Autoimmune hypophysitis is characterized by an enlargement of the pituitary gland and a deficiency in one or more pituitary hormones. The causative of auto antigens remain unknown. A companion video demonstrates a protocol for preparing a mouse pituitary immunogen, which will be used here to create a mouse model resembling human autoimmune lymphocytic hypophysitis.
The pituitary immunogen is injected into a susceptible mouse strain. Then the injection is repeated seven days later after 14 days, blood is collected for biomarker studies. On day 28, pituitary glands are harvested from sacrificed mice sectioned and stained to determine the extent of leukocyte infiltration.
Autoimmune hypophysitis is an increasingly recognized disease that can be difficult to distinguish from other non hormone secreting masses that arise in the cellica. Although the disease was first described about 50 years ago, the auto antigens recognized by the patient's immune system in autoimmune hypophysitis have remained elusive. In 2008, we published the development of a mouse model that closely recapitulates the human disease it was designed to mimic.
This mouse model can be used to discover the pituitary auto antigens involved and to learn more about the pathogenesis and natural histories of hypophysitis. The model is based on the injection of mouse pituitary proteins into a susceptible strain like the SJL strain. These proteins need to be mixed with a strong adjuvant in order to create a local inflammatory environment that eventually induces the appearance of pituitary autoreactive lymphocytes.
First in the lymph nodes draining the injection site and then in the pituitary gland. The model requires the injection of relatively large amounts of pituitary proteins, a total of two milligram per mouse. This suggests that the cosset auto antigen or auto antigens are present in minute amount within the fertility gland Injection of a properly prepared emerge is critical for the induction of the disease because it translates into a slow, continuous release of pituitary proteins at the injection sites, ultimately supporting the generation and activation of pituitary specific lymphocytes.
In the companion video, a method for preparing pituitary immunogen is presented in that video. Pituitary glands isolated from euthanized mice are homogenized and clarified by low speed centrifugation. The the protein mixture is assessed for quantity by colormetric, BCA assay and quality by poly acrylamide gel electrophoresis.
The aqueous protein homogenate is then mixed with an oily adjuvant containing inactive mycobacterium tuberculosis bacilli to produce the pituitary emulsion. After preparing the emulsion as demonstrated in the companion video, remove the micro emulsifying needle from the one milliliter syringe and attach a 25 gauge needle. 100 microliters of the pituitary emulsion is required for each mouse to be injected.
After anesthetizing a mouse with Everton, ensure there is no response to a toe pinch, then inject 50 microliters of the emulsion subcutaneously in the left dorsal hind leg region. Ensure that the tip of the needle is completely under the skin and that no emulsion oozes out of the injection site. Next, inject 50 microliters in the right inguinal region.
Again, ensure that nothing oozes out. Note that the emulsion is whitish and can be difficult to distinguish in the context of the white background of the SGL L mouse fur following injection. Return the mouse to its cage and monitor its recovery.
Monitor the injected mice daily for the first three days after injection to ensure that no extreme distress occurs. The adjuvant is pro-inflammatory since it contains inactivated m tuberculosis. Bacilli on day seven, repeat the injection on opposite anatomical sides.
Inject 50 microliters in the right dorsal hin leg region, and 50 microliters in the left inguinal region. Monitor the mouse as before experimental outcomes will be assessed after an immune response has occurred. Blood is the tissue most commonly used to assess experimental outcomes while the mice are still alive.
Blood is routinely drawn 14 days after the first immunization to measure the serum levels of pituitary antibodies or other markers like cytokines and chemokines. To collect blood from a live mouse, hold the animal securely by the scruff of the neck and locate the back of the jawbone. Once this location is clear prick the submandibular vascular bundle with a four millimeter lancet.
Blood will begin to emerge from the pierced area. Collect six drops of blood into a micro centrifuge tube. Clean the pierced area with a wet paper towel to stop the bleeding.
Place the blood tubes in a centrifuge and spin it 2000 G for 20 minutes. This centrifugation separates the blood cells, which will end up in the pellet from the serum snat. Transfer the serum to a new micro centrifuge tube.
Store the serum at minus 80 degrees Celsius until it is needed. It takes approximately one month for the immunized mice to develop full-blown autoimmune hypophysitis. Therefore, mice are typically sacrificed 28 days after the first immunization.
After cutting the skull, lift the brain and flip it backward to expose the pituitary gland. A diseased pituitary gland appears swollen and more firmly adherent to the surrounding meninges. Move the tip of sharp forceps around the pituitary gland to break the meninges loose.
Once the pituitary moves freely on this phen bone, grab one end of the gland with forceps and carefully lift it off this phen bone. Place the tissue in a micro centrifuge tube containing one milliliter of betted solution, a zinc based fixative and fix the gland overnight at room temperature. The next day, transfer the pituitary to a piece of lens paper.
Wrap the pituitary gland in the lens paper and place it in a histology cassette. Process the specimen by incubating and increasing concentrations of alcohol and then xylene after processing. Remove the lens paper and place the pituitary in a metal mold.
Then cover the pituitary with paraffin. Place the tissue cassette on top of the mold and fill it with additional paraffin. Finally, put the assembly on ice to let the paraffin solidify.
Once the paraffin has solidified, use a microtome to cut five micron thick sections. Pick up the sections on a glass slide. Collect at least five non-consecutive sections on the same slide from each mouse pituitary gland.
Stain the slides with hematin and eosin using an automated tissue staining station. Examine the slides with a light microscope to collect the results. The cardinal feature of a pituitary gland that has developed hypophysitis is its infiltration with lymphocytes which appear as small blue cells that percolate through the pituitary parenchyma in isolation or forming clusters.
Lymphocytes are associated with various degrees of destruction of the normal endocrine cells of the pituitary. The section with the most severe lymphocytic infiltration and destruction of the pituitary gland is chosen for scoring by two investigators blind to the experimental conditions. Scoring is based on a subjective estimate of the extent of the damage.
A score of zero indicates a normal pituitary gland or lymphocytes and other hematopoietic mononuclear cells are rarely seen. A score of one indicates that the mononuclear infiltration occupies between two and 20%of the total anterior pituitary area. A score of two indicates the mononuclear infiltration occupies between 20 and 30%of the total anterior pituitary area.
A score of three indicates the mononuclear infiltration occupies between 30 and 50%of the total anterior pituitary area. A score of four indicates that the mononuclear infiltration occupies between 50 and 90%of the total anterior pituitary area, and a score of five indicates the mononuclear infiltration occupied nearly the entire anterior pituitary area. Once mastered, the injection of the immunogen into one mouse can be performed in 15 minutes.
The collection of peripheral blood from one mouse can be accomplished in five minutes and the preparation of slides for histopathology in six hours while attempting the immunization procedure. It's important to pay close attention to the health of the mouse following anesthesia and injection of the immunogen. In addition to pit pitu histopathology.
This procedure can be used to collect a vast array of experimental outcomes using various methods like assessment of the invoice subpopulations in the draining lymph nodes by flow cytometry. The technique of immunization with a tissue extract for the induction and autoimmune disease paves the way for researchers in the field of autoimmunity to discover the key events in disease Pathogenesis. After watching this video, it should have a good understanding of how to immunize mice for inducing experimental autoimmune hypophysitis and of how to evaluate its incidents and severity.
Don't forget that working with microtone blades can be hazard. Those, and therefore precautions such as wearing gloves and working in good lighting conditions should always be taken.
