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 applications6,7,11. For example, when supercharging or superdrainage is applied to a long flap, which can resolve distal ischemic necrosis, predicting the outcome in the flap territory is possible; hence, it is possible to increase the survival of flaps by applying these techniques to clinical practice (Figure 2). In the case of cadaver dissection, gross anatomic and angiographic evaluations can be performed simultaneously, maximizing the cadaver&#39;s use12.
The silicone rubber injection compound is easier to handle than other radiopaque materials. Mixing the silicone rubber injection compound and diluent beforehand and adding the curing agent directly before injection are the primary processes involved. As with other imaging methods, care should be taken to avoid spillage during injection. To avoid spillage, we recommend using a secure tie and a three-way stopcock with a catheter after cannulation. If there is spillage, the specimen may become contaminated with the radiopaque agent, lowering its quality as an angiographic image. As discussed, the silicone rubber injection compound is advantageous in that it solidifies quickly after mixing it in, after about 15 min. However, the experiment is typically finished within this time. Therefore, we recommend mixing in only the amount needed for one rat at a time, even when performing multiple experiments. Performance of X-ray imaging after compound solidification should be done before the denaturation of the fresh tissue.
The molecular size of the silicone rubber injection compound is small enough to pass through the capillaries; therefore, there is no need to inject the agent into the vein separately, which is another advantage. On the contrary, the lead oxide-gelatin mixture or barium-gelatin mixture must be injected into veins separately for venous visualization. However, they cannot pass through valves in the veins, so it is necessary to perform serial cannulations and injections. The physiologic antegrade flow from artery to vein can be observed when using silicone rubber injection compounds, yet this characteristic is not observed when using other contrast agents.
Both arteries and veins can be contrasted with the silicone rubber injection compound and can be distinguished according to the continuity of flow and diameter (Figure 3). In general, veins show a relatively larger diameter.
One of the disadvantages of performing a 3D analysis using the soft tissue X-ray system is that it is more difficult than using CT angiography8. Although stereoscopic radiography can be achieved using the soft tissue X-ray system, it does not produce the high-quality 3D images seen in CT angiography13. However, it is still superior for visualizing fine vascular structures. This is particularly noticeable when using the soft tissue X-ray system rather than the plain X-ray system (Figure 3). At present, real-time CT angiography is used to observe 3D changes with time flow14,15. 3D real-time images cannot be achieved using only the soft tissue X-ray system, but real-time angiography can be achieved through serial imaging using the mobile C-arm system during the injection. Although its image quality is weaker than that of the soft tissue X-ray system for visualizing fine vascular structures, it can enable evaluation of dynamic changes in real time. We actively recommend this method to researchers who cannot utilize micro-CT or wish to conduct basic research on fine vascular structures such as small veins that are not effectively visualized by micro-CT.

