This study is supported by the American Venous Forum (AVF) 2023 Basic Science research grant and Surmodics, Inc.

The following protocol follows the University of Michigan (UMICH) Institutional Animal Care and Use Committee (IACUC) policies and guidelines, the U.S. Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training, and the ARRIVE guidelines 2.0. The UMICH Animal Welfare Assurance agreement is compliant with OLAW and USDA and is fully accredited by AAALAC International. This protocol was approved with the ID number PRO00010841. Male outbred Sprague Dawley rats from Charles River Laboratories, weighing 450 g and 15 weeks of age, were used.
1. Rat anesthesia
<ol>
	<li>Remove animals from their cage and place them in an anesthetic pre-operative induction gas chamber at 5% isoflurane and 100% oxygen at a flow rate of 0.8 to 1 L/min (vaporizer-controlled) to induce general anesthesia.</li>
	<li>Sedate rats in the anesthetic induction chamber, remove them from the chamber, weigh, lubricate their eyes with sterile ophthalmic ointment, and then place them in dorsal recumbency on a warm water circulating heating device. Confirm adequate depth of anesthesia using pedal reflex (firm toe pinch).</li>
	<li>Shave the ventral abdomen with electric clippers. Maintain general anesthesia at 2.5% isoflurane and 100% oxygen at 0.8 to 1 L/min with a non-rebreathing circuit through a nosecone.</li>
</ol>
2. Rat ultrasound scanning
<ol>
	<li>Monitor physiological assessments, including respiratory rate, heart rate, and temperature, during imaging.</li>
	<li>For abdominal imaging of the Inferior Vena Cava (IVC), apply conducting gel and orient the transducer in a transverse position. While presetting the peripheral vascular, use two-dimensional imaging mode or B-mode, lower the linear probe (10 MHz) on the center of the abdomen, adjusting depth with pressure until the abdominal vessels come into view.</li>
	<li>Differentiate IVC from the abdominal aorta by assessing compressibility and by assessing flow using both color and pulsed-wave Doppler mode with a high-frequency linear probe (10 mHz).
	<ol>
		<li>When applying compression with the ultrasound probe, differentiate the two as the IVC is compressible, whereas the aorta maintains its shape and patency.</li>
	</ol>
	</li>
	<li>Assess the direction and velocity of blood flow by using both color and pulsed-wave Doppler. The color Doppler window represents the flow toward the probe in red and flows away from the probe in blue. Switch the instrument to pulse-wave Doppler mode (Duplex mode) to assess blood flow direction and velocity over time. Acquire necessary flow images by using the ultrasound machine&#39;s automated image capture tool (The exact specifications for image acquisition may vary between ultrasound systems).
	<ol>
		<li>Use the following assessment method: the aorta has blood flow toward the probe and a triphasic waveform, whereas the IVC has spontaneous flow away from the probe with a much lower amplitude.</li>
		<li>If the proximal IVC displays a normal waveform, yet a nonphasic or absent (flat) waveform is detected distally, explore the possibility of a venous obstruction between these points of examination.</li>
	</ol>
	</li>
	<li>Once the IVC is located, identify the mid-section, which is in the middle third portion, inferior to the renal veins and superior to the IVC bifurcation, most likely where the lumbar veins are located.</li>
	<li>Measure at the mid-section IVC the wall-to-wall diameter in the transverse view using the cross-sectional B-mode image, recording the widest diameter of the vessel. Imaging acquisition may vary in different ultrasound machines. Store data using compact disks (CDs) in DICOM files.</li>
</ol>
3. Rat micro-surgery and recovery
<ol>
	<li>Clean the abdomen with gauze-soaked chlorhexidine scrub and chlorhexidine solution 3x to ensure an aseptic surgical condition. Administer buprenorphine extended-release injectable suspension, 0.65 mg/kg subcutaneous (SC), as an analgesic for rats since it does not interfere with the biomarker panels of inflammatory molecules for protein analysis.</li>
	<li>Place sterilized cling film as a surgical wrap to cover the rat thorax and abdominal area, microscope knobs (magnification and focus), and eyepiece.</li>
	<li>Make a ventral midline incision (3 cm) approximately 2 cm below the xiphoid process with iris scissors through the skin and abdominal wall, exposing the abdominal contents. Use sterile saline-soaked 2 inch x 2 inch gauze to reflect the intestines to the animal&#39;s right side.</li>
	<li>For IVC exposure, perform a blunt dissection using a sterile cotton-tipped applicator. Place a wire speculum in the incision, allowing IVC visualization.</li>
	<li>Cauterize all IVC lumbar branches using a low-temperature fine tip cautery, from the renal veins to the iliac bifurcation, and ligate side branches with 7-0 non-absorbable polypropylene sutures.</li>
	<li>Place a proximal curved vascular micro-clip on the IVC, which is separated from the aorta and just inferior to the renal veins. Place a straight microclip on the distal IVC, which is separated from the aorta and superior to the IVC bifurcation.</li>
	<li>Place an 8-0 nylon U stitch suture caudal to the left renal vein centered on the anterior surface of the IVC.</li>
	<li>Insert a 0.014 mm sharpened guidewire with the venoplasty balloon backloaded, retrograde to blood flow into the infrarenal IVC, caudal to the curved micro-clip.</li>
	<li>Advance the balloon into the mid-IVC using the Seldinger technique. Remove the sharpened guidewire and inflate the venoplasty balloon for 3 min with a 10% to 15% IVC overstretch using a 20 mL inflation syringe to generate positive pressure over a range of 0-30 Atm. Flush all systems with sterile saline before IVC canulation to avoid air embolism. 
	NOTE: A 2.8 mm IVC would undergo venoplasty to 3.22 mm (15% overstretch). The required inflation pressure to reach the desired overstretch is determined by the characteristics of the balloon and is available on the manufacturer&#39;s packaging.</li>
	<li>Tighten the U stitch upon venoplasty balloon deflation and removal. Remove the micro-clips.</li>
	<li>Close the laparotomy site in a two-layer fashion. Use a 5-0 polyglactin absorbable synthetic suture in a continuous pattern to close both the abdominal wall and skin.</li>
	<li>Recover rats in an individual cage, observe post-operatively (30 min) under a heating lamp (minimum distance - 24 inch away from the cage), and then return to their original housing units.</li>
	<li>For sham animals from each experimental group, perform only the dissection, without the IVC branches ligation, cauterization, and cannulation.</li>
	<li>For post-thrombotic conditions, place a proximal straight vascular micro-clip on the IVC, which is separated from the aorta, just inferior to the renal veins, for 24 h. Use the same dissection and closing techniques.
	<ol>
		<li>Check for the ultrasound signs of IVC occlusion, which include the visualization of the IVC clip, confirmation of distal IVC without flow, and common iliac veins distended. Follow-up timepoints can be customized to fit the study&#39;s specifications.</li>
		<li>To observe the physiologic changes that are being modeled, select timepoints in the first 72 h for early thrombi formation and from 3 to 7 days postprocedural for later thrombi resolution. Later timepoints tested in this model include 7 to 28 days5,13.</li>
	</ol>
	</li>
	<li>Use the techniques in steps 3.5-3.9 for post-thrombotic balloon venoplasty with the following caveats: A higher insufflator pressure might be required to achieve the desired overstretch since the IVC diameter increases in post-thrombotic conditions.</li>
	<li>Perform euthanasia under the recommendations set forth by the current American Veterinary Medical Association Guidelines on Euthanasia for rodents by performing two confirmatory methods (blood removal and vital organ removal) to ensure the animal will not revive.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66820/66820fig01.jpg" /> 
Figure 1:&#160;Distribution of transabdominal duplex measures of transverse IVC (mid-infrarenal section) diameter. The measurement was done by animal weight. No significant correlation was found between the weight and the IVC diameter. <a href="https://www.jove.com/files/ftp_upload/66820/66820fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Sample preparation
<ol>
	<li>Perform confirmatory tissue transfer of drug-coated venoplasty balloons using high-performance liquid chromatography (HPLC) assay technique.</li>
	<li>Dissect the IVC and aorta and remove en bloc for formalin-fixed paraffin embedding and histological analysis.</li>
	<li>For protein analysis, use RIPA buffer in a diluting concentration of 60,000 &#181;g/g of sample, process with a tissue homogenizer in low-temperature room settings at 4 &#176;C, and store at -80 &#176;C after processing.</li>
</ol>

