non-invasive anterior cruciate ligament injury for initiating post-traumatic osteoarthritis in mice

Impact of Non-Invasive ACL Injury and Surgery on Protease Activity in Mice

Osteoarthritis is a pervasive health issue that affects millions of people in the United States1. Post-traumatic osteoarthritis (PTOA) is a subset of OA that is initiated by a joint injury such as anterior cruciate ligament (ACL) rupture, meniscus injury, or intra-articular fracture2. The proportion of symptomatic OA patients that can be classified as PTOA is at least 12%3, and this etiology typically affects a younger population than idiopathic OA4. Mouse models of OA are crucial tools for investigating disease etiology and potential OA treatments on a much shorter timeline (4-12 weeks in mouse models compared to 10-20 years in humans). However, the methods to initiate OA in mice commonly involve invasive surgical techniques such as ACL transection5,6, removal or destabilization of the medial meniscus5,7,8,9,10,11,12,13,14,15,16, or a combination of the two17,18,19, which do not reproduce clinically relevant injury conditions. Surgical models also exacerbate inflammation in the joint due to disruption of the joint capsule, which could accelerate OA progression.
Non-invasive knee injury mouse models provide the opportunity to study biological and biomechanical changes at early time points post-injury and may yield more clinically relevant results20. Our lab has established a non-invasive injury model that uses a single externally applied tibial compression overload to induce anterior cruciate ligament (ACL) rupture in mice21,22,23,24. This non-invasive injury method is able to produce an aseptic joint injury without disrupting the skin or joint capsule.
Fluorescence reflectance imaging (FRI) is an optical imaging method that involves exciting a target with infrared light at a specific wavelength and quantifying the reflected light emitted at another wavelength. Commercially available protease-specific probes can be injected into animal models and FRI can then be used to quantify protease activity at specific sites such as the knee joint. This method has been widely used for in vivo detection of biological activities such as inflammation. The probes used for this application are fluorescently quenched until they encounter relevant proteases. Those proteases will then break an enzyme cleavage site on the probes, after which they will produce a near-infrared fluorescent signal. These probes and this imaging method have been extensively validated and used in studies of cancer25,26,27,28 and atherosclerosis29,30,31,32, and our group has used them for studies of the musculoskeletal system to measure markers of inflammation and matrix degradation23,24,33.
Together, non-invasive joint injury combined with in vivo FRI and protease activatable probes provide a unique ability to track inflammation and protease activity following a traumatic joint injury. This analysis can be done as early as hours or even minutes after injury, and the same animal can be assessed multiple times to study the time course of protease activity in the joint. Importantly, this imaging method may not be feasible when combined with surgical models of OA, since disruption of the skin and joint capsule results in a fluorescence signal that would confound the signal from within the joint.

Author Spotlight: Investigating Early Events and Long-Term Effects of ACL Injuries for Osteoarthritis Progression

Non-Invasive Compression-Induced Anterior Cruciate Ligament (ACL) Injury and In Vivo Imaging of Protease Activity in Mice

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 appropriate models of your study, determining what techniques to use to assess OA progression, and at what time point you will perform this 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.

Watch this Scientific Journal Video about Non-Invasive Anterior Cruciate Ligament Injury for Initiating Post-Traumatic Osteoarthritis in Mice at JoVE.com

Non-Invasive Anterior Cruciate Ligament Injury for Initiating Post-Traumatic Osteoarthritis in Mice

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, tear the force reading of the load cell and set the displacement of the actuator to zero.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 one to two Newton and tighten the set screw to hold the mouse in position. Next, apply a single compressive load to a target force of 12 to 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 the 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 retro-orbital sinus and pull the needle from the eye socket. Finally, apply ointment to the injected eye.

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51 Views.  University of California Davis Health. 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, tear the force reading of the load cell and set the displacement of the actuator to zero.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 th...