The authors would like to acknowledge Dr. Ellen M. Fox for her work regarding this model of local anaphylaxis. This work was supported by the Uniformed Services University of the Health Sciences grant number R073UE.

1. Sensitize Mice
<ol>
	<li>Prepare an OVA stock solution by diluting OVA in 1x PBS to a final concentration of 20 mg/ml. Freeze down aliquots in cryovials and store at -80 &#176;C for future use.</li>
	<li>Bring one or more aliquots of 20 mg/ml OVA stock to room temperature. Dilute OVA in 1x PBS to a final concentration of 1 mg/ml in a 5 ml polystyrene round bottom tube with a cap. Vortex briefly to mix.</li>
	<li>While holding the polystyrene tube containing the 1 mg/ml OVA on a vortex set at medium speed, add an equal volume of thoroughly homogenized Imject Alum (40 mg/ml) dropwise while continuing to vortex the tube to produce a 1:1 ratio of Alum to immunogen.</li>
	<li>Place cap on tube and tape tube to vortex. Turn vortex on lowest setting and allow OVA to adsorb to Alum for 30 min.
	<ol>
		<li>Do not allow a large amount of time to pass between preparing OVA/Alum and sensitizing mice. If sensitization cannot be completed directly after OVA/Alum preparation, store OVA/Alum at room temperature. Vortex for 1 min before using.</li>
	</ol>
	</li>
	<li>Load a 1 ml insulin syringe with OVA/Alum mixture. Cap the syringe with 27 gauge needle.</li>
	<li>Inject 100 &#956;l of OVA/Alum into the peritoneum of BALB/c mice for a dose per mouse of 50 &#956;g of OVA and 2 mg of Alum.</li>
	<li>For a negative control, sensitize another group of mice to PBS. Prepare a 1:1 mixture of Alum and PBS in same manner as listed above, omitting OVA.</li>
	<li>Sensitize all mice (OVA and PBS control) once per week for 3 weeks for a total of 3 injections. After final injection, wait at least 2 weeks before performing the local anaphylaxis assay.</li>
</ol>
2. Local Anaphylaxis Assay
<ol>
	<li>Bring OVA stock solution to room temperature. In a 1.5 ml microcentrifuge tube, dilute 20 mg/ml OVA stock to a final concentration of 5 mg/ml with PBS (1:4 dilution of OVA in 1X PBS). Vortex briefly to mix.</li>
	<li>Anesthetize mice using RC2 Rodent Anesthesia System (or comparable vaporized delivery system). Fill reservoir with isoflurane, and turn on O2 airflow. Place mice in induction chamber and adjust isoflurane control dial to 5% to initiate anesthetization. Once mice are fully anesthetized, indicated by lack of mobility, adjust isoflurane control dial to 3% to maintain deep anesthetization. The purpose of anesthesia in this procedure is to immobilize the animal. The ear and tail vein injections do not cause significant pain or distress to the animal.&#160; 
	NOTE: Isoflurane can have negative effects on the human reproductive system. Be sure to use in a well-ventilated room and attach a scavenging canister to the induction chamber to neutralize waste gas. If you are pregnant while using isoflurane, wear a carbon filter mask to minimize exposure.</li>
	<li>Load 10 &#956;l of 5 mg/ml OVA into a 3/10 cc insulin syringe capped with a 31 G needle. Load another 3/10 cc insulin syringe with 10 &#956;l of 1x PBS.</li>
	<li>Using a dissecting microscope, inject 10 &#956;l of OVA into the right ear of an anesthetized mouse for a challenge dose of 50 &#956;g of OVA. Other doses, such as 20 &#956;g, are also acceptable.
	<ol>
		<li>To stabilize the ear for injection, wrap scotch tape (sticky side up) around a 15 ml conical tube. Secure the dorsal side of the right ear to the tape. Insert the needle bevel side up between the two leaves of the ear. Slowly inject OVA into the center of the ear tissue, taking care not to hit any blood vessels.</li>
	</ol>
	</li>
	<li>Using the same method described above, inject 10 &#956;l of PBS into the left ear as a negative control.</li>
	<li>Wait 3 min.
	<ol>
		<li>During this time, place the mouse in a restrainer and hold the tail under a heat lamp or in a warm water bath for 1 min to dilate the blood vessels.</li>
	</ol>
	</li>
	<li>Using a 1 ml insulin syringe capped with a 27 G needle, slowly inject 200 &#956;l of 0.5% Evans Blue dye into the tail vein of the mouse. Rapid injection may destroy the vessel structure before the entire 200 &#956;l is administered. If this occurs, attempt to inject the remaining volume of dye into the vein on the opposite side of the tail.</li>
	<li>Wait 10 min.</li>
	<li>Euthanize the mouse and remove both ears using forceps and surgical scissors.
	<ol>
		<li>Grab the edges of the ear with forceps, making sure that no pressure is applied to the injection site at the center of the ear. Remove as much ear tissue as possible by cutting close to, but not through, the ridge of cartilage present at the base of the ear. Small amounts of fur may be present on the excised ear; this will not affect the assay.</li>
	</ol>
	</li>
	<li>Aliquot 700 &#956;l of formamide into 1.5 ml microcentrifuge tubes. Prepare one tube per ear (two tubes per mouse). 
	NOTE: Formamide is teratogenic, mutagenic, and an irritant. Wear gloves, eye protection, and face protection. Pregnant women should use caution when handling as formamide may be fetotoxic.</li>
	<li>Add ears to formamide, cap the tubes, and incubate overnight in a dry heat bath set to 63 &#176;C. Ears may still appear slightly blue after incubation; however the majority of the dye will be extracted into the formamide.</li>
	<li>Add 300 &#956;l of each sample, in duplicate, to a 96 well plate. Avoid transferring fur if it is present in the sample. Ensure that there are no bubbles present in the wells, as they will affect the absorbance reading.</li>
	<li>Add 300 &#956;l of formamide, in duplicate, to the 96 well plate. These wells will be used as blanks.</li>
	<li>Use a spectrophotometer to measure absorbance at 620 nm. Subtract formamide wells from each sample reading. 
	NOTE: All experiments were performed under protocols approved by the Uniformed Services University Institutional Animal Care and Use Committee.</li>
</ol>

