Intra-Articular MI injection causes measurable changes in pain related behavior, which plays an important role in evaluating the success of modeling The procedure reproducible and it's time efficient. In addition, the dose accelerated are contra label falls small as well as light mammals. And demonstrating the procedure will be Zuxiang Chen, a grad student from our laboratory.
Place the anesthetized rat in the supine position and remove the fur from one knee. Disinfect the exposed skin with alcohol and position the knee at a 90 degree angle to reveal the white patella tendon below the patella. Apply pressure to the patellar tendon with a fingertip to find the gap beneath the patella.
And holding the needle perpendicular to the joint, insert a 26 gauge needle into the junction of the gap and the lateral patellar tendon. When the needle is in place, inject 50 microliters of MIA solution into the joint cavity before slowly retracting the needle. Then wrap a piece of gauze around the injection site to minimize reflux and leakage.
At 1, 7, 14, 21, 28, and 35 days after injection place one injected rat in an elevated 17 by 11 by 13 centimeter plastic with a wire mesh base suspended 50 centimeters above a table, in a quiet environment Give the rat 30 minutes to adapt to the environment before pressing a Von Frey Needle perpendicular on the planter's surface of one hind paw. Increasing the pressure gradually and linearly until paw lifting or paw licking occurs. When paw withdrawal occurs, use a force lower than the previous threshold to ensure that the threshold is the minimum withdrawal force.
When each rat has been tested at least four times, at least three to five minutes apart, record the minimal force eliciting a paw withdrawal reflex and average the data as the MWT of the rats. To test the TWL place one injected rat into each compartment of a 60 by 20 by 14 centimeter plexiglass box. Set down to a three millimeter thick glass plate in a quiet room with a constant temperature and give the rats 30 minutes to adapt to the testing environment.
At the end of the orientation period calibrate the thermal stimulus via an infrared radiometer and set the desired infrared intensity to 70 units. Place the infrared emitter detector on the container directly under the center of the paw being tested, and press start. The timer will start automatically.
As soon as movement of the paw occurs the controller will automatically turn off the infrared light and stop the timer. Record the reaction time when paw withdrawal and paw licking is observed. After repeating the test at least four times for each rat average the data as the TWL of the rats.
To assess the gate pattern of the MIA treated rats, first use the knobs at both ends of a walking compartment to adjust the length of the compartment to 61 centimeters. Next, place a rat into the walking compartment and train the rat to make uninterrupted runs for at least five step cycles at a speed of 18 centimeters per second. When the rat has been trained, capture at least five second videos of continuous movement of each rat with a high speed digital video camera mounted below the transparent treadmill belt.
After capturing at least three uninterrupted runs five minutes apart, draw a bounding box to define the boundary of the animal walking image in the imaging software and enter the run speed for each video. Then start the automatic analysis by the software. After processing, the software will output several spreadsheets reporting the gate indices, including the stance, swing, breaking, propulsion, cadence and step sequence.
MIA induces mechanical allodynia and thermal hyperalgesia in a dose-dependent manner. Remarkably, the decrease of MWT reaches a peak from 21 days to 28 days before rebounding suggesting that joint repair may occur at this stage. Note that the MWT of the three milligram MIA group is still at a low level at these time points.
The change of TWL is roughly consistent with that observed for the MWT. At 1.5 milligrams of MIA, the levels of the gait parameters including the total paw area and the unit stride length are significantly reduced in the MIA group. After 28 days suggesting that MIA induces osteoarthritis related joint pain in rats.
Histopathological analysis by Makin Scoring reveals clear cartilage degeneration collagen disruption and matrix disorganization in the MIA group. In addition, treatment with 1.5 milligrams of MIA induces a significant up regulation of MMP 13 and collagen type 10 and significant downregulation of type two collagen. It's important to find the right junction.
No resistance should be filled when the needle reaches atricular space. This technique facilitated as a production of a stable and reproducible OA pain model making an important contribution to the development of drugs for reducing pain and increasing function. Another side to MI has an extremely objective effect on the hail membrane and there for mask and the gloves should always be worn when preparing this solution.
