The authors would like to thank Autumn Peterson for assistance with mouse photography, Daniel Thoresen for assistance with mice, and Drs. Nicole Ward and Abdelmadjid Belkadi for assistance with surgical denervation. These studies were supported by funding from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (grants R00AR059796 and R01AR065409); the University of Michigan Department of Dermatology; the Biological Sciences Scholars Program; the Center for Organogenesis; the University of Michigan Comprehensive Cancer Center; and the John S. and Suzanne C. Munn Cancer Fund. S.C.P. was supported by funding from the National Institute of General Medical Sciences (grant T32 GM007315). This work was also supported by the NIH Intramural Research Program, Center for Cancer Research, National Cancer Institute.

All procedures described in this protocol were performed in accordance with regulations established by the University of Michigan Unit for Laboratory Animal Medicine.
1. Induce Genetic Recombination in Mice
Note: The Gli1tm3(cre/ERT2)Alj/J mouse strain (Gli1-CreERT2)13 enables targeting of tamoxifen-induced genetic recombination to TD epithelia. Cross this strain with B6.129S4-Gt(ROSA)26Sortm1Sor/J reporter mice (<...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenic+inflammation+and+the+peripheral+nervous+system+in+host+defense+and+immunopathology.">Neurogenic inflammation and the peripheral nervous system in host defense and immunopathology.</a> Nat Neurosci. 15, 1063-1067 (2012).</li><li>Gautron, L., Elmquist, J. K., Williams, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+control+of+energy+balance:+translating+circuits+to+therapies.">Neural control of energy balance: translating circuits to therapies.</a> Cell. 161, 133-145 (2015).</li><li>Peterson, S. 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M., Lanigan, S. W., Rein, G. <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+psychoneuroimmunology+in+the+pathogenesis+of+psoriasis.">The role of psychoneuroimmunology in the pathogenesis of psoriasis.</a> Cutis. 46, 314-316 (1990).</li><li>Dewing, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remission+of+psoriasis+associated+with+cutaneous+nerve+section.">Remission of psoriasis associated with cutaneous nerve section.</a> Arch Dermatol. 104, 220-221 (1971).</li><li>Brownell, I., Guevara, E., Bai, C. B., Loomis, C. A., Joyner, A. 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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+touch+dome+defines+an+epidermal+niche+specialized+for+mechanosensory+signaling.">The touch dome defines an epidermal niche specialized for mechanosensory signaling.</a> Cell Reports. 3, 1759-1765 (2013).</li><li>Xiao, Y., 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=Neural+Hedgehog+signaling+maintains+stem+cell+renewal+in+the+sensory+touch+dome+epithelium.">Neural Hedgehog signaling maintains stem cell renewal in the sensory touch dome epithelium.</a> Proc Natl Acad Sci USA. 112, 7195-7200 (2015).</li><li>English, K. B., Van Norman, D. K. -. V., Horch, K. <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+chronic+denervation+in+type+I+cutaneous+mechanoreceptors+(Haarscheiben).">Effects of chronic denervation in type I cutaneous mechanoreceptors (Haarscheiben).</a> Anat Rec. 207, 79-88 (1983).</li><li>Burgess, P. R., English, K. B., Horch, K. W., Stensaas, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+in+the+regeneration+of+type+I+cutaneous+receptors.">Patterning in the regeneration of type I cutaneous receptors.</a> J Physiol. 236, 57-82 (1974).</li><li>Soriano, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generalized+lacZ+expression+with+the+ROSA26+Cre+reporter+strain.">Generalized lacZ expression with the ROSA26 Cre reporter strain.</a> Nat Genet. 21, 70-71 (1999).</li><li>Wright, M. 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=Atoh1++progenitors+maintain+the+Merkel+cell+population+in+embryonic+and+adult+mice.">Atoh1+ progenitors maintain the Merkel cell population in embryonic and adult mice.</a> J Cell Biol. 208, 367-379 (2015).</li><li>Kumar, A., Brockes, J. 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Nerves serve crucial functions not only in sensation, but also in mammalian organ development, maintenance and regeneration13,24-27. As nerves have recently been implicated in diverse skin disorders, the techniques described here can be used to study the requirement for innervation in a variety of animal disease models. Indeed, the unilateral denervation technique allows for the direct comparison of skin with either intact or disrupted nerves from the same mouse. This provides an ideal internal control to comp...

