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. H., 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=Adrenergic+nerves+activate+an+angio-metabolic+switch+in+prostate+cancer.">Adrenergic nerves activate an angio-metabolic switch in prostate cancer.</a> Science. 358 (6361), 321-326 (2017).</li><li>Amit, 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=Loss+of+p53+drives+neuron+reprogramming+in+head+and+neck+cancer.">Loss of p53 drives neuron reprogramming in head and neck cancer.</a> Nature. 578 (7795), 449-454 (2020).</li><li>Renz, B. W., 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=Cholinergic+signaling+via+muscarinic+receptors+directly+and+indirectly+suppresses+pancreatic+tumorigenesis+and+cancer+stemness.">Cholinergic signaling via muscarinic receptors directly and indirectly suppresses pancreatic tumorigenesis and cancer stemness.</a> Cancer Discovery. 8 (11), 1458-1473 (2018).</li><li>Lucido, C. T., 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=Innervation+of+cervical+carcinoma+is+mediated+by+cancer-derived+exosomes.">Innervation of cervical carcinoma is mediated by cancer-derived exosomes.</a> Gynecologic Oncology. 154 (1), 228-235 (2019).</li><li>Peterson, S. 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=Basal+cell+carcinoma+preferentially+arises+from+stem+cells+within+hair+follicle+and+mechanosensory+niches.">Basal cell carcinoma preferentially arises from stem cells within hair follicle and mechanosensory niches.</a> Cell Stem Cell. 16 (4), 400-412 (2015).</li><li>Maronde, E., Stehle, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mammalian+pineal+gland:+Known+facts,+unknown+facets.">The mammalian pineal gland: Known facts, unknown facets.</a> Trends in Endocrinology &amp; Metabolism. 18 (4), 142-149 (2007).</li><li>Yamazaki, S., 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=Ontogeny+of+circadian+organization+in+the+rat.">Ontogeny of circadian organization in the rat.</a> Journal of Biological Rhythms. 24 (1), 55-63 (2009).</li><li>Huang, J., 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=S100++cells:+A+new+neuro-immune+cross-talkers+in+lymph+organs.">S100+ cells: A new neuro-immune cross-talkers in lymph organs.</a> Scientific Reports. 3 (1), 1114 (2013).</li><li>Dieguez, H. H., 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=Melatonin+protects+the+retina+from+experimental+nonexudative+age-related+macular+degeneration+in+mice.">Melatonin protects the retina from experimental nonexudative age-related macular degeneration in mice.</a> Journal of Pineal Research. 68 (4), 12643 (2020).</li><li>Liu, H., 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=Bilateral+superior+cervical+ganglionectomy+attenuates+the+progression+of+&#946;-aminopropionitrile-induced+aortic+dissection+in+rats.">Bilateral superior cervical ganglionectomy attenuates the progression of &#946;-aminopropionitrile-induced aortic dissection in rats.</a> Life Sciences. 193, 200-206 (2018).</li><li>Zhang, W., 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=The+role+of+the+superior+cervical+sympathetic+ganglion+in+ischemia+reperfusion-induced+acute+kidney+injury+in+rats.">The role of the superior cervical sympathetic ganglion in ischemia reperfusion-induced acute kidney injury in rats.</a> Frontiers in Medicine. 9, 792000 (2022).</li><li>Zhang, W., 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=Superior+cervical+ganglionectomy+alters+gut+microbiota+in+rats.">Superior cervical ganglionectomy alters gut microbiota in rats.</a> American Journal of Translational Research. 14 (3), 2037-2050 (2022).</li><li>Wang, X., 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=&#946;-Adrenergic+signaling+induces+Notch-mediated+salivary+gland+progenitor+cell+control.">&#946;-Adrenergic signaling induces Notch-mediated salivary gland progenitor cell control.</a> Stem Cell Reports. 16 (11), 2813-2824 (2021).</li><li>Boyd, A., Aragon, I. V., Abou Saleh, L., Southers, D., Richter, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cAMP-phosphodiesterase+4+(PDE4)+controls+&#946;-adrenoceptor-+and+CFTR-dependent+saliva+secretion+in+mice.">The cAMP-phosphodiesterase 4 (PDE4) controls &#946;-adrenoceptor- and CFTR-dependent saliva secretion in mice.</a> Biochemical Journal. 478 (10), 1891-1906 (2021).</li><li>Smith, B., Butler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+long-term+propranolol+on+the+salivary+glands+and+intestinal+serosa+of+the+mouse.">The effects of long-term propranolol on the salivary glands and intestinal serosa of the mouse.</a> The Journal of Pathology. 124 (4), 185-187 (1978).</li><li>Sucharov, C. 