<ol>
	<li>Kojima, 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=Mast+cells+and+Bbasophils+are+selectively+activated+in+vitro+and+in+vivo+through+CD200R3+in+an+IgE-independent+manner.">Mast cells and Bbasophils are selectively activated in vitro and in vivo through CD200R3 in an IgE-independent manner.</a> The Journal of Immunology. 179 (10), 7093-7100 (2007).</li><li>Parikh, S. A., Cho, S. H., Oh, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preformed+enzymes+in+mast+cell+granules+and+their+potential+role+in+allergic+rhinitis.">Preformed enzymes in mast cell granules and their potential role in allergic rhinitis.</a> Current Allergy and Asthma Reports. 3 (3), 266-272 (2003).</li><li>Amin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+mast+cells+in+allergic+inflammation.">The role of mast cells in allergic inflammation.</a> Respiratory Medicine. 106 (1), 9-14 (2012).</li><li>Nakaya, M., Takeuchi, N., Kondo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+localization+of+histamine+receptor+subtypes+in+human+inferior+turbinates.">Immunohistochemical localization of histamine receptor subtypes in human inferior turbinates.</a> Annals of Otology, Rhinology &amp; Laryngology. 113 (7), 552-557 (2004).</li><li>Ramsdell, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+Trypan+Blue+to+demonstrate+the+immediate+skin+reaction+in+rabbits+and+guinea+pigs.">The use of Trypan Blue to demonstrate the immediate skin reaction in rabbits and guinea pigs.</a> The Journal of Immunology. 15 (4), 305-311 (1928).</li><li>Chase, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+sensitization+of+animals+with+simple+chemical+compounds:+X.+Antibodies+inducing+immediate-type+skin+reactions.">Studies on the sensitization of animals with simple chemical compounds: X. Antibodies inducing immediate-type skin reactions.</a> The Journal of Experimental Medicine. 86 (6), 489-514 (1947).</li><li>Goose, J., Blair, A. M. J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passive+cutaneous+anaphylaxis+in+the+rat,+induced+with+two+homologous+reagin-like+antibodies+and+its+specific+inhibition+with+disodium+cromoglycate.">Passive cutaneous anaphylaxis in the rat, induced with two homologous reagin-like antibodies and its specific inhibition with disodium cromoglycate.</a> Immunology. 16, 749-760 (1969).</li><li>Ovary, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passive+cutaneous+anaphylaxis+in+the+mouse.">Passive cutaneous anaphylaxis in the mouse.</a> The Journal of Immunology. 81 (4), 355-357 (1958).</li><li>Ovary, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immediate+reactions+in+the+skin+of+experimental+animals+provoked+by+antibody-antigen+interactoin.">Immediate reactions in the skin of experimental animals provoked by antibody-antigen interactoin.</a> Progress in Allergy. 5, 459-508 (1958).</li><li>Akiyama, 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=Quantitiative+evaluation+of+passive+cutaneous+anaphylaxis+(PCA)+using+a+hand-held+spectrophotometer.">Quantitiative evaluation of passive cutaneous anaphylaxis (PCA) using a hand-held spectrophotometer.</a> Biological &amp; Pharmaceutical Bulletin. 19 (8), 1112-1114 (1996).</li><li>Teshima, R., 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=Simple+spectrophotometric+analysis+of+passive+and+active+ear+cutaneous+anaphylaxis+in+the+mouse.">Simple spectrophotometric analysis of passive and active ear cutaneous anaphylaxis in the mouse.</a> Toxicology Letters. 95 (2), 109-115 (1998).</li></ol>