Rodent Venoplasty Balloon Model for Deep Vein Treatment Studies

<ol>
	<li>Prandoni, P., 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=The+long-term+clinical+course+of+acute+deep+venous+thrombosis.">The long-term clinical course of acute deep venous thrombosis.</a> Ann Inter Med. 125 (1), 1-7 (1996).</li><li>Prandoni, P., Kahn, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-thrombotic+syndrome:+prevalence,+prognostication+and+need+for+progress.">Post-thrombotic syndrome: prevalence, prognostication and need for progress.</a> Br J Haematol. 145 (3), 286-295 (2009).</li><li>Gloviczki, P., 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=The+2023+Society+for+Vascular+Surgery,+American+Venous+Forum,+and+American+Vein+and+Lymphatic+Society+clinical+practice+guidelines+for+the+management+of+varicose+veins+of+the+lower+extremities.+Part+II.">The 2023 Society for Vascular Surgery, American Venous Forum, and American Vein and Lymphatic Society clinical practice guidelines for the management of varicose veins of the lower extremities. Part II.</a> J Vas Surg Venous Lymph Diso. 12 (1), 101670 (2024).</li><li>Henke, P., Sharma, S., Wakefield, T., Myers, D., Obi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+from+experimental+post-thrombotic+syndrome+and+potential+for+novel+therapies.">Insights from experimental post-thrombotic syndrome and potential for novel therapies.</a> Transl Res. 225, 95-104 (2020).</li><li>Diaz, J. A., 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=Choosing+a+mouse+model+of+venous+thrombosis:+A+consensus+assessment+of+utility+and+application.">Choosing a mouse model of venous thrombosis: A consensus assessment of utility and application.</a> Arterioscl Thrombo Vasc Biol. 39 (3), 311-318 (2019).</li><li>Lee, Y. U., Lee, A. Y., Humphrey, J. D., Rausch, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+and+biomechanical+changes+in+a+mouse+model+of+venous+thrombus+remodeling.">Histological and biomechanical changes in a mouse model of venous thrombus remodeling.</a> Biorheology. 52 (3), 235-245 (2015).</li><li>Xie, H., 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=Correspondence+of+ultrasound+elasticity+imaging+to+direct+mechanical+measurement+in+aging+DVT+in+rats.">Correspondence of ultrasound elasticity imaging to direct mechanical measurement in aging DVT in rats.</a> Ultrasound Med Biol. 31 (10), 1351-1359 (2005).</li><li>Moini, 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=Venoplasty+and+stenting+in+post-thrombotic+syndrome+and+non-thrombotic+iliac+vein+lesion.">Venoplasty and stenting in post-thrombotic syndrome and non-thrombotic iliac vein lesion.</a> Minim Invasive Ther Allied Technol. 29 (1), 35-41 (2020).</li><li>Chaitidis, N., 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=Management+of+post-thrombotic+syndrome:+A+comprehensive+review.">Management of post-thrombotic syndrome: A comprehensive review.</a> Curr Pharma Des. 28 (7), 550-559 (2022).</li><li>Dangas, G. D., 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=In-stent+restenosis+in+the+drug-eluting+stent+era.">In-stent restenosis in the drug-eluting stent era.</a> J Am Coll Cardiol. 56 (23), 1897-1907 (2010).</li><li>Negl&#233;n, P., Berry, M. A., Raju, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endovascular+surgery+in+the+treatment+of+chronic+primary+and+post-thrombotic+iliac+vein+obstruction.">Endovascular surgery in the treatment of chronic primary and post-thrombotic iliac vein obstruction.</a> Euro J Vas Endovas Surg. 20 (6), 560-571 (2000).</li><li>Sharifi, M., Mehdipour, M., Bay, C., Smith, G., Sharifi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endovenous+therapy+for+deep+venous+thrombosis:+The+TORPEDO+trial.">Endovenous therapy for deep venous thrombosis: The TORPEDO trial.</a> Catheter Cardiovasc Interv. 76 (3), 316-325 (2010).</li><li>Nicklas, J. M., Gordon, A. E., Henke, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolution+of+deep+venous+thrombosis:+Proposed+immune+paradigms.">Resolution of deep venous thrombosis: Proposed immune paradigms.</a> Int J Mol Sci. 21 (6), 2080 (2020).</li><li>Li, J., Kim, S. G., Blenis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapamycin:+One+drug,+many+effects.">Rapamycin: One drug, many effects.</a> Cell Metabol. 19 (3), 373-379 (2014).</li><li>Chen, X., 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=Rapamycin+regulates+Akt+and+ERK+phosphorylation+through+mTORC1+and+mTORC2+signaling+pathways.">Rapamycin regulates Akt and ERK phosphorylation through mTORC1 and mTORC2 signaling pathways.</a> Mol Carcinogen. 49 (6), 603-610 (2010).</li><li>Diaz, J. A., Farris, D. M., Wrobleski, S. K., Myers, D. D., Wakefield, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inferior+vena+cava+branch+variations+in+C57BL/6+mice+have+an+impact+on+thrombus+size+in+an+IVC+ligation+(stasis)+model.">Inferior vena cava branch variations in C57BL/6 mice have an impact on thrombus size in an IVC ligation (stasis) model.</a> J Thromb Haemo. 13 (4), 660-664 (2015).</li><li>Sch&#246;nfelder, T., J&#228;ckel, S., Wenzel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+deep+vein+thrombosis.">Mouse models of deep vein thrombosis.</a> Gef&#228;sschirurgie. 22 (1), 28-33 (2017).</li><li>de Barros, R. S. 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=Morphometric+analysis+of+rat+femoral+vessels+under+a+video+magnification+system.">Morphometric analysis of rat femoral vessels under a video magnification system.</a> J Vasc Bras. 16 (1), 73-76 (2017).</li></ol>

No conflicts of interest were declared.

The VBM can be performed on rats &#8805; 250 g using balloons in the 2.5-3.5 mm range. For rats exceeding 750 g, the technique is limited by a higher amount of adipose tissue in the abdominal cavity and organ size rather than changes in IVC diameter. From our experience, we have found that 10 or more animals per group are needed to obtain statistical significance between the groups for vein wall protein analysis or activity assays and histology. Periodic temperature control and respiration rate surveillance during surgical interventions are highly important to guarantee the success of the procedure, as well as diligent postsurgical management.
VBM is safe (91.2% survival rate, 83/91 procedures performed) and reproducible with an average procedure time of 30 min. Procedure-related morbidity includes anesthetic complications (1%, 1/91), opioid adverse effects (1%, 1/91), bleeding (2.1%, 2/91), and thrombosis (4.3%, 4/91). IVC bleeding was significantly reduced during the model development by using a retrograde IVC canulation approach with the insertion point next to the infrarenal IVC, which offers a broader angle for wire insertion with less interference by the abdominal organs. Also, during the insertion and insufflation phases, we recommend a surgical assistant to guarantee the correct administered pressure, inflation time and sharpened guide wire retraction. Upon deflation, correct retrieval of the venoplasty balloon needs to be ensured to prevent the IVC wall from being torn at the insertion point. Pressure measurements are followed from the manufacturer to avoid balloon hyperinflation and rupture. These measurements overall contribute to a more stable approach, decreasing bleeding complications and improving animal survival.
IVC bleeding can also be mitigated with the following precautions. First, verify the complete cauterization of back branches and ligations of side branches. This also guarantees the reproducibility of the findings based on previous rodent IVC ligation-stasis analysis5,16,17. Second, verify the correct occlusion with the proximal and distal clips to guarantee complete hemostatic control. Finally, verify the complete occlusion of the U-stitch suture upon balloon removal before removing the IVC clips. If bleeding is present,&#160;gentle pressure over the IVC with a cotton-tipped applicator until hemostatic control is achieved will usually stop the bleeding. Additional hemostatic control agents, such as absorbable gelatin powder, can be used in case of bleeding.
Thrombotic complications should be minimal if the balloon inflation, deflation, and the U-stitch suture closing time is less than 5 min. Anticoagulation can be administered if necessary. To date, there is no completely percutaneous procedure to mimic the minimally invasive balloon venoplasties that are currently performed in humans. The size of the access vessel (rat femoral vein) is significantly smaller (0.81 mm on average)18 than the outer diameter of the smallest commercially available 4F sheath (1.61 mm) that would be required for a completely endovascular procedure. Limitations include the need for skilled microsurgeons and the need for specialized equipment.
In conclusion, the VBM model closely simulates the clinical scenario of deep venous venoplasty following DVT. It is a valuable tool for studying the endovascular effect of venoplasty balloons on the IVC with different drug alternatives at multiple time points. Future applications for this technique include the preclinical testing of other medical devices, including stents, thrombectomy catheters, balloon coatings, and novel pharmacological therapeutics.

