Department of Pathology, The Johns Hopkins University
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Tzou, S., Landek-Salgado, M. A., Kimura, H., Caturegli, P. Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis. J. Vis. Exp. (46), e2181, doi:10.3791/2181 (2010).
Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1. It has traditionally been considered a rare disease but reporting has increased markedly in recent years. Hypophysitis, in fact, develops not uncommonly as a "side effect" in cancer patients treated with antibodies that block inhibitory receptors expressed on T lymphocytes, such as CTLA-42 and PD-1 receptors. Autoimmune hypophysitis can be induced experimentally by injecting mice with pituitary proteins mixed with an adjuvant3. In this video article we demonstrate how to extract proteins from mouse pituitary glands and how to prepare them in a form suitable for inducing autoimmune hypophysitis in SJL mice.
The first step in this experimental protocol is to collect a large number of mouse pituitary glands. The second step is to homogenize the glands to prepare a protein extract. The third step is to emulsify these proteins with an oily mixture.
Step 1: Collection and storage of mouse pituitary glands
The mouse pituitary gland sits at the base of the skull in a depression of the sphenoid bone called the sella turcica, surrounded laterally by the trigeminal nerves and anteriorly by the optic chiasm. The gland is composed of a larger anterior lobe and smaller posterior and intermediate lobes, and weighs on average 1.9 mg. Pituitary glands are collected from mice of mixed strains, sexes, and ages, destined to be euthanized by the animal care facility.
To isolate the pituitary gland, the mouse is euthanized, the skin above the skull dissected, and the skull bone cut open with scissors. The brain is lifted to expose the pituitary gland, which is then isolated from the surrounding dura mater, scooped out of the sphenoid bone and stored in a plastic tube on dry ice.
In general we use 300 mouse pituitary glands for one protein preparation, a number that can be collected by one person in 9 hours.
Once the collection is completed, the tube containing the pituitary glands is stored at -80 °C until ready for the next step.
Step 2. Extraction of mouse pituitary proteins
Step 3. Preparation of the emulsion for immunization
Despite the fact that the first patient with autoimmune hypophysitis was reported in 19624, the pathogenic pituitary autoantigens remain to be identified5. Animal models can be very useful to identify these autoantigens, aiding the discovery of biomarkers that are translatable to patient care. This article has demonstrated a straightforward procedure for preparing pituitary proteins in a form that is adequate for inducing autoimmune hypophysitis in experimental animals.
No conflicts of interest declared.
This work was supported by NIH grant DK080351 to PC.
|15-ml Falcon tubes|
|phosphate buffered saline|
|protease inhibitor cocktail|
|BCA assay||Pierce, Thermo Scientific||23225|
|Complete Freund’s Adjuvant (CFA)||Sigma-Aldrich||F-5881|
|Mycobacterium tuberculosis||BD Biosciences||231141|
|2.5 mL Hamilton gastight syringe|
|22-gauge micro-emulsifying needle|