Anterior Cruciate Ligament Transection and Synovial Fluid Lavage in a Rodent Model to Study Joint Inflammation and Posttraumatic Osteoarthritis

Our research investigates how injury to the anterior crusade ligament drives inflammation in progression to post-traumatic osteoarthritis, focusing on the inflammatory mechanism involved. Our protocol offers a straightforward, consistent, and reliable method to induce post-traumatic osteoarthritis and monitor the inflammatory response after injury. The ACLT procedure and methodology for monitoring inflammation provides the experimental setup to further investigate the inflammatory mechanisms contributing to post-traumatic osteoarthritis and possible treatment interventions.

To begin, apply eye lubricant to the anesthetized mouse. Shave the fur over the knee, covering the front and lateral sides from mid-shin to mid-thigh using small hair clippers. Check the mouse for anesthesia depth by ensuring it is unresponsive to the toe pinch reflex.

Then disinfect the exposed skin by applying an antibacterial skin cleanser. Administer 0.05 milligrams per kilogram of buprenorphine subcutaneously for preemptive analgesia. Now, position the mouse on its dorsal side under a dissection microscope, ensuring the operative knee faces upward within the microscope's viewing field.

Position the leg with the knee flexed at approximately 90 degrees, and the patella facing upward. Use surgical tape to keep the knee in the flexed position. Adjust the microscope to focus on the patella.

After making an incision over the patella and exposing the patellar tendon, flex the knee to about 120 degrees. Use the non-dominant hand to keep it flexed and maintain retraction of the skin edges for better visualization of the patellar tendon. Identify the medial border of the patellar tendon, and make an incision along it with a number 11 blade, extending from the midpoint to the superior pole of the patella to open the joint capsule.

Use blunt tip forceps to gently grasp the patellar tendon, lifting it upwards slightly, and shift it laterally to dislocate the patella and fully expose the knee joint. Locate the infra-patellar fat pad. Then use blunt tip forceps to shift its medial portion and expose the anterior cruciate ligament, while preserving fat pad integrity.

While maintaining the knee at 120 degrees flexion, identify the anterior cruciate ligament, or ACL, extending from the lateral femoral condyle to the tibial plateau. Then, use microsurgical scissors to transect the ACL, ensuring the surrounding cartilage, meniscus, and ligaments remain intact. Confirm ACL transection by performing an anterior-posterior drawer test.

Flex the knee to 90 degrees, stabilize the proximal tibia, and gently push the distal femur backward with blunt tip forceps. Reposition the patella and patellar tendon by lifting and shifting medially. Then close the joint capsule with a single stitch using absorbable 6-O sutures.

Close the skin with two to three stitches, using absorbable 6-O sutures. Expose the knee joint of the euthanized mouse by making an anterior longitudinal incision over the knee. Dissect through the skin and underlying tissues to fully reveal the patellar tendon and patella.

With a number 11 blade, open the joint capsule along the medial side of the patellar ligament, extending from its midpoint to the superior edge of the kneecap. Then gently displace the patella laterally to access the joint space, taking care not to damage surrounding structures. Flex the knee to approximately 120 degrees to optimize the joint cavity position for synovial fluid collection.

Now use a P-10 pipette to perform serial lavages of the knee joint with 2.5 microliters of room temperature PBS, and add to a tube prefilled with 100 microliters of PBS for a final volume of 120 microliters. Then, centrifuge the diluted knee lavage fluid at four degrees Celsius for five minutes at 240G to separate the cellular components from the supernatant. Knee size increased significantly at one day post ACLT before progressively declining back to baseline levels.

Leukocyte recruitment in synovial fluid increased sharply at one day post ACLT, and remained elevated through the first week before returning to baseline. Interleukin 1 beta levels also spiked significantly at one day post-surgery, and rapidly decreased to near baseline levels by one week. Both interleukin 6 and TNF alpha increased significantly on the first day after surgery and remained elevated one week post-surgery before returning to baseline.

Matrix-metalloproteinase-9 levels increased dramatically at one day, and then declined steadily to baseline by four weeks post-injury. Micro CT analysis revealed significantly higher osteoarthritis scores in ACLT-injured knees compared to controls at eight weeks post-injury. Histological evaluation demonstrated substantial cartilage degradation in ACLT knees versus controls at eight weeks.