We thank Yasuyo Kimura, Megumi Nagahiro, and Saeko Tokunaga for the excellent technical support in animal experiments.

All procedures were performed in accordance with the Kumamoto University animal care guidelines (approval reference no. M30-040/A2020-105), which conform to the US National Institutes of Health Guide for the Care and Use of Laboratory Animals (publication no. 85-23, revised 2011).
1. Preparation
<ol>
	<li>Prepare the perfusion apparatus and vasodilator buffer (4 mg/L papaverine hydrochloride, 4 g/L; adenosine, 1 g/L; heparin, 1 U/mL in phosphate-buffered saline (PBS)). 
	NOTE: The reflux device and vasodilator reagents are the same as those reported by Weyers et al.5.</li>
	<li>Connect the 22 G catheter, 2 mL extension tube, and three-way stopcock (Figure 1A). 
	NOTE: Adjust the gauge according to the size of the animal. For adult C57BL/6 mice, 22 G is optimal.</li>
	<li>Fill the pressure perfusion apparatus with the vasodilator buffer (Figure 1A). 
	&#8203;NOTE: Avoid the formation of bubbles to prevent disruption in the filling of the contrast medium.</li>
</ol>
2. Perfusion
<ol>
	<li>Inject 1 U/g heparin in PBS into the intraperitoneal cavity 30 min before the operation.</li>
	<li>Completely anesthetize the mouse with isoflurane and euthanize it by cervical dislocation.</li>
	<li>After decapitation, make a midline incision in the sternum and fix the open thorax with pins. 
	NOTE: To avoid leakage of the contrast, avoid injuring the diaphragm.</li>
	<li>Cut the ascending aorta and remove the heart.</li>
	<li>Remove the lung and expose the descending aorta. 
	NOTE: Do not injure the descending aorta.</li>
	<li>Cut the descending aorta diagonally to expose the cross-section (Figure 1B). 
	NOTE: Do not peel off the aorta; a diagonal section is better for catheter insertion.</li>
	<li>Insert the 22 G catheter into the descending aorta while running the vasodilation buffer. 
	NOTE: Insertion of the catheter while running the vasodilation buffer avoids air contamination.</li>
	<li>Pin the root of the catheter (Figure 1C).</li>
	<li>Make a knot to prevent leakage due to backflow.</li>
	<li>Perfuse a warmed vasodilating solution (papaverine hydrochloride, 4 g/L; adenosine, 1 g/L; heparin, 1 U/mL) for 3 min at a fixed pressure between 13 and 15 kPa.</li>
	<li>Perfuse a solution of 4% paraformaldehyde in (PBS) for 3 min. 
	NOTE: The success of fixation can be confirmed by the movement of the foot (Figure 1D).</li>
	<li>Prepare the contrast medium just before perfusion. 
	NOTE: Adjust the dilution rate according to the sample; for adult mice, mix the stain and diluent in a ratio of 1:1.</li>
	<li>Stop the perfusion and fill the extension tube with 2 mL of diluted contrast medium (Figure 1E). 
	NOTE: The contrast medium should be injected slowly to avoid injury to the blood vessels.</li>
	<li>Perfuse the contrast medium at a fixed pressure between 13 and 15 kPa.
	<ol>
		<li>To visualize arteries, check the toenail to confirm that the contrast medium has reached the artery (Figure 1F).</li>
		<li>To visualize all vessels, check the inferior vena cava of the diaphragm to confirm complete circulation of the contrast medium. 
		NOTE: In the beginning, the contrast contains the vasodilating solution; hence, its proper circulation is essential.</li>
	</ol>
	</li>
	<li>Close the three-way stopcock and remove the tube (Figure 1G). 
	NOTE: If the three-way stopcock is not closed, the contrast will flow backward.</li>
	<li>Incubate the sample overnight at 4 &#176;C.</li>
	<li>Remove the skin and fix it in 10% formaldehyde solution.</li>
</ol>
3. Visualization
NOTE: Visualization protocols differ depending on the CT scanner. A microfocus X-ray CT scanner was used in this protocol. It is necessary to optimize the imaging method according to each CT scanner.
<ol>
	<li>Fix the sample in a 50 mL tube containing PBS.</li>
	<li>Place the sample tube on the table.</li>
	<li>Scan the sample with a voltage of 50 kV and a current of 600 &#181;A, ensuring a focus-to-center distance of 75.2 mm. 
	NOTE: A dimension of 1 voxel was 28.7 &#181;m x 28.7 &#181;m x 28.7 &#181;m in this setting.</li>
	<li>Load the acquired image data with Fiji, an open-source platform for biological-image analysis.</li>
	<li>Determine the muscle voxel value using the gastrocnemius muscle.
	<ol>
		<li>Choose the gastrocnemius muscle by using the rectangle tool.</li>
		<li>Check the mean and standard deviation (SD) from the histogram (Analyze | Histogram).</li>
		<li>Define the muscle voxel density as mean + 2SD of the gastrocnemius muscle.</li>
	</ol>
	</li>
	<li>Set the muscle voxel density as the lower threshold level (Image | adjust | Threshold | Set | lower threshold level). 
	NOTE: The vascular area and bone area remain in the binarized data after the threshold is set.</li>
</ol>

<ol>
	<li>Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis.">Angiogenesis.</a> Annual Review of Medicine. 57, 1-18 (2006).</li><li>Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+in+cancer,+vascular,+rheumatoid+and+other+disease.">Angiogenesis in cancer, vascular, rheumatoid and other disease.</a> Nature Medicine. 1 (1), 27-31 (1995).</li><li>Kochi, 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=Characterization+of+the+arterial+anatomy+of+the+murine+hindlimb:+functional+role+in+the+design+and+understanding+of+ischemia+models.">Characterization of the arterial anatomy of the murine hindlimb: functional role in the design and understanding of ischemia models.</a> PLoS One. 8 (12), 84047 (2013).</li><li>Limbourg, A., 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=Evaluation+of+postnatal+arteriogenesis+and+angiogenesis+in+a+mouse+model+of+hind-limb+ischemia.">Evaluation of postnatal arteriogenesis and angiogenesis in a mouse model of hind-limb ischemia.</a> Nature Protocols. 4 (12), 1737-1746 (2009).</li><li>Weyers, J. J., Carlson, D. D., Murry, C. E., Schwartz, S. M., Mahoney, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrograde+perfusion+and+filling+of+mouse+coronary+vasculature+as+preparation+for+micro+computed+tomography+imaging.">Retrograde perfusion and filling of mouse coronary vasculature as preparation for micro computed tomography imaging.</a> Journal of Visualized Experiments: JoVE. (60), e3740 (2012).</li><li>Arima, Y., 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=Evaluation+of+collateral+source+characteristics+with+3-dimensional+analysis+using+micro-X-ray+computed+tomography.">Evaluation of collateral source characteristics with 3-dimensional analysis using micro-X-ray computed tomography.</a> Journal of the American Heart Association. 7 (6), 007800 (2018).</li><li>Tian, T., Yang, Z., Li, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+clearing+technique:+Recent+progress+and+biomedical+applications.">Tissue clearing technique: Recent progress and biomedical applications.</a> Journal of Anatomy. 238 (2), 489-507 (2021).</li><li>Susaki, E. A., 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=Advanced+CUBIC+protocols+for+whole-brain+and+whole-body+clearing+and+imaging.">Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging.</a> Nature Protocols. 10 (11), 1709-1727 (2015).</li><li>Fernandez, M., 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=Angiogenesis+in+liver+disease.">Angiogenesis in liver disease.</a> Journal of Hepatology. 50 (3), 604-620 (2009).</li><li>Li, S., 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=Angiogenesis+in+pancreatic+cancer:+current+research+status+and+clinical+implications.">Angiogenesis in pancreatic cancer: current research status and clinical implications.</a> Angiogenesis. 22 (1), 15-36 (2019).</li></ol>

The authors have no conflicts of interest to declare.