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=&#946;-Adrenergic+receptor+antagonism+in+mice:+A+model+for+pediatric+heart+disease.">&#946;-Adrenergic receptor antagonism in mice: A model for pediatric heart disease.</a> Journal of Applied Physiology. 115 (7), 979-987 (2013).</li><li>Ding, C., Walcott, B., Keyser, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+alpha1-+and+beta1-adrenergic+modulation+of+lacrimal+gland+function+in+the+mouse.">The alpha1- and beta1-adrenergic modulation of lacrimal gland function in the mouse.</a> Investigative Ophthalmology &amp; Visual Science. 48 (4), 1504-1510 (2007).</li><li>Grisanti, L. A., 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=Prior+&#946;-blocker+treatment+decreases+leukocyte+responsiveness+to+injury.">Prior &#946;-blocker treatment decreases leukocyte responsiveness to injury.</a> JCI Insight. 5 (9), 99485 (2019).</li><li>Alito, A. E., 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+nervous+system+regulation+of+murine+immune+responses+as+assessed+by+local+surgical+sympathetic+and+parasympathetic+denervation.">Autonomic nervous system regulation of murine immune responses as assessed by local surgical sympathetic and parasympathetic denervation.</a> Acta Physiologica, Pharmacologica et Therapeutica Latinoamericana. 37 (3), 305-319 (1987).</li><li>Yun, H., Lathrop, K. L., Hendricks, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+central+role+for+sympathetic+nerves+in+herpes+stromal+keratitis+in+mice.">A central role for sympathetic nerves in herpes stromal keratitis in mice.</a> Ophthalmology &amp; Visual Science. 57 (4), 1749-1756 (2016).</li><li>Haug, S. R., Heyeraas, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+sympathectomy+on+experimentally+induced+pulpal+inflammation+and+periapical+lesions+in+rats.">Effects of sympathectomy on experimentally induced pulpal inflammation and periapical lesions in rats.</a> Neuroscience. 120 (3), 827-836 (2003).</li><li>Savastano, L. E., 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=A+standardized+surgical+technique+for+rat+superior+cervical+ganglionectomy.">A standardized surgical technique for rat superior cervical ganglionectomy.</a> Journal of Neuroscience Methods. 192 (1), 22-33 (2010).</li><li>Garcia, J. B., Romeo, H. E., Basabe, J. C., Cardinali, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+superior+cervical+ganglionectomy+on+insulin+release+by+murine+pancreas+slices.">Effect of superior cervical ganglionectomy on insulin release by murine pancreas slices.</a> Journal of the Autonomic Nervous System. 22 (2), 159-165 (1988).</li><li>Ziegler, K. A., 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=Local+sympathetic+denervation+attenuates+myocardial+inflammation+and+improves+cardiac+function+after+myocardial+infarction+in+mice.">Local sympathetic denervation attenuates myocardial inflammation and improves cardiac function after myocardial infarction in mice.</a> Cardiovascular Research. 114 (2), 291-299 (2017).</li><li>Getsy, P. M., Coffee, G. A., Hsieh, Y. H., Lewis, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+superior+cervical+ganglia+modulate+ventilatory+responses+to+hypoxia+independently+of+preganglionic+drive+from+the+cervical+sympathetic+chain.">The superior cervical ganglia modulate ventilatory responses to hypoxia independently of preganglionic drive from the cervical sympathetic chain.</a> Journal of Applied Physiology. 131 (2), 836-857 (2021).</li><li>Dieguez, H. H., 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=Superior+cervical+gangliectomy+induces+non-exudative+age-related+macular+degeneration+in+mice.">Superior cervical gangliectomy induces non-exudative age-related macular degeneration in mice.</a> Disease Models &amp; Mechanisms. 11 (2), 031641 (2018).</li><li>Zhang, B., 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=Hyperactivation+of+sympathetic+nerves+drives+depletion+of+melanocyte+stem+cells.">Hyperactivation of sympathetic nerves drives depletion of melanocyte stem cells.</a> Nature. 577 (7792), 676-681 (2020).</li><li>Pirzgalska, R. 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=Sympathetic+neuron-associated+macrophages+contribute+to+obesity+by+importing+and+metabolizing+norepinephrine.">Sympathetic neuron-associated macrophages contribute to obesity by importing and metabolizing norepinephrine.</a> Nature Medicine. 23 (11), 1309-1318 (2017).</li><li>Kajimura, D., Paone, R., Mann, J. J., Karsenty, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foxo1+regulates+Dbh+expression+and+the+activity+of+the+sympathetic+nervous+system+in+vivo.">Foxo1 regulates Dbh expression and the activity of the sympathetic nervous system in vivo.</a> Molecular Metabolism. 3 (7), 770-777 (2014).</li></ol>

