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><tbody><tr><td>1-Fluoro-2,4-dinitrobenzene (DNFB)</td><td>Merck</td><td>200-734-3</td><td>1-Fluoro-2,4-dinitrobenzene, &amp;ge;99%</td></tr><tr><td>Acetone</td><td>Sinopharm Chemical Reagent Co. LTD</td><td>10000418</td><td>&amp;ge;99.5%</td></tr><tr><td>Aluminum foil&amp;nbsp;</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 &amp;mu;l</td></tr><tr><td>Pipettor</td><td>Eppendorf AG</td><td>3123000250</td><td>20 - 200 &amp;mu;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

Research

JoVE Journal

Immunology and Infection

3.3K Views.  Yangzhou University. Dit model kan niet alleen worden gebruikt om de pathogenese van ACD te onderzoeken, maar is ook toepasbaar voor de preklinische evolutie van nieuwe therapeutische methoden, die een belangrijke betekenis heeft. Dit is een stabiel en efficiënt diermodel met veel voordelen, zoals een korte experimentele periode, eenvoudige bediening, duidelijke symptomen en een hoog slagingspercentage. Ons experimentele protocol is zeer gedetailleerd en eenvoudig te bedienen.Zorg ervoor dat u elke stap v...

Video: A Mouse Ear Model for Allergic Contact Dermatitis Evaluation

Murine Model of Allergen Induced Asthma

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, with the prevalence increasing.2 As with other diseases, animal models of allergic airway disease greatly facilitate understanding of the underlying pathophysiology, help identify potential therapeutic targets, and allow preclinical testing of possible new therapies. Models of allergic airway disease have been developed in several animal species, but murine models are particularly attractive due to the low cost, ready availability, and well-characterized immune systems of these animals.3 Availability of a variety of transgenic strains further increases the attractiveness of these models.4 Here we describe two murine models of allergic airway disease, both employing ovalbumin as the antigen. Following initial sensitization by intraperitoneal injection, one model delivers the antigen challenge by nebulization, the other by intratracheal delivery. These two models offer complementary advantages, with each mimicking the major features of human asthma.5
The major features of acute asthma include an exaggerated airway response to stimuli such as methacholine (airway hyperresponsiveness; AHR) and eosinophil-rich airway inflammation. These are also prominent effects of allergen challenge in our murine models,5,6 and we describe techniques for measuring them and thus evaluating the effects of experimental manipulation. Specifically, we describe both invasive7 and non-invasive8 techniques for measuring airway hyperresponsiveness as well as methods for assessing infiltration of inflammatory cells into the airways and the lung. Airway inflammatory cells are collected by bronchoalveolar lavage while lung histopathology is used to assess markers of inflammation throughout the organ. These techniques provide powerful tools for studying asthma in ways that would not be possible in humans.

Experimental mouse models of allergic asthma offer new possibilities for studying disease pathogenesis and developing new therapeutics. These models are well suited to measuring factors governing the allergic immune response, airway inflammation, and pulmonary pathophysiology.

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, ...

Whole-mount Confocal Microscopy for Adult Ear Skin: A Model System to Study Neuro-vascular Branching Morphogenesis and Immune Cell Distribution

Here, we present a protocol of a whole-mount adult ear skin imaging technique to study comprehensive three-dimensional neuro-vascular branching morphogenesis and patterning, as well as immune cell distribution at a cellular level. The analysis of peripheral nerve and blood vessel anatomical structures in adult tissues provides some insights into the understanding of functional neuro-vascular wiring and neuro-vascular degeneration in pathological conditions such as wound healing. As a highly informative model system, we have focused our studies on adult ear skin, which is readily accessible for dissection. Our simple and reproducible protocol provides an accurate depiction of the cellular components in the entire skin, such as peripheral nerves (sensory axons, sympathetic axons, and Schwann cells), blood vessels (endothelial cells and vascular smooth muscle cells), and inflammatory cells. We believe this protocol will pave the way to investigate morphological abnormalities in peripheral nerves and blood vessels as well as the inflammation in the adult ear skin under different pathological conditions.

Here, we describe a high resolution whole-mount imaging method in the entire adult mouse ear skin, which enables us to visualize branching morphogenesis and patterning of peripheral nerves and blood vessels, as well as immune cell distribution.

Here, we present a protocol of a whole-mount adult ear skin imaging technique to study comprehensive three-dimensional neuro-vascular branching ...

Developmental Biology

Measuring Local Anaphylaxis in Mice

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. Exposure to an allergen in a sensitized individual can result in clinical symptoms that vary from minor erythema to life threatening anaphylaxis. In the laboratory, various animal models have been developed to understand the mechanisms driving allergic responses. Herein, we describe a detailed method for measuring changes in vascular permeability to quantify localized allergic responses. The local anaphylaxis assay was first reported in the 1920s, and has been adapted from the technique published by Kojima et al. in 20071. In this assay, mice sensitized to OVA are challenged in the left ear with vehicle and in the right ear with OVA. This is followed by an intravenous injection of Evans Blue dye. Ten min after injecting Evans Blue, the animal is euthanized and the dye that has extravasated into the ears is extracted overnight in formamide. The absorbance of the extracted dye is then quantified with a spectrophotometer. This method reliably results in a visual and quantifiable manifestation of a local allergic response.

