This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (No. 2020R1A4A3079570), the Ministry of Education (No. 2021R1I1A1A01051425), and the Industrial Strategic Technology Development Program (No. 20014873) funded by the Ministry of Trade, Industry &#38; Energy, Republic of Korea.

All animal research procedures were approved and performed in accordance with the Laboratory Animals Welfare Act, the Guide for the Care and Use of Laboratory Animals, and the Guidelines and Policies for Animal Experiments provided by the Institutional Animal Care and Use Committee (IACUC) at the College of Medicine of The Catholic University of Korea (approval number: CUMC-2021-0176-05). The present study utilized 3 month old male New Zealand white (NZW) rabbits weighing 3.5-4.0 kg, which were maintained under standard conditions in individual cages. The rabbits were fed either a normal diet or a 0.5% cholesterol-enriched diet supplemented with 50,000 U of vitamin D2 (see Table of Materials). The experimental design and analysis methods for the induction of the AVS rabbit model are depicted in Figure 1.
1. Preparation for the operation
<ol>
	<li>Ensure that all the medical and surgical instruments (see Table of Materials) are sterilized at the beginning of the operation.</li>
	<li>Prepare the dilation balloon catheter set following the steps below.
	<ol>
		<li>Connect the in-deflation device filled with a mixture of saline and commercially available contrast medium (1:1) to the luer lock part of the balloon catheter (see Table of Materials).</li>
		<li>Fill the balloon with inflation solution, and remove any air from the balloon catheter. 
		&#8203;NOTE: For the present study, the inflation solution consisted of 30% iodixanol with 0.9% saline (see Table of Materials).</li>
		<li>Verify proper balloon inflation by purging the balloon lumen with the inflation solution.</li>
	</ol>
	</li>
</ol>
2. Surgical procedure for the aortic valve injury
<ol>
	<li>Administer an intramuscular injection of tiletamine &#38; zolazepam (15 mg/kg) and xylazine (5 mg/kg) (see Table of Materials) to anesthetize the animal. 
	NOTE: Before administering the anesthesia, the rabbits were pre-treated with a subcutaneous glycopyrrolate injection (0.05 mg/kg) as a pre-anesthetic anticholinergic agent. The adequate anesthesia level was determined by a combination of criteria, including a lack of response to a toe pinch and a steady respiratory rate.</li>
	<li>Insert a 24 G intravenous (IV) catheter into the marginal auricular vein, and connect an infusion set with heparinized saline (100 U/kg heparin).</li>
	<li>Connect the rabbit with a multiparameter veterinary monitor (see Table of Materials) to continuously monitor the vital signs, such as the oxygen saturation signal (SpO2), temperature, and blood pressure. 
	NOTE: For the SpO2 monitoring, attach the SpO2 sensor to the rabbit&#39;s tongue. For the temperature monitoring, insert the probe into the rabbit&#39;s rectum. For the blood pressure monitoring, place the cuff on the forelimb.</li>
	<li>Place the rabbit in a supine position on an operating table equipped with a C-arm fluoroscopy (see Table of Materials), and remove the hair from the ventral neck area using animal hair clippers (Figure 2A).</li>
	<li>Sterilize the incision area with iodine, and cover the rabbit with surgical towels.</li>
	<li>Position the rabbit&#39;s heart at the center of the C-arm image. 
	NOTE: All researchers must wear protective gear with attached thermoluminescent dosimeters (TLDs) to reduce radiation exposure while performing the C-arm-guided surgery.
	<ol>
		<li>Turn on the C-arm, and select the fluoroscopic mode for cardiac imaging.</li>
		<li>Adjust the position of the rabbit to ensure that the heart is at the center of the imaging field.</li>
	</ol>
	</li>
	<li>Make a longitudinal incision of approximately 3 cm in the skin of the neck, and use surgical scissors to cut the fascia and fat tissue.</li>
	<li>Expose the left common carotid artery (LCCA) by carefully separating the muscles until approximately 3-3.5 cm of the LCCA is exposed (Figure 2B).</li>
	<li>Ligate the LCCA with a 3-0 silk suture (see Table of Materials) at the top and end of the exposed LCCA to stop the blood flow.</li>
	<li>Insert a 22 G IV catheter into the LCCA, and introduce a guide wire (0.016 in, see Table of Materials) into the left ventricle (LV) through the IV catheter, ensuring that the tip of the catheter is properly positioned in the imaging field of the C-arm. 
	NOTE: When inserting the IV catheter, carefully loosen the ligature suture on the way down to the aortic valve to allow for the advancement of the catheter.</li>
	<li>Withdraw the IV catheter, leaving the guide wire, and place a 4-F sheath (see Table of Materials) over the guide wire into the LCCA to introduce the balloon catheter (Figure 2C). 
	NOTE: After replacing the IV catheter with the sheath, any trapped air should be removed from the sheath device.</li>
	<li>Carefully insert the 8 mm balloon catheter over the guidewire into the aortic valve under C-arm fluoroscopic guidance (Figure 2D).</li>
	<li>Place the balloon catheter tip approximately 1-2 cm distal to the aortic valve, and inflate the balloon by purging the inflation solution with a pressure inflator at 6 atm.</li>
	<li>Advance the balloon into the LV apex, and pull it back into the LV outlet. Repeat this procedure five times, and then deflate the balloon (Figure 2E, F).</li>
	<li>Repeat steps 2.8-2.9 three times to ensure adequate valve injury.</li>
	<li>Withdraw the balloon catheter and guidewire. Slowly remove the sheath from the LCCA, and immediately tie the LCCA with the suture on the way down to the aortic valve.</li>
	<li>Clean the incision area with saline to remove the blood clots, and inspect the punctured site for arterial bleeding.</li>
	<li>Close the muscle and skin with a 3-0 non-absorbable suture, and sterilize all the sides of the wound with iodine.</li>
</ol>
3. Post-operative care
<ol>
	<li>Remove the monitoring patches and clips, and keep the rabbit in an intensive care incubator. 
	NOTE: After surgery, the rabbits were closely observed for 1 day in an intensive care incubator and then moved to a home cage.</li>
	<li>Manage post-operative pain with 5 mg/kg tramadol and 3 mg/kg ketoprofen and administer antibiotics (4 mg/kg gentamicin) twice daily for 3 days via subcutaneous injection (see Table of Materials). 
	NOTE: Post-operative pain management should adhere to veterinary and IACUC guidelines (e.g., opioid, NSAID, local anesthetic or combination).</li>
	<li>Feed a 0.5% cholesterol-enriched diet with 50,000 U of vitamin D2 (HC + VitD2) for 8 weeks.</li>
</ol>
4. Echocardiography
<ol>
	<li>After 8 weeks of balloon injury, anesthetize the rabbit using the same procedure described in step 2.1.</li>
	<li>Visualize the aortic valves using two-dimensional transthoracic views, and record M-mode images in short-axis and long-axis views.
	<ol>
		<li>Place the rabbit in a supine position on an echo table.</li>
		<li>Shave the chest area using clippers and hair removal cream.</li>
		<li>Apply ultrasound transducer gel (see Table of Materials) to the chest.</li>
		<li>Adjust the transducer to obtain the parasternal long-axis view and the parasternal short-axis view of the aortic valve.</li>
		<li>Use M-mode imaging to record images of the aortic valve in both long-axis and short-axis views, and save the images for later analysis.</li>
	</ol>
	</li>
</ol>
5. Histological analysis
<ol>
	<li>After the echocardiography, euthanize the rabbit by administering an intravenous injection of potassium chloride (KCl, 3 g/20 mL, 1 mL).</li>
	<li>Open the thoracic cavity, harvest the heart with the ascending aorta7, and place it on ice in phosphate-buffered saline (PBS).</li>
	<li>Immediately immerse the heart in a 4% paraformaldehyde (PFA) solution, and embed it in a paraffin block (see Table of Materials).</li>
	<li>Cut the paraffin-embedded heart block into 4 &#181;m thick sections using a microtome, and stain the sections with Masson&#39;s trichrome (MT), Alizarin Red, and von Kossa (see Table of Materials) to assess the collagen deposition and valve calcification, respectively8,9.</li>
</ol>

