Name: separation of rat epidermis and dermis with thermolysin to detect site-specific inflammatory mrna and protein
Uploaded: 2021-09-29T13:00:00
Description: Watch this Scientific Journal Video about Separation of Rat Epidermis and Dermis with Thermolysin to Detect Site-Specific Inflammatory mRNA and Protein at JoVE.com

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

This protocol follows the animal care guidelines of Oklahoma State University Center for Health Sciences IACUC (#2016-03).
1. Carrageenan-induced inflammation (C-II)
<ol>
	<li>Anesthetize male and/or female Sprague Dawley rats (200&#8211;250 g; 8&#8211;9 weeks old) with isoflurane (or injectable anesthetic).</li>
	<li>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...

<ol>
	<li>Choi, J. E., Di Nardo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+neurogenic+inflammation.">Skin neurogenic inflammation.</a> Seminars in Immunopathology. 40 (3), 249-259 (2018).</li><li>Manti, S., Brown, P., Perez, M. K., Piedimonte, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+neurotrophins+in+inflammation+and+allergy.">The role of neurotrophins in inflammation and allergy.</a> Vitamins and Hormones. 104, 313-341 (2017).</li><li>Sch&#228;kel, K., Sch&#246;n, M. 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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dermal-epidermal+separation+methods:+research+implications.">Dermal-epidermal separation methods: research implications.</a> Archives of Dermatological Research. 310 (1), 1-9 (2018).</li><li>Baumberger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+the+separation+of+epidermis+from+dermis+and+some+physiologic+and+chemical+properties+of+isolated+epidermis.">Methods for the separation of epidermis from dermis and some physiologic and chemical properties of isolated epidermis.</a> Journal of the National Cancer Institute. 2, 413-423 (1942).</li><li>Felsher, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+adherence+of+the+epidermis+to+the+corium.">Studies on the adherence of the epidermis to the corium.</a> Journal of Investigative Dermatology. 8, 35-47 (1947).</li><li>Einbinder, J. M., Walzer, R. A., Mandl, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal-dermal+separation+with+proteolytic+enzymes.">Epidermal-dermal separation with proteolytic enzymes.</a> Journal of Investigative Dermatology. 46, 492-504 (1966).</li><li>Rakhorst, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucosal+keratinocyte+isolation:+a+short+comparative+study+on+thermolysin+and+dispase.">Mucosal keratinocyte isolation: a short comparative study on thermolysin and dispase.</a> International Association of Oral and Maxillofacial Surgeons. 35 (10), 935-940 (2006).</li><li>Tschachler, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sheet+preparations+expose+the+dermal+nerve+plexus+of+human+skin+and+render+the+dermal+nerve+end+organ+accessible+to+extensive+analysis.">Sheet preparations expose the dermal nerve plexus of human skin and render the dermal nerve end organ accessible to extensive analysis.</a> Journal of Investigative Dermatology. 122 (1), 177-182 (2004).</li><li>Walzer, C., Benathan, M., Frenk, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermolysin+treatment-a+new+method+for+dermo-epidermal+separation.">Thermolysin treatment-a new method for dermo-epidermal separation.</a> Journal of Investigative Dermatology. 92, 78-81 (1989).</li><li>Anderson, M. B., Miller, K. E., Schechter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+rat+epidermis+and+dermis+following+thermolysin+separation:+PGP+9.5+and+Nav+1.8+localization.">Evaluation of rat epidermis and dermis following thermolysin separation: PGP 9.5 and Nav 1.8 localization.</a> Society for Neuroscience Abstract. 584 (9), (2010).</li><li>Ibitokun, B. O., Anderson, M. B., Miller, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+corneal+epithelium+from+the+stroma+using+thermolysin:+evaluation+of+corneal+afferents.">Separation of corneal epithelium from the stroma using thermolysin: evaluation of corneal afferents.</a> Society for Neuroscience Abstract. , 584 (2010).</li><li>Nawani, P., Anderson, M., Miller, K. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carrageenan-induced+thermal+hyperalgesia+in+the+mouse:+role+of+nerve+growth+factor+and+the+mitogen-activated+protein+kinase+pathway.">Carrageenan-induced thermal hyperalgesia in the mouse: role of nerve growth factor and the mitogen-activated protein kinase pathway.</a> Brain Research. 876 (1-2), 48-54 (2000).</li><li>Hoffman, E. M., Miller, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+inhibition+of+glutaminase+reduces+carrageenan+induced+Fos+expression+in+the+superficial+dorsal+horn+of+the+rat.">Peripheral inhibition of glutaminase reduces carrageenan induced Fos expression in the superficial dorsal horn of the rat.</a> Neuroscience Letters. 472 (3), 157-160 (2010).</li><li>Crosby, H. A., Ihnat, M., Spencer, D., Miller, K. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixative+composition+alters+distributions+of+immunoreactivity+for+glutaminase+and+two+markers+of+nociceptive+neurons+Nav1.8+and+TRPV1,+in+the+rat+dorsal+ganglion.">Fixative composition alters distributions of immunoreactivity for glutaminase and two markers of nociceptive neurons Nav1.8 and TRPV1, in the rat dorsal ganglion.</a> Journal of Histochemistry and Cytochemistry. 58 (4), 329-344 (2010).</li><li>Hoffman, E. M., Zhang, Z., Schechter, R., Miller, K. 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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiparameter+flow+cytometric+characterization+of+epidermal+cell+suspensions+prepared+from+normal+and+hyperproliferative+human+skin+using+an+optimized+thermolysin-trypsin+protocol.">Multiparameter flow cytometric characterization of epidermal cell suspensions prepared from normal and hyperproliferative human skin using an optimized thermolysin-trypsin protocol.</a> Archives of Dermatological Research. 288 (4), 203-210 (1996).</li><li>Sato, J. D., Kan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Media+for+culture+of+mammalian+cells.">Media for culture of mammalian cells.</a> Current Protocols in Cell Biology. , (2001).</li><li>Gragnani, A., Sobral, C. S., Ferreira, L. 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Animal. 35 (6), 318-326 (1999).</li><li>Fassina, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autolysis+of+thermolysin.+Isolation+and+characterization+of+a+folded+three-fragment+complex.">Autolysis of thermolysin. Isolation and characterization of a folded three-fragment complex.</a> European Journal of Biochemistry. 156 (2), 221-228 (1986).</li><li>Petho, G., Reeh, P. 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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 &deg;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...