Deep vein obstructions from unresolved thrombi are a common consequence of deep vein thrombosis (DVT) and are one of the causes of post-thrombotic syndrome (PTS), costing billions of dollars in healthcare and significantly impacting the quality of life1,2,3. PTS is a unique inflammatory pathology with consequent vein wall fibrosis4 not fully captured in small animal DVT models5,6,7, and more importantly, previous models cannot evaluate the response to therapeutic interventions. Thrombotic venous obstructions are often treated by stenting and balloon venoplasty8,9; such interventions are hampered by a high reintervention rate due to restenosis and occlusion10,11,12.
Despite the availability of drug-coated balloons to address arterial-specific stenosis/occlusions, none exist for deep veins. The molecular mechanism behind restenosis is poorly understood in veins and has no direct therapy4. Therefore, our goal is to present an animal model that can test locally delivered therapeutics via venoplasty balloons simulating deep vein post-thrombotic obstructions. This model reproduces the post-thrombotic condition and evaluates how different types of venoplasty balloons affect the vein wall in vivo.

<table><tbody><tr><td>3-0 DemeCRYL</td><td>DemeTech</td><td>G183019F4P</td><td>12 per box</td></tr><tr><td>5-0 DemeCRYL</td><td>DemeTech</td><td>G285017B0P</td><td>12 per box</td></tr><tr><td>7-0 DemeLENE</td><td>DemeTech</td><td>PM197011F13M</td><td>12 per box</td></tr><tr><td>8-0 DemeLON</td><td>DemeTech</td><td>NL868007C7P</td><td>12 per box</td></tr><tr><td>Angled Clip Applier</td><td>SCANLAN</td><td>3795-01A</td><td>7-1/4&amp;Prime; (18.5 cm)</td></tr><tr><td>Baxter 0.9% Sodium Chloride Irrigation Bottle</td><td>Baxter</td><td>SKU: 195-7124</td><td>1000mL Bottle</td></tr><tr><td>Bovie Cautery Low Temperature Fine Tip</td><td>Symmetry Surgical Inc</td><td>AA00</td><td>10/bx</td></tr><tr><td>Buprenorphine</td><td>Fidelis Animal Health</td><td>SKU- 099114</td><td>1.3mg/mL, 3mL</td></tr><tr><td>CD (Sprague Dawley) Rats</td><td>Charles River</td><td>Strain Code 001</td><td>Male, 400 gm</td></tr><tr><td>Clip-It Traceable Timer</td><td>TRACEABLE PRODUCTS</td><td>Cat. No. 5046</td><td></td></tr><tr><td>Cotton-Tip Applicator 3&quot;</td><td>Dukal Corporation</td><td>922-00037BX100</td><td>1000/BOX</td></tr><tr><td>Enzymatic Surgical Instrument Detergent and Presoak</td><td>Medline</td><td>MDS88000B9</td><td>1 Gal</td></tr><tr><td>Eye Lube</td><td>Optixcare</td><td>SKU-065441</td><td>20gm&amp;nbsp;</td></tr><tr><td>Gaymar T/Pump Stryker Localized Warming/Cooling Therapy System</td><td>Stryker</td><td>TP700</td><td></td></tr><tr><td>Heifetz Microclips 12mmx2.25mm</td><td>SCANLAN</td><td>3795-20</td><td>Straight</td></tr><tr><td>Heifetz Microclips 12mmx2.25mm</td><td>SCANLAN</td><td>3795-28</td><td>Curved</td></tr><tr><td>Heifetz Microclips 8mmx1.75mm&amp;nbsp;</td><td>SCANLAN</td><td>3795-14</td><td>Straight</td></tr><tr><td>Heifetz Microclips 8mmx1.75mm&amp;nbsp;</td><td>SCANLAN</td><td>3795-18</td><td>Curved</td></tr><tr><td>Inflation device&amp;nbsp;</td><td>Merit Medical</td><td>IN4130</td><td>BasixCOMPAK with Analog Display</td></tr><tr><td>Isoflurane</td><td>MFR VETONE</td><td>SKU-501017</td><td>100mL, Liquid for Inhalation</td></tr><tr><td>Microsurgery kit containing tweezers and forceps</td><td>Customized</td><td></td><td></td></tr><tr><td>Oxygen, E-tank</td><td>Medline</td><td>MDM1630</td><td></td></tr><tr><td>Sharpened Guidewire</td><td>Customized</td><td></td><td></td></tr><tr><td>Sterile Glad Press-n-Seal</td><td>Glad</td><td>SKU # PSS-140</td><td>ETO Exposed</td></tr><tr><td>Sterile Nonwoven Gauze Sponges 2s 4ply 4x4&amp;nbsp;</td><td>Medline</td><td>SKU PRM21444H</td><td>100Ct</td></tr><tr><td>Surgical microscope&amp;nbsp;</td><td>Zeiss</td><td>S100 / OPMI pico</td><td></td></tr><tr><td>Surgical Scrub &amp;amp; Handwash</td><td>Vetoquinol</td><td>NDC: 17030-003-16</td><td>16 oz</td></tr><tr><td>Ultrasound Gel</td><td>Medline</td><td>CTR001305</td><td>8.5 oz, 12/CS</td></tr><tr><td>Ultrasound system</td><td>Siemens</td><td>ACUSON Antares</td><td>3-D, Color, Continuous wave (CW), Pulsed wave (PW), Power CW/CW Spectral</td></tr><tr><td>Venoplasty Balloon</td><td>Customized</td><td></td><td></td></tr></tbody></table>

