Name: non-invasive compression-induced anterior cruciate ligament (acl) injury and in vivo imaging of protease activity in mice
Uploaded: 2023-09-29T14:10:00.000+00:00
Duration: 6 min 27 s
Description: Watch this Scientific Journal Video about Author Spotlight: Investigating Early Events and Long-Term Effects of ACL Injuries for Osteoarthritis Progression at JoVE.com

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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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Joint+loads+resulting+in+ACL+rupture:+Effects+of+age,+sex,+and+body+mass+on+injury+load+and+mode+of+failure+in+a+mouse+model.">Joint loads resulting in ACL rupture: Effects of age, sex, and body mass on injury load and mode of failure in a mouse model.</a> Journal of Orthopaedic Research. 35 (8), 1754-1763 (2017).</li><li>Murata, K., 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=Controlling+joint+instability+delays+the+degeneration+of+articular+cartilage+in+a+rat+model.">Controlling joint instability delays the degeneration of articular cartilage in a rat model.</a> Osteoarthritis Cartilage. 25 (2), 297-308 (2017).</li><li>Murata, K., 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=Controlling+Abnormal+joint+movement+inhibits+response+of+osteophyte+formation.">Controlling Abnormal joint movement inhibits response of osteophyte formation.</a> Cartilage. 9 (4), 391-401 (2018).</li><li>Yardeni, T., Eckhaus, M., Morris, H. 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><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&amp;nbsp;</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&amp;nbsp;</td><td>Perkin Elmer</td><td></td><td></td></tr><tr><td>Materials testing systems&amp;nbsp;</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&amp;nbsp;</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.

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

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

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.

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 ...

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609 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.

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

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

Muscle strains are one of the most common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and physical exam alone, however the clinical presentation can vary greatly depending on the extent of injury, the patient's pain tolerance, etc. In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Biological markers, such as serum creatine kinase levels, are typically elevated with muscle injury, but their levels do not always correlate with the loss of force production. This is even true of histological findings from animals, which provide a "direct measure" of damage, but do not account for all the loss of function. Some have argued that the most comprehensive measure of the overall health of the muscle in contractile force. Because muscle injury is a random event that occurs under a variety of biomechanical conditions, it is difficult to study. Here, we describe an in vivo animal model to measure torque and to produce a reliable muscle injury. We also describe our model for measurement of force from an isolated muscle in situ. Furthermore, we describe our small animal MRI procedure.

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

An in vivo animal model of injury is described. The method takes advantage of the subcutaneous position of the fibular nerve. Velocity, timing of muscle activation, and arc of motion are all pre-determined and synchronized using commercial software. Post injury changes are monitored in vivo using MR imaging/spectroscopy.

Muscle strains are one of the most  common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and  physical ...

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

Non-invasive Assessment of Microvascular and Endothelial Function

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of cardiovascular disease. Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. Percent capillary recruitment is assessed by dividing the increase in capillary density induced by postocclusive reactive hyperemia (postocclusive reactive hyperemia capillary density minus baseline capillary density), by the maximal capillary density (observed during passive venous occlusion). Percent perfused capillaries represents the proportion of all capillaries present that are perfused (functionally active), and is calculated by dividing postocclusive reactive hyperemia capillary density by the maximal capillary density. Both percent capillary recruitment and percent perfused capillaries reflect the number of functional capillaries. The forearm blood flow (FBF) technique provides accepted non-invasive measures of endothelial function: The ratio FBFmax/FBFbase is computed as an estimate of vasodilation, by dividing the mean of the four FBFmax values by the mean of the four FBFbase values. Forearm vascular resistance at maximal vasodilation (FVRmax) is calculated as the mean arterial pressure (MAP) divided by FBFmax. Both the capillaroscopy and forearm techniques are readily acceptable to patients and can be learned quickly.
The microvascular and endothelial function measures obtained using the methodologies described in this paper may have future utility in clinical patient cardiovascular risk-reduction strategies. As we have published reports demonstrating that microvascular and endothelial dysfunction are found in initial stages of hypertension including prehypertension, microvascular and endothelial function measures may eventually aid in early identification, risk-stratification and prevention of end-stage vascular pathology, with its potentially fatal consequences.

Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. The forearm blood flow technique provides accepted non-invasive measures of endothelial function.

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of ...

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical deficiencies that impair function and stability. Current options for the treatment of partial ACL tears range from nonoperative, conservative management to multiple surgical options, such as: thermal modification, single-bundle repair, complete reconstruction, and reconstruction of the damaged portion of the native ligament. Few studies, if any, have demonstrated any single method for management to be consistently superior, and in many cases patients continue to demonstrate persistent instability and other comorbidities.
The goal of this study is to identify a potential cell source for utilization in the development of a tissue engineered patch that could be implemented in the repair of a partially torn ACL. A novel protocol was developed for the expansion of cells derived from patients undergoing ACL reconstruction. To isolate the cells, minced hACL tissue obtained during ACL reconstruction was digested in a Collagenase solution. Expansion was performed using DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). The cells were then stored at -80 &#186;C or in liquid nitrogen in a freezing medium consisting of DMSO, FBS and the expansion medium. After thawing, the hACL derived cells were then seeded onto a tissue engineered scaffold, PLAGA (Poly lactic-co-glycolic acid) and control Tissue culture polystyrene (TCPS). After 7 days, SEM was performed to compare cellular adhesion to the PLAGA versus the control TCPS. Cellular morphology was evaluated using immunofluorescence staining. SEM (Scanning Electron Microscope) micrographs demonstrated that cells grew and adhered on both PLAGA and TCPS surfaces and were confluent over the entire surfaces by day 7. Immunofluorescence staining showed normal, non-stressed morphological patterns on both surfaces. This technique is promising for applications in ACL regeneration and reconstruction.

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

For future applications as a patch to repair partial tears of the Anterior Cruciate Ligament (ACL), human ACL derived cells were isolated from tissue obtained during reconstructive procedures, expanded in vitro and grown on tissue engineered scaffolds. Cellular adhesion and morphology was then performed to confirm biocompatibility on scaffold surface.

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical ...

Bioengineering

Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without intervention. Current treatment strategies include ligament reconstruction with either autograft or allograft, which each have their associated limitations. Thus, there is interest in designing a tissue-engineered graft for use in ACL reconstruction. We describe the fabrication of an electrospun polymer graft for use in ACL tissue engineering. This polycaprolactone graft is biocompatible, biodegradable, porous, and is comprised of aligned fibers. Because an animal model is necessary to evaluate such a graft, this paper describes an intra-articular athymic rat model of ACL reconstruction that can be used to evaluate engineered grafts, including those seeded with xenogeneic cells. Representative histology and biomechanical testing results at 16 weeks postoperatively are presented, with grafts tested immediately post-implantation and contralateral native ACLs serving as controls. The present study provides a reproducible animal model with which to evaluate tissue engineered ACL grafts, and demonstrates the potential of a regenerative medicine approach to treatment of ACL rupture.

Animal models are important tools for the evaluation of tissue-engineered grafts. This paper presents the protocol for preparing an electrospun biodegradable polymer graft for use in anterior cruciate ligament tissue engineering, as well as a surgical protocol for implantation in a rat model.

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without ...

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent evidence indicates a potential inflammatory component of the disease, with both T-cells and monocytes/macrophages potentially associated with the pathogenesis of OA. Further studies postulated an important role for subsets of both inflammatory cell lineages, such as Th1, Th2, Th17, and T-regulatory lymphocytes, and M1, M2, and synovium-tissue-resident macrophages. However, the interaction between the local synovial and systemic inflammatory cellular response and the structural changes in the joint is unknown. To fully understand how T-cells and monocytes/macrophages contribute towards OA, it is important to be able to quantitively identify these cells and their subsets simultaneously in synovial tissue, secondary lymphatic organs and systemically (the spleen and bone marrow). Nowadays, the different inflammatory cell subsets can be identified by a combination of cell-surface markers making multi-color flow cytometry a powerful technique in investigating these cellular processes. In this protocol, we describe detailed steps regarding the harvest of synovial tissue and secondary lymphatic organs as well as generation of single cell suspensions. Furthermore, we present both an extracellular staining assay to identify monocytes/macrophages and their subsets as well as an extra- and intra-cellular staining assay to identify T-cells and their subsets within the murine spleen, bone marrow, lymph nodes and synovial tissue. Each step of this protocol was optimized and tested, resulting in a highly reproducible assay that can be utilized for other surgical and non-surgical OA mouse models.

Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis.

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent ...

A Non-Invasive Method for Generating the Cyclic Loading-Induced Intra-Articular Cartilage Lesion Model of the Rat Knee

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely correlated with a history of articular cartilage damage and ligament avulsion. Surgical animal models of OA of the knee play an important role in understanding the onset and progression of post-traumatic OA and aid in the development of novel therapies for this disease. However, non-surgical models have been recently considered to avoid traumatic inflammation that could affect the evaluation of the intervention. 
In this study, an intra-articular cartilage lesion rat model induced by in vivo cyclic compressive loading was developed, which allowed researchers to (1) determine the optimal magnitude, speed, and duration of load that could cause focal cartilage damage; (2) assess post-traumatic spatiotemporal pathological changes in chondrocyte vitality; and (3) evaluate the histological expression of destructive or protective molecules that are involved in the adaptation and repair mechanisms against joint compressive loads. This report describes the experimental protocol for this novel cartilage lesion in a rat model.

