This work (2017R1A2B1006403) was supported by the Mid-Career Researcher Program through a National Research Foundation grant funded by the Korean government (Ministry of Science and ICT).

All procedures, including animal subjects, have been approved by the Institutional Animal Care and Use Committees of Seoul National University Hospital (IACUC No. 10-0184). This protocol is optimized for research on flap vasculature. This example is based on a four-territory flap model in our previous reports.
1. Establishing a Flap Condition
NOTE: It is important to generate a vascular change in a rat flap model 4 to 5 days before visible estimation6,7.
<ol>
	<li>Use 7-week-old male Sprague-Dawley rats weighing 200 - 250 g.</li>
	<li>Anesthetize the rats using isoflurane at 3 - 5% for induction and 2 - 2.5% for maintenance. Perform a toe pinch withdrawal reflection test to confirm that the depth of anesthesia is sufficient.&#160;&#160;Inject meloxicam 5mg/kg subcutaneously for pain relief.</li>
	<li>Shave the trunk using an animal hair clipper and hair removal cream (thioglycolic acid, 80%). Prepare a sterile surgical field with 10% povidone-iodine and a sterile drape to maintain a sterile condition throughout the procedures. Apply a vet ointment to the eyes to prevent dryness.&#160;Maintain all instruments in sterile condition.</li>
	<li>Establish the appropriate the flap condition.
	<ol>
		<li>Mark a circumferential skin flap design from the lower abdomen to the back, measuring 4 x 12 cm. Locate the center of the flap halfway between the xiphoid process and the penis (Figure 1).</li>
		<li>Make the incision as marked using a surgical blade.</li>
		<li>Dissect the flap using scissors, including the skin and panniculus carnosus.</li>
		<li>Dissect around the vascular pedicle [bilateral deep circumflex iliac (DCI) vessels and bilateral superficial inferior epigastric (SIE) vessels] at the lower abdomen and expose the vascular pedicle using a surgical loupe and microsurgical instruments.</li>
		<li>Maintain or ligate the vessels depending on the desired conditions.</li>
		<li>Divide the flap along the dorsal midline using a surgical blade or scissors.</li>
		<li>Lay the flap in its original position and fix it with a skin stapler.</li>
		<li>Apply a topical ointment to the surgical wound for 3 days and provide postoperative analgesia by administering meloxicam at a dose of 5 mg/kg orally once per day for 3 days.</li>
		<li>Confirm that the rat regains sufficient consciousness to maintain sternal recumbency.&#160;Return the rat into the cage and move it to the housing area. Apply an Elizabethan collar to each rats.</li>
	</ol>
	</li>
</ol>
2. Preparation of the Instruments
<ol>
	<li>Prepare a 24G catheter and a three-way stopcock.</li>
	<li>Prepare mosquito forceps, small scissors, a surgical scalpel, and a surgical blade.</li>
	<li>Prepare the angiographic agent (silicone rubber injection compound).
	<ol>
		<li>Blend the color agent compound with the diluent in the sterile specimen collection cup. Ensure an equal quantity by weight: 15 mL of color agent compound and 15 mL of MV diluent in one rat (Sprague-Dawley rat, 200 - 250 g).</li>
		<li>Add the curing agent per 5% weight or volume of the mixture solution immediately before injection: 1.5 mL of curing agent in one rat (Sprague-Dawley rat, 200 - 250 g).</li>
	</ol>
	</li>
</ol>
3.&#160;Rat Artery Preparation
<ol>
	<li>Use isoflurane to anesthetize the rats (3 - 5% for induction and 2 - 2.5% for maintenance).&#160;Perform a toe pinch withdrawal reflection test to confirm that the depth of anesthesia is sufficient.</li>
	<li>Shave the neck using an animal hair clipper and hair removal cream (thioglycolic acid, 80%).&#160;</li>
	<li>Expose the common carotid artery10.
	<ol>
		<li>Make a 2-cm midline incision between the scapulae.</li>
		<li>Dissect more deeply using mosquito forceps and blunt scissors until the salivary gland complex is exposed.</li>
		<li>Retract the salivary gland and bluntly dissect the omohyoid muscle longitudinally.</li>
		<li>Dissect around the common carotid artery.</li>
	</ol>
	</li>
	<li>Hook the cephalic and caudal sides of the common carotid artery with black silk and affix it.
	<ol>
		<li>Make a tie on proximal suture and keep traction to maintain engorgement of the artery.</li>
		<li>Prepare one silk suture at the caudal side for securing of 24G catheter.</li>
	</ol>
	</li>
</ol>
4. Cannulation
<ol>
	<li>Cannulate the prepared carotid artery using a 24G catheter.</li>
	<li>Tighten the pre-made tie in the caudal side and be careful to not remove the catheter during injection.</li>
	<li>Prepare the curing agent (step 1.3.2).</li>
	<li>Connect the three-way stopcock securely to the inserted catheter.
	<ol>
		<li>Confirm regurgitated blood into the catheter by adding negative pressure using an empty syringe.</li>
	</ol>
	</li>
</ol>
5. Injection
<ol>
	<li>Inject the silicone rubber injection compound until the color of the eye and foot has changed. 
	&#8203;NOTE: The color change should appear as the injected fluid progresses (injection amount is approximately 25 - 30 mL for each rat).</li>
	<li>Lock the three-way stopcock and wait until the agent solidifies.
	<ol>
		<li>Be careful not to contaminate with the agent, especially when removing the syringe from the three-way stopcock. Use a protective barrier such as gauze or vinyl to separate the injection space from its surroundings. 
		CAUTION: Any contamination makes it difficult to analyze the angiographic image because the compound is radio-opaque.</li>
		<li>Confirm the cessation of heartbeats and respiration. Stop the anesthesia.</li>
		<li>Observe the rate of hardness with the remaining agent as a reference (approximately 15 min needed).</li>
	</ol>
	</li>
</ol>
6. Harvesting of Specimen
<ol>
	<li>Make an incision using a surgical blade to the panniculus carnosus 1 cm outside the flap to prevent damage to any vascular structure inside the flap.</li>
	<li>Dissect along the previously dissected plane from step 1.4 (under the panniculus carnosus plane) and harvest the tissue including the flap and vascular pedicle using scissors (the vascular structure is included in the flap).</li>
	<li>Ligate the pedicle of the flap using a 5-0 silk suture and separate the flap from the body. Be careful not to damage the vascular structure.</li>
</ol>
7. Capturing the Angiographic Image
<ol>
	<li>Spread out the specimen, ensuring that it does not fold, and gently place it on the surgical drape using forceps.</li>
	<li>Take a radiography image.
	<ol>
		<li>Transfer the specimen lying on the film cassette to the sample loading space.</li>
		<li>Set the soft tissue X-ray system to 60 kVp, 5 mA, and 5 s exposure.</li>
	</ol>
	</li>
	<li>Develop the film in a darkroom using an automatic development machine.</li>
	<li>Scan the film at the highest resolution possible.</li>
</ol>
8. Analyzing the Image6,7,11
<ol>
	<li>Distinguish the arteries and veins based on the continuity of flow and diameter.
	<ol>
		<li>Start from the inflow of the pedicle artery and focus on the target vessel being examined.</li>
		<li>Measure the diameters with software by first opening the image.
		<ol>
			<li>Click the Straight button and draw a line on the scale bar that is the same length.</li>
			<li>Open the Analyze | Set scale menu and enter the value of the scale bar into Known distance.</li>
			<li>Click the Straight button and draw a line onto the vessel of which the diameter needs to be measured.</li>
			<li>Open the Analyze | Measure menu and confirm the Length.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Analyze the vascular pattern considering the flap survival area.</li>
</ol>