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 border="0" cellpadding="0" cellspacing="0" style="width:877px;" width="875">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:339px;">MICROFIL Silicone Rubber Injection Compounds</td>
			<td style="width:151px;">Flow Tech Inc.</td>
			<td style="width:115px;">MV-112</td>
			<td style="width:273px;">White color agent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:339px;">MICROFIL Silicone Rubber Injection Compounds</td>
			<td style="width:151px;">Flow Tech Inc.</td>
			<td style="width:115px;">MV-117</td>
			<td style="width:273px;">Orange color agent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:339px;">MICROFIL Silicone Rubber Injection Compounds</td>
			<td style="width:151px;">Flow Tech Inc.</td>
			<td style="width:115px;">MV-120</td>
			<td style="width:273px;">Blue color agent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:339px;">MICROFIL Silicone Rubber Injection Compounds</td>
			<td style="width:151px;">Flow Tech Inc.</td>
			<td style="width:115px;">MV-122</td>
			<td style="width:273px;">Yellow color agent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:339px;">MICROFIL Silicone Rubber Injection Compounds</td>
			<td style="width:151px;">Flow Tech Inc.</td>
			<td style="width:115px;">MV-130</td>
			<td style="width:273px;">Red color agent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:339px;">MICROFIL Silicone Rubber Injection Compounds</td>
			<td style="width:151px;">Flow Tech Inc.</td>
			<td style="width:115px;">MV-132</td>
			<td style="width:273px;">Clear agent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:339px;">MICROFIL Silicone Rubber Injection Compounds</td>
			<td style="width:151px;">Flow Tech Inc.</td>
			<td style="width:115px;">MV-Diluent</td>
			<td style="width:273px;">Diluent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:339px;">MICROFIL CP-101 For Cast Corrosion Preparations</td>
			<td style="width:151px;">Flow Tech Inc.</td>
			<td style="width:115px;">CP-101</td>
			<td style="width:273px;">Curing agent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:339px;">SOFTEX X-ray film photographing inspection equipment</td>
			<td style="width:151px;">SOFTEX</td>
			<td style="width:115px;">CMB-2</td>
			<td style="width:273px;">Soft tissue x-ray system</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:339px;">Film&#160;</td>
			<td style="width:151px;">Fujifilm</td>
			<td style="width:115px;">Industrial X-ray Film (FR 12x16.5cm)</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:339px;">Automatic Development Machine</td>
			<td style="width:151px;">Fujifilm</td>
			<td style="width:115px;">FPM 2800</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:339px;">Rat&#160;</td>
			<td style="width:151px;"></td>
			<td style="width:115px;"></td>
			<td style="width:273px;">Sprague-Dawley rat weighing 200-250 g</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:339px;">Three-way stopcock</td>
			<td style="width:151px;"></td>
			<td style="width:115px;"></td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:339px;">24-guage catheter</td>
			<td style="width:151px;"></td>
			<td style="width:115px;"></td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:339px;">Image J</td>
			<td style="width:151px;">National Institutes of Health&#160;</td>
			<td style="width:115px;"></td>
			<td style="width:273px;">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 was the control, group 2 was supercharged with the ipsilateral superficial inferior epigastric artery (SIEA), group 3 was supercharged with the contralateral SIEA, and group 4 was supercharged with the contralateral DCIA. As a result, angiographs of each flap showed different patterns. If the entire flap was divided into four zones as the standard with the main vessel charging the flap territory, the angiographic agent has reached the main vessel that charges the next distal zone beyond the supercharging artery. The dilated choke vein was also observed, but not seen in normal skin angiography (Figure 2).
In other instances, silicone rubber injection angiography clearly showed fine vascular structures. Here, the method is advantageous because the rat vein is so thin and not easily visualized with angiography. High-quality angiographic images displayed dilated choke veins even in specific conditions looking at arterial supply and venous drainage from different sources (e.g., an artery such as the ipsilateral DCIA or vein such as the contralateral superficial inferior epigastric vein) (Figure 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57732/57732fig1.jpg" /> 
Figure 1: Schema of the entire procedure. This panel shows the preparation of the desired flap condition beforehand and the performance of angiography 4 - 5 days later, using the following process: angiographic agent preparation, injection, specimen harvesting, and image capturing. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57732/57732fig1large.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/57732/57732fig2.jpg" /> 
Figure 2: Typical angiography of flaps on postoperative day 4 in the rat skin flap. The normal skin represents the original four vascular territory flaps in the lower trunk area. The flap includes four vascular territories, including the bilateral deep circumflex iliac (DCI) and superficial inferior epigastric (SIE) vessel territories (yellow square). All groups have a common vascular pedicle, the deep circumflex iliac artery and vein (DCIA&#38;V). Additionally, each flap has a different supercharging artery. Group 2 is supercharged with the ipsilateral superficial inferior epigastric artery (SIEA), group 3 is supercharged with the contralateral SIEA, and group 4 is supercharged with the contralateral DCIA. Group 4 shows different survived flap areas and angiographic patterns. Because the supercharging artery is distal to the original vascular pedicle (DIEA&#38;V), the more distally located major vessel is contrasted by the agent (yellow arrow). Finally, the dilated choke veins that connect the vascular territories are visible (white arrowhead). i = ipsilateral; c = contralateral. Scale bar = 1 cm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57732/57732fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57732/57732fig3.jpg" /> 
Figure 3: Other representative results. The flap shown is supplied by the ipsilateral deep circumflex iliac artery (DCIA) and drained by the contralateral superficial inferior epigastric vein (SIEV). (Upper left) This panel shows the gross photography of the harvested flap. The ipsilateral side shows congestive change, but flap necrosis does not occur. The mid-portion and distal two-thirds of the flap show a complete survival. (Lower left) Angiography shows detailed fine vascular structure including the dilated choke vein, which connects the adjacent vascular territory. (Right) Magnified angiography distinguishes arteries (red arrowheads) and veins (blue arrowheads) by the continuity and diameter of vessels. In general, veins show larger diameters than arteries. DCIA = the deep circumflex iliac artery; SIEV = the superficial inferior epigastric vein; the white arrowhead = the dilated choke vein. Scale bar = 1 cm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57732/57732fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Two-Dimensional X-Ray Angiography to Examine Fine Vascular Structure Using a Silicone Rubber Injection Compound at JoVE.com

