The research outlined in this manuscript was supported by South Carolina SmartState&#174; Endowed Chair in Drug Discovery Endowment funds (PMW),&#160;the MUSC Division of Laboratory Animal Resources, and the MUSC Drug Discovery Core. This publication was also supported by the National Center for Advancing Translational Sciences of the National Institutes of Health under Grant Numbers TL1 TR001451 &#38; UL1 TR001450, as well as the National Institute of Dental &#38; Craniofacial Research of the National Institutes of Health under Award Number R01DE029637.

All methods described here have been approved by the Institutional Animal Care and Use Committee of the Medical University of South Carolina. The study followed the principle of 3R.
1. Intra-articular injection preparations
<ol>
	<li>Allow Dunkin-Hartley guinea pigs to acclimate to facility for at least one week prior to starting the experiment. 
	NOTE: 5- (n=2), 9- (n=1), and 12- (n=2) month-old male guinea pigs were used. Males display an accelerated development and more severe phenotype of OA.</li>
	<li>Shave the knee area with an electric razor. 
	NOTE: Be careful of the nipples medially.</li>
	<li>Anesthetize guinea pig in an isoflurane chamber delivering 3-5% isoflurane in O2 mixture (flow rate 2.5 L/min) and then transfer the guinea pig to a nose cone connected to a non-rebreathing anesthesia circuit. Adjust isoflurane to maintain surgical plane of anesthesia during the injection, typically with an oxygen flow rate of 0.5-1 L/min and 1-3% isoflurane. 
	NOTE: Intra-articular injections cause mild, momentary pain. Animals are anesthetized during the procedure to prevent perception of painful stimuli and improve injection accuracy. In the presented study, the administration of analgesic agents, including nonsteroidal anti-inflammatory drugs, would interfere with OA progression21. Due to the momentary pain, provided anesthesia and potential of analgesics to confound the model, analgesics were not administered unless animals displayed side effects, including limping and signs of pain on joint palpation after injection. Investigators should consider the use of analgesics for routine injections. Analgesics are recommended when side effects occur. Analgesic regimens should be discussed with the institutional veterinarian and approved by the IACUC prior to initiating studies.</li>
	<li>Ensure guinea pig is at appropriate anesthesia depth by lack of toe pinch response.</li>
	<li>Place sterile ocular lubricant on both eyes to prevent desiccation and injury.</li>
	<li>Dilute betadine with sterile water to 10%.</li>
	<li>Dilute 200 proof ethanol with sterile water to 70% ethanol.</li>
	<li>Prepare solutions for injection, in a biosafety cabinet to maintain sterility. In the presented protocol, a sterile vehicle (1x phosphate buffered saline) was utilized to inject both knees. Solutions can be changed based on research objectives. 
	NOTE: Make sure to dilute fresh solutions for injection immediately before the injection session to ensure sterility. Any unused solutions should be discarded at the end of each injection session.</li>
	<li>Fill sterile one-time-use insulin syringes with solutions for injection. Take care to utilize the smallest volume achievable to prevent overloading the joint space with volume. In the present study, 50 &#181;L was used.</li>
	<li>Place guinea pig and nose cone on a clean surface with a heating pad for thermal support and padding under the head to elevate it slightly.</li>
	<li>Don surgical gown, hair net, sterile gloves, and mask while performing the injection procedure.</li>
	<li>Pour 10% betadine onto a cotton ball and wipe both knee areas.</li>
	<li>Pour 70% ethanol onto a cotton ball and wipe both knee areas.</li>
	<li>Repeat 1.12 and 1.13 two more times. 
	NOTE: For demonstration purposes, the corresponding video shows cleaning the knee and injection site once with 10% betadine and 70% ethanol. The injection site was subsequently cleaned using circular motions two more times, alternating these solutions. Three serial scrubs with alternating scrub solutions and alcohol are recommended to achieve aseptic technique.</li>
</ol>
2. Intra-articular injection
<ol>
	<li>Place the guinea pig in supine position for entirety of the procedure.</li>
	<li>Don a new pair of sterile gloves and palpate the knee joint. 
	NOTE: In the presented protocol and video, autoclaved nitrile gloves were utilized. Sterile gloves, including either autoclaved nitrile gloves or surgical gloves, should be utilized for aseptic technique.</li>
