Measurement of pain in pre-clinical models of arthritis is difficult, but it is essential for the evaluation of both regenerative and analgesic treatments. Our protocol is sensitive and reproducible for the measurement of both evoked and spontaneous pain behaviors. And it is simple and relatively inexpensive.
Arthritis is the largest contributor to disability, and pain is the major cause of disability. Treatment of arthritis is currently inadequate. Pre-clinical models are an important part of treatment development.
Demonstrating this procedure will be Christopher Dorman, a technician, and Sandra Frizelle, a staff scientist from my laboratory. To prevent the needle from being inserted too deeply and exiting the joint posteriorly, cover up a point 5 inch needle with a sheath of polyethylene tubing until only 2.5 millimeters of the needle protrude out of the sheath. Next, shave the fur from the knee of the anesthetized mouse to be injected, and sterilize the exposed skin with povidone iodine scrub.
Then insert the sheathed needle into the joint at the midline through the patellar tendon just inferior to the lower edge of the patella, and inject the substance of interest. Before assessing the evoked joint pain, use an altimeter level 4 to practice applying seven-hundred gram forces per centimeter squared of consistent and firm pressure. The device will emit 4 beeps when the appropriate level of pressure is applied.
When the appropriate pressure can reliably be applied, scruff the mouse at the neck with the index finger and thumb tightly enough to prevent casual movement. And use the fourth and fifth fingers to restrain the tail. The mouse should be able to make discrete, brief movements with effort and when stimulated, but should not be able to move freely or continuously.
Administer repeated firm palpations concurrently to the lateral and medial sides of each knee with the thumb and index finger of the free hand at approximately the joint line once per second for 1 minute. And have an observer count the number of times the animal vocalizes and fights to escape the restraint during this 1 minute pressure application period. The sum of the bites and vocalizations is considered the Evoked Pain Score.
Open the advanced dynamic weight bearing software program, and select Mice"as the sensor type. Prior to each experiment, select full calibration"under the calibration menu. And enter zero"for the tare value.
And the weight of the foam pad, medium density fiber board, plastic beaker, and water as the calibration weight. Select next"and tare"And cover the sensor pad with the foam pad, medium density fiber board, and plastic beaker of water. When all of the materials have been placed on the sensor pad, select gain preview"And adjust the calibration weight until it is evenly distributed across the individual sensors.
When the value of the calibration indicator is above ninety-five, select gain definition"to produce a normalized mean for the individual sensors and to confirm the integrity of the sensors. Then, select next"and save the calibration file in the calibration folder. Select new"and enter the project and animal names for the first mouse to be tested.
Select browse"and open the calibration file that was created for the project. Then, select next"confirm that enable video capture"is selected, and select ok"Next, weigh the animal. Select capture"and record the weight in the software.
After selecting ok"place the mouse in the chamber for 5 minutes. And select launch"The software will record the animal's activity, capture a video, and synchronize with the sensor data, and automatically partition the video into analyzable and non-analyzable segments. At the end of the assay period, select easy scoring"and independent pause setting"And select fast forward"to move to the first analyzable segment.
If a stable zone does not have the correct paw identified, hold the cursor over that zone, and press the appropriate shortcut key corresponding to the correct body part. The advanced dynamic weight bearing software will automatically calculate the percentage of analyzed time spent, and percentage of body weight placed on each of the 4 limbs. Then select results"export"and yes"to export the first 5 summary lines of data into the appropriate spreadsheet program.
For each additional animal in the same group, select new"and enter a unique animal name without changing the project name. Then capture each animal's activity in the chamber as just demonstrated. Evoked Pain Scores for acute inflammatory care-a-gene-a induced arthritis, chronic inflammatory complete f-roy-nes add-jav-ent induced arthritis, and chronic non-inflammatory cole-ag-in-a induced arthritis increase over those observed for naive un-injected animals for all three types of joint pain.
Females, but not males with chronic degenerative joint pain, demonstrate similar reductions in advance dynamic weight bearing measures compared to naive mice in acute and chronic inflammatory joint pain. Males with acute inflammatory joint pain transfer more weight to the 4 limbs than do naive animals. Weight bearing on the 4 limbs increases in female mice compared to naive animals, but not in males with chronic degenerative joint pain.
In both males and females with chronic degenerative joint pain, there is no difference in time spend on the left hind limbs compared to naive animals. And females with chronic degenerative joint pain spend more time on the non-arthritic right hind limb compared to un-injected mice. Finally, male mice with acute inflammatory joint pain, and females with chronic degenerative joint pain, spend significantly more time on their 4 limbs than do naive animals.
Correct identification of the posterior of the animal's feet in relation to it's body is very important. Focus on the posterior of the animal rather than it's head. With this method, we are able to test the affectiveness of novel, new inter-articular analgesics.