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. Trypsin, pancreatin, elastase, keratinase, collagenase, pronase, dispase, and thermolysin are enzymes that have been used for epidermal-dermal separation4,7. Trypsin and other broad scale proteolytic enzymes are active at 37&ndash;40 &deg;C but must be monitored carefully to prevent dissociation of epidermal layers. Dispase cleaves the epidermis at the lamina densa, but requires 24 h for separation in the cold4,8 or shorter timepoints at 37 &deg;C4,9. A limiting feature of all these techniques is the potential disruption of tissue morphology and loss of integrity of mRNA and protein.
To maintain the integrity of mRNA and protein, a skin separation method should be carried out in the cold for a short period of time. In evaluating skin separation techniques for inflammation studies, thermolysin is an effective enzyme to separate the epidermis from dermis at cold temperatures4. Thermolysin is active at 4 &deg;C, cleaves epidermal hemidesmosomes from the lamina lucida, and separates the epidermis from dermis within 1&ndash;3 h4,8,10. The goal of this report is to optimize the use of thermolysin for separation of inflamed rat epidermis from dermis to detect mRNA and protein levels for inflammatory mediators and neurotrophic factors. Several preliminary reports have been presented11,12,13,14,15. The objective of this manuscript is to describe an optimal skin separation technique using thermolysin and demonstrate the detection of 1) markers of inflammation, 2) interleukin-6 (IL-6) mRNA, and 3) nerve growth factor (NGF) mRNA and protein in the epidermis of rats with carrageenan-induced inflammation (C-II)16,17. A preliminary report using the complete Freund&rsquo;s adjuvant model indicates that NGF mRNA and protein levels increase early during inflammation15. In mice, skin sensitization with the topical application of oxazolone causes an early rise in the IL-6 mRNA using in situ hybridization36. Both IL-6 and NGF have been implicated in C-II18,19, but there have been no reports describing mRNA or protein levels for IL-6 or NGF specifically from the epidermis during the acute stages of C-II.
The thermolysin technique is inexpensive and straightforward to perform. Furthermore, thermolysin separation of the epidermis from dermis allows for mRNA, western blot, and immunohistochemical analysis of inflammatory mediators and neurotrophic factors during the process of inflammation15. Investigators should be able to easily use this technique in both preclinical and clinical studies of skin inflammation.

