Assessment of Knee Hyperalgesia in Mice using Pressure Application Measurement

We developed translationally relevant pain assays in mice with progressive experimental knee osteoarthritis to enhance understanding of mechanisms underlying joint pain. Human with osteoarthritis also have lowered pain pressure thresholds. Method is robust and reproducible and offers medium throughput approach for testing the potential analogistic effects of systemically or intraarticularly administered novel compounds.

I am interested in studying anxiety associated with progressive experimental OA.We are developing assays to study correlation of anxiety with OA pain and to enable exploring underlying mechanisms. To begin, connect a cable from the PAM device to the computer. Connect the second cable from the device to the force transducer.

Now, switch on the computer and launch the PAM software. Set the maximum pressure value in the software to 450 grams. Next, leave the mice in the testing room for 15 to 30 minutes until they acclimatize and settle down.

Confirm the mouse is calm and still while restrained. If it is not, return it to the cage and delay testing by a few days. Cradle the mouse in the left hand, restraining the back and firmly holding the tail down with the fourth and fifth fingers.

Loop the index finger of the right hand through the tie of the transducer and insert the finger with the transducer into a transparent plastic bag. Now, with the right thumb and middle finger, hold the right leg. Gently pin the paw down onto the thenar of the left hand using the middle finger.

Ensure the knee is positioned at approximately 90 degrees flexion at the start of testing. Next, touch the lateral side of the knee with the right thumb and the medial side of the transducer with the index finger. Apply pressure against the knee slowly, then apply increasing force at the rate of 30 grams per second.

Continue increasing the pressure constantly until reaching 450 grams per second or until the mouse shows pain-related behaviors, such as vocalization, muscle twitching, body wriggling, or whisker movement. Once the threshold has been reached, retract the finger with the transducer. Record the pressure displayed on the screen.

Intraarticular injection of Pam3CSK4 in the mice resulted in knee hyperalgesia in a dose-dependent fashion, peaking four hours after injection and returning to baseline by 24 hours. Intraarticular injection of lidocaine at the four-hour peak reversed Pam3CSK4-induced knee hyperalgesia within 30 minutes. DMM surgery caused pronounced knee hyperalgesia two weeks after surgery, slowly recovering through week 16.

Sham-operated mice also developed pronounced knee hyperalgesia after surgery, but knee hyperalgesia recovered faster. Intraarticular injection of lidocaine four weeks after DMM surgery resulted in an immediate reversal of knee hyperalgesia within 30 minutes with the effect diminishing by four hours.