The authors have nothing to disclose.

Numerous markers of allergic disease are used to assess the strength of allergic responses following allergen challenge, including changes in levels of circulating histamine, Th2 cytokine production, and cell recruitment into bronchoalveolar fluid in the setting of airway challenge. While monitoring changes in these parameters is important for studying allergic reactions, biological markers do not always correlate with clinical allergic disease. The local anaphylaxis assay described in this protocol provides reproducible measurement of localized vascular permeability as a correlate for localized allergic disease. By using blue dye extravasation, this protocol enables both visualization and relative quantification of a localized allergic reaction. In contrast to allergy assays that rely on clinical scoring, the primary outcome of this assay (blue dye extravasation as measured by spectrophotometric absorbance) is not easily subjected to observer bias. Another advantage is that animals are euthanized shortly after challenge, limiting any discomfort potentially caused by the allergen challenge and dye administration. The alternative use of a hand-held spectrophotometer described by Akiyama et al.10 allows for the continuous measurement of changes in vascular permeability. However, Evans Blue dye is toxic, and injection will ultimately lead to the animal needing to be euthanized.
While this assay has a number of strengths, it does have the limitation of being somewhat technically challenging. Reliable results are dependent on consistent and successful ear and tail vein injections. If the entire challenge dose is not administered into the ear, if ear injection results in obvious puncture of a blood vessel, or if the Evans Blue intravenous injection is not complete, the animal should be excluded from analysis. In our laboratory, investigators have had to practice the intradermal ear injections for several weeks before developing proficiency. Additionally, it is important to run proper controls for the assay. Specifically, PBS injection should routinely be used as an internal control, as animals seem to respond slightly differently to damage caused by needle insertion. The PBS reading gives a baseline measure for changes in vascular permeability induced by the injection itself. In lieu of using intradermal ear injections, the assay may also be applied to the back skin of mice. Investigators may find this route to be less technically challenging, and, in addition, it allows for the testing of multiple antigens simultaneously.
The local anaphylaxis assay is extremely versatile, as the challenge dose can be manipulated depending on the experiment. If it is necessary to look at changes in allergic responses, it may be beneficial to use a lower OVA challenge dose, such as 20 &#956;g. This allows for increased sensitivity in monitoring changes in absorbance, as it will be less likely that the saturation point for cell activation will be reached.
Additionally, this assay can be used to study passive cutaneous anaphylaxis (PCA). In this setting, antigen-specific IgE is injected into one ear and vehicle into the other. 24 hr later, the animal is given an intravenous challenge of antigen and Evans Blue dye. The PCA model can reduce the time it takes to complete the experiment, the number of animals used, and allow investigators to look at the specific effects of IgE cross-linking.
Despite taking longer to perform, there are some advantages to using the active cutaneous anaphylaxis (ACA) model described in this paper. Sensitization through weekly exposure to antigen mimics the process by which individuals develop allergic disease, as sensitization is usually systemic and people generally have both allergen-specific IgE and IgG in circulation. While IgE cross-linking is the primary means of mast cell degranulation, mast cells also respond to IgG cross-linking. Therefore, the ACA model allows for researchers to investigate mechanisms of allergic responses in addition to only IgE-mediated disease. In conclusion, while the method of sensitization used is at the discretion of the investigator, the local anaphylaxis assay is versatile and can be used with a variety of sensitization methodologies.
Ear swelling, a commonly used marker for assessing allergic responses, can also be measured after OVA challenge. We have found that ears challenged with OVA have significantly more swelling than ears challenged with PBS at 1 hr post injection. In contrast to the Evans Blue test, we have observed that ear thickness measurements are less sensitive than dye extravasation and exhibit greater variability between investigators as ear thickness can be altered by compression of the ear when using measuring devices.
Mouse strains other than BALB/c may be used for the local anaphylaxis assay; however absorbance readings will vary slightly depending on the strain used. Because of their propensity to develop strong Th2 responses, BALB/c mice generally produce reliable, reproducible readings with this assay.
This assay allows for the quantification of localized allergic responses in animals sensitized to a variety of antigens. Antigen selection is flexible, as the assay has been successfully performed with common allergens, such as OVA and Keyhole Limpet Hemocyanin (KLH). Furthermore, in lieu of sensitization, a local allergic reaction can be elicited in a na&#239;ve animal by injecting antibodies that cross-link IgE or IgE receptors on basophils and mast cells, or by ligating non-IgE activation receptors on these cells such as CD200R31.

