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.

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.
<ol>
	<li>Open the software compatible with the load frame instrument (see Table of Materials) and select an existing control file or create a new file.</li>
	<li>Turn on the power of the actuator.</li>
	<li>In the calibration menu, tare the force reading of the load cell and set the displacement of the actuator to 0.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Injury is typically indicated with a sound (&#34;click&#34; or &#34;crunch&#34;) 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.</li>
	<li>Confirm the ACL injury using an anterior-posterior drawer test35,36 or comparable assessment of joint instability.</li>
	<li>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.</li>
</ol>
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&#39;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.
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Let the cream stand for ~1 min, then use wipes to remove the cream and fur from the leg. Repeat if necessary.</li>
	<li>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.</li>
</ol>
3. Preparation of the probe solution
<ol>
	<li>If necessary, dilute the fluorescence activatable probe according to the manufacturer&#39;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.</li>
	<li>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. 
	&#8203;NOTE: The solution can be stored at 2-8 &#176;C in a location that is protected from light for up to 12 months.</li>
</ol>
4. Retro-orbital injection
NOTE: Refer to Yardeni et al. regarding the details of this procedure37.
<ol>
	<li>Use 1%-4% inhaled isoflurane in oxygen to anesthetize mice and place the mouse on its side with the snout in a nose cone.</li>
	<li>Use ~29 G insulin syringes for injection of probe solution (prepared in step 3).</li>
	<li>Keep the syringe covered before use to prevent exposure to light.</li>
	<li>When administering the injection:
	<ol>
		<li>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.</li>
		<li>With the non-injecting hand, gently pull back the skin around the eye to stabilize the head and cause the eye to protrude.</li>
		<li>Angle the syringe parallel to the body of the mouse.</li>
		<li>Gently advance the syringe past the eye until it meets rigid resistance; do not attempt to push past this point.</li>
		<li>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.</li>
		<li>Apply saline or eye ointment to the injected eye. 
		&#8203;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.</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Open the compatible software (see Table of Materials) on the imaging system computer; the &#34;Acquisition Control Panel&#34; will appear.</li>
	<li>To warm up the system, click on Initialize and wait until the temperature light turns green.</li>
	<li>Click on Imaging Wizard, and ensure that the &#34;Imaging Wizard&#34; window will appear.</li>
	<li>Click on Filter Pair, and ensure the setting is on &#39;Epi-Illumination&#39;, then press Next.</li>
	<li>To select the correct excitation/emission settings, find the probe of interest from the pulldown list. If one can&#39;t find the correct probe, find the Name &#39;Input Ex/Em&#39; 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.</li>
	<li>Choose Mouse for &#34;Imaging Subject&#34;. In &#34;Exposure Parameters&#34;, ensure the Auto Settings is checked, and the Fluorescent and Photograph options are selected. Select D-22.6 cm in the checklist of &#34;Field of View&#34;. Press Next.</li>
	<li>The imaging setting can be seen and modified on the right panel of the &#34;Acquisition Control Panel&#34;. 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.</li>
	<li>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).</li>
	<li>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.</li>
</ol>

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

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D., Huizing, M., Hoogstraten-Miller, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retro-orbital+injections+in+mice.">Retro-orbital injections in mice.</a> Laboratory Animals (NY). 40 (5), 155-160 (2011).</li><li>Kokubun, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+changing+the+joint+kinematics+of+knees+with+a+ruptured+anterior+cruciate+ligament+on+the+molecular+biological+responses+and+spontaneous+healing+in+a+rat+model.">Effect of changing the joint kinematics of knees with a ruptured anterior cruciate ligament on the molecular biological responses and spontaneous healing in a rat model.</a> The American Journal of Sports Medicine. 44 (11), 2900-2910 (2016).</li><li>Bhatti, F. U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+non-invasively+induced+post-traumatic+osteoarthritis+in+mice.">Characterization of non-invasively induced post-traumatic osteoarthritis in mice.</a> Antioxidants (Basel). 11 (9), 1783 (2022).</li><li>Steel, C. D., Stephens, A. L., Hahto, S. M., Singletary, S. J., Ciavarra, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+lateral+tail+vein+and+the+retro-orbital+venous+sinus+as+routes+of+intravenous+drug+delivery+in+a+transgenic+mouse+model.">Comparison of the lateral tail vein and the retro-orbital venous sinus as routes of intravenous drug delivery in a transgenic mouse model.</a> Laboratory Animals (NY). 37 (1), 26-32 (2008).</li></ol>

The authors have nothing to disclose.

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...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10x Phosphate-Buffered Saline</td>
			<td>Tissue Protech</td>
			<td>PBS01-32R</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Air Anesthetia System</td>
			<td></td>
			<td></td>
			<td>Isoflurane vaporizor with induction chamber and nose cone</td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td></td>
			<td></td>
			<td>Analgesic post-injury&#160;</td>
		</tr>
		<tr>
			<td>Depilatory Cream</td>
			<td>Veet</td>
			<td>B001KYPZ4G</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Fixtures</td>
			<td></td>
			<td></td>
			<td>Custom-made knee fixture, ankle fixture, and platform</td>
		</tr>
		<tr>
			<td>IVIS Spectrum</td>
			<td>Perkin Elmer</td>
			<td>124262</td>
			<td>Can also use comparable optical imaging system</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimberly-Clark Corporation</td>
			<td>06-666</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Living Image software&#160;</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Materials testing systems&#160;</td>
			<td>TA Instruments</td>
			<td></td>
			<td>Electroforce 3200 or equivalent</td>
		</tr>
		<tr>
			<td>ProSense680</td>
			<td>Perkin Elmer</td>
			<td>NEV10003</td>
			<td>Can also use other probes such as OsteoSense, MMPSense, Cat K, AngioSense, etc.</td>
		</tr>
		<tr>
			<td>Sterile Syringe with Needle</td>
			<td>Spectrum Chemical Mfg. Corp.</td>
			<td>550-82231-CS</td>
			<td>Covidien 1 mL TB Syringe with 28 G x 1/2 in. Needle, Sterile or equivalent</td>
		</tr>
		<tr>
			<td>Uniaxial load cell</td>
			<td>TA Instruments</td>
			<td></td>
			<td>20 N capacity</td>
		</tr>
		<tr>
			<td>Vortex-Genie 2</td>
			<td>Scientific Industries, Inc.</td>
			<td>SI-0236</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>WinTest software&#160;</td>
			<td>TA Instruments</td>
			<td></td>
			<td>compatible with Electroforce 3200</td>
		</tr>
	</tbody>
</table>

non-invasive compression-induced anterior cruciate ligament (acl) injury and in vivo imaging of protease activity in mice

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.

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.

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...

Watch this Scientific Journal Video about Author Spotlight: Investigating Early Events and Long-Term Effects of ACL Injuries for Osteoarthritis Progression at JoVE.com

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.

Traumatic joint injuries such as anterior cruciate ligament (ACL) rupture or meniscus tears commonly lead to post-traumatic osteoarthritis (PTOA) within ...

non-invasive-compression-induced-anterior-cruciate-ligament-acl

Research

JoVE Journal

Medicine

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.

Fluorescence Reflectance Imaging for Quantification of Protease Activity in Mice Joints

To begin, lace 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 radient efficiency and average radient efficiency for each knee joint. If the radient efficiency is also calculated on the contralateral legs, normalize the data by dividing the radient efficiency 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 radient efficiency compared to contralateral joints, and joints from uninjured mice. A 30 to 80%greater radiant efficiency 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.

470 Views.  University of California Davis Health.  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.

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

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