This work was supported by R01 HL163065 and W81XWH1810518. We extend our appreciation to the dedicated staff at the Animal Research Core. We also wish to express our gratitude to Carmen Arsuaga for her invaluable expertise and vigilant care throughout the study.

The study protocol was approved by the Institutional Animal Care and Use Committee of Nationwide Children&#39;s Hospital Abigail Wexner Research Institute (AR22-0004). All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health.
1. Animal preparation
<ol>
	<li>Have a veterinary team evaluate the sheep 1 week prior to catheterization, including a physical exam and analysis of vital signs, to ensure the animal can safely receive anesthesia.</li>
	<li>Fast the animal overnight, or up to 12 h prior to the procedure, to limit the risk of aspiration of stomach contents upon induction of anesthesia.</li>
	<li>Press the on button on the control panel to turn on the C-arm and 3D angiography system (Figure 1A). Wait until the system is fully loaded. 
	NOTE: Ensure the fluoroscopy is paused until ready to image and all personnel are wearing protective lead.</li>
	<li>Prep heparinized saline to use in the procedure by adding 1 mL of heparin (1,000 USP units/mL concentration) to 1,000 mL of 0.9% saline.</li>
	<li>Shave the left side of the neck and scrub with alcohol. Administer sedation by injecting a combination of ketamine (4 mg/kg), butorphanol (0.1 mg/kg), and diazepam (0.5 mg/kg) into the left jugular vein.</li>
	<li>Place the sedated sheep on a hospital bed and position it in sternal recumbency for intubation. Intubate with a 9-14 mm Endotracheal (ET) tube, based on the sheep&#39;s size, by depressing the tongue and epiglottis with a laryngoscope and inserting the ET tube into the trachea.</li>
	<li>Position the sheep in a right lateral position. Attach the ET tube to a ventilator and mechanically ventilate with 100% Oxygen at 1-3 L/min.</li>
	<li>Maintain anesthesia with 1-3% inhaled isoflurane. Set the respiratory rate at 15-30 breaths/min and end tidal volume at 8-10 mL/kg.</li>
	<li>Place standard monitoring equipment, including a blood pressure cuff on the right front leg, an ear clip to monitor oxygen saturation on the right ear, a temperature probe into the esophagus, and an end tidal CO2 monitor on the ET tube. Shave the wool from the caudal aspect of each hoof between the dewclaws and heel. Place electrocardiogram (ECG) nodes and secure the ECG nodes with tape.</li>
	<li>Lubricate both eyes by applying ophthalmic ointment and insert an orogastric tube to ensure gas and food evacuation.</li>
	<li>Establish an IV line in the left IJV to allow for the administration of propofol constant-rate infusion (CRI) (20-40 mg&#8729;kg-1&#8729;h-1), maintenance fluids (10 mL&#8729;kg-1&#8729;h-1), and the saline bolus.</li>
	<li>Position the sheep in left lateral recumbency. Shave the right side of the neck to gain access to the catheterization site (Figure 2A). Swab the area with at least 2 alcohol scrubs and 2 chlorhexidine scrubs.</li>
	<li>Disconnect from the monitoring equipment and ventilator and move the sheep to the catheterization laboratory table. Again, situate the animal in a left lateral recumbent position (Figure 3A).</li>
	<li>Reconnect to the ventilator and monitoring equipment (ECG leads, temperature probe, blood pressure cuff, pulse oximeter).</li>
	<li>Maintain anesthesia during the procedure by administering inhaled isoflurane 1-3% with 100% O2, and/or propofol CRI (20-40 mg&#8729;kg-1&#8729;h-1). 
	NOTE: Assess the plane of anesthesia by gauging animal movement, response to painful stimuli, respiratory rate, pulse rate, and blood pressure. Make adjustments in sedation as appropriate, such as by using a 5-10 mL bolus of propofol to induce a deeper plane of anesthesia.</li>
	<li>Measure the sheep&#39;s width at the area of the heart using large calipers. Divide the width by 2 to set the pressure transducer.</li>
	<li>Aseptically clean the surgical site and drape it in a sterile fashion (Figure 2B,C).</li>
</ol>
2. Catheterization
<ol>
	<li>Move the C-Arm from the parked position to the sheep&#39;s chest and raise the table as needed. Press buttons 7 and 3 on the control panel, and then hold the Start button to use the preprogrammed settings to autoposition the table and the C-Arm on the left side of the table (Figure 1A).</li>
	<li>Access the right IJV using a 21 G micro puncture needle and 10 cc Luer Slip syringe; access the IJV in a cranial/caudal direction through the skin while pulling back on the syringe plunger. Ensure that blood is aspirated to confirm that the needle is in the vessel (Figure 2A, B).</li>
	<li>Carefully disconnect the syringe while holding the needle steady.</li>
	<li>Insert a 0.018 inch Stainless Steel (SS) Wire Guide through the needle into the vessel approximately halfway. Remove the needle from over the SS wire.</li>
	<li>Place a 5-French (Fr) dilator over the SS wire and into the vessel. Remove the inner piece of the dilator and SS wire. Feed a 0.038 inch guidewire through the dilator into the vessel about halfway and remove the dilator.</li>
	<li>Use an 11-blade scalpel to cut the skin above the vein where the wire enters. Feed a 9-Fr sheath over the guidewire and into the vessel. Remove the inner sheath section and guidewire.</li>
	<li>Confirm proper sheath placement by aspirating blood and then flushing the sheath with heparinized saline.</li>
	<li>Repeat steps 2.2-2.7 so that there are two 9-Fr sheaths in the right IJV.</li>
	<li>Administer 150 U/kg of heparin through the IV to prevent clotting.</li>
	<li>Use the foot pedal to start fluoroscopy (Figure 1B). Insert a Judkins Right (JR) catheter through the sheath, following the thoracic IVC across the diaphragm into the abdominal IVC.</li>
	<li>Thread a Rosen wire through the JR catheter until it reaches the abdominal IVC and the tip emerges from the JR catheter. While holding the Rosen wire in place, gently remove the JR catheter.</li>
	<li>Repeat steps 2.10-2.11 with the second sheath.</li>
	<li>Thread a 7-Fr Multitrack catheter over each Rosen wire.</li>
	<li>Under fluoroscopy guidance, place one multitrack angiographic catheter in the abdominal IVC for contrast injection.</li>
	<li>Using fluoroscopy, place another multitrack angiographic catheter into the specific region of interest (e.g., the center of the graft) for pressure measurement (Figure 2C).</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66724/66724fig01.jpg" /> 
Figure 1: Control panel. (A) 3D angiography system control panel (B) Fluoroscopy foot pedals <a href="https://www.jove.com/files/ftp_upload/66724/66724fig01large.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/66724/66724fig02v3.jpg" /> 
Figure 2: Animal catheterization. (A) The key surgical site, prepped for catheterization. (B) Technique to visualize the right internal jugular vein (black arrows). (C) Two multitrack angiographic catheters are placed through the right internal jugular vein (blue arrow: pressure measurement in the graft; red arrow: contrast injection into abdominal IVC; white arrow: stiff wires). Abbreviation: IVC = inferior vena cava. <a href="https://www.jove.com/files/ftp_upload/66724/66724fig02largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Gathering the predata
<ol>
	<li>Connect the multitrack centered in the region of interest to the pressure transducer using a three-way stopcock. With the stopcock open to the multitrack, pull back with a 10 mL syringe until air bubbles are removed and blood is seen.</li>
	<li>With the 10 mL syringe flipped upside-down, return the blood to the sheep with care not to push any air back into the multitrack. Flush the multitrack with heparinized saline.</li>
	<li>Flip the off position of the stopcock to the syringe so that the pressure transducer and the multitrack are open to each other.</li>
	<li>Prepare the contrast injector by adding a contrast agent. The minimal volume for a 3D angiogram is 60 mL and the total contrast cannot exceed 5 mL/kg or 250 mL.</li>
	<li>Connect the contrast injector to the multitrack centered in the abdominal IVC. Using the contrast injector, turn the knob counterclockwise slowly to pull air bubbles from the multitrack until blood is seen. Turn the knob clockwise to push contrast forward slowly into the multitrack.</li>
	<li>Use fluoroscopy to confirm when the contrast has reached the tip of the multitrack.</li>
	<li>Take the total contrast used for the 3D angiogram and divide by 5 to get mL/s. Set the rate rise to 0 and 600 psi.</li>
	<li>Set the C-arm to the preprogrammed mode by clicking the Program button on the right top of the screen and the 3D DSA 110 8&#39;&#39; button (3D Angiography with an SID of 110 cm and Field View at 8 inches).</li>
	<li>Move all objects and people away from the front or sides of the table. Initiate the C-Arm program by clicking the button numbered 3 on the control panel. Position the target region (e.g., midgraft) at the center of the x-y plane (Figure 3A-C).</li>
	<li>Progress to the second program by clicking the button numbered 4 on the control panel. Adjust the height of the table accordingly to center the region of interest.</li>
	<li>Press the button numbered 5 and take the test image.</li>
	<li>Pretest the C-Arm&#39;s range of motion by clicking the Confirm Conditions button | Start (Figure 3D,E).</li>
	<li>Ask the anesthesiologist to hold ventilation and take a 3D rotational angiogram with contrast injection by initiating the program with the middle acquisition pedal. Concurrently measure and record the mean pressure of the target region.</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66724/66724fig03.jpg" /> 
Figure 3: C-arm positioning and range of motion. (A) Sheep positioning for the beginning of the procedure (B) First position for 3D angiogram program (C) C-arm moved in the xy-axis (D) C-arm moved in the z-axis (E) C-arm completing test-spin with full range of motion. <a href="https://www.jove.com/files/ftp_upload/66724/66724fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Administering the saline bolus and collecting data
<ol>
	<li>Prep 20 mL/kg of 0.9% saline.</li>
	<li>To prepare the bolus pressurized bag, add a 1,000 mL bag of 0.9% saline into a pressurized bag unit. Use a second unit if necessary to achieve the total volume to be administered.</li>
	<li>Squeeze the inflation bulb until the pressure gauge rises into the green zone, immediately before the red line (pressure 250-300 mmHg). Flush saline through the line and remove air bubbles.</li>
	<li>Connect the pressurized saline bag to a 9-Fr sheath and maintain 250-300 mmHg to maintain a constant speed of the bolus. Allow it to flow until the sheep has received a bolus equivalent to 20 mL/kg or the mean pressure reaches 15 mmHg.</li>
	<li>While the bolus is flowing, record intravascular pressures of the target region every minute.</li>
	<li>Have the C-Arm ready for a second 3D rotational angiography by repeating steps 3.9-3.12. As soon as the bolus ends, before the pressure begins to drop, take the second 3D angiography and concurrent intravascular pressure measurement by initiating as described in step 3.13.</li>
</ol>
5. Recovery
<ol>
	<li>Following imaging completion, place the C-Arm back in the preprogrammed parked position by entering number 77 and holding the Start button until the C-Arm positions itself.</li>
	<li>Remove the multitrack angiography catheters and Rosen wires.</li>
	<li>Remove both sheaths while applying direct pressure over insertion sites with a hemostasis patch for at least 7 min to stop bleeding.</li>
	<li>Wrap a sterile roll of gauze around patches and neck such that the wrap is secure to maintain pressure, but not too tight to risk cutting off circulation or preventing breathing.</li>
	<li>Turn off anesthetics (isoflurane and/or propofol CRI).</li>
	<li>Maintain sheep on the ventilator with 100% O2 until they consistently breathe on their own. 
	NOTE: Signs that the sheep is waking up include movement, blinking, response to painful stimuli, jaw tone or attempts to chew, and breathing without assistance from a ventilator.</li>
	<li>Once the sheep is able to breathe on their own, extubate (remove the ET tube) and remove the orogastric tube.</li>
	<li>Remove all monitoring equipment and transfer the sheep to a hospital bed. Move it back to the housing room.</li>
	<li>Assist the sheep with staying in sternal recumbency or when attempting to stand until the sheep is able to keep balance on their own. Prevent them from running into walls. 
	NOTE: Monitor the animals closely during recovery. If any signs of pain or distress are observed, consult the veterinary team.</li>
	<li>Once they appear awake enough, give small amounts of hay or grain.</li>
</ol>
6. Analysis of data
<ol>
	<li>Export the original raw 3D angiography data from the angiography imaging software in DICOM file format.</li>
	<li>Launch the DICOM viewer software. Drag and drop the 3D angiography file into the viewer to open (Figure 4A).</li>
	<li>Within the DICOM viewer software, select the 3D MPR (Multi-Planar Reconstruction) tool to generate a 3D reconstructed view of the angiography data. This will present three distinct 2D views from three different angles: axial, sagittal (Figure 4B), and coronal (Figure 4C) planes.</li>
	<li>Adjust the placement and orientation of the target region in the sagittal and coronal planes to achieve the desired vertical position by placing the target region at the center and rotating the direction of the reference lines on each plane with a hand tool (Figure 4D).</li>
	<li>Utilize the pencil tool within the DICOM viewer to outline the vessel wall in the axial view of the target region (Figure 4E). The software automatically calculates and shows both the area and perimeter (Circumference) of the region in the middle of the axial view.</li>
	<li>Utilize equation (1) to calculate compliance, where A is the cross-sectional area (cm2) and P is the pressure (mmHg): 
	<img alt="Equation 1" src="/files/ftp_upload/66724/66724eq01.jpg" style="margin: auto;" />&#160; &#160; &#160; (1)</li>
	<li>Employ equation (2) to calculate distensibility where C is the circumference (cm) and P is the pressure (mmHg): 
	<img alt="Equation 2" src="/files/ftp_upload/66724/66724eq02.jpg" style="margin: auto;" />&#160; &#160;(&#8203;2)</li>
</ol>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66724/66724fig04.jpg" /> 
Figure 4: Data analysis in DICOM viewer. (A) Raw data of 3D angiogram loaded into DICOM viewer. (B) The sagittal section of the graft. (C) The coronal section. (D) A true cross-section is visualized after adjusting the angle on the sagittal and coronal sections. (E) The pencil tool is used to outline the target vessel to make circumference and cross-sectional area measurements. <a href="https://www.jove.com/files/ftp_upload/66724/66724fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

3D Angiography for Venous Compliance Assessment in Sheep

<ol>
	<li>Hagler, D. J., 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=Fate+of+the+Fontan+connection:+Mechanisms+of+stenosis+and+management.">Fate of the Fontan connection: Mechanisms of stenosis and management.</a> Congenit Heart Dis. 14 (4), 571-581 (2019).</li><li>Nezerati, R. M., Eifert, M. B., Dempsey, D. K., Cosgriff-Hernandez, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+vascular+grafts+with+improved+compliance+matching+to+native+vessels.">Electrospun vascular grafts with improved compliance matching to native vessels.</a> J Biomed Mater Res B Appl Biomater. 103 (2), 313-323 (2015).</li><li>Bates, O., Semple, T., Krupickova, S., Bautisa-Rodriguez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Case+report+of+a+Gore-Tex+TCPC+conduit+dissection+causing+severe+stenosis.">Case report of a Gore-Tex TCPC conduit dissection causing severe stenosis.</a> Eur Heart J Case Rep. 5 (11), 1-6 (2021).</li><li>Sathananthan, G., 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=Clinical+importance+of+Fontan+Circuit+thrombus+in+the+adult+population:+Significant+association+with+increased+risk+of+cardiovascular+events.">Clinical importance of Fontan Circuit thrombus in the adult population: Significant association with increased risk of cardiovascular events.</a> Can J Cardiol. 35 (12), 1807-1814 (2019).</li><li>Kumar, P., Bhatia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computed+tomography+in+the+evaluation+of+Fontan+Circulation.">Computed tomography in the evaluation of Fontan Circulation.</a> J Cardiovasc Imaging. 29 (2), 108-122 (2021).</li><li>Abbott, W. M., Megerman, J., Hasson, J. E., L'Italien, G., Warnock, D. F. <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+compliance+mismatch+on+graft+patency.">Effect of compliance mismatch on graft patency.</a> J Vasc Surg. 5 (2), 376-382 (1987).</li><li>Weston, M. W., Rhee, K., Tarbell, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compliance+and+diameter+mismatch+affect+the+wall+shear+rate+distribution+near+an+end-to-end+anastomosis.">Compliance and diameter mismatch affect the wall shear rate distribution near an end-to-end anastomosis.</a> J Biomech. 29 (2), 187-198 (1996).</li><li>Ballyk, P. D., Walsh, C., Butany, J., Ojha, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compliance+mismatch+may+promote+graft-artery+intimal+hyperplasia+by+altering+suture-line+stress.">Compliance mismatch may promote graft-artery intimal hyperplasia by altering suture-line stress.</a> J Biomech. 31 (3), 229-237 (1997).</li><li>Lemson, M. S., Tordoir, J. H. M., Daemen, M. J. A. P., Kitslaar, P. J. E. H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intimal+hyperplasia+in+vascular+grafts.">Intimal hyperplasia in vascular grafts.</a> Eur J Vasc Endovasc Surg. 19 (4), 336-350 (2000).</li><li>Blum, K. M., 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=Tissue+engineered+vascular+grafts+transform+into+autologous+neovessels+capable+of+native+function+and+growth.">Tissue engineered vascular grafts transform into autologous neovessels capable of native function and growth.</a> Commun Med. 2, 3 (2022).</li><li>Turner, M. E., 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=Tissue+engineered+vascular+grafts+are+resistant+to+the+formation+of+dystrophic+calcification.">Tissue engineered vascular grafts are resistant to the formation of dystrophic calcification.</a> Nat Commun. 15, 2187 (2024).</li></ol>

This study was sponsored in part by Gunze Ltd.

Compliance and distensibility are key properties for blood vessel function, serving as indicators of potential complications and interventions. Precisely quantifying and comparing changes in these parameters is important to assess graft efficacy. Our in vivo method overcomes the limitations of ex vivo analysis and maintains comparable results. Comparing our in vivo data to the ex vivo data presented by Blum et al., both methods demonstrate marked differences between the synthetic graft...

Patients with critical cardiac anomalies require reconstructive surgery. Most reconstructive operations require the use of prosthetic materials, including vascular grafts. Potential conduits to bridge this space include synthetic or biologic materials. Initially, homografts were used as the Fontan conduit but have since been abandoned due to a high incidence of calcification and acute phase incidents1. Currently, synthetic vascular grafts derived from inorganic polymers are used. There remains a challenge that these grafts are less compliant than the native vessel into which they are implanted and have long-term complications, such as stenosis, occlusion, and calcification1,2,3,4,5.
The structure of synthetic vascular grafts lends itself to mechanical tensile strength, leading to their invariably lower compliance compared to native tissue2. Vascular compliance, which defines the vessel&#39;s change in volume over a change in pressure, serves as an indicator of a vessel&#39;s responsiveness to mechanical loads. The difference between the graft material and native vessel properties creates a compliance mismatch, which has been demonstrated to disrupt blood flow patterns, resulting in areas of recirculation and flow separation2,6,7,8,9. This phenomenon alters the shear stress on the endothelial wall and induces intimal hyperplasia2,7,8,9. Such responses can lead to graft-related complications, necessitating graft replacement or re-intervention6.
As vascular compliance assumes a key role in determining graft outcomes, the accurate measurement of this property is essential. The current gold standard for measuring vascular compliance is ex vivo tubular biaxial mechanical testing. This method involves excising a graft or vessel of interest, connecting it to latex tubes, and pressurizing it to assess circumferential stress-stretch behavior across various pressures. Compliance is determined by comparing the pressure with a measurement of the inner diameter10. However, ex vivo methods have some disadvantages. When evaluating the functionality of implanted grafts using the ex vivo method, sacrificing the animals and explanting the grafts are necessary, making it impossible to conduct prolonged examinations. Therefore, we have developed an in vivo compliance measurement protocol.
Our group focuses on developing tissue-engineered vascular grafts (TEVGs) for use in the Fontan surgery to ameliorate the congenital heart defect hypoplastic left heart syndrome (HLHS). Recent developments in the field of congenital heart surgery have improved postoperative outcomes, leading to longer life expectancies. This makes the long-term properties and success of the implanted vascular conduit increasingly crucial. Currently, no animal model of HLHS exists so we evaluate our grafts in an accelerated large animal inferior vena cava (IVC) interposition graft model. While this model does not attempt to create the flow of the Fontan circulation, it effectively recapitulates the unique hemodynamic conditions. Our recent use of this in vivo protocol demonstrated significant differences in graft compliance between our TEVG and conventional expanded polytetrafluoroethylene (PTFE) grafts11. As this previous study did not focus on methodology, we have conducted additional experiments detailing this novel in vivo method.
We implanted the synthetic graft currently serving as the standard of care, comprised of expanded polytetrafluoroethylene (PTFE), in Dorset sheep study animals and compared it to the native IVC in surgically na&#239;ve control animals. This protocol was performed on the PTFE group 5-7 years postimplantation of a PTFE conduit and unoperated control animals of varying ages. Thus, in subsequent sections describing the protocol and representative results, we will occasionally refer to the region of interest as, for example, the middle of the graft (midgraft) region of the IVC interposition graft.
This protocol allows us to analyze the in vivo compliance of the PTFE conduit, known to be non-compliant at a long-term time point, with the native vein. We chose to compare the clinical standard material, PTFE, with the native unoperated vein. We selected a long-term time point because the PTFE conduit is known to remain non-compliant and is prone to calcify, further reducing its compliance11. We opted to conduct all comparisons in vivo as systemic hemodynamic changes are accurately reflected in measurements obtained through in vivo methods. From this comparison, we found that this protocol is able to confirm the non-compliance of PTFE and obtain measurements of in vivo venous compliance in a safe and reproducible manner. This method has been successfully implemented in a published study to demonstrate statistically significant differences between PTFE conduits and tissue-engineered vascular grafts (TEVGs) in vivo11.
The overall goal of this protocol is to calculate compliance and distensibility of the thoracic IVC in an ovine large animal model using in vivo measurements from a survival procedure. To this end, we visualized and measured the changes in circumference and cross-sectional area of thoracic IVC to a fluid bolus. We simultaneously measured the intravascular change in pressure and used these measurements to calculate compliance and distensibility. Using 3D angiography imaging allows us multiple advantages, including the ability to adjust the view of the image post capture to ensure our measurements are taken from a cross-section of the vein, as well as allow us to measure multiple locations along the vessel. The three areas of interest in this study were the midgraft region, as well as the two adjacent anastomosis sites of the PTFE graft, and the comparable areas in the native IVC. By conducting experiments in vivo, there are advantages in evaluating the functionality of grafts within the actual flow of blood and surrounded by tissues and organs. The measurements obtained through this method are believed to reflect the actual functionality of the grafts in a living organism.
The protocol is divided into six main sections including preprocedure preparation of the sheep, catheterization, collection of baseline pre bolus data, collection of study data, animal recovery, and data analysis. In the animal preparation section, we discuss sedation, initiating anesthesia, and the placement of monitoring equipment used during the catheterization procedure. In the second section, we explain the process of placing the two catheter sheaths needed for data acquisition. For this protocol, both sheaths are placed in the right internal jugular vein (IJV) to allow two multitrack catheters to be introduced into the vessel. One will be positioned in the region of interest to record the change in pressure, and the other will be placed lower in the vein for contrast injection. Once the catheters are placed, a baseline pre bolus 3D angiograph is taken for comparison. Study data collection begins with preparing the saline bolus in a pressurized bag system for administration, providing the saline bolus with recording intravascular pressures, and taking the post bolus 3D angiograph. We then describe the process to facilitate the recovery of the sheep after the protocol. Lastly, we discuss the method to obtain the proper images and cross-sectional measurements for analysis and statistical comparison.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.035&#34; x 260 cm Rosen Curved Wire Guide</td>
			<td>Cook Medical</td>
			<td>G01253</td>
			<td>Guide for holding placement swapping caths (Multi-track, IVUS, etc)</td>
		</tr>
		<tr>
			<td>0.035&#34;x 150 cm Glidewire</td>
			<td>Terumo</td>
			<td>GR3507</td>
			<td>Guide for JR cath</td>
		</tr>
		<tr>
			<td>0.9% Sodium Chloride Saline</td>
			<td>Baxter Healthcare Corporation</td>
			<td>NCH pharmacy</td>
			<td>For diluting norepinpherine, pressure monitoring</td>
		</tr>
		<tr>
			<td>10.0 Endotracheal tube</td>
			<td>Coviden</td>
			<td>86117</td>
			<td>To secure airway</td>
		</tr>
		<tr>
			<td>16 G IV catheter</td>
			<td>BD</td>
			<td>382259</td>
			<td>To administer fluids and anesthetic drugs</td>
		</tr>
		<tr>
			<td>22 G IV catheter</td>
			<td>BD</td>
			<td>381423</td>
			<td>For invasive blood pressure</td>
		</tr>
		<tr>
			<td>5Fr x .35&#34; JR2.5</td>
			<td>Cook Medical&#160;</td>
			<td>G05035</td>
			<td>Guide for rosen wire</td>
		</tr>
		<tr>
			<td>70% isopropyl alcohol</td>
			<td>Aspen Vet</td>
			<td>11795782</td>
			<td>Topical cleaning solution</td>
		</tr>
		<tr>
			<td>7Fr x 100 cm Multi-track</td>
			<td>B. Braun</td>
			<td>615001</td>
			<td>Collecting pressure, Administering contrast to specific intravascular location</td>
		</tr>
		<tr>
			<td>9Fr Introducer sheath</td>
			<td>Terumo</td>
			<td>RSS901</td>
			<td>Access catheter through skin into vessel for wires to pass through</td>
		</tr>
		<tr>
			<td>ACT cartridge</td>
			<td>Abbot Diagnostics</td>
			<td>03P86-25</td>
			<td>Activated Clotting Time</td>
		</tr>
		<tr>
			<td>Angiographic syringe w/ filling spike</td>
			<td>Guerbet</td>
			<td>900103S</td>
			<td>For contrast injector</td>
		</tr>
		<tr>
			<td>Bag decanter</td>
			<td>Advance Medical Designs, LLC</td>
			<td>10-102</td>
			<td>Punctures saline bag to pour and fill sterile bowl with saline</td>
		</tr>
		<tr>
			<td>Butorphanol</td>
			<td>Zoetis</td>
			<td>NCH pharmacy</td>
			<td>Sedation drug: Concentration 10 mg/mL, Dosage 0.1 mg/kg</td>
		</tr>
		<tr>
			<td>Cath Research Pack</td>
			<td>Cardinal Health</td>
			<td>SAN33RTCH6</td>
			<td>Cath pack with misc. supplies</td>
		</tr>
		<tr>
			<td>Cetacaine</td>
			<td>Cetylite</td>
			<td>220</td>
			<td>Topical anesthetic spray</td>
		</tr>
		<tr>
			<td>Chloraprep</td>
			<td>BD</td>
			<td>930825</td>
			<td>Topical cleaning solution</td>
		</tr>
		<tr>
			<td>Chlorhexidine 2% solution</td>
			<td>Vedco INC</td>
			<td>VINV-CLOR-SOLN</td>
			<td>Topical cleaning solution</td>
		</tr>
		<tr>
			<td>Conform stretch bandage</td>
			<td>Coviden</td>
			<td>2232</td>
			<td>Neck wrap to prevent bleeding</td>
		</tr>
		<tr>
			<td>Connection tubing</td>
			<td>Deroyal</td>
			<td>77-301713</td>
			<td>Connects t-port to fluid/drug lines</td>
		</tr>
		<tr>
			<td>Diazepam</td>
			<td>Hospira Pharmaceuticals</td>
			<td>NCH pharmacy</td>
			<td>Sedation drug: Concentration 5 mg/mL, Dosage 0.5 mg/kg</td>
		</tr>
		<tr>
			<td>EKG monitoring dots</td>
			<td>3M</td>
			<td>2570</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluid administration set</td>
			<td>Alaris</td>
			<td>2420-0007</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluid warming set</td>
			<td>Carefusion</td>
			<td>50056</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemcon Patch</td>
			<td>Tricol Biomedical</td>
			<td>1102</td>
			<td>Patch for hemostasis</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Hospira, Inc</td>
			<td>NCH pharmacy</td>
			<td>Angicoagulant: 1,000 USP units/mL</td>
		</tr>
		<tr>
			<td>Infinix-i INFX-8000C</td>
			<td>Toshiba Medical Systems</td>
			<td>2B308-124EN*E</td>
			<td>Interventional angiography system</td>
		</tr>
		<tr>
			<td>Invasive pressure transducer</td>
			<td>Medline</td>
			<td>23DBB538</td>
			<td>For invasive blood pressure</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter Healthcare Corporation</td>
			<td>NCH pharmacy</td>
			<td>Anesthetic used in prep room</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Hospira Pharmaceuticals</td>
			<td>NCH pharmacy</td>
			<td>Sedation drug: Concentration 100 mg/mL, Dosage 4 mg/kg</td>
		</tr>
		<tr>
			<td>Lubricating Jelly</td>
			<td>MedLine</td>
			<td>MDS0322273Z</td>
			<td>ET tube lubricant</td>
		</tr>
		<tr>
			<td>Micropuncture Introducer Set</td>
			<td>Cook Medical</td>
			<td>G47945</td>
			<td>Access through skin into vessel</td>
		</tr>
		<tr>
			<td>Needle &#38; syringes</td>
			<td>Cardinal Health</td>
			<td>309604</td>
			<td>For sedation</td>
		</tr>
		<tr>
			<td>Norepinpherine Bitartrate Injection, USP</td>
			<td>Baxter Healthcare Corporation</td>
			<td>NCH pharmacy</td>
			<td>1 mg/mL</td>
		</tr>
		<tr>
			<td>Optiray 320</td>
			<td>Liebel-Flarsheim Company, LLC</td>
			<td>NCH pharmacy</td>
			<td>Contrast&#160;</td>
		</tr>
		<tr>
			<td>Optixcare</td>
			<td>Aventix</td>
			<td>OPX-4252</td>
			<td>Corneal lubricant</td>
		</tr>
		<tr>
			<td>OsiriX MD</td>
			<td>Pixmeo SARL</td>
			<td>-</td>
			<td>DICOM Viewer and Analysis software</td>
		</tr>
		<tr>
			<td>Pressure infusor bag</td>
			<td>Carefusion</td>
			<td>64-10029</td>
			<td>To maintain invasive blood pressure</td>
		</tr>
		<tr>
			<td>Propofol</td>
			<td>Fresenius Kabi</td>
			<td>NCH pharmacy</td>
			<td>Anesthetic drug: Concentration 10 mg/mL, Dosage 20-45 mg&#183;kg-1&#183;h-1</td>
		</tr>
		<tr>
			<td>Silk suture 3-0</td>
			<td>Ethicon</td>
			<td>C013D</td>
			<td>To secure IV catheter&#160;</td>
		</tr>
		<tr>
			<td>SoftCarry Stretcher</td>
			<td>Four Flags Over Aspen</td>
			<td>SSTR-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Stomach tube</td>
			<td>Jorgensen Lab, INC</td>
			<td>J0348R</td>
			<td>To release gastric juices and gas and prevent bloat</td>
		</tr>
		<tr>
			<td>T-port</td>
			<td>Medline</td>
			<td>DYNDTN0001</td>
			<td>Connects to IV catheter</td>
		</tr>
		<tr>
			<td>Urine drainage bag</td>
			<td>Coviden</td>
			<td>3512</td>
			<td>Connects to stomach tube to collect gastric juices</td>
		</tr>
		<tr>
			<td>Warming blanket</td>
			<td>Jorgensen Lab, INC</td>
			<td>J1034B</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantifying inferior vena cava compliance and distensibility in an in vivo ovine model using 3d angiography

Synthetic vascular grafts overcome some challenges of allografts, autografts, and xenografts but are often more rigid and less compliant than the native vessel into which they are implanted. Compliance matching with the native vessel is emerging as a key property for graft success. The current gold standard for assessing vessel compliance involves the vessel's excision and ex vivo biaxial mechanical testing. We developed an in vivo method to assess venous compliance and distensibility that better reflects natural physiology and takes into consideration the impact of a pressure change caused by flowing blood and by any morphologic changes present. 
This method is designed as a survival procedure, facilitating longitudinal studies while potentially reducing the need for animal use. Our method involves injecting a 20 mL/kg saline bolus into the venous vasculature, followed by the acquisition of pre and post bolus 3D angiograms to observe alterations induced by the bolus, concurrently with intravascular pressure measurements in target regions. We are then able to measure the circumference and the cross-sectional area of the vessel pre and post bolus. 
With these data and the intravascular pressure, we are able to calculate the compliance and distensibility with specific equations. This method was used to compare the inferior vena cava's compliance and distensibility in native unoperated sheep to the conduit of sheep implanted with a long-term expanded polytetrafluorethylene (PTFE) graft. The native vessel was found to be more compliant and distensible than the PTFE graft at all measured locations. We conclude that this method safely provides in vivo measurements of vein compliance and distensibility.

Author Spotlight: Improved Imaging for Neovascular Development in Congenital Heart Disease Research

Quantifying Inferior Vena Cava Compliance and Distensibility in an In Vivo Ovine Model Using 3D Angiography

We aim to improve long-term outcomes for congenital-heart-disease patients using a regenerative medicine approach. Our tissue-engineered vascular graft develops into a neo vessel comprised of the patient's own cells. Our goal is to make comparisons between our graft, the native vein, and the clinical standard polytetrafluoroethylene or PTFE.We have established that our neo vessel displays growth capacity and that it approaches native vessel functionality. Using this method, we've also recently demonstrated that the neo vessel retains compliance and distensibility at a long-term time point, and is resistant to the formation of dystrophic calcification. Using 3D angiography allows us to image the entire path of the thoracic inferior vena cava in our ovine large-animal model.In addition to allowing us to view the morphology of the vessel, it also allows for post-capture orientation adjustments to ensure we're obtaining measurements from a true cross-section. Our in-vivo method allows us to determine the capacity for vessel compliance and distensibility in its native context. Additionally, it allows us to take longitudinal measurements of the same study animal, which is necessary to track changes in neo-vessel development and remodeling.

We have successfully performed this procedure with over 25 sheep. Importantly, there were no instances of morbidity and mortality related to this procedure. All the sheep exhibited uncomplicated recoveries. These representative results were taken from three sheep implanted with PTFE grafts and three unoperated native sheep. Figure 5 provides the intravascular pressure measurements taken from both groups of study animals during the protocol. These values are important for the compliance and d...

Read this Scientific Article Protocol about Author Spotlight: Improved Imaging for Neovascular Development in Congenital Heart Disease Research at JoVE.com

This protocol allows for the in vivo quantification of venous compliance and distensibility using catheterization and 3D angiography as a survival procedure allowing for a variety of potential applications.

Synthetic vascular grafts overcome some challenges of allografts, autografts, and xenografts but are often more rigid and less compliant than the native ...

quantifying-inferior-vena-cava-compliance-distensibility-an-vivo

Research

JoVE Journal

Bioengineering

448 Aufrufe.  Nationwide Children's Hospital. Unser Ziel ist es, die langfristigen Ergebnisse für Patienten mit angeborenen Herzfehlern mit einem regenerativen Medizinansatz zu verbessern. Unser Tissue-Engineering-Gefäßtransplantat entwickelt sich zu einem Neo-Gefäß, das aus patienteneigenen Zellen besteht. Unser Ziel ist es, Vergleiche zwischen unserem Transplantat, der nativen Vene, und dem klinischen Standard Polytetrafluorethylen oder PTFE anzustellen.Wir haben festgestellt, dass unser Ne...

Serie: Autor im Rampenlicht: Verbesserte Bildgebung für die neovaskuläre Entwicklung in der Forschung zu angeborenen Herzfehlern

Catheterization of an Anesthetized Sheep for 3D Angiogram Analysis

To begin, place a sedated sheep in the left lateral recumbency position on the catheterization table. Press buttons 7 and 3 on the control panel to move the C-arm to the sheep and adjust the table. Then auto-position the C-arm towards the left side of the table.Next, use a 21-gauge puncture needle on a 10-cubic centimeter Luer syringe to access the internal jugular vein. Carefully disconnect the syringe while holding the needle steady. Now insert a 0.018-inch stainless steel wire guide through the needle into the vessel.Remove the needle from over the wire. Place a 5 French dilator over the wire and into the vessel. After removing the inner piece of the dilator and wire, feed a 0.038-inch guide wire through the dilator into the vessel and remove the dilator.With an 11 blade scalpel, cut the skin above the vein just where the wire enters. Feed a 9 French sheath over the guide wire and into the vessel. Then remove the inner sheath section and guide wire.Aspirate the blood to confirm sheath placement. Flush the sheath with heparinized saline. After inserting two 9 French sheaths in the right internal jugular vein, inject 150 units per kilogram of heparin through the IV to prevent clotting.Now, use the foot pedal to start fluoroscopy. Insert a JR catheter through the sheath, following the thoracic IVC across the diaphragm into the abdominal IVC. Thread a Rosen wire through the JR catheter until it reaches the abdominal IVC and the tip emerges from the JR catheter.While holding the Rosen wire in place, gently remove the JR catheter. When the second sheath has similarly been inserted, thread a 7 French multi-track catheter over each Rosen wire. Now place one multi-track angiographic catheter in the abdominal IVC for contrast injection.Place another multi-track angiographic catheter into the specific region of interest for pressure measurement.

Acquisition of  Pre&#45;and Post&#45;Bolus 3D Angiograms and Intravascular Pressure from a Catheterized Sheep for Measurement of the Vessel Circumference

To begin, connect a multi-track angiographic catheter in the region of interest of an anesthetized catheterized sheep to a pressure transducer with a three-way stopcock. Then flush the multi-track with heparinized saline. Flip the off position on the stopcock to the syringe so that the pressure transducer and the multi-track are open to each other.Connect the contrast injector to the multi-track centered in the abdominal IVC. Turn the knob of the contrast injector counterclockwise slowly to pull air bubbles from the multi-track until blood is seen. Then turn the knob clockwise to push contrast forward slowly into the multi-track.Confirm when the contrast has reached the tip of the multi-track with a fluoroscope. Divide the total contrast used for the 3D angiogram by five to obtain the rate of injection in milliliters per second. Then set the rate rise to 0 in 600 pounds per square inch.Next, to set the C-arm to the pre-programmed mode, click the Program button, then press the 3D-110 8"button. Move all objects and people away from the front or sides of the table. Click on button 3 on the control panel to initiate the C-arm.Position the target at the center of the xy plane. Click button 4 on the control panel to move onto the second program. Then adjust the height of the table to center the region of interest.Next, press button 5 to take the test image. click on the Confirm Conditions icon, followed by Start to pretest the C-arm's range of motion. Use the middle acquisition pedal to initiate the program and take a 3D rotational angiogram.Simultaneously, measure and record the mean pressure of the target region. Connect a pressurized saline bag to a 9 French sheath. Allow it to flow until the sheep has received a bolus equivalent to 20 milliliters per kilogram.Record the intravascular pressures of the target region every minute while the bolus is flowing. Perform the second 3D angiography with concurrent intravascular pressure measurement. Once imaging is complete, press the number 77 and hold the Start button to place the C-arm back in its pre-programmed to position.Remove the multi-track angiography catheters and Rosen wires from the sheep. Then remove both sheaths. Apply pressure with a hemostasis patch over the insertion sites for at least seven minutes to control bleeding.For data analysis, export the original raw 3D angiography data from the angiography imaging software in a DICOM file format. Launch the DICOM Viewer software. Then drag and drop the 3D angiography file into the Viewer to open.Select the 3D Multiplanar Reconstruction tool to generate a 3D reconstructed view of the angiography data. Three distinct 2D views from the axial, sagittal, and coronal planes can now be seen. Now adjust the placement and orientation of the target region in the sagittal and coronal planes to place the target region at the center.Use the Hand tool to rotate the direction of the reference lines on each page. With the Pencil tool, outline the vessel wall in the axial view of the target region. The software will automatically calculate and show both the area and perimeter of the region in the middle of the axial view.The intravascular pressures of sheep with and without PTFE grafts were obtained. A change in circumference was seen in the native sheep in response to bolus. The native sheep vessels were more compliant and distensible than the PTFE grafts.