This study was supported by the National Nature Science Foundation of China (No. 81974572 and No.82274523) and the Beijing University of Chinese Medicine (No.202310026002).

This protocol has received ethical approval from the Beijing University of Chinese Medicine (No.2023033103-1183). Twelve male Sprague-Dawley rats (weight, 208 g &#177; 5 g) were used in this protocol and divided into four groups: NC group (n = 3), OA group (n = 3), C. acnes group (n = 3) and OA + C. acnes group (n = 3).
1. Developing the acne model
<ol>
	<li>Measure and record the thickness of rat ears using an electronic vernier caliper (see Table of Materials) before modeling.
	<ol>
		<li>Place the outer jaw of the electronic vernier caliper at the midpoint of the outer edge of the ear and extend it into the ear canal. Record the thickness of 2/3 points and the thickness of 1/2 points along the line. Measure each point 3x and calculate the average values of the thickness of two points to be considered the thickness of the ear.</li>
	</ol>
	</li>
	<li>Tilt the rat&#39;s head to one side, make the ear face upwards, and apply 50 &#181;L of oleic acid (see Table of Materials) to the ventral and dorsal sides of the ear evenly, once a day, for 25 days.</li>
	<li>From day 21 to day 25, daily prior to the application of oleic acid, anesthetize the rats using 4% isoflurane and then inject 50 &#181;L of 1 &#215; 107 CFU C. acnes suspension (see Table of Materials) intradermally into the ventral surface of the ear using a 1 mL syringe equipped with a 34 G or 36 G needle (see Table of Materials). 
	NOTE: Take care to avoid blood vessels in the ear when injecting intradermally. A micro-syringe with a 34 G or 36 G needle (see Table of Materials) is required. To ensure proper intradermal injection, the needle should be punctured at an angle of 10 to 15 degrees, with a depth of approximately 1 mm.</li>
	<li>On day 25, measure and record the thickness of the ears, as described in step 1.1. 
	NOTE: The OA group was only smeared by oleic acid as described in step 1.2 and injected with saline from the 21st to the 25th day. The C. acnes group was only injected intradermally by C. acnes suspension, as described in step 1.3. The OA + C. acnes group was treated with oleic acid and C. acnes suspension, as described in steps 1.2 and 1.3.</li>
</ol>
2. Tissue collection and analysis
<ol>
	<li>On day 26, after the rats are humanely euthanized using an institutionally approved method, drill fixed size on bilateral ears with an 8 mm ring drill.</li>
	<li>Immerse the ear tissues in 4% paraformaldehyde for 24 h, followed by paraffin-embedding and sectioning into slices 5 &#181;m thick for hematoxylin and eosin (HE) staining20. Observe the sections under a light microscope and record the pathological changes for follow-up evaluation by a pathologist. Assign scores to the pathological changes according to Table 1 (also see Figure 1).</li>
	<li>Deparaffinize, rehydrate, and heat the sections for antigen retrieval. Inactivate endogenous peroxidases by incubating with 0.3% H2O2 in methanol for 15 min at room temperature.</li>
	<li>Wash the sections with phosphate-buffered saline (PBS) and then incubate them with 3% BSA for 30 min at room temperature.</li>
	<li>Incubate the sections with TNF-&#945; monoclonal antibody (dilution 1:800) (see Table of Materials) at 4 &#176;C overnight.</li>
	<li>Wash the sections with PBS again and incubate with horseradish peroxidase-conjugated secondary antibody (see Table of Materials) for 50 min.</li>
	<li>After another PBS wash, apply 3,3&#39;-diaminobenzidine (DAB) mixture to the slides and monitor the staining process under a microscope, stopping the reaction with PBS when the desired intensity is reached.</li>
	<li>Counterstain the sections with Meyer&#39;s hematoxylin for 1 min, rinse them with running water, and dehydrate and mount the sections. Observe the sections under a microscope and record the pathological changes for follow-up evaluation by a pathologist.</li>
</ol>
3. Statistical analysis
<ol>
	<li>Perform statistical analysis.</li>
	<li>Present data as mean &#177; standard deviation. Access the normality of all the data using the Shapro-Wilke test. If it follows a normal distribution, use one-way ANOVA to assess significant differences. For non-normally distributed data, apply the Kruskal-Wallis test followed by Dunn&#39;s multiple comparisons. A p-value less than 0.05 is considered statistically significant.</li>
</ol>

Establishing a Compound Acne Model in Rats Using Oleic Acid and C. acnes

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	<li>Cooper, A. J., Harris, V. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modern+management+of+acne.">Modern management of acne.</a> The Medical Journal of Australia. 206 (1), 41-45 (2017).</li><li>Vos, T., 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=Years+lived+with+disability+(YLDs)+for+1160+sequelae+of+289+diseases+and+injuries+1990-2010:+a+systematic+analysis+for+the+Global+Burden+of+Disease+Study+2010.">Years lived with disability (YLDs) for 1160 sequelae of 289 diseases and injuries 1990-2010: a systematic analysis for the Global Burden of Disease Study 2010.</a> Lancet. 380 (9859), 2163-2196 (2012).</li><li>Layton, A. M., Thiboutot, D., Tan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reviewing+the+global+burden+of+acne:+how+could+we+improve+care+to+reduce+the+burden.">Reviewing the global burden of acne: how could we improve care to reduce the burden.</a> The British Journal of Dermatology. 184 (2), 219-225 (2021).</li><li>Hazarika, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acne+vulgaris:+new+evidence+in+pathogenesis+and+future+modalities+of+treatment.">Acne vulgaris: new evidence in pathogenesis and future modalities of treatment.</a> Journal of Dermatological Treatment. 32 (3), 277-285 (2021).</li><li>Das, S., Reynolds, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+acne+pathogenesis:+implications+for+therapy.">Recent advances in acne pathogenesis: implications for therapy.</a> American Journal of Clinical Dermatology. 15 (6), 479-488 (2014).</li><li>Dr&#233;no, B., 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=Cutibacterium+acnes+(Propionibacterium+acnes)+and+acne+vulgaris:+a+brief+look+at+the+latest+updates.">Cutibacterium acnes (Propionibacterium acnes) and acne vulgaris: a brief look at the latest updates.</a> Journal of the European Academy of Dermatology and Venereology: JEADV. 32, 5-14 (2018).</li><li>Marples, R. R., Downing, D. T., Kligman, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+free+fatty+acids+in+human+surface+lipids+by+Corynebacterium+acnes.">Control of free fatty acids in human surface lipids by Corynebacterium acnes.</a> The Journal of Investigative Dermatology. 56 (2), 127-131 (1971).</li><li>Katsuta, Y., Iida, T., Hasegawa, K., Inomata, S., Denda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+oleic+acid+on+epidermal+barrier+and+calcium+influx+into+keratinocytes+is+associated+with+N-methyl+D-aspartate-type+glutamate+receptors.">Function of oleic acid on epidermal barrier and calcium influx into keratinocytes is associated with N-methyl D-aspartate-type glutamate receptors.</a> The British Journal of Dermatology. 160 (1), 69-74 (2009).</li><li>Kurokawa, I., 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=New+developments+in+our+understanding+of+acne+pathogenesis+and+treatment.">New developments in our understanding of acne pathogenesis and treatment.</a> Experimental Dermatology. 18 (10), 821-832 (2009).</li><li>Clayton, R. W., 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=Homeostasis+of+the+sebaceous+gland+and+mechanisms+of+acne+pathogenesis.">Homeostasis of the sebaceous gland and mechanisms of acne pathogenesis.</a> The British Journal of Dermatology. 181 (4), 677-690 (2019).</li><li>Cong, T. X., 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=From+pathogenesis+of+acne+vulgaris+to+anti-acne+agents.">From pathogenesis of acne vulgaris to anti-acne agents.</a> Archives of dermatological research. 311 (5), 337-349 (2019).</li><li>Ji, 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=Analgesic+and+anti-inflammatory+effects+and+mechanism+of+action+of+borneol+on+photodynamic+therapy+of+acne.">Analgesic and anti-inflammatory effects and mechanism of action of borneol on photodynamic therapy of acne.</a> Environmental Toxicology and Pharmacology. 75, 103329 (2020).</li><li>Ou-Yang, X. L., 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=Nontargeted+metabolomics+to+characterize+the+effects+of+isotretinoin+on+skin+metabolism+in+rabbit+with+acne.">Nontargeted metabolomics to characterize the effects of isotretinoin on skin metabolism in rabbit with acne.</a> Frontiers in Pharmacology. 13, 963472 (2022).</li><li>Zhu, Z., 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=Skin+microbiome+reconstruction+and+lipid+metabolism+profile+alteration+reveal+the+treatment+mechanism+of+Cryptotanshinone+in+the+acne+rat.">Skin microbiome reconstruction and lipid metabolism profile alteration reveal the treatment mechanism of Cryptotanshinone in the acne rat.</a> Phytomedicine. 101, 154101 (2022).</li><li>Jang, Y. H., 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=HR-1+mice:+A+new+inflammatory+acne+mouse+model.">HR-1 mice: A new inflammatory acne mouse model.</a> Annals of Dermatology. 27 (3), 257-264 (2015).</li><li>De Young, L. M., Young, J. M., Ballaron, S. J., Spires, D. A., Puhvel, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intradermal+injection+of+Propionibacterium+acnes:+a+model+of+inflammation+relevant+to+acne.">Intradermal injection of Propionibacterium acnes: a model of inflammation relevant to acne.</a> The Journal of Investigative Dermatology. 83 (5), 394-398 (1984).</li><li>Chen, T., 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=A+skin+lipidomics+study+reveals+the+therapeutic+effects+of+Tanshinones+in+a+rat+model+of+acne.">A skin lipidomics study reveals the therapeutic effects of Tanshinones in a rat model of acne.</a> Frontiers in Pharmacology. 12, 675659 (2021).</li><li>Cao, J., Xu, M., Zhu, L., Xiao, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viaminate+ameliorates+Propionibacterium+acnes-induced+acne+via+inhibition+of+the+TLR2/NF-&#954;B+and+MAPK+pathways+in+rats.">Viaminate ameliorates Propionibacterium acnes-induced acne via inhibition of the TLR2/NF-&#954;B and MAPK pathways in rats.</a> Naunyn-Schmiedeberg's Archives of Pharmacology. 396 (7), 1487-1500 (2023).</li><li>Kolar, S. L., 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=Propionibacterium+acnes-induced+immunopathology+correlates+with+health+and+disease+association.">Propionibacterium acnes-induced immunopathology correlates with health and disease association.</a> JCI insight. 4 (5), e124687 (2019).</li><li>Zhong, C., 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=Inhibition+of+protein+glycosylation+is+a+novel+pro-angiogenic+strategy+that+acts+via+activation+of+stress+pathways.">Inhibition of protein glycosylation is a novel pro-angiogenic strategy that acts via activation of stress pathways.</a> Nature Communications. 11 (1), 6330 (2020).</li><li>O'Neill, A. M., Gallo, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-microbiome+interactions+and+recent+progress+into+understanding+the+biology+of+acne+vulgaris.">Host-microbiome interactions and recent progress into understanding the biology of acne vulgaris.</a> Microbiome. 6 (1), 177 (2018).</li><li>Moradi Tuchayi, S., 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=Acne+vulgaris.">Acne vulgaris.</a> Nature Reviews. Disease Primers. 1, 15029 (2015).</li><li>American Academy of Dermatology. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=American+Academy+of+Dermatology+invitational+symposium+on+comedogenicity.">American Academy of Dermatology invitational symposium on comedogenicity.</a> Journal of the American Academy of Dermatology. 20 (2), 272-277 (1989).</li><li>Katsambas, A. D., Cunliffe, W. J., Zoubolis, C. C., Zouboulis, C. C., Katsambas, A. D., Kligman, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+aspects+of+acne+vulgaris.">Clinical aspects of acne vulgaris.</a> Pathogenesis and treatment of acne and rosacea. , 213-221 (2014).</li><li>Williams, H. C., Dellavalle, R. 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All authors declare that they have no conflicts of interest.

