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

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 transection^5,6, removal or destabilization of the medial meniscus^{5,7,8,9,10,11,12,13,14,15,16}, or a combination of the two^17,18,19, 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 mice^21,22,23,24. 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 cancer^25,26,27,28 and atherosclerosis^29,30,31,32, and our group has used them for studies of the musculoskeletal system to measure markers of inflammation and matrix degradation^23,24,33.

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 instrument (see Table of Materials), but could be adapted for similar systems.

1. Open the software compatible with the load frame instrument (see Table of Materials) and select an existing control file or create a new file.
2. Turn on the power of the actuator.
3. In the calibration menu, tare the force reading of the load cell and set the displacement of the actuator to 0.
4. Use 1%-4% inhaled isoflurane in oxygen to anesthetize the mice and ensure the animals are fully anesthetized by toe pinch and/or tail pinch.
5. Place the mouse in a prone position on the platform. Position the lower leg vertically between two loading fixtures (Figure 1) (see Table of Materials). Fit the foot into the cutout of the top fixture and the knee in the cup of the bottom fixture.
6. Manually adjust the height of the bottom fixture to apply a preload of 1-2 N (monitored in real-time on the computer screen) and tighten the set screw to hold the position. The preload is necessary to hold the leg in the correct position prior to applying the injury load.
7. Apply a single compressive load to a target force (~12-15 N) or target displacement (~1.5-2.0 mm).
   NOTE: Applying the load at a slower loading rate (~1 mm/s) will provide a greater level of real-time monitoring and control but will likely result in an avulsion failure of the ACL. Applying a faster loading rate (~200 mm/s) will be more likely to produce a mid-substance ACL injury²². If a tibial fracture or other excessive injury is determined to have occurred, euthanize the animal using an IACUC-approved method prior to the animal recovering from anesthesia.
   1. Set the loading rate in the software control file and confirm using the force-displacement data.
      NOTE: Bone fracture during tibial compression loading is typically not a concern since the fracture force (~20 N) is considerably higher than the ACL injury force. However, this should be monitored with palpation, and imaging (i.e., x-ray) can be used to confirm that no tibial fractures have occurred.
8. Injury is typically indicated with a sound ("click" or "crunch") and a release of force that is identifiable on the force-displacement plots (Figure 1C). If a slower loading rate is used, stop the compressive load immediately after injury to prevent further loading and possible damage to other joint tissues.
   NOTE: ACL injury typically occurs at 8-15 N depending on body mass³⁴. It is important to set a target force greater than the anticipated ACL injury force.
9. Confirm the ACL injury using an anterior-posterior drawer test^35,36 or comparable assessment of joint instability.
10. Administer an animal weight-dependent dose (e.g., 0.05-0.1 mg/kg SC or IP of buprenorphine, see Table of Materials) to mice post-injury, with duration and dose as recommended and approved by the home institution IACUC.
    NOTE: NSAIDs may alter the progression of PTOA after injury, so it is not recommended that NSAIDs are used for pain management in this injury model unless it is a specific aim of the study.

2. Animal preparation for FRI imaging

NOTE: For optical imaging, animal fur (particularly dark fur) is highly effective at blocking, absorbing, and scattering light, therefore, fur must be removed as much as possible from the area around the knee joints before imaging. A depilatory cream is typically more effective for fur removal than clippers. Nude or hairless mice don't require fur removal. However, fur removal is necessary for most commonly used mouse strains (e.g., C57BL/6). If possible, feed mice with low-fluorescence purified food before imaging. Standard mouse chow contains chlorophyll, which auto-fluoresces with a wavelength of around 700 nm and may affect data collection from the near-infrared FRI system.

1. Anesthetize mice with 1%-4% inhaled isoflurane in oxygen. Keep mice on a heating pad as much as possible and apply eye ointment to prevent irritation of the eyes.
2. Use a cotton swab to apply depilatory cream (see Table of Materials) onto the anterior (cranial) aspect of the legs of the mice around the knee joint.
3. Let the cream stand for ~1 min, then use wipes to remove the cream and fur from the leg. Repeat if necessary.
4. Once the knee joints are fully exposed without any fur covering the area, clean the legs with alcohol wipes to remove any remaining depilatory cream.
   NOTE: Depilatory cream can be used on the same mice multiple times throughout a study, but these applications should be at least one week apart to prevent unnecessary irritation of the skin.

3. Preparation of the probe solution

1. If necessary, dilute the fluorescence activatable probe according to the manufacturer's instructions in sterile 1x phosphate-buffered saline (PBS). One vial of the commercially available probe (see Table of Materials) typically contains 20 nmol in 0.15 mL of 1x PBS. To dilute the solution in the vial, add 1.35 mL of 1x PBS to make 20 nmol in 1.5 mL of 1x PBS.
   NOTE: After dilution, one vial can be used to image ten mice when injecting 0.15 mL per mouse.
2. Vortex the solution with a minimum speed (~2000 rpm) for 30 s to ensure that the probe is dissolved in solution, and then centrifuge briefly to ensure that all liquid is out of the lid.
   ​NOTE: The solution can be stored at 2-8 °C in a location that is protected from light for up to 12 months.

4. Retro-orbital injection

NOTE: Refer to Yardeni et al. regarding the details of this procedure³⁷.

1. Use 1%-4% inhaled isoflurane in oxygen to anesthetize mice and place the mouse on its side with the snout in a nose cone.
2. Use ~29 G insulin syringes for injection of probe solution (prepared in step 3).
3. Keep the syringe covered before use to prevent exposure to light.
4. When administering the injection:
   1. Inject on the inside of the eye (lacrimal caruncula), and ensure that the bevel of the syringe is angled towards the eye. For right-handed people, it is recommended to inject into the right eye of the mouse with the animal facing right.
   2. With the non-injecting hand, gently pull back the skin around the eye to stabilize the head and cause the eye to protrude.
   3. Angle the syringe parallel to the body of the mouse.
   4. Gently advance the syringe past the eye until it meets rigid resistance; do not attempt to push past this point.
   5. Slowly inject the probe solution into the retro-orbital sinus, then slowly pull the needle out of the eye socket. If no solution comes out with the needle, the injection is successful.
   6. Apply saline or eye ointment to the injected eye.
      ​NOTE: Based on the documentation provided with the imaging probes, the optimal imaging time is typically between 1 and 2 days after injection of the probe solution. If possible, it is recommended to do an initial time screening to determine the optimal imaging time for each specific application. Mice will metabolize the injected probe within approximately 7 days, after which a new dose of probe solution will need to be injected if additional time points are desired.

5. Fluorescence reflectance imaging

NOTE: The procedures in this section are specific to a commercially available optical imaging system (see Table of Materials) . Similar imaging can be performed with comparable systems.

1. Anesthetize mice with 1%-4% inhaled isoflurane in oxygen and place the animal supine in the imaging system with the snout in a nose cone.
2. Position the mouse with the lower legs extended so that the knees are pointed slightly in the air (it may be necessary to tape down the feet). It is critical that a consistent positioning is used for all animals.
3. Open the compatible software (see Table of Materials) on the imaging system computer; the "Acquisition Control Panel" will appear.
4. To warm up the system, click on Initialize and wait until the temperature light turns green.
5. Click on Imaging Wizard, and ensure that the "Imaging Wizard" window will appear.
6. Click on Filter Pair, and ensure the setting is on 'Epi-Illumination', then press Next.
7. To select the correct excitation/emission settings, find the probe of interest from the pulldown list. If one can't find the correct probe, find the Name 'Input Ex/Em' and manually type in the value of Excitation Peak and Emission Peak based on the property of the probe that is to be used (e.g., for Excitation Peak, enter 675, and for Emission Peak, enter 720). Click on Next.
8. Choose Mouse for "Imaging Subject". In "Exposure Parameters", ensure the Auto Settings is checked, and the Fluorescent and Photograph options are selected. Select D-22.6 cm in the checklist of "Field of View". Press Next.
9. The imaging setting can be seen and modified on the right panel of the "Acquisition Control Panel". Ensure all settings are correct, and press the Acquire Sequence button. After the image shows up, confirm that the image had adequate exposure. If not, change the exposure time setting and click on Acquire Sequence again.
10. To analyze the image, position a region of interest (ROI) circle with consistent size over each knee joint on the black and white image (this prevents biased positioning based on areas of fluorescent signal).
11. Calculate total radiant efficiency and/or average radiant efficiency for each knee joint. If the radiant efficiency is also calculated on the contralateral legs, normalize the data by dividing the radiant efficiency measurement for the injured leg by the radiant efficiency measurement of the contralateral leg.
    NOTE: If a region of interest with consistent area is used for all knees, both total radiant efficiency and average radiant efficiency will yield similar results. Using average radiant efficiency is recommended if regions of interest with different sizes are used. Normalizing radiant efficiency data from the injured joint with the data from the uninjured contralateral knee will provide an internal control to account for any differences in the amount of probe injected and the delivery efficiency between different animals.

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 mice^20,21,24,33. 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...

Materials

Name	Company	Catalog Number	Comments
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