<ol>
	<li>Taylor, G. I., Minabe, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Angiosomes+of+the+Mammals+and+Other+Vertebrates.">The Angiosomes of the Mammals and Other Vertebrates.</a> Plastic and Reconstructive Surgery. 89 (2), 181-215 (1992).</li><li>Taylor, G. I., Palmer, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vascular+territories+(angiosomes)+of+the+body:+experimental+study+and+clinical+applications.">The vascular territories (angiosomes) of the body: experimental study and clinical applications.</a> British Journal of Plastic Surgery. 40 (2), 113-141 (1987).</li><li>Geddes, C. R., Morris, S. F., Neligan, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perforator+flaps:+evolution,+classification,+and+applications.">Perforator flaps: evolution, classification, and applications.</a> Annals of Plastic Surgery. 50 (1), 90-99 (2003).</li><li>Saint-Cyr, M., Schaverien, M. V., Rohrich, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perforator+Flaps:+History,+Controversies,+Physiology,+Anatomy,+and+Use+in+Reconstruction.">Perforator Flaps: History, Controversies, Physiology, Anatomy, and Use in Reconstruction.</a> Plastic and Reconstructive Surgery. 123 (4), 132-145 (2009).</li><li>Lie, K. H., Taylor, G. I., Ashton, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+peroxide+priming+of+the+venous+architecture:+a+new+technique+that+reveals+the+underlying+anatomical+basis+for+venous+complications+of+DIEP,+TRAM,+and+other+abdominal+flaps.">Hydrogen peroxide priming of the venous architecture: a new technique that reveals the underlying anatomical basis for venous complications of DIEP, TRAM, and other abdominal flaps.</a> Plastic and Reconstructive Surgery. 133 (6), 790-804 (2014).</li><li>Chang, H., Nobuaki, I., Minabe, T., Nakajima, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Three+Different+Supercharging+Procedures+in+a+Rat+Skin+Flap+Model.">Comparison of Three Different Supercharging Procedures in a Rat Skin Flap Model.</a> Plastic and Reconstructive Surgery. 113 (1), 277-283 (2004).</li><li>Chang, H., Minn, K. W., Imanishi, N., Minabe, T., Nakajima, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Venous+Superdrainage+on+a+Four-Territory+Skin+Flap+Survival+in+Rats.">Effect of Venous Superdrainage on a Four-Territory Skin Flap Survival in Rats.</a> Plastic and Reconstructive Surgery. 119 (7), 2046-2051 (2007).</li><li>Rozen, W. M., Stella, D. L., Ashton, M. W., Phillips, T. J., Taylor, G. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+CT+angiography:+a+new+technique+for+imaging+microvascular+anatomy.">Three-dimensional CT angiography: a new technique for imaging microvascular anatomy.</a> Clinical Anatomy. 20 (8), 1001-1003 (2007).</li><li>Taylor, G. I., Chubb, D. P., Ashton, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=True+and+"choke"+anastomoses+between+perforator+angiosomes:+part+i.+anatomical+location.">True and "choke" anastomoses between perforator angiosomes: part i. anatomical location.</a> Plastic and Reconstructive Surgery. 132 (6), 1447-1456 (2013).</li><li>Feng, J., Fitz, 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=Catheterization+of+the+carotid+artery+and+jugular+vein+to+perform+hemodynamic+measures,+infusions+and+blood+sampling+in+a+conscious+rat+model.">Catheterization of the carotid artery and jugular vein to perform hemodynamic measures, infusions and blood sampling in a conscious rat model.</a> Journal of Visualized Experiments. (95), e51881 (2015).</li><li>Park, S. O., Cho, J., Imanishi, N., Chang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+distal+venous+drainage+on+the+survival+of+four-territory+flaps+with+no+pedicle+vein:+Results+from+a+rat+model.">Effect of distal venous drainage on the survival of four-territory flaps with no pedicle vein: Results from a rat model.</a> Journal of Plastic, Reconstructive &amp; Aesthetic Surgery. 71 (3), 410-415 (2018).</li><li>Park, S. O., Chang, H., Imanishi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+free+serratus+anterior+artery+perforator+flap-A+case+report+and+anatomic+study.">The free serratus anterior artery perforator flap-A case report and anatomic study.</a> Microsurgery. 36 (4), 339-344 (2016).</li><li>Imanishi, N., Nakajima, H., Minabe, T., Chang, H., Aiso, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomical+relationship+between+arteries+and+veins+in+the+paraumbilical+region.">Anatomical relationship between arteries and veins in the paraumbilical region.</a> British Journal of Plastic Surgery. 56 (6), 552-556 (2003).</li><li>Schaverien, M., Saint-Cyr, M., Arbique, G., Rohrich, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-+and+four-dimensional+arterial+and+venous+anatomies+of+the+thoracodorsal+artery+perforator+flap.">Three- and four-dimensional arterial and venous anatomies of the thoracodorsal artery perforator flap.</a> Plastic and Reconstructive Surgery. 121 (5), 1578-1587 (2008).</li><li>Schaverien, M., Saint-Cyr, M., Arbique, G., Hatef, D., Brown, S. A., Rohrich, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-+and+Four-Dimensional+Computed+Tomographic+Angiography+and+Venography+of+the+Anterolateral+Thigh+Perforator+Flap.">Three- and Four-Dimensional Computed Tomographic Angiography and Venography of the Anterolateral Thigh Perforator Flap.</a> Plastic and Reconstructive Surgery. 121 (5), 1685-1696 (2008).</li></ol>

The authors have nothing to disclose.

Silicone rubber injection compound angiography can be performed easily, does not require expensive equipment, and offers many advantages. In contrast to the preoperative and intraoperative evaluations of patients, experiments using animals and cadavers can provide details on specific conditions, enabling more diverse and in-depth studies. The flap model using rats is particularly valuable to clinicians because changes in various contexts can be observed before clinical applications6,<su...

Examining vascular structures such as arteries and veins is an important area of interest, particularly in reconstructive surgery. In this field, flap surgery is widely performed. Therefore, angiographic imaging is actively used to study the flap territory, angiosome, and vascular supply of fresh tissue1. Specifically, there have been continuous efforts to observe the fine vasculature, including fine vessels such as perforators (vessels emerging from deep vessels reaching the skin), and choke vessels (connecting vessels between adjacent angiosomes)2. These two types of vessels are important in the perforator flap reconstruction field and are the main focus of the research3,4.
Various materials are used in angiography. First, there is India ink, which is helpful in observing the gross anatomy of blood vessels. However, it is radiolucent, so angiographic images cannot be obtained. The more commonly-used radiopaque materials are lead oxide and barium. However, toxicity is a crucial drawback of lead oxide, and it is inconvenient to use when mixed with water because of its powdered form. Barium is free from toxicity; however, it is not very feasible, as it should be used after dilution. Both of these radiopaque materials cannot cross capillaries; therefore, if a whole vascular structure must be analyzed, it is necessary to inject them into the artery and vein separately5. In addition, the two materials cause dye leakage during anatomical dissection, so they should be combined with gelatin. Lead oxide-gelatin and barium-gelatin mixtures take at least one day to solidify1,6,7.
Computed tomography (CT) angiography is another widely-used method and can aid in viewing three-dimensional (3D) structures8. However, veins cannot be visualized effectively5. In this modality, clear visualization of the fine vasculature such as choke veins is difficult, except when using specific equipment. The need for more expensive equipment can be a disadvantage, so CT angiography cannot be utilized in all laboratories. By contrast, the soft tissue X-ray system is relatively cheap and can operate more easily. This system is optimal for viewing soft tissues and can provide higher quality soft tissue images than the simple X-ray system. Although the soft tissue X-ray system itself cannot show 3D images, it can help visualize fine vascular structures more clearly than CT angiography. Therefore, we have used the soft tissue X-ray system in many experiments, particularly in various flap models and basic anatomy2,9.
Finally, the use of silicone rubber injection compound angiography has numerous advantages. Because various color agents are prepared, it can be injected and display distinguishable colors such as India ink. Therefore, simultaneously studying the gross anatomy and angiography is possible. It can both pass through capillaries and allow veins to be visualized, making examinations of fine vascular structures possible. Unlike the gelatin mixture, the silicone rubber injection compound solidifies within a short time period, approximately 15 minutes, without any additional procedures. The entire process is summarized in the schematic image in Figure 1.