Over the past few years, there has been a widening appreciation for the influence of nerves on diseases not typically regarded as classical neuropathies1-4. In the skin, recent experimental evidence has suggested that sensory nerves can modulate diverse pathologies ranging from psoriasis to cancer5-9. This has been demonstrated using techniques such as surgical denervation and pharmacological inhibition of neural function in rodents. In the case of psoriasis, these studies have provided a mechanistic framework for understanding why human psoriatic plaques regress following loss of neural function7,10-12.
Cutaneous nerves can also affect gene expression13,14 and are critical for mechanosensing in normal skin15. In particular, touch dome (TD) epithelia are comprised of a patch of columnar epidermal cells in juxtaposition with neuroendocrine Merkel cells innervated by slowly adapting type 1 (SA1) nerve fibers16-18. TDs mediate light touch sensation and have been shown to display Hedgehog pathway activity5,19. TD maintenance depends on innervation20,21, as nerves secrete Hedgehog ligands to sustain normal TDs and their associated Merkel cells19. In addition, innervation promotes Hedgehog-dependent tumor formation from TD epithelia5. Together, these studies reinforce the notion that intricate molecular interactions occurring between nerves and the surrounding cells in their niche are crucial for normal TD physiology as well as disease.
To interrogate the nature of these interactions, we describe here a series of in vivo techniques for manipulating gene expression in the TD, as well as for harvesting skin biopsies for TD visualization after lineage tracing. Finally, we describe procedures for performing unilateral surgical denervation, wherein nerves are removed from one side of the mouse dorsal skin, while leaving the contralateral side intact as a sham internal control. Several weeks after surgery, denervated and sham control skin are compared to assess changes that occur when nerves are ablated. Although these techniques are described in the context of normal TDs, the denervation procedure has been used to examine the requirement for nerves in mouse models of psoriasis6, wound healing13 and tumorigenesis5. Finally, since the skin is amenable to repeated biopsies, this provides an opportunity to monitor the long-term fates of labeled cells or to assess disease progression over multiple time points.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alcohol prep pads</td>
			<td>PDI</td>
			<td>B339</td>
			<td></td>
		</tr>
		<tr>
			<td>AnaSed (Xylazine)</td>
			<td>Lloyd</td>
			<td>NADA 139-236</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody, anti-Keratin 8</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>TROMA-I</td>
			<td>rat antibody, use at 1:500 concentration</td>
		</tr>
		<tr>
			<td>Antibody, anti-Keratin 17</td>
			<td>Cell Signaling</td>
			<td>#4543</td>
			<td>rabbit antibody, use at 1:1,000 concentration</td>
		</tr>
		<tr>
			<td>Antibody, anti-Neurofilament</td>
			<td>Cell Signaling</td>
			<td>C28E10</td>
			<td>rabbit antibody, use at 1:500 concentration</td>
		</tr>
		<tr>
			<td>Betadine prep pads</td>
			<td>Medline</td>
			<td>MDS093917</td>
			<td></td>
		</tr>
		<tr>
			<td>Carprofen (Rimadyl)</td>
			<td>Zoetis</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cordless rechargable clipper</td>
			<td>Wahl</td>
			<td>trimmer model 8900</td>
			<td></td>
		</tr>
		<tr>
			<td>Corn Oil</td>
			<td>Sigma-Aldrich</td>
			<td>C8267</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM1860</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>ThermoFisher Scientific</td>
			<td>D1306</td>
			<td>use at 1:1000 concentration</td>
		</tr>
		<tr>
			<td>Deoxycholate</td>
			<td>Sigma-Aldrich</td>
			<td>D6750</td>
			<td></td>
		</tr>
		<tr>
			<td>Depilatory Cream</td>
			<td>Nair</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethylforamide</td>
			<td>Sigma-Aldrich</td>
			<td>319937</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>G5882</td>
			<td></td>
		</tr>
		<tr>
			<td>ImmEdge Pen</td>
			<td>Vector Laboratories</td>
			<td>H-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine HCl</td>
			<td>Hospira</td>
			<td>NDC 0409-2051-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro cover glass</td>
			<td>VWR</td>
			<td>48404-454</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Slides</td>
			<td>VWR</td>
			<td>48311-703</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Neutral Buffered Formalin</td>
			<td>VWR</td>
			<td>BDH0502-4LP</td>
			<td></td>
		</tr>
		<tr>
			<td>6-0 nylon sutures</td>
			<td>DemeTECH</td>
			<td>NL166012F4P</td>
			<td></td>
		</tr>
		<tr>
			<td>Octylphenyl-polyethylene glycol</td>
			<td>Sigma-Aldrich</td>
			<td>I8896</td>
			<td></td>
		</tr>
		<tr>
			<td>O.C.T. Compound</td>
			<td>Sakura Tissue-Tek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>Pottasium ferrocyanide</td>
			<td>Sigma-Aldrich</td>
			<td>P9387</td>
			<td></td>
		</tr>
		<tr>
			<td>Pottasium ferricyanide</td>
			<td>Sigma-Aldrich</td>
			<td>702587</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>P9791</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>Sigma-Aldrich</td>
			<td>S5136</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>84097</td>
			<td></td>
		</tr>
		<tr>
			<td>Tamoxifen</td>
			<td>Sigma-Aldrich</td>
			<td>T5648-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra fine forceps</td>
			<td>Dumont</td>
			<td>0103-5-PO</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield</td>
			<td>Vector Laboratories</td>
			<td>H1000</td>
			<td></td>
		</tr>
		<tr>
			<td>X-gal</td>
			<td>Roche</td>
			<td>10 651 745 001</td>
			<td>Disolve in dimethylforamide to create 50x stock prior to use</td>
		</tr>
	</tbody>
</table>

