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 free access to food and water. An experimental flow diagram is shown in Figure 1.
1. Animal preparation
<ol>
	<li>Start the modeling after 1 week of acclimatization to the environment.</li>
	<li>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.</li>
	<li>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:&#160;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.</li>
	<li>Weigh the mouse and compare the weight changes among each group.</li>
</ol>
2. Abdominal sensitization stimulation
<ol>
	<li>Ensure the full recovery of any minor injury to the abdomen skin induced by shaving. Apply abdominal sensitization 2 days after shaving (day 2).</li>
	<li>Prepare the 0.5% DNFB solution: dilute DNFB with an acetone:olive oil mixture at a 4:1 ratio (e.g., 400 &#181;L of acetone mixed with 100 &#181;L 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.</li>
	<li>Apply 25 &#181;L of the 0.5% DNFB solution to the skin of the shaved area on the abdomen of the mice with a pipettor (Figure 2B).</li>
	<li>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.</li>
	<li>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.</li>
	<li>Wear gloves when handling the DNFB solution as it is strongly irritating to human skin.</li>
</ol>
3. Ear sensitization stimulation
<ol>
	<li>Prepare a 0.2% DNFB solution as above, the vehicle solution (a 4:1 mixture of acetone and olive oil), and pure water.</li>
	<li>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.</li>
	<li>On days 4, 6, 8, and 10, use a pipettor to apply 20 &#181;L 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).</li>
	<li>Wait until the DNFB solution is dry and place the mice back in the cage (about 30 s).</li>
	<li>Wear gloves when handling the DNFB solution.</li>
</ol>
4. Recording mouse weight and ACD symptoms
<ol>
	<li>Weigh the mouse every day, start&#160;on day 1, and compare with its corresponding weight on day 0;&#160;evaluate the effect of ACD on the body weight of mice as weight change (g) &#177; standard error of the mean (SEM).</li>
	<li>Take high resolution photos of the mouse ears to record ACD clinical symptoms every 2 days, starting on day 1.</li>
</ol>
5. Measurement of the auricle thickness
<ol>
	<li>Measure the auricle thickness every 2 days, starting on day 1. Measure and record both ears in detail.</li>
	<li>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.</li>
	<li>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 (&#181;m) &#177; standard error of the mean (SEM).</li>
</ol>
6. Evaluation of the degree of inflammatory swelling
<ol>
	<li>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.</li>
	<li>Immobilize the mice with a fixator:&#160;open the lid of the fixator (see Table of Materials),&#160;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.</li>
	<li>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.</li>
	<li>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: &#8203;Put the mouse on the table and gently hold it&#160;to expose the ear region for image acquisition. Shortly after injection with the Evans blue dye solution and observing&#160;corresponding indications, use cervical dislocation to euthanize the mouse.</li>
</ol>

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F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+chemical-induced+innate+immunity+in+allergic+contact+dermatitis.">Mechanisms of chemical-induced innate immunity in allergic contact dermatitis.</a> Allergy. 66 (9), 1152-1163 (2011).</li><li>Kimber, I., Basketter, D. A., Gerberick, G. F., Dearman, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allergic+contact+dermatitis.">Allergic contact dermatitis.</a> International Immunopharmacology. 2 (2-3), 201-211 (2002).</li><li>Vocanson, M., Hennino, A., Rozieres, A., Poyet, G., Nicolas, J. 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Use of C14 labelled 2:4-dinitrochlorobenzene in guinea pigs.</a> The Journal of Investigative Dermatology. 31 (2), 97-102 (1958).</li><li>Knop, J., Riechmann, R., Macher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+suppressor+mechanism+in+allergic+contact+dermatitis.+IV.+Selective+inhibition+of+suppressor+T-lymphocytes+by+serum+obtained+from+Corynebacterium+parvum+treated+mice.">Modulation of suppressor mechanism in allergic contact dermatitis. IV. Selective inhibition of suppressor T-lymphocytes by serum obtained from Corynebacterium parvum treated mice.</a> The Journal of Investigative Dermatology. 77 (6), 469-473 (1981).</li><li>Rubic-Schneider, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GPR91+deficiency+exacerbates+allergic+contact+dermatitis+while+reducing+arthritic+disease+in+mice.">GPR91 deficiency exacerbates allergic contact dermatitis while reducing arthritic disease in mice.</a> Allergy. 72 (3), 444-452 (2017).</li><li>Stampf, J. 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C., Kaidbey, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+photoallergic+contact+dermatitis:+a+mouse+model.">Experimental photoallergic contact dermatitis: a mouse model.</a> The Journal of Investigative Dermatology. 79 (3), 147-152 (1982).</li><li>Qiu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dysregulated+sebum-microbial+metabolite-IL-33+axis+initiates+skin+inflammation+in+atopic+dermatitis.">A dysregulated sebum-microbial metabolite-IL-33 axis initiates skin inflammation in atopic dermatitis.</a> The Journal of Experimental Medicine. 219 (10), e2021397 (2022).</li><li>Kim, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-inflammatory+activities+of+Dictamnus+dasycarpus+Turcz.,+root+bark+on+allergic+contact+dermatitis+induced+by+dinitrofluorobenzene+in+mice.">Anti-inflammatory activities of Dictamnus dasycarpus Turcz., root bark on allergic contact dermatitis induced by dinitrofluorobenzene in mice.</a> Journal of Ethnopharmacology. 149 (2), 471-477 (2013).</li><li>Zhou, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+6'-acetylpaeoniflorin+on+dinitrochlorobenzene-induced+allergic+contact+dermatitis+in+BALB/c+mice.">Effect of 6'-acetylpaeoniflorin on dinitrochlorobenzene-induced allergic contact dermatitis in BALB/c mice.</a> Immunologic Research. 64 (4), 857-868 (2016).</li><li>Donglang, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+on+different+skin+pruritus+mouse+models.">Comparative study on different skin pruritus mouse models.</a> Frontiers in Medicine. 8, 630237 (2021).</li><li>Yang, N., Shao, H., Deng, J., Liu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Network+pharmacology-based+analysis+to+explore+the+therapeutic+mechanism+of+Cortex+Dictamni+on+atopic+dermatitis.">Network pharmacology-based analysis to explore the therapeutic mechanism of Cortex Dictamni on atopic dermatitis.</a> Journal of Ethnopharmacology. 304, 116023 (2023).</li></ol>

