Name: Induction of Experimental Autoimmune Hypophysitis in SJL Mice (Video) | JoVE Journal
Uploaded: 2010-12-17T17:00:00.000+00:00
Duration: 10 min 38 s
Description: Learn to induce experimental autoimmune hypophysitis in SJL mice using pituitary immunogen injections. This protocol enables histopathological assessment of disease severity through leukocyte infiltration analysis.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

This work was supported by NIH grant DK080351 to PC, and by a generous gift of the Terry and Kristine Gore family.

Experimental Protocol
Once the pituitary immunogen is prepared (see companion article), it is injected subcutaneously into SJL mice (The Jackson Laboratories, stock 000686). A second identical injection is repeated 7 days later. Mice are then used to collect in vivo and post-mortem outcomes.
Step 1. Subcutaneous injection of pituitary immunogen into SJL mice
Mice are first anesthetized by injecting 0.5 ml of 20 mg/ml 2,2,2-Tribromoethanol (Avertin, TCI America, T-1420) intraperitoneally. Mice are then injected subcutaneously with 100 &mu;l of the pituitary emulsion that, as indicated in the companion JoVE 2181 article, contains 1 mg of pituitary extract and 0.25 mg of complete Freund's adjuvant. The emulsion is injected in the left dorsal hind leg region ((50 &mu;L ) and the right inguinal region (50 &mu;L). The pituitary emulsion is injected again on day 7 into the opposite sites (50 &mu;l in the right dorsal hind leg region and 50 &mu;l in the left inguinal region). Mice are monitored daily for the first 3 days and then weekly until the day of sacrifice, which is typically 28 days after the first immunization.
Step 2. Collection of in vivo outcomes: Blood assays
The tissue most commonly used to assess experimental outcomes while the mice are still alive is blood. 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.
Blood is collected from live mice as follows. Mice are held securely by the scruff of the neck and the back of the jawbone is located. Once the back of the jawbone is located, 4 mm lancets are used to pierce the submandibular vascular bundle. After blood is collected, place pressure on the on the area using an alcohol swab to avoid a hematoma. Collected blood is centrifuged at 2,000g for 20 minutes, and stored at -80&deg;C until used.
Step 3. Collection of post-mortem outcomes: Pituitary histopathology
Mice are typically sacrificed 28 days after the first immunization. The cardinal feature necessary to establish the diagnosis of EAH is the infiltration of the pituitary gland by hematopoietic mononuclear cells.
Mice are euthanized by placing them in a sealed plastic chamber that is perfused with CO2. Mice are kept in the chamber for a total of 8 minutes. After euthanasia, the pituitary gland is collected as described in the companion article. A diseased pituitary gland appears swollen and more firmly adherent to the surrounding dura mater. Sharp forceps are used to loosen the meninges surrounding the pituitary. Once the pituitary moves freely on the sphenoid bone, one end of the pituitary gland is grasped with forceps and the pituitary gland is carefully lifted. The gland is then placed in a microcentrifuge tube containing Beckstead's fixative5. The pituitary gland is fixed overnight, wrapped in lens paper, and placed in a cassette. The glands are then processed using the following protocol:
 <table border="1">
		<tbody>
 <tr>
 <td>Station Number</td>
 <td>Reagent</td>
 <td>Time in Minutes</td>
 <td>Temperature (Celsius)</td>
 </tr>
 <tr>
 <td>1</td>
 <td>70% alcohol</td>
 <td>5</td>
 <td>25</td>
 </tr>
 <tr>
 <td>2</td>
 <td>70% alcohol</td>
 <td>15</td>
 <td>25</td>
 </tr>
 <tr>
 <td>3</td>
 <td>95% alcohol</td>
 <td>15</td>
 <td>25</td>
 </tr>
 <tr>
 <td>4</td>
 <td>95% alcohol</td>
 <td>15</td>
 <td>25</td>
 </tr>
 <tr>
 <td>5</td>
 <td>100% alcohol</td>
 <td>15</td>
 <td>25</td>
 </tr>
 <tr>
 <td>6</td>
 <td>100% alcohol</td>
 <td>7.5</td>
 <td>25</td>
 </tr>
 <tr>
 <td>7</td>
 <td>100% alcohol</td>
 <td>7.5</td>
 <td>25</td>
 </tr>
 <tr>
 <td>8</td>
 <td>xylene</td>
 <td>20</td>
 <td>25</td>
 </tr>
 <tr>
 <td>9</td>
 <td>xylene</td>
 <td>20</td>
 <td>25</td>
 </tr>
 <tr>
 <td>10</td>
 <td>xylene</td>
 <td>20</td>
 <td>25</td>
 </tr>
 <tr>
 <td>11</td>
 <td>paraffin</td>
 <td>30</td>
 <td>58</td>
 </tr>
 <tr>
 <td>12</td>
 <td>paraffin</td>
 <td>5</td>
 <td>58</td>
 </tr>
 <tr>
 <td>13</td>
 <td>paraffin</td>
 <td>5</td>
 <td>58</td>
 </tr>
 </tbody>
 </table>
 After processing, pituitary glands are embedded in paraffin and at least 5 nonconsecutive sections (5-&mu;m thick) are cut from each gland. After sectioning, the glands are stained with hematoxylin and eosin, and analyzed to quantify the mononuclear infiltration. The section with the most severe infiltration is selected for scoring. The severity is scored based on a subjective estimate of the extent of anterior pituitary replacement or destruction by immune cells: grade 0, no disease; grade 1, 2%-20% involvement; grade 2, 20%-30%; grade 3, 30%-50%; grade 4, 50%-90%; grade 5, greater than 90%. All sections are scored by two individuals blinded to the source of the sections.

<ol><li>Tzou, S. C., Lupi, I., Landek, M., Gutenberg, A., Tzou, Y. M., Kimura, H., Pinna, G., Rose, N. R., Caturegli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autoimmune+Hypophysitis+of+SJL+mice%3A+Clinical+Insights+from+a+New+Animal+Model%5BTitle%5D)+AND+%22Endocrinology%22%5BJournal%5D)">Autoimmune Hypophysitis of SJL mice: Clinical Insights from a New Animal Model.</a> Endocrinology. , (2008).</li>
<li>Twarog, F. J., Rose, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+production+of+thyroid+autoantibodies+in+mice%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">The production of thyroid autoantibodies in mice.</a> J Immunol. 101, 242-2450 (1968).</li>
<li>Patrick, J., Lindstrom, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autoimmune+response+to+acetylcholine+receptor%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Autoimmune response to acetylcholine receptor.</a> Science. 180, 871-872 (1973).</li>
<li>Caturegli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autoimmune+hypophysitis%3A+an+underestimated+disease+in+search+of+its+autoantigen%28s%29%5BTitle%5D)+AND+%22J+Clin+Endocrinol+Metab%22%5BJournal%5D)">Autoimmune hypophysitis: an underestimated disease in search of its autoantigen(s).</a> J Clin Endocrinol Metab. 92, 2038-2040 (2007).</li>
<li>Beckstead, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+simple+technique+for+preservation+of+fixation-sensitive+antigens+in+paraffin-embedded+tissues%3A+addendum%5BTitle%5D)+AND+%22J+Histochem+Cytochem%22%5BJournal%5D)">A simple technique for preservation of fixation-sensitive antigens in paraffin-embedded tissues: addendum.</a> J Histochem Cytochem. 43, 345-345 (1995).</li>
<li>Levine, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Allergic+adenophypophysitis%3A+new+experimental+disease+of+the+pituitary+gland%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Allergic adenophypophysitis: new experimental disease of the pituitary gland.</a> Science. 158, 1190-1191 (1967).</li>
<li>Yoon, J. W., Choi, D. S., Liang, H. C., Baek, H. S., Ko, I. Y., Jun, H. S., Gillam, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induction+of+an+organ-specific+autoimmune+disease%2C+lymphocytic+hypophysitis%2C+in+hamsters+by+recombinant+rubella+virus+glycoprotein+and+prevention+of+disease+by+neonatal+thymectomy%5BTitle%5D)+AND+%22Journal+of+Virology%22%5BJournal%5D)">Induction of an organ-specific autoimmune disease, lymphocytic hypophysitis, in hamsters by recombinant rubella virus glycoprotein and prevention of disease by neonatal thymectomy.</a> Journal of Virology. 66, 1210-1214 (1992).</li>
<li>Davis, J. R., McMahon, R. F., Lowenstein, P. R., Castro, M. G., Lincoln, G. A., McNeilly, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adenovirus-mediated+gene+transfer+in+the+ovine+pituitary+gland+is+associated+with+hypophysitis%5BTitle%5D)+AND+%22J+Endocrinol%22%5BJournal%5D)">Adenovirus-mediated gene transfer in the ovine pituitary gland is associated with hypophysitis.</a> J Endocrinol. 173, 265-271 (2002).</li>
<li>Rotondo, F., Quintanar-Stephano, A., Asa, S. L., Lombardero, M., Berczi, I., Scheithauer, B. W., Horvath, E., Kovacs, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adenohypophysitis+in+rat+pituitary+allografts%5BTitle%5D)+AND+%22Int+J+Exp+Pathol%22%5BJournal%5D)">Adenohypophysitis in rat pituitary allografts.</a> Int J Exp Pathol. , (2010).</li>
</ol>

No conflicts of interest declared.

Four techniques have been reported to induce hypophysitis in experimental animals. The technique described in this article, that is the injection of pituitary proteins mixed with Freund s adjuvant, is the oldest one, dating back to 19676, and has been improved more recently1. Other techniques are the injection of soluble rubella virus glycoproteins in 19927, the stereotactic injection of an adenovirus directly into the pituitary gland in 20028, and the transplantation of pitu...