Allergic responses, characterized by the activation of mast cells and basophils, are driven by the cross-linking of IgE and release of proinflammatory mediators. A quantitative assessment of allergic responses can be achieved by using Evans Blue dye to monitor changes in vascular permeability after allergen challenge.

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. ...

Measuring Changes in Tactile Sensitivity in the Hind Paw of Mice Using an Electronic von Frey Apparatus

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain perception in rodents. Withdrawal thresholds can be measured first at baseline and then following drug, venom, injury, allergen,&#160;or otherwise evoked inflammation by applying an accurate force on very specific areas of the skin. An electronic von Frey apparatus allows precise assessment of mouse hind paw withdrawal thresholds that are not limited by the available filament sizes in contrast to classical von Frey measurements. The ease and rapidity of measurements allow for incorporation of assessment of tactile sensitivity outcomes in diverse models of rapid-onset inflammatory and neuropathic pain as multiple measurements can be taken within a short time period. Experimental measurements for individual rodent subjects can be internally controlled against individual baseline responses and exclusion criteria easily established to standardize baseline responses within and across experimental groups. Thus, measurements using an electronic von Frey apparatus represent a useful modification of the well-established classical von Frey filament-based assays for rodent mechanical allodynia that may also be applied to other nonhuman mammalian models.

Electronic von Frey measurements are an effective and improved alternative to classical von Frey filaments, as they allow rapid and precise measurement of changes in rodent mechanical withdrawal thresholds to continuously applied pressure stimuli before and after an inflammatory event.

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain ...

Neuroscience

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...

Cheek Injection Model for Simultaneous Measurement of Pain and Itch-related Behaviors

Itch was defined as &#34;an unpleasant cutaneous sensation that provokes a desire to scratch&#34;&#160;by Rothman in 1941. In mouse models, scratch bouts are typically counted to evaluate itch induced by pruritogens. However, previous reports have shown that algesic substances also induce scratching behaviors in a mouse neck injection model, which is the most common test used for scratching behaviors. This finding makes it difficult to study itch in mice. &#160;In contrast, capsaicin, a common algogen, reduced scratching behaviors in some neck injection experiments. Therefore, the effect of pain on scratching behaviors remains unclear. It is thus necessary to develop a method to concurrently investigate itch and pain sensation using behavioral tests. Here, a cheek injection model is introduced which can be used to simultaneously measure pain- and itch-related behaviors. In this model, pruritogens induce scratching behaviors while algesic substances induce wiping behaviors. Using this model, lysophosphatidic acid (LPA), an itch mediator found in cholestatic patients with itch, is shown to exclusively induce itch but not pain. However, in mouse models, LPA has been reported to be both a pruritogen and an algogen. Investigation into the effects of LPA in a mouse cheek injection model showed that LPA only induced scratching, but not wiping behaviors. This indicates that LPA acts as a pruritogen similarly in mice and humans, and demonstrates the utility of a cheek injection model for itch research.

Typically, the mouse neck injection model is used for evaluate pruritogen-induced scratch behaviors. However, the model provides information only on itch, not pain. Here, a cheek injection model is introduced in mice which can be used to simultaneously measure pain and itch-related behaviors.

Itch was defined as &#34;an unpleasant cutaneous sensation that provokes a desire to scratch&#34;&#160;by Rothman in 1941. In mouse models, scratch bouts ...

Analysis of Granulocyte-Macrophage Colony-Stimulating Factor-Producing T Helper Cells in a Mouse Model of Contact Hypersensitivity

Parallel to traditional Th1/Th2/Th17/Treg lineages, granulocyte-macrophage colony-stimulating factor-producing T helper (Th-GM) cells have been identified as a distinct subset of T helper cells (GM-CSF+ IFN-&#947;- IL-17A- IL-22- effector CD4+ T cells) in human and mice. Contact hypersensitivity (CHS) is considered an excellent animal model for allergic contact dermatitis (ACD) in human, manifesting an intact T cell-mediated immune response. To provide a standardized and comprehensive assay to analyze the Th-GM cell subset in the T cell-dependent immune response in vivo, a murine CHS model was induced by sensitization/challenge with a reactive, low-molecular-weight, organic hapten, 2,4-dinitrofluorobenzene (DNFB). The Th-GM subset in effector CD4+ T cells generated upon immunization with the hapten was analyzed by flow cytometry. We found that Th-GM was mainly expanded in lesions and draining lymph nodes in the DNFB-induced CHS mouse model. This method can be applied to further study the biology of Th-GM cells and pharmacological research of therapeutic strategies centered on GM-CSF in various conditions, such as ACD.