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>Anti-Tyrosine Hydroxylase Antibody</td>
			<td>EMD Millipore</td>
			<td>AB152</td>
			<td></td>
		</tr>
		<tr>
			<td>Artificial Tears Lubricant Ophthalmic Ointment</td>
			<td>Akorn</td>
			<td>59399-162-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Curity 2 x 2 Inch Gauze Sponge 8 Ply, Sterile</td>
			<td>Covidien</td>
			<td>1806</td>
			<td></td>
		</tr>
		<tr>
			<td>Derf Needle Holder</td>
			<td>Thomas Scientific</td>
			<td>1177K00</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5/45 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #7b Forceps</td>
			<td>Fine Science Tools</td>
			<td>11270-20</td>
			<td></td>
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			<td>ETHILON Nylon Suture</td>
			<td>Ethicon</td>
			<td>698H</td>
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			<td>Fine Scissors - ToughCut</td>
			<td>Fine Science Tools</td>
			<td>14058-09</td>
			<td></td>
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		<tr>
			<td>Hypoallergenic Surgical Tape</td>
			<td>3M Blenderm</td>
			<td>70200419342</td>
			<td></td>
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		<tr>
			<td>Induction Chamber, 2 Liter</td>
			<td>VetEquip</td>
			<td>941444</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter</td>
			<td>1001936060</td>
			<td></td>
		</tr>
		<tr>
			<td>Nair</td>
			<td>Church &#38; Dwight Co., Inc</td>
			<td>40002957</td>
			<td>chemical hair removing agent</td>
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			<td>NORADRENALINE RESEARCH ELISA</td>
			<td>Labor Diagnostika Nord (Rocky Mountain Diagnostics)</td>
			<td>BA E-5200</td>
			<td></td>
		</tr>
		<tr>
			<td>NSG Mouse</td>
			<td>Jackson Laboratory</td>
			<td>JAX:005557</td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone-Iodine Swabstick</td>
			<td>PDI</td>
			<td>S41350</td>
			<td></td>
		</tr>
		<tr>
			<td>Webcol Alcohol Preps</td>
			<td>Covidien</td>
			<td>5110</td>
			<td></td>
		</tr>
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</table>