	<li>Manually flex the knee to 90&#176;.</li>
	<li>Move finger distal to the patella to locate the groove of the distal aspect of joint space by flexing and extending the hindlimb. 
	NOTE: The patella can be palpated in this position as a small, firm structure located directly over the joint space. The tibia can be felt as a bony structure distal to the patella. Once the location of the tibia and patella are determined, the joint, felt as a groove, is between them, distal to the patella and proximal to the tibia.</li>
	<li>Insert the insulin needle carefully on the midline distal to the patella within the joint space. The needle should be inserted 1-2 mm below the skin to enter the joint space. 
	NOTE: The largest access window for the joint space while the knee is flexed is on the anterior aspect of the limb on midline, directly distal to the patella. Injecting on midline in the anterior-to-posterior direction will aid in accurately injecting into the joint space without penetrating boney structures. Accurately injecting into the joint space can be achieved using a lateral-to-medial approach, although the access window is narrower especially when the knee is flexed.</li>
	<li>Inject 50 &#181;L of the solution into the joint slowly. Ensure that the needle inserts easily, and contents are injected without resistance. 
	NOTE: Make sure not to insert the needle too deep as it can cause joint or bone damage and result in unwanted inflammation and/or pain. If the groove corresponding to the joint space is not found, the needle could penetrate the femur, patella, or tibia. Therefore, it is beneficial to confidently palpate the groove corresponding to the joint space to prevent peri-articular injections or injuries associated with penetrating boney structures. If a bubble develops at the injection site under the skin, the injection was too shallow and the fluid has entered the subcutaneous space. Depending on the properties of the agent utilized, the drug may enter the joint space via diffusion, or another injection attempt may be needed.</li>
	<li>Once done discard the needle into sharps bin. 
	NOTE: For practice and training purposes, inject liquid containing a dye into the joint space of a cadaver in a similarly sized rodent or guinea pig. Then, dissect the joint to confirm the location of the injection.</li>
	<li>Massage the knee by flexing and extending the joint a few times promote diffusion of the drug within the joint space.</li>
	<li>Repeat steps 2.1-2.5 once on the contralateral limb with 1x PBS solution.</li>
</ol>
3. Recovery from intra-articular injection
<ol>
	<li>Turn off isoflurane and maintain 100% flow by oxygen until the animal regains consciousness.</li>
	<li>Place the animal on a heating pad for thermal support until ambulatory.</li>
	<li>Apply an ice pack to the knee for 30 s with a paper towel as a barrier to help decrease swelling from the injection.</li>
	<li>Assess animal gait when ambulatory prior to returning them to housing. 
	NOTE: If any gait abnormalities are noted, analgesics and supportive care may be warranted. It is advisable to assess their gait again several hours after recovery from anesthesia to ensure normal mobility.</li>
</ol>
4. Micro computed tomography (&#181;CT) scan
<ol>
	<li>For the tissue harvest, establish a surgical plane of anesthesia with 100% oxygen and 5% isoflurane mixture.</li>
	<li>Confirm a surgical plane of anesthesia with the lack of response to a toe-pinch stimulus. Humanely euthanize the animal via administration of &#8805; 150 mg/kg of pentobarbital intravenously according to institutional policies and approved animal use protocol. 
	NOTE: In the presented protocol, each of the five guinea pig received one injection in both knees. Animals were anesthetized and humanely euthanized one week after the injection.</li>
	<li>Harvest both hindlimbs by dissecting the skin away from the surrounding musculature.</li>
	<li>Next, disarticulate the hindlimb with Rongeurs at the mid-shaft of the femur and proximal to the ankle. 
	NOTE: The scanning bed and specimen holder used was unable to accommodate the entire hindlimb of an adult guinea pig. Large specimen holders are commercially available for larger specimen sizes.</li>
	<li>Place the tissues in neutral buffered formalin solution for 72 h for fixation before performing &#181;CT.</li>
