An Adoptive Transfer Model of Rheumatoid Arthritis in Mice

The scope of this research is to establish a rapid and stable animal model of rheumatoid arthritis for study of pathogenesis and the molecular mechanism. Current rheumatoid arthritis animal models face limitation in experimental practicality, including prolonged induction timelines, suboptimal cost-effectiveness, and the inconsistent disease phenotype reproducibility. Here, we developed a novel rheumatoid arthritis model by adoptively transferring SKG mouse CD4+ T cells into wild type C57BL/6 mice, achieving a high incidence with 100% success within 14 days, which is a lot faster than conventional methods. This cost-effective, highly reproducible system uniquely enables precise immune mechanism analysis and therapy testing.

[Narrator] To begin, immerse euthanized SKG mice in 75% alcohol for five minutes to disinfect. Locate the spleen in the abdominal cavity and the lymph nodes in the inguinal and popliteal regions. Use sterile forceps and scissors to carefully dissect the spleen and lymph nodes and immediately transfer them into pre-chilled PBS. Place the spleen and lymph nodes in separate Petri dishes. Then press the tissues through a 70 micrometer cell strainer using the plunger of a sterile syringe while gradually adding 10 to 12 milliliters of pre-chilled PBS to form a uniform cell suspension. Transfer the cell suspension into a 15 milliliter centrifuge tube. Then centrifuge the tube at 300 G for seven minutes at four degrees Celsius. Discard the supernatant and retain the cell pellet. Adjust the cell concentration to 10 to the power of eight cells per milliliter using an appropriate amount of buffer provided in the CD4+ T cell isolation kit. Then mix 10 microliters of cell suspension with trypan blue to assess cell viability. Now, transfer 100 microliters of the cell suspension into a new tube. Pipette 10 microliters of biotin antibody cocktail into the tube. Mix thoroughly and incubate on ice for 15 minutes. Re-suspend the beads by vortexing at maximum speed. Add 10 microliters of the streptavidin bead suspension to the tube, mix well, and incubate on ice for 15 minutes. Next, add 2.5 milliliters of the kit buffer to the tube and place it on a magnetic separation rack for five minutes. Carefully pour the liquid containing the target cells into a new sterile tube. Centrifuge the tube at 300 G for five minutes at four degrees Celsius. Then discard the supernatant and retain the cell pellet. Now, add enough sterile PBS to adjust the cell concentration to 2 million cells per milliliter and keep the suspension on ice for later use. For adoptive transfer of CD4+ T cells, first, gently clean the inner canthus of an anesthetized C57BL/6 mouse with a sterile cotton swab. Immobilize the mouse by hand. Then draw 200 microliters of CD4+ T cell suspension into a one milliliter syringe. Insert the needle at a 10 to 15 degree angle into the retro-orbital sinus, ensuring accurate placement. Inject the suspension slowly and evenly over 10 to 15 seconds. After withdrawing the needle, press the retro-orbital sinus area gently with a sterile cotton swab for three to five seconds to prevent bleeding. Now place the mouse in a quiet, dry, and clean cage for monitoring until it fully regains consciousness with stable breathing and no abnormal behavior. Record the infusion details and label the model and control group mice, ensuring four animals per group to avoid confusion. Inject CD4+ T cells to the model mice while leaving the control mice untreated. On day four, weigh the mannan powder and dissolve it in sterile PBS to a concentration of 100 milligrams per milliliter. Hold the mouse securely to expose its abdomen, then disinfect the skin using iodophor. Locate the injection site approximately one centimeter to the side of the abdominal midline. Mix the mannan solution thoroughly. Draw 20 to 30 milligrams of the solution into a one milliliter syringe. Insert the needle at a 45 degree angle into the peritoneal cavity, and inject slowly to ensure even distribution. Withdraw the needle slowly and press the injection site gently with a sterile cotton swab for a few seconds. Transfer the mouse into a quiet and clean cage and observe for five to 10 minutes to ensure there is no leakage, abdominal distension, or abnormal breathing. Record the injection details thoroughly for each mouse. The incidence rate in the model group reached 100% and clinical scores for joint swelling significantly increased over six weeks with a temporary relief observed during the second week. Significant thickening and swelling were observed in the forelimb and hindlimb joints of model group mice. Ankle joint pathology in model group mice revealed pronounced synovial thickening, bone discontinuity, and marked inflammatory cell aggregation compared to controls. Serum analysis revealed that the model group had significantly higher levels of IL-6, IL-10, TNF, IL-17, and IFN-gamma compared to the control group. In the spleen, mRNA levels of TBX21 and IL-17 were significantly elevated in the model group compared to controls, indicating increased Th1 and Th17 cell activity.