This report introduces a sophisticated method to visualize blood vessels in the lower body. There are several critical steps in this process: the first is preperfusion before the injection of contrast medium. If enough blood is not removed, the contrast will not fill the system. Additionally, the inclusion of air bubbles disturbs the filling of the contrast; thus, the air in the circuit must be completely removed. Moreover, because the contrast medium does not solidify immediately after injection, the sample should not be excessively moved.
This method is useful to evaluate the increased formation of blood vessels and circulation, such as collateral circulation. Conversely, as a limitation, it is difficult to evaluate narrowed blood vessels, as distinguishing between stenosis and an artificial reduction in contrast medium is difficult. Additionally, it is challenging to evaluate blood vessels in bones, as the separation of blood and bone is difficult.
An alternative method for 3D visualization is immunostaining. Using the tissue clearing technique, several methods are available for 3D imaging7. Immunostaining is advantageous as it allows the staining of specific proteins using antibodies. A recent report challenges whole-body imaging based on immunostaining8; however, CT-based imaging does not require any tissue-clearing pretreatment. 
This method enables the visualization of all vessels below the diaphragm, including the abdominal organs. Angiogenesis in the abdominal organs has a strong impact on maintaining homeostasis and developing diseases9,10. As this protocol was optimized for the evaluation of the lower extremity vessels, organ-specific priming would enable the visualization of angiogenesis associated with any factor, such as inflammation or tumors.

Blood vessels are complex networks with tree-like structures. Angiogenesis and new vascular formation play essential roles in the maintenance of organ homeostasis1. Angiogenesis is regulated for the treatment of ischemic and malignant diseases2. It is therefore essential to understand the underlying mechanisms of angiogenesis. Murine hind-limb vessels are often used as a useful model for vascular research3; ipsilateral ligation of the iliac or femoral artery is a known hind-limb ischemia model used to assess angiogenesis and vascular remodeling in physiological and pathological angiogenesis4. However, the evaluation of angiogenesis is mainly performed by section staining, and methods for evaluating 3D vascular morphology are particularly limited.
Compared with section staining, CT enables 3D visualization. Recently, Weyers et al. reported a sophisticated protocol suitable for CT imaging, enabling the visualization of the murine adult coronary circulatory system5. We modified their method to create a sample preparation method suitable for CT imaging of the lower extremity blood vessels6. Here, a radiation-opaque resin is injected through the descending aorta, and vessels in the lower extremities are filled with dye. By adjusting the time of dye injection, arterial-specific filling is also possible, and samples can be obtained with any micro-X-ray computed tomography device. This contrast method provides a basic technique for the 3D evaluation of murine blood vessels below the diaphragm and in abdominal organs and the lower extremities.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL syringe</td>
			<td>TERUMO</td>
			<td>SS-01T</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Formalin Solution</td>
			<td>Fujifilm-Wako</td>
			<td>068-03841</td>
			<td></td>
		</tr>
		<tr>
			<td>10x phosphate-buffered saline (-) (PBS)</td>
			<td>Fujifilm-Wako</td>
			<td>163-25265</td>
			<td>Prepare 1x PBS</td>
		</tr>
		<tr>
			<td>22 G catheter (22 G S5 x 1&#34; V(F))</td>
			<td>MEDIKIT</td>
			<td>HP2140</td>
			<td>Only catheter is used.</td>
		</tr>
		<tr>
			<td>23 G needle</td>
			<td>TERUMO</td>
			<td>NN-2325R</td>
			<td>Use as a pin</td>
		</tr>
		<tr>
			<td>4% paraformaldehyde in PBS</td>
			<td>Fujifilm-Wako</td>
			<td>163-20145</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL syringe</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5-0 Suture with needle</td>
			<td>Alfresa Pharma Corporation</td>
			<td>ER1205SB45</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine</td>
			<td>Sigma-aldrich</td>
			<td>A9251-5G</td>
			<td>For vasodilating solution</td>
		</tr>
		<tr>
			<td>Dumont #55 Forceps</td>
			<td>FST</td>
			<td>No.11255-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Extension tube</td>
			<td>TOP</td>
			<td>X2-FL50</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50 mL tube</td>
			<td>CORNING</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>Graefe Forceps</td>
			<td>FST</td>
			<td>No.11051-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin Sodium 5,000 units/5 mL</td>
			<td>Mochida Co. Ltd.</td>
			<td>224122458</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Fujifilm-Wako</td>
			<td>099-06571</td>
			<td></td>
		</tr>
		<tr>
			<td>Microfil Injection Compounds</td>
			<td>Flow Tech Inc.</td>
			<td>MV-117</td>
			<td>Mix liquid MV-Compound (stain) and MV-Diluent 1: 1</td>
		</tr>
		<tr>
			<td>Papaverine hydrochloride</td>
			<td>Fujifilm</td>
			<td>164-18002</td>
			<td>For vasodilating solution</td>
		</tr>
		<tr>
			<td>Small Animal Anesthetizer</td>
			<td>Muromachi Kikai Co. Ltd.</td>
			<td>MK-A100ecoW-ST</td>
			<td></td>
		</tr>
		<tr>
			<td>Spring Scissors - Angled to Side</td>
			<td>FST</td>
			<td>No.15006-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scissors - Sharp-Blunt</td>
			<td>FST</td>
			<td>No.14001-12</td>
			<td></td>
		</tr>
		<tr>
			<td>three-way cock</td>
			<td>TERUMO</td>
			<td>TS-TR1K</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipette</td>
			<td>SAMCO SCIENTIFIC</td>
			<td>SM262-1S</td>
			<td>Use for mixing contrast medium</td>
		</tr>
		<tr>
			<td>X-ray CT scanner</td>
			<td>Toshiba IT &#38; Control Systems Corporation</td>
			<td>TOSHIBA TOSCANNER 32300 FPD</td>
			<td></td>
		</tr>
	</tbody>
</table>

