On study day 0, mice are sensitized with aliquots of 150 µL of a 5% oxazolone solution epicutaneously on their shaved abdomens. On day 7, the right ear of each mouse is challenged with 3% oxazolone solution (10 µL on the front and 10 µL on the back). Left ears are painted on both sides with an ethanol/acetone mixture.
Mice develop swelling within 24 to 48 hours of antigen challenge.
Body weights are taken on study days 0, 7, and 8 (prior to termination). Ear caliper measurements are taken on days 7 (baseline) and 8 using a Digitrix II micrometer (Fowler & NSK). On day 8 (24 hours post-antigen challenge), animals are necropsied. Measurement of the DTH reaction can be done grossly by comparing the ear core weight difference between normal versus antigen-injected ears (weight is proportional to edema). A 7-mm disc is collected by cork borer (Fisher Scientific) from the pinna of each ear and weighed.
Tissues are examined microscopically by a board certified veterinary pathologist (Dr. Alison Bendele) and scored according to these methods.
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Chemokines such as IL-8, monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1a (MIP-1a), and macrophage migration inhibitory factor (MIF) have been found to be involved in the recruitment of leukocytes to the DTH reaction site. Cytokine inhibitors, such as anti-IL-16, have been shown to minimize the DTH response. Other compounds that effectively inhibit the DTH reaction are dexamethasone (a potent steroid that induces lympholysis) and cyclosporine-A (CsA), which inhibit the action and growth of T-lymphocytes.