<ol>
	<li>Aliev, G., Burnstock, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Watanabe+rabbits+with+heritable+hypercholesterolaemia:+A+model+of+atherosclerosis.">Watanabe rabbits with heritable hypercholesterolaemia: A model of atherosclerosis.</a> Histology and Histopathology. 13 (3), 797-817 (1998).</li><li>Cimini, M., Boughner, D. R., Ronald, J. A., Aldington, L., Rogers, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+aortic+valve+sclerosis+in+a+rabbit+model+of+atherosclerosis:+An+immunohistochemical+and+histological+study.">Development of aortic valve sclerosis in a rabbit model of atherosclerosis: An immunohistochemical and histological study.</a> Journal of Heart Valve Disease. 14 (3), 365-375 (2005).</li><li>Drolet, M. C., Couet, J., Arsenault, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+aortic+valve+sclerosis+or+stenosis+in+rabbits:+role+of+cholesterol+and+calcium.">Development of aortic valve sclerosis or stenosis in rabbits: role of cholesterol and calcium.</a> Journal of Heart Valve Disease. 17 (4), 381-387 (2008).</li><li>Sider, K. L., Blaser, M. C., Simmons, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+calcific+aortic+valve+disease.">Animal models of calcific aortic valve disease.</a> International Journal of Inflammation. 2011, 364310 (2011).</li><li>Honda, S., 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=A+novel+mouse+model+of+aortic+valve+stenosis+induced+by+direct+wire+injury.">A novel mouse model of aortic valve stenosis induced by direct wire injury.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 34 (2), 270-278 (2014).</li><li>Niepmann, S. T., 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=Graded+murine+wire-induced+aortic+valve+stenosis+model+mimics+human+functional+and+morphological+disease+phenotype.">Graded murine wire-induced aortic valve stenosis model mimics human functional and morphological disease phenotype.</a> Clinical Research in Cardiology. 108 (8), 847-856 (2019).</li><li>Robbins, N., Thompson, A., Mann, A., Blomkalns, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+excision+of+murine+aorta;+A+versatile+technique+in+the+study+of+cardiovascular+disease.">Isolation and excision of murine aorta; A versatile technique in the study of cardiovascular disease.</a> Journal of Visualized Experiments. (93), e52172 (2014).</li><li>Wirrig, E. E., Gomez, M. V., Hinton, R. B., Yutzey, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=COX2+inhibition+reduces+aortic+valve+calcification+in+vivo.">COX2 inhibition reduces aortic valve calcification in vivo.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 35 (4), 938-947 (2015).</li><li>Jung, S. 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=Spatiotemporal+dynamics+of+macrophage+heterogeneity+and+a+potential+function+of+Trem2(hi)+macrophages+in+infarcted+hearts.">Spatiotemporal dynamics of macrophage heterogeneity and a potential function of Trem2(hi) macrophages in infarcted hearts.</a> Nature Communications. 13 (1), 4580 (2022).</li><li>Freeman, R. V., Otto, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrum+of+calcific+aortic+valve+disease:+Pathogenesis,+disease+progression,+and+treatment+strategies.">Spectrum of calcific aortic valve disease: Pathogenesis, disease progression, and treatment strategies.</a> Circulation. 111 (24), 3316-3326 (2005).</li><li>Lindman, B. R., 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=Calcific+aortic+stenosis.">Calcific aortic stenosis.</a> Nature Reviews Disease Primers. 2, 16006 (2016).</li><li>Cuniberti, L. 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=Development+of+mild+aortic+valve+stenosis+in+a+rabbit+model+of+hypertension.">Development of mild aortic valve stenosis in a rabbit model of hypertension.</a> Journal of the American College of Cardiology. 47 (11), 2303-2309 (2006).</li><li>Marechaux, S., 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=Identification+of+tissue+factor+in+experimental+aortic+valve+sclerosis.">Identification of tissue factor in experimental aortic valve sclerosis.</a> Cardiovascular Pathology. 18 (2), 67-76 (2009).</li><li>Hara, T., 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=Progression+of+calcific+aortic+valve+sclerosis+in+WHHLMI+rabbits.">Progression of calcific aortic valve sclerosis in WHHLMI rabbits.</a> Atherosclerosis. 273, 8-14 (2018).</li></ol>

The authors have no conflicts of interest to declare with this work.

Animal AVS models are commonly used to study the pathological aspects of AVS, including the initiation and progression of AVS. This protocol introduces a new rabbit AVS model induced by a direct balloon injury to the aortic valve. In this study, the aortic valve injury model showed significant leaflet thickening and calcification. Compared to the mild AVS model induced by dietary supplementation, the aortic valve in the direct balloon injury model was selectively injured, leading to thickened cusps and restricted motion,...

It is increasingly recognized that the use of appropriate animal models can contribute to a better understanding of the pathological mechanisms underlying aortic valve stenosis (AVS) due to the lack of access to reliable sources of diseased human aortic valves associated with the progression of aortic stenosis (AS). Among the various animal models for studying AVS, rabbits are one of the most commonly used large-animal AVS models, and the AVS rabbit model is induced either through cholesterol/vitamin D2 supplementation or genetic manipulation1,2,3,4.
Although rabbit AVS models have provided significant insight into the development and progression of AVS, it still remains challenging to induce AVS consistently and reproducibly, as seen in our preliminary experiments.
In addition to diet-induced and genetically susceptible animal models, a new model of AVS has been established through direct mechanical injury in mice5,6. The mechanical injury model successfully induces aortic stenosis and represents a simple and reproducible AVS model in wild-type mice. To the best of our knowledge, there have been no prior studies examining the effects of a mechanical injury on the aortic valve in rabbit models. Thus, this study provides a new procedure for inducing AVS in male New Zealand white rabbits through a direct balloon injury to the aortic valve, which can accurately mimic the condition of valvular aortic stenosis. This protocol includes step-by-step descriptions of the preparation, the surgical procedure, and the post-operative care, which are useful for inducing reproducible AVS rabbit models.

<table><tbody><tr><td>3-0 Silk suture</td><td>AILEE</td><td>SK312</td><td></td></tr><tr><td>4% paraformaldehyde(PFA)</td><td>Intron</td><td>IBS-BP031-2</td><td></td></tr><tr><td>Alizarin red Solution</td><td>Millpore</td><td>TMS-008-C</td><td></td></tr><tr><td>ASAHI SION BLUE&amp;nbsp;</td><td>ASAHI</td><td></td><td>Guide wire</td></tr><tr><td>Back Table Cover</td><td>Yuhan kimberly</td><td>80101-30</td><td></td></tr><tr><td>Balloon In-deflation Device</td><td>Demax Medical</td><td>DID30s</td><td></td></tr><tr><td>Bionet Veterinary monitor</td><td>BIONET</td><td>BM3 VET</td><td></td></tr><tr><td>C-Arm</td><td>SIEMENS Healthcare GmbH</td><td>Cios alpha</td><td></td></tr><tr><td>Certified Rabbit Diet</td><td>Purina</td><td>5322</td><td>4.7% Hydrogenated Coconut Oil, 0.5% Cholesterol, &amp;amp; 1% Molasse</td></tr><tr><td>Curadle Smart Incubator</td><td>Autoelex</td><td>CS-CV206</td><td>Intensive Care Unit (ICU)</td></tr><tr><td>Ergocalciferol</td><td>Sigma-aldrich&amp;nbsp;</td><td>E5750</td><td>Vitamin D2</td></tr><tr><td>Fechtner conjunctiva forceps titanium</td><td>WORLD PRECISSION Instrument</td><td>WP1820</td><td></td></tr><tr><td>Forceps</td><td>HEBU</td><td>HB203</td><td></td></tr><tr><td>Gentamicin</td><td>Shin Poong</td><td></td><td></td></tr><tr><td>Glycopyrrolate&amp;nbsp;</td><td>SamChunDang</td><td></td><td></td></tr><tr><td>Greenflex NS</td><td>DAI HAN PHARM</td><td></td><td>Normal saline 500 mL</td></tr><tr><td>Hematoxylin solution</td><td>Sigma-aldrich&amp;nbsp;</td><td>HT1079-1 SET</td><td></td></tr><tr><td>Heparin</td><td>JW pharmaceutical</td><td></td><td>25,000 U</td></tr><tr><td>Infusion set for single use</td><td>SWOON MEDICAL</td><td></td><td></td></tr><tr><td>Iodine</td><td>Green pharmaceutical</td><td></td><td></td></tr><tr><td>Iodixanol</td><td>GE Healthcare</td><td>Visipaque</td><td>Inflation solution (contrast agent)</td></tr><tr><td>IV catheter 22 G</td><td>BD&amp;nbsp;</td><td>382423</td><td></td></tr><tr><td>IV catheter 24 G</td><td>BD</td><td>382412</td><td></td></tr><tr><td>Ketoprofen</td><td>SamChunDang</td><td></td><td></td></tr><tr><td>Luer-Lok syringe 10 mL</td><td>Becton Dickinson Medical</td><td></td><td></td></tr><tr><td>Luer-Lok syringe 3 mL</td><td>Becton Dickinson Medical</td><td></td><td></td></tr><tr><td>Microscope</td><td>OLYMPUS</td><td>SZ61</td><td></td></tr><tr><td>Microtome</td><td>ThermoFisher Scientific</td><td>HM 325</td><td></td></tr><tr><td>MT stain kit</td><td>Sigma-aldrich</td><td>HT15-1kt</td><td></td></tr><tr><td>Needel holder</td><td>Solco</td><td>009-1304</td><td></td></tr><tr><td>Needle Holder with Lock and Suture</td><td>JEUNGDO BIO &amp;amp; PLANT</td><td>H-1222-18</td><td></td></tr><tr><td>Paraffin</td><td>LK LABKOREA</td><td>H06-660-107</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>10010-023</td><td></td></tr><tr><td>Potassium chloride 40</td><td>Daihan Pharm</td><td></td><td>KCl</td></tr><tr><td>Prelude Ideal Hydrophilic Sheath</td><td>MERIT MEDICAL</td><td>PID4F11018SS</td><td>Sheath 4F</td></tr><tr><td>PTA Balloon Dilatation catheter</td><td>Boston Scientific</td><td>H749-3903280208-0</td><td>Balloon catheter 8.0 mm</td></tr><tr><td>Rompun</td><td>Elanco</td><td></td><td>Xylaxine</td></tr><tr><td>sterile Gauze</td><td>DAE HAN Medical</td><td></td><td>10 cm x 20 cm&amp;nbsp;</td></tr><tr><td>Surgical Gloves</td><td>Ansell</td><td>Ansell</td><td></td></tr><tr><td>Surgical Gown</td><td>Yuhan kimberly</td><td>90002-02</td><td></td></tr><tr><td>Surgical Scissors</td><td>Nopa, Germany</td><td>AC020/16</td><td></td></tr><tr><td>Surgical Tape</td><td>3M micopore</td><td>1530-1</td><td></td></tr><tr><td>Syringe 1 mL</td><td>Shin Chang Medical</td><td></td><td></td></tr><tr><td>Syringe 10 mL</td><td>Shin Chang Medical</td><td></td><td></td></tr><tr><td>Tissue cassette</td><td>Scilav korea</td><td>Cas3003</td><td></td></tr><tr><td>Transducer gel&amp;nbsp;</td><td>SUNGHEUNG</td><td>SH102</td><td></td></tr><tr><td>Tridol</td><td>Yuhan Corp.</td><td></td><td>Tramadol HCl</td></tr><tr><td>Ultrasound system</td><td>Philps</td><td>Affiniti 50</td><td></td></tr><tr><td>Von Kossa stain kit</td><td>Abcam</td><td>ab105689</td><td></td></tr><tr><td>Zoletil 50</td><td>Virbac korea</td><td></td><td>Tiletamine &amp;amp; zolazepam</td></tr></tbody></table>

