Minimum Erythematous Dose Assay: A Method for Measuring UV Sensitivity in Mouse Models

Overview

This video describes a method to measure UV sensitivity in a humanized mouse model—a mouse model that mimics human skin. We determine the minimal erythematous dose (MED) of UV radiation that can cause erythema or edema on the skin. The method can help to find out the efficacy of drugs used to reduce UV sensitivity.

Protocol

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

Determination of UV Sensitivity by Calculation of "Minimal Erythematous Dose" (MED)

1. Use animals that have been pre-treated with either vehicle or forskolin. Anesthetize animals with intraperitoneal injection of a standard mixture of ketamine and xylazine.
2. Prepare a piece of UV-occlusive tape for MED testing. To generate holes in the tape, use a heavy-duty hole punch with a 1 cm² circular cut out (Figures 1A and B). Having holes of a defined size and symmetric arrangement in the tape facilitates recognition of skin changes after irradiation. Over each hole in the tape, apply a small but easily detachable piece of tape that can be removed at defined times during UV exposure to allow administration of different UV doses.
3. Once animals are adequately sedated, place the tape on the dorsal surface. Eye lubricant should always be utilized under anesthesia.
4. Turn on the UV source consisting of two Westinghouse F15T8UV-B lamps with a peak output of 313 nm and a range of 280-370 nm. Allow the lamp to equilibrate to a constant UV output as measured by a UV photometer with a UVB sensor (generally takes a few minutes for the lamps to warm up).
5. Based on the UV transmission rate as measured by the UV Photometer, calculate UV exposure time for each desired dose. For example, our lamp's UVB output measures 2.4 mW/cm². Therefore, to administer 5 kJ/m², the skin would need to be exposed to 208 sec (which is 3 min and 28 sec) of UVB radiation, as calculated below:
   
6. Place sedated animals (each with occlusive tape in place) ventral surface down to ensure even UV exposure. To administer the chosen doses of UV radiation, sequentially remove the small occlusive tapes covering the holes to expose 1 cm² areas of skin to the correct doses of radiation. Therefore, using the above example, if 40 kJ/m² is the largest dose in the experiment, then the animal would be under the lamp for 27 min and 47 sec total and the skin in the 40 kJ/m² condition would have no overlying tape the entire time. However, tape overlying the 5 kJ/m² condition would be removed when there is 208 sec remaining in the exposure. Timing of tape removal should be such that each condition ends simultaneously.
7. After UV exposure, peel off the tape from the dorsal skin carefully, taking care not to rip the skin with sudden or overly-forceful movements. Place animals in a warm quiet place to allow recovery from anesthesia.
8. Monitor mice for 24-48 hr to look for discreet areas of erythema (redness) or edema (swelling) corresponding to the anatomic sites exposed to the specific dose of UV irradiation. Document skin findings photographically.
9. MED value corresponds to the minimum dose of UV that causes inflammation as defined by erythema and/or edema of the entire exposed circle of skin. Note that pigmentation of the skin can challenge determination of MED, however, erythema and edema can still generally be accurately assessed, thanks in part to the defined shape of the apertures in the tape during UV exposure.

Representative Results

Figure 1. Forskolin-induced melanization protects against UV-mediated inflammation as determined by minimal erythematous dose (MED) testing. (A,B) Position of UV occlusive tape and UVB doses of animals treated twice daily for 5 days (A,C) or once daily for 15 days (B,D,E) with either vehicle or forskolin. The last topical treatment was applied 48 hr prior to irradiation. Dorsal skin was exposed to various doses of UVB by using UV-occlusive tape with punched-out 1 cm2 circular apertures, and varying exposure times to yield the appropriate dose. After irradiation, circles of exposed skin were labeled with a pen in some experiments. MED's, defined by erythema and/or edema of the entire circle of exposed skin to a particular dose, were determined 48 hr after exposure. The MED ± SD results are reported as kJ/m2 UVB, * p≤0.001. (E) Skin color reflectometry and MED values for tyrosine-deficient K14-Scf albino extension mice treated for 10 days with vehicle

Materials

Name	Company	Catalog Number	Comments
Xylazine	Anased Injection	LA04612	Shenandoah, Iowa, USA
Ketamine HCl, USP	Putney	NDC 26637-411-01	St. Joseph, MO, USA
Ethanol	Decon Labs.	2705
Depilatory cream, Nair	Church Dwight	JF-11 4381322	Princeton, NJ
Germicidal Hg Lamp UV-B	Westinghouse	F15T8UV-B
Radiometer photometer	International light	1LT400A	Peabody, MA,USA
Isothesia, Isoflurane , USP	Butler Schein	NCD 11695-6776-1	Dublin, OH, USA
Chromameter	Konica Minolta	CR-400	Ramsey, NJ, USA
Data Processor for Chromameter CR-400	Konica Minolta	DR-400	Ramsey, NJ, USA