sample preparation for computed tomography-based three-dimensional visualization of murine hind-limb vessels

Blood vessels are complex networks with tree-like structures, and vascular networks are essential for maintaining both circulation and maintaining organ function. Clarifying the mechanism of blood vessel formation is therefore extremely useful for elucidating developmental processes and pathological mechanisms. Murine hind-limb vessels are often used as a model for physiological and pathological angiogenesis. Evaluation is mainly performed via a two-dimensional method using tissue sections. However, methods for evaluating three-dimensional (3D) vascular morphology are particularly limited. This paper introduces a method for visualizing murine hind-limbs using computed tomography (CT). Radiation-opaque resin is injected through the descending aorta, and whole vessels are filled with dye. By adjusting the time of dye injection, arterial-specific filling is also possible, and samples can be obtained with any micro-X-ray CT device. This contrast method provides a basic technique for the 3D evaluation of murine blood vessels in the lower extremities. Furthermore, this method can be used to visualize all blood vessels below the diaphragm and evaluate blood vessels in the abdominal organs.

All vessels in the lower extremities can be visualized if this protocol is correctly performed (Figure 2A). In a hind-limb ischemia model, the non-ligated femoral artery runs parallel to the femoral vein (Figure 2B), and a ligated femoral artery can be confirmed by the interruption of contrast media (Figure 2C). The results revealed the development of collateral vessels (Figure 2D). Collateral circulation is formed between the arteries proximal to the ligated artery and the artery in the lower leg region and on the ventral and dorsal sides of the femoral artery. The inferior gluteal artery-beginning on the dorsal side of the pelvis and running on the lateral side of the thigh-expands robustly on the ischemic side.
Contrast-filled vessels are filled with the contrast medium (Figure 2E); disruption in the contrast indicates mixing of noncontrast media (e.g., blood, vasodilator buffer, or bubbles) or insufficient perfusion of the contrast (Figure 2F). Vasodilatation and fixation would not work well if the blood vessels have shrunk. Although CT imaging can only visualize the contrast medium, it is possible to view the arteries on the surface of the body by macroscopic or stereomicroscopic observation (Figure 2G). Thus, it is easier to assess defects using the contrast medium (Figure 2H).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63009/63009fig01.jpg" /> 
Figure 1: Outline of the procedure. (A) Pressure perfusion apparatus and the 22 G catheter connected via a 2 mL extension tube and three-way stopcock.&#160;(B) The ascending aorta is cut diagonally to expose the cross-section (yellow arrow).&#160;(C) The catheter was fixed using two pins (yellow arrows).&#160;(D) The fixed lower limbs extend upon fixation (yellow arrows).&#160;(E) Injection of contrast through the three-way stopcock. The injection direction is indicated by a yellow arrow. (F) The hind-limb nail is filled with the contrast (yellow arrow).&#160;(G) The stopcock is closed and removed from the perfusion apparatus. <a href="https://www.jove.com/files/ftp_upload/63009/63009fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63009/63009fig02.jpg" /> 
Figure 2: Images of vessels. (A) Whole image of the hind-limb bones and vessels. (B) Femoral artery (red arrow) and vein (blue arrow). (C) Ligated femoral artery (yellow arrow). The periphery is interrupted by the obstruction (yellow dotted line).&#160;(D) Collateral vessels in the ligated side (yellow arrows). The yellow dotted line represents the interrupted femoral artery. (E) A well-filled sample of the saphenous artery (red arrow) and vein (blue arrow).&#160;(F) Inadequate perfusion leads to interruption of saphenous vessels (yellow dotted line). (G) Stereomicroscopic observation of a representative sample. The right femoral artery (yellow arrow) is filled with contrast medium.&#160;(H) Stereomicroscope observation of a failed sample. The right femoral artery (yellow arrow) lacks contrast medium. Scale bars = 1 mm (B-F), 2 mm (G, H), 10 mm (A). <a href="https://www.jove.com/files/ftp_upload/63009/63009fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Sample Preparation for Computed Tomography-based Three-dimensional Visualization of Murine Hind-limb Vessels at JoVE.com

