Minimum Erythematous Dose Assay: A Method for Measuring UV Sensitivity in Mouse Models
Transcrição
– Melanocytes produce a dark pigment called eumelanin that absorbs harmful UV radiations, protecting the skin. To measure UV sensitivity, begin by taking an anesthetized mouse model. Prep the mouse by removing its dorsal fur to expose the epidermal layer. Fasten a piece of UV-blocking tape bearing a set number of holes on the exposed skin. Fix small detachable tapes over all the holes, except one, to mask the opening.
Now, subject the mouse to UV radiation for the desired time duration. During the exposure period, unmask the remaining holes sequentially at predetermined time points. This facilitates the successively exposed skin patches to receive decreasing doses of UV radiation. The holes allow the localized penetration of UV rays into the mouse's epidermis.
Upon exposure, the epidermal cells release pro-inflammatory molecules. These molecules diffuse towards the dermis and dilate the dermal blood vessels causing erythema or skin redness. Subsequently, the leukocytes infiltrate the dermis and cause edema or swelling in the exposed area.
At the experimental endpoint, stop the radiation and remove the tape to observe erythema and edema. In the following protocol, we will measure UV sensitivity in a humanized mouse model treated with forskolin – a melanin producing drug – by determining the Minimum Erythematous Dose.
– To determine the Minimal Erythematous Dose, or MED, each animal will be exposed to a variety of UV doses. Begin by preparing a piece of UV-occlusive tape, using a heavy-duty hole punch with a 1 square centimeter circular cutout to punch holes in the tape. Over each hole in the tape, apply a small but easily detachable piece of tape that can be removed for UV exposure. Place the tape on the dorsal surface of the anesthetized mouse.
If the animal's eyes are open, apply artificial tears to prevent injury from drying and UV exposure. Turn on the UV source consisting of two UVB lamps with a peak output of 313 nanometers and a range of 280 to 370 nanometers. Allow the lamp to equilibrate to a constant UV output before using a UV photometer with the UVB sensor to verify the output.
Based on the UV transmission rate as measured by the UV photometer, calculate the UV exposure time for each desired dose. For example, your lamp's UVB output measures 2.4 milliwatts per square centimeter. Therefore, to administer 5 kilojoules per square meter, the skin would need to be exposed to 208 seconds of UVB radiation as calculated here.
Next, place the sedated animal's ventral surface down to ensure even UV exposure. To administer the chosen dose of radiation, sequentially remove the small pieces of tape covering the holes at the appropriate time.
For example, if 40 kilojoules per square meter is the largest dose, the mouse would be under the lamp for 27 minutes and 47 seconds and the hole for this dose would be exposed from the beginning. However, for the 5 kilojoules per square meter condition, the tape would be removed with 208 seconds of exposure time remaining. After UV exposure, carefully remove the tape and place the animals in a warm quiet place to recover from anesthesia before returning them to their cages.
The Minimal Erythematous Dose, or 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. Monitor the mouse for 24 to 48 hours to look for discrete areas of erythema or edema in the UV exposed areas.
Sometimes, residual fur stubble confounds interpretation of the MED testing by masking underlying skin erythema or edema. Wetting the skin with 70% ethanol makes the skin much more interpretable. Document any skin findings photographically and then return the mice to the animal facility.
