Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Presented here is a protocol for the separation of epidermis from dermis to evaluate inflammatory mediator production. Following inflammation, rat hind paw epidermis is separated from the dermis by thermolysin at 4 °C. The epidermis is then used for mRNA analysis by RT-PCR and protein evaluation by western blot and immunohistochemistry.

Abstract

Easy-to-use and inexpensive techniques are needed to determine the site-specific production of inflammatory mediators and neurotrophins during skin injury, inflammation, and/or sensitization. The goal of this study is to describe an epidermal-dermal separation protocol using thermolysin, a proteinase that is active at 4 °C. To illustrate this procedure, Sprague Dawley rats are anesthetized, and right hind paws are injected with carrageenan. Six and twelve hours after injection, rats with inflammation and naïve rats are euthanized, and a piece of hind paw, glabrous skin is placed in cold Dulbecco's Modified Eagle Medium. The epidermis is then separated at the basement membrane from the dermis by thermolysin in PBS with calcium chloride. Next, the dermis is secured by microdissection forceps, and the epidermis is gently teased away. Toluidine blue staining of tissue sections show that the epidermis is separated cleanly from the dermis at the basement membrane. All keratinocyte cell layers remain intact, and the epidermal rete ridges along with indentations from dermal papillae are clearly observed. Qualitative and real-time RT-PCR is used to determine nerve growth factor and interleukin-6 expression levels. Western blotting and immunohistochemistry are finally performed to detect amounts of nerve growth factor. This report illustrates that cold thermolysin digestion is an effective method to separate epidermis from dermis for evaluation of mRNA and protein alterations during inflammation.

Introduction

Evaluation of inflammatory mediators and neurotrophic factors from the skin can be limited due to the heterogeneity of cell types found in the inflamed dermis and epidermis1,2,3. Several enzymes, chemical, thermal, or mechanical techniques involving separation of the two layers or for performing cell dissociation for evaluation have been reviewed recently4. Acid, alkali, neutral salt, and heat can divide the epidermis from dermis quickly, but cellular and extracellular swelling often occurs5,6.....

Protocol

This protocol follows the animal care guidelines of Oklahoma State University Center for Health Sciences IACUC (#2016-03).

1. Carrageenan-induced inflammation (C-II)

  1. Anesthetize male and/or female Sprague Dawley rats (200–250 g; 8–9 weeks old) with isoflurane (or injectable anesthetic).
  2. Check the depth of anesthesia by touching the cornea and lightly pinching the left hind paw. When the animal is appropriately anesthetized, no corneal or paw response will be o.......

Representative Results

Carrageenan injection into the rat hind paw caused classic symptoms of inflammation such as redness and edema16,17. The swelling of the hind paw was measured with mechanical calipers20. A baseline value of the thickness of the paw was obtained for each rat before carrageenan treatment and measured again at 6 h and 12 h. Paw thickness was increased significantly compared to the baseline values (Figure 1).

<.......

Discussion

The study determined that the epidermis of rat hind paw glabrous skin was easily separated from dermis using thermolysin (0.5 mG/mL) in PBS with 1 mM calcium chloride at 4 °C for 2.5 h. Histological evaluation indicated that the epidermis was separated from the dermis at the basement membrane and that the epidermal rete ridges were intact. Thermolysin is an extracellular metalloendopeptidase produced by Gram-positive (Geo)Bacillus thermoproteolyticus24. Its activity is stable.......

Acknowledgements

Funding for this research was provided by National Institutes of Health NIH-AR047410 (KEM)

....

Materials

NameCompanyCatalog NumberComments
λ-carrageenanMillipore Sigma22049Subcutaneous injection of carrageenan induces inflammation
7500 Fast Real-Time PCR SystemThermo Fisher Scientific4351107For RT-PCR analysis
Calcium chloride (CaCl2), anhydrousMillipore Sigma499609Prevents autolysis of thermolysin
Crystal Mount Aqueous Mounting MediumMillipore SigmaC0612Aqueous mounting medium after toluidine blue staining
Donkey anti-Mouse Alexa Fluor 555Thermo Fisher ScientificA-31570Secondary antibody for immunohistochemistry
Donkey anti-Rabbit IgG, Alexa Fluor 488Thermo Fisher ScientificA-21206Secondary antibody for immunohistochemistry
Dulbecco's Modified Eagle MediumThermo Fisher Scientific11966-025To maintain tissue integrity
Ethylenediaminetetraacetic acidMillipore SigmaE6758Stops thermolysin reaction
Moloney Murine Leukemia Virus (M-MLV) Reverse transcriptasePromegaM1701For complementary DNA synthesis
Mouse anti-NGF Antibody (E-12)Santa Cruz Biotechnologysc-365944For neurotrophin immunohistochemistry
ProLong Gold Antifade MountantThermo Fisher ScientificP36930To retard immunofluorescence quenching
Rabbit anti-PGP 9.5Cedarlane LabsCL7756APFor intraepidermal nerve staining
SAS Sprague Dawley RatCharles RiverStrain Code 400Animal used for inflammation studies
Shandon M-1 Embedding MatrixThermo Fisher Scientific1310TSTissue embedding matrix for tinctorial- and immuno-histochemistry
SimpliAmp Thermal CyclerThermo Fisher ScientificA24811For RT-PCR analysis
SYBR Select Master MixThermo Fisher Scientific4472908For RT-PCR analysis
ThermolysinMillipore SigmaT7902From Geobacillus stearothermophilus
Toluidine BlueMillipore Sigma89640For tinctorial staining for brightfield microscopy
TRIzol ReagentThermo Fisher Scientific15596026For total RNA extraction for RTPCR

References

  1. Choi, J. E., Di Nardo, A. Skin neurogenic inflammation. Seminars in Immunopathology. 40 (3), 249-259 (2018).
  2. Manti, S., Brown, P., Perez, M. K., Piedimonte, G. The role of neurotrophins in inflammation and allergy. Vitamins and Hormone....

Explore More Articles

EpidermisDermisThermolysinSkin InflammationInflammatory MediatorsNeurotrophinsWound HealingSkin DiseaseChronic WoundsBurnsEpithelial SurfacesGI TractCorneaSprague Dawley RatLambda CarrageenanEdemaGlabrous Hind PawMicrodissection ForcepsDMEMStratum Corneum

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved