids the side effects associated with pharmacologic inhibition of the sympathetic nervous system.Additionally, it is less invasive than previously described methods because it avoids dissection of the carotid artery. When performing this technique, it may be difficult to enter the right space where the ganglion resides and differentiate it from the fat. Identifying the landmark and practice is therefore essential.The anatomy appears slightly different when in motion and undissected, so visual demonstration provides a better guide on how to identify the anatomical landmark which is key for this technique. Begin by applying topical ophthalmic ointment to the anesthetized mouse to prevent ocular injuries and dryness. Place the mouse under a dissecting microscope on its dorsal side and maintain inhalation anesthesia using a precision vaporizer and nose cone.Secure both forelimbs with hypoallergenic tape. Clean the shaved ventral aspect of the neck with povidone iodine and then wipe with 70%alcohol. After repeating the cleaning process two more times, place a sterile drape over the surgical site.Then use small scissors and make a 1.5 centimeter midline skin incision on the ventral aspect of the neck from approximately two millimeters below the chin to two millimeters above the sternal notch. Separate the skin from the underlying fascia by inserting pointed scissors under the skin on each side and spreading. Then retract the edges of the skin laterally with forceps to expose the underlying fascia and submandibular salivary glands.Pull the submandibular glands caudally with forceps to reveal the underlying muscles. Next, locate the junction of the posterior belly of the digastric muscle and omohyoid muscle. Insert the tip of 45 degree 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 open with the 45 degree angled forceps and expand this opening wider by performing spreading maneuvers with a pair of curved forceps in the other hand. Locate the superior cervical ganglion or SCG on the lateral wall of the revealed space. 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 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 base of the ganglion and check for bleeding by looking for blood pooling. Once done, move the salivary glands back to their normal anatomic positions. Approximate and close the skin using simple interrupted 5-0 nylon sutures.During the dissection, the right anterior jugular vein coursed alongside the trachea's lateral border. When P0 tdTomato transgenic mouse with red fluorescent neurons was examined under a fluorescent microscope, the fluorescent vagus nerve was observed coursing laterally to the common carotid artery and the fluorescent SCG was observed at the bifurcation of the common carotid artery lateral to the anterior jugular vein. The resected SCG in a transgenic mouse was confirmed by its red fluorescence compared to the non-fluorescent SCG control.The immunofluorescent staining confirmed a tyrosine hydroxylase, a marker for adrenergic nerves. After superior cervical ganglionectomy, ptosis, that is the drooping of the eyelid, was observed, which is a sign of Horner syndrome. Immunofluorescence staining for a tyrosine hydroxylase on the right submandibular salivary gland following right superior cervical ganglionectomy confirmed the successful ablation of the adrenergic signaling with absent tyrosine hydroxylase nerve staining.In contrast, the left control submandibular gland maintained its adrenergic input and intact a tyrosine hydroxylase nerve staining which was also confirmed by quantification. ELISA quantification of norepinephrine in the salivary gland confirmed a significant reduction in norepinephrine expression in the submandibular gland on the side of superior cervical ganglionectomy in contrast to the control sham surgery side. Be patient and gentle when removing the SCG.Avulse it slowly in a piecemeal fashion. Otherwise, the carotid artery connected to the SCG may easily be injured resulting in bleeding.

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The Application of 1% Methylene Blue Dye As a Single Technique in Breast Cancer Sentinel Node Biopsy

In this study, we injected 1% methylene blue dye (MBD) into the subareolar or peritumoral space of the breast. In the case of breast conserving surgery ...

An Orthotopic Resectional Mouse Model of Pancreatic Cancer

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer ...

A Murine Ommaya Xenograft Model to Study Direct-Targeted Therapy of Leptomeningeal Disease

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers ...

Robot Assisted Distal Pancreatectomy with Celiac Axis Resection (DP-CAR) for Pancreatic Cancer: Surgical Planning and Technique

Robot Assisted Distal Pancreatectomy with Celiac Axis Resection DP-CAR for Pancreatic Cancer: Surgical Planning and Technique

Malignant pancreatic tumors involving the celiac artery can be resected with a distal pancreatectomy, splenectomy and celiac axis resection (DP-CAR), ...

Magnetic Resonance-Guided High Intensity Focused Ultrasound Generated Hyperthermia: A Feasible Treatment Method in a Murine Rhabdomyosarcoma Model

Magnetic resonance-guided high intensity focused ultrasound (MRgHIFU) is an established method for producing localized hyperthermia. Given the real-time ...