A Technique to Assess the Immune Response against an Antigen by Inducing Skin Inflammation

Overview

This video demonstrates an in vivo technique to assess a test antigen by inducing skin inflammation in a murine model. The test antigen—a hapten in an oil/acetone solution—is topically applied to the skin of the mouse ear, and inflammation of the skin is assessed after an adequate period.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Induction of Skin Inflammation in pIL1-DsRed Mice

1. Anesthetize mouse by intraperitoneal (i.p.) injection of an anesthetic cocktail containing 100 mg/kg ketamine, 10 mg/kg xylazine, and 1 mg/kg acepromazine. Adequately anesthetized mice do not show hind paw withdrawal to toe pinch.
2. Apply hair-removal cream to the dorsal surface of both ears of the mouse. Wait 30 sec to 1 min. Wipe ear surface with water-wetted cotton and allow to air-dry.
3. Measure the ear thickness using a micrometer and replace the mouse in a separate cage. Observe until recovery from anesthesia (~ 15 - 30 min). To resolve skin inflammation induced by the hair removal product, allow the mouse to rest for 3 days before further experimentation is carried out.
4. Prepare 1.25% (w/v) oxazolone (OX) by dissolving 16.7 mg of OX in 1 ml acetone and mixing 750 µl of OX/acetone solution with 250 µl of olive oil. Prepare vehicle solution by mixing 750 µl of acetone with 250 µl of olive oil.
5. Re-anesthetize mouse by intraperitoneal (i.p.) injection of anesthetic cocktail (1.1). Measure the ear thickness as before.
6. Apply 12.5 µl of 1.25% OX onto each side of the right ear. Apply 12.5 µl of acetone/olive oil vehicle solution onto each side of the left ear.
7. Keep the mouse separate from cage-mates until fully recovered to prevent insult to its ears by other mice, then replace the mouse in its original cage and return it to the animal housing facility.
   NOTE: Hair removal may result in minor skin inflammation following by change of ear thickness. This minor inflammation will be resolved 3 days later confirmed by normal ear thickness. After topical application for 24 hr, OX-treated ears show significant (p < 0.01) swelling compared to vehicle solution alone-treated ears.

Materials

Name	Company	Catalog Number	Comments
Ketamine hydrochloride	Hospira	NDC 0409-2051-05
Xylazine	LLOYD Laboratory	NADA #139-236
Acepromazine	Boehringer Ingelheim	ANADA 200-361
Hair-removal cream	Church & Dwight
Acetone	Fisher Scientific	A16P4
Oxazolone	Sigma-Aldrich	E0753