cutaneous surgical denervation:  a method for testing the requirement for nerves in mouse models of skin disease

Cutaneous somatosensory nerves function to detect diverse stimuli that act upon the skin. In addition to their established sensory roles, recent studies have suggested that nerves may also modulate skin disorders including atopic dermatitis, psoriasis and cancer. Here, we describe protocols for testing the requirement for nerves in maintaining a cutaneous mechanosensory organ, the touch dome (TD). Specifically, we discuss methods for genetically labeling, harvesting and visualizing TDs by whole-mount staining, and for performing unilateral surgical denervation on mouse dorsal back skin. Together, these approaches can be used to directly compare TD morphology and gene expression in denervated as well as sham-operated skin from the same animal. These methods can also be readily adapted to examine the requirement for nerves in mouse models of skin pathology. Finally, the ability to repeatedly sample the skin provides an opportunity to monitor disease progression at different stages and times after initiation.

By generating mice expressing tamoxifen-inducible Gli1-CreERT2 and a LacZ reporter allele, it is possible to visualize TD epithelia and track the fates of these cells over time. The entire denervation procedure typically can be completed within 1 hr per mouse and should cause minimal distress to the animal.
Our previous studies have indicated that nerves are crucial for maintaining both normal TDs as well as th...

Watch this Scientific Journal Video about Cutaneous Surgical Denervation:  A Method for Testing the Requirement for Nerves in Mouse Models of Skin Disease at JoVE.com

Cutaneous Surgical Denervation:  A Method for Testing the Requirement for Nerves in Mouse Models of Skin Disease

This article includes detailed protocols for genetic labeling of mouse skin, surgical denervation, skin biopsy and visualizing labeled epithelia by whole-mount &#946;-galactosidase staining. These methods can be used to test the requirement for nerves in mouse models of normal and pathological skin.

Cutaneous somatosensory nerves function to detect diverse stimuli that act upon the skin. In addition to their established sensory roles, recent studies ...

