Visualization of Knee Joint Degeneration after Non-invasive ACL Injury in Rats

Non-invasive ACL Injury

1. Wear proper personal protection equipment. You may use a respiratory mask, but it is not mandatory for this protocol.
2. Anesthetize the rats using an induction chamber with 5% isoflurane and 1 L/min oxygen. Maintain the flow of anesthesia using via a nose cone with 1 - 3% isoflurane and 500 mL/min of oxygen. If the apparatus is not set up on a backdraft or downdraft table, ensure that waste gas is scavenged using a tabletop system and charcoal filters.
3. Perform a toe-pinch to ensure an adequate depth of anesthetic has been reached. Note that it is not necessary to apply eye lubricant the protocol is performed quickly (< 3 min) and there is minimal risk of cornea dryness.
4. Position the right hindlimb at 30° of dorsiflexion and 100° of knee flexion while providing room for anterior subluxation of the tibia relative to the femur.
5. Rigidly mount the top knee stage to a linear actuator.
6. Position the flexed knee on the bottom stage, which is mounted directly above a load cell.
7. Induce ACL injury using a single load of tibial compression at a speed of 8 mm/s.
8. ACL injury is noted by a release of compressive force. This is monitored via a custom program.
9. Post-injury, while the animal is still under the plane of anesthesia, perform a Lachman's test to clinically confirm an ACL rupture has occurred. A Lachman's test is a clinical test used to evaluate the integrity of the ACL by assessing sagittal plan stability. While stabilizing the femur, pull the tibia forward (in an anterior direction) to assess the amount of motion. An intact ACL will produce a 'firm end-feel' where the researcher will not be able to translate the tibia forward. An injured ACL will produce a 'soft or mushy end-feel,' indicative of a torn ACL.
10. Palpate the femur and tibia to detect any gross bone damage. If no contraindications are identified, transfer the animal to its cage and allow it to recover. During this time, monitor the animal to ensure it doesn't display any signs of pain, such as a reluctance to move, vocalization, or abnormal posturing.

µCT imaging of joint degeneration

2-D images are obtained using scanner settings of 70 kV, current 85.5 µA (Figure 2B). Data is collected every 0.6° rotation step at a resolution of 11.5 µm through a complete 180°. Cross-sectional images are reconstructed using a smoothed back-projection algorithm and on the stack of reconstructed images (Figure 2C). Trabecular structure is then analyzed by segmentation in software, whereby a 1.53 mm sphere is centered in the epiphyseal plate of the medial and lateral tibial plateaus and femur to determine trabecular thickness (µm), trabecular separation (µm) and trabecular number (1/mm).^5,6

1. At 4 weeks post-ACL injury, euthanize the rat with prolonged exposure to CO₂ in the induction chamber.
2. Extend and secure ACL injured hindlimb in a custom 3D-printed device (Figure 2A).
3. Acquire images using µCT.
4. Obtain frontal plane radiographs to determine joint space. narrowing (between the femoral condyle and tibial plateau [measured in mm]) compared to the non-injured limb.
5. Obtain 2-D images using the following scanner settings: 70 kV and current 85.5 µA.
6. Collect the data every 0.6° rotation step at a pixel size of 11.5 µm through a complete 180°.
7. Reconstruct cross-sectional images using a smoothed back-projection algorithm on the stack of reconstructed images.
8. To ensure a consistent region of interest is measured, place a 1.53 mm sphere in the epiphyseal plate of the medial and lateral tibial plateaus and femur to determine trabecular thickness (µm), trabecular separation (µm), and trabecular number (1/mm).

Figure 2: A) Custom printed device to hold hindlimb during μCT, B) 2-D images, and C) 3-D μCT.