rodent inferior vena cava venoplasty balloon model

Balloon venoplasty is a commonly used clinical technique to treat deep vein stenosis and occlusion as a consequence of trauma, congenital anatomic abnormalities, acute deep vein thrombosis (DVT), or stenting. Chronic deep venous obstruction is histopathologically characterized by thrombosis, fibrosis, or both. Currently, no direct treatment is available to target these pathological processes. Therefore, a reliable in vivo animal model to test novel interventions is necessary. The rodent survival inferior vena cava (IVC) venoplasty balloon model (VBM) allows the study of balloon venoplasty in non-thrombotic and post-thrombotic conditions across multiple time points. The local and systemic effect of coated and uncoated venoplasty balloons can be quantified via tissue, thrombus, and blood assays such as real-time polymerase chain reaction (RT-PCR), western blot, enzyme-linked immunosorbent assay (ELISA), zymography, vein wall and thrombus cellular analysis, whole blood and plasma assays, and histological analysis. The VBM is reproducible, replicates surgical human interventions, can identify local vein wall-thrombi protein changes, and allows multiple analyses from the same sample, decreasing the number of animals required per group.

The VBM is a model that evaluates the effect of venoplasty balloons in complete hemostasis control. First, we quantified the IVC (mid-infrarenal section) diameter, as shown in Figure 1, for correct balloon sizing. Second, the developed step-by-step microsurgical technique is illustrated in Figure 2, and we also show examples of the critical steps in Figure 3. Notice the retrograde approach used for the IVC canulation with the sharpened guide wire.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66820/66820fig02.jpg" /> 
Figure 2: Microsurgical technique for the testing of venoplasty balloons in rats. The technique used is described here. <a href="https://www.jove.com/files/ftp_upload/66820/66820fig02large.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/66820/66820fig03.jpg" /> 
Figure 3: In vivo images of the deployment technique. (A) Hemostatic control. (B) U-stitch on infrarenal IVC. (C) Wire insertion through U-stitch with 3 mm sirolimus-coated balloon.&#160;(D) Balloon venoplasty retrograde deployment (Sirolimus coated). Abbreviations: IVC = inferior vena cava; AO = Aorta; CBV = Coated balloon venoplasty.&#160;<a href="https://www.jove.com/files/ftp_upload/66820/66820fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Other normal venous vessel measurements and distinctions between the IVC and the aorta are highlighted in Figure 4. Thrombotic ultrasound findings are included in Figure 5. Finally, a comparison between the diameter of preoperative ultrasound and post-thrombotic measurements can be observed in Figure 6. Average pulsed-wave Doppler flow velocity (mm/s) in the inferior vena cava before and after clip placement of the vessel is shown in Figure 7.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66820/66820fig04.jpg" /> 
Figure 4: Representative transabdominal duplex images, with examples of a transverse IVC diameter calculation. (A) Proximal IVC. (B) Mid-infrarenal IVC. (C) Distal IVC. Color Doppler analysis of (D) Mid-infrarenal IVC and (E) Abdominal aorta. <a href="https://www.jove.com/files/ftp_upload/66820/66820fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66820/66820fig05.jpg" /> 
Figure 5. Images showing pre- and post-clip placement images. (A)&#160;Post clip placement ultrasound findings confirming IVC occlusion. (B) Proximal and distal aorta flow. (C) Proximal IVC flow. (D) Common iliac veins. <a href="https://www.jove.com/files/ftp_upload/66820/66820fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/66820/66820fig06.jpg" /> 
Figure 6. Ultrasound images. Ultrasound changes before and after 15 min, 30 min, and 45 min, confirming IVC occlusion. <a href="https://www.jove.com/files/ftp_upload/66820/66820fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/66820/66820fig07.jpg" /> 
Figure 7: Average pulsed-wave Doppler flow velocity. Mean flow velocity (mm/s) in the inferior vena cava before and after clip placement (**: P &#8804; 0.01, Mann-Whitney test performed). Error bars show standard error. <a href="https://www.jove.com/files/ftp_upload/66820/66820fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To analyze the outcome of different venoplasty interventions with novel coatings, we recommend the measurement of effective proteins which can demonstrate the on-target effect of the drug of interest. For the sirolimus-coated venoplasty balloon used here, we measured phosphorylated AKT (p-AKT) protein levels of rat IVC lysates, confirming mTOR pathway inhibition since p-AKT is a downstream protein that decreases in response to sirolimus14,15. We can observe in Figure 8 how, after coated-balloon venoplasty (CBV), p-AKT is significantly decreased compared to the sham group.
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/66820/66820fig08.jpg" /> 
Figure 8: Protein p-AKT levels. Protein levels of rat IVC lysates of sham vs. coated-balloon venoplasty (CBV) in normal conditions confirming on target effect with mTOR pathway inhibition using sirolimus venoplasty coated balloons. (**: P &#8804; 0.01, Mann-Whitney test performed). Error bars show standard error.&#160;<a href="https://www.jove.com/files/ftp_upload/66820/66820fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Author Spotlight: Utilizing Venoplasty Balloon Model in Rodents to Simulate Surgical Interventions for Deep Veins

