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.

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 ...

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

Research

JoVE Journal

Medicine

593 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 ...

Controlled Cervical Laceration Injury in Mice

Use of genetically modified mice enhances our understanding of molecular mechanisms underlying several neurological disorders such as a spinal cord injury (SCI). Freehand manual control used to produce a laceration model of SCI creates inconsistent injuries often associated with a crush or contusion component and, therefore, a novel technique was developed. Our model of cervical laceration SCI has resolved inherent difficulties with the freehand method by incorporating 1) cervical vertebral stabilization by vertebral facet fixation, 2) enhanced spinal cord exposure, and 3) creation of a reproducible laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm in depth without associated contusion. Compared to the standard methods of creating a SCI laceration such as freehand use of a scalpel or scissors, our method has produced a consistent lesion. This method is useful for studies on axonal regeneration of corticospinal, rubrospinal, and dorsal ascending tracts.

A novel technique to create a reproducible in vivo model of cervical spinal cord laceration injury in the mouse is described. This technique is based on spine stabilization by fixation of the cervical facets and laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm.

Use of genetically modified mice enhances our  understanding of molecular mechanisms underlying several neurological disorders  such as a spinal cord ...

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

Intravital imaging has emerged as an indispensable tool in biological research. In the process, many imaging techniques have been developed to study different biological processes in animals non-invasively. However, a major technical limitation in existing intravital imaging modalities is the inability to combine non-invasive, longitudinal imaging with single-cell resolution capabilities. We show here how transplantation into the anterior chamber of the eye circumvents such significant limitation offering a versatile experimental platform that enables non-invasive, longitudinal imaging with cellular resolution in vivo. We demonstrate the transplantation procedure in the mouse and provide representative results using a model with clinical relevance, namely pancreatic islet transplantation. In addition to enabling direct visualization in a variety of tissues transplanted into the anterior chamber of the eye, this approach provides a platform to screen drugs by performing long-term follow up and monitoring in target tissues. Because of its versatility, tissue/cell transplantation into the anterior chamber of the eye not only benefits transplantation therapies, it extends to other in vivo applications to study physiological and pathophysiological processes such as signal transduction and cancer or autoimmune disease development.

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

A new approach combining intraocular transplantation and confocal microscopy enables longitudinal, non-invasive real-time imaging with single-cell resolution within grafted tissues in vivo. We demonstrate how to transplant pancreatic islets into the anterior chamber of the mouse eye.

Intravital imaging has emerged as an indispensable tool in biological research.  In the process, many imaging techniques have been developed to study ...

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to return to normal in a large majority of patients2, which is mainly due to current lack of clinical treatment for acute stroke. This necessitates understanding the physiological effects of cerebral ischemia on brain tissue over time and is a major area of active research. Towards this end, experimental progress has been made using rats as a preclinical model for stroke, particularly, using non-invasive methods such as 18F-fluorodeoxyglucose (FDG) coupled with Positron Emission Tomography (PET) imaging3,10,17. Here we present a strategy for inducing cerebral ischemia in rats by middle cerebral artery occlusion (MCAO) that mimics focal cerebral ischemia in humans, and imaging its effects over 24 hr using FDG-PET coupled with X-ray computed tomography (CT) with an Albira PET-CT instrument. A VOI template atlas was subsequently fused to the cerebral rat data to enable a unbiased analysis of the brain and its sub-regions4. In addition, a method for 3D visualization of the FDG-PET-CT time course is presented. In summary, we present a detailed protocol for initiating, quantifying, and visualizing an induced ischemic stroke event in a living Sprague-Dawley rat in three dimensions using FDG-PET.

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Brain damage resulting from cerebral ischemia may be non-invasively imaged and studied in rats using pre-clinical positron emission tomography coupled with the injectable radioactive probe, 18F-fluorodeoxyglucose. Further, the use of modern software tools that include volume of interest (VOI) brain templates dramatically increase the quantitative information gleaned from these studies.

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to ...

Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of care consisting of surgical debulking and combination chemotherapy consisting of platinum and taxane compounds, almost 90% of patients recur within a few years. In these patients the development of chemoresistant disease limits the efficacy of currently available chemotherapy agents and therefore contributes to the high mortality. To discover novel therapy options that can target recurrent disease, appropriate animal models that closely mimic the clinical profile of patients with recurrent ovarian cancer are required. The challenge in monitoring intra-peritoneal (i.p.) disease limits the use of i.p. models and thus most xenografts are established subcutaneously. We have developed a sensitive optical imaging platform that allows the detection and anatomical location of i.p. tumor mass. The platform includes the use of optical reporters that extend from the visible light range to near infrared, which in combination with 2-dimensional X-ray co-registration can provide anatomical location of molecular signals. Detection is significantly improved by the use of a rotation system that drives the animal to multiple angular positions for 360 degree imaging, allowing the identification of tumors that are not visible in single orientation. This platform provides a unique model to non-invasively monitor tumor growth and evaluate the efficacy of new therapies for the prevention or treatment of recurrent ovarian cancer.

We describe a non-invasive animal imaging platform that allows the detection, quantification, and monitoring of ovarian cancer growth and recurrence. This intra-peritoneal xenograft model mimics the clinical profile of patients with ovarian cancer.

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic mechanisms that contribute to the onset and progression of intestinal diseases. The identification of new molecules that promote these pathologies has led to a flurry of activity focused on the development of potential new therapies to inhibit their function. As a result, various pre-clinical mouse models with an intact immune system and stromal microenvironment are now heavily used. Here we describe three experimental protocols to test the efficacy of new therapeutics in pre-clinical models of (1) acute mucosal damage, (2) chronic colitis and/or colitis-associated colon cancer, and (3) sporadic colorectal cancer. We also outline procedures for serial endoscopic examination that can be used to document the therapeutic response of an individual tumor and to monitor the health of individual mice. These protocols provide complementary experimental platforms to test the effectiveness of therapeutic compounds shown to be well tolerated by mice.

We describe methods for longitudinal monitoring of the efficacy of therapeutics for the treatment of colonic pathologies in mice using a rigid endoscope. This protocol can be readily used for the characterization of the therapeutic response of an individual tumor in live mice and also for monitoring potential disease relapse.

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic ...