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 &lt;i&gt;C. acnes&lt;/i&gt;

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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. 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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 &lt;i&gt;C. acnes&lt;/i&gt; 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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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.

1.3K 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

Summary