	<li>Open &#181;CT scan software and place sample with formalin in a compatible container that will fit into the &#181;CT specimen folder while maintaining tissue in the field of vision.</li>
	<li>Calibrate &#181;CT machine for dark field and light field exposures according to manufacturer recommendations.</li>
	<li>Scan the sample with Al+Cu filter at 18 &#181;m. Use rotation step 0.7&#176; for 360&#176; with offset camera. 
	NOTE: The scan automatically saves.</li>
</ol>
5. Image processing for evaluating bone microarchitectural parameters
<ol>
	<li>Download and install &#181;CT reconstruction software for the reconstruction of &#181;CT images.</li>
	<li>Select the software folder and double click to open the software.</li>
	<li>Select one slice from the &#181;CT images by clicking on an image slice.</li>
	<li>Choose the reconstruction file destination. Select Browse and create a new folder named Recon. The selected file format should be BMP(8).</li>
	<li>Check Misalignment Compensation. 
	NOTE: Usually, the estimation is close to correct, but it can be manually adjusted to shift the overlapping images so that the right and left edges align as closely as possible.</li>
	<li>Under Settings, apply Smoothing, Beam Hardening, CS Rotation, and Ring Artifacts algorithms. 
	NOTE: It can be helpful to choose preview image to determine the clarity before reconstructing. The fine-tuning setting can also be helpful to determine which settings are the best.</li>
	<li>Select Start to begin processing the reconstruction.</li>
</ol>
6. Collection of microarchitectural data from reconstructed images
<ol>
	<li>Download and install Dataviewer.</li>
	<li>Select VOI and orient the sample to align vertically for easier analysis at a later time.</li>
	<li>Save edited VOI as a new folder.</li>
	<li>Download and install CTAnalyser for the bone property analysis of microarchitectural parameters. 
	NOTE: The free version of CTAnalyser is limited in functionality, so is recommended to obtain a full license.</li>
	<li>Split the analysis to subchondral plate and trabecular bone by saving them as separate range of images. 
	NOTE: Splitting the analysis is not necessary, but because subchondral plate and trabecular bone have different features, separate analyses can assist with robust measurements and comparison.</li>
	<li>Select the range of images to analyze, starting with subchondral plate by clicking on the image slice that you want to start with.</li>
	<li>Select the region of interest for each image to ensure that it is encompassing the bone by clicking on the region of interest tab.</li>
	<li>Select the Binary Selection tab. Adjust the histogram so that the background and the bone is completely separate.</li>
	<li>Select the Bone Mineral Density (BMD) tab. Save that data into a new analysis data folder.</li>
	<li>Select Custom Processing and go to Internal tab.</li>
	<li>First perform Thresholding and select Automatic Otsu, then Run.</li>
	<li>Then select Despeckle and choose Remove black speckles, then Run.</li>
	<li>Repeat Despeckle and choose Remove white speckles, then Run.</li>
	<li>Choose 3D analysis and select Basic values and Additional values.</li>
	<li>Repeat steps 6.2.2-6.4.5 to reset the image for trabecular bone analysis. 
	NOTE: Make sure the output file is in a new folder with the same file as the BMD data.</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targets,+models+and+challenges+in+osteoarthritis+research.">Targets, models and challenges in osteoarthritis research.</a> Dis Model Mech. 8 (1), 17-30 (2015).</li><li>Vazquez-Portalatin, N., Breur, G. J., Panitch, A., Goergen, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+of+ultrasound-guided+intra-articular+injections+in+guinea+pig+knees.">Accuracy of ultrasound-guided intra-articular injections in guinea pig knees.</a> Bone Joint Res. 4 (1), 1-5 (2015).</li><li>Nie, C., Wang, Z., Liu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+depression+on+fracture+healing+and+osteoblast+differentiation+in+rats.">The effect of depression on fracture healing and osteoblast differentiation in rats.</a> Neuropsychiatr Dis Treat. 14, 1705-1713 (2018).</li><li>Jonsson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-CT+and+deep+learning:+Modern+techniques+and+applications+in+insect+morphology+and+neuroscience.">Micro-CT and deep learning: Modern techniques and applications in insect morphology and neuroscience.</a> Front Insect Sci. 3, (2023).</li></ol>