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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Research

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Cancer Research

A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model

The intravenous injection of C1498 cells into syngeneic or congenic mice has been performed since 1941. These injections result in the development of acute leukemia. However, the nature of this disease has not been well documented in the literature. Here, we provide a technical protocol for characterizing C1498 cells in vitro and for determining the nature of the induced leukemia in vivo. The first part of this procedure is focused on determining the hematopoietic lineage and the stage of differentiation of cultured C1498 cells. To achieve this, multi-parametric flow cytometric staining is used to detect hematopoietic cell markers. Immunofluorescence microscopy, cytochemistry and a May-Gr&#252;nwald Giemsa staining are then performed to assess the expression of myeloperoxidase, the activity of esterases and cellular morphology, respectively. The second part of this protocol is dedicated to describing the leukemia disease that is induced in vivo. The latter can be achieved by determining the frequencies of leukemic and inherent cells in the blood, hematopoietic organs (e.g., bone marrow and spleen) and non-lymphoid tissues (e.g., the liver and lungs) using specific staining and flow cytometry analyses. The nature of the leukemia is then confirmed using May-Gr&#252;nwald Giemsa staining and staining for specific esterases in the bone marrow. Here, we present the results that were obtained using this protocol in age-matched C1498- and PBS-injected mice.

This manuscript provides a technical procedure that can be used to characterize C1498 cell cultures in vitro and the acute leukemia induced in mice after their injection. Phenotypic and functional analyses are performed using flow cytometry, immunofluorescence microscopy, cytochemistry and May-Gr&#252;nwald Giemsa staining.

The intravenous injection of C1498 cells into syngeneic or congenic mice has been performed since 1941. These injections result in the development of ...

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study end-points. Thus, studies focused on elucidating mechanisms of late-stage de novo metastasis are compromised, as are studies examining efficacy of anti-cancer therapies targeting mediators of metastasis in the adjuvant setting. Numerous murine mammary cancer models have been developed via targeted expression of dominant oncoproteins to mammary epithelial cells yielding models variably mimicking histopathologic and transcriptome-defined breast cancer subtypes common in women1. While much has been learned regarding the biology of mammary carcinogenesis with these models, their utility in identifying molecules regulating growth of late-stage metastasis are compromised as mice are typically euthanized at earlier time points due to significant primary tumor burden. Moreover, since a significant percentage of women diagnosed with breast cancer receive adjuvant therapy after surgical resection of primary tumors and prior to presence of detectable metastatic disease, preclinical models of de novo metastasis are urgently needed as platforms to evaluate new therapies aimed at targeting metastatic foci. To address these deficiencies, we developed a murine model of de novo mammary cancer metastasis, wherein primary mammary tumors are surgically resected, and metastatic foci subsequently develop over a 115 day post-surgical period. This long latency provides a tractable model to identify functionally significant regulators of metastatic progression in mice lacking primary tumor, as well as a model to evaluate preclinical therapeutic efficacy of agents aimed at blocking functionally significant molecules aiding metastatic tumor survival and growth.

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

Pre-clinical models evaluating adjuvant therapy targeting breast cancer metastasis are lacking. To address this, we developed a murine model of de novo pulmonary mammary adenocarcinoma metastasis, wherein therapies administered in the adjuvant setting (post surgical resection of primary tumors) can be evaluated for efficacy in impacting previously seeded pulmonary metastases.

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study ...

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified to secrete human Prostate Specific Antigen (PSA), and the procedure for the confirmation of tumor implantation. In brief, mice are anesthetized using injectable drugs and are made to lay in the dorsal position. Urine is vacated from the bladder and 50 &#181;L of poly-L-lysine (PLL) is slowly instilled at a rate of 10 &#181;L/20 s using a 24 G IV catheter. It is left in the bladder for 20 min by stoppering the catheter. The catheter is removed and PLL is vacated by gentle pressure on the bladder. This is followed by instillation of the murine bladder cancer cell line (1 x 105 cells/50 &#181;L) at a rate of 10 &#181;L/20 s. The catheter is stoppered to prevent premature evacuation. After 1 h, the mice are revived with a reversal drug, and the bladder is vacated. The slow instillation rate is important, as it reduces vesico-ureteral reflux, which can cause tumors to occur in the upper urinary tract and in the kidneys. The cell line should be well re-suspended to reduce clumping of cells, as this can lead to uneven tumor sizes after implantation.
This technique induces tumors with high efficiency. Tumor growth is monitored by urinary PSA secretion. PSA marker monitoring is more reliable than ultrasound or fluorescence imaging for the detection of the presence of tumors in the bladder. Tumors in mice generally reach a maximum size that negatively impacts health by about 3 - 4 weeks if left untreated. By monitoring tumor growth, it is possible to differentiate mice that were cured from those that were not successfully implanted with tumors. With only end-point analysis, the latter may be mistakenly assumed to have been cured by therapy.

This protocol describes the generation of murine orthotopic bladder tumors in female C57BL/6J mice and the monitoring of tumor growth.

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified ...

A Mouse Model of Fatigue Induced by Peripheral Irradiation

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.

