An objective laboratory method presented here may help to study the contact hypersensitivity reaction in mice, which mimics allergic contact dermatitis in humans. The contact hypersensitivity reaction can be measured not only by ear edema, but also by various laboratory tests that allow for the study of cellular mechanisms involved in this reaction. The more in-contact hypersensitivity model can test various and environmental factors and new substances to show if tested factors modulate T-cell dependent immune responses, which can be used to implement new therapies.
Begin by shaving the mouse skin with a razor blade and labeling the paw. Six hours before the induction of contact hypersensitivity, or CHS, apply gray soap with water and shave a two-centimeter-square area on the chest and abdomen of the mouse. On the same day, sensitize the mice by applying 150 microliters of freshly prepared 5%hapten on the previously shaved spot.
In the control mouse, only apply the vehicle. Dry the hapten spot for 30 seconds before placing the animal back into the cage. On day four, measure the ear thickness of an anesthetized mouse using a micrometer at zero hour by an observer unaware of the experimental groups.
Then, apply 10 microliters of freshly prepared 04%hapten on both sides of the ears in test and control groups and allow it to dry for 30 seconds. On day five, 24 hours after the previous 0.4%hapten application, repeat the measurement of the ear thickness for the 24-hour measurement. 24 hours after the ear thickness measurement, when the mouse is still in deep anesthesia, cut off the ears as close to the skull as possible using scissors.
At the distal side of the ears, Make a six-millimeter diameter punch using a biopsy punch. Measure the weight of each ear biopsy on the analytical balance, and express it in milligrams. For the myeloperoxidase, or MPO, assay, put the ear biopsies in a two-milliliter microcentrifuge tube with stainless steel beads and add 500 microliters of the prepared buffer.
Homogenize the biopsies for 10 minutes in a homogenizer and then cool down the sample for 15 minutes at 4 degrees Celsius before homogenizing it again for 10 minutes. Take out the beads once biopsies are homogenized. Freeze the homogenates at minus 20 degrees Celsius for 30 minutes.
Thaw and vortex the samples, and repeat the homogenization and freezing procedure three times. Once done, centrifuge the homogenate at 3, 000 G for 30 minutes at 4 degrees Celsius. Harvest the supernatant with a pipette to measure the MPO activity, and express it in units per one-milligram protein.
To perform a vascular permeability test, sensitize mice on day zero, and on day four, directly challenge by applying hapten on the ears using the previously mentioned procedure. 23 hours later, intravenously inject 8.3 microliters per gram body weight of 1%Evans blue dye in Dulbecco's phosphate buffered saline, or DPBS, to the anesthetized mouse. After one hour, again anesthetize the mouse to collect the ear biopsies, as described earlier.
To extract the dye from the tissue, incubate the biopsies in the tubes containing one milliliter of formamide at 37 degrees Celsius in an atmosphere of 5%carbon dioxide for 18 hours. After collecting ear biopsies, when the mouse is still under deep anesthesia, remove the eyeball with tweezers. Put gentle pressure on the mouse and collect blood from the retro-orbital sinus into the tube for serum collection.
Invert the tube six times and wait 30 minutes for the blood to clot. Then, centrifuge the tube at 1, 300 to 2, 000 G for 10 minutes at room temperature. Sensitize the donor mice with TNCB hapten on day zero using the previously mentioned procedure On day four, after disinfecting the skin, isolate the axillary and inguinal lymph nodes, or ALNs, from the anesthetized mouse using forceps, followed by isolating the spleen.
Mash the tissue between the frosted ends of two microscope slides and pass the cell suspension through a cell strainer with a pore size of 70 micrometers. Then, wash the cells with DPBS supplemented with 1%fetal bovine serum and centrifuge them at 300 G for 10 minutes at four degrees Celsius. Decant the supernatant and resuspend the remaining cell pellet in one to five milliliters of DPBS.
Prepare a 1:1 mixture of ALNs and spleens to achieve a concentration up to 7.0 times 10 to the seventh cells in 200 microliters of DBPS before intravenously injecting the mixture of CHS-effector cells in an anesthetized mouse. Measure the ear thickness at zero hour and after 24 hours of the challenge. The data showed that mice sensitized to TNCB and challenged in the ears four days later developed significantly increased ear swelling, compared to sham-sensitized and then similarly challenged control mice.
The increase in ear edema in the test group resulted in an increased ear weight, MPO activity, interferon gamma concentration in the ear extracts, edematous dermis changes in the histological examination, and ear vascular permeability The TNP-specific IgG1 antibodies were elevated in the sera of the test mice when compared with the control animals. The animals that received the CHS-effector cells from donors previously sensitized with TNCB showed significantly increased ear swelling, compared to animals that have not received the cells. The most critical moment is to trigger contact hypersensitivity.
Hapten solution is very volatile and light-sensitive, so it must be quickly applied to the skin of the animals. Additional tests can be performed on the isolated effector cells. For example, cell cultures can be established to assess the ability to proliferate in the presence of antigens.