<table><tbody><tr><td>MICROFIL Silicone Rubber Injection Compounds</td><td>Flow Tech Inc.</td><td>MV-112</td><td>White color agent</td></tr><tr><td>MICROFIL Silicone Rubber Injection Compounds</td><td>Flow Tech Inc.</td><td>MV-117</td><td>Orange color agent</td></tr><tr><td>MICROFIL Silicone Rubber Injection Compounds</td><td>Flow Tech Inc.</td><td>MV-120</td><td>Blue color agent</td></tr><tr><td>MICROFIL Silicone Rubber Injection Compounds</td><td>Flow Tech Inc.</td><td>MV-122</td><td>Yellow color agent</td></tr><tr><td>MICROFIL Silicone Rubber Injection Compounds</td><td>Flow Tech Inc.</td><td>MV-130</td><td>Red color agent</td></tr><tr><td>MICROFIL Silicone Rubber Injection Compounds</td><td>Flow Tech Inc.</td><td>MV-132</td><td>Clear agent</td></tr><tr><td>MICROFIL Silicone Rubber Injection Compounds</td><td>Flow Tech Inc.</td><td>MV-Diluent</td><td>Diluent</td></tr><tr><td>MICROFIL CP-101 For Cast Corrosion Preparations</td><td>Flow Tech Inc.</td><td>CP-101</td><td>Curing agent</td></tr><tr><td>SOFTEX X-ray film photographing inspection equipment</td><td>SOFTEX</td><td>CMB-2</td><td>Soft tissue x-ray system</td></tr><tr><td>Film&amp;nbsp;</td><td>Fujifilm</td><td>Industrial X-ray Film (FR 12x16.5cm)</td><td></td></tr><tr><td>Automatic Development Machine</td><td>Fujifilm</td><td>FPM 2800</td><td></td></tr><tr><td>Rat&amp;nbsp;</td><td></td><td></td><td>Sprague-Dawley rat weighing 200-250 g</td></tr><tr><td>Three-way stopcock</td><td></td><td></td><td></td></tr><tr><td>24-guage catheter</td><td></td><td></td><td></td></tr><tr><td>Image J</td><td>National Institutes of Health&amp;nbsp;</td><td></td><td>https://imagej.nih.gov/ij/</td></tr></tbody></table>

two-dimensional x-ray angiography to examine fine vascular structure using a silicone rubber injection compound

Angiography is an essential tool for the study of vascular structures in various research fields. The aim of this study is to introduce a simple angiographic method for examining the fine vascular structure of unfixed, fresh tissue using a silicone rubber injection compound and soft tissue X-ray system. This study is especially focused on flap territories used in reconstructive surgery. This study employs angiography with a silicone rubber injection compound in various experimental conditions using Sprague-Dawley rats. First, 15 mL of MV compound and 15 mL of diluent is mixed. Then, 1.5 mL of the curing agent is prepared, and a 24G catheter is cannulated in the common carotid artery of the rat. A three-way stopcock is then connected to a catheter, and the radiopaque agent, after being mixed with the prepared curing agent, is injected immediately without spillage. Finally, as the agent solidifies, the specimen is harvested, and an angiographic image is obtained using a soft tissue X-ray system. This method indicates that high-quality angiography showing fine vascular structures can be easily and simply obtained within in a short period of time.

Through following this protocol, the flap vascularity of the Sprague-Dawley rat was examined. A circumferential skin flap from the lower abdomen to the back that measured 4 x 12 cm was marked based on our previous reports. Each specimen was in a different vascular condition.
All the flaps were elevated based on the deep circumflex iliac artery (DCIA) and vein and then supercharged with arteries from various locations. Group 1 wa...

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Two-Dimensional X-Ray Angiography to Examine Fine Vascular Structure Using a Silicone Rubber Injection Compound

This study presents a simple two-dimensional angiographic method to examine fine vascular structures using a silicone rubber injection compound and soft tissue X-ray system.

Angiography is an essential tool for the study of vascular structures in various research fields. The aim of this study is to introduce a simple ...

two-dimensional-x-ray-angiography-to-examine-fine-vascular-structure

Research

JoVE Journal

Medicine

5.7K Views.  Seoul National University College of Medicine. This study presents a simple two-dimensional angiographic method to examine fine vascular structures using a silicone rubber injection compound and soft tissue X-ray system.

Video: Two-Dimensional X-Ray Angiography to Examine Fine Vascular Structure Using a Silicone Rubber Injection Compound

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 ...

