Our lab uses mouse models of post-traumatic osteoarthritis to investigate early processes following joint injuries such as anterior cruciate ligament or ACL ruptures, and how these early events can lead to tissue degeneration and osteoarthritis progression. In particular, we're interested in learning how exercise or rest after an injury can affect long-term joint health. There are many animal models of osteoarthritis available, and all of them are useful for investigating certain research questions.
Some of the experimental challenges in this field are selecting the the appropriate models of your study, determining what techniques to use to assess OA progression, and at what time point you will perform these analysis. This knee injury method is very quick and easy to perform. Using noninvasive compression induced injury also allows us to study the joint response at early time points.
And since there's no surgical procedure, we can use in vivo optical imaging to track proteus activity in their joint. In the future, we plan to use different genetic strains of mice, some that are vulnerable to osteoarthritis and some that are resistant to osteoarthritis to determine how certain aspects of the inflammatory response contribute to either joint healing or joint degeneration following injury. To begin, open the software compatible with the load frame instrument and select an existing control file for analysis.
Turn on the actuator. In the Calibration menu, tare the force reading of the load cell and set the displacement of the actuator to 0. After ensuring the mouse is fully anesthetized by toe or tail pinch, place it in a prone position on the platform.
Position the lower leg vertically between two loading fixtures. Fit the foot into the cutout of the top fixture and the knee in the cup of the bottom fixture. Manually adjust the height of the bottom fixture to apply a preload of 1-2 Newton, and tighten the set screw to hold the mouse in position.
Next, apply a single compressive load to a target force of 12-15 Newton. Set the loading rate in the control file and confirm using the force displacement data. If a slower loading rate is applied, stop the compressive load immediately after injury to avoid any damage to other joint tissues.
Prepare a 29-gauge insulin syringe to inject fluorescence activatable probe solution. Place the anesthetized mouse on its side with the snout in a nose cone. With a non-injecting hand, gently retract the skin around the eye to stabilize the head and protrude the eye.
Using the injecting hand, angle the bevel of the syringe towards the eye. Angle the syringe parallel to the mouse's body, and proceed by cautiously advancing the syringe beyond the eye until rigid resistance is met. Slowly inject the probe solution into the retroorbital sinus and pull the needle from the eye socket.
Finally, apply ointment to the injected eye. To begin, place the anesthetized mouse supine in the imaging system with the snout in a nose cone. Position the mouse such that the lower legs are extended and the knees are pointed slightly in the air.
Open the compatible software on the imaging system computer. From the acquisition control panel, click on Initialize to warm up the system and wait until the temperature light turns green, then click on Imaging Wizard. After clicking on Filter Pair, ensure that the setting is configured for epi illumination.
Then press Next. From the pull down list, find the probe of interest to select the correct excitation and emission settings and click Next. Choose Mouse for Imaging Subject.
In Exposure Parameters, ensure the Auto Settings is checked and the Fluorescence and Photograph options are selected. In the checklist of Field of View, select D to 22.6 centimeters and press Next. After confirming all settings are correct, press the Acquire Sequence button and confirm that the image got adequate exposure.
To analyze the image, place a region of interest circle of consistent size over each knee joint on the black and white image. Calculate the total radiant deficiency and average radiant deficiency for each knee joint. If the radiant deficiency is also calculated on the contralateral legs, normalize the data by dividing the radiant deficiency measurement for the injured leg by that of the contralateral leg.
Fluorescence reflectance imaging showed significantly greater protease activity in the injured joints of mice compared to the uninjured mice. Injured joints showed 43%greater average radiant deficiency compared to contralateral joints and joints from uninjured mice. A 30-80%greater radiant deficiency was observed in injured joints compared to contralateral joints at two weeks post-injury.
In contrast, surgically operated joints exhibited 300%greater radiant efficiency at week four compared to contralateral joints, suggesting a notable confounding effect of surgery.
