This video article demonstrates a method to visualize the autoimmune reaction in skin of gluten sensitive Reeses Maccas with dermatitis herpetiformis. This is achieved by first identifying animals with dermatitis that cannot be linked with other, more commonly diagnosed causes of dermatitis from these idiopathic cases plus age matched healthy controls. Skin samples are obtained from the affected area to evaluate the tissue architecture, inflammation, as well as presence of epidermal transglutaminase.
Autoantibodies collected samples are fixed in zinc formin, and 2%paraldehyde hematin in EOSIN or HNE. Staining is then performed to evaluate the extent of inflammation and histopathology and immunofluorescent Staining is performed to evaluate epidermal transglutaminase autoantibody deposition into deep epidermis Dermatitis is herpetiformis is a chronic inflammatory condition characterized by an autoimmune reaction between IGA and epidermal transglutaminase DH develops in a very small portion of gluten sensitive celiac patients. The results of this study indicate that DH can also develop in a small fraction of gluten sensitive Resus monkeys.
Our results corroborate validity of recently developed Resus Meac model of gluten sensitivity as presented. Results show that clinical manifestations of this condition are not confined only to small intestine. A typical protocol involves one animal with idiopathic dermatitis and one age-matched healthy control.
After appropriate anesthesia and site preparation using sterile technique, place a 4.0 millimeter miltex punch dermal biopsy instrument against the animal skin while applying slight pressure. Rotate the instrument 180 degrees clockwise and counterclockwise until the biopsy punch transects the dermal layers into the subcutaneous tissue. Repeat for a second biopsy using forceps.
Grasp the transected portion of skin and free it from the subcutaneous tissue. Place the first sample which will be processed for h and e staining in zinc formin. Then place the second sample which will be evaluated by immunofluorescence microscopy in 2%Paraform aldehyde using a three eighth circle cutting needle with three dash zero nylon suture.
Close the skin defect in cruciate pattern. Repeat this process to obtain two to three biopsy samples from each animal. Administer postoperative analgesia as described in detail in the written protocol to examine IGA and TTG two presence and localization.
Begin by incubating the samples that were placed in 2%power formaldehyde for four hours at room temperature to fix them following fixation. Remove the para formaldehyde and wash with PBS for 30 to 60 minutes on a rocker platform. Repeat twice for a total of three washes, then remove PBS and replace with 30%sucrose for at least four hours and up to two weeks.
Next, place the sample in OCT freezing medium and submerge it in a bath of two methyl butane and dry ice until the clear OCT becomes solid white indicating it has frozen completely. Then place the blocks at minus 80 degrees Celsius for at least 20 minutes. This ensures the tissue is frozen solid so that the blade slices through it without crushing it.
The tissue can be stored long term in OCT at minus 80 degrees Celsius. After sectioning and mounting the sections on slides, remove the OCT and to permeate the tissue. Submerge the slides in a Copeland jar containing PBS with 0.2%fish skin gelatin and 0.1%triton X 100.
Then place the jar on a rocker for 20 minutes after 20 minutes, wash the slides for 10 minutes in PBS with 0.2%fish skin gelatin, then incubate with 10%normal goat serum diluted in PBS with 0.2%fish skin gelatin for 40 minutes at room temperature in a humidified slide chamber. Incubate slides with anti resus IGA and TTG two primary antibodies each separately for one hour. Next, holding the slide by its edges, gently squirt it with P-B-S-F-S-G Triton X 100 using a transfer pipette.
Then place it in a Copeland jar containing the same buffer. Then place the jar on a rocker for 10 minutes. Repeat the wash once.
Then perform a wash with P-B-S-F-S-G for 10 minutes. Incubate with isotype specific Alexa floor labeled secondary antibodies for 40 minutes following the incubation. Wash the slide as before.
Then add the nuclear DNA stain to pro three to anti quenching mounting medium at a dilution of one to 2000. Apply a drop of the medium directly to each slide. Then place a cover slip over the tissue seal with nail polish.
Place the slide in the dark and allow it to dry. To visualize the IGA and TTG two staining, perform imaging with a confocal laser scanning microscope equipped with the appropriate lasers for the secondary antibodies used for evaluation of non labeled tissue record. Differential interference contrast, DIC images.
DIC is a channel within the microscope that resolves the tissue on a gray scale. This makes it possible to better determine cellular architecture to perform HNE staining. The sample that was placed in zinc formin is first blocked in paraffin and sectioned onto slides.
The slides are then baked at 60 degrees Celsius overnight and placed in an automatic stainer, which stains the slides with HNE when the staining is complete. Inspect the stained biopsy sections under a light microscope equipped with the digital camera using Forex and 40 x magnification. Record three to four images for each magnification Biopsy samples from skin lesions of Reuss Maca were obtained and stained as described in this video to study the effect of withdrawal of dietary gluten on dermatitis here.
Inguinal region of one maca is shown before dietary gluten was withdrawn. When this animal was placed on a gluten-free diet, the dermatitis disappeared and was no longer detectable as can be seen here. Dermatitis reappeared after reintroduction of dietary gluten to evaluate the tissue architecture.
H and e staining was performed prior to withdraw of gluten. The epithelium was thickened with seven to 15 layers of epithelial cells and a thick superficial crust of hyperkeratotic cells infiltrated with degenerating neutrophils. Neutrophils also appeared inside the epithelium and were mixed with moderate numbers of lymphocytes, plasma cells and histiocytes around the ad nexa in the upper dermis after withdrawal of dietary gluten for nine weeks.
Some hyperkeratosis and thickened epithelium were still present, however, only with minimal lymphoplasmacytic infiltration in the upper dermis for comparison. Normal abdominal skin with no epithelial thickening and no lymphoplasmacytic infiltration of the upper dermis is shown here. Confocal microscopy images of the same maca skin biopsies collected before withdrawal of dietary gluten, colocalization of IGA shown in green and ttg two shown in red appears in yellow around the area of dermal papillae indicating the presence of autoantibodies against epidermal transglutaminase.
The to pro three nuclear DNA marker is shown in blue after nine weeks of gluten-free diet. IGA was no longer observed within the epithelium indicating the absence of autoantibodies against epidermal transglutaminase, which would be shown in yellow. Some subepithelial iga, A shown in green was still present the to pro three nuclear DNA marker is shown in blue in other Maccas, including an animal with unrelated idiopathic dermatitis.
Autoantibodies were not detected. The IGA staining is in green ttg two in red and to pro three nuclear DNA marker is in blue. The ttg two positive cells are present inside the epithelium.
A few IGA positive cells beneath the epithelium. While no extensive spectral overlap between the red and green markers is seen indicating the absence of autoantibodies against epidermal transglutaminase. In this study, we use confocal microscopy to visualize the presence of IGA autoantibodies against epidermal transglutaminase.
While attempting the procedure, it is important to remember that most of the cases of dermatitis are not caused by gluten sensitivity. Therefore, other methods including the serum, anti gladin, anti tissue transglutaminase, antibody detection and careful clinical evaluation need to be performed prior. Despite that prevalence of dermatitis, herpetiformis is lower than prevalence of gluten sensitive enteropathy presented.
Results provide foundation for future research, assuming that additional animals with this condition will be identified.
