The overall goal of the following experiments is to generate and verify a mouse model for autoimmune hepatitis, A human autoimmune liver disease that is characterized by a chronic inflammation of the liver interface, hepatitis, hyper gamma globin, and the production of hallmark autoantibodies animals are first infected with an adenovirus construct expressing the disease causing auto antigen CYP 2D six to induce local inflammation within the liver. Next liver lymphocytes are isolated by liver perfusion and differential density centrifugation. Ultimately, liver damage and auto antigen specific T-cell identification can be assessed by immunohistochemistry and intracellular cytokine staining respectively.
The main advantage of this technique over existing methods is that our autoimmune hepatitis model is characterized by a chronic inflammation of the liver and the auto antigen specific immune response that is almost identical to the one observed in human individuals suffering from type two autoimmune hepatitis. Our model can help to answer key questions in the autoimmune liver disease field, such as how is the progressive destruction of the liver driven in patients with autoimmune hepatitis. Generally in vials method, we struggle with it doing intravenous infection and blood collection via the ophthalmic venous sinus as well as the perfusion of the liver while the porter wearing well needs practice.
After anesthetizing, hold the mouse by the back of the neck and tighten the loose skin of the head with the thumb and middle finger. Note how in this position, the eye protrude slightly due to the traction of the skin adjacent to the eye. Now place the needle into the medial canthus at the junction of one of the eyelids closest to the animal's nose, and slowly inject 100 microliters of the 80 2D six virus solution.
Then slightly withdraw the needle and close the eyelids. Begin by first filling a five milliliter syringe with cold PBS and attaching a 27 gauge needle to the syringe. Then after euthanizing the mouse, make an incision with scissors about one centimeter away from the hind legs of the animal through the skin and muscle layers cut on both sides of the animal with scissors working up to the diaphragm.
And when the diaphragm is reached, pull the skin backwards and cut the diaphragm away from the ribs with scissors and dissect the vena cva. At this point in the dissection, move the stomach and intestines off to the side, exposing the portal vein Perfusing the liver is the trickiest part of the procedure, especially when the liver is chronically inflamed or even fibrotic. The mouse is quite small, so it needs a steady hand to puncture the portal vein accurately.
Furthermore, it's important that the PBS is applied slowly, otherwise the tissue can be damaged. Insert the previously prepared needle into the portal vein and then gently apply pressure to the plunger of the syringe to let the PBS slowly wash the blood out of the liver when the perfusion is complete. Remove the liver by grabbing onto the diaphragm with a pair of forceps and using the scissors to cut the diaphragm away from the body.
Finally, transfer the organ to a Petri dish and remove the diaphragm, gallbladder, and any other tissue still connected to the liver. Note that the liver from the animal with autoimmune hepatitis is fibrotic and the individual lobes are fused. Begin by filling a Petri dish with 10 milliliters of fresh PPS and placing it on ice.
Transfer the perfused liver to the dish and using scissors, cut the tissue into small pieces. Next, pour the tissue and PBS into a 70 micrometer cell strainer and using a glass pestle, press the liver pieces through the strainer. Now carefully pour 10 milliliters of cold collagenase buffer over the tissue pieces, continuing to use the pestle as necessary.
Spin down the cells at 30 times G for three minutes at four degrees Celsius. Transfer the supernatant to a fresh tube, leaving five millimeters of fluid above the pellet centrifuge at 650 times G for 10 minutes at four degrees Celsius. Discard all but the last three millimeters of the supernatant.
Re suspend the pellet in 20 milliliters of per call buffer, and then after spinning down the cells at 600 times G for 20 minutes at four degrees Celsius, discard the supernatant and resuspend the pellet by flicking the tube in persistent autoimmune hepatitis developing after adenovirus 2D six infection of wild type mice in the CYP 2D six model. The liver exhibits and apparently changed morphology as observed here in the right hand figure. In particular, the liver lobes are fused as indicated by black arrowheads and hyperphagic nodules indicated by black arrows appear scattered over the entire liver and a massive capsular fibrosis is visible infection with a control.
Adenovirus not expressing human CYP 2D six has no effect on the liver morphology as seen in the figure on the left. Further extensive and persistent cellular infiltrations appear in the periportal and parenchymal regions of the livers of adenovirus 2D six infected but not adenovirus control infected mice. Note the significant infiltrations of mononuclear cells present only in the adenovirus 2D six infected mice as indicated by the single black arrows.Here.
The triple arrows indicate larger cellular infiltrations in the liver parenchyma bridging neighboring portal tracts. In this figure, it can be observed how fibrosis predominantly develops in the subcapsular region with some collagen bundles protruding into the parenchyma. Note the multiple layers of collagen disposition under the liver capsule as indicated by the triple black arrows with some bundles protruding into the parenchyma as indicated by the single arrows, as well as the presence of large clusters of infiltrating cells as indicated by the black arrow heads.
A third feature of persistent autoimmune hepatitis in adenovirus 2D six infected mice is the predominant generation of liver homing human CYP 2D six, specific CD four and CD eight T cells as seen in these representative dot plots of intracellular cytokine staining, overnight stimulation of liver lymphocytes with the immunodominant CD four or CD eight, human CYP 2D six peptide elicits CD four positive or CD eight positive T-cell specific interferon gamma expression respectively. While attempting this procedure, it's important to remember that the optimal perfusion of the liver is a critical step in order to avoid contamination of liver lymphocytes by peripheral blood monocle cells. Following this procedure, other methods like the isolation of primary hepatic steroid cells can be performed in order to study the activation status and the migratory potential of T cells After its development.
This animal model should pave the way for researchers in the field of autoimmune liver diseases to explore key factors driving the chronic inflammation of the liver, and to evaluate possible treatments for patients.
