A Mouse Ear Model for Allergic Contact Dermatitis Evaluation

Podsumowanie

Here, we describe the methods for inducing allergic contact dermatitis in mouse ears by 1-fluoro-2,4-dinitrobenzene (DNFB) and how to evaluate the severity of allergic contact dermatitis.

Streszczenie

Skin is the human body's first line of defense and one of the most exposed organs to environmental chemicals. Allergic contact dermatitis (ACD) is a common skin disease that manifests as a local rash, redness, and skin lesions. The occurrence and development of ACD are influenced by both genetic and environmental factors. Although many scholars have constructed a series of models of ACD in recent years, the experimental protocols of these models are all different, which makes it difficult for readers to establish them well. Therefore, a stable and efficient animal model is of great significance to further study the pathogenesis of atopic dermatitis. In this study, we detail a modeling method using 1-fluoro-2,4-dinitrobenzene (DNFB) to induce ACD-like symptoms in the ears of mice and describe several methods for assessing the severity of dermatitis during modeling. This experimental protocol has been successfully applied in some experiments and has a certain promotional role in the field of ACD research.

Wprowadzenie

Allergic contact dermatitis (ACD) is a common skin disease that is characterized by eczema-like symptoms at the contact site, edema and erythema in moderate cases, and papules, erosion, exudation, or even massive scars in severe cases¹. It affects up to 20% of the population and can affect people of any age². ACD often occurs in individuals who have been exposed to allergens repeatedly and can be caused by the individual's immune response to one or more allergens in their home or workplace³. Type IV delayed hypersensitivity is considered the main type of immune response in ACD⁴. In areas of the skin that have been repeatedly exposed to allergens, circulating memory T cells accumulate in large numbers and induce immune and inflammatory responses^3,5,6. The purpose of this work is to propose a reliable laboratory technique for further investigation of immunological and inflammatory responses in the development of ACD.

The onset of ACD is usually due to contact hypersensitivity caused by repeated exposure to chemicals. Numerous researchers have developed various ACD animal models in house mice^7,8, guinea pigs^9,10, and other animals over the course of the last few decades, in order to simulate the onset of the disease. Most of the experimental methods consist of two stages: abdominal sensitization (induction) and providing stimuli on the back or the ear lobe (stimulation). Commonly used chemical substances mainly include 1-fluoro-2,4-dinitrobenzene (DNFB)/1-chloro-2,4-dinitrobenzene (DNCB)^8,9,11, oxazolone¹², urushiol¹³, etc. Among them, DNFB and DNCB are the most widely used, first reported in October 1958¹⁰. The nickel sensitization model¹⁴ and the photoallergic contact dermatitis model¹⁵ are also frequently used.

We present an experimental method for building the ACD model. This method is summarized and optimized on the basis of previous studies and upon comparison with multiple experiments. Compared with other ACD models, this model has some advantages, such as small individual differences, short experimental periods, a small amount of chemical stimulation, etc. In addition, this study is applicable to mice, which are not only economical but also have more options for gene knockout or transgenic mice preparation¹⁶. We also describe the various methods used to monitor ACD progress in the experiment, such as measuring ear thickness, using Evans blue dye to measure inflammatory exudation, etc. This model can not only analyze mouse ears, blood, spleen, and other samples by laboratory means to explore the pathogenesis of ACD, but also is applicable for the preclinical evaluation of new therapeutic methods, which has a certain promotional significance.

Protokół

All the care and treatment of the mice were in accordance with the guidelines established by the Institutional Animal Care and Use Committee of Yangzhou University and were approved by the Institutional Animal Care and Use Committee under the project license SYXK(SU)2022-0044. BALB/c male and female mice aged 6-8 weeks were used in this study. Each group consisted of six mice (see Table of Materials). Cages were placed in a temperature-controlled chamber (22 ± 2 °C, 12 h light/dark cycle) with free access to food and water. An experimental flow diagram is shown in Figure 1.

1. Animal preparation

1. Start the modeling after 1 week of acclimatization to the environment.
2. Use an ultraviolet lamp and 75% alcohol disinfectant to clean and disinfect the environment and countertops prior to manipulating the mice.
   NOTE: In order to avoid the influence of external factors, marking the mice for identification cannot be performed on the mouse ear; staining on the back or on the tail can be used as an alternative.
3. Use a small cotton swab to apply soapy water to the abdomen of the mice (approximately 1-2 cm² in size). Shave the area in the direction of hair growth with a blade or shaver (see Table of Materials) at the beginning of modeling (day 0; Figure 2A).
   NOTE: The use of a straight razor blade for hair removal requires a skilled operator. If not performed correctly, it might cause skin irritation. Consider using depilatory cream, clippers, or a safety razor for hair removal.
4. Weigh the mouse and compare the weight changes among each group.