<table>
	<tbody>
		<tr>
			<td>&#955;-carrageenan</td>
			<td>Millipore Sigma</td>
			<td>22049</td>
			<td>Subcutaneous injection of carrageenan induces inflammation</td>
		</tr>
		<tr>
			<td>7500 Fast Real-Time PCR System</td>
			<td>Thermo Fisher Scientific</td>
			<td>4351107</td>
			<td>For RT-PCR analysis</td>
		</tr>
		<tr>
			<td>Calcium chloride (CaCl2), anhydrous</td>
			<td>Millipore Sigma</td>
			<td>499609</td>
			<td>Prevents autolysis of thermolysin</td>
		</tr>
		<tr>
			<td>Crystal Mount Aqueous Mounting Medium</td>
			<td>Millipore Sigma</td>
			<td>C0612</td>
			<td>Aqueous mounting medium after toluidine blue staining</td>
		</tr>
		<tr>
			<td>Donkey anti-Mouse Alexa Fluor 555</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-31570</td>
			<td>Secondary antibody for immunohistochemistry</td>
		</tr>
		<tr>
			<td>Donkey anti-Rabbit IgG, Alexa Fluor 488</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-21206</td>
			<td>Secondary antibody for immunohistochemistry</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>11966-025</td>
			<td>To maintain tissue integrity</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid</td>
			<td>Millipore Sigma</td>
			<td>E6758</td>
			<td>Stops thermolysin reaction</td>
		</tr>
		<tr>
			<td>Moloney Murine Leukemia Virus (M-MLV) Reverse transcriptase</td>
			<td>Promega</td>
			<td>M1701</td>
			<td>For complementary DNA synthesis</td>
		</tr>
		<tr>
			<td>Mouse anti-NGF Antibody (E-12)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-365944</td>
			<td>For neurotrophin immunohistochemistry</td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>Thermo Fisher Scientific</td>
			<td>P36930</td>
			<td>To retard immunofluorescence quenching</td>
		</tr>
		<tr>
			<td>Rabbit anti-PGP 9.5</td>
			<td>Cedarlane Labs</td>
			<td>CL7756AP</td>
			<td>For intraepidermal nerve staining</td>
		</tr>
		<tr>
			<td>SAS Sprague Dawley Rat</td>
			<td>Charles River</td>
			<td>Strain Code 400</td>
			<td>Animal used for inflammation studies</td>
		</tr>
		<tr>
			<td>Shandon M-1 Embedding Matrix</td>
			<td>Thermo Fisher Scientific</td>
			<td>1310TS</td>
			<td>Tissue embedding matrix for tinctorial- and immuno-histochemistry</td>
		</tr>
		<tr>
			<td>SimpliAmp Thermal Cycler</td>
			<td>Thermo Fisher Scientific</td>
			<td>A24811</td>
			<td>For RT-PCR analysis</td>
		</tr>
		<tr>
			<td>SYBR Select Master Mix</td>
			<td>Thermo Fisher Scientific</td>
			<td>4472908</td>
			<td>For RT-PCR analysis</td>
		</tr>
		<tr>
			<td>Thermolysin</td>
			<td>Millipore Sigma</td>
			<td>T7902</td>
			<td>From Geobacillus stearothermophilus</td>
		</tr>
		<tr>
			<td>Toluidine Blue</td>
			<td>Millipore Sigma</td>
			<td>89640</td>
			<td>For tinctorial staining for brightfield microscopy</td>
		</tr>
		<tr>
			<td>TRIzol Reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>15596026</td>
			<td>For total RNA extraction for RTPCR</td>
		</tr>
	</tbody>
</table>

separation of rat epidermis and dermis with thermolysin to detect site-specific inflammatory mrna and protein

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 &deg;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 na&iuml;ve 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.

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).
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Separation of Rat Epidermis and Dermis with Thermolysin to Detect Site-Specific Inflammatory mRNA and Protein

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 &#176;C. The epidermis is then used for mRNA analysis by RT-PCR and protein evaluation by western blot and immunohistochemistry.

Easy-to-use and inexpensive techniques are needed to determine the site-specific production of inflammatory mediators and neurotrophins during skin ...

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.

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