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

Published: September 29, 2023

doi:

¹Lawrence J. Ellison Musculoskeletal Research Center, Department of Orthopaedic Surgery,University of California Davis Health

Summary

Non-invasive ACL injury is a reliable and clinically relevant method for initiating post-traumatic osteoarthritis (PTOA) in mice. This injury method also allows for in vivo quantification of protease activity in the joint at early time points post-injury using protease-activatable near infrared probes and fluorescence reflectance imaging.

Abstract

Traumatic joint injuries such as anterior cruciate ligament (ACL) rupture or meniscus tears commonly lead to post-traumatic osteoarthritis (PTOA) within 10-20 years following injury. Understanding the early biological processes initiated by joint injuries (e.g., inflammation, matrix metalloproteinases (MMPs), cathepsin proteases, bone resorption) is crucial for understanding the etiology of PTOA. However, there are few options for in vivo measurement of these biological processes, and the early biological responses may be confounded if invasive surgical techniques or injections are used to initiate OA. In our studies of PTOA, we have used commercially available near-infrared protease activatable probes combined with fluorescence reflectance imaging (FRI) to quantify protease activity in vivo following non-invasive compression-induced ACL injury in mice. This non-invasive ACL injury method closely recapitulates clinically relevant injury conditions and is completely aseptic since it does not involve disrupting the skin or the joint capsule. The combination of these injury and imaging methods allows us to study the time course of protease activity at multiple time points following a traumatic joint injury.

Introduction

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.

Protocol

All procedures described have been approved by the Institutional Animal Care and Use Committee at the University of California Davis. 3-month-old male C57BL/6J mice were used for the present study. 1. Non-invasive ACL injury NOTE: ACL injury produced by an externally applied compressive load is a simple and reproducible method that closely recapitulates ACL injury conditions in humans. This protocol is written for a commercially available load frame i…

Representative Results

After applying a single compressive force (1 mm/s until injury) on the lower legs of 3-month-old male C57BL/6J mice, ACL injury was consistently induced in all mice. The average compressive force at knee injury was approximately 10 N (Figure 1). FRI analysis showed significantly greater protease activity in the injured joints of mice subjected to non-invasive ACL injury at 7 days following injury (Figure 2). FRI analysis of knee joint…

Discussion

This protocol has established and rigorously described a reproducible, non-invasive method for inducing ACL injury in mice²⁰^,²¹^,²⁴^,³³. This simple and efficient injury method can be performed in just a few minutes, which facilitates high-throughput studies of PTOA. This injury method also closely recapitulates injury conditions relevant to human ACL injury. Surgical methods used to induce OA in…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases, part of the National Institutes of Health, under Award Number R01 AR075013.

Materials

10x Phosphate-Buffered Saline	Tissue Protech	PBS01-32R	or equivalent
Air Anesthetia System			Isoflurane vaporizor with induction chamber and nose cone
Buprenorphine			Analgesic post-injury
Depilatory Cream	Veet	B001KYPZ4G	or equivalent
Fixtures			Custom-made knee fixture, ankle fixture, and platform
IVIS Spectrum	Perkin Elmer	124262	Can also use comparable optical imaging system
Kimwipes	Kimberly-Clark Corporation	06-666	or equivalent
Living Image software	Perkin Elmer
Materials testing systems	TA Instruments		Electroforce 3200 or equivalent
ProSense680	Perkin Elmer	NEV10003	Can also use other probes such as OsteoSense, MMPSense, Cat K, AngioSense, etc.
Sterile Syringe with Needle	Spectrum Chemical Mfg. Corp.	550-82231-CS	Covidien 1 mL TB Syringe with 28 G x 1/2 in. Needle, Sterile or equivalent
Uniaxial load cell	TA Instruments		20 N capacity
Vortex-Genie 2	Scientific Industries, Inc.	SI-0236	or equivalent
WinTest software	TA Instruments		compatible with Electroforce 3200

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Lin, Y., Christiansen, B. A. Non-Invasive Compression-Induced Anterior Cruciate Ligament (ACL) Injury and In Vivo Imaging of Protease Activity in Mice. J. Vis. Exp. (199), e65249, doi:10.3791/65249 (2023).