Name: use of micro x-ray computed tomography with phosphotungstic acid preparation to visualize human fibromuscular tissue
Uploaded: 2019-09-05T14:30:00
Description: Watch this Scientific Journal Video about Use of Micro X-ray Computed Tomography with Phosphotungstic Acid Preparation to Visualize Human Fibromuscular Tissue at JoVE.com

This study was supported by a faculty research grant of Yonsei University College of Medicine (6-2018-0099). The authors thank the people who very generously donated their bodies to Yonsei University College of Medicine. We are grateful to Jun Ho Kim and Jong Ho Bang for their technical support (staff members in the Surgical Anatomy Education Centre at the Yonsei University College of Medicine). We are also grateful to Genoss Co., Ltd. for the high-quality microCT scanning system used in this research.

All cadavers utilized in this study were legally donated to the Surgical Anatomy Education Centre at Yonsei University College of Medicine.
1. Obtaining samples
<ol>
	<li>Draw an incision line on the cadaver with a colored pencil to indicate the cutting area for sample harvesting. Check that the incision line drawn extends medially to a medial canthus, laterally to a lateral canthus, superiorly to a superior border of the lower eyelid, and inferiorly to 1 cm below the line from the orbital r...

<ol>
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We implemented microCT with PTA preparation in the examination of human soft tissues. Briefly, specimens are harvested and fixed in formalin for a few days, followed by dehydration in serial ethanol solutions. Placing the sample into the PTA solution directly after formalin fixation can result in some tissue cracking due to rapid dehydration. Therefore, serial dehydration is needed before PTA staining. Next, the samples are stained using PTA solution for about a week. MicroCT scanning, 3D reconstruction, and analysis can...

Manual dissection and histological observation are typically used to examine human tissues, such as muscles and connective tissues. However, manual dissection can easily damage delicate structures, and histological observation provides limited information about flat cross-sectional surfaces1,2. Therefore, improved methods are needed to examine tissues more precisely and efficiently.
Conventional computed tomography (CT) is generally used in clinical practice, but it lacks the ability to distinguish small structures2,3. Micro X-ray CT (microCT) is an effective tool for obtaining three-dimensional (3D) information of small structures from specimens, without destroying them. However, microCT has limited applications because only dense tissues can be visualized clearly; it cannot be used to differentiate soft tissues. To overcome this limitation, staining agents can be used. Contrast-enhancing agents, like phosphotungstic acid (PTA), phosphomolybdic acid, and Lugol&#8217;s iodine, improve the soft tissue contrast rate during scanning4,5. Several studies comparing these agents suggest that PTA demonstrates good performance and is easy to handle6,7,8.
The orbicularis retaining ligament (ORL) is a delicate structure around the orbit, which can be easily damaged during conventional observation9. We examined and successfully retrieved 3D information on this structure using microCT with PTA as a contrast agent. This method can be applied to studies on other human tissues, such as the heart and liver, with appropriate modifications10,11,12.

<table>
	<tbody>
		<tr>
			<td>12 Tungsto(&#8549;)phosphoric acid n-hydrate 
			Phosphotungstic acid</td>
			<td>Junsei</td>
			<td>84220-0410</td>
			<td>PTA powder</td>
		</tr>
		<tr>
			<td>CTvox</td>
			<td>Bruker</td>
			<td>ver 2.7</td>
			<td>3D recon software</td>
		</tr>
		<tr>
			<td>Nrecon</td>
			<td>Bruker</td>
			<td>ver 1.7.0.4</td>
			<td>Reconstruction software</td>
		</tr>
		<tr>
			<td>Skyscan</td>
			<td>Bruker</td>
			<td>1173</td>
			<td>MicroCT scanner</td>
		</tr>
		<tr>
			<td>Tconv</td>
			<td>Bruker</td>
			<td>ver 2.0</td>
			<td>File resizing software</td>
		</tr>
	</tbody>
</table>

use of micro x-ray computed tomography with phosphotungstic acid preparation to visualize human fibromuscular tissue

Manual dissection and histological observation are common methods used to investigate human tissues. However, manual dissection can damage delicate structures&#160;while processing and histological observation provide limited information through cross-sectional imaging. Micro X-ray computed tomography (microCT) is an effective tool for obtaining three-dimensional information without damaging specimens. However, it shows limited efficiency in differentiating soft tissue parts. Use of contrast-enhancing agents, like phosphotungstic acid (PTA), can solve this problem by improving soft tissue contrast. We implemented microCT with PTA to investigate the human orbicularis retaining ligament (ORL), which is a delicate structure in the orbit area. In this method, harvested specimens are fixed in formalin, dehydrated in serial ethanol solutions, and stained with a PTA solution. After staining, microCT scanning, 3D reconstruction, and analysis are performed. Skin, ligaments, and muscles can be clearly visualized using this method. The specimen size and duration of staining are essential features of the method. The suitable specimen thickness was about 5&#8211;7 mm, above which the process was slowed, and the optimum duration was 5&#8211;7 days, below which an empty hole in the central area occasionally occurred. To maintain the location and direction of small pieces during cutting, sewing on the same region of each part is recommended. Furthermore, preliminary analyses of the anatomical structure are needed to correctly identify each piece. Parafilm can be used to prevent drying, but care should be taken to prevent specimen distortion. Our multidirectional observation showed that the ORL is composed of a multilayered meshwork of continuous plates, rather than thread-like fibers, as reported previously. These results suggest that microCT scanning with PTA is useful for examining specific compartments within complex structures of human tissue. It may be helpful in the analyses of cancer tissues, nerve tissues, and various organs, like the heart and liver.