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

Nanyang Technological University

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

Guide Wire Assisted Catheterization and Colored Dye Injection for Vascular Mapping of Monochorionic Twin Placentas

Monochorionic (MC) twin pregnancies are associated with significantly higher morbidity and mortality rates than dichorionic twins. Approximately 50% of MC twin pregnancies develop complications arising from the shared placenta and associated vascular connections1. Severe twin-to-twin syndrome (TTTS) is reported to account for approximately 20% of these complications2,3. Inter-twin vascular connections occur in almost all MC placentas and are related to the prognosis and outcome of these high-risk twin pregnancies. The number, size and type of connections have been implicated in the development of TTTS and other MC twin conditions. Three types of inter-twin vascular connections occur: 1) artery to vein connections (AVs) in which a branch artery carrying deoxygenated blood from one twin courses along the fetal surface of the placenta and dives into a placental cotyledon. Blood flows via a deep intraparenchymal capillary network into a draining vein that emerges at the fetal surface of the placenta and brings oxygenated blood toward the other twin. There is unidirectional flow from the twin supplying the afferent artery toward the twin receiving the efferent vein; 2) artery to artery connections (AAs) in which a branch artery from each twin meets directly on the superficial placental surface resulting in a vessel with pulsatile bidirectional flow, and 3) vein to vein connections (VVs) in which a branch vein from each twin meets directly on the superficial placental surface allowing low pressure bidirectional flow. In utero obstetric sonography with targeted Doppler interrogation has been used to identify the presence of AV and AA connections4. Prenatally detected AAs that have been confirmed by postnatal placental injection studies have been shown to be associated with an improved prognosis for both twins5. Furthermore, fetoscopic laser ablation of inter-twin vascular connections on the fetal surface of the shared placenta is now the preferred treatment for early, severe TTTS. 
Postnatal placental injection studies provide a valuable method to confirm the accuracy of prenatal Doppler ultrasound findings and the efficacy of fetal laser therapy6. Using colored dyes separately hand-injected into the arterial and venous circulations of each twin, the technique highlights and delineates AVs, AAs, and VVs. This definitive demonstration of MC placental vascular anatomy may then be correlated with Doppler ultrasound findings and neonatal outcome to enhance our understanding of the pathophysiology of MC twinning and its sequelae. Here we demonstrate our placental injection technique.

Vascular mapping of monochorionic (MC) twin placentas after birth provides a means for detailed demonstration of vascular connections between the twins&#x2019; circulations. 
Imbalance of these connections is thought to play a pivotal role in the development of complications of MC twinning including twin-to-twin transfusion syndrome.

Monochorionic (MC) twin pregnancies are associated with significantly higher morbidity and mortality rates than dichorionic twins. Approximately 50% of MC ...

X-ray Dose Reduction through Adaptive Exposure in Fluoroscopic Imaging

X-ray fluoroscopy is widely used for image guidance during cardiac intervention. However, radiation dose in these procedures can be high, and this is a significant concern, particularly in pediatric applications. Pediatrics procedures are in general much more complex than those performed on adults and thus are on average four to eight times longer1. Furthermore, children can undergo up to 10 fluoroscopic procedures by the age of 10, and have been shown to have a three-fold higher risk of developing fatal cancer throughout their life than the general population2,3.
We have shown that radiation dose can be significantly reduced in adult cardiac procedures by using our scanning beam digital x-ray (SBDX) system4-- a fluoroscopic imaging system that employs an inverse imaging geometry5,6 (Figure 1, Movie 1 and Figure 2). Instead of a single focal spot and an extended detector as used in conventional systems, our approach utilizes an extended X-ray source with multiple focal spots focused on a small detector. Our X-ray source consists of a scanning electron beam sequentially illuminating up to 9,000 focal spot positions. Each focal spot projects a small portion of the imaging volume onto the detector. In contrast to a conventional system where the final image is directly projected onto the detector, the SBDX uses a dedicated algorithm to reconstruct the final image from the 9,000 detector images.
For pediatric applications, dose savings with the SBDX system are expected to be smaller than in adult procedures. However, the SBDX system allows for additional dose savings by implementing an electronic adaptive exposure technique. Key to this method is the multi-beam scanning technique of the SBDX system: rather than exposing every part of the image with the same radiation dose, we can dynamically vary the exposure depending on the opacity of the region exposed. Therefore, we can significantly reduce exposure in radiolucent areas and maintain exposure in more opaque regions. In our current implementation, the adaptive exposure requires user interaction (Figure 3). However, in the future, the adaptive exposure will be real time and fully automatic.
We have performed experiments with an anthropomorphic phantom and compared measured radiation dose with and without adaptive exposure using a dose area product (DAP) meter. In the experiment presented here, we find a dose reduction of 30%.

