Name: two-dimensional x-ray angiography to examine fine vascular structure using a silicone rubber injection compound
Uploaded: 2019-01-07T14:00:00
Description: 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

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 estimation...

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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.

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	<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>
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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...

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 ...

This method can help answer key questions in the reconstructive surgery field about how to perform the perforative lift surgery. Main advantage of this technique is that the variation of fine vascular structures, such as choke vessels, can be performed easily under various vascular flap conditions. To establish a flap condition, confirm the appropriate level of sedation by a lack of response to toe pinch in an anesthetized 200 to 250 gram 7 week old male Sprague-Dawley rat and mark a 4 by 12 centimeter circumferential skin flap design from the lower abdomen to the back.With the center of the flap halfway between the xiphoid process and the penis. Use a surgical blade to make the incision as marked and use scissors to dissect the flap, including the skin and panniculus carnosus. Dissect around the vascular pedicle at the lower abdomen and use a microscope or surgical loop to expose the vascular tissue.Ligate the vessels and divide the flap along the dorsal midline. The return the flap to its original position and secure the flap with the skin stapler. It is important to make flaps that have obvious vascular territories to allow changes to be easily made to the vascular condition of the flap as required for doing experimental protocol.On the day of the procedure, make a 2 centimeter midline skin incision between the scapulae and use mosquito forceps and blunt scissors to expose the salivary gland complex. After longitudinal dissection of the omohyoid muscle, dissect around the common carotid artery and use black silk to retract the cephalic and caudal sides of the artery. Make a tie on the proximal suture and keep traction to maintain engorgement of the artery and loosely place the caudal suture.The insert a 24 gauge catheter into the prepared artery and secure the catheter with the caudal suture. Connect a three way stopcock to the catheter and use an empty syringe to apply negative pressure to the catheter confirming a back flow of blood into the catheter. Next, inject 25 to 30 milliliters of freshly prepared silicone rubber injection compound into the catheter until the colors of the eye and foot have changed.When all of the compound has been injected, lock the three way stopcock and allow the agent to solidify for about 15 minutes. Then place a protective barrier around the syringe of compound so as not to contaminate the radio opaque agent. And remove the syringe from the stopcock.To harvest the specimen, use a surgical blade to make an incision in the panniculus carnosus one centimeter away from the flap to prevent damage to any vascular structures inside the flap and dissect around the flap below the panniculus carnosus plane. Then use scissors to harvest the tissue including the flap and and vascular pedicle within the flap and use a five o silk suture to ligate the pedicle of the flap, separating the flap from the body. Silicone rubber injection angiography clearly reveals fine vascular structures, allowing the acquisition of high-quality angiographic images of the dilated choke veins even under otherwise difficult imaging conditions.For example, in this representative experiment, it can be observed that all of the flaps were elevated based on the deep circumflex iliac artery and vein before being supercharged with arteries from various locations. If an entire flap is divided into four zones as the standard with the main vessel charging the flap territory, the angiographic agent that reaches the main vessel charges the next distal zone beyond the supercharging artery. The dilated choke vein can also be observed, even though it was not seen in the normal skin angiography.Once mastered, this technique can be completed in one hour per rat if it is performed properly. While attempting this procedure, it is important to remember not to contaminate or spill the angiographic agent during the injection. After watching this video, you should have a good understanding of how to make a specific condition flap and to perform an angiography.

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