The authors report no conflicts of interest in this work.

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 ...

a-mouse-ear-model-for-allergic-contact-dermatitis-evaluation

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Immunology and Infection

2.8K Views.  Yangzhou University. 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.

Video: A Mouse Ear Model for Allergic Contact Dermatitis Evaluation

Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis

Tissue transglutaminase 2 (tTG2) is an intestinal digestive enzyme which deamidates already partially digested dietary gluten e.g. gliadin peptides. In genetically predisposed individuals, tTG2 triggers autoimmune responses that are characterized by the production of tTG2 antibodies and their direct deposition into small intestinal wall 1,2. The presence of such antibodies constitutes one of the major hallmarks of the celiac disease (CD). Epidermal transglutaminase (eTG) is another member of the transglutaminase family that can also function as an autoantigen in a small minority of CD patients. In these relatively rare cases, eTG triggers an autoimmune reaction (a skin rash) clinically known as dermatitis herpetiformis (DH). Although the exact mechanism of CD and DH pathogenesis is not well understood, it is known that tTG2 and eTG share antigenic epitopes that can be recognized by serum antibodies from both CD and DH patients 3,4.
In this study, the confocal microscopy examination of biopsy samples from skin lesions of two rhesus macaques (Macaca mulatta) with dermatitis (Table 1, Fig. 1 and 2) was used to study the affected tissues. In one animal (EM96) a spectral overlap of IgA and tTG2 antibodies (Fig. 3) was demonstrated. The presence of double-positive tTG2+IgA+ cells was focused in the deep epidermis, around the dermal papillae. This is consistent with lesions described in DH patients 3. When EM96 was placed on a gluten-free diet, the dermatitis, as well as tTG2+IgA+ deposits disappeared and were no longer detectable (Figs. 1-3). Dermatitis reappeared however, based on re-introduction of dietary gluten in EM96 (not shown). In other macaques including animal with unrelated dermatitis, the tTG2+IgA+ deposits were not detected. Gluten-free diet-dependent remission of dermatitis in EM96 together with presence of tTG2+IgA+ cells in its skin suggest an autoimmune, DH-like mechanism for the development of this condition. This is the first report of DH-like dermatitis in any non-human primate.

Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis

Dermatitis herpetiformis (DH) is a chronic inflammatory condition characterized by an autoimmune reaction between IgA and epidermal transglutaminase (eTG). DH develops in a very small portion of gluten-sensitive and/or celiac patients. The results of this study indicate that DH can also develop in a rhesus monkey host with symptoms of idiopatic dermatitis.

Tissue transglutaminase 2 (tTG2) is an intestinal digestive enzyme which deamidates already partially digested dietary gluten e.g. gliadin peptides. In ...

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3.
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.

We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection.

