Measuring Local Anaphylaxis in Mice

Summary

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.

Abstract

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.

Introduction

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 “immediate reaction”. 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 “late phase response” 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 ear^5,6. 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 reactions^5,9. Numerous dyes have been utilized to assess allergic responses, including Trypan Blue, Pontamine Sky Blue, Evans Blue, Geigy Blue 536, and India Ink^5,6,9. 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 dye^7-9. 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 FcεRI. Spectrophotometric techniques for measuring dye extravasation into the tissue were developed for passive cutaneous anaphylaxis (PCA) in the rat¹⁰ and mouse¹¹ in the 1990’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.

Protocol

1. Sensitize Mice

1. 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 °C for future use.
2. 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.
3. 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.
4. Place cap on tube and tape tube to vortex. Turn vortex on lowest setting and allow OVA to adsorb to Alum for 30 min.
   1. 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.
5. Load a 1 ml insulin syringe with OVA/Alum mixture. Cap the syringe with 27 gauge needle.
6. Inject 100 μl of OVA/Alum into the peritoneum of BALB/c mice for a dose per mouse of 50 μg of OVA and 2 mg of Alum.
7. 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.
8. 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.

2. Local Anaphylaxis Assay

1. 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.
2. 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. 
   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.
3. Load 10 μl 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 μl of 1x PBS.
4. Using a dissecting microscope, inject 10 μl of OVA into the right ear of an anesthetized mouse for a challenge dose of 50 μg of OVA. Other doses, such as 20 μg, are also acceptable.
   1. 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.
5. Using the same method described above, inject 10 μl of PBS into the left ear as a negative control.
6. Wait 3 min.
   1. 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.
7. Using a 1 ml insulin syringe capped with a 27 G needle, slowly inject 200 μl of 0.5% Evans Blue dye into the tail vein of the mouse. Rapid injection may destroy the vessel structure before the entire 200 μl is administered. If this occurs, attempt to inject the remaining volume of dye into the vein on the opposite side of the tail.
8. Wait 10 min.
9. Euthanize the mouse and remove both ears using forceps and surgical scissors.
   1. 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.
10. Aliquot 700 μl 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.
11. Add ears to formamide, cap the tubes, and incubate overnight in a dry heat bath set to 63 °C. Ears may still appear slightly blue after incubation; however the majority of the dye will be extracted into the formamide.
12. Add 300 μl 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.
13. Add 300 μl of formamide, in duplicate, to the 96 well plate. These wells will be used as blanks.
14. 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.

Results

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 administrat...

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
BALB/c Mice	The Jackson Laboratory	651
PBS pH 7.4	Quality Biologic	114-058-101
Ovalbumin	Sigma	A5503-10G
Imject Alum	Thermo Scientific	77161	Mix thoroughly before use
Evans Blue Dye	Sigma	E2129
Formamide	Sigma	295876	99%+ Spectrophotometric grade
Isoflurane, USP	Phoenix	NDC 57319-474-06
1cc Insulin syringes	BD	329654
3/10 cc Insulin syringe with 31 G needle	Terumo	NDC 100861
27 G Needles	BD	305109
Forceps	F.S.T.	11000-12
Surgical scissors	F.S.T.	14070-12
5 ml Polystyrene round-bottom tubes	BD Falcon	352058
1.5 ml Microcentrifuge tubes	Medical Supply Partners	15-1151
15 ml Conical tubes	BD Falcon	352097
Flat-bottom 96 well plate	Costar	3590
Scotch tape
RC2 Rodent Anesthesia System	VetEquip	922100
Vortex Genie 2	Scientific Industries	SI-0236	Model G560 with 3 inch platform