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/pubmed?term=((Angiogenesis%5BTitle%5D)+AND+%22Annual+Review+of+Medicine%22%5BJournal%5D)">Angiogenesis.</a> Annual Review of Medicine. 57, 1-18 (2006).</li>
<li>Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Angiogenesis+in+cancer%2C+vascular%2C+rheumatoid+and+other+disease%5BTitle%5D)+AND+%22Nature+Medicine%22%5BJournal%5D)">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/pubmed?term=((Characterization+of+the+arterial+anatomy+of+the+murine+hindlimb%3A+functional+role+in+the+design+and+understanding+of+ischemia+models%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">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/pubmed?term=((Evaluation+of+postnatal+arteriogenesis+and+angiogenesis+in+a+mouse+model+of+hind-limb+ischemia%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">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/pubmed?term=((Retrograde+perfusion+and+filling+of+mouse+coronary+vasculature+as+preparation+for+micro+computed+tomography+imaging%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%3A+JoVE%22%5BJournal%5D)">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/pubmed?term=((Evaluation+of+collateral+source+characteristics+with+3-dimensional+analysis+using+micro-X-ray+computed+tomography%5BTitle%5D)+AND+%22Journal+of+the+American+Heart+Association%22%5BJournal%5D)">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/pubmed?term=((Tissue+clearing+technique%3A+Recent+progress+and+biomedical+applications%5BTitle%5D)+AND+%22Journal+of+Anatomy%22%5BJournal%5D)">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/pubmed?term=((Advanced+CUBIC+protocols+for+whole-brain+and+whole-body+clearing+and+imaging%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">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/pubmed?term=((Angiogenesis+in+liver+disease%5BTitle%5D)+AND+%22Journal+of+Hepatology%22%5BJournal%5D)">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/pubmed?term=((Angiogenesis+in+pancreatic+cancer%3A+current+research+status+and+clinical+implications%5BTitle%5D)+AND+%22Angiogenesis%22%5BJournal%5D)">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><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&quot; 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 &amp;amp; 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 ...

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2.3K 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

Contrast Enhanced Vessel Imaging using MicroCT

Microscopic computed tomography (microCT) offers high-resolution volumetric imaging of the anatomy of living small animals. However, the contrast between different soft tissues and body fluids is inherently poor in micro-CT images 1. Under these circumstances, visualization of blood vessels becomes a nearly impossible task. To overcome this and to improve the visualization of blood vessels exogenous contrast agents can be used. Herein, we present a methodology for visualizing the vascular network in a rodent model. By using a long-acting aqueous colloidal polydisperse iodinated blood-pool contrast agent, eXIA 160XL, we optimized image acquisition parameters and volume-rendering techniques for finding blood vessels in live animals. Our findings suggest that, to achieve a superior contrast between bone and soft tissue from vessel, multiple-frames (at least 5-8/ frames per view), and 360-720 views (for a full 360&deg; rotation) acquisitions were mandatory. We have also demonstrated the use of a two-dimensional transfer function (where voxel color and opacity was assigned in proportion to CT value and gradient magnitude), in visualizing the anatomy and highlighting the structure of interest, the blood vessel network. This promising work lays a foundation for the qualitative and quantitative assessment of anti-angiogenesis preclinical studies using transgenic or xenograft tumor-bearing mice.

Contrast enhanced small animal vessel imaging by microCT is a rapid, cost-effective and high-throughput technique for serial in situ examination for tumor development, for analyzing the network of blood vessels that nourish them, and for following the response of tumors to preclinical therapeutic intervention(s).

Microscopic computed tomography (microCT) offers high-resolution volumetric imaging of the anatomy of living small animals. However, the contrast between ...

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 ...

Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). Animal models are necessary for studying the etiopathology and potential therapies of cerebrovascular diseases. Imaging the vasculature in large animals is relatively easy. However, developing vessel imaging methods of murine brain disease models is desirable due to the cost and availability of genetically-modified mouse lines. Imaging the murine cerebral vascular tree is a challenge. In humans and larger animals, the gold standard for assessing the angioarchitecture at the macrovascular (conductance) level is x-ray catheter contrast-based angiography, a method not suited for small rodents.
In this article, we present a method of cerebrovascular casting that produces a durable skeleton of the entire vascular bed, including arteries, veins, and capillaries that may be analyzed using many different modalities. Complete casting of the microvessels of the mouse cerebrovasculature can be difficult; however, these challenges are addressed in this step-by-step protocol. Through intracardial perfusion of the vascular casting material, all vessels of the body are casted. The brain can then be removed and clarified using the organic solvent methyl salicylate. Three dimensional imaging of the brain blood vessels can be visualized simply and inexpensively with any conventional brightfield microscope or dissecting microscope. The casted cerebrovasculature can also be imaged and quantified using micro-computed tomography (micro-CT)1. In addition, after being imaged, the casted brain can be embedded in paraffin for histological analysis.
The benefit of this vascular casting method as compared to other techniques is its broad adaptation to various analytic tools, including brightfield microscopic analysis, CT scanning due to the radiopaque characteristic of the material, as well as histological and immunohistochemical analysis. This efficient use of tissue can save animal usage and reduce costs. We have recently demonstrated application of this method to visualize the irregular blood vessels in a mouse model of adult BAVM at a microscopic level2, and provide additional images of the malformed vessels imaged by micro-CT scan. Although this method has drawbacks and may not be ideal for all types of analyses, it is a simple, practical technique that can be easily learned and widely applied to vascular casting of blood vessels throughout the body.

In this article, we present a simple, practical technique for cerebrovascular casting that is easy to perform and can be utilized to image the vascular tree of the adult mouse brain.

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). ...

Neuroscience

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 ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

A Volumetric Method for Quantification of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor determining patient outcome and is therefore frequently taken as a study endpoint. However, in small animal studies on SAH, quantification of cerebral vasospasm is a major challenge. Here, an ex vivo method is presented that allows quantification of volumes of entire vessel segments, which can be used as an objective measure to quantify cerebral vasospasm. In a first step, endovascular casting of the cerebral vasculature is performed using a radiopaque casting agent. Then, cross-sectional imaging data are acquired by micro computed tomography. The final step involves 3-dimensional reconstruction of the virtual vascular tree, followed by an algorithm to calculate center lines and volumes of the selected vessel segments. The method resulted in a highly accurate virtual reconstruction of the cerebrovascular tree shown by a diameter-based comparison of anatomical samples with their virtual reconstructions. Compared with vessel diameters alone, the vessel volumes highlight the differences between vasospastic and non-vasospastic vessels shown in a series of SAH and sham-operated mice.

The goal of this article is to present a method that allows a 3-dimensional reconstruction of the cerebrovascular tree in mice after micro computed tomography and determination of volumes of entire vessel segments that can be used to quantify cerebral vasospasm in murine models of subarachnoid hemorrhage.

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor ...

Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of &#946;-aminopropionitrile-induced Aortic Aneurysm and Dissection

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of aortic disease exist, in vivo imaging of the vasculature has been limited. In recent years, micro-computerized tomography (micro-CT) has emerged as a preferred modality for imaging both large and small vessels both in vivo and ex vivo. In conjunction with a method of vascular casting, we have successfully used micro-CT to characterize the frequency and distribution of aortic pathology in &#946;-aminopropionitrile-treated C57/Bl6 mice. Technical limitations of this method include variations in the quality of the perfusion introduced by poor animal preparation, the application of proper methodologies for vessel size quantification, and the non-survivability of this procedure. This article details a methodology for the intravascular perfusion of a lead-based radiopaque silicone rubber for the quantitative characterization of aortopathy in a mouse model of aneurysm and dissection. In addition to visualizing aortic pathology, this method may be used for examining other vascular beds in vivo or vascular beds removed post-mortem.

This article describes a detailed methodology of using a radiopaque lead-based silicone rubber to perfuse the murine vasculature for aortic diameter quantification in a mouse model of aortic aneurysm and dissection.

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of ...

Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard therapy, a considerable number of CLI patients are not suited for either surgical or endovascular revascularization. Angiogenic therapies are emerging as an option for these patients but are currently still under investigation. Before application in humans, those therapies must be tested in animal models and its mechanisms must be clearly understood. An animal model of hindlimb ischemia (HLI) has been developed by the ligation and excision of the distal external iliac and femoral arteries and veins in mice. A comprehensive panel of tests was assembled to assess the effects of ischemia and putative angiogenic therapies at functional, histologic and molecular levels. Laser Doppler was used for the flow measurement and functional assessment of perfusion. Tissue response was evaluated by the analysis of capillary density after staining with the anti-CD31 antibody on histological sections of gastrocnemius muscle and by measurement of collateral vessel density after diaphonization. Expression of angiogenic genes was quantified by RT-PCR targeting selected angiogenic factors exclusively in endothelial cells (ECs) after laser capture microdissection from mice gastrocnemius muscles. These methods were sensitive in identifying differences between ischemic and non-ischemic limbs and between treated and non-treated limbs. This protocol provides a reproducible model of CLI and a framework for testing angiogenic therapies.