Despite recent advancements in symptomatic treatment of OA, there is a complete lack of therapeutic agents that prevent onset or delay progression of OA24. Currently, the only cure for severe OA is joint replacement, which is costly, invasive, and can result in patient morbidity and mortality25. As a result, there is a dire need for continued research with animal models of OA and the sustained development of novel therapeutics. Several animal models are available to study d...

Osteoarthritis (OA) impacts 32.5 million US adults. It is caused by progressive loss of articular cartilage, mild inflammation of the tissues in and around the joints, and formation of osteophytes and bone cysts1,2. Symptoms typically manifest in the later stages of the disease, with current treatments providing only palliative relief as well as having systemic side effects. The lack of disease-modifying drugs stems from a poor understanding of the underlying mechanisms of the disease3. As a result, there is a critical and ongoing medical need for improved agents to treat OA. 
Several animal models of OA are available that examine different components of the disease processes4. While several surgical models exist, including transection of the anterior cruciate ligament and destabilization of the medial meniscus, these are invasive and require a high level of technical skill5. Chemically induced models are comparatively less invasive procedures typically used to study OA pain mechanisms6. One such widely used mouse model involves OA induction by an intra-articular knee injection of monosodium iodoacetate (MIA). This model generates a reproducible, robust, and rapid pain-like phenotype that can be graded by altering MIA dosage7. Technical details of inducing this model have been previously described7. Translation of this technique to larger rodents, like guinea pigs, is difficult due to their anatomical differences. Some differences include increased musculature surrounding the adjacent bones and joint space in the guinea pig and an articulating fibula and tibia compared to distal fusion seen in mice8. Dunkin-Hartley guinea pigs, a widely available guinea pig strain, are an established OA animal model as they naturally develop this disease, thereby offering a robust model for investigating the effects of novel therapeutics administered by intra-articular injection on disease progression9. Dunkin-Hartley guinea pigs start developing OA at three months, with males displaying an accelerated development and more severe phenotype10. In guinea pigs, OA progresses with age, and at 12 months, associated pathology is apparent on imaging11. Spontaneous OA models, like the Dunkin-Hartley model, do not require any intervention to induce OA and thus recapitulate the development and progression of the disease phenotype in humans, thereby providing a powerful translational model10. Furthermore, the spontaneous development of OA allows for the internal control when novel therapeutics are administered unilaterally in a single knee of a given animal. This internal control minimizes the effects of inter-animal variabilities when analyzing data and may help reduce overall animal numbers. 
X-ray Micro Computed Tomography (&#181;CT) analysis is a powerful tool that allows for quantitative assessment of OA severity12. &#181;CT involves scanning multiple, high-resolution X-ray images, obtained from a rotating sample or rotating X-ray source and detector13. Then, three- dimensional (3D) volumetric data is reconstructed in the form of stacked image slices14. Because mineralized bone has excellent contrast on &#181;CT, this modality can be used to assess 3D features and perform quantitative analyses of changes associated with OA15,16,17. &#181;CT offers several advantages over more widely used tools, including histopathology and gait analyses. In contrast to histologic assessment of one or few sections of tissues, &#181;CT scans the entire joint and offers a more wholistic assessment of OA lesions18. While gait analysis can discern symptomatic changes in joint function over time, joint changes develop long before functional changes associated with OA. &#181;CT can provide a more sensitive measure of OA development prior to the onset of lameness. Two particularly relevant quantitative measurements include bone mineral density and trabecular thickness as both increase throughout the progression of OA19,20. It can be helpful to split the analysis into subchondral plate and trabecular bone, as they have different features, to achieve more robust measurements and comparisons.
The overall goal of this method is to help researchers successfully perform intra-articular injections on guinea pigs. The presented protocol utilized five-(n=2), nine- (n=1), and 12- (n=2) month-old intact, male Dunkin-Hartley guinea pigs; procedures can be extrapolated to other guinea pig strains and ages requiring intra-articular knee injections. In spontaneous models of OA, like the Dunkin-Hartley model, disease progression and response to serial treatment is often monitored over long periods of times, spanning weeks to months9. This extended protocol results in multiple intra-articular injections, and thus it is important to have proper injection technique to prevent adverse events, including pain, lameness, or infections, all of which can impact animal welfare and confound study results while necessitating additional animals on study. The presented protocol describes methodology of intra-articular injections in guinea pigs and subsequent analysis of &#181;CT data.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>200 Proof Ethanol</td>
			<td>Decon Laboratories</td>
			<td>2701</td>
			<td>sterilizing agent</td>
		</tr>
		<tr>
			<td>3D.SUITE software</td>
			<td>Bruker</td>
			<td></td>
			<td>&#956;-CT analyzing software</td>
		</tr>
		<tr>
			<td>Betadine Surgical Scrub</td>
			<td>Avrio Health</td>
			<td>67618-151-16</td>
			<td>sterilizing agent</td>
		</tr>
		<tr>
			<td>Insulin syringe with needle</td>
			<td>Ulticare</td>
			<td>91008</td>
			<td>to perform injections</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Piramal</td>
			<td>803249</td>
			<td>anesthesize animal</td>
		</tr>
		<tr>
			<td>Neutral Buffered Formalin</td>
			<td>Fisher Scientific</td>
			<td>23-427098</td>
			<td>Fix tissue</td>
		</tr>
		<tr>
			<td>Nrecon Software</td>
			<td>Bruker</td>
			<td></td>
			<td>&#956;-CT reconstruction software</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Cytiva</td>
			<td>SH30258.01</td>
			<td>control and diluting agent</td>
		</tr>
		<tr>
			<td>SkyScan 1176</td>
			<td>Bruker</td>
			<td></td>
			<td>to scan samples&#160;</td>
		</tr>
	</tbody>
</table>

micro-computed tomography analysis of the knee in aged dunkin-hartley guinea pigs after intra articular injection

The purpose of this protocol is to guide researchers in performing a palpation-guided technique of intra-articular knee injection in guinea pigs and assessment using micro-computed tomography. Dunkin-Hartley guinea pigs are robust models for osteoarthritis research as they spontaneously develop osteoarthritis in their knees. Intra-articular drug delivery is a common method to study the effects of an investigational drug in vivo. In humans, therapeutic agents administered via intra-articular injection can offer pain relief and delay further progression of osteoarthritis. As with any species, the introduction of a needle into a joint space has the potential to cause injury, which can result in pain, lameness, or infection. Such adverse events can compromise animal welfare, confound study results, and necessitate additional animals to achieve study objectives. As such, it is imperative to develop proper injection techniques to prevent complications, especially in longitudinal studies that require multiple, repeated intra-articular injections. Using the presented methodology, five guinea pigs received bilateral knee injections under general anesthesia. Seven days after injection, animals were humanely euthanized for analysis of osteoarthritis severity. No adverse events occurred following anesthesia or knee injections, including limping, pain, or infection. X-ray micro-computed tomography analysis of the knee can detect pathologic changes associated with osteoarthritis. Micro-computed tomography data indicates osteoarthritis is more severe in older animals, as indicated by increased bone mineral density and trabecular thickness with age. These results are consistent with histologic changes and Modified Mankin scores, an established and widely used scoring system to assess arthritis severity in these same animals. This protocol can be utilized to refine intra-articular injections in guinea pigs.

Before performing intra-articular injections on live animals, the above protocol was practiced on three rat cadavers to ensure correct injection location. During the practice sessions, 50 &#181;L of 70% new methylene blue dye was injected into both knee joints using the methodology described above. This equates to six practice injections. After injections, the knee joint was dissected by incising through the cranial aspect of the joint space, distal to the patella and through the patellar ligament, to visualize the joint...

Dunkin-Hartley guinea pigs are an established animal model for osteoarthritis research. Such studies may benefit from intra-articular injections for various reasons, including investigating novel agents or treating disease. We describe a methodology for intra-articular knee injections in Guinea pigs and subsequent micro-computed tomography analysis assessing arthritis-associated knee changes.

Micro-Computed Tomography Analysis of the Knee in Aged Dunkin-Hartley Guinea Pigs after Intra Articular Injection

The purpose of this protocol is to guide researchers in performing a palpation-guided technique of intra-articular knee injection in guinea pigs and ...

micro-computed-tomography-analysis-knee-aged-dunkin-hartley-guinea

Research

JoVE Journal

Medicine

190 Views.  Medical University of South Carolina. Dunkin-Hartley guinea pigs are an established animal model for osteoarthritis research. Such studies may benefit from intra-articular injections for various reasons, including investigating novel agents or treating disease. We describe a methodology for intra-articular knee injections in Guinea pigs and subsequent micro-computed tomography analysis assessing arthritis-associated knee changes. 

Video: Micro-Computed Tomography Analysis of the Knee in Aged Dunkin-Hartley Guinea Pigs after Intra Articular Injection

Retrograde Perfusion and Filling of Mouse Coronary Vasculature as Preparation for Micro Computed Tomography Imaging

Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for imaging vasculature, few are able to visualize the vascular network as a whole while extending to a resolution that includes the smaller vessels1,2. Additionally, many vascular casting techniques destroy the surrounding tissue, preventing further analysis of the sample3-5. One method which circumvents these issues is micro-Computed Tomography (&mu;CT). &mu;CT imaging can scan at resolutions &lt;10 microns, is capable of producing 3D reconstructions of the vascular network, and leaves the tissue intact for subsequent analysis (e.g., histology and morphometry)6-11. However, imaging vessels by ex vivo &mu;CT methods requires that the vessels be filled with a radiopaque compound. As such, the accurate representation of vasculature produced by &mu;CT imaging is contingent upon reliable and complete filling of the vessels. In this protocol, we describe a technique for filling mouse coronary vessels in preparation for &mu;CT imaging.
Two predominate techniques exist for filling the coronary vasculature: in vivo via cannulation and retrograde perfusion of the aorta (or a branch off the aortic arch) 12-14, or ex vivo via a Langendorff perfusion system 15-17. Here we describe an in vivo aortic cannulation method which has been specifically designed to ensure filling of all vessels. We use a low viscosity radiopaque compound called Microfil which can perfuse through the smallest vessels to fill all the capillaries, as well as both the arterial and venous sides of the vascular network. Vessels are perfused with buffer using a pressurized perfusion system, and then filled with Microfil. To ensure that Microfil fills the small higher resistance vessels, we ligate the large branches emanating from the aorta, which diverts the Microfil into the coronaries. Once filling is complete, to prevent the elastic nature of cardiac tissue from squeezing Microfil out of some vessels, we ligate accessible major vascular exit points immediately after filling. Therefore, our technique is optimized for complete filling and maximum retention of the filling agent, enabling visualization of the complete coronary vascular network &#x2013; arteries, capillaries, and veins alike.

Visualization of the coronary vessels is critical to advancing our understanding of cardiovascular diseases. Here we describe a method for perfusing murine coronary vasculature with a radiopaque silicone rubber (Microfil), in preparation for micro-Computed Tomography (&mu;CT) imaging.

Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for ...

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to stimulate microcirculation. The choice of delivery method for these agents is important because the route of administration profoundly affects the bioactivity and efficacy of these agents1,2. In this article, we demonstrate how to locally administer a substance in ischemic hindlimbs by using a catheterized osmotic pump. This pump can deliver a fixed volume of aqueous solution continuously for an allotted period of time. We also present our mouse model of unilateral hindlimb ischemia induced by ligation of the common femoral artery proximal to the origin of profunda femoris and epigastrica arteries in the left hindlimb. Lastly, we describe the in vivo cannulation and ligation of the infrarenal abdominal aorta and perfusion of the hindlimb vasculature with Microfil, a silicone radiopaque casting agent. Microfil can perfuse and fill the entire vascular bed (arterial and venous), and because we have ligated the major vascular conduit for exit, the agent can be retained in the vasculature for future ex vivo imaging with the use of small specimen micro-CT3.

We show here the in vivo insertion of an osmotic pump for constant local drug delivery and the creation of hindlimb ischemia in a mouse model. Moreover, the hindlimb vasculature is perfused with Microfil, a silicone radiopaque agent, to prepare for micro-computed tomography (micro-CT) imaging.

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft tissue filler as it is readily available, easily obtained, inexpensive, and inherently biocompatible.1 However, despite its burgeoning popularity, fat grafting is hampered by unpredictable results and variable graft survival, with published retention rates ranging anywhere from 10-80%. 1-3
To facilitate investigations on fat grafting, we have therefore developed an animal model that allows for real-time analysis of injected fat volume retention. Briefly, a small cut is made in the scalp of a CD-1 nude mouse and 200-400 &#181;l&#160;of processed lipoaspirate is placed over the skull. The scalp is chosen as the recipient site because of its absence of native subcutaneous fat, and because of the excellent background contrast provided by the calvarium, which aids in the analysis process. Micro-computed tomography (micro-CT) is used to scan the graft at baseline and every two weeks thereafter. The CT images are reconstructed, and an imaging software is used to quantify graft volumes.
Traditionally, techniques to assess fat graft volume have necessitated euthanizing the study animal to provide just a single assessment of graft weight and volume by physical measurement ex vivo. Biochemical and histological comparisons have likewise required the study animal to be euthanized. This described imaging technique offers the advantage of visualizing and objectively quantifying volume at multiple time points after initial grafting without having to sacrifice the study animal. The technique is limited by the size of the graft able to be injected as larger grafts risk skin and fat necrosis. This method has utility for all studies evaluating fat graft viability and volume retention. It is particularly well-suited to providing a visual representation of fat grafts and following changes in volume over time.

Fat grafting is an essential technique for reconstructing soft tissue deficits. However, it remains an unpredictable procedure characterized by variable graft survival. Our goal was to devise a mouse model that utilizes a novel imaging method to compare volume retention between differing techniques of fat graft preparation and delivery.

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

Using Micro-computed Tomography for the Assessment of Tumor Development and Follow-up of Response to Treatment in a Mouse Model of Lung Cancer

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse models of lung cancer are being used to study the mechanism of cancer development and to experiment with various therapeutic strategies. However, the absence of a real-time technique to identify the development of tumor nodules in mice lungs and to monitor the changes in their size in response to various experimental and therapeutic interventions hampers the ability to obtain an accurate description of the course of the disease and its timely response to treatments. In this study, a method using a micro-computed tomography (CT) scanner for the detection of the development of lung tumors in a mouse model of lung adenocarcinoma is described. Next, we show that monthly follow-up with micro-CT can identify dynamic changes in the lung tumor, such as the appearance of additional nodules, increase in the size of previously detected nodules, and decrease in the size or complete resolution of nodules in response to treatment. Finally, the accuracy of this real-time assessment method was confirmed with end-point histological quantification. This technique paves the way for planning and conducting more complex experiments on lung cancer animal models, and it enables us to better understand the mechanisms of carcinogenesis and the effects of different treatment modalities while saving time and resources.

We describe a method for the detection of tumor nodule development in the lungs of an adenocarcinoma mouse model using micro-computed tomography and its use for monitoring changes in nodule size over time and in response to treatment. The accuracy of the assessment was confirmed with end-point histological quantification.

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse ...

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of stroke than relying on indirect measurements, and will be a potent and robust vascular research tool. We use an optical imaging approach that labels thrombi with a molecular imaging thrombus marker&#160;&#8212; a Cy5.5 near-infrared fluorescent (NIRF) probe that is covalently linked to the fibrin strands of the thrombus by the fibrin-crosslinking enzymatic action of activated coagulation factor XIIIa during the process of clot maturation. A micro-computed tomography (microCT)-based approach uses thrombus-seeking gold nanoparticles (AuNPs) functionalized to target the major component of the clot: fibrin. This paper describes a detailed protocol for the combined in vivo microCT and ex vivo NIRF imaging of thromboemboli in a mouse model of embolic stroke. We show that in vivo microCT and fibrin-targeted glycol-chitosan AuNPs (fib-GC-AuNPs) can be used for visualizing both in situ thrombi and cerebral embolic thrombi. We also describe the use of in vivo microCT-based direct thrombus imaging to serially monitor the therapeutic effects of tissue plasminogen activator-mediated thrombolysis. After the last imaging session, we demonstrate by ex vivo NIRF imaging the extent and the distribution of residual thromboemboli in the brain. Finally, we describe quantitative image analyses of microCT and NIRF imaging data. The combined technique of direct thrombus imaging allows two independent methods of thrombus visualization to be compared: the area of thrombus-related fluorescent signal on ex vivo NIRF imaging vs. the volume of hyperdense microCT thrombi in vivo.

This protocol describes the application of combined near-infrared fluorescent (NIRF) imaging and micro-computed tomography (microCT) for visualizing cerebral thromboemboli. This technique allows the quantification of thrombus burden and evolution. The NIRF imaging technique visualizes fluorescently labeled thrombus in excised brain, while the microCT technique visualizes thrombus inside living animals using gold-nanoparticles.

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of ...

Oleic Acid-Injection in Pigs As a Model for Acute Respiratory Distress Syndrome

The acute respiratory distress syndrome is a relevant intensive care disease with an incidence ranging between 2.2% and 19% of intensive care unit patients. Despite treatment advances over the last decades, ARDS patients still suffer mortality rates between 35 and 40%. There is still a need for further research to improve the outcome of patients suffering from ARDS. One problem is that no single animal model can mimic the complex pathomechanism of the acute respiratory distress syndrome, but several models exist to study different parts of it. Oleic acid injection (OAI)-induced lung injury is a well-established model for studying ventilation strategies, lung mechanics and ventilation/perfusion distribution in animals. OAI leads to severely impaired gas exchange, deterioration of lung mechanics and disruption of the alveolo-capillary barrier. The disadvantage of this model is the controversial mechanistic relevance of this model and the necessity for central venous access, which is challenging especially in smaller animal models. In summary, OAI-induced lung injury leads to reproducible results in small and large animals and hence represents a well-suited model for studying ARDS. Nevertheless, further research is necessary to find a model that mimics all parts of ARDS and lacks the problems associated with the different models existing today.

In this article, we present a protocol to induce acute lung injury in pigs by central-venous injection of oleic acid. This is an established animal model for studying the acute respiratory distress syndrome (ARDS).

The acute respiratory distress syndrome is a relevant intensive care disease with an incidence ranging between 2.2% and 19% of intensive care unit ...

Osteoarthritis Pain Model Induced by Intra-Articular Injection of Mono-Iodoacetate in Rats

The current animal models of osteoarthritis (OA) can be divided into spontaneous models and induced models, both of which aim to simulate the pathophysiological changes of human OA. However, as the main symptom in the late stage of OA, pain affects the patients&#39; daily life, and there are not many available models. The mono-iodoacetate (MIA)-induced model is the most widely used OA pain model, mainly used in rodents. MIA is an inhibitor of glyceraldehyde-3-phosphate dehydrogenase, which causes chondrocyte death, cartilage degeneration, osteophyte, and measurable changes in animal behavior. Besides, expression changes of matrix metalloproteinase (MMP) and pro-inflammatory cytokines (IL1 &#946; and TNF &#945;) can be detected in the MIA-induced model. Those changes are consistent with OA pathophysiological conditions in humans, indicating that MIA can induce a measurable and successful OA pain model. This study aims to describe the methodology of intra-articular injection of MIA in rats and discuss the resulting pain-related behaviors and histopathological changes.

This study describes the method of intra-articular injection of mono-iodoacetate in rats and discusses the resulting pain-related behaviors and histopathological changes, which provide references for future applications.

The current animal models of osteoarthritis (OA) can be divided into spontaneous models and induced models, both of which aim to simulate the ...

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 ...

Tissue Preparation Techniques for Contrast-Enhanced Micro Computed Tomography Imaging of Large Mammalian Cardiac Models with Chronic Disease

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional properties of diseased tissue is critical to determine their interactions with arrhythmic behavior. Microscale tissue remodeling, below the clinical resolution, is emerging as an important source of lethal arrhythmia, with high prevalence in young adults. Challenges remain in obtaining high imaging contrast at sufficient microscale resolution for preclinical models, such as large mammalian whole hearts. Moreover, tissue composition-selective contrast enhancement for three-dimensional high-resolution imaging is still lacking. Non-destructive imaging using micro-computed tomography shows promise for high-resolution imaging. The objective was to alleviate sufferance from X-ray over attenuation in large biological samples. Hearts were extracted from healthy pigs (N = 2), and sheep (N = 2) with either induced chronic myocardial infarction and fibrotic scar formation or induced chronic atrial fibrillation. Excised hearts were perfused with: a saline solution supplemented with a calcium ion quenching agent and a vasodilator, ethanol in serial dehydration, and hexamethyldisilizane under vacuum. The latter reinforced the heart structure during air-drying for 1 week. Collagen-dominant tissue was selectively bound by an X-ray contrast-enhancing agent, phosphomolybdic acid. Tissue conformation was stable in air, permitting long-duration microcomputed tomography acquisitions to obtain high-resolution (isotropic 20.7 &#181;m) images. Optimal contrast agent loading by diffusion showed selective contrast enhancement of the epithelial layer and sub-endocardial Purkinje fibers in healthy pig ventricles. Atrial fibrillation (AF) hearts showed enhanced contrast accumulation in the posterior walls and appendages of the atria, attributed to greater collagen content. Myocardial infarction hearts showed increased contrast selectively in regions of cardiac fibrosis, which enabled the identification of interweaving surviving myocardial muscle fibers. Contrast-enhanced air-dried tissue preparations enabled microscale imaging of the intact large mammalian heart and selective contrast enhancement of underlying disease constituents.

Here, we present a protocol to obtain high resolution micro computed tomography images of healthy and pathological large mammalian whole hearts with collagen-selective contrast enhancement.

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional ...

DUCT: Double Resin Casting followed by Micro-Computed Tomography for 3D Liver Analysis

The liver is the biggest internal organ in humans and mice, and high auto-fluorescence presents a significant challenge for assessing the three-dimensional (3D) architecture of the organ at the whole-organ level. Liver architecture is characterized by multiple branching lumenized structures, which can be filled with resin, including vascular and biliary trees, establishing a highly stereotyped pattern in the otherwise hepatocyte-rich parenchyma. This protocol describes the pipeline for performing double resin casting micro-computed tomography, or &#34;DUCT&#34;. DUCT entails injecting the portal vein and common bile duct with two different radiopaque synthetic resins, followed by tissue fixation. Quality control by clearing one lobe, or the entire liver, with an optical clearing agent, allows for pre-screening of suitably injected samples. In the second part of the DUCT pipeline, a lobe or the whole liver can be used for micro-computed tomography (microCT) scanning, (semi-)automated segmentation, and 3D rendering of the portal venous and biliary networks. MicroCT results in 3D coordinate data for the two resins allowing for qualitative as well as quantitative analysis of the two systems and their spatial relationship. DUCT can be applied to postnatal and adult mouse liver and can be further extended to other tubular networks, for example, vascular networks and airways in the lungs.

Double resin casting micro-computed tomography, or DUCT, enables visualization, digitalization, and segmentation of two tubular systems simultaneously to facilitate 3D analysis of organ architecture. DUCT combines ex vivo injection of two radiopaque resins followed by micro-computed tomography scanning and segmentation of the tomographic data.