a rabbit aortic valve stenosis model induced by direct balloon injury

Animal models are emerging as an important tool to understand the pathologic mechanisms underlying aortic valve stenosis (AVS) because of the lack of access to reliable sources of diseased human aortic valves. Among the various animal models, AVS rabbit models are one of the most commonly used in large animal studies. However, traditional AVS rabbit models require a long-term period of dietary supplementation and genetic manipulation to induce significant stenosis in the aortic valve, limiting their use in experimental studies. To address these limitations, a new AVS rabbit model is proposed, in which stenosis is induced by a direct balloon injury to the aortic valve. The present protocol describes a successful technique for inducing AVS in New Zealand white (NZW) rabbits, with step-by-step procedures for the preparation, the surgical procedure, and the post-operative care. This simple and reproducible model offers a promising approach for studying the initiation and progression of AVS and provides a valuable tool for investigating the underlying pathological mechanisms of the disease.

Rabbit AVS model induced by aortic valve injury 
To induce the rabbit AVS model, male NZW rabbits weighing 3.5-4.0 kg were used for this study. According to the surgical procedures described in step 2 (Figure 2), the AVS model was established by aortic valve injury, which resulted in mechanical aortic valve degeneration and calcification. The control group included rabbits fed with a 0.5% cholesterol-enriched diet (high-cholesterol, HC) and 50,000 U of vitamin D2 (VitD2...

Watch this Scientific Journal Video about A Rabbit Aortic Valve Stenosis Model Induced by Direct Balloon Injury at JoVE.com

A Rabbit Aortic Valve Stenosis Model Induced by Direct Balloon Injury

An appropriate animal model is needed to understand the pathologic mechanisms underlying aortic valve stenosis (AVS) and to evaluate the efficacy of therapeutic interventions. The present protocol describes a new procedure for developing the AVS rabbit model via a direct balloon injury in vivo.

Animal models are emerging as an important tool to understand the pathologic mechanisms underlying aortic valve stenosis (AVS) because of the lack of ...

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951 Views.  The Catholic University of Korea. Jaarlijkse modellen zijn in opkomst als een belangrijk hulpmiddel om de pathologische mechanismen te begrijpen die ten grondslag liggen aan aortaklepstenose vanwege het gebrek aan toegang tot betrouwbare bronnen van zieke menselijke aortakleppen. Traditionele konijnenmodellen voor aortaklepstenose vereisen langdurige voedingssuppletie en genetische manipulatie om significante stenose in de aortaklep te induceren, waardoor het gebruik ervan in experimentele studies wordt beperkt. Deze techniek...

Video: A Rabbit Aortic Valve Stenosis Model Induced by Direct Balloon Injury

Microsurgical Venous Pouch Arterial-Bifurcation Aneurysms in the Rabbit Model: Technical Aspects

For ruptured human cerebral aneurysms endovascular embolization has become an equivalent alternative to aneurysm clipping.1 However, large clinical trials have shown disappointing long-term results with unacceptable high rates of aneurysm recanalization and delayed aneurysm rupture.2 To overcome these problems, animal experimental studies are crucial for the development of better endovascular devices.3-5
Several animal models in rats, rabbits, canines and swine are available.6-8 Comparisons of the different animal models showed the superiority of the rabbit model with regard to hemodynamics and comparability of the coagulation system and cost-effectiveness.9-11 
The venous pouch arterial bifurcation model in rabbits is formed by a venous pouch sutured into an artificially created true bifurcation of both common carotid arteries (CCA). The main advantage of this model are true bifurcational hemodynamics.12 The major drawbacks are the sofar high microsurgical technical demands and high morbidity and mortality rates of up to 50%.13 These limitations have resulted in less frequent use of this aneurysm model in the recent years. These shortcomings could be overcome with improved surgical procedures and modified peri- and postoperative analgetic management and anticoagulation.14-16 Our techniques reported in this paper demonstrate this optimized technique for microsurgical creation of arterial bifurcation aneurysms.

