Name: a mouse ear model for allergic contact dermatitis evaluation
Uploaded: 2023-03-24T14:55:00
Description: Watch this Scientific Journal Video about A Mouse Ear Model for Allergic Contact Dermatitis Evaluation at JoVE.com

This work was supported by the National Natural Science Foundation of China (NSFC) to N.-N. Y. (81904212);&#160;Jiangsu Traditional Chinese Medicine Science and Technology Project (YB201995); and the Special Funding Project for Postdoctoral Researchers in China (2020T130562).

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 &#177; 2 &#176;C, 12 h light/dark cycle) with ...

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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...

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&#39;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 responses3,5,6. 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 mice7,8, guinea pigs9,10, 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1-Fluoro-2,4-dinitrobenzene (DNFB)</td>
			<td>Merck</td>
			<td>200-734-3</td>
			<td>1-Fluoro-2,4-dinitrobenzene, &#8805;99%</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Sinopharm Chemical Reagent Co. LTD</td>
			<td>10000418</td>
			<td>&#8805;99.5%</td>
		</tr>
		<tr>
			<td>Aluminum foil&#160;</td>
			<td>Cleanwrap</td>
			<td>CF-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Evans blue dye</td>
			<td>Solarbio</td>
			<td>314-13-6</td>
			<td>Dye content approx. 80%</td>
		</tr>
		<tr>
			<td>Mouse fixator</td>
			<td>ZHUYANBANG</td>
			<td>GEGD-SM1830</td>
			<td></td>
		</tr>
		<tr>
			<td>Olive oil</td>
			<td>Solarbio</td>
			<td>8001-25-0</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>Pipet tip</td>
			<td>Biofount</td>
			<td>FT-200</td>
			<td>10 - 200 &#956;l</td>
		</tr>
		<tr>
			<td>Pipettor</td>
			<td>Eppendorf AG</td>
			<td>3123000250</td>
			<td>20 - 200 &#956;l</td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>Shanghai Gillette Co. LTD</td>
			<td>74-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Vernier calipers</td>
			<td>Delixi Electric</td>
			<td>DECHOTVCS1200</td>
			<td></td>
		</tr>
	</tbody>
</table>

a mouse ear model for allergic contact dermatitis evaluation

Skin is the human body&#39;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.

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...

Watch this Scientific Journal Video about A Mouse Ear Model for Allergic Contact Dermatitis Evaluation at JoVE.com

A Mouse Ear Model for Allergic Contact Dermatitis Evaluation

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.

Skin is the human body&#39;s first line of defense and one of the most exposed organs to environmental chemicals. Allergic contact dermatitis (ACD) is a ...

This model can not only be used to explore the pathogenesis of ACD, but also is applicable for the preclinical evolution of new therapeutic methods, which has an important meaning. This is a stable and efficient animal model with many advantages, such as a short experimental period, easy operation, obvious symptoms, and a high success rate. Our experimental protocol is very detailed and easy to operate.Ensure to follow each step of the protocol correctly. Before starting the modeling, acclimatize the mice to the environment for one week with free access to food and water. On the day of modeling, using an ultraviolet lamp and 75%alcohol, clean and disinfect the environment and countertops.Apply soapy water to the abdomen of the mice using a cotton swab, and shave the area in the direction of hair growth with a blade or shaver. Weigh the mouse and compare the weight changes among each group. Before performing abdominal sensitization stimulation, ensure the full recovery of any minor injury to the mouse abdomen skin induced by shaving.Prepare 0.5%DNFB solution and dilute it with acetone to olive oil mixture at a 4 to 1 ratio. Using a pipette gun, blow and mix 20 times to thoroughly mix the DNFB solution. Apply 25 microliters of the 0.5%DNFB solution over the middle of the abdominal shaving area with a pipetter, and lightly spread with the smooth side of the pipetter tip to disperse it uniformly.After 30 seconds of DNFB stimulation, place the mouse in an empty cage without bedding to prevent the mouse from rubbing off the DNFB solution. When the solution is completely dry, return the mouse to its original cage with free access to food and water. For ear sensitization stimulation, orient the mouse body and make the outside border of the auricle face downward to prevent the solution from entering the ear canal during DNFB stimulation.On days 4, and 10, using a pipetter, apply 20 microliters of the vehicle, or 0.2%DNFB solution slowly and uniformly to the inner surface of the left auricle, and gently distribute the solution using the smooth side of the pipetter. Leave the right ear untreated. Wait until the DNFB solution is dry and place the mouse back in the cage.After abdominal and ear sensitization stimulation, weigh the mice every two days starting on day one. Compare the weight of each mouse to day zero, and evaluate the effect of ACD on the body weight of the mice as weight changes. Capture high-resolution photos of the mouse ears to record ACD clinical symptoms every two days, starting on day one.Using vernier calipers, measure the auricle thickness at the same time each day for accurate results. 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 for both ears.Record the average of the three data and evaluate the ear swelling in micrometers. To evaluate the degree of inflammatory swelling on day 11, prepare 0.5%Evans Blue dye solution and dilute it with PBS. To immobilize the mouse with a fixator, open the lid, hold the mouse tail, make the head of the mouse face the fixator, and allow 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. Wipe the tail repeatedly with an alcohol cotton ball, or soak it in warm water for 30 seconds, and gently pinch the root of the tail to fill, and expand the veins on both sides.Slowly inject Evans Blue Dye solution into the mouse tail vein using a 1 mm insulin needle under the irradiation of a cold light source. Wait for 15 minutes to take pictures of the mouse ears. Mouse ears of the DNFB group displayed evident clinical symptoms comparable to ACD, with sensitive areas showing the typical symptoms of redness, dryness, erosion, and exudation.However, ear administration of pure water or solvent control did not produce any symptoms similar to DNFB group. In the DNFB group, compared to the untreated right ear, The thickness of the left ear increased significantly after DNFB stimulation. There was no significant difference in the control and vehicle groups.The left ear of the DNFB group mice turned dark blue after the injection of Evans Blue dye, which was visually different from the right ear. The left and right ears of mice in control and vehicle groups were approximately the same color. Mouse weight gain was slightly slowed by DNFB, or simple vehicle stimulation, but did not result in significant weight loss.Repeated DNFB stimulation in the mouse ear resulted in spleen enlargement and an increase in the spleen index. In contrast, the spleen index of mice in the vehicle group did not change significantly. Ensure the DINFB solution is fully mixed and evenly applied to the mouse ear without flowing into the ear canal.This is the key to the success of modeling.

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