As the methodology and evaluation criteria for an acne model were not clear, this protocol aimed to provide a reference for researchers studying acne. Due to the small size of the mouse ear and the interference caused by growing hair on the back, the use of depilatory cream and a shaver, the ear of the rat may be a better alternative to establish a rodent model of acne. We had attempted to create the acne model on the back skin of rats, but the effect was not as good as those observed on the rat ear.
<p class="jove_c...

Acne vulgaris is a common chronic skin disease characterized by the presence of comedones, papules, and pustules on the face, neck, and chest, which, in severe cases, may progress to nodules, cysts, and permanent scars1. Epidemiological studies report that acne impacts 9.4% of the global population while its resulting symptoms pose severe physical and psychosocial challenges2,3.
The pathogenesis of acne is multifactorial, including four critical processes: excess sebum production, comedone formation, follicular colonization by skin microbiota, and the release of inflammatory mediators around the pilosebaceous unit4,5. Increased sebum secretion, resulting in the accumulation of excess unsaturated free fatty acids (UFFAs), contributes to the abnormal reproduction of skin microbiota, one of which is Cutibacterium acnes, as shown in genomic studies6. Meanwhile, skin microbiota decomposes the sebum and increases the concentration of UFFAs, leading to a vicious circle7. In addition, excess UFFAs cause hyperkeratinization in hair follicles, which in turn triggers acne8. Skin microbiota and increased sebum both activate Toll-like receptors (TLRs) to produce multiple proinflammatory cytokines such as interleukin (IL)-1 and TNF-&#945;9,10. This cascade of inflammation, coupled with increased sebum and microbial overgrowth, culminates in pronounced hyperkeratosis of the hair follicle and the onset of acne11.
The rapid development in the field of acne research and related clinical requirements has driven the creation of a series of animal models of acne inflammation on the rat ear, the backs of rats or mice, and rabbit ear12,13,14,15. The methods include intradermal injection of C. acnes,&#160;the application of oils on the skin to simulate the abnormal secretion of the sebaceous glands and hyperkeratinization, or a combination of both to accelerate the skin inflammation to form acne16,17,18,19. However, the usage and dosage of chemical agents and biological agents vary among previous studies, which may confuse researchers intending to establish an appropriate acne model. This study aimed to establish an easy-to-operate and effective method to form a compound acne rodent model and provide a model reference for researchers studying acne vulgaris.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anaero-indicator&#160;</td>
			<td>Mitsubishi, Japan</td>
			<td>C-22</td>
			<td></td>
		</tr>
		<tr>
			<td>AnaeroPack</td>
			<td>Mitsubishi, Japan</td>
			<td>C-11, C-41</td>
			<td></td>
		</tr>
		<tr>
			<td>Columbia blood agar plate</td>
			<td>BeNa Cuture Collection, China</td>
			<td>BNCC330605</td>
			<td></td>
		</tr>
		<tr>
			<td>Constant temperature incubator</td>
			<td>SHANGCHENG, China</td>
			<td>303-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton swab</td>
			<td>HYNAUT, China</td>
			<td>_</td>
			<td></td>
		</tr>
		<tr>
			<td>Cutibacterium acnes</td>
			<td>BeNa Cuture Collection, China</td>
			<td>BNCC330605</td>
			<td></td>
		</tr>
		<tr>
			<td>Dako REAL EnVision Detection System, Peroxidase/DAB+, Rabbit/Mouse</td>
			<td>DAKO, Denmark</td>
			<td>K5007</td>
			<td></td>
		</tr>
		<tr>
			<td>disposable sterile injection needle</td>
			<td>Zhejiang Oujian Medical Apparatus, China</td>
			<td>_</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic scale</td>
			<td>JINXUAN, China</td>
			<td>A017</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic vernier caliper</td>
			<td>Deli, China</td>
			<td>DL90150</td>
			<td></td>
		</tr>
		<tr>
			<td>Oleic acid (Analytical reagent, AR)</td>
			<td>Fangzheng, China</td>
			<td>_</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride injection</td>
			<td>CR Double CRANE, China</td>
			<td>Y2212241</td>
			<td></td>
		</tr>
		<tr>
			<td>SPSS Statistics</td>
			<td>IBM, USA</td>
			<td>26.0</td>
			<td></td>
		</tr>
		<tr>
			<td>sterile hypodermic syringes</td>
			<td>Shandong weigao group medical polymer, China</td>
			<td>_</td>
			<td></td>
		</tr>
		<tr>
			<td>TNF Alpha Monoclonal antibody</td>
			<td>Proteintech Group,int, USA</td>
			<td>60291-1-lg</td>
			<td></td>
		</tr>
	</tbody>
</table>

a rat model of compound acne

Acne vulgaris is a prevalent chronic skin condition characterized by the presence of comedones, papules, and pustules on the face, neck, and chest. To simulate the inflammation of acne vulgaris, this protocol details an approach to establish a compound acne rodent model by inducing acne inflammation in rat ears using oleic acid and Cutibacterium acnes (C. acnes).&#160;Rats were randomly divided into four groups: the normal control group (NC), ears treated with oleic acid group (OA), ears treated with C. acnes group (C. acnes), ears treated with oleic acid and C. acnes (OA + C. acnes). To mimic excessive sebum production, oleic acid was smeared on the ears of rats in OA and OA + C. acnes groups for 25 days.
From days 21 to 25, C. acnes suspension was injected intradermally into the ears of rats in the C. acnes and OA + C. acnes groups to aggravate the acne inflammation. Ear thickness was measured weekly as a gauge of inflammation severity. Gross observation, hematoxylin and eosin staining, and immunohistochemistry (IHC) were conducted and the results showed that the ears of the OA group and the OA + C. acnes group were thickened and indurated, accompanied by erythema and the presence of comedones. Additionally, papules were observed in C. acnes and OA + C. acnes groups. The histopathology exhibited hyperkeratinization and expanded infundibulum of the hair follicles in OA and OA + C. acnes groups. Infiltration of inflammatory cells and abscesses were found in the dermis of C. acnes and OA + C. acnes groups. The IHC results confirmed increased levels of tumor necrosis factor (TNF)-&#945; in the dermis of C. acnes and OA + C. acnes groups. All the above results collectively indicated the successful establishment of the compound acne rodent model.

Author Spotlight: Development and Evaluation of a Compound Acne Rodent Model Using C. acnes and Oleic Acid

A Rat Model of Compound Acne

This study aims to establish an easy-to-operate and effective method to form a compound acne rodent model. We confirm that this compound acne rodent model is better than the models only induced by Cutibacterium acnes or oleic acids. Some researchers only adopted a single methodology to cause acne inflammation such as applying oils or injecting microbes.However, studies show that interplay between both exacerbates acne inflammation. This protocol aims to provide a method and evaluation criteria for an acne model that combines C.acnes with oleic acids. Our findings show that the compound model induced by oils and C.acnes may be more consistent with the clinical lesion of acne compared to smearing oleic acid alone or injecting C.acnes alone.Furthermore, we try to establish criteria for evaluating acne inflammation.

Thickness and skin appearance 
From day 7 to day 21, the ears in the OA group and the OA + C. acnes group were significantly thicker than that of the NC and C. acnes groups. On day 25, the ears in the OA group, C. acnes group, and OA + C. acnes group were significantly thicker than those of the NC group (p &#60; 0.05). The average thickness of the ears during the experiment is shown in Figure 2 and Table 2.</...

This protocol outlines the procedure for inducing acne inflammation in rat skin with oleic acid and Cutibacterium acnes.

Acne vulgaris is a prevalent chronic skin condition characterized by the presence of comedones, papules, and pustules on the face, neck, and chest. To ...

