皮膚外科神経切除：皮膚病のマウスモデルにおける神経のための要件をテストする方法
Summary
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.
Abstract
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.
Introduction
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.
Protocol
Representative Results
Discussion
神経は感覚ではなく、哺乳動物の臓器の開発、保守及び再生13,24-27だけでなく重要な機能を果たします。神経が最近多様な皮膚疾患に関与しているように、ここで説明される技術は、動物疾患モデルの様々な神経支配の要件を研究するために使用することができます。確かに、一方的除神経の技術は、同じマウスから無傷または破壊神経のいずれかで皮膚の直接比較を可能にします。…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| 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 |
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