We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

Cancer cells invade nerves through a process termed perineural invasion (PNI), in which cancer cells proliferate and migrate in the nerve microenvironment. This type of invasion is exhibited by a variety of cancer types, and very frequently is found in pancreatic cancer. The microscopic size of nerve fibers within mouse pancreas renders the study of PNI difficult in orthotopic murine models. Here, we describe a heterotopic in vivo model of PNI, where we inject syngeneic pancreatic cancer cell line Panc02-H7 into the murine sciatic nerve. In this model, sciatic nerves of anesthetized mice are exposed and injected with cancer cells. The cancer cells invade in the nerves proximally toward the spinal cord from the point of injection. The invaded sciatic nerves are then extracted and processed with OCT for frozen sectioning. H&amp;E and immunofluorescence staining of these sections allow quantification of both the degree of invasion and changes in protein expression. This model can be applied to a variety of studies on PNI given its versatility. Using mice with different genetic modifications and/or different types of cancer cells allows for investigation of the cellular and molecular mechanisms of PNI and for different cancer types. Furthermore, the effects of therapeutic agents on nerve invasion can be studied by applying treatment to these mice.

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

We describe an in vivo murine model of perineural invasion by injecting syngeneic pancreatic cancer cells into the sciatic nerve. The model allows for quantification of the extent of nerve invasion, and supports investigation of the cellular and molecular mechanisms of perineural invasion.

Cancer cells invade nerves through a process termed perineural invasion (PNI), in which cancer cells proliferate and migrate in the nerve ...

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 (BCS), a separate incision in the lower axilla hairline was made to find the sentinel nodes (SNs). In mastectomy, the SNs were identified through the same mastectomy incision. The SNs were described as blue nodes or nodes with lymphatic blue channels. An anatomical landmark in the axilla was used to facilitate SNs identification. The SNs metastases were evaluated by intraoperative frozen section analysis and histopathology examination as it is a gold standard. Here, we described the MBD as the lone technique in breast cancer sentinel node biopsy (SNB) which could be useful when radioisotope tracer or patent or isosulfan blue dye (PBD) cannot be provided.

In Indonesia, sentinel node biopsy (SNB) is not routinely performed for breast cancer surgery because of the limitation to provide radioisotope tracer and isosulfan or patent blue dye (PBD). To overcome this obstacle, we applied 1% methylene blue dye (MBD) as a single agent to map the sentinel nodes (SNs).

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 (PC). To address this deficiency, we describe a mouse model involving orthotopic implantation of PC followed by distal pancreatectomy and splenectomy. The model has been demonstrated to be safe and suitably flexible for the study of various therapeutic approaches in adjuvant and neo adjuvant settings.
In this model, a pancreatic tumor is first generated by implanting a mixture of human pancreatic cancer cells (luciferase-tagged AsPC-1) and human cancer associated pancreatic stellate cells into the distal pancreas of Balb/c athymic nude mice. After three weeks, the cancer is resected by re-laparotomy, distal pancreatectomy and splenectomy. In this model, bioluminescence imaging can be used to follow the progress of cancer development and effects of resection/treatments. Following resection, adjuvant therapy can be given. Alternatively, neoadjuvant treatment can be given prior to resection.
Representative data from 45 mice are presented. All mice underwent successful distal pancreatectomy/splenectomy with no issues of hemostasis. A macroscopic proximal pancreatic margin greater than 5 mm was achieved in 43 (96%) mice. The technical success rate of pancreatic resection was 100%, with 0% early mortality and morbidity. None of the animals died during the week after resection.
In summary, we describe a robust and reproducible technique for a surgical resection model of pancreatic cancer in mice which mimics the clinical scenario. The model may be useful for the testing of both adjuvant and neoadjuvant treatments.

In the clinical context, patients with localized pancreatic cancer will undergo pancreatectomy followed by adjuvant treatment. This protocol reported here aims to establish a safe and effective method of modelling this clinical scenario in nude mice, through orthotopic implantation of pancreatic cancer followed by distal pancreatectomy and splenectomy.