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Research

JoVE Journal

Medicine

Inducing Acne Inflammation in Rat Skin with Oleic Acid and Cutibacterium acnes

To begin, take an electronic Vernier caliper, position its outer jaw at the midpoint of the ear's outer edge, and extend it into the ear canal. Measure and record the thickness at two thirds and one half points along the line. Then, tilt the rat's head to one side, orienting the ear upwards.Apply 50 microliters of oleic acid evenly to both the ventral and dorsal sides of the ear, once daily, for 25 days. Using a one milliliter syringe equipped with a 34 or 36 gauge needle, inject 50 microliters of cutibacterium acnes suspension intradermally into the ventral surface of the ear. On day 25, measure and record the thickness of the ears.After cutting the ears off the euthanized rat, drill fixed size holes in the ears using an eight millimeter ring drill. After preparing and staining the ear sections, observe them under a light microscope. Record the pathological changes for follow-up evaluation by a pathologist, and assign scores to these changes according to the table shown here.The ears in the oleic acid and oleic acid with cutibacterium acnes groups showed thickening, induration, erythema, scales, and comedones with occasional bleeding due to scratching. Histopathological analysis revealed parakeratosis, hyperkeratosis and hypertrophy in the oleic acid and oleic acid with cutibacterium acnes groups. Pathological scoring indicated increased severity in these groups compared to the control group.Immunohistochemistry showed higher expression of TNF alpha in the dermis of the cutibacterium acnes, and oleic acid with cutibacterium acnes groups.

Chemical-Induced Skin Carcinogenesis Model Using Dimethylbenz[a]Anthracene and 12-O-Tetradecanoyl Phorbol-13-Acetate (DMBA-TPA)

Cancer is one of the most devastating human diseases. Experimental cancer models are important to gain insight into the complex interplay of different cell types and genes in promoting tumor progression and to provide a platform for testing the efficacy of different therapeutic approaches. One of the most commonly used experimental inflammatory cancer models is the DMBA-TPA two-stage skin carcinogenesis model. Tumor formation is induced in this model by the topical application of two different chemicals, 7,12-dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoyl phorbol-13-acetate (TPA), that together cause papilloma formation in the skin. As the primary outcome is papilloma formation in the skin, the model is an ideal, reliable, and reproducible way to address both tumor initiation (tumor-free survival) and tumor progression (number and size of visible tumors). The effects of the DMBA-TPA treatment are transmitted via an inflammatory mechanism, which makes this model especially suitable for studying the role of the immune system in tumor formation. However, this model is restricted to the skin and other surfaces where the chemicals can be applied on. A detailed protocol is provided in this article to use the model successfully.

Chemical-Induced Skin Carcinogenesis Model Using Dimethylbenz[a]Anthracene and 12-O-Tetradecanoyl Phorbol-13-Acetate DMBA-TPA

Two-stage skin carcinogenesis is induced by two topically applied chemicals. A mutagen 7,12-dimethylbenz[a]anthracene) causes mutations in the epidermal cells and a continuous application of general growth stimulator 12-O-tetradecanoyl phorbol-13-acetate accelerates skin papilloma formation.

Cancer is one of the most devastating human diseases. Experimental cancer models are important to gain insight into the complex interplay of different ...

Cancer Research

Resolving Water, Proteins, and Lipids from In Vivo Confocal Raman Spectra of Stratum Corneum through a Chemometric Approach

Development of this in vivo confocal Raman spectroscopic method enables the direct measurement of water, proteins, and lipids with depth resolution in human subjects. This information is very important for skin-related diseases and characterizing skin care product performance. This protocol illustrates a method for confocal Raman spectra collection and the subsequent analysis of the spectral dataset leveraging chemometrics. The goal of this method is to establish a standard protocol for data collection and provide general guidance for data analysis. Preprocessing (e.g., removal of outlier spectra) is a critical step when processing large datasets from clinical studies. As an example, we provide guidance based on prior knowledge of a dataset to identify the types of outliers and develop specific strategies to remove them. A principal component analysis is performed, and the loading spectra are compared with spectra from reference materials to select the number of components used in the final multivariate curve resolution (MCR) analysis. This approach is successful for extracting meaningful information from a large spectral dataset.

Here, we present a protocol for collection of confocal Raman spectra from human subjects in clinical studies combined with chemometric approaches for spectral outlier removal and the subsequent extraction of key features.