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Effects of Exposure of Formaldehyde to a Rat Model of Atopic Dermatitis Induced by Neonatal Capsaicin Treatment

Atopic dermatitis is chronically relapsing pruritic eczema and prevails around the world especially in developed countries. Complex interactions between genetic and environmental factors are known to play an important role in the pathophysiology of atopic dermatitis. However, we still lack a detailed picture of the pathogenesis of this disease. Thus, it is of importance to develop appropriate animal models for elucidating the progression of atopic dermatitis. Moreover, investigating the effect of environmental factors such as air pollutants on atopic dermatitis expands understanding of the disease. Here, we describe a method for inducing atopic dermatitis in rats with neonatal capsaicin treatment and a protocol for exposure of a constant concentration of formaldehyde to rats to reveal effects on the development of atopic dermatitis in infantile and adolescent periods. These protocols have been successfully applied to several experiments and can be used for other substances.

We describe here methods for neonatal capsaicin treatment to induce atopic dermatitis in rats and exposure of vaporized formaldehyde to investigate the effects of formaldehyde inhalation on atopic dermatitis.

Atopic dermatitis is chronically relapsing pruritic eczema and prevails around the world especially in developed countries. Complex interactions between ...

A Controlled Mouse Model for Neonatal Polymicrobial Sepsis

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse polymicrobial sepsis can be induced by injecting cecal slurry intraperitoneally into day of life 7 mice and monitoring them for the following week. Presented here are the detailed steps necessary for the implementation of this neonatal sepsis model. This includes making a homogeneous cecal slurry stock, diluting it to a weight- and litter-adjusted dose, an outline of the monitoring schedule, and a definition of observed health categories used to define humane endpoints. The generation of a homogeneous cecal slurry stock from pooled donors allows for the administration into many litters over time, reducing the variation between donors, and preventing the use of potentially toxic glycerol. The monitoring strategy used allows for the anticipation of survival outcome and the identification of mice that would later progress to death, allowing for an earlier identification of the humane endpoint. Two main behavioral features are used to define the health scores, namely, the ability of the neonatal mice to right themselves when placed on their back and their level of mobility. These criteria could potentially be applied to address humane endpoints in other studies of neonatal disease in mice, as long as a pilot study is performed to confirm accuracy. In conclusion, this approach provides a standardized method to model newborn sepsis in mice, while providing resources to assess animal welfare used to define early humane endpoints for challenged animals.

This protocol provides the necessary steps to establish and evaluate neonatal sepsis in 7-day-old mice.

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse ...

Isolation and Quantitative Evaluation of Brush Cells from Mouse Tracheas

Tracheal brush cells are cholinergic chemosensory epithelial cells poised to transmit signals from the airway lumen to the immune and nervous systems. They are part of a family of chemosensory epithelial cells which include tuft cells in the intestinal mucosa, brush cells in the trachea, and solitary chemosensory and microvillous cells in the nasal mucosa. Chemosensory cells in different epithelial compartments share key intracellular markers and a core transcriptional signature, but also display significant transcriptional heterogeneity, likely reflective of the local tissue environment. Isolation of tracheal brush cells from single cell suspensions is required to define the function of these rare epithelial cells in detail, but their isolation is challenging, potentially due to the close interaction between tracheal brush cells and nerve endings or due to airway-specific composition of tight and adherens junctions. Here, we describe a procedure for isolation of brush cells from mouse tracheal epithelium. The method is based on an initial separation of tracheal epithelium from the submucosa, allowing for a subsequent shorter incubation of the epithelial sheet with papain. This procedure offers a rapid and convenient solution for flow cytometric sorting and functional analysis of viable tracheal brush cells.

Brush cells are rare cholinergic chemosensory epithelial cells found in the na&#239;ve mouse trachea. Due to their limited numbers, ex vivo evaluation of their functional role in airway immunity and remodeling is challenging. We describe a method for isolation of tracheal brush cells by flow cytometry.

Tracheal brush cells are cholinergic chemosensory epithelial cells poised to transmit signals from the airway lumen to the immune and nervous systems. ...

Contact-Free Co-Culture Model for the Study of Innate Immune Cell Activation During Respiratory Virus Infection

