Name: Author Spotlight: Exploring Plasticity of Sympathetic Neurons
Uploaded: 2024-07-05T13:40:00
Description: Peripheral nerve injuries are common, and full functional recovery after injury is achieved in only 10% of patients. The sympathetic nervous system plays ...

This work was supported by the NIH National Institute of Neurological Disorders and Stroke under award number K01NS124912 and in part by a developmental grant from the NIH-funded Emory Specialized Center of Research Excellence in Sex Differences U54AG062334 and the Medical Scientist Training Program of Emory University School of Medicine. Thank you to David Kim, postbaccalaureate, for sectioning sciatic nerves and to HaoMin SiMa, research specialist, for 3D printing a phone mount for our stereo microscope that allowed for the filming of&#160;the video.

All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Emory University (under the IACUC protocol number PROTO201700371). Four adult female wild-type C57BL/6J mice, aged 14 weeks and weighing between 16-21 g, were used in this study. The details of the reagents and equipment used here are listed in the Table of Materials.
1. Presurgical preparation
<ol>
	<li>Autoclave the surgical tools: 1 pair of sharp scissors, 2 fine-t...

Targeting Mouse Sympathetic Neurons: A Lumbar Sympathectomy Approach

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O., Del Rey, A., Sorkin, E., Da Prada, M., Keller, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoregulation+mediated+by+the+sympathetic+nervous+system.">Immunoregulation mediated by the sympathetic nervous system.</a> Cell Immunol. 48 (2), 346-355 (1979).</li><li>Straka, 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=Postnatal+development+and+distribution+of+sympathetic+innervation+in+mouse+skeletal+muscle.">Postnatal development and distribution of sympathetic innervation in mouse skeletal muscle.</a> Int J Mol Sci. 19 (7), 1935 (2018).</li><li>Geng, 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=Pgc-1&#945;+plays+a+functional+role+in+exercise-induced+mitochondrial+biogenesis+and+angiogenesis+but+not+fiber-type+transformation+in+mouse+skeletal+muscle.">Pgc-1&#945; plays a functional role in exercise-induced mitochondrial biogenesis and angiogenesis but not fiber-type transformation in mouse skeletal muscle.</a> Am J Physiol Cell Physiol. 298 (3), C572-C579 (2010).</li><li>Lin, J., Handschin, C., Spiegelman, B. 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F., 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=Recovery+of+sympathetic+nerve+function+after+lumbar+sympathectomy+is+slower+in+the+hind+limbs+than+in+the+torso.">Recovery of sympathetic nerve function after lumbar sympathectomy is slower in the hind limbs than in the torso.</a> Neural Regen Res. 12 (7), 1177 (2017).</li><li>Holmberg, K., Shi, T. -. J. S., Albers, K. M., Davis, B. M., H&#246;kfelt, T. <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+peripheral+nerve+lesion+and+lumbar+sympathectomy+on+peptide+regulation+in+dorsal+root+ganglia+in+the+ngf-overexpressing+mouse.">Effect of peripheral nerve lesion and lumbar sympathectomy on peptide regulation in dorsal root ganglia in the ngf-overexpressing mouse.</a> Exp Neurol. 167 (2), 290-303 (2001).</li><li>Thoenen, H., Tranzer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+sympathectomy+by+selective+destruction+of+adrenergic+nerve+endings+with+6-hydroxydopamine.">Chemical sympathectomy by selective destruction of adrenergic nerve endings with 6-hydroxydopamine.</a> Naunyn Schmiedebergs Arch. Exp. Pathol. Pharmakol. 261, 271-288 (1968).</li><li>Thoenen, H., Tranzer, J. P., H&#228;usler, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chemical sympathectomy with 6-hydroxydopamine. New Aspects of Storage and Release Mechanisms of Catecholamines. , 130-143 (1970).</li><li>Xie, 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=Localized+sympathectomy+reduces+mechanical+hypersensitivity+by+restoring+normal+immune+homeostasis+in+rat+models+of+inflammatory+pain.">Localized sympathectomy reduces mechanical hypersensitivity by restoring normal immune homeostasis in rat models of inflammatory pain.</a> J Neuroscience. 36 (33), 8712-8725 (2016).</li><li>Zhu, X., Xie, W., Zhang, J., Strong, J. A., Zhang, J. -. 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M., Dintzis, S. M., Montine, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Comparative anatomy and histology: A mouse, rat, and human atlas. , 119 (2017).</li><li>Hweidi, S. A., Lee, S., Wolf, 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+sympathectomy+on+microvascular+anastomosis+in+the+rat.">Effect of sympathectomy on microvascular anastomosis in the rat.</a> Microsurgery. 6 (2), 9-96 (1985).</li><li>Navarro, X., Kennedy, W. 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The lumbar sympathetic ganglia are very small structures located behind many critical abdominal organs and large vessels. Therefore, this procedure requires significant precision and accuracy. Much of the difficulty lies in identifying the sympathetic ganglia intraoperatively. It is suggested that the learner first be able to identify the ganglia in a mouse cadaver prior to attempting this procedure in a live mouse. Troubleshooting will often need to occur when identifying the sympathetic ganglia after the diversion of t...

