JoVE Logo
Faculty Resource Center

Sign In





Representative Results






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

Published: June 26th, 2016



1Dermatology, Cell and Developmental Biology, University of Michigan, 2Dermatology Branch, National Cancer Institute, National Institutes of Health
* These authors contributed equally

This article includes detailed protocols for genetic labeling of mouse skin, surgical denervation, skin biopsy and visualizing labeled epithelia by whole-mount β-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 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.

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.


Log in or to access full content. Learn more about your institution’s access to JoVE content here

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 (<.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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 M....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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

  1. Magnon, C., et al. Autonomic nerve development contributes to prostate cancer progression. Science. 341, 1236361 (2013).
  2. Zhao, C. M., et al. Denervation suppresses gastric tumorigenesis. Sci Transl Med. 6, 115 (2014).
  3. Chiu, I. M., von Hehn, C. A., Woolf, C. J. Neurogenic inflammation and the peripheral nervous system in host defense and immunopathology. Nat Neurosci. 15, 1063-1067 (2012).
  4. Gautron, L., Elmquist, J. K., Williams, K. W. Neural control of energy balance: translating circuits to therapies. Cell. 161, 133-145 (2015).
  5. Peterson, S. C., et al. Basal cell carcinoma preferentially arises from stem cells within the hair follicle and mechanosensory niches. Cell Stem Cell. 16, 400-412 (2015).
  6. Ostrowski, S. M., Belkadi, A., Loyd, C. M., Diaconu, D., Ward, N. L. Cutaneous denervation of psoriasiform mouse skin improves acanthosis and inflammation in a sensory neuropeptide-dependent manner. J Invest Dermatol. 131, 1530-1538 (2011).
  7. Ward, N. L., et al. Botulinum neurotoxin A decreases infiltrating cutaneous lymphocytes and improves acanthosis in the KC-Tie2 mouse model. J Invest Dermatol. 132, 1927-1930 (2012).
  8. Roggenkamp, D., et al. Epidermal nerve fibers modulate keratinocyte growth via neuropeptide signaling in an innervated skin model. J Invest Dermatol. 133, 1620-1628 (2013).
  9. Riol-Blanco, L., et al. Nociceptive sensory neurons drive interleukin-23-mediated psoriasiform skin inflammation. Nature. 510, 157-161 (2014).
  10. Zanchi, M., et al. Botulinum toxin type-A for the treatment of inverse psoriasis. J Eur Acad Dermatol Venereol. 22, 431-436 (2008).
  11. Farber, E. M., Lanigan, S. W., Rein, G. The role of psychoneuroimmunology in the pathogenesis of psoriasis. Cutis. 46, 314-316 (1990).
  12. Dewing, S. B. Remission of psoriasis associated with cutaneous nerve section. Arch Dermatol. 104, 220-221 (1971).
  13. Brownell, I., Guevara, E., Bai, C. B., Loomis, C. A., Joyner, A. L. Nerve-derived sonic hedgehog defines a niche for hair follicle stem cells capable of becoming epidermal stem cells. Cell Stem Cell. 8, 552-565 (2011).
  14. Liao, X. H., Nguyen, H. Epidermal expression of Lgr6 is dependent on nerve endings and Schwann cells. Exp Dermatol. 23, 195-198 (2014).
  15. Lumpkin, E. A., Marshall, K. L., Nelson, A. M. The cell biology of touch. J Cell Biol. 191, 237-248 (2010).
  16. Maricich, S. M., et al. Merkel cells are essential for light-touch responses. Science. 324, 1580-1582 (2009).
  17. Reinisch, C. M., Tschachler, E. The touch dome in human skin is supplied by different types of nerve fibers. Ann Neurol. 58, 88-95 (2005).
  18. Doucet, Y. S., Woo, S. H., Ruiz, M. E., Owens, D. M. The touch dome defines an epidermal niche specialized for mechanosensory signaling. Cell Reports. 3, 1759-1765 (2013).
  19. Xiao, Y., et al. Neural Hedgehog signaling maintains stem cell renewal in the sensory touch dome epithelium. Proc Natl Acad Sci USA. 112, 7195-7200 (2015).
  20. English, K. B., Van Norman, D. K. -. V., Horch, K. Effects of chronic denervation in type I cutaneous mechanoreceptors (Haarscheiben). Anat Rec. 207, 79-88 (1983).
  21. Burgess, P. R., English, K. B., Horch, K. W., Stensaas, L. J. Patterning in the regeneration of type I cutaneous receptors. J Physiol. 236, 57-82 (1974).
  22. Soriano, P. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet. 21, 70-71 (1999).
  23. Wright, M. C., et al. Atoh1+ progenitors maintain the Merkel cell population in embryonic and adult mice. J Cell Biol. 208, 367-379 (2015).
  24. Kumar, A., Brockes, J. P. Nerve dependence in tissue, organ, and appendage regeneration. Trends Neurosci. 35, 691-699 (2012).
  25. Rinkevich, Y., et al. Clonal analysis reveals nerve-dependent and independent roles on mammalian hind limb tissue maintenance and regeneration. Proc Natl Acad Sci USA. 111, 9846-9851 (2014).
  26. Ueno, H., et al. Dependence of corneal stem/progenitor cells on ocular surface innervation. Invest Ophthalmol Vis Sci. 53, 867-872 (2012).
  27. Westphalen, C. B., et al. Long-lived intestinal tuft cells serve as colon cancer-initiating cells. J Clin Invest. 124, 1283-1295 (2014).
  28. Uhmann, A., et al. The Hedgehog receptor Patched controls lymphoid lineage commitment. Blood. 110, 1814-1823 (2007).
  29. Lau, J., et al. Temporal control of gene deletion in sensory ganglia using a tamoxifen-inducible Advillin-Cre-ERT2 recombinase mouse. Mol Pain. 7, 100 (2011).

This article has been published

Video Coming Soon

JoVE Logo


Terms of Use





Copyright © 2024 MyJoVE Corporation. All rights reserved