Sample Preparation for Computed Tomography-based Three-dimensional Visualization of Murine Hind-limb Vessels

Here, we describe a visualization and quantification method for murine hind-limb vessels using micro-X-ray computed tomography.

Blood vessels are complex networks with tree-like structures, and vascular networks are essential for maintaining both circulation and maintaining organ ...

sample-preparation-for-computed-tomography-based-three-dimensional

Research

JoVE Journal

Medicine

2.2K Views.  National Cerebral and Cardiovascular Center Research Institute. Here, we describe a visualization and quantification method for murine hind-limb vessels using micro-X-ray computed tomography. 

Video: Sample Preparation for Computed Tomography-based Three-dimensional Visualization of Murine Hind-limb Vessels

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher frequency of fractures 1. As these fractures often occur in regions that have lost normal sensory function, the patient is at a greater risk of fracture-dependent pathologies, including death. SCI-dependent loss in both bone mineral density (BMD, grams/cm2) and bone mineral content (BMC, grams) has been attributed to mechanical disuse 2, aberrant neuronal signaling 3 and hormonal changes 4. The use of rodent models of SCI-induced osteoporosis can provide invaluable information regarding the mechanisms underlying the development of osteoporosis following SCI as well as a test environment for the generation of new therapies 5-7 (and reviewed in 8). Mouse models of SCI are of great interest as they permit a reductionist approach to mechanism-based assessment through the use of null and transgenic mice. While such models have provided important data, there is still a need for minimally-invasive, reliable, reproducible, and quantifiable methods in determining the extent of bone loss following SCI, particularly over time and within the same cohort of experimental animals, to improve diagnosis, treatment methods, and/or prevention of SCI-induced osteoporosis.
An ideal method for measuring bone density in rodents would allow multiple, sequential (over time) exposures to low-levels of X-ray radiation. This study describes the use of a new whole-animal scanner, the IVIS Lumina XR (Caliper Instruments) that can be used to provide low-energy (1-3 milligray (mGy)) high-resolution, high-magnification X-ray images of mouse hind limb bones over time following SCI. Significant bone density loss was seen in the tibiae of mice by 10 days post-spinal transection when compared to uninjured, age-matched control (na&iuml;ve) mice (13% decrease, p&lt;0.0005). Loss of bone density in the distal femur was also detectable by day 10 post-SCI, while a loss of density in the proximal femur was not detectable until 40 days post injury (7% decrease, p&lt;0.05). SCI-dependent loss of mouse femur density was confirmed post-mortem through the use of Dual-energy X-ray Absorptiometry (DXA), the current "gold standard" for bone density measurements. We detect a 12% loss of BMC in the femurs of mice at 40 days post-SCI using the IVIS Lumina XR. This compares favorably with a previously reported BMC loss of 13.5% by Picard and colleagues who used DXA analysis on mouse femurs post-mortem 30 days post-SCI 9. Our results suggest that the IVIS Lumina XR provides a novel, high-resolution/high-magnification method for performing long-term, longitudinal measurements of hind limb bone density in the mouse following SCI.