An optimized technique for the microsurgical creation of arterial bifurcation aneurysms mimicking bifurcation human cerebral aneurysms is described. A venous pouch is sutured into an artificially created true bifurcation of both common carotid arteries. Facilitated microsurgical techniques and aggressive postoperative anticoagulation and analgesia lead to minimized morbidity rates and high aneurysm patency rates.

For ruptured human cerebral aneurysms endovascular embolization has become an equivalent alternative to aneurysm clipping.1 However, large clinical trials ...

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 ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

The Rabbit Model of Accelerated Atherosclerosis: A Methodological Perspective of the Iliac Artery Balloon Injury

Acute coronary syndrome resulting from coronary occlusion following atherosclerotic plaque development and rupture is the leading cause of death in the industrialized world. New Zealand White (NZW) rabbits are widely used as an animal model for the study of atherosclerosis. They develop spontaneous lesions when fed with atherogenic diet; however, this requires long time of 4 - 8 months. To further enhance and accelerate atherogenesis, a combination of atherogenic diet and mechanical endothelial injury is often employed. The presented procedure for inducing atherosclerotic plaques in rabbits uses a balloon catheter to disrupt the endothelium in the left iliac artery of NZW rabbits fed with atherogenic diet. Such mechanical damage caused by the balloon catheter induces a chain of inflammatory reactions initiating neointimal lipid accumulation in a time dependent fashion. Atherosclerotic plaque following balloon injury show neointimal thickening with extensive lipid infiltration, high smooth muscle cell content and presence of macrophage derived foam cells. This technique is simple, reproducible and produces plaque of controlled length within the iliac artery. The whole procedure is completed within 20 - 30 min. The procedure is safe with low mortality and also offers high success in obtaining substantial intimal lesions. The procedure of balloon catheter induced arterial injury results in atherosclerosis within two weeks. This model can be used for investigating the disease pathology, diagnostic imaging and to evaluate new therapeutic strategies.

Animal models of atherosclerosis are essential to understand the mechanism and to investigate newer approaches to prevent plaque development or rupture, a leading cause of death in the industrialized world. This protocol uses a combination of balloon injury and cholesterol rich diet to induce atherosclerotic plaques in rabbit iliac artery.

Acute coronary syndrome resulting from coronary occlusion following atherosclerotic plaque development and rupture is the leading cause of death in the ...

Balloon-based Injury to Induce Myointimal Hyperplasia in the Mouse Abdominal Aorta

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine balloon denudation model, which is comparable with established vessel injury models in large animals. The aorta denudation model with balloon catheters mimics the clinical setting and leads to comparable pathobiological and physiological changes. Briefly, after performing a horizontal incision in the aorta abdominalis, a balloon catheter will be inserted into the vessel, inflated, and introduced retrogradely. Inflation of the balloon will lead to intima injury and overdistension of the vessel. After removing the catheter, the aortic incision will be closed with single stiches. The model shown in this article is reproducible, easy to perform, and can be established quickly and reliably. It is especially suitable for evaluating expensive experimental therapeutic agents, which can be applied in an economical fashion. By using different knockout-mouse strains, the impact of different genes on MH development can be assessed.

This article demonstrates a murine model to study the development of myointimal hyperplasia (MH) after aortic balloon injury.

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine ...