We are developing a dynamic adaptive exposure technique using our scanning beam digital X-ray system. Rather than exposing an object uniformly, the exposure is adapted depending on the opacity of the object. Here we show an experiment on an anthropomorphic phantom that resulted in a dose saving of 30%.

X-ray fluoroscopy is widely used for image guidance during cardiac intervention. However, radiation dose in these procedures can be high, and this is a ...

Segmentation and Measurement of Fat Volumes in Murine Obesity Models Using X-ray Computed Tomography

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are associated with obesity, though the precise underlying mechanisms that contribute to the disease are currently being delineated.2,3 Several small animal models of obesity have been developed and are employed in a variety of studies.4 A critical component to these experiments involves the collection of regional and/or total animal fat content data under varied conditions.
Traditional experimental methods available for measuring fat content in small animal models of obesity include invasive (e.g. ex vivo measurement of fat deposits) and non-invasive (e.g. Dual Energy X-ray Absorptiometry (DEXA), or Magnetic Resonance (MR)) protocols, each of which presents relative trade-offs. Current invasive methods for measuring fat content may provide details for organ and region specific fat distribution, but sacrificing the subjects will preclude longitudinal assessments. Conversely, current non-invasive strategies provide limited details for organ and region specific fat distribution, but enable valuable longitudinal assessment. With the advent of dedicated small animal X-ray computed tomography (CT) systems and customized analytical procedures, both organ and region specific analysis of fat distribution and longitudinal profiling may be possible. Recent reports have validated the use of CT for in vivo longitudinal imaging of adiposity in living mice.5,6 Here we provide a modified method that allows for fat/total volume measurement, analysis and visualization utilizing the Carestream Molecular Imaging Albira CT system in conjunction with PMOD and Volview software packages.

Fat content analysis is routinely conducted in studies utilizing murine obesity models. Emerging methods in small animal CT imaging and analysis are providing for longitudinal detail rich fat content analysis. Here we detail step by step procedures for performing small animal CT imaging, analysis, and visualization.

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are ...

Intranasal Administration of CNS Therapeutics to Awake Mice

Intranasal administration is a method of delivering therapeutic agents to the central nervous system (CNS). It is non-invasive and allows large molecules that do not cross the blood-brain barrier access to the CNS. Drugs are directly targeted to the CNS with intranasal delivery, reducing systemic exposure and thus unwanted systemic side effects1. Delivery from the nose to the CNS occurs within minutes along both the olfactory and trigeminal neural pathways via an extracellular route and does not require drug to bind to any receptor or axonal transport2. Intranasal delivery is a widely publicized method and is currently being used in human clinical trials3.
Intranasal delivery of drugs in animal models allows for initial evaluation of pharmacokinetic distribution and efficacy. With mice, it is possible to administer drugs to awake (non-anesthetized) animals on a regular basis using a specialized intranasal grip. Awake delivery is beneficial because it allows for long-term chronic dosing without anesthesia, it takes less time than with anesthesia, and can be learned and done by many people so that teams of technicians can dose large numbers of mice in short periods. Efficacy of therapeutics administered intranasally in this way to mice has been demonstrated in a number of studies including insulin in diabetic mouse models 4-6 and deferoxamine in Alzheimer's mouse models. 7,8
The intranasal grip for mice can be learned, but is not easy and requires practice, skill, and a precise grip to effectively deliver drug to the brain and avoid drainage to the lung and stomach. Mice are restrained by hand using a modified scruff in the non-dominant hand with the neck held parallel to the floor, while drug is delivered with a pipettor using the dominant hand. It usually takes 3-4 weeks of acclimating to handling before mice can be held with this grip without a stress response. We have prepared this JoVE video to make this intranasal delivery technique more accessible.

A method to intranasally administer drugs to awake mice for the purpose of targeting the brain is described. This method allows for repeat dosing over long periods using intranasal administration of drug without anesthesia, and nose-to-brain delivery with minimal systemic exposure.

Intranasal administration is a method of delivering  therapeutic agents to the central nervous system (CNS). It is  non-invasive and allows large ...

Dual-phase Cone-beam Computed Tomography to See, Reach, and Treat Hepatocellular Carcinoma during Drug-eluting Beads Transarterial Chemo-embolization

The advent of cone-beam computed tomography (CBCT) in the angiography suite has been revolutionary in interventional radiology. CBCT offers 3 dimensional&#160;(3D) diagnostic imaging in the interventional suite and can enhance minimally-invasive therapy beyond the limitations of 2D angiography alone. The role of CBCT has been recognized in transarterial chemo-embolization (TACE) treatment of hepatocellular carcinoma (HCC). The recent introduction of a CBCT technique: dual-phase CBCT (DP-CBCT) improves intra-arterial HCC treatment with drug-eluting beads (DEB-TACE). DP-CBCT can be used to localize liver tumors with the diagnostic accuracy of multi-phasic multidetector computed tomography (M-MDCT) and contrast enhanced magnetic resonance imaging (CE-MRI) (See the tumor), to guide intra-arterially guidewire and microcatheter to the desired location for selective therapy (Reach the tumor), and to evaluate treatment success during the procedure (Treat the tumor). The purpose of this manuscript is to illustrate how DP-CBCT is used in DEB-TACE to see, reach, and treat HCC.

Dual-phase cone-beam computed tomography (DP-CBCT) is a useful intraprocedural imaging technique for transarterial chemo-embolization treatment with drug-eluting beads of hepatocellular carcinoma. DP-CBCT has been used to perform three major steps in oncologic interventional radiology: tumor localization (see), navigation and intraprocedural catheter guidance (reach), and intraprocedural evaluation of treatment success (treat).

The advent of cone-beam computed tomography (CBCT) in the angiography suite has been revolutionary in interventional radiology. CBCT offers 3 ...

Real-time X-ray Imaging of Lung Fluid Volumes in Neonatal Mouse Lung

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting and sustaining this phenotype is critically important in normal alveolar growth and gas exchange throughout life. Several in vitro studies have characterized the role of key regulatory proteins, signaling molecules, and steroid hormones that can influence the rate of lung fluid clearance. However, in vivo examinations must be performed to evaluate whether these regulatory factors play important physiological roles in regulating perinatal lung liquid absorption. As such, the utilization of real time X-ray imaging to determine perinatal lung fluid clearance, or pulmonary edema, represents a technological advancement in the field. Herein, we explain and illustrate an approach to assess the rate of alveolar lung fluid clearance and alveolar flooding in C57BL/6 mice at post natal day 10 using X-ray imaging and analysis. Successful implementation of this protocol requires prior approval from institutional animal care and use committees (IACUC), an in vivo small animal X-ray imaging system, and compatible molecular imaging software.

We present a protocol to assess the rate of alveolar fluid clearance or pulmonary edema in neonatal mouse lung using X-ray imaging technology.

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting ...

Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The simple murine AVF model was combined with intraluminal administration of drug solution to the venous endothelium at the same time as fistula creation. Technical aspects of this model are discussed. Under general anesthesia, an abdominal incision is made and the aorta and inferior vena cava (IVC) are exposed. The infra-renal aorta and IVC are dissected for clamping. After proximal and distal clamping, the puncture site is exposed and a 25 G needle is used to puncture both walls of the aorta and into the IVC. Immediately after the puncture, a reporter gene-expressing viral vector was infused in the IVC via the same needle, followed by 15 min of incubation. The intraluminal administration method enabled more robust viral gene delivery to the venous endothelium compared to administration by the external route. This novel method of delivery will facilitate studies that explore the role of the endothelium in AVF maturation and enable intraluminal drug delivery at the time of surgical operation.

After puncturing the aorta through the inferior vena cava (IVC) to create an aorto-caval fistula in the mouse, solution containing a drug is infused into the IVC via the same needle, followed by incubation. This method enables more robust drug delivery to the venous endothelium compared to the external route.

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The ...

Using the Sleeve Technique in a Mouse Model of Aortic Transplantation - An Instructional Video

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the aorta in mice using the sleeve method was short and easy averaging 20 min, permitting studies of iso/allo grafts. The following article describes the aortic transplantation procedure used in our laboratory. The mice were anesthetized with a mixture of 1.5% volume isoflurane and 100% oxygen through a face mask. At this point, the segment of the aorta between the renal arteries and its bifurcation was separated from the vena cava, freely prepared and clampedat the proximal and distal segments with a single silk suture. Prior to the removal of the aorta, a saline solution containing heparin was injected into the inferior vena cava. Then the aorta was cut between the clamps and a saline heparin solution was used to flush the lumen. The sleeve technique with monofilament sutures was used in order to transplant the abdominal aorta in the orthotopic position.

We present an orthotopic aortic transplantation model using the sleeve technique in mice. It is a very rapid anastomosis method, which can be employed in studies of vascular disease.

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the ...

Ultrasound-guided Intracardiac Injection of Human Mesenchymal Stem Cells to Increase Homing to the Intestine for Use in Murine Models of Experimental Inflammatory Bowel Diseases

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have immunosuppressive potential and have been shown to suppress inflammation in mouse models of intestinal inflammation, even though the route of administration can limit their homing and effectiveness 1,3,4,5. Local application of MSCs to colonic injury models has shown greater efficacy at ameliorating inflammation in the colon. However, there is paucity of data on techniques to enhance the localization of human bone marrow-derived MSCs (hMSCs) to the small intestine, the site of inflammation in the SAMP-1/YitFc (SAMP) model of experimental Crohn&#39;s disease. This work describes a novel technique for the ultrasound-guided intracardiac injection of hMSCs in SAMP mice, a well-characterized spontaneous model of chronic intestinal inflammation. Sex- and age-matched, inflammation-free AKR/J (AKR) mice were used as controls. To analyze the biodistribution and the localization, hMSCs were transduced with a lentivirus containing a triple reporter. The triple reporter consisted of firefly luciferase (fl), for bioluminescent imaging; monomeric red fluorescent protein (mrfp), for cell sorting; and truncated herpes simplex virus thymidine kinase (ttk), for positron emission tomography (PET) imaging. The results of this study show that 24 h after the intracardiac administration, hMSCs localize in the small intestine of SAMP mice as opposed to inflammation-free AKR mice. This novel, ultrasound-guided injection of hMSCs in the left ventricle of SAMP mice ensures a high success rate of cell delivery, allowing for the rapid recovery of mice with minimal morbidity and mortality. This technique could be a useful method for the enhanced localization of MSCs in other models of small-intestinal inflammation, such as TNF&#916;RE6. Future studies will determine if the increased localization of hMSCs by intra-arterial delivery can lead to increased therapeutic efficacy.

Murine studies in models of colonic inflammation have demonstrated that a small percentage (1 - 5%) of mesenchymal stem cells (MSC) injected intravenously or intraperitoneally home to the inflamed colon1,2. This study shows that ultrasound-guided intracardiac injections of MSCs result in increased localization to the intestine.

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have ...

Improved Registration of 3D CT Angiography with X-ray Fluoroscopy for Image Fusion During Transcatheter Aortic Valve Implantation

The fusion of 3D anatomical models derived from high-fidelity pre-interventional computed tomography angiography (CTA), and x-ray (XR) fluoroscopy to facilitate anatomical guidance is of huge interest for complex cardiac interventions like TAVI procedures with cerebral protection. Co-registration of CTA and XR has been introduced either based on additional intraoperative non-/contrast-enhanced cone-beam computed tomography (CBCT) or two separate aortograms. With the related increase of radiation exposure and/or contrast agent (CA) dose, a potential additional risk for the patient is introduced. Here, we propose a modified co-registration approach making use of arteriograms of the iliofemoral arteries, routinely performed during the femoral puncture and sheath introduction. On-the-fly refinement of the co-registration during the on-going procedure enables accurate co-registration without any additional angiograms, thus reducing CA, XR dose and procedure time, while simultaneously improving operator confidence and procedure safety.

The aim of this study was to improve the co-registration for image fusion (IF) of pre-interventional CT data with real-time x-ray (XR) fluoroscopy during transfemoral transcatheter aortic valve implantation (TAVI).

The fusion of 3D anatomical models derived from high-fidelity pre-interventional computed tomography angiography (CTA), and x-ray (XR) fluoroscopy to ...