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 that cause LMD are breast and lung cancers and melanoma. Patients diagnosed with LMD have a very poor prognosis and generally survive for only a few weeks or months. One possible reason for the lack of efficacy of systemic therapy against LMD is the failure to achieve therapeutically effective concentrations of drug in the CSF because of an intact and relatively impermeable blood-brain barrier (BBB) or blood-CSF barrier across the choroid plexus. Therefore, directly administering drugs intrathecally or intraventricularly may overcome these barriers. This group has developed a model that allows for the effective delivery of therapeutics (i.e., drugs, antibodies, and cellular therapies) chronically and the repeated sampling of CSF to determine drug concentrations and target modulation in the CSF (when the tumor microenvironment is targeted in mice). The model is the murine equivalent of a magnetic resonance imaging-compatible Ommaya reservoir, which is used clinically. This model, which is affixed to the skull, has been designated as the &#34;Murine Ommaya.&#34; As a therapeutic proof of concept, human epidermal growth factor receptor 2 antibodies (clone 7.16.4) were delivered into the CSF via the Murine Ommaya to treat mice with LMD from human epidermal growth factor receptor 2-positive breast cancer. The Murine Ommaya increases the efficiency of drug delivery using a miniature access port and prevents the wastage of excess drug; it does not interfere with CSF sampling for molecular and immunological studies. The Murine Ommaya is useful for testing novel therapeutics in experimental models of LMD.

Here, we describe a murine xenograft model that functionally resembles an Ommaya reservoir in patients. We developed the Murine Ommaya to study novel therapeutics for the universally fatal 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

Malignant pancreatic tumors involving the celiac artery can be resected with a distal pancreatectomy, splenectomy and celiac axis resection (DP-CAR), relying on collateral flow to the liver through the gastroduodenal artery (GDA). In the current manuscript, the technical conduct of robotic DP-CAR is outlined. The greater curve of the stomach is mobilized with care to avoid sacrificing the gastroepiploic vessels. The stomach and liver are retracted cephalad to facilitate dissection of the porta hepatis. The hepatic artery (HA) is dissected and encircled with a vessel loop. The gastroduodenal artery (GDA) is carefully preserved. The common HA is clamped and triphasic flow in the proper HA via the GDA is confirmed using intra-operative ultrasound. A retropancreatic tunnel is made over the superior mesenteric vein (SMV). The pancreas is divided with an endovascular stapler at the neck. The inferior mesenteric vein (IMV) and splenic vein are ligated. The HA is stapled proximal to the GDA. The entire specimen is retracted laterally with further dissection cephalad to expose the superior mesenteric artery (SMA). The SMA is then traced back to the aorta. The dissection continues cephalad along the aorta with the bipolar energy device used to divide the crural fibers and celiac nerve plexus. The specimen is mobilized from the patient's right to left until the origin of the celiac axis is identified and oriented towards the left. The trunk is circumferentially dissected and stapled. Additional dissection with hook cautery and the bipolar energy device fully mobilizes the pancreatic tail and spleen. The specimen is removed from the left lower quadrant extraction site and one drain is left in the resection bed. A final intra-operative ultrasound of the proper HA confirms pulsatile, triphasic flow in the artery and liver parenchyma. The stomach is inspected for evidence of ischemia. Robotic DP-CAR is safe, feasible and when used in conjunction with multi-modality therapy, offers potential for long-term survival in selected patients.

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

We present our operative approach to robot assisted distal pancreatectomy, splenectomy, and celiac axis resection (DP-CAR), demonstrating that the procedure is safe and feasible with proper planning, patient selection, and surgeon experience.

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 imaging and acoustic energy modulation, this modality enables precise temperature control within a defined area. Many thermal applications are being explored with this noninvasive, nonionizing technology, such as hyperthermia generation, to release drugs from thermosensitive liposomal carriers. These drugs can include chemotherapies such as doxorubicin, for which targeted release is desired due to the dose-limiting systemic side effects, namely cardiotoxicity. Doxorubicin is a mainstay for treating a variety of malignant tumors and is commonly used in relapsed or recurrent rhabdomyosarcoma (RMS). RMS is the most common solid soft tissue extracranial tumor in children and young adults. Despite aggressive, multimodal therapy, RMS survival rates have remained the same for the past 30 years. To explore a solution for addressing this unmet need, an experimental protocol was developed to evaluate the release of thermosensitive liposomal doxorubicin (TLD) in an immunocompetent, syngeneic RMS mouse model using MRgHIFU as the source of hyperthermia for drug release.

Presented here is a protocol to use controlled hyperthermia, generated by magnetic resonance-guided high intensity focused ultrasound, to trigger drug release from temperature-sensitive liposomes in a rhabdomyosarcoma mouse model.

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