Here, we present a simple and standardized method of analyzing the granulocyte-macrophage colony-stimulating factor-producing T helper subset in vivo.

Parallel to traditional Th1/Th2/Th17/Treg lineages, granulocyte-macrophage colony-stimulating factor-producing T helper (Th-GM) cells have been identified ...

Contact Hypersensitivity as a Murine Model of Allergic Contact Dermatitis

Contact hypersensitivity (CHS) is an experimental model of allergic contact dermatitis (ACD) that can be studied in mice. This study aims to present an objective laboratory method that may help to study the CHS reaction in mice, which can be measured and quantified by various tests. To induce CHS, on day "0", mice were sensitized on a previously shaved spot by abdominal skin painting with the hapten 2,4,6-trinitrochlorobenzene (TNCB) in an acetone-ethanol mixture, whereas negative control mice were sham sensitized with vehicle alone-acetone-ethanol mixture. On day "4", the baseline ear thickness was measured with a micrometer prior to the elicitation of CHS (challenge) by painting both ears with diluted TNCB both in the test and control groups. After 24 h, the ear swelling was measured with a micrometer. CHS is an example of a T cell-mediated immune response that causes swelling in inflamed tissue, peaking 24 h after the skin challenge with the same hapten. An increase in ear edema correlated with augmented ear weight, myeloperoxidase (MPO) activity, pro-inflammatory cytokine concentration in the ear extracts, increased thickening of the edematous dermis in the histological examination, and ear vascular permeability. There was also an increase in the concentration of TNP-specific IgG1 antibodies in the sera of the test group when compared with the control mice. Additionally, CHS can be successfully transferred with the CHS-effector cells obtained from donors previously sensitized with TNCB. The CHS-effector cells were administered intravenously into na&#239;ve recipient mice, which were subsequently challenged with the same diluted hapten. Ear swelling was measured with a micrometer 24 h later.

Contact hypersensitivity (CHS) is a murine experimental model of allergic contact dermatitis (ACD). CHS is based on sensitization with reactive hapten by painting the shaved skin of the chest and abdomen, with a subsequent ear skin challenge with a diluted hapten, causing a swelling reaction that is assessed in various ways.

Contact hypersensitivity (CHS) is an experimental model of allergic contact dermatitis (ACD) that can be studied in mice. This study aims to present an ...

Minimum Erythematous Dose Assay: A Method for Measuring UV Sensitivity in Mouse Models

Source: Amaro-Ortiz, A. et al. <a href="https://www.jove.com/v/50670"> Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model</a>. J. Vis. Exp. (2013)
This video describes a method to measure UV sensitivity in a humanized mouse model&#8212;a mouse model that mimics human skin. We determine the minimal erythematous dose (MED) of UV radiation that can cause erythema or edema on the skin. The method can help to find out the efficacy of drugs used to reduce UV sensitivity.

Source: Amaro-Ortiz, A. et al.  Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model. J. Vis. Exp. ...

JoVE Encyclopedia of Experiments

Cancer Research

Skin Cancer

Animal model studies

Malassezia-Induced Skin Inflammation and Fungal Burden Analysis in a Murine Model

To initiate a Malassezia infection, confirm a lack of response to pedal reflex in an anesthetized six- to eight-week-old female C57/Black/6 mouse, and apply ointment to the animal&#39;s eyes. Use a caliper to measure the thickness of two different areas of both ears to allow calculation of the average thickness per ear. Then, apply a small piece of tape to the skin of one ear before quickly removing the tape from the ear skin five times using a new piece of tape each time.
After stripping, use a sterile pipette to apply 100 microliters of the Malassezia olive oil suspension to the dorsal side of each ear, and inject 200 microliters of sterile pre-warmed 2% glucose solution subcutaneously into the nuchal fold to support metabolism and rehydration. Then, allow the mouse to recover on a heating pad for 30 minutes with monitoring before returning the animal to its cage.
At the appropriate experimental time points, use a caliper to measure two different areas of each mouse ear to allow calculation of the average thickness per ear. To measure the fungal burden in the infected skin, add 500 microliters of sterile 0.05% NP40 and distilled water in an autoclaved 5-milliliter diameter steel ball to one 2-milliliter microcentrifuge tube per ear, and weigh each tube on a balance.
Next, harvest the ears from each animal at the base, and place the tissues into individual tubes of NP40. Weigh each tube on the balance to determine the weight of each ear sample before homogenizing the ear tissues for six minutes at 25 hertz. Then, dispense 100 microliters of each sample onto m-Dixon agar plates and distribute the suspensions homogeneously across the agar before their upside-down incubation at 30 degrees Celsius.

A Mouse Ear Model for Allergic Contact Dermatitis Evaluation

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