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.

From day 7 to day 25, the ears in the OA group and the OA + C. acnes group were thickened and indurated, accompanied by erythema, scales, and comedones. The rats were observed to be scratching their ears from time to time, which caused bleeding. On day 25, papules were found in the ears of the C. acnes group and the OA + C. acnes group (Figure 3).

Histopathology
Parakeratosis and hyperkeratosis were observed in the hair follicles, along with hypertrophy of the granular layer and spinous layer, and dilation of the hair follicle infundibulum in the ears of the OA group and the OA + C. acnes group. The dermis of the C. acnes and OA + C. acnes groups showed mixed inflammatory cell infiltration, including lymphocytes and neutrophils. Also, the dermis exhibited a significant presence of erythrocyte extravasation and abscesses in C. acnes and OA + C. acnes (Figure 4). As shown in Figure 5, the pathological scoring revealed an increase in the C. acnes group and OA group compared to the NC group and the score was significantly higher in the OA + C. acnes group than in the NC and OA groups (p < 0.05).

IHC
As shown in Figure 6, TNF-α was highly expressed in the dermis of the C. acnes and OA + C. acnes groups.

Figure 1: Sample images of pathological scores. Inflammatory cells are marked with red arrows. Refer to Table 1 for the scoring assignment. (A) Score = 0, no horn in infundibulum; (B) score = 1, epithelial hyperplasia and a dense, consistent accumulation of horn distending the follicular canal, though not severe; (C) score = 1, a small number of inflammatory cells around the follicles; (D) score = 2, widespread infiltration of inflammatory cells in the dermis; (E) score = 3, accumulation of dense horn that expands the infundibulum and extends into the sebaceous ducts, resembling the comedones of clinical cases; (F) presence of abscesses around the infundibulum that can potentially result in papules or pustule. Scale bars = 100 µm. Please click here to view a larger version of this figure.

Figure 2: The changing trends of ear thickness from day 0 to day 25. The NC group is indicated by the red line, the OA group is indicated by the blue line, the C. acnes group is indicated by the green line, and the OA and C. acnes group is indicated by the yellow line. Please click here to view a larger version of this figure.

Figure 3: Gross appearance of ears. The comedones and papules are denoted by red arrows. Please click here to view a larger version of this figure.

Figure 4: Histology of rat ears. (▲) Hyperkeratosis; (■) parakeratosis; (•) expanded infundibulum of the hair follicle; (♦) Infiltration of inflammatory cells. Please click here to view a larger version of this figure.

Figure 5: Pathological score. @ indicates a statistically significant difference between the NC group and the C. acnes group; # indicates a significant difference between the NC group and the OA + C. acnes group; * indicates a significant difference between the OA + C. acnes group and the OA group (p < 0.05). Please click here to view a larger version of this figure.

Figure 6: IHC images of TNF-α expression in rat ears. (♦) Infiltration of inflammatory cells. Please click here to view a larger version of this figure.

Score	Characteristics
0	Non-comedogenic. (see Figure 1 A)
1	Epithelial hyperplasia and a dense, consistent accumulation of horn beginning to distend the follicular canal. (see Figure 1 B)
1	The presence of a small number of inflammatory cells, especially around the follicles or sebaceous ducts. (see Figure 1 C)
2	Widespread infiltration of a large number of inflammatory cells. (see Figure 1 D)
3	The presence of accumulation of dense horn that expands the infundibulum and extends into the sebaceous ducts, along with notable hyperplasia of follicular epithelium. (see Figure 1 E)
3	The presence of abscesses. (see Figure 1 F)

Table 1: Pathological score assignment. Sample images are shown in Figure 1.

Time	NC group (mm)	OA group (mm)	C. acnes group (mm)	OA + C. acnes group (mm)
Day 0	0.4000±0.0250	0.4167±0.0144	0.4500±0.0000	0.4667±0.0382
Day 7	0.5250±0.0661	0.7667±0.0878*▲	0.4500±0.0000	0.7917±0.0764*▲
Day 14	0.5083±0.0289	0.8167±0.1233*▲	0.6083±0.0382	0.9333±0.0804*▲
Day 21	0.5250±0.0433	1.0667±0.2241*▲	0.5583±0.0144	0.9417±0.0722*▲
Day 25	0.5750±0.0500	0.8250±0.1392*	0.9500±0.1639*	1.0583±0.0804*

Table 2: Thickness of the ears. Data are displayed as mean ± standard deviation. Symbol "^*" indicates a statistically significant difference compared with the NC group; Symbol "^▲" indicates a statistically significant difference compared with the C. acnes group (p < 0.05).