The early interactions between the nasal epithelial layer and the innate immune cells during viral infections remains an under-explored area. The significance of innate immunity signaling in viral infections has increased substantially as patients with respiratory infections who exhibit high innate T cell activation show a better disease outcome. Hence, dissecting these early innate immune interactions allows the elucidation of the processes that govern them and may facilitate the development of potential therapeutic targets and strategies for dampening or even preventing early progression of viral infections. This protocol details a versatile model that can be used to study early crosstalk, interactions, and activation of innate immune cells from factors secreted by virally infected airway epithelial cells. Using an H3N2 influenza virus (A/Aichi/2/1968) as the representative virus model, innate cell activation of co-cultured peripheral blood mononuclear cells (PBMCs) has been analyzed using flow cytometry to investigate the subsets of cells that are activated by the soluble factors released from the epithelium in response to the viral infection. The results demonstrate the gating strategy for differentiating the subsets of cells and reveal the clear differences between the activated populations of PBMCs and their crosstalk with the control and infected epithelium. The activated subsets can then be further analyzed to determine their functions as well as molecular changes specific to the cells. Findings from such a crosstalk investigation may uncover factors that are important for the activation of vital innate cell populations, which are beneficial in controlling and suppressing the progression of viral infection. Furthermore, these factors can be universally applied to different viral diseases, especially to newly emerging viruses, to dampen the impact of such viruses when they first circulate in na&#239;ve human populations.

This protocol details an investigation of the early interactions between virally infected nasal epithelial cells and innate cell activation. Individual subsets of immune cells can be distinguished based on their activation in response to viral infections. They can then be further investigated to determine their effects on early antiviral responses.

The early interactions between the nasal epithelial layer and the innate immune cells during viral infections remains an under-explored area. The ...

Therapeutic Evaluation of Fecal Microbiota Transplantation in an Interleukin 10-Deficient Mouse Model

With the development of microecology in recent years, the relationship between intestinal bacteria and inflammatory bowel disease (IBD) has attracted considerable attention. Accumulating evidence suggests that dysbiotic microbiota plays an active role in triggering or worsening the inflammatory process in IBD and that fecal microbiota transplantation (FMT) is an attractive therapeutic strategy since transferring a healthy microbiota to IBD patient could restore the appropriate host-microbiota communication. However, the molecular mechanisms are unclear, and the efficacy of FMT has not been very well established. Thus, further studies in animal models of IBD are necessary. In this method, we applied FMT from wild-type C57BL/6J mice to IL-10 deficient mice, a widely used mouse model of colitis. The study elaborates on collecting fecal pellets from the donor mice, making the fecal solution/suspension, administering the fecal solution, and monitoring the disease. We found that FMT significantly mitigated the cardiac impairment in IL-10 knockout mice, underlining its therapeutic potential for IBD management.

The interaction of genetic susceptibility, mucosal immunity, and intestinal microecological environment is involved in the pathogenesis of inflammatory bowel disease (IBD). In this study, we applied fecal microbiota transplantation to IL-10 deficient mice and investigated its impact on colonic inflammation and heart function.

With the development of microecology in recent years, the relationship between intestinal bacteria and inflammatory bowel disease (IBD) has attracted ...

Canine Intestinal Organoids in a Dual-Chamber Permeable Support System

The permeable support system is typically used in conjunction with traditional two-dimensional (2D) cell lines as an in vitro tool for evaluating the oral permeability of new therapeutic drug candidates. However, the use of these conventional cell lines has limitations, such as altered expression of tight junctions, partial cell differentiation, and the absence of key nuclear receptors. Despite these shortcomings, the Caco-2 and MDCK models are widely accepted and validated for the prediction of human in vivo oral permeability.
Dogs are a relevant translational model for biomedical research due to their similarities in gastrointestinal anatomy and intestinal microflora with humans. Accordingly, and in support of parallel drug development, the elaboration of an efficient and accurate in vitro tool for predicting in vivo drug permeability characteristics both in dogs and humans is highly desirable. Such a tool could be the canine intestinal organoid system, characterized by three-dimensional (3D), self-assembled epithelial structures derived from adult stem cells.
The (1) Permeable Support Seeding Protocol describes the experimental methods for dissociating and seeding canine organoids in the inserts. Canine organoid isolation, culture, and harvest have been previously described in a separate set of protocols in this special issue. Methods for general upkeep of the canine intestinal organoid monolayer are discussed thoroughly in the (2) Monolayer Maintenance Protocol. Additionally, this protocol describes methods to assess the structural integrity of the monolayer via transepithelial electrical resistance (TEER) measurements and light microscopy. Finally, the (3) Permeability Experimental Protocol&#160;describes the tasks directly preceding an experiment, including in vitro validation of experimental results.
Overall, the canine organoid model, combined with a dual-chamber cell culture technology, overcomes limitations associated with 2D experimental models, thereby improving the reliability of predictions of the apparent oral permeability of therapeutic drug candidates both in the canine and human patient.

Here, we present a protocol describing the culture of canine intestinal organoids in a dual-chamber, permeable support system. Organoid seeding in the permeable supports, the monolayer maintenance, and subsequent drug permeability experiments are described.

The permeable support system is typically used in conjunction with traditional two-dimensional (2D) cell lines as an in vitro tool for evaluating the oral ...