Establishment of Psoriasis Mouse Model by Imiquimod

Summary

Here, we present a highly efficacious and stable mouse model of psoriasis established through the utilization of imiquimod. Additionally, we detail the methodologies employed to assess the severity of psoriasis.

Abstract

Psoriasis is a common, chronic, relapsing, and inflammatory skin condition characterized by thickened skin infiltration, scales, and erythema. The pathogenesis of the disease is highly complex, involving the interplay of various factors such as genetics, immunity, and environmental factors. To investigate the mechanisms underlying psoriasis, researchers have developed multiple animal models of the disease. However, the variability in methodologies has presented challenges for constructing these models consistently. In this study, we established and comprehensively evaluated a highly efficient and stable psoriasis mouse model using imiquimod (IMQ). 8-week-old SPF-grade female C57BL/6J mice were used to establish the psoriasis mouse model. After one week of acclimatization, mice with irritated or damaged skin in the depilated areas were excluded. Starting on day two, 5% IMQ cream (62.5 mg/d) was applied daily to the depilated neck and back regions for seven consecutive days. During the experiment, daily measurements of body weight, skin photography, skin scoring, and behavioral observations were conducted. Post-experiment, spleen index determination, skin histological analysis, serum cytokine detection, and skin mRNA level detection were performed. The results showed that mice in the IMQ group developed typical psoriasis-like symptoms, including erythema, scales, and skin thickening, accompanied by increased scratching behavior, weight loss, elevated spleen index, and increased skin thickness. And significant changes in the levels of TNF-α, IL-23, and IL-17A in serum, as well as the mRNA levels of IL-23, IL-17A, IL-17F, and IFN-γ in the skin. Serum cytokine levels (TNF-α, IL-23, IL-17A) and skin mRNA expression of pro-inflammatory markers (IL-23, IL-17A, IL-17F, IFN-γ) were notably upregulated. In contrast, the control group exhibited no such alterations. By optimizing the IMQ induction duration and standardizing the administration protocol, while dynamically monitoring skin changes, this study aims to provide a standardized framework for researchers to construct reliable psoriasis models.

Introduction

Psoriasis is a common, chronic, relapsing, and inflammatory systemic skin disease, with plaque psoriasis being the most predominant clinical manifestation¹. Its clinical characteristics typically include skin infiltration, thickness, scales, and erythema², and it can also trigger a variety of complications, including psoriatic arthritis, depression, metabolic syndrome, and cardiovascular diseases¹. Additionally, psoriasis is often accompanied by chronic pruritus³. Globally, psoriasis impacts 2% to 3% of the population⁴, and its incidence is on the rise. Therefore, establishing reliable animal models that closely mimic the pathological features of human psoriasis is crucial for uncovering its pathogenesis and developing new therapeutic strategies.

In the field of dermatological research, psoriasis has garnered significant attention due to its complex pathogenesis, which involves the interplay of genetic, immunological, and environmental factors, presenting considerable challenges for in-depth study¹. Current research indicates that the onset of psoriasis is primarily mediated by T cells, which, upon activation, stimulate keratinocytes to overproliferate and alter the secretion of cytokines during their differentiation process⁵. In this process, the Interleukin (IL)-23/IL-17 axis plays a pivotal role. IL-23 promotes the differentiation and activation of Th17 cells, which produce cytokines such as IL-17A and IL-17F, playing a significant role in the development of psoriasis^6,7,8. Currently, the pathogenesis of psoriasis encompasses numerous molecular alterations. These involve cytokines such as tumor necrosis factor-α (TNF-α), the IL-23/IL-17 axis, IL-37, intracellular signaling pathways like nuclear factor-κB (NF-κB), and Janus kinase-signal transduction and transcriptional activator (JAK-STAT). Moreover, it is closely associated with molecules related to sugar, the tricarboxylic acid cycle (TCA), amino acids, lipid metabolism pathways, as well as epigenetic regulatory factors, including DNA methylation and histone modification. These targets are intricately intertwined and interact with one another, jointly driving the onset and progression of psoriasis⁹.