Development of this in vivo confocal Raman spectroscopic method enables the direct measurement of water, proteins, and lipids with depth resolution in ...

Chemistry

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls.
The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as &#34;bio-scaffolds&#34; for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications.
In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct.

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

Bioengineering

Effects of Exposure of Formaldehyde to a Rat Model of Atopic Dermatitis Induced by Neonatal Capsaicin Treatment

Atopic dermatitis is chronically relapsing pruritic eczema and prevails around the world especially in developed countries. Complex interactions between genetic and environmental factors are known to play an important role in the pathophysiology of atopic dermatitis. However, we still lack a detailed picture of the pathogenesis of this disease. Thus, it is of importance to develop appropriate animal models for elucidating the progression of atopic dermatitis. Moreover, investigating the effect of environmental factors such as air pollutants on atopic dermatitis expands understanding of the disease. Here, we describe a method for inducing atopic dermatitis in rats with neonatal capsaicin treatment and a protocol for exposure of a constant concentration of formaldehyde to rats to reveal effects on the development of atopic dermatitis in infantile and adolescent periods. These protocols have been successfully applied to several experiments and can be used for other substances.

We describe here methods for neonatal capsaicin treatment to induce atopic dermatitis in rats and exposure of vaporized formaldehyde to investigate the effects of formaldehyde inhalation on atopic dermatitis.

Atopic dermatitis is chronically relapsing pruritic eczema and prevails around the world especially in developed countries. Complex interactions between ...

Immunology and Infection

In Vitro Model of Human Cutaneous Hypertrophic Scarring using Macromolecular Crowding

It has been shown that in vivo tissues are highly crowded by proteins, nucleic acids, ribonucleoproteins, polysaccharides, etc. The following protocol applies a macromolecular crowding (MMC) technique to mimic this physiological crowding through the addition of neutral polymers (crowders) to cell cultures in vitro. Previous studies using Ficoll or dextran as crowders demonstrate that the expression of collagen I and fibronectin in WI38 and WS-1 cell lines are significantly enhanced using the MMC technique. However, this technique has not been validated in primary hypertrophic scar-derived human skin fibroblasts (hHSFs). As hypertrophic scarring arises from the excessive deposition of collagen, this protocol aims to construct a collagen-rich in vitro hypertrophic scar model by applying the MMC technique with hHSFs. This optimized MMC model has been shown to possess more similarities with in vivo scar tissue compared to traditional 2-dimensional (2-D) cell culture systems. In addition, it is cost-effective, time-efficient, and ethically desirable compared to animal models. Therefore, the optimized model reported here offers an advanced &#8220;in vivo-like&#8221; model for hypertrophic scar-related studies.

This protocol describes the use of macromolecular crowding to create an in vitro human hypertrophic scar tissue model that resembles in vivo conditions. When cultivated in a crowded macromolecular environment, human skin fibroblasts exhibit phenotypes, biochemistry, physiology, and functional characteristics resembling scar tissue.

It has been shown that in vivo tissues are highly crowded by proteins, nucleic acids, ribonucleoproteins, polysaccharides, etc. The following protocol ...

Cultivating a Three-dimensional Reconstructed Human Epidermis at a Large Scale

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process and the characterization of the model is described. Neonatal primary keratinocytes are grown submerged on permeable polycarbonate inserts and lifted to the air-liquid interface three days after seeding. After fourteen days of stimulation with defined growth factors and ascorbic acid in high calcium culture medium, the model is fully differentiated. Histological analysis revealed a completely stratified epidermis, mimicking the morphology of native human skin. To characterize the model and its barrier functions, protein levels and localization specific for early-stage keratinocyte differentiation (i.e., keratin 10), late-stage differentiation (i.e., involucrin, loricrin, and filaggrin) and tissue adhesion (i.e., desmoglein 1), were assessed by immunofluorescence. The tissue barrier integrity was further evaluated by measuring transepithelial electrical resistance. Reconstructed human epidermis was responsive to proinflammatory stimuli (i.e., lipopolysaccharide and tumor necrosis factor alpha), leading to increased cytokine release (i.e., interleukin 1 alpha and interleukin 8). This protocol represents a straightforward and reproducible in vitro method to cultivate reconstructed human epidermis as a tool to assess environmental effects and a broad range of skin-related studies.

This protocol describes a straightforward method to cultivate three-dimensional reconstituted human epidermis in a reproducible and robust manner. Additionally, it characterizes the structure-function relationship of the epidermal barrier model. The biological responses of the reconstituted human epidermis upon proinflammatory stimuli are also presented.

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process ...

A Mouse Ear Model for Allergic Contact Dermatitis Evaluation

Skin is the human body&#39;s first line of defense and one of the most exposed organs to environmental chemicals. Allergic contact dermatitis (ACD) is a common skin disease that manifests as a local rash, redness, and skin lesions. The occurrence and development of ACD are influenced by both genetic and environmental factors. Although many scholars have constructed a series of models of ACD in recent years, the experimental protocols of these models are all different, which makes it difficult for readers to establish them well. Therefore, a stable and efficient animal model is of great significance to further study the pathogenesis of atopic dermatitis. In this study, we detail a modeling method using 1-fluoro-2,4-dinitrobenzene (DNFB) to induce ACD-like symptoms in the ears of mice and describe several methods for assessing the severity of dermatitis during modeling. This experimental protocol has been successfully applied in some experiments and has a certain promotional role in the field of ACD research.

Here, we describe the methods for inducing allergic contact dermatitis in mouse ears by 1-fluoro-2,4-dinitrobenzene (DNFB) and how to evaluate the severity of allergic contact dermatitis.

Skin is the human body&#39;s first line of defense and one of the most exposed organs to environmental chemicals. Allergic contact dermatitis (ACD) is a ...

Visualizing and Quantifying Pharmaceutical Compounds within Skin using Coherent Raman Scattering Imaging

Cutaneous pharmacokinetics (cPK) after topical formulation application has been a research area of particular interest for regulatory and drug development scientists to mechanistically understand topical bioavailability (BA). Semi-invasive techniques, such as tape-stripping, dermal microdialysis, or dermal open-flow microperfusion, all quantify macroscale cPK. While these techniques have provided vast cPK knowledge, the community lacks a mechanistic understanding of active pharmaceutical ingredient (API) penetration and permeation at the cellular level. 
One noninvasive approach to address microscale cPK is coherent Raman scattering imaging (CRI), which selectively targets intrinsic molecular vibrations without the need for extrinsic labels or chemical modification. CRI has two main methods-coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS)-that enable sensitive and selective quantification of APIs or inactive ingredients. CARS is typically utilized to derive structural skin information or visualize chemical contrast. In contrast, the SRS signal, which is linear with molecular concentration, is used to quantify APIs or inactive ingredients within skin stratifications. 
Although mouse tissue has commonly been utilized for cPK with CRI, topical BA and bioequivalence (BE) must ultimately be assessed in human tissue before regulatory approval. This paper presents a methodology to prepare and image ex vivo skin to be used in quantitative pharmacokinetic CRI studies in the evaluation of topical BA and BE. This methodology enables reliable and reproducible API quantification within human and mouse skin over time. The concentrations within lipid-rich and lipid-poor compartments, as well as total API concentration over time are quantified; these are utilized for estimates of micro- and macroscale BA and, potentially, BE.

A coherent Raman scattering imaging methodology to visualize and quantify pharmaceutical compounds within the skin is described. This paper describes skin tissue preparation (human and mouse) and topical formulation application, image acquisition to quantify spatiotemporal concentration profiles, and preliminary pharmacokinetic analysis to assess topical drug delivery.

Cutaneous pharmacokinetics (cPK) after topical formulation application has been a research area of particular interest for regulatory and drug development ...

2.1K Views.  Capital Medical University.  This protocol outlines the procedure for inducing acne inflammation in rat skin with oleic acid and Cutibacterium acnes.

Establishing a Compound Acne Model in Rats Using Oleic Acid and C. acnes

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