Cutane Chirurgische denervatie: een methode voor de Eis Testen op Zenuwen in muismodellen van huidziekte

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 neuropathies^1-4. In the skin, recent experimental evidence has suggested that sensory nerves can modulate diverse pathologies ranging from psoriasis to cancer^5-9. 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 function^7,10-12.

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Protocol

Alle in dit protocol beschreven procedures werden uitgevoerd in overeenstemming met de door de Universiteit van Michigan Unit Laboratory Animal Medicine regelgeving.

1. Laten genetische recombinatie in Muizen

Opmerking: De Gli1 ^{TM3 (CRE / ERT2) Alj} / J muizenstam (Gli1-CreERT2) ¹³ maakt de gerichtheid van tamoxifen geïnduceerde genetische recombinatie te epitheel TD. Steek deze stam met B6.129S4-Gt (ROSA) 26Sor ^tm1Sor / J reporter muizen (LacZ) ²² tot Gli1 te genereren; LacZ dieren TD cellen door whole-mount kleuring hieronder te ....

Results

Door het genereren van muizen die tamoxifen-induceerbare Gli1-CreERT2 en LacZ reporter allel, kan men TD epithelia visualiseren en volgen het lot van deze cellen in de tijd. De gehele denervatie procedure meestal binnen 1 uur per muis kan worden ingevuld en moet een minimale nood aan het dier te veroorzaken.

Onze vorige studies hebben aangetoond dat zenuwen cruciaal voor het handhaven van zowel normale TD en .......

Discussion

Zenuwen cruciale functies dienen niet alleen in gevoel, maar ook in zoogdier orgaanontwikkeling, onderhoud en regeneratie ^13,24-27. Zoals zenuwen zijn onlangs betrokken bij diverse huidaandoeningen, kan de hier beschreven technieken worden gebruikt om de vereiste innervatie bestuderen verschillende dierlijke ziektemodellen. Inderdaad, de eenzijdige denervatie techniek maakt de directe vergelijking van de huid met hetzij intacte of verstoord zenuwen van dezelfde muis. Dit is een ideale interne controle compens.......

Materials

List of materials used in this article

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 1:1,000 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