The protocol presents a survival rodent model to test venoplasty balloon (VB) interventions in non-thrombotic, thrombotic, and post-thrombotic deep veins.

Rodent Inferior Vena Cava Venoplasty Balloon Model

Balloon venoplasty is a commonly used clinical technique to treat deep vein stenosis and occlusion as a consequence of trauma, congenital anatomic ...

rodent-inferior-vena-cava-venoplasty-balloon-model

Nanyang Technological University

Research

JoVE Journal

Medicine

551 Views.  University of Michigan. The protocol presents a survival rodent model to test venoplasty balloon (VB) interventions in non-thrombotic, thrombotic, and post-thrombotic deep veins.

Video: Author Spotlight: Utilizing Venoplasty Balloon Model in Rodents to Simulate Surgical Interventions for Deep Veins

Electrolytic Inferior Vena Cava Model (EIM) of Venous Thrombosis

Animal models serve a vital role in deep venous thrombosis (DVT) research in order to study thrombus formation, thrombus resolution and to test potential therapeutic compounds (1). New compounds to be utilized in the treatment and prevention of DVT are currently being developed. The delivery of potential therapeutic antagonist compounds to an affected thrombosed vein has been problematic. In the context of therapeutic applications, a model that uses partial stasis and consistently generates thrombi within a major vein has been recently established. The Electrolytic Inferior vena cava Model (EIM) is mouse model of DVT that permits thrombus formation in the presence of continuous blood flow. This model allows therapeutic agents to be in contact with the thrombus in a dynamic fashion, and is more sensitive than other models of DVT (1). In addition, this thrombosis model closely simulates clinical situations of thrombus formation and is ideal to study venous endothelial cell activation, leukocyte migration, venous thrombogenesis, and to test therapeutic applications (1). The EIM model is technically simple, easily reproducible, creates consistent thrombi sizes and allows for a large sample (i.e. thrombus and vein wall) which is required for analytical purposes.

Electrolytic Inferior Vena Cava Model EIM of Venous Thrombosis

The electrolytic induction of endothelial activation to the internal surface of the Inferior Vena Cava results in venous type thrombus formation due to endothelial activation and partial blood stasis, two components of Virchow's triad.

Animal models serve a vital role in deep venous thrombosis (DVT) research in order to study thrombus formation, thrombus resolution and to test potential ...

Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. The long-term clinical results showed excellent patency rates, however, with significant incidence of stenosis. To investigate the cellular and molecular mechanisms of vascular neotissue formation and prevent stenosis development in tissue engineered vascular grafts (TEVGs), we developed a mouse model of the graft with approximately 1 mm internal diameter. First, the TEVGs were assembled from biodegradable tubular scaffolds fabricated from a polyglycolic acid nonwoven felt mesh coated with &#949;-caprolactone and L-lactide copolymer. The scaffolds were then placed in a lyophilizer, vacuumed for 24 hr, and stored in a desiccator until cell seeding. Second, bone marrow was collected from donor mice and mononuclear cells were isolated by density gradient centrifugation. Third, approximately one million cells were seeded on a scaffold and incubated O/N. Finally, the seeded scaffolds were then implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. The implanted grafts demonstrated excellent patency (&#62;90%) without evidence of thromboembolic complications or aneurysmal formation. This murine model will aid us in understanding and quantifying the cellular and molecular mechanisms of neotissue formation in the TEVG.

To improve our knowledge of cellular and molecular neotissue formation, a murine model of the TEVG was recently developed. The grafts were implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. This model achieves similar results to those achieved in our clinical investigation, but over a far shortened time-course.

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. ...

Vascular Balloon Injury and Intraluminal Administration in Rat Carotid Artery

The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and cellular mechanisms involved in vascular smooth muscle dedifferentiation, neointima formation and vascular remodeling. Male Sprague-Dawley rats are the most frequently employed animals for this model. Female rats are not preferred as female hormones are protective against vascular diseases and thus introduce a variation into this procedure. The left carotid is typically injured with the right carotid serving as a negative control. Left carotid injury is caused by the inflated balloon that denudes the endothelium and distends the vessel wall. Following injury, potential therapeutic strategies such as the use of pharmacological compounds and either gene or shRNA transfer can be evaluated. Typically for gene or shRNA transfer, the injured section of the vessel lumen is locally transduced for 30 min with viral particles encoding either a protein or shRNA for delivery and expression in the injured vessel wall. Neointimal thickening representing proliferative vascular smooth muscle cells usually peaks at 2 weeks after injury. Vessels are mostly harvested at this time point for cellular and molecular analysis of cell signaling pathways as well as gene and protein expression. Vessels can also be harvested at earlier time points to determine the onset of expression and/or activation of a specific protein or pathway, depending on the experimental aims intended. Vessels can be characterized and evaluated using histological staining, immunohistochemistry, protein/mRNA assays, and activity assays. The intact right carotid artery from the same animal is an ideal internal control. Injury-induced changes in molecular and cellular parameters can be evaluated by comparing the injured artery to the internal right control artery. Likewise, therapeutic modalities can be evaluated by comparing the injured and treated artery to the control injured only artery.

This protocol uses a balloon catheter to cause an intraluminal injury on the rat carotid artery and henceforth elicit neointimal hyperplasia. This is a well-established model for studying the mechanisms of vascular remodeling in response to injury. It is also widely used to determine the validity of potential therapeutic approaches.

The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and ...

A Rat Carotid Balloon Injury Model to Test Anti-vascular Remodeling Therapeutics

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is also a valuable model system to test, and to evaluate therapeutics and drugs that negate maladaptive remodeling in the vessel. The injury, or barotrauma, in the vessel lumen caused by an inflated balloon via an inserted catheter induces subsequent neointimal growth, often leading to hyperplasia or thickening of the vessel wall that narrows, or obstructs the lumen. The method described here is sufficiently sensitive, and the results can be obtained in relatively short time (2 weeks after the surgery). The efficacy of the drug or therapeutic against the induced-remodeling can be evaluated either by the post-mortem pathological and histomorphological analysis, or by ultrasound sonography in live animals. In addition, this model system has also been used to determine the therapeutic window or the time course of the administered drug. These studies can leadto the development of a better administrative strategy and a better therapeutic outcome. The procedure described here provides a tool for translational studies that bring drug and therapeutic candidates from bench research to clinical applications.

The rat carotid balloon injury model described below allows researchers to evaluate drugs or therapeutics that negate injury-induced arterial hyperplasia. Detailed pre-surgical preparation, surgical procedure, and post-surgical cares of the animal are described.

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is ...

Stenosis of the Inferior Vena Cava: A Murine Model of Deep Vein Thrombosis

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus formation in veins remain obscure. Lack of mobility (e.g., after surgery or long-haul flights) is one of the main factors leading to DVT. The pathophysiological consequence of the lack of mobility is blood flow stagnation in venous valves. Here, a model is described that mimics such flow disturbance as a thrombosis-driving factor. In this model, partial flow restriction (stenosis) in the inferior vena cava (IVC) is created. Closure of about 90% of the IVC lumen for 48 h results in development of thrombi structurally similar to those in humans. The similarities are: i) most of the thrombus volume is red, i.e., consists of red blood cells and fibrin, ii) presence of a white part (lines of Zahn), iii) non-denuded endothelial monolayer, iv) elevated plasma D-Dimer levels, and v) possibility to prevent thrombosis by low molecular weight heparin. Limitations include variable size of thrombi and the fact that a certain percentage of wild-type mice (0 - 35%) may not produce a thrombus. In addition to visual observation and measurement, thrombi may be visualized by non-invasive technologies, such as ultrasonography, which allows for monitoring the dynamics of thrombus development. At shorter time points (1 - 6 h), intravital microscopy may be applied to directly observe events (e.g., recruitment of cells to the vessel wall) preceding thrombus formation. Use of this method by several teams around the world has made it possible to uncover basic mechanisms of DVT initiation and identify potential targets that might be beneficial for its prevention.

We describe here stenosis in the inferior vena cava as a murine model of deep vein thrombosis. This model recapitulates blood flow restriction, one of the major triggers of venous thrombosis in humans.

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus ...

Immunology and Infection

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 ...