Type I hypersensitivity is mediated by antigen-induced cross-linking of IgE on the surface of mast cells and basophils. This results in cellular degranulation and the release of vasoactive and proinflammatory mediators such as histamine, tryptase, and platelet-activating factor2. Following the release of preformed mediators during degranulation, mast cells synthesize and release prostaglandins and leukotrienes, which further increase vascular permeability3. The initial clinical response occurs rapidly and is referred to as an &#8220;immediate reaction&#8221;. In the skin, a wheal-and-flare response is readily visible within minutes of antigen challenge. Depending on the dose of the challenge, it is possible to observe a &#8220;late phase response&#8221; a few hr later. Late phase swelling is due to localized edema and leukocyte recruitment into the tissues2. Histamine, generally considered to be the major mediator taking part in immediate allergic responses, acts on histamine receptor 1 (HR1) expressed on vessels and histamine receptor 2 (HR2) expressed on smooth muscle. The combined effect increases blood flow and vascular permeability at the site of inflammation4.
A variety of animal models of allergy have been developed in order to study the mechanisms involved in allergic inflammation, including models of allergic asthma, systemic anaphylaxis, and local anaphylaxis. Intravenous dye administration has been used to measure localized allergic responses in animal models for almost a century, with publications describing this technique dating back to the 1920s5. Rabbits and guinea pigs were the first animal models used to test immediate hypersensitivity reactions, and the most sensitive responses were generally found in the ear5,6. The assay was later validated for use in rats7 and mice8.
Historically, a variety of experimental methods have been used, including injection of antigen prior to injection of dye, injection of dye prior to injection of antigen, and simultaneous injection of dye and antigen. Intravenous dye administration as a means for measuring allergic responses is a versatile assay as it can be used for measuring active, passive, and reverse passive reactions5,9. Numerous dyes have been utilized to assess allergic responses, including Trypan Blue, Pontamine Sky Blue, Evans Blue, Geigy Blue 536, and India Ink5,6,9. A solution of 0.5% Evans Blue is currently the standard dye used for measuring allergic responses in the skin.
The anaphylactic response to challenge is transient; maximum intensity is reached within 10 - 15 min of dye injection, and no reaction is visible if dye is administered more than 30 min after challenge, regardless of the animal species used9. Quantification of dye extravasation was originally obtained by measuring wheal size as indicated by the blue dye7-9. Additionally, counts of degranulated mast cells can be quantified by excising skin tissue from the site of the reaction and staining with toluidine blue7. Mast cell degranulation is often used as a marker for cutaneous, IgE-mediated allergic responses, as mast cells are the main local cell population expressing the high affinity IgE receptor Fc&#949;RI. Spectrophotometric techniques for measuring dye extravasation into the tissue were developed for passive cutaneous anaphylaxis (PCA) in the rat10 and mouse11 in the 1990&#8217;s.
The following local anaphylaxis assay protocol was adapted from Kojima et al.1, and utilizes chicken egg ovalbumin (OVA) as the antigen for eliciting allergic responses. However, antigens other than OVA may be used if desired. The assay then uses Evans Blue dye to monitor changes in vascular permeability that occur due to mast cell IgE cross-linking and histamine release.

<table><tbody><tr><td>BALB/c Mice</td><td>The Jackson Laboratory</td><td>651</td><td></td></tr><tr><td>PBS pH 7.4</td><td>Quality Biologic</td><td>114-058-101</td><td></td></tr><tr><td>Ovalbumin</td><td>Sigma</td><td>A5503-10G</td><td></td></tr><tr><td>Imject Alum</td><td>Thermo Scientific</td><td>77161</td><td>Mix thoroughly before use</td></tr><tr><td>Evans Blue Dye</td><td>Sigma</td><td>E2129</td><td></td></tr><tr><td>Formamide</td><td>Sigma</td><td>295876</td><td>99%+ Spectrophotometric grade</td></tr><tr><td>Isoflurane, USP</td><td>Phoenix</td><td>NDC 57319-474-06</td><td></td></tr><tr><td>1cc Insulin syringes</td><td>BD</td><td>329654</td><td></td></tr><tr><td>3/10 cc Insulin syringe with 31 G needle</td><td>Terumo</td><td>NDC 100861</td><td></td></tr><tr><td>27 G Needles</td><td>BD</td><td>305109</td><td></td></tr><tr><td>Forceps</td><td>F.S.T.</td><td>11000-12</td><td></td></tr><tr><td>Surgical scissors</td><td>F.S.T.</td><td>14070-12</td><td></td></tr><tr><td>5 ml Polystyrene round-bottom tubes</td><td>BD Falcon</td><td>352058</td><td></td></tr><tr><td>1.5 ml Microcentrifuge tubes</td><td>Medical Supply Partners</td><td>15-1151</td><td></td></tr><tr><td>15 ml Conical tubes</td><td>BD Falcon</td><td>352097</td><td></td></tr><tr><td>Flat-bottom 96 well plate</td><td>Costar</td><td>3590</td><td></td></tr><tr><td>Scotch tape</td><td></td><td></td><td></td></tr><tr><td>RC&lt;sup&gt;2&lt;/sup&gt; Rodent Anesthesia System</td><td>VetEquip</td><td>922100</td><td></td></tr><tr><td>Vortex Genie 2</td><td>Scientific Industries</td><td>SI-0236</td><td>Model G560 with 3 inch platform</td></tr></tbody></table>

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.

Animals that have undergone the assay successfully will have skin and eyes that appear blue. PBS sensitized animals should not react to either PBS or OVA challenge, therefore both ears should remain white (Figure 1A). In OVA sensitized animals, the ear receiving the PBS challenge (left) should be either completely white or lightly blue in a localized manner at the site of injection. The ear receiving the OVA challenge (right) should become progressively darker blue during the 10 min after dye administration (Figure 1B).
PBS sensitized animals will have negligible absorbance readings for both the PBS and OVA challenge (Figure 2A). For OVA sensitized animals, PBS injection generally results in a mean absorbance reading of 0.13 and a standard deviation of 0.04. Conversely, a 50 &#956;g OVA challenge results in a mean absorbance reading of 0.58 and a standard deviation of 0.18 (Figure 2B). The amount of OVA used for challenge may be adjusted for individual experiments if necessary. For example, a 20 &#956;g OVA challenge results in a mean absorbance of 0.30 and a standard deviation of 0.10 (Figure 2C), however a challenge less than 20 &#956;g may not give reliable results. Changes in OVA concentration must be optimized and validated prior to use.
Results may be graphically paired, with PBS absorbance compared to OVA absorbance. Alternatively, results may be plotted as a single value, with the PBS absorbance subtracted from the OVA absorbance for each individual animal.
<img alt="Figure 1" src="/files/ftp_upload/52005/52005fig1highres.jpg" /> 
Figure 1: Ear pigmentation in PBS and OVA sensitized animals. (A). Ear pigmentation in PBS sensitized animals. The left ear (challenged with PBS) and the right ear (challenged with 50&#160;&#956;g OVA) show little to no dye extravasation. (B). Ear pigmentation in OVA sensitized animals. There is no allergic response in the left ear (challenged with PBS), as indicated by an absence of blue dye extravasation. The right ear (challenged with 50 &#956;g OVA) shows a bright blue coloring indicative of increased vascular permeability, a direct result of cell activation in the ear tissue.
<img alt="Figure 2" src="/files/ftp_upload/52005/52005fig2highres.jpg" /> 
Figure 2: Representative O.D. values for OVA sensitized animals. O.D. values derived from spectrophotometric measurements taken at 620 nm. (A). 50 &#956;g OVA challenge in PBS sensitized animals. O.D. values for both PBS and OVA challenge are negligible. (B). 50 &#956;g OVA challenge in OVA sensitized animals. PBS challenge results in O.D. values &#60;0.2 and OVA challenge results in O.D. values &#62;0.3. (C). 20 &#956;g OVA challenge. PBS challenge results in O.D. values &#60;0.2. The right ears have slightly reduced O.D. values due to a smaller challenge dose of OVA used.

Watch this Scientific Journal Video about Measuring Local Anaphylaxis in Mice at JoVE.com

Measuring Local Anaphylaxis in Mice

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. ...

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

19.2K Views.  Uniformed Services University of the Health Sciences. 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.

Video: Measuring Local Anaphylaxis in Mice

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, ...

Assessing Changes in Volatile General Anesthetic Sensitivity of Mice after Local or Systemic Pharmacological Intervention

One desirable endpoint of general anesthesia is the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is less straightforward than it is in human patients. A widely used behavioral surrogate for hypnosis in rodents is the loss of righting reflex (LORR), or the point at which the animal no longer responds to their innate instinct to avoid the vulnerability of dorsal recumbency. We have developed a system to assess LORR in 24 mice simultaneously while carefully controlling for potential confounds, including temperature fluctuations and varying gas flows. These chambers permit reliable assessment of anesthetic sensitivity as measured by latency to return of the righting reflex (RORR) following a fixed anesthetic exposure. Alternatively, using stepwise increases (or decreases) in anesthetic concentration, the chambers also enable determination of a population's sensitivity to induction (or emergence) as measured by EC50 and Hill slope. Finally, the controlled environmental chambers described here can be adapted for a variety of alternative uses, including inhaled delivery of other drugs, toxicology studies, and simultaneous real-time monitoring of vital signs.

Loss of the righting reflex has long served as a standard behavioral surrogate for unconsciousness, also called hypnosis, in laboratory animals. Alterations in volatile anesthetic sensitivity caused by pharmacological interventions can be detected with a carefully controlled high-throughput assessment system, which may be adapted for delivery of any inhaled therapeutic.

One desirable endpoint of general anesthesia is  the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is  less ...

Medicine

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 Reversible, Non-invasive Method for Airway Resistance Measurements and Bronchoalveolar Lavage Fluid Sampling in Mice

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated procedures to obtain such measurements in the same animal are generally not feasible.  Here, we demonstrate protocols for obtaining from mice repeated measurements of AHR and bronchoalveolar lavage fluid samples.  Mice were challenged intranasally seven times over 14 days with a potent allergen or sham treated.  Prior to the initial challenge, and within 24 hours following each intranasal challenge, the same animals were anesthetized, orally intubated and mechanically ventilated.  AHR, assessed by comparing dose response curves of respiratory system resistance (RRS) induced by increasing intravenous doses of acetylcholine (Ach) chloride between sham and allergen-challenged animals, were determined.  Afterwards, and via the same intubation, the left lung was lavaged so that differential enumeration of airway cells could be performed.  These studies reveal that repeated measurements of AHR and BAL fluid collection are possible from the same animals and that maximal airway hyperresponsiveness and airway eosinophilia are achieved within 7-10 days of initiating allergen challenge.  This novel technique significantly reduces the number of mice required for longitudinal experimentation and is applicable to diverse rodent species, disease models and airway physiology instruments.  

Repeated measurements of rodent respiratory physiology and sampling of airway inflammatory cells are desirable, but generally not feasible. Here we describe a repeatable method for orally intubating mice that permits repeated measurements of airway hyperreactivity and sampling of airway inflammatory cells.

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated ...

Biology

Antigenic Liposomes for Generation of Disease-specific Antibodies

Antibody responses provide critical protective immunity to a wide array of pathogens. There remains a high interest in generating robust antibodies for vaccination as well as understand how pathogenic antibody responses develop in allergies and autoimmune disease. Generating robust antigen-specific antibody responses is not always trivial. In mouse models, it often requires multiple rounds of immunizations with adjuvant that leads to a great deal of variability in the levels of induced antibodies. One example is in mouse models of peanut allergies where more robust and reproducible models that minimize mouse numbers and the use of adjuvant would be beneficial. Presented here is a highly reproducible mouse model of peanut allergy anaphylaxis. This new model relies on two key factors: (1) antigen-specific splenocytes are adoptively transferred from a peanut-sensitized mouse into a na&#239;ve recipient mouse, normalizing the number of antigen-specific memory B- and T-cells across a large number of mice; and (2) recipient mice are subsequently boosted with a strong multivalent immunogen in the form of liposomal nanoparticles displaying the major peanut allergen (Ara h 2). The major advantage of this model is its reproducibility, which ultimately lowers the number of animals used in each study, while minimizing the number of animals receiving multiple injections of adjuvant. The modular assembly of these immunogenic liposomes provides relatively facile adaptability to other allergic or autoimmune models that involve pathogenic antibodies.

Described is the preparation of antigenic liposomal nanoparticles and their use in stimulating B-cell activation in vitro and in vivo. Consistent and robust antibody responses led to the development of a new peanut allergy model. The protocol for generating antigenic liposomes can be extended to different antigens and immunization models.

Antibody responses provide critical protective immunity to a wide array of pathogens. There remains a high interest in generating robust antibodies for ...

Mouse Body Temperature Measurement Using Infrared Thermometer During Passive Systemic Anaphylaxis and Food Allergy Evaluation

Mouse body temperature measurement is of paramount importance for investigating allergies and anaphylactic symptoms. Rectal probes for temperature readings is common, and they have been proven to be accurate and invaluable in this regard. However, this method of temperature measurement requires the mice to be anesthetized in order to insert the probe without injury to the animal. This limits the ability to observe other phenotypes of the mouse simultaneously. In order to investigate other phenotypes while measuring temperatures, rectal probes are not ideal, and another method is desired. Here, we introduce a noninvasive method of temperature measurement that foregoes the requirement for mouse anesthesia while maintaining equal reliability to rectal probes in measuring body temperature. We use an infrared thermometer that detects body surface temperatures at ranges between 2 and 150 mm. This method of body temperature measurement is successful in reliably replicating temperature change trends during passive system anaphylaxis experiments in mice. We show that body surface temperatures are about 2.0 &#176;C lower than rectal probe measurements, but the degree of temperature drop follows the same trend. Furthermore, we use the same technique to observe mice in a food allergy model to evaluate temperature and activity levels simultaneously.

Here we present a new method to accurately measure body temperature differences in passive systemic anaphylaxis (PSA) and food allergy mouse models using an infrared thermometer. This procedure has been accurately duplicated in previous PSA results.

Mouse body temperature measurement is of paramount importance for investigating allergies and anaphylactic symptoms. Rectal probes for temperature ...

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 ...

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.

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 ...