The detailed reconstruction of the ORL was achieved by microCT with PTA preparation (Figure 4). The ligamentous fibromuscular structure extending obliquely between the dermis and periosteum was distinctly observed (Figure 4A). In the coronal view (Figure 4B), the amount and complexity of fibers increased laterally. In the horizontal view (Figure 4C), an elaborate meshwork with an arborized formation was...

Watch this Scientific Journal Video about Use of Micro X-ray Computed Tomography with Phosphotungstic Acid Preparation to Visualize Human Fibromuscular Tissue at JoVE.com

Use of Micro X-ray Computed Tomography with Phosphotungstic Acid Preparation to Visualize Human Fibromuscular Tissue

Micro X-ray computed tomography is effective in obtaining three-dimensional information from undamaged human specimens but has limited success in observing soft tissues. The use of phosphotungstic acid contrast agent can resolve this issue. We implemented this contrast agent to examine human delicate fibromuscular tissues (the orbicularis retaining ligament).

Manual dissection and histological observation are common methods used to investigate human tissues. However, manual dissection can damage delicate ...

We implement microCT with phosphotungstic acid to enhance the soft tissue contrast in the non-invasive investigation of the human orbicularis retaining ligament, a delicate structure within the orbit area. This protocol has been optimized to aid researchers in the non-invasive investigation of human soft tissues and is simple to perform with a reasonable amount of expense. To obtain the samples, first use a colored pencil to indicate the cutting area on the skin for the sample harvesting.Confirm that the marked line extends medially to a medial campus, laterally to a lateral campus, superiorly to a superior border of the lower eyelid, and inferiorly to one centimeter below the line from the orbital rim. Next, use a blade to cut the facial tissues along the marked line, making sure that the cut is deep enough for the blade tip to touch the bone. The harvested sample must include the skin, subcutaneous tissue, muscle, fat, and periosteum.Upon collection, immediately fix the sample in 10%formalin for five to seven days at room temperature. At the end of the fixation period, slice the sample in three five to seven-millimeter thick pieces. Use a needle and black thread to sew the superolateral side of each sample such that the direction of the sample can be determined at later time points.Dehydrate the sample in an ascending ethanol concentration series for one day per concentration, leaving the samples in the 70%ethanol solution until they're staining. One week before the microcomputer tomography scanning is scheduled, add 2.1 grams of PTA to 210 milliliters of 70%ethanol and place the solution on a shaker at 55 to 60 rotations per minute. Prepare three 70-milliliter plastic containers for each sliced piece.When the dye has dissolved into the alcohol solution, fill the containers with the solution. Place one specimen into each container of 1%PTA, and shake the samples at 55 to 60 rotations per minute for five to seven days. When the staining is complete, store the samples in fresh 70%ethanol for one week to prepare for the scanning.For microCT scanning, wrap the samples in Parafilm to prevent drying and place a sample onto the scanner tray. Set the scanner's source voltage to 70 kilovolts, the source current to 114 microamps, the aluminum filter to 0.5 millimeters, the image pixel size to 20 squared micrometers, the pixels to 2, 240 by 2, 240, the exposure to 500 milliseconds, and the rotation step to 0.3 degrees. Then, initiate the scanning.When the scanning is complete, open the reconstruction software and select Actions and Open Dataset to launch the scanned files. Select the Settings tab and set the ring artifacts reduction to seven and the beam-hardening correction. To begin the reconstruction, select Start.The final data will be stored in the designated folder. At the end of the reconstruction, open the file resizing software, and select Source data set to launch the reconstructed files. Select jpg on the Destination data set tab and select the 1/2 Resizing option with the Quality option of No interpolation Then adjust the slide bar to 100 in the Image compression tab and start the conversion.For 3D reconstruction, open the 3D volume rendering software, and select Actions and Load Volume Data. To adjust the brightness and contrast level, modify the shape transfer function in the histogram in the Transfer Function Editor tab. Next select Options and Lighting, and select the Shadows and Surface Lighting icons to achieve a realistic modeling tone.Click and drag, and right-click and drag to move and rotate the 3D image to find the best view, scrolling to zoom in or out as desired. Click Shift and drag to slide the place to view the sectional images. Turn on the Light icon and adjust the lighting indication bar to find the best lighting for viewing, then turn off the Light icon and close the Lighting tab.In the Options tab, select Show and Clipping Box to hide the box for the final image. Then, select Actions and Save Image to store the image. This detailed reconstruction of the ORL was achieved by microCT with PTA preparation as demonstrated, allowing visualization of a ligamentous fibromuscular structure extending obliquely between the dermis and the periosteum.In the coronal view, the amount and complexity of the fibers increases laterally. In horizontal view, an elaborate meshwork with an arborized formation is observed. In the sagittal view, the thicknesses of the ORL fibers decreases inferiorly.Overall, this multi-directional observation proves that the ORL is made up of a multilayered meshwork of continuous plates with variations in the number and thickness of the fibers depending on their location. When the duration of the staining is insufficient compared to the volume of a specimen, the final image may include an empty hole in the central area of the specimen. Other agents beside PTA can be used and different agents have different merits or staining features interests.Therefore, it is useful to optimize the staining region according to your experiment.

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