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

<ol>
	<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.

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.</...

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

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

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 ...

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.

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2.0K Views.  Capital Medical University. 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 ...

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

Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain barrier1. The barrier selectively excludes molecules from crossing between the blood and the brain based upon their size and charge. This function can impede the delivery of therapeutics for neurological disorders. A number of chemotherapeutic drugs, for example, will not effectively cross the blood-brain barrier to reach tumor cells2. Thus, improving the delivery of drugs across the blood-brain barrier is an area of interest.
The most prevalent methods for enhancing the delivery of drugs to the brain are direct cerebral infusion and blood-brain barrier disruption3. Direct intracerebral infusion guarantees that therapies reach the brain; however, this method has a limited ability to disperse the drug4. Blood-brain barrier disruption (BBBD) allows drugs to flow directly from the circulatory system into the brain and thus more effectively reach dispersed tumor cells. Three methods of barrier disruption include osmotic barrier disruption, pharmacological barrier disruption, and focused ultrasound with microbubbles. Osmotic disruption, pioneered by Neuwelt, uses a hypertonic solution of 25% mannitol that dehydrates the cells of the blood-brain barrier causing them to shrink and disrupt their tight junctions. Barrier disruption can also be accomplished pharmacologically with vasoactive compounds such as histamine5 and bradykinin6. This method, however, is selective primarily for the brain-tumor barrier7. Additionally, RMP-7, an analog of the peptide bradykinin, was found to be inferior when compared head-to-head with osmotic BBBD with 25% mannitol8. Another method, focused ultrasound (FUS) in conjunction with microbubble ultrasound contrast agents, has also been shown to reversibly open the blood-brain barrier9. In comparison to FUS, though, 25% mannitol has a longer history of safety in human patients that makes it a proven tool for translational research10-12.
In order to accomplish BBBD, mannitol must be delivered at a high rate directly into the brain's arterial circulation. In humans, an endovascular catheter is guided to the brain where rapid, direct flow can be accomplished. This protocol models human BBBD as closely as possible. Following a cut-down to the bifurcation of the common carotid artery, a catheter is inserted retrograde into the ECA and used to deliver mannitol directly into the internal carotid artery (ICA) circulation. Propofol and N2O anesthesia are used for their ability to maximize the effectiveness of barrier disruption13. If executed properly, this procedure has the ability to safely, effectively, and reversibly open the blood-brain barrier and improve the delivery of drugs that do not ordinarily reach the brain 8,13,14.

Blood-brain barrier disruption aids the delivery of certain drugs to the brain. Mannitol delivered intra-arterially shrinks cells surrounding blood vessels in order to physically disrupt the barrier.

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain ...

A Model of Chronic Nutrient Infusion in the Rat

Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.

A protocol for chronic infusions of glucose and Intralipid in rats is described. This model can be used to study the impact of nutrient excess on organ function and physiological parameters.

Chronic exposure to excessive levels of nutrients is postulated to  affect the function of several organs and tissues and to contribute to the  ...

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and &#8220;pearls of wisdom&#8221;/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

Intrarenal Injection of Escherichia coli in a Rat Model of Pyelonephritis

Pyelonephritis is a bacterial infection of the kidney and is most commonly caused by Escherichia coli. Recurrent infections can cause significant renal inflammation and fibrosis ultimately resulting in declining kidney function. Before improved clinical management and prevention of pyelonephritis can be instituted, a reliable animal model must be established in order to study the mechanisms of progression, recurrence, and therapeutic efficacy. The transurethral infection model closely mimics human pyelonephritis but exhibits considerable variation due to its reliance on urethral reflux to transport the bacteria to the kidney. Herein, a detailed surgical protocol for performing bacterial injections into the rat renal pelvis is provided and confirmed by non-invasive Magnetic Resonance Imaging (MRI). Using this protocol, animals receive direct exposure to a desired concentration of E. coli bacteria and can fully recover from the surgical procedure with adequate post-operative care. This facilitates subsequent longitudinal MRI assessments of the experimental animal models for comparison with saline (sham) controls. Using this direct delivery approach, the severity of infection is controllable and applicable for mechanistic studies of progression as well as development of novel treatment strategies.

Intrarenal Injection of Escherichia coli in a Rat Model of Pyelonephritis

This manuscript describes a rat surgical model of pyelonephritis using direct intra-renal infection by Escherichia coli into the renal pelvis. The experimental procedure can be utilized to study the pathogenesis of pyelonephritis as well as the associated inflammation and fibrosis.

Pyelonephritis is a bacterial infection of the kidney and is most commonly caused by Escherichia coli. Recurrent infections can cause significant renal ...

A Model of Free Tissue Transfer: The Rat Epigastric Free Flap

Free tissue transfer has been increasingly used in clinical practice since the 1970s, allowing reconstruction of complex and otherwise untreatable defects resulting from tumor extirpation, trauma, infections, malformations or burns. Free flaps are particularly useful for reconstructing highly complex anatomical regions, like those of the head and neck, the hand, the foot and the perineum. Moreover, basic and translational research in the area of free tissue transfer is of great clinical potential. Notwithstanding, surgical trainees and researchers are frequently deterred from using microsurgical models of tissue transfer, due to lack of information regarding the technical aspects involved in the operative procedures. The aim of this paper is to present the steps required to transfer a fasciocutaneous epigastric free flap to the neck in the rat.
This flap is based on the superficial epigastric artery and vein, which originates from and drain into the femoral artery and vein, respectively. On average the caliber of the superficial epigastric vein is 0.6 to 0.8 mm, contrasting with the 0.3 to 0.5 mm of the superficial epigastric artery. Histologically, the flap is a composite block of tissues, containing skin (epidermis and dermis), a layer of fat tissue (panniculus adiposus), a layer of striated muscle (panniculus carnosus), and a layer of loose areolar tissue.
Succinctly, the epigastric flap is raised on its pedicle vessels that are then anastomosed to the external jugular vein and to the carotid artery on the ventral surface of the rat's neck. According to our experience, this model guarantees the complete survival of approximately 70 to 80% of epigastric flaps transferred to the neck region. The flap can be evaluated whenever needed by visual inspection. Hence, the authors believe this is a good experimental model for microsurgical research and training.

This paper describes the steps required to raise a fasciocutaneous epigastric free flap and transfer it to the neck in the rat.

Free tissue transfer has been increasingly used in clinical practice since the 1970s, allowing reconstruction of complex and otherwise untreatable defects ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Acupuncture in a Rat Model of Asthma

A high platform can fix rats without restriction and completely expose the acupoints on the back during acupuncture manipulation. This article describes methods for the fabrication of the high platform, establishes a rat model of asthma and measures changes in respiratory function using a noninvasive and real-time whole-body plethysmography (WBP) system.

A high platform can fix rats without restriction and completely expose the acupoints on the back during acupuncture manipulation. This article describes ...

Retinal Pathophysiological Evaluation in a Rat Model

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal detachment, breakdown of the blood-retinal barrier (BRB), and retinal angiogenesis. An in vivo rat model is a valuable experimental tool to examine the changes in the structure and function of the retina. We propose three different experimental techniques in the rat model to identify morphological changes of retinal cells, retinal vasculature, and compromised BRB. Retinal histology is used to study the morphology of various retinal cells. Also, quantitative measurement is performed by retinal cell count and thickness measurement of different retinal layers. A BRB breakdown assay is used to determine the leakage of extraocular proteins from the plasma to vitreous tissue due to the breakdown of BRB. Fluorescence angiography is used to study angiogenesis and leakage of blood vessels by visualizing retinal vasculature using FITC-dextran dye.

Diabetic retinopathy is one of the leading causes of blindness. Histology, blood-retinal barrier breakdown assay, and fluorescence angiography are valuable techniques to understand the pathophysiology of the retina, which could further enhance the efficient drug screening against diabetic retinopathy.

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal ...

Development of a Uterosacral Ligament Suspension Rat Model

Pelvic organ prolapse (POP) is a common pelvic floor disorder (PFD) with the potential to significantly impact a woman's quality of life. Approximately 10%-20% of women undergo pelvic floor repair surgery to treat prolapse in the United States. PFD cases result in an overall $26.3 billion annual cost in the United States alone. This multifactorial condition has a negative impact on the quality of life and yet the treatment options have only dwindled in the recent past. One common surgical option is uterosacral ligament suspension (USLS), which is typically performed by affixing the vaginal vault to the uterosacral ligament in the pelvis. This repair has a lower incidence of complications compared to those with mesh augmentation, but is notable for a relatively high failure rate of up to 40%. Considering the lack of standard animal models to study pelvic floor dysfunction, there is an urgent clinical need for innovation in this field with a focus on developing cost-effective and accessible animal models. In this manuscript, we describe a rat model of USLS involving a complete hysterectomy followed by fixation of the remaining vaginal vault to the uterosacral ligament. The goal of this model is to mimic the procedure performed on women to be able to use the model to then investigate reparative strategies that improve the mechanical integrity of the ligament attachment. Importantly, we also describe the development of an in situ tensile testing procedure to characterize interface integrity at chosen time points following surgical intervention. Overall, this model will be a useful tool for future studies that investigate treatment options for POP repair via USLS.

Pelvic organ prolapse affects millions of women worldwide and yet some common surgical interventions have failure rates as high as 40%. The lack of standard animal models to investigate this condition impedes progress. We propose the following protocol as a model for uterosacral ligament suspension and in vivo tensile testing.

Pelvic organ prolapse (POP) is a common pelvic floor disorder (PFD) with the potential to significantly impact a woman's quality of life. Approximately ...

A Protocol for Constructing a Rat Wound Model of Type 1 Diabetes

A single high dose of streptozotocin injection followed by full-thickness skin excision on the dorsum of rats is a common method for constructing animal models of type 1 diabetic wounds. However, improper manipulation can lead to model instability and high mortality in rats. Unfortunately, there are few existing guidelines on type 1 diabetic wound modeling, and they lack detail and do not present specific reference strategies. Therefore, this protocol details the complete procedure for constructing a type 1 diabetic wound model and analyzes the progression and angiogenic characteristics of the diabetic wounds. Type 1 diabetic wound modeling involves the following steps: preparation of the streptozotocin injection, induction of type 1 diabetes mellitus, and construction of the wound model. The wound area was measured on day 7 and day 14 after wounding, and the skin tissues of the rats were extracted for histopathological and immunofluorescence analysis. The results revealed that type 1 diabetes mellitus induced by 55 mg/kg streptozotocin was associated with lower mortality and a high success rate. The blood glucose levels were relatively stable after 5 weeks of induction. The diabetic wound healing rate was significantly lower than that of normal wounds on day 7 and day 14 (p &#60; 0.05), but both could reach more than 90% on day 14. Compared with the normal group, the epidermal layer closure of diabetic wounds on day 14 was incomplete and had delayed re-epithelialization and significantly lower angiogenesis (p &#60; 0.01). The type 1 diabetic wound model constructed based on this protocol has the characteristics of chronic wound healing, including poor closure, delayed re-epithelialization, and decreased angiogenesis compared to normal rat wounds.

The streptozotocin-induced diabetic wound model in male SD rats is currently the most widely used model for studying wound healing in type I diabetes mellitus. This protocol describes the methods used to construct this model. It also presents and addresses potential challenges and examines the progression and angiogenic characteristics of diabetic wounds.

A single high dose of streptozotocin injection followed by full-thickness skin excision on the dorsum of rats is a common method for constructing animal ...