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

A longitudinal examination of bone loss in the femurs and tibiae of adult mice was performed following spinal cord injury using sequential low-dose X-ray scans. Tibia bone loss was detected throughout the study, while bone loss in the femur was not detected until 40 days post injury.

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher ...

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 ...

A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation (VCA) Research

Vascularized Composite Allotransplantation (VCA) such as hand and face transplants represent a viable treatment option for complex musculoskeletal trauma and devastating tissue loss. Despite favorable and highly encouraging early and intermediate functional outcomes, rejection of the highly immunogenic skin component of a VCA and potential adverse effects of chronic multi-drug immunosuppression continue to hamper widespread clinical application of VCA. Therefore, research in this novel field needs to focus on translational studies related to unique immunologic features of VCA and to develop novel immunomodulatory strategies for immunomodulation and tolerance induction following VCA without the need for long term immunosuppression.
This article describes a reliable and reproducible translational large animal model of VCA that is comprised of an osteomyocutaneous flap in a MHC-defined swine heterotopic hind limb allotransplantation. Briefly, a well-vascularized skin paddle is identified in the anteromedial thigh region using near infrared laser angiography. The underlying muscles, knee joint, distal femur, and proximal tibia are harvested on a femoral vascular pedicle. This allograft can be considered both a VCA and a vascularized bone marrow transplant with its unique immune privileged features. The graft is transplanted to a subcutaneous abdominal pocket in the recipient animal with a skin component exteriorized to the dorsolateral region for immune monitoring.
Three surgical teams work simultaneously in a well-coordinated manner to reduce anesthesia and ischemia times, thereby improving efficiency of this model and reducing potential confounders in experimental protocols. This model serves as the groundwork for future therapeutic strategies aimed at reducing and potentially eliminating the need for chronic multi-drug immunosuppression in VCA.

A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation VCA Research

Vascularized Composite Allotransplantations (VCA) have become a clinical reality. However, broad clinical application of VCA is limited by chronic multi-drug immunosuppression. The authors present a reliable and reproducible large animal model to translate novel immunomodulatory strategies that can minimize or potentially eliminate the need of immunosuppression in VCA.

Vascularized Composite Allotransplantation (VCA) such as hand and face transplants represent a viable treatment option for complex musculoskeletal trauma ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Orthotopic Hind Limb Transplantation in the Mouse

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and translational research in the field of transplantation medicine. A vast array of reagents is available exclusively in this setting, including mono- and polyclonal antibodies for both diagnostic and interventional applications. In addition, a vast number of genotyped, inbred, transgenic, and knock out strains allow detailed investigation of the individual contributions of humoral and cellular components to the complex interplay of an immune response and make the mouse the gold standard for immunological research. 
Vascularized Composite Allotransplantation (VCA) delineates a novel field of transplantation using allografts to replace "like with like" in patients suffering traumatic or congenital tissue loss. This surgical methodological protocol shows the use of a non-suture cuff technique for super-microvascular anastomosis in an orthotopic mouse hind limb transplantation model. The model specifically allows for comparison between established paradigms in solid organ transplantation with a novel form of transplants consisting of various different tissue components. Uniquely, this model allows for the transplantation of a viable vascularized bone marrow compartment and niche that have the potential to exert a beneficial effect on the balance of immune acceptance and rejection. This technique provides a tool to investigate alloantigen recognition and allograft rejection and acceptance, as well as enables the pursuit of functional nerve regeneration studies to further advance this novel field of transplantation.

This novel model for orthotopic hind limb transplantation in the mouse, applying a non-suture cuff technique for super-microvascular anastomosis, provides a powerful tool for in&#160;vivo mechanistic immunological research related to vascularized composite allotransplantation (VCA).

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Cone Beam Intraoperative Computed Tomography-based Image Guidance for Minimally Invasive Transforaminal Interbody Fusion

Transforaminal lumbar interbody fusion (TLIF) is commonly used for the treatment of spinal stenosis, degenerative disc disease, and spondylolisthesis. Minimally invasive surgery (MIS) approaches have been applied to this technique with an associated decrease in estimated blood loss (EBL), length of hospital stay, and infection rates, while preserving outcomes with traditional open surgery. Previous MIS TLIF techniques involve significant fluoroscopy that subjects the patient, surgeon, and operating room staff to non-trivial levels of radiation exposure, particularly for complex multi-level procedures. We present a technique that utilizes an intraoperative computed tomography (CT) scan to aid in placement of pedicle screws, followed by traditional fluoroscopy for confirmation of cage placement. Patients are positioned in the standard fashion and a reference arc is placed in the posterior superior iliac spine (PSIS) followed by intraoperative CT scan. This allows for image-guidance-based placement of pedicle screws through a one-inch skin incision on each side. Unlike traditional MIS-TLIF that requires significant fluoroscopic imaging during this stage, the operation can now be performed without any additional radiation exposure to the patient or operating room staff. After completion of the facetectomy and discectomy, final TLIF cage placement is confirmed with fluoroscopy. This technique has the potential to decrease operative time and minimize total radiation exposure.

The purpose of this article is to provide image-guidance for minimally invasive transforaminal interbody fusion.

Transforaminal lumbar interbody fusion (TLIF) is commonly used for the treatment of spinal stenosis, degenerative disc disease, and spondylolisthesis. ...

Preclinical Model of Hind Limb Ischemia in Diabetic Rabbits

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular disease is the development of ischemia. In severe cases, patients can develop critical limb ischemia in which they experience constant pain and an increased risk of limb amputation. Current therapies for peripheral ischemia include bypass surgery or percutaneous interventions such as angioplasty with stenting or atherectomy to restore blood flow. However, these treatments often fail to the continued progression of vascular disease or restenosis or are contraindicated due to the overall poor health of the patient. A promising potential approach to treat peripheral ischemia involves the induction of therapeutic neovascularization to allow the patient to develop collateral vasculature. This newly formed network alleviates peripheral ischemia by restoring perfusion to the affected area. The most frequently employed preclinical model for peripheral ischemia utilizes the creation of hind limb ischemia in healthy rabbits through femoral artery ligation. In the past, however, there has been a strong disconnect between the success of preclinical studies and the failure of clinical trials regarding treatments for peripheral ischemia. Healthy animals typically have robust vascular regeneration in response to surgically induced ischemia, in contrast to the reduced vascularity and regeneration in patients with chronic peripheral ischemia. Here, we describe an optimized animal model for peripheral ischemia in rabbits that includes hyperlipidemia and diabetes. This model has reduced collateral formation and blood pressure recovery in comparison to a model with a higher cholesterol diet. Thus, the model may provide better correlation with human patients with compromised angiogenesis from the common co-morbidities that accompany peripheral vascular disease.

We describe a surgical procedure used to induce peripheral ischemia in rabbits with hyperlipidemia and diabetes. This surgery acts as a preclinical model for conditions experienced in peripheral artery disease in patients. Angiography is also described as a means to measure the extent of introduced ischemia and recovery of perfusion.

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular ...

High-Resolution Three-Dimensional Imaging of the Footpad Vasculature in a Murine Hindlimb Gangrene Model

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering from its most severe form, chronic limb-threatening ischemia (CLTI), face substantial impairments to daily living, including chronic pain, limited walking distance without pain, and nonhealing wounds. Preclinical models have been developed in various animals to study PAD, but mouse hindlimb ischemia remains the most widely used. There can be significant variation in response to ischemic insult in these models depending on the mouse strain used and the site, number, and means of arterial disruption. This protocol describes a unique method combining femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase (NOS) inhibitor to reliably induce footpad gangrene in Friend Virus B (FVB) mice that resembles the tissue loss of CLTI. While traditional means of assessing reperfusion such as laser Doppler perfusion imaging (LDPI) are still recommended, intracardiac perfusion of the lipophilic dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) is used to label the vasculature. Subsequent whole-mount confocal laser scanning microscopy allows for high-resolution, three-dimensional (3D) reconstruction of footpad vascular networks that complements traditional means of assessing reperfusion in hindlimb ischemia models.

The present protocol describes a unique, clinically relevant model of peripheral arterial disease that combines femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase inhibitor to induce hindlimb gangrene in FVB mice. Intracardiac DiI perfusion is then used for high-resolution, three-dimensional imaging of the footpad vasculature.

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering ...