The overall goal of the following experiment is to measure UV sensitivity by determining the minimal erythema dose in fair skin mice pretreated with four skin. This is achieved by first preparing the dorsal skin of mice to be irradiated by shearing and ablation. Next topical treatments of forskolin are applied to the dorsal skin.
Then the mice are exposed to different doses of UV irradiation to determine the effects of four mediate melon on UV induced inflammation. Results are obtained that show that topically applied forline robustly increases skin pigmentation and that animals with pharmacologically enhanced me are UV resistant as determined by MED. These are proof of concept studies that show that it is possible to pharmacologically induce a tan in an individual incapable of tanning because of a defect in a very important melanocyte gene called the melanocortin one receptor or MC one R In human populations there loss of function of mc one R correlates with UV sensitivity, sunburning tendencies, and inability to tan and later in life up up to a fourfold risk of melanoma.
We chose four scullin as a drug for these studies because it's able to bypass a defective melanocortin receptor signaling pathway and trick the melanocytes into thinking they have a good mc one R signaling pathway, and therefore they make a lot of melanin, which gets deposited in the skin and protects the animals against UV radiation. Though we know this method provide insight into skin that can and somber. It might also be important for cancer prevention.
Since we know that UV radiation is the major carcinogen for the great majority of skin cancers and enhancing implementation of the skin protects against UV injury One four performing intraperitoneal injections. Be sure to aspirate after needle placement and before injection of contents to ensure that the needle has not penetrated a blood vessel intestines or the urinary bladder IP injections should be given on the right side to avoid the season. After preparing the forline extract and briefly anesthetizing the mouse by isof fluorine.
Inhalation, according to the text protocol, administer additional anesthesia to the animal with an IP injection of ketamine and xylazine. Perform a toe pinch to verify sedation. Use electric shears outfitted with a 0.25 millimeter surgical preparatory head to shear the dorsal fur.
Use a depilatory cream to remove the stubble. Then to avoid burning or otherwise damaging the skin, immediately use water soaked gauze pads to gently wipe the dorsal skin until all the cream has been removed. Use soft paper towels to dry the animal before placing it in a clean cage on a warming pad to recover.
In general, mice should be briefly anesthetized to facilitate topical administration of forskolin or control solutions using inhalation isof fluorine under fume hood. Begin with an isof fluorine saturated paper towel in an isof fluorine, saturated litted clear glass jar. After attaching a form fitted nylon air permeable filter anesthetized the mouse by placing it on top of the filter just long enough to suppress voluntary muscular movements with the sedated animal on a clean absorbent bench pad.
After performing a toe pinch transfer 400 microliters of crude forline extract onto the back of the animal by dripping it onto the skin. Then using the side of the tip to smear the extract over the entire surface of the exposed dorsal skin. Return the mouse to its cage and carefully observe it until it recovers from anesthesia to prevent non pigment.
Cyclic A MP effects from confounding UV sensitivity experiments. Discontinue all topical treatments two days prior to UV exposure to carry out reflective colorimetry. After briefly anesthetizing the mouse with isof fluorine inhalation.
Calibrate a colorimeter by placing the portable head on the standardized white surface provided with the device. Place the portable measuring head flush with the dorsal skin of the animal, ensuring that the one square centimeter round aperture is completely pressed onto the skin. Take at least three separate measurements in different areas of the dorsal skin.
Calculate the mean L score, plus or minus standard deviation per animal and per treatment group. To determine 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 one 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 lamb to equilibrate to a constant UV output before using a UV photometer with a 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 lamps UVB output measures 2.4 milliwatts per square centimeter. Therefore, to administer five kilojoules per square meter, the skin would need to be exposed to 208 seconds of UVB radiation as calculated here. Next place a 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 five 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 discreet areas of erythema or edema in the UV exposed areas.
Sometimes residual first 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. In these experiments, C 56 black six mice were generated on anodic anodic or anodic backgrounds incorporating the K 14 stem cell factor transgene, which allows the mice to retain melanocytes in the epidermis and as a result, deposit melanin.
In the epidermis shown here, cohorts of fair skin extension mice were treated for five days topically with twice daily doses of either vehicle or 40%crude colus fors coly root extract. The effects of topical treatments on epidermal pigmentation were determined both by visual inspection and by reflective color.Imagery. Application of the root extract was associated with robust epidermal darkening in the K 14 SCF transgenic background, but not in genetically matched non transgenic animals, suggesting that inter follicular epidermal melanocytes must be present in order for pharmacologically induced melanin to be deposited.
In the epidermis, though the root extract is deeply colored because of the presence of plants phytochemicals besides four galin, skin darkening cannot be solely due to a dying effect from the drug. As non transgenic animals fail to exhibit skin darkening with the root extract as demonstrated here when topically applied twice daily, the melanin effects of forskolin were noted in as little as two days. Although maximal darkening was evident after several more days shown in this figure by font mason and melanin staining of dorsal skin sections.
The degree of melan is dose dependent animals that were treated with four skin once per day for 15 days or twice per day for five days experience similar amounts of skin darkening. In addition, there were no obvious toxic effects from four skin exposure in either treatment group. Thus, we concluded that the five day accelerated treatment promoted safe mein of the dorsal skin as effectively as the standard once per day treatment.
This figure demonstrates that as measured by MED testing, forline induced epidermal mein resulted in profound UV protection, whereas the mean MED for K 14 SCF extension mice treated twice per day for five days with vehicle was 5.0 plus or minus 0.0 kilojoules per meter square Average MED for cohorts treated with topical four galin was greater than 30.0 plus or minus 0.0 kilojoules per meter square. In fact, a dose of 30.0 kilojoules per meter square was insufficient to generate erythema. In this experiment using standard force Glen dosing, the average MED for treated K 14 SCF extension mice was 50.0 plus or minus 7.1 kilojoules per square meter.
Importantly, fours Kline pretreatment did not affect the MED of animals incapable of melon either because of the lack of K 14 SCF mediated epidermal melanocytes, or because of tyrosinase deficiency. Since fours Kline applications were discontinued two days prior to UV exposure and were not continued after UV exposure, we conclude that non pigmentary CAMP effects did not play a role in MED results. Rather, the data suggests that epidermal mein was the mechanism by which forline induced UV protection in this model.
Once mastered this technique allows easy and reproducible assessment of skin pigmentation response and ultraviolet sensitivity. In animal model application of the forland takes only minutes a day and the bulk of the experimental time really is with the UV radiation and MED testing. While attempting this procedure, it is important to remember that the skin gradually face back to its baseline fair complexion pharmacologic.
Induction of melanin is very similar to nain response in that the skin will stay dark as long as it is stimulated, but then that tan work its way out of the skin over the next several weeks when the stimulus is removed.