<table><tbody><tr><td>SJL mice (The Jackson Laboratories)</td><td></td><td>stock 000686</td><td></td></tr><tr><td>2,2,2-Tribrom&amp;#8211;thanol (Avertin)</td><td>TCI America</td><td>T-1420</td><td></td></tr><tr><td>Beckstead&amp;rsquo;s fixative</td><td></td><td></td><td></td></tr></tbody></table>

induction of experimental autoimmune hypophysitis in sjl mice

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse models allow us to study how diseases unfold, often providing a good replica of the same processes occurring in humans. For some autoimmune diseases, like type 1A diabetes, there are models (the NOD mouse) that spontaneously develop a disease similar to the human counterpart. For many other autoimmune diseases, however, the model needs to be induced experimentally. A common approach in this regard is to inject the mouse with a dominant antigen derived from the organ being studied. For example, investigators interested in autoimmune thyroiditis inject mice with thyroglobulin2, and those interested in myasthenia gravis inject them with the acetylcholine receptor3. If the autoantigen for a particular autoimmune disease is not known, investigators inject a crude protein extract from the organ targeted by the autoimmune reaction. For autoimmune hypophysitis, the pathogenic autoantigen(s) remain to be identified4, and thus a crude pituitary protein preparation is used. In this video article we demonstrate how to induce experimental autoimmune hypophysitis in SJL mice.

This video shows how to induce autoimmune hypophysitis in SJL mice and how to assess its severity by histopathology.

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

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.

induction-of-experimental-autoimmune-hypophysitis-in-sjl-mice

Research

JoVE Journal

Immunology and Infection

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T (TEM) lymphocytes. Here we demonstrate how to activate antigen-specific TEM cells, induce adoptive DTH in Lewis rats and monitor the inflammatory response.

Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats

Autoimmune hypophysitis can be reproduced in mice by injecting an extract of mouse pituitary proteins.

Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis

The video demonstrates the induction of experimental autoimmune encephalomyelitis in a mouse model by injecting a combination of myelin oligodendrocyte glycoproteins and inactivated Mycobacterium tuberculosis. The model enables researchers to study disease mechanisms and test therapeutic interventions.

Induction of Experimental Autoimmune Encephalomyelitis in a Mouse Model

In this video, a mouse model of multiple sclerosis is established using experimental autoimmune encephalomyelitis (EAE). The mouse is injected with an emulsion containing a neuronal peptide and an adjuvant, triggering an immune response that activates autoreactive T cells. This is followed by an injection of pertussis toxin (PTX) to increase blood-brain barrier permeability, allowing the autoreactive T-cells to infiltrate the brain. This infiltration initiates a signaling cascade that...

The transfer of bm12 lymphocytes into a C57BL/6 recipient is an established model of systemic lupus erythematosus. Here we describe how to initiate disease using this model and how to characterize T follicular helper cells, germinal center B cells and plasma cells by flow cytometry.

The bm12 Inducible Model of Systemic Lupus Erythematosus (SLE) in C57BL/6 Mice

This manuscript describes the methods for induction and scoring of the experimental autoimmune encephalomyelitis (EAE) model, together with the assessment of immune cell distribution and mRNA cytokine levels in lymph nodes, spleen, blood and spinal cord using flow cytometry and quantitative PCR, respectively, at various disease phases.

Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

This report outlines a simple approach to successfully induce experimental autoimmune neuritis (EAN) using the myelin protein zero (P0)180-199 peptide in combination with Freund's complete adjuvant and pertussis toxin. We present a sophisticated paradigm capable of accurately assessing the extent of functional deficits and neuropathology that occur in this EAN.

A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments

We describe an in vivo immunization, translational hepatitis model in BALB/c mice that can be utilized to study the pathogenesis of drug-induced autoimmune hepatitis including sex differences seen in this disease. We will describe how this model demonstrates reproducible analyses using in vivo and in vitro experimental techniques.

Induction of Drug-Induced, Autoimmune Hepatitis in BALB/c Mice for the Study of Its Pathogenic Mechanisms

Several types of animal models of Hashimoto's thyroiditis have been established, as has spontaneous autoimmune thyroiditis in the NOD mouse. H-2h4 mice are a simple and reliable model for HT induction. This article describes this approach and evaluates the pathological process for a better understanding of the SAT murine model.

Generation of a Mouse Spontaneous Autoimmune Thyroiditis Model

Experimental autoimmune encephalomyelitis is one of the most widely used murine models of multiple sclerosis. In the current protocol, C57BL/6J mice of both sexes are immunized with myelin oligodendrocyte glycoprotein peptide, resulting mainly in ascending paresis of the tail and limbs. Here we discuss the protocol of EAE induction and evaluation.

Modeling Multiple Sclerosis in the Two Sexes: MOG35-55-Induced Experimental Autoimmune Encephalomyelitis

Here, we report a protocol establishing a rheumatoid arthritis (RA) mouse model through adoptive transfer of CD4+ T cells from SKG mice, providing a rapid and reliable experimental tool for investigating the immunological mechanisms, pathological progression, and development of new treatments for RA.

An Adoptive Transfer Model of Rheumatoid Arthritis in Mice

Learn to induce experimental autoimmune hypophysitis in SJL mice using pituitary immunogen injections. This protocol enables histopathological assessment of disease severity through leukocyte infiltration analysis.

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Explore More Videos