In current psoriasis research, a variety of animal models are employed, including spontaneous models, allogeneic transplant models, transgenic or gene knockout models, and chemically induced models, among others¹⁰. These animal models play an essential role in the study of psoriasis, with scientists having utilized multiple species such as mice, guinea pigs, and zebrafish to construct psoriasis models^11,12,13. However, these models all have certain limitations, including genetic complexity, species differences, ethical concerns, and cost considerations¹⁴.

Imiquimod (IMQ), as an agonist of Toll-like receptors (TLRs), can trigger skin inflammation and immune cell infiltration, while also promoting the proliferation of keratinocytes and the enhancement of dermal blood vessels¹³. A study has reported that the topical application of IMQ in humans can induce psoriasis¹⁵. Clinical observations have also revealed that IMQ exacerbates psoriasis symptoms in patients⁷. In mice psoriasis models, continuous administration of a 5% concentration of IMQ can reproduce the cutaneous manifestations of human psoriasis, such as erythema and scales. Additionally, other symptoms closely mimic those of human plaque psoriasis, including epidermal changes like acanthosis and parakeratosis, neoangiogenesis, and so on. The psoriasis mouse models induced by IMQ are also able to simulate chronic pruritus⁴. There is also an infiltration of inflammatory cells consisting of T cells, neutrophils, and dendritic cells in psoriasis mouse models^2,16. The IMQ-induced skin lesions mainly rely on the TNF-α-IL-23/IL-17 axis, indicating that IMQ-based psoriasis mouse models can be utilized to further investigate human psoriasis⁹.

The IMQ-induced psoriasis mouse model employed in this study is suitable for mouse strains such as C57BL/6 and BALB/c, which have Th1 and Th2 tendencies, respectively⁷. This model is stable and highly efficient, with the advantages of low cost, short experimental duration, simple operation, distinct symptoms, and a high success rate. In the experiment, we provided a detailed account of several methods employed to monitor the progression of psoriasis. These methods encompassed skin scoring and behavioral testing. Additionally, we further validated the experimental findings through the utilization of histopathological analysis, enzyme-linked immunosorbent assay (ELISA), and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). This model is not only suitable for exploring the pathogenesis of psoriasis but also for preclinical evaluation of new therapeutic approaches, making it a valuable tool in research.

Protocol

The animal use protocol listed below has been reviewed and approved by the Guangxi University of Chinese Medicine Institutional Welfare and Ethical Committee (DW20191220-153). The animal certificate of the purchased mice was SCXK (Xiang) 2024-0009. In this study, 8-week-old C57BL/6J female mice, SPF grade, were randomly divided into groups, each group containing 6 mice. The cage is placed in a temperature-controlled room (23 ± 2 °C, 12 h light/dark cycle) with free access to food and water. The flow chart of the experiment is shown in Figure 1. Except for not being induced with IMQ cream, the mice in the control group received the same treatments as those in the IMQ group in all other respects.

1. Animal preparation

1. Start modeling after adapting the animal to the environment for a week. Before starting the experiment, meticulously disinfect the experimental table using a 75% ethanol solution.
2. Using a marker pen, clearly mark the tails of the mice, ensuring their individual identification.
3. Use the shaver to shave the hair of the necks and backs of all mice for an area of about 4 cm x 3 cm (day 0; Figure 2A). Use a cotton swab to apply an appropriate amount of depilatory cream to the shaved skin and spread it evenly (Figure 2B).
4. After applying the depilatory cream, wait for about 15 s. Leave the depilatory cream on the mouse's skin surface for an optimal 15-20 s. Wipe gently and quickly.
5. Use a new cotton swab to soak it in water and repeatedly wipe in the direction from the mouse's tail to the head to clean the hair. Gently dry the local skin with medical absorbent paper.
6. Examine the skin condition of mice 24 h after depilation and exclude those with damaged depilation areas.

2. IMQ-induced psoriasis mouse model

1. Ensure that the slight skin damage caused by shaving or depilating is fully recovered.
2. Starting from the 2^nd day, apply 5% IMQ cream at a dosage of 62.5 mg⁷. To do this, pre-fill the entire 1 mL syringe with IMQ cream in advance and administer the drug to the mouse according to the volume corresponding to 62.5 mg of IMQ per day to the depilated skin of the mouse using a flat-handled spoon (Figure 2C). During application, ensure that the IMQ cream is spread as evenly as possible over the depilated area, and the application area is smaller than the depilated area.
3. After smearing the drug, place the mouse in an empty cage and wait for 15 min. Put the mouse back into the feeding cage after the drug is completely absorbed. Perform the drug application continuously for 7 days.

3. Body weight analysis of psoriasis mice

1. Place a clean beaker on an electronic balance and tare it. Then, gently place the mouse inside and note the stable readings displayed on the balance.
2. From day 1 (the day preceding the initial application of IMQ), weigh the mice daily until day 8. Compare the weight of each mouse with its corresponding weight on day 1.

4. Skin characterization of psoriasis mice

1. Place the mice on a white background with a ruler. Keep the mice's bodies straight without shrinking their heads or making large swings (Figure 3).
2. From day 1, take a high-resolution photograph of the administration site of the mice under the same lighting intensity every day and record the skin characterization of the mice.
3. During each photography session, keep the distance between the camera and the mice consistent, as well as the camera's magnification, and position the ruler perpendicular to the camera.

5. Skin scoring

1. Score the severity of psoriasis dermatitis in mice using the modified Psoriasis Area and Severity Index (PASI)¹⁷ every day from day 1 to day 8. Employ three researchers using a double-blind method to independently score erythema, scales, and skin thickness on a scale of 0 to 4, taking the arithmetic mean of the three. For PASI scoring rules, check Table 1.
   PASI score = erythema + scale + thickness

6. Behavioral observation of mice

1. In the same quiet, normally lit environment at room temperature (as in step 4), place each mouse in an individual acrylic box (10 cm x 10 cm) with a small amount of corn cob padding at the bottom.
2. Use black partitions to separate each box and block the line of sight. After a 15 min adaptation period, video-record the animals' behavior for 1 h.
3. Record behavioral videos at a fixed time each day from day 1 to day 8. Position the camera lens perpendicular to the acrylic box and ensure it fully captures the mouse's activities inside.
4. Record instances where the mouse lifts its hind paw to scratch the neck and back and then lowers it as one itch behavior.

7. Collecting the blood and measuring the levels of cytokines in the serum by ELISA

1. Remove the eyeballs of the mice quickly with forceps and collect the blood on day 9.
2. Leave the blood to stand at 4 °C for 4 h. Separate the blood by using a low-temperature centrifuge at 3000 x g for 30 min at 4 °C. Collect the serum and store it at -80 °C for future use.
3. Quantify the serum TNF-α, IL-17A, and IL-23 levels using the relevant ELISA kit according to the manufacturer's protocol¹⁸.

8. Sampling the skin and the spleen

1. According to the regulations of the relevant animal experiment ethics committee, dislocate the cervical vertebrae of mice for euthanasia after blood collection at day 9.
2. After euthanasia, cut the skin lesions on the mice's backs into two portions. Immerse one portion in a 5 mL centrifuge tube with a 4% paraformaldehyde solution for fixation and preservation. Separate and package the other portion of skin tissues and store them at -80 °C for future use.
3. Use sterilized scissors to cut open the skin along the midline of the mouse's abdomen, expose the abdominal cavity, and find and completely remove the spleen. Wash the samples with sterile normal saline, then put them into normal saline, weigh them, and record the data.