2. Abdominal sensitization stimulation

1. Ensure the full recovery of any minor injury to the abdomen skin induced by shaving. Apply abdominal sensitization 2 days after shaving (day 2).
2. Prepare the 0.5% DNFB solution: dilute DNFB with an acetone:olive oil mixture at a 4:1 ratio (e.g., 400 µL of acetone mixed with 100 µL of olive oil; see Table of Materials). Use a pipette gun to blow and mix 20 times to thoroughly mix the DNFB solution. Before each administration of DNFB solution to the mouse, blow and mix it three to five times.
   NOTE: Prepare the solution before use and wrap it in aluminum foil to protect it from direct sunlight.
3. Apply 25 µL of the 0.5% DNFB solution to the skin of the shaved area on the abdomen of the mice with a pipettor (Figure 2B).
4. Dribble the DNFB solution over the middle of the abdominal shaving area and lightly spread with the smooth side of the pipettor tip to uniformly disperse it.
5. At 30 s after DNFB stimulation, place the mice in empty cages without bedding to prevent them from rubbing off the DNFB solution. When the DNFB solution is completely dry (about 2 min), return the mice to their original cage.
6. Wear gloves when handling the DNFB solution as it is strongly irritating to human skin.

3. Ear sensitization stimulation

1. Prepare a 0.2% DNFB solution as above, the vehicle solution (a 4:1 mixture of acetone and olive oil), and pure water.
2. Orient the mouse body and make the outside border of the auricle face downward during the entire operation to prevent the solution from entering the ear canal during DNFB stimulation.
3. On days 4, 6, 8, and 10, use a pipettor to apply 20 µL of the 0.2% DNFB solution or vehicle solution slowly and uniformly to the inner surface of the left auricles of the mice. To avoid DNFB solution from entering the ear canal, use the smooth side of the pipettor tip to gently distribute the DNFB solution during administration. Leave the right ears untreated (Figure 2C).
4. Wait until the DNFB solution is dry and place the mice back in the cage (about 30 s).
5. Wear gloves when handling the DNFB solution.

4. Recording mouse weight and ACD symptoms

1. Weigh the mouse every day, start on day 1, and compare with its corresponding weight on day 0; evaluate the effect of ACD on the body weight of mice as weight change (g) ± standard error of the mean (SEM).
2. Take high resolution photos of the mouse ears to record ACD clinical symptoms every 2 days, starting on day 1.

5. Measurement of the auricle thickness

1. Measure the auricle thickness every 2 days, starting on day 1. Measure and record both ears in detail.
2. Use vernier calipers (see Table of Materials) to measure the auricle thickness at the same time each day for accurate results (Figure 3A). Stop the vernier calipers from continuing inward clamping when there is a slight blockage, to prevent tissue damage to the mouse ear. Keep the position fixed and record the data.
3. Collect the thickness from three different locations on each auricle (Figure 3B). Record the average of the three data as a valid value. Evaluate the ear swelling in micrometers (µm) ± standard error of the mean (SEM).

6. Evaluation of the degree of inflammatory swelling

1. Prepare 0.5% Evans blue dye (see Table of Materials) solution: dilute Evans blue dye with phosphate buffered saline (PBS) on day 11. Wear a lab coat and gloves at all times, as Evans blue dye is slightly toxic to humans.
2. Immobilize the mice with a fixator: open the lid of the fixator (see Table of Materials), hold the mouse tail, make the head of the mouse face the fixator, and make the mouse instinctively climb into the fixator. Cover the lid, make the mouse tail come out of the hole on the lid, and adjust the length of the fixator to expose the whole mouse tail.
3. Wipe the tail repeatedly with an alcohol cotton ball or soak it in warm water for 30 s, and gently pinch the root of the tail to fill and expand the veins on both sides. Perform the injection under the irradiation of a cold light source.
4. Slowly inject Evans blue dye solution into the mouse tail vein using a 1 mm insulin needle. Wait for 15 min and then take pictures of the mouse ears.
   NOTE: ​Put the mouse on the table and gently hold it to expose the ear region for image acquisition. Shortly after injection with the Evans blue dye solution and observing corresponding indications, use cervical dislocation to euthanize the mouse.

Wyniki

Under repeated DNFB stimulation, the mouse ears of the DNFB group displayed evident clinical symptoms comparable to ACD, with sensitive areas showing the typical symptoms of redness, dryness, and even erosion and exudation. However, ear administration of pure water (control group) or solvent control (vehicle group) did not produce similar symptoms (Figure 4).

Meanwhile, in the DNFB group, compared to the untreated right ear, the thickness of the left ear increased...

Dyskusje

The protocol described here for inducing ACD-like symptoms in the ears of mice can be used to study the pathophysiology of ACD and as a screening tool for the development of new drugs.

There are two key steps to establish an ACD model: initial sensitization, and subsequent stimulation. The abdomen is usually the site of initial sensitization, but the subsequent stimulation site was chosen slightly differently. Previous studies have shown that most scholars choose to use chemical sensitizers su...

Materiały

Name	Company	Catalog Number	Comments
1-Fluoro-2,4-dinitrobenzene (DNFB)	Merck	200-734-3	1-Fluoro-2,4-dinitrobenzene, ≥99%
Acetone	Sinopharm Chemical Reagent Co. LTD	10000418	≥99.5%
Aluminum foil	Cleanwrap	CF-2
Evans blue dye	Solarbio	314-13-6	Dye content approx. 80%
Mouse fixator	ZHUYANBANG	GEGD-SM1830
Olive oil	Solarbio	8001-25-0	500 ml
Pipet tip	Biofount	FT-200	10 - 200 μl
Pipettor	Eppendorf AG	3123000250	20 - 200 μl
Razor blade	Shanghai Gillette Co. LTD	74-S
Vernier calipers	Delixi Electric	DECHOTVCS1200