Patch Angioplasty in the Rat Aorta or Inferior Vena Cava

Pericardial patches are commonly used in vascular surgery to close vessels. To facilitate studies of the neointimal hyperplasia that forms on the patch, we developed a rat model of patch angioplasty that can be used in either a vein or an artery, creating a patch venoplasty or a patch arterioplasty, respectively. Technical aspects of this model are discussed. The infra-renal IVC or aorta are dissected and then clamped proximally and distally. A 3 mm venotomy or arteriotomy is performed in the infrarenal inferior vena cava or aorta of 6 to 8 week-old Wistar rats. A bovine pericardial patch (3 mm x 1.5 mm x 0.6 mm) is then used to close the site using a 10-0 nylon suture. Compared to arterial patches, venous patches show increased neointimal thickness on postoperative day 7. This novel model of pericardial patch angioplasty can be used to examine neointimal hyperplasia on vascular biomaterials, as well as to compare the differences between the arterial and venous environments.

We have established a model of pericardial patch angioplasty that can be used in either small-diameter veins or arteries. This model can be used to compare venous and arterial neointimal hyperplasia formation.

Pericardial patches are commonly used in vascular surgery to close vessels. To facilitate studies of the neointimal hyperplasia that forms on the patch, ...

A Rat Model of Orthotopic Liver Transplantation Using a Novel Magnetic Anastomosis Technique for Suprahepatic Vena Cava Reconstruction

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep learning curve. The introduction of the cuff technique for anastomosis of the portal vein (PV) and infrahepatic vena cava (IHVC) has significantly simplified the transplant procedure in rats. However, due to the short anterior wall of the recipients' suprahepatic vena cava (SHVC), the cuff technique is very difficult to use for the reconstruction of the SHVC. Most researchers in this field still use the hand-suture technique for SHVC reconstruction, which makes it the bottleneck step in rat orthotopic liver transplantation. The magnetic anastomosis technique (i.e., magnamosis) is a method of connecting two vessels using the attractive force between two magnets. Our recent study has shown that the magnetic anastomosis technique is superior to the hand-suture technique for SHVC reconstruction in rats. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using the novel magnetic anastomosis technique. In this model, the reconstruction of the PV and IHVC was performed by the standard cuff technique, while the reconstruction of the bile duct (BD) was performed by a stent technique. The hepatic re-arterialization was not performed. The magnetic anastomosis technique made SHVC reconstruction much easier and significantly shortened the anphepatic phase. After a reasonable learning curve, even researchers without advanced microsurgical skills can produce reliable and reproducible results using this rat model of OLT.

The reconstruction of the suprahepatic vena cava (SHVC) remains a difficult step in rat orthotopic liver transplantation. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using a novel magnetic anastomosis technique.

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep ...

Murine Model of Central Venous Stenosis using Aortocaval Fistula with an Outflow Stenosis

Central venous stenosis is an important entity contributing to arteriovenous fistula (AVF) failure. A murine AVF model was modified to create a partial ligation of the inferior vena cava (IVC) in the outflow of the fistula, mimicking central venous stenosis. Technical aspects of this model are introduced. The aorta and IVC are exposed, following an abdominal incision. The infra-renal aorta and IVC are dissected for proximal clamping, and the distal aorta is exposed for puncture. The IVC at the midpoint between the left renal vein and the aortic bifurcation is carefully dissected to place an 8-0 suture beneath the IVC. After clamping the aorta and IVC, an AVF is created by puncturing the infra-renal aorta through both walls into the IVC with a 25 G needle, followed by ligating a 22 G intra-venous (IV) catheter and IVC together. The catheter is then removed, creating a reproducible venous stenosis without occlusion. The aorta and IVC are unclamped after confirming primary hemostasis. This novel model of central vein stenosis is easy to perform, reproducible, and will facilitate studies on AVF failure.

An aortocaval fistula was created by puncturing the murine infra-renal aorta through both walls into the inferior vena cava and was followed by creation of a stenosis in its outflow via partial ligation of the inferior vena cava. This reproducible model can be used to study central venous stenosis.

Central venous stenosis is an important entity contributing to arteriovenous fistula (AVF) failure. A murine AVF model was modified to create a partial ...

A Rabbit Venous Interposition Model Mimicking Revascularization Surgery using Vein Grafts to Assess Intimal Hyperplasia under Arterial Blood Pressure

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor because of the acceleration of intimal hyperplasia due to the exposure to arterial blood pressure. The present protocol is designed for the establishment of experimental venous intimal hyperplasia by interposing rabbit jugular veins to the ipsilateral carotid arteries. The protocol does not require surgical procedures deep in the body trunk and the extent of the incision is limited, which is less invasive for the animals, allowing long-term observation after implantation. This simple procedure enables researchers to investigate strategies to attenuate the progression of intimal hyperplasia of the implanted vein grafts. Using this protocol, we reported the effects transduction of microRNA-145 (miR-145), which is known to control the phenotype of vascular smooth muscle cells (VSMCs) from the proliferative to the contractile state, into harvested vein grafts. We confirmed the attenuation of intimal hyperplasia of vein grafts by transducing miR-145 before implantation surgery through the phenotype change of the VSMCs. Here we report a less invasive experimental platform to investigate the strategies that can be used to attenuate intimal hyperplasia of vein grafts in revascularization surgeries.

The present protocol aims to experimentally create venous intimal hyperplasia by subjecting veins to arterial blood pressure for developing strategies to attenuate venous intimal hyperplasia following revascularization surgery using vein grafts.

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor ...