A Rat Model of Compound Acne

Summary

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

Abstract

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). 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)-α 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.

Introduction

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 challenges^2,3.

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 unit^4,5. 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-α^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 ear^12,13,14,15. The methods include intradermal injection of C. acnes, 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 acne^16,17,18,19. 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.

Protocol

This protocol has received ethical approval from the Beijing University of Chinese Medicine (No.2023033103-1183). Twelve male Sprague-Dawley rats (weight, 208 g ± 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

1. Measure and record the thickness of rat ears using an electronic vernier caliper (see Table of Materials) before modeling.
   1. 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.
2. Tilt the rat's head to one side, make the ear face upwards, and apply 50 µL of oleic acid (see Table of Materials) to the ventral and dorsal sides of the ear evenly, once a day, for 25 days.
3. From day 21 to day 25, daily prior to the application of oleic acid, anesthetize the rats using 4% isoflurane and then inject 50 µL of 1 × 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.
4. 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.

2. Tissue collection and analysis

1. 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.
2. Immerse the ear tissues in 4% paraformaldehyde for 24 h, followed by paraffin-embedding and sectioning into slices 5 µm 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).
3. 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.
4. Wash the sections with phosphate-buffered saline (PBS) and then incubate them with 3% BSA for 30 min at room temperature.
5. Incubate the sections with TNF-α monoclonal antibody (dilution 1:800) (see Table of Materials) at 4 °C overnight.
6. Wash the sections with PBS again and incubate with horseradish peroxidase-conjugated secondary antibody (see Table of Materials) for 50 min.
7. After another PBS wash, apply 3,3'-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.
8. Counterstain the sections with Meyer'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.

3. Statistical analysis

1. Perform statistical analysis.
2. Present data as mean ± 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's multiple comparisons. A p-value less than 0.05 is considered statistically significant.

Results

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 < 0.05). The average thickness of the ears during the experiment is shown in Figure 2 and Table 2.

Discussion

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.

Materials

Name	Company	Catalog Number	Comments
Anaero-indicator	Mitsubishi, Japan	C-22
AnaeroPack	Mitsubishi, Japan	C-11, C-41
Columbia blood agar plate	BeNa Cuture Collection, China	BNCC330605
Constant temperature incubator	SHANGCHENG, China	303-0
Cotton swab	HYNAUT, China	_
Cutibacterium acnes	BeNa Cuture Collection, China	BNCC330605
Dako REAL EnVision Detection System, Peroxidase/DAB+, Rabbit/Mouse	DAKO, Denmark	K5007
disposable sterile injection needle	Zhejiang Oujian Medical Apparatus, China	_
Electronic scale	JINXUAN, China	A017
Electronic vernier caliper	Deli, China	DL90150
Oleic acid (Analytical reagent, AR)	Fangzheng, China	_
Sodium chloride injection	CR Double CRANE, China	Y2212241
SPSS Statistics	IBM, USA	26.0
sterile hypodermic syringes	Shandong weigao group medical polymer, China	_
TNF Alpha Monoclonal antibody	Proteintech Group,int, USA	60291-1-lg