Universal Hand-held Three-dimensional Optoacoustic Imaging Probe for Deep Tissue Human Angiography and Functional Preclinical Studies in Real Time

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously attaining anatomical, functional and molecular contrast in deep optically opaque tissues. While enormous potential has been recently demonstrated in the application of optoacoustics for small animal research, vast efforts have also been undertaken in translating this imaging technology into clinical practice. We present here a newly developed optoacoustic tomography approach capable of delivering high resolution and spectrally enriched volumetric images of tissue morphology and function in real time. A detailed description of the experimental protocol for operating with the imaging system in both hand-held and stationary modes is provided and showcased for different potential scenarios involving functional and molecular studies in murine models and humans. The possibility for real time visualization in three dimensions along with the versatile handheld design of the imaging probe make the newly developed approach unique among the pantheon of imaging modalities used in today&#8217;s preclinical research and clinical practice.

We provide herein a detailed description of the experimental protocol for imaging with a newly developed hand-held optoacoustic (photoacoustic) system for three-dimensional functional and molecular imaging in real time. The demonstrated powerful performance and versatility may define new application areas of the optoacoustic technology in preclinical research and clinical practice.

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously ...

Bioengineering

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 ...

Vascular Casting of Adult and Early Postnatal Mouse Lungs for Micro-CT Imaging

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and behave by observing the surface of a tissue. This method provides a means to visualize the complex 3-dimensional vascular architecture of the lung.
To accomplish this, a catheter is inserted into the pulmonary artery and the vasculature is simultaneously flushed of blood and chemically dilated to limit resistance. Lungs are then inflated through the trachea at a standard pressure and the polymer compound is infused into the vascular bed at a standard flow rate. Once the entire arterial network is filled and allowed to cure, the lung vasculature may be visualized directly or imaged on a micro-CT (&#181;CT) scanner.
When performed successfully, one can appreciate the pulmonary arterial network in mice ranging from early postnatal ages to adults. Additionally, while demonstrated in the pulmonary arterial bed, this method can be applied to any vascular bed with optimized catheter placement and endpoints.

The aim of this technique is ex vivo visualization of pulmonary arterial networks of early postnatal and adult mice through lung inflation and injection of a radio-opaque polymer-based compound via the pulmonary artery. Potential applications for casted tissues are also discussed.

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and ...

Developmental Biology

3D Imaging of Soft-Tissue Samples using an X-ray Specific Staining Method and Nanoscopic Computed Tomography

We demonstrate a laboratory-based method combining X-ray microCT and nanoCT with a specific X-ray stain, which targets the cell cytoplasm. The described protocol is easy to apply, fast and suitable for larger soft-tissue samples. The presented methodology enables the characterization of crucial tissue structures in three dimensions and is demonstrated on a whole mouse kidney. The multiscale approach allows to image the entire mouse kidney and supports the selection of further volumes of interest, which are acquired with higher resolutions ranging into the nanometer range. Thereby, soft-tissue morphology with a similar detail level as the corresponding histological light microscopy images is reproduced. Deeper insights into the 3D configuration of tissue structures are achieved without impeding further investigations through histological methods.

A protocol for 3D visualization of microscopic tissue structures by using an X-ray specific staining method designed for X-ray computed tomography is presented.

We demonstrate a laboratory-based method combining X-ray microCT and nanoCT with a specific X-ray stain, which targets the cell cytoplasm. The described ...

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being used to evaluate the structure and function of plant xylem network in three dimensions (3D) (e.g. Brodersen et al. 2010; 2011; 2012a,b). HRCT imaging is based on the same principles as medical CT systems, but a high intensity synchrotron x-ray source results in higher spatial resolution and decreased image acquisition time. Here, we demonstrate in detail how synchrotron-based HRCT (performed at the Advanced Light Source-LBNL Berkeley, CA, USA) in combination with Avizo software (VSG Inc., Burlington, MA, USA) is being used to explore plant xylem in excised tissue and living plants. This new imaging tool allows users to move beyond traditional static, 2D light or electron micrographs and study samples using virtual serial sections in any plane. An infinite number of slices in any orientation can be made on the same sample, a feature that is physically impossible using traditional microscopy methods.
Results demonstrate that HRCT can be applied to both herbaceous and woody plant species, and a range of plant organs (i.e. leaves, petioles, stems, trunks, roots). Figures presented here help demonstrate both a range of representative plant vascular anatomy and the type of detail extracted from HRCT datasets, including scans for coast redwood (Sequoia sempervirens), walnut (Juglans spp.), oak (Quercus spp.), and maple (Acer spp.) tree saplings to sunflowers (Helianthus annuus), grapevines (Vitis spp.), and ferns (Pteridium aquilinum and Woodwardia fimbriata). Excised and dried samples from woody species are easiest to scan and typically yield the best images. However, recent improvements (i.e. more rapid scans and sample stabilization) have made it possible to use this visualization technique on green tissues (e.g. petioles) and in living plants. On occasion some shrinkage of hydrated green plant tissues will cause images to blur and methods to avoid these issues are described. These recent advances with HRCT provide promising new insights into plant vascular function.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique that can be used to study the structure and function of plant vasculature in 3D. We demonstrate how HRCT facilitates exploration of xylem networks across a wide range of plant tissues and species.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being ...

Biology

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 ...

Time-Resolved, Dynamic Computed Tomography Angiography for Characterization of Aortic Endoleaks and Treatment Guidance via 2D-3D Fusion-Imaging

In the United States, more than 80% of all abdominal aortic aneurysms are treated by endovascular aortic aneurysm repair (EVAR). The endovascular approach warrants good early results, but adequate follow-up imaging after EVAR is imperative to maintain long-term positive outcomes. Potential graft-related complications are graft migration, infection, fraction, and endoleaks, with the last one being the most common. The most frequently used imaging after EVAR is computed tomography angiography (CTA) and duplex ultrasound. Dynamic, time-resolved computed tomography angiography (d-CTA) is a reasonably new technique to characterize the endoleaks. Multiple scans are done sequentially around the endograft during acquisition that grants good visualization of the contrast passage and graft-related complications. This high diagnostic accuracy of d-CTA can be implemented into therapy via image fusion and reduce additional radiation and contrast material exposure.
This protocol describes the technical aspects of this modality: patient selection, preliminary image review, d-CTA scan acquisition, image processing, qualitative and quantitative endoleak characterization. The steps of integrating dynamic CTA into intra-operative fluoroscopy using 2D-3D fusion-imaging to facilitate targeted embolization are also demonstrated. In conclusion, time-resolved, dynamic CTA is an ideal modality for endoleak characterization with additional quantitative analysis. It can reduce radiation and iodinated contrast material exposure during endoleak treatment by guiding interventions.

Time-Resolved, Dynamic Computed Tomography Angiography for Characterization of Aortic Endoleaks and Treatment Guidance via 2D-3D Fusion-Imaging

Dynamic computed tomography angiography (CTA) imaging provides additional diagnostic value in characterizing aortic endoleaks. This protocol describes a qualitative and quantitative approach using time-attenuation curve analysis to characterize endoleaks. The technique of integrating dynamic CTA imaging with fluoroscopy using 2D-3D image fusion is illustrated for better image guidance during treatment.

In the United States, more than 80% of all abdominal aortic aneurysms are treated by endovascular aortic aneurysm repair (EVAR). The endovascular approach ...

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.

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 ...

Two-Dimensional X-Ray Angiography to Examine Fine Vascular Structure Using a Silicone Rubber Injection Compound

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Chapters in this video

Contrast Enhanced Vessel Imaging using MicroCT

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

Universal Hand-held Three-dimensional Optoacoustic Imaging Probe for Deep Tissue Human Angiography and Functional Preclinical Studies in Real Time

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

Vascular Casting of Adult and Early Postnatal Mouse Lungs for Micro-CT Imaging

3D Imaging of Soft-Tissue Samples using an X-ray Specific Staining Method and Nanoscopic Computed Tomography

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of β-aminopropionitrile-induced Aortic Aneurysm and Dissection

Time-Resolved, Dynamic Computed Tomography Angiography for Characterization of Aortic Endoleaks and Treatment Guidance via 2D-3D Fusion-Imaging

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