The overall goal of this procedure is to examine the influence of nerves on normal and pathological skin. This method can be used to answer key questions in normal skin biology, such as whether cutaneous nerves affect homeostasis and disease. The main advantage of this technique is that you can compare intact and denervated skin samples from the same animal.Visual demonstration of this technique is critical as nerves can be difficult to recognize and remove with minimal damage to the surrounding tissues. First, anesthetize the mouse and check that it has reached the proper plane of sedation using a toe pinch. Also, confirm that the mouse exhibits normal breathing and heartbeat.Now set the animal on a warming pad in an aseptic surgical area. Using an electric clipper, carefully remove the hair from the dorsal side where the biopsy will be taken. Then wipe the shaved area using Betadyne and alcohol wipes.Ensure that all the hair clippings are removed from the site. Now outline the biopsy site using a black marker. To obtain longitudinal sections of hair follicles, the longer edge of the biopsy should run in an anterior to posterior direction parasetidal to the dorsal midline.Using a scalpel, make a full thickness excision without damaging the underlying muscular fascia. Bleeding is typically minimal. The biopsy sample should include the epidermis, dermis, subcutaneous fat, and panniculus carnosus.Flatten the excised skin sample on a dry paper towel, dermis side down. Trim away the excess paper towel, and store the sample on cold TBS for up to an hour if other samples need to be collected. Returning to the mouse, suture the biopsy site using 60 nylon sutures about three millimeters apart.Then monitor the mouse in a recovery cage until it regains consciousness. Use analgesics in accordance with designated institutional animal care, and follow their guidelines if the mouse appears distressed. Within seven to 10 days of surgery, remove the sutures.Proceed with processing the biopsies for histology or whole-mount staining. Anesthetize the animal, shave the entire dorsal skin, and clean the area similar to the biopsy procedure. Now make an incision using a sterile scalpel along the dorsal midline from the base of the neck to roughly half a centimeter above the tail.Using sterile blunt forceps, gently retract the skin on the left side away from the flank to visualize the underlying tissue from the scapular fat pads near the neck to just above the hind limb. Now identify the dorsal cutaneous nerve under a dissecting light microscope. These appear as white strands that travel caudally through the translucent fascia of the trunk wall before making sharp bends and entering the loose connective tissue underneath the skin.Next, using ultrafine forceps, remove the nerve exclusively from the left side of the animal located at anatomical sites T3 to T12 by plucking them from where their segments bend at the trunk wall to their entry sites into the skin. Orient the forceps vertically and grasp the nerves about half a centimeter below their bend sites. Once grabbed, pull upwards to cause the nerves to stretch and separate from the surrounding tissue to remove them.Avoid damaging the adjacent blood vessels. Be sure to keep the tissue moist throughout the procedure by periodically applying drops of 0.9%still saline solution. Continue removing all nerves extending from the trunk wall to the skin, but do not disrupt the nerves within the dense fascia of the trunk wall.Next, remove any nerves from the exposed skin flap. These fibers comprise the distal branches of the dorsal cutaneous nerve and appear as white branching strands located sporadically within the connective tissue on the dermal side of the skin flap. To remove these fine branches, position the forceps roughly parallel to the dermal surface, grasp the nerve and pluck upwards.Remove all the visible nerves in this fashion. Do not puncture the blood vessels and skin. During this step, it's important to avoid rupturing neighboring blood vessels.If you find a nerve with an adjacent blood vessel, follow it eventually to an area where there is no adjacent vessel and remove it by plucking. Now, loosen the skin on the right side of the dorsal midline incision, but do not remove any nerves. This will serve as the contralateral sham operative control.Finally, suture up the animal and monitor post-operatively as before. Later, to confirm the stable loss of nerves, weeks after surgery, gently prick the denervated area using a sterile hypodermic needle. Note whether the animal responds, typically by shuddering or turning its head.If the skin area has been stably denervated, the animal will exhibit little or no response. Harvest skin biopsies from the area of no response and the contralateral sham side for matched experimental and control samples. From the biopsies, it is important to sample multiple histological sections to identify potential differences in touch dome abundance or morphology between the sham and denervated specimens.The glebe one cree ERT2 mouse strain allows targeting of tamoxifen-induced genetic recombination to areas of the skin that display high hedgehog pathway activity such as touch dome epithelia. These mice were crossed to induce abolaxi reporter mice to visualize the touch dome epithelia. Nodes are crucial for maintaining both normal touch domes as well as their associated Merkel cells.This is demonstrated experimentally by the gradual loss of normal touch dome morphology as well as loss of keratin eight deposited cells following denervation. Nerves are also crucial for promoting hedgehog signaling in the touch dome as monitored by Glebe one cree ERE2 expressions, and home-mount beta collective sided staining from both sham and denervated skin. After watching this video, you should have a good understanding of how to surgically ablate dorsal cutaneous nerves in order to test whether these nerves are involved in normal skin function or if they modulate disease.Once mastered, this technique can be performed routinely and reliably with minimal stress to the animal. Following this procedure, methods such as amino fluorescent staining can be used to confirm whether nerves are fully ablated from the skin, and whether gene expression is being affected.

Research

JoVE Journal

Medicine

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&beta; using a Whole Blood Stimulation Assay

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&#946; using a Whole Blood Stimulation Assay

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues ...

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis (ALS)

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis ALS

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 ...

A Simple Method of Mouse Lung Intubation

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. ...

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

A Mouse Fetal Skin Model of Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

Surgical Models of Gastroesophageal Reflux with Mice

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to ...

Inducing Ischemia-reperfusion Injury in the Mouse Ear Skin for Intravital Multiphoton Imaging of Immune Responses

Ischemia-reperfusion injury (IRI) occurs when there is transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent ...

Repeated Measurement of Respiratory Muscle Activity and Ventilation in Mouse Models of Neuromuscular Disease

Accessory respiratory muscles help to maintain ventilation when diaphragm function is impaired. The following protocol describes a method for repeated ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...