Here, we present the cyclic loading-induced intra-articular cartilage lesion model of the rat knee, generated by 60 cyclic compressions over 20 N, resulting in damage to the femoral condylar cartilage in rats.

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely ...

Destabilization of the Medial Meniscus and Cartilage Scratch Murine Model of Accelerated Osteoarthritis

Osteoarthritis is the most prevalent musculoskeletal disease in people over 45, leading to an increasing economic and societal cost. Animal models are used to mimic many aspects of the disease. The present protocol describes the destabilization and cartilage scratch model (DCS) of post-traumatic osteoarthritis. Based on the widely used destabilization of the medial meniscus (DMM) model, DCS introduces three scratches on the cartilage surface. The current article highlights the steps to destabilize the knee by transecting the medial meniscotibial ligament followed by three intentional superficial scratches on the articular cartilage. The possible analysis methods by dynamic weight-bearing, microcomputed tomography, and histology are also demonstrated. While the DCS model is not recommended for studies that focus on the effect of osteoarthritis on the cartilage, it enables the study of osteoarthritis development in a shorter time window, with special focus on (1) osteophyte formation, (2) osteoarthritic and injury pain, and (3) the effect of cartilage damage in the whole joint.

The present protocol describes the controlled microblade scratches on the surface of the articular cartilage after destabilizing the mouse knee by cutting the medial miniscotibial ligament. This animal model presents an accelerated form of osteoarthritis (OA) suitable for studying osteophyte formation, osteosclerosis, and early-stage pain.

Osteoarthritis is the most prevalent musculoskeletal disease in people over 45, leading to an increasing economic and societal cost. Animal models are ...

Visualization of Knee Joint Degeneration after Non-invasive ACL Injury in Rats

Source: Lindsey K. Lepley1,2, Steven M. Davi1, Timothy A. Butterfield3,4 and Sina Shahbazmohamadi5,
1Department of Kinesiology, University of Connecticut, Storrs, CT; 2Department of Orthopaedic Surgery, University of Connecticut Health Center, Farmington, CT; 3Department of Rehabilitation Sciences, University of Kentucky, Lexington, KY; 4Center for Muscle Biology, Department of Physiology, University of Kentucky, Lexington, KY; 5Biomedical Engineering Department, University of Connecticut, Storrs, CT
Anterior cruciate ligament (ACL) injury to the knee dramatically increases the risk of post-traumatic osteoarthritis (PTOA), as approximately one-third of individuals will demonstrate radiographic PTOA within the first decade following ACL injury. Though ACL reconstruction (ACLR) successfully restores knee joint stability, ACLR and current rehabilitation techniques do not prevent the onset of PTOA. Therefore, ACL injury represents the ideal model to study the development of PTOA after traumatic joint injury.
Rat models have been used extensively to study the onset and effect of ACL injury on PTOA. The most widely used model of ACL injury is ACL transection, which is an acute model that surgically destabilizes the joint. Though practical, this model does not faithfully mimic human ACL injury due to the invasive and non-physiological injury procedures that mask the native biological response to injury. To improve the clinical translation of our results, we have recently developed a novel non-invasive model of ACL injury where the ACL is ruptured through a single load of tibial compression. This injury closely replicates injury conditions relevant to humans and is highly reproducible.
Visualization of joint degeneration through micro-computed tomography (&#181;CT) provides several major advancements over traditional OA staining techniques, including rapid, high-resolution, non-destructive 3D imaging of whole joint degeneration. The goal of this demonstration is to introduce the state of the art non-invasive ACL injury in a rodent model and use &#181;CT to quantify knee joint degeneration.

Knee Joint Degeneration after Non-invasive Anterior Cruciate Ligament Injury

Source: Lindsey K. Lepley1,2, Steven M. Davi1, Timothy A. Butterfield3,4 and Sina Shahbazmohamadi5,
1Department of Kinesiology, University of Connecticut, ...

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

Summary

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Chapters in this video

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Non-invasive Assessment of Microvascular and Endothelial Function

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

A Non-Invasive Method for Generating the Cyclic Loading-Induced Intra-Articular Cartilage Lesion Model of the Rat Knee

Destabilization of the Medial Meniscus and Cartilage Scratch Murine Model of Accelerated Osteoarthritis

Visualization of Knee Joint Degeneration after Non-invasive ACL Injury in Rats