Peripheral nerve injuries (PNIs) can lead to motor, sensory, and sympathetic deficits in distal tissue targets that rarely fully functionally recover1. PNI research has often focused on the&#160;motor and sensory regeneration; however, nearly one-quarter of the rat sciatic nerve consists of unmyelinated sympathetic axons2.&#160;The role of sympathetic innervation in the peripheral tissues, nevertheless, is not fully understood3. The sympathetic nervous system plays a major role in maintaining bodily homeostasis, participating in immune regulation, thermoregulation, vascular tone, mitochondrial biogenesis, and more4,5,6,7,8,9,10,11. When sympathetic innervation at the neuromuscular junction is lost, persistent muscle weakness and synaptic instability are observed despite the maintenance of motoneuron innervation12. This sympathetic regulation of synaptic transmission at the neuromuscular junction has been shown to decline with aging13,14, which contributes to sarcopenia, defined as the age-dependent reduction in muscle mass, force, and power15. A better understanding of the role of sympathetic innervation of peripheral tissues is necessary for the development of therapies that will optimize functional outcomes for patients with PNIs and other forms of sympathetic dysfunction.
A sympathectomy is a powerful experimental tool that will allow for investigations of the role of sympathetic innervation in distal target tissues. Specifically, removal of the L2-L5 level sympathetic ganglia removes a majority of the sympathetic innervation to the lower limbs, which is especially useful for investigators interested in the sciatic nerve.
This protocol details the removal of L2-L5 level postganglionic sympathetic neurons from a mouse as a survival surgery. This procedure requires rodent microsurgical skills and familiarity with mouse anatomy, and when performed effectively, does not cause any visible phenotypic differences. A surgical lumbar sympathectomy has been used in rodent research, more so in rats than in mice16,17,18,19,20,21; however, a detailed protocol describing the protocol does not currently exist. Previous studies utilizing the lumbar sympathectomy have primarily focused on the role of sympathetic innervation in the pain response, which is generally attenuated by sympathectomy in various nerve injury models. Fewer studies have used this technique in mice22, likely due to the smaller size of anatomic landmarks, as the use of surgical sympathectomy was believed to be &#34;virtually not practicable&#34; in small animals23,24. Localized sympathectomies in the form of microsympathectomies have also been utilized in rodent models, also mostly in the context of pain behaviors25,26,27. The microsympathectomy, in contrast to the total lumbar sympathectomy, utilizes a dorsal approach through which a segment of the gray ramus to a specific spinal nerve is disconnected and removed, allowing for a very targeted sympathectomy that will avoid wider spread side effects.
Because mouse models are critical for many studies requiring genetic manipulation, this procedure will have versatile applications beyond the breadth of peripheral nerve injuries as well. Using a transabdominal approach, the lumbar sympathetic ganglia can be reliably visualized and resected from the mouse with no apparent adverse effects. Although protocols for the chemical destruction of postganglionic sympathetic neurons are available, such as the use of 6-hydroxydopamine (6-OHDA)23,24, this surgical procedure allows for anatomically specific targeting of the postganglionic lumbar sympathetic ganglia. The use of a surgical sympathectomy also avoids the nonspecific and dose-dependent concerns related to pharmacological methods28,29.
The use of chemical sympathectomies via administration of 6-OHDA was described in 1967 as a simple way to achieve selective destruction of adrenergic nerve endings since surgical sympathectomies in small animals were not favored23,24. 6-OHDA is a catecholaminergic neurotoxin that is endogenously formed in patients with Parkinson&#39;s disease, and its toxicity is derived from its ability to form free radicals and inhibit the electron transport chain in mitochondria30,31. Through norepinephrine uptake-1 transport mechanisms, 6-OHDA is able to accumulate within noradrenergic neurons, such as postganglionic sympathetic neurons28. Eventually, the neuron is destroyed by 6-OHDA; however, terminals in the peripheral nervous system do regenerate, with the restoration of functional activity even when the amine levels are still reduced. Different dosage thresholds are also present for different organs in response to 6-OHDA, and higher doses of 6-OHDA have been shown to exhibit more nonspecific effects, extending its neurotoxic consequences to non-catecholamine-containing neurons and even non-neuronal cells. Aside from noradrenergic neurons, dopaminergic neurons are affected by 6-OHDA as well29, making the chemical sympathectomy ultimately less specific to postganglionic sympathetic neurons than the surgical sympathectomy.
Therefore, a surgical lumbar sympathectomy enables the targeted ablation of the sympathetic innervation to the lower limbs, which can be combined with a variety of experimental techniques and genetic manipulations in the mouse to study how the sympathetic nervous system contributes to various injury and disease states.

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	<tbody>
		<tr>
			<td>5-0 absorable suture</td>
			<td>CP Medical</td>
			<td>421A</td>
			<td></td>
		</tr>
		<tr>
			<td>5-0 nylon suture</td>
			<td>Med-Vet International</td>
			<td>MV-661</td>
			<td></td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>E7023-4L</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia Induction Chamber</td>
			<td>Kent Scientific VetFlo</td>
			<td>VetFlo-0530XS</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia Vaporizer</td>
			<td>Kent Scientific VetFlo</td>
			<td>13-005-202</td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine</td>
			<td>HealthyPets</td>
			<td>BET16OZ</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J mice</td>
			<td>Jackson Laboratory</td>
			<td>#000664</td>
			<td></td>
		</tr>
		<tr>
			<td>Chicken anti-neurofilament-heavy</td>
			<td>Abcam</td>
			<td>ab72996</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM1850</td>
			<td></td>
		</tr>
		<tr>
			<td>Data Analysis Software</td>
			<td>Prism</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine-tipped tweezers</td>
			<td>World Precision Instruments</td>
			<td>500233</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent microscope</td>
			<td>Nikon</td>
			<td>Ti-E</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-chicken 488</td>
			<td>Invitrogen</td>
			<td>A32931</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit 647</td>
			<td>Invitrogen</td>
			<td>A21245</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Braintree Scientific</td>
			<td>39DP</td>
			<td></td>
		</tr>
		<tr>
			<td>Image Analysis Software</td>
			<td>Fiji</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging Software</td>
			<td>Nikon</td>
			<td>NIS-Elements</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Med-Vet International</td>
			<td>RXISO-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Meloxicam</td>
			<td>Med-Vet International</td>
			<td>RXMELOXIDYL32</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle driver</td>
			<td>Roboz Surgical Store</td>
			<td>RS-7894</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>Abcam</td>
			<td>ab7481</td>
			<td></td>
		</tr>
				<tr>
			<td>Ophthalmic ointment</td>
			<td>Refresh</td>
			<td>Refresh P.M.</td>
			<td></td>
		</tr>
		<tr>
			<td>Phox2bCre:tdTomato mutant mice</td>
			<td>Jackson Laboratory&#160;</td>
			<td>#016223, #007914</td>
			<td></td>
		</tr>
		<tr>
			<td>Pilocarpine hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>P6503</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-tyrosine hydroxylase</td>
			<td>Abcam</td>
			<td>ab112</td>
			<td></td>
		</tr>
		<tr>
			<td>Small straight scissors&#160;</td>
			<td>Fine Science Tools</td>
			<td>14084-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile cotton swabs 2x2</td>
			<td>Dynarex</td>
			<td>3252</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile cotton tipped applicators</td>
			<td>Dynarex</td>
			<td>4301</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile drape</td>
			<td>Med-Vet International</td>
			<td>DR4042</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile saline solution</td>
			<td>Med-Vet International</td>
			<td>1070988-BX</td>
			<td></td>
		</tr>
		<tr>
			<td>ThCre:mTmG mutant mice</td>
			<td>Mutant Mouse Resource and Research Centers</td>
			<td>strain #017262-UCD</td>
			<td>Jackson Laboratory, strain #007576</td>
		</tr>
		<tr>
			<td>ThCre:tdTomato mutant mice</td>
			<td>European Mouse Mutant Archive</td>
			<td>strain #00254</td>
			<td>Jackson Laboratory, strain #007914</td>
		</tr>
	</tbody>
</table>

surgical lumbar sympathectomy in mice

Peripheral nerve injuries are common, and full functional recovery after injury is achieved in only 10% of patients. The sympathetic nervous system plays many critical roles in maintaining bodily homeostasis, but it has rarely been studied in the context of peripheral nerve injury. The extent of postganglionic sympathetic neuronal functions in distal targets in the periphery is currently unclear. To better explore the role of sympathetic innervation of peripheral targets, a surgical &#34;knock-out&#34; model provides an alternative approach. Although this can be achieved chemically, chemical destruction of postganglionic sympathetic neurons can be nonspecific and dose-dependent. The use of a surgical lumbar sympathectomy in mice, once thought to be &#34;virtually not practicable&#34; in small animals, allows for specific targeting of postganglionic sympathetic neurons that innervate the hind limbs. This manuscript describes how to surgically remove the L2-L5 lumbar sympathetic ganglia from a mouse as a survival surgery, which reliably decreases the hind paw sweat response and the number of sympathetic axons in the sciatic nerve.

Author Spotlight: Exploring Plasticity of Sympathetic Neurons

Surgical Lumbar Sympathectomy in Mice

Our research is focused on the plasticity of sympathetic neurons. Some of the questions that we're interested in include how postganglionic sympathetic axons regenerate compared to other neuron types, as well as how sympathetic innervation regulates the function of distal tissues that they innervate. The sympathetic nervous system is well known to be responsible for a wide range of homeostatic mechanisms.More recently, it was discovered that the sympathetic nervous system is also important for maintaining the integrity and function of neuromuscular junctions. This is a fundamental finding that requires a re-imagining of the classic nerve-muscle interface. We've established that postganglionic lumbar sympathetic neurons do not regenerate in response to classic nerve regeneration interventions such as electrical stimulation and conditioning lesions.There's something innately different about this class of neurons compared to motor and sensory neurons. Using a surgical lumbar sympathectomy allows for anatomically-specific ablation of the postganglionic sympathetic system that enervates the lower limbs. Although chemical sympathectomies are possible, using a surgical method limits off-target effects as well as the potential for regeneration.Mainstream use of the surgical lumbar sympathectomy has allowed for investigations into the role of sympathetic innervation in exercise-induced metabolic changes in muscle. This protocol can also be adapted to extract these ganglia for neuronal cell culture, as well as for retrograde tracing experiments in postmortem animals.

This protocol describes the surgical removal of postganglionic lumbar sympathetic neurons from a mouse. Two mice received lumbar sympathectomies, and two mice served as controls. To achieve a successful surgical lumbar sympathectomy, adequate visualization of at least the L2 and L3 bilateral lumbar sympathetic ganglia must be achieved, as seen in Figure 1. Removal of the L4 and L5 ganglia would achieve complete sympathetic denervation of the lower body; however, visualization of the lower ga...

This manuscript presents a protocol for surgically removing the postganglionic lumbar sympathetic neurons from a mouse. This procedure will facilitate a multitude of studies aimed at investigating the role of sympathetic innervation in distal tissue targets.

Peripheral nerve injuries are common, and full functional recovery after injury is achieved in only 10% of patients. The sympathetic nervous system plays ...

Research

JoVE Journal

Neuroscience

Surgical Disruption of Sympathetic Neurons in Mice for Peripheral Nerve Study

To begin, position the anesthetized mouse on the surgical table. Cover the mouse with a sterile surgical drape, having a central hole. Using a pair of sharp scissors and fine tipped tweezers, create a midline incision from approximately one millimeter above the level of the pubic synthesis to approximately two millimeters below the ribs.Identify the midline fascia between the bilateral rectus abdominis muscles. Using the tweezers, lift the abdominal muscles away from the underlying organs, and cut along the linea alba to enter the abdominal cavity. Using 5/0 sutures, retract the abdominal muscles and skin laterally.Lay a sterile cotton square, saturated in sterile saline on the drape lying superiorly and to the left of the incision. With two sterile cotton tipped applicators, push the colon and small intestine partially out onto the saline soaked cotton square, ensuring that the cecum and appendix are out of the abdominal cavity. Cover the exposed intestines with another saline saturated cotton square.Employing closed tweezers, deflect the descending colon to the left to reveal the abdominal aorta and inferior vena cava. Next with a pair of tweezers, lift the abdominal vessels by surrounding connective tissue and carefully place a 5/0 nylon suture through the connective tissue. After suture insertion, deflect the abdominal vessels to the left.Using tweezers bluntly dissect any overlying fascia to visualize a triangle, with two sides consisting of the deflected abdominal vessels and the third side defined by the mouse's midline. Next, bluntly dissect any overlying fascia to reveal the ganglia. Within this triangle, visualize a quadrangle bordered by the left renal artery superiorly, the animal's midline and abdominal vessels laterally, and the left testicular or ovarian artery inferiorly.Carefully blunt dissect with tweezers to enter this quadrangle and identify the bilateral L2 ganglia. Then using a pair of tweezers, grab the inferior aspect of the visible bilateral ganglia and pull upwards. After achieving the required hemostasis, remove the suture holding the abdominal vessels in place.Using two sterile cotton tipped applicators, carefully replace diverted intestines into the abdominal cavity. With a 5/0 absorbable suture, perform a running stitch to approximate the abdominal muscles. Finally, using 5/0 nylon suture, close the skin with simple interrupted stitches.Upon removal of the ganglia, the sweating response due to pilocarpine decreased significantly by postoperative day seven. Tyrosine hydroxylase staining indicated that surgical lumbar sympathectomy reduced the number of sympathetic axons in the sciatic nerve by postoperative day 21, whereas the neurofilament heavy chain density was largely unaffected.