Here, a critical hindlimb ischemia experimental model is presented followed by a battery of functional, histologic and molecular tests&#160;to assess the effectiveness of angiogenic therapies.&#8203;

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard ...

Monitoring Functionality and Morphology of Vasculature Recruited by Factors Secreted by Fast-growing Tumor-generating Cells

The subcutaneous matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic factors. In this method, desired factors are introduced into cold-liquid ECM-mimic gel which, after subcutaneous injection, solidifies to form an environment mimicking the cancer milieu. This matrix permits the penetration of host cells, such as endothelial cells, and therefore, the formation of vasculature.
Herein we propose a new modified matrigel plug assay, which can be exploited to illustrate the angiogenic potential of a pool of factors secreted by cancer cells, as opposed to a specific factor (e.g., bFGF and VEGF) or agent. The plug containing ECM-mimic gel is utilized to introduce the host (i.e., mouse) with a pool of factors secreted to the C.M. of fast-growing tumor-generating glioblastoma cells. We have previously described an extensive comparison of the angiogenic potential of U-87 MG human glioblastoma and its dormant-derived clone, in this system model, showing induced angiogenesis in the U-87 MG parental cells. The C.M. is prepared by filtering collected media from confluent tissue culture plates of either cell line following 48 hr incubation. Hence, it contains only factors secreted by the cells, without the cells themselves. Described here is the combination of two imaging modalities, microbubbles contrast-enhanced ultrasound imaging and intravital fibered-confocal endomicroscopy, for an accurate, real-time characterization of the extent, morphology and functionality of newly-formed blood vessels within the plugs.

We describe a matrigel plug assay to illustrate angiogenic potential of a pool of factors secreted by cancer cells using two complementary imaging modalities, ultrasound and endomicroscopy. The matrigel, an extracellular matrix (ECM)-mimic gel, is utilized to introduce the host (mouse) with angiogenic factors secreted to the conditioned media (C.M.).&#160;

The subcutaneous matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic factors. In this method, desired ...

Biology

Rapid Whole-Mount High-Resolution Imaging of Small Animal Vasculature for Quantitative Studies

In small animal models of cardiovascular development and diseases, subject-specific computational simulations of blood flow&#160;enable quantitative assessments of hemodynamic metrics that are difficult to measure experimentally. Computational fluid dynamic simulations shed light on the critical roles of mechanics in cardiovascular function and disease progression. Acquiring high-quality volumetric images of the vessels of interest is central to the accuracy and reproducibility of morphological measurement and flow quantitation results. This study proposes a rapid, cost-effective, and accessible method for whole-mount high-resolution imaging of small animal vasculature using light-sheet fluorescence microscopy. The modified iDISCO+ (immunolabeling-enabled three-dimensional imaging of solvent-cleared organs) light-sheet sample preparation protocol involves (1) labeling vasculature with a fluorescent agent, (2) preserving the sample, and (3) rendering the sample transparent. Unlike classical iDISCO+, which uses immunohistochemical staining, the authors label vascular endothelium with FITC-tagged poly-L-lysine, an affordable non-specific fluorescent dye that is highly resistant to photo-bleaching, in a process termed &#34;endo-painting.&#34; The rapid labeling reduces sample preparation time from approximately four weeks to less than 3 days. Furthermore, the use of minimally hazardous solvent ethyl cinnamate (ECi) as the clearing agent and imaging solution makes the samples safer to handle and compliant with a wider range of imaging facilities. The proposed protocol is applied to obtain highly resolved light-sheet fluorescence microscopy image stacks of the cardiovascular system in chick embryos ranging from day 3 (HH18) to day 8 (HH34). This study further demonstrates the suitability of this method for vascular quantitation through 3D reconstruction and computational hemodynamic modeling of a day 5 (HH 26) chick embryo.

This protocol introduces a rapid method for quantitative whole-mount three-dimensional vascular imaging using light-sheet fluorescence microscopy. The efficacy of the method is demonstrated using the pharyngeal arch artery system of the chick embryo model, with hemodynamic forces quantified via computational fluid dynamics.

In small animal models of cardiovascular development and diseases, subject-specific computational simulations of blood flow&#160;enable quantitative ...