Our protocol is an easy-to-use and inexpensive epidermal-dermal separation technique for determining the site-specific production of inflammatory mediators and neurotrophins during skin inflammation. The main advantage of this technique is that enzymatic separation of the epidermis from the dermis is performed at four degrees Celsius, maintaining the integrity of mRNA and protein. This method provides insight into the types of mediators that sensitize primary afferent terminals during acute and chronic inflammation for the development of novel therapeutic interventions.
Wound healing, skin disease, chronic wounds, and burns could benefit from this research. And this method could be applied to other epithelial surfaces, such as the GI tract and cornea. The optimal separation time may differ slightly between labs.
A preliminary experiment comparing different time points combined with a 2D morphology evaluation is recommended to determine the quality of the separation. After confirming a lack of response to pedal reflex in an anesthetized eight to nine-week-old Sprague Dawley rat, subcutaneously inject the right glabrous hind paw with 100 microliters of 1X Lambda carrageenan diluted in PBS. Use calipers to measure the hind paw metatarsal thickness immediately before and at 6 and 12 hours after injection to determine the volume of edema in response to the stimulus.
At the appropriate experimental endpoint, use a sharp scalpel to harvest a one by two millimeter piece of glabrous hind paw skin from the injected paw and use microdissection forceps to transfer the skin into one milliliter of cold DMEM in a microcentrifuge tube on ice for 15 to 60 minutes. While the tissue is incubating, add one milliliter of freshly prepared activated thermolysin to 10 wells of a 24-well cell culture plate on ice. At the end of the incubation, use the microdissection forceps to transfer one skin sample into each well of activated thermolysin, stratum corneum side up without immersing the samples in the solution.
It is critical that the skin is not immersed in the thermolysin and that the stratum corneum faces up to achieve an effective separation. After two to three hours, use the microdissection forceps to immerse one skin sample in seven to eight milliliters of four degrees Celsius DMEM in a Petri dish to allow more room for the epidermis to separate from the dermis. Use forceps to gently brush the epidermis around the perimeter of the skin until the near translucent epidermis is observed at the borders of the sample.
When the epidermis noticeably separates from the dermis, carefully grasp each layer of skin with a single pair of forceps and slowly pull the epidermis from the dermis, then evaluate the translucence of the isolated epidermis by light microscopy to make sure it is optically consistent. If the layers have been properly separated, place the epidermal and dermal tissues into individual containers of five millimolar EDTA in DMEM at four degrees Celsius for 30 minutes. After thermolysin deactivation, fix a portion of the epidermis in an appropriate fixative for one hour at room temperature with agitation.
At the end of the incubation, place the fixed tissue in 10%sucrose in PBS for one hour at room temperature with agitation, before freezing the sample in a suitable tissue embedding matrix for sectioning. Use a cryostat to obtain 14 micrometer thick cross-sections and thaw mount the sections onto gelatin-coated glass microscope slides. After drying on a slide warmer, stain the samples with a working solution of toluidine blue for 90 seconds.
Afterward, gently wash toluidine blue from the slides with PBS and oppose coverslips with an aqueous mounting medium, then observe the epidermis with Brightfield microscopy at a 50 to 250X magnification. Carrageenan injection into the rat hind paw causes classic symptoms of inflammation, such as redness and edema at both 6 and 12-hour post-injection. Brightfield microscopy can be used to evaluate the effectiveness of thermolysin separation of the epidermis and dermis of the rat glabrous hind paw skin.
Indeed, toluidine blue staining of the epidermal cross-sections shows that the epidermis is separated from the dermis at the baseline membrane, but that the epidermal Rete ridges and the indentations from the dermal papilla remain intact. Further, all of the keratinocyte cell layers are observed. Western blotting of the thermolysin separated epidermis produces consistent results, indicating stable protein levels during the technique at four degrees Celsius.
Very little NGF protein is detected in the naive rat epidermis, but NGF protein levels are significantly upregulated after six hours of carrageenan-induced inflammation. 12 hours after the injection, NGF levels are reduced compared to those observed at six hours, but remain elevated relative to controls. As expected from the Western blot analysis, NGF immunoreactivity is not detected at naive control epidermis tissue samples, but at six hours post carrageenan-induced inflammation, NGF immunoreactivity is observed in most of the keratinocytes of the stratum granulosum and stratum lucidum.
A few cells in the stratum spinosum are also NGF immunoreactive. When using thermolysin to isolate the intact epidermis, it is important to remember that the optimal separation time must be empirically determined by the end user. Various proteomic and molecular techniques can be used to validate the expression of specific proteins and/or primary gene transcripts to confirm that this procedure does not alter the basal expression.
This technique of epidermal-dermal separation will allow researchers to answer additional questions regarding the site-specific expression of neurotrophins and inflammatory mediators in different cutaneous inflammatory models. Picric acid and paraformaldehyde are hazardous. Work with both chemicals in a fume hood, use personal protective clothing and gloves and wash your face and hands after handling.
