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 cha....

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.

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

List of materials used in this article

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