This model can not only be used to explore the pathogenesis of ACD, but also is applicable for the preclinical evolution of new therapeutic methods, which has an important meaning. This is a stable and efficient animal model with many advantages, such as a short experimental period, easy operation, obvious symptoms, and a high success rate. Our experimental protocol is very detailed and easy to operate.
Ensure to follow each step of the protocol correctly. Before starting the modeling, acclimatize the mice to the environment for one week with free access to food and water. On the day of modeling, using an ultraviolet lamp and 75%alcohol, clean and disinfect the environment and countertops.
Apply soapy water to the abdomen of the mice using a cotton swab, and shave the area in the direction of hair growth with a blade or shaver. Weigh the mouse and compare the weight changes among each group. Before performing abdominal sensitization stimulation, ensure the full recovery of any minor injury to the mouse abdomen skin induced by shaving.
Prepare 0.5%DNFB solution and dilute it with acetone to olive oil mixture at a 4 to 1 ratio. Using a pipette gun, blow and mix 20 times to thoroughly mix the DNFB solution. Apply 25 microliters of the 0.5%DNFB solution over the middle of the abdominal shaving area with a pipetter, and lightly spread with the smooth side of the pipetter tip to disperse it uniformly.
After 30 seconds of DNFB stimulation, place the mouse in an empty cage without bedding to prevent the mouse from rubbing off the DNFB solution. When the solution is completely dry, return the mouse to its original cage with free access to food and water. For ear sensitization stimulation, orient the mouse body and make the outside border of the auricle face downward to prevent the solution from entering the ear canal during DNFB stimulation.
On days 4, and 10, using a pipetter, apply 20 microliters of the vehicle, or 0.2%DNFB solution slowly and uniformly to the inner surface of the left auricle, and gently distribute the solution using the smooth side of the pipetter. Leave the right ear untreated. Wait until the DNFB solution is dry and place the mouse back in the cage.
After abdominal and ear sensitization stimulation, weigh the mice every two days starting on day one. Compare the weight of each mouse to day zero, and evaluate the effect of ACD on the body weight of the mice as weight changes. Capture high-resolution photos of the mouse ears to record ACD clinical symptoms every two days, starting on day one.
Using vernier calipers, measure the auricle thickness at the same time each day for accurate results. Stop the vernier calipers from continuing inward clamping when there is a slight blockage to prevent tissue damage to the mouse ear. Keep the position fixed and record the data for both ears.
Record the average of the three data and evaluate the ear swelling in micrometers. To evaluate the degree of inflammatory swelling on day 11, prepare 0.5%Evans Blue dye solution and dilute it with PBS. To immobilize the mouse with a fixator, open the lid, hold the mouse tail, make the head of the mouse face the fixator, and allow the mouse instinctively climb into the fixator.
Cover the lid. Make the mouse tail come out of the hole on the lid and adjust the length of the fixator to expose the whole mouse tail. Wipe the tail repeatedly with an alcohol cotton ball, or soak it in warm water for 30 seconds, and gently pinch the root of the tail to fill, and expand the veins on both sides.
Slowly inject Evans Blue Dye solution into the mouse tail vein using a 1 mm insulin needle under the irradiation of a cold light source. Wait for 15 minutes to take pictures of the mouse ears. Mouse ears of the DNFB group displayed evident clinical symptoms comparable to ACD, with sensitive areas showing the typical symptoms of redness, dryness, erosion, and exudation.
However, ear administration of pure water or solvent control did not produce any symptoms similar to DNFB group. In the DNFB group, compared to the untreated right ear, The thickness of the left ear increased significantly after DNFB stimulation. There was no significant difference in the control and vehicle groups.
The left ear of the DNFB group mice turned dark blue after the injection of Evans Blue dye, which was visually different from the right ear. The left and right ears of mice in control and vehicle groups were approximately the same color. Mouse weight gain was slightly slowed by DNFB, or simple vehicle stimulation, but did not result in significant weight loss.
Repeated DNFB stimulation in the mouse ear resulted in spleen enlargement and an increase in the spleen index. In contrast, the spleen index of mice in the vehicle group did not change significantly. Ensure the DINFB solution is fully mixed and evenly applied to the mouse ear without flowing into the ear canal.
This is the key to the success of modeling.