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.

Some researchers only adopt a single methodology to cause acne inflammation, such as applying oils or injecting microbes^14,16. However, studies have shown that increased sebum secretion will promote the excessive proliferation of C. acnes in clogged follicles, while C. acnes in turn stimulates the abnormal secretion of sebaceous glands; the interplay between both may exacerbate acne inflammation. Therefore, the compound model induced by oils and C. acnes may be more consistent with the clinical lesions of acne^10,21,22. To objectively assess the pathological alterations across different experimental groups, we devised a scoring system inspired by prior research, which includes the keratin accumulation and the resulting enlargement of hair follicle canals and epithelial proliferation, the infiltration of inflammatory cells in the dermis, and even the further development of abscess (Table 1 and Figure 1)^16,23. Utilizing this scoring method, along with histopathological and immunohistochemical (IHC) analyses, we found that the application of C. acnes and oleic acid to the ears led to more significant dermal disorder and inflammation. These findings mirror the symptoms observed in moderate cases of human acne, underscoring the value of this composite model in mimicking the development of acne (Figure 4 and Figure 5)²⁴.

To better evaluate the model, it is necessary to record the thickness of the ears before applying oil and C. acnes. Considering that the external and internal thickness of the ear are different, we recorded two specific points to ensure that the results were better to represent the thickness of the ears and two operators collaborated while measuring the ears. When applying oleic acid to the ear, attention should be paid to the fact that the fluidity of oleic acid makes it easily flow into the ear canal and irritate the rats; hence, it is necessary to tilt the head of rats to avoid oleic acid going into the ear canal.

Comedones were observed on day 7, and the lesions continued to be aggravated until the end of the experiment. The stimulation with C. acnes is another important factor affecting acne. C. acnes not only promotes lipid synthesis in sebaceous glands but also reacts with inflammatory cells around the hair follicle and sebaceous glands in the dermis to induce inflammatory factors; this further aggravates inflammatory response^6,25. Therefore, we use intradermal injection of C. acnes to accelerate the inflammation. Considering that the ears of rats are extremely thin, micro needles are needed to avoid piercing them. 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. In addition, when one operator carries out the intradermal injection, another operator must fix the head of the rats to prevent the ears from being pierced. To prevent hemorrhage, the injection site must avoid the superficial blood vessels of the rats. After being injected with C. acnes, the ears were significantly rougher, redder, and more swollen and found with papules (Figure 3).

The applications of sebum/sebum components and C. acnes vary in different experiments. Some researchers adopted the method of injection with C. acnes once and then application with sebum for seven days, which induced abscess, erythema, and induration on the back of rats¹⁹. Some other researchers injected C. acnes for 4 days and smeared sebum for 30 days on the ears of rats¹⁸. There is no unified standard because acne inflammation is related to the application of sebum/sebum components, the activity and concentration of C. acnes, and the tissues for the model. In this experiment, instead of artificial sebum, oleic acid was used for simulating sebum, considering that oleic acid not only induces keratinization of hair follicles, stimulates epidermis inflammation, but also promotes the growth of C. acnes^6,7,8. In the experiment, 7 days after the application of oleic acid, the rats' ears showed redness and swelling. From day 7 to day 14, the rat ears began to display scales and comedones. From day 15 to day 21, comedones increased, but no further acne symptoms were observed. On day 21, we began to intradermally inject 1 × 10⁷ CFU C. acnes, a widely used approach documented in previous studies¹⁹.

At first, the effects were not obvious, but later, we found papules in the ears of the rats injected with C. acnes. Given that the ear thickness did not show significant variation over time among the groups, we decided to terminate the experiment on day 25 (Figure 2 and Table 2). This protocol still has some limitations. The small size of the ear area may affect follow-up drug treatment. C. acnes is not the only microbiota involved in the pathological process of acne, so it may be better to use a variety of bacteria to induce acne to mimic the clinical condition²⁶.

Materials

List of materials used in this article

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