9. Histological analyses of skin sections

1. Remove the skin tissue from the paraformaldehyde and place it in the embedding box.
2. Perform the following steps on the skin: Use gradient alcohol from 75% to 100% (from low to high concentration), with three tanks of 100% alcohol. Leave it in each tank for 30 min at room temperature. Use two tanks of xylene. Leave it in each tank for 30 min at room temperature. Use three tanks of paraffin wax with a melting point of 56-58 °C. Leave it in each tank for 1 h at room temperature.
3. Embed the treated skin tissue: Place the wax-impregnated skin tissue upright in a paraffin mold. Pour the melted paraffin into the mold when the embedding paraffin temperature is 5-7 °C higher than the melting point of paraffin. Quickly move it into the freezing table to cool and solidify the wax block rapidly.
4. Freeze the wax block to 0 °C, remove it from the embedding box, fix it on the slicer's clamping table, and slice it to a thickness of 3 µm.
5. Use tweezers to pick up the slices and blanch them in warm water at 45 °C. Attach the section to the center of the smooth part of the slide and mark the pathological number at the frosted area with a pencil. Tilt the slide at 45° for a moment to allow the moisture to flow out, then place it in an oven at 62 °C and bake for 30 min.
6. HE staining: Place the slices in xylene for 5 min, then in another tank of xylene for 5 min, in anhydrous ethanol for 1 min, in 95% ethanol for 1 min, in 85% ethanol for 1 min, and in 75% ethanol for 1 min. Rinse with tap water for 1 min, stain with hematoxylin solution for 5 min, and wash off the hematoxylin solution under running water for 1 min.
7. Soak in 1% hydrochloric acid ethanol for 3 s, wash with water for 20 s, use warm water for blue return for 5 min, rinse with running water for 2 min, stain with 0.5% eosin for 1 min, soak in 95% ethanol for 2 s, then in 95% ethanol for 2 min, in anhydrous ethanol for 4 min, in another tank of anhydrous ethanol for 4 min, in xylene for 4 min, in another tank of xylene for 4 min, and in the last tank of xylene for 4 min. After air-drying, seal it with neutral gum.
8. Observe the sections under a microscope and randomly take pictures at three positions at a magnification of 400. For each picture, randomly measure the epidermal thickness at five locations using Image J software. Record and analyze the data.

10. Measuring the mRNA levels of IL-17A, IL-17F, IL-23 and IFN-γ in the skin by RT-qPCR

1. Take the skin tissue stored in a refrigerator at -80 °C, with a weight of approximately 100 mg, and extract RNA by the Trizol method.
2. Reverse transcribe into cDNA according to the reverse transcription kit (VAZYME, China).
3. Configure the PCR reaction system according to the manual and conduct Real-Time PCR using a fluorescence quantitative PCR instrument under the specified reaction procedure (Table 2) and primer sequence (Table 3).
4. Collect real-time fluorescence quantitative PCR data, calculate and analyze the results using the qPCR algorithm (2-ΔΔCt method).

Results

After continuous administration of IMQ, mice in the IMQ group displayed back skin clinical symptoms reminiscent of psoriasis (Figure 4), with the IMQ-induced area presenting the typical symptoms of erythema, scales, and thickness (Figure 5). In contrast, the control group did not exhibit any symptoms akin to those of the IMQ group. Compared to the control group, erythema and minor scales began to emerge in the IMQ group from day 3 (Figure 5A...

Discussion

Our research utilizes IMQ to induce psoriasis-like skin lesions, thereby constructing a psoriasis model. This model can be used to study the mechanisms underlying the development of Psoriasis and to provide insights for the development of treatments for the condition. In the study by van der Fits⁷, when initially administering IMQ induction, the induction time was set at either 5 or 6 days. Nevertheless, Jabeen et al. suggested that the severity of IMQ-induced psoriasis models would reach a maximu...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
5% Imiquimod Cream	Sichuan Medshine Pharmaceutical Co.	40220602
75% Ethanol	XUE HUAN	20240401
Analytical Balance	sartorius	SQP
Depilatory Cream	Reckitt Benckiser	20240612 08X
Electronic Scale	Yingheng Technology	7915
Medical Tissue Paper	JD	20221208
Shaver	HORD shenjian	AUX-C6S
Sterilized Cotton Swab	Beilun River	20230728