Arterial Pouch Microsurgical Bifurcation Aneurysm Model in the Rabbit

Endovascular treatment for intracranial aneurysms gained importance over the past decades, consequently there is an increased need of testing endovascular devices. Animal models respecting rheological, hemodynamic and aneurysm wall conditions are highly warranted. Therefore, the aim of the present study was to design a novel standardized and reproducible surgical technique to create autologous arterial pouch bifurcation aneurysms with non-modified and modified wall conditions in rabbits.
Bifurcation aneurysms were created by end-to-side anastomosis of the right on the left common carotid artery, both serving as parent arteries for the arterial pouch, which was microsurgically sewn on. Grafts were taken from the proximal right common carotid artery, either for the control (n = 7, immediate autologous re-implantation) or modified (n = 7, incubated with 100 international units elastase for 20 minutes before autologous re-implantation) group. Pouch and parent artery patency were controlled by fluorescence angiography immediately after creation. At follow-up (28 days), all rabbits underwent contrast enhanced magnetic resonance angiography and fluorescence angiography followed by aneurysm harvesting, macroscopic and histological evaluation.
A total of 16 female New Zealand White rabbits were operated upon. Two animals died prematurely. At follow-up, 85.72% of all aneurysms remained patent. Both groups revealed an increase in aneurysm size over time; this was more pronounced in the control group (6.48 &#177; 1.81 mm3 at time of creation vs. 19.85 &#177; 6.40 mm3 at follow-up, p = 0.037) than in the modified group (8.03 &#177; 1.08 mm3 at time of creation vs. 20.29 &#177; 6.16 mm3 at follow-up, p = 0.054).
Our findings demonstrate the adequacy of this new rabbit model which allows for the creation of bifurcation aneurysms with different wall conditions in a microsurgical approach. Given the excellent long-term patency and the property of aneurysm growth over time, this model may serve as an important tool for preclinical evaluation of novel endovascular therapies.

Developing and testing endovascular devices for intracranial aneurysm treatment is still of great importance. Most aneurysm models used today miss either the important characteristics of an arterial degenerated wall or the hemodynamics of a true bifurcation. Therefore, we aimed to design a novel arterial pouch bifurcation model in rabbits.

Endovascular treatment for intracranial aneurysms gained importance over the past decades, consequently there is an increased need of testing endovascular ...

Neuroscience

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the luminal surface area in arteries thereby reducing adequate blood flow to organs and distal tissues. Clinically, revascularization procedures such as balloon angioplasty with or without stent placement aim to restore blood flow. Although these procedures reestablish blood flow by reducing plaque burden, they damage the vessel wall, which initiates the arterial healing response. The prolonged healing response causes arterial restenosis, or re-narrowing, ultimately limiting the long-term success of these revascularization procedures. Therefore, preclinical animal models are integral for analyzing the pathophysiological mechanisms driving restenosis, and provide the opportunity to test novel therapeutic strategies. Murine models are cheaper and easier to operate on than large animal models. Balloon or wire injury are the two commonly accepted injury modalities used in murine models. Balloon injury models in particular mimic the clinical angioplasty procedure and cause adequate damage to the artery for the development of restenosis. Herein we describe the surgical details for performing and histologically analyzing the modified, pressure-controlled rat carotid artery balloon injury model. Additionally, this protocol highlights how local periadventitial application of therapeutics can be used to inhibit neointimal hyperplasia. Lastly, we present light sheet fluorescence microscopy as a novel approach for imaging and visualizing the arterial injury in three-dimensions.

The rat carotid artery balloon injury mimics the clinical angioplasty procedure performed to restore blood flow in atherosclerotic vessels. This model induces the arterial injury response by distending the arterial wall, and denuding the intimal layer of endothelial cells, ultimately causing remodeling and an intimal hyperplastic response.

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the ...

An Image Guided Transapical Mitral Valve Leaflet Puncture Model of Controlled Volume Overload from Mitral Regurgitation in the Rat

Mitral regurgitation (MR) is a widely prevalent heart valve lesion, which causes cardiac remodeling and leads to congestive heart failure. Though the risks of uncorrected MR and its poor prognosis are known, the longitudinal changes in cardiac function, structure and remodeling are incompletely understood. This knowledge gap has limited our understanding of the optimal timing for MR correction, and the benefit that early versus late MR correction may have on the left ventricle. To investigate the molecular mechanisms that underlie left ventricular remodeling in the setting of MR, animal models are necessary. Traditionally, the aorto-caval fistula model has been used to induce volume overload, which differs from clinically relevant lesions such as MR. MR represents a low-pressure volume overload hemodynamic stressor, which requires animal models that mimic this condition. Herein, we describe a rodent model of severe MR in which the anterior leaflet of the rat mitral valve is perforated with a 23G needle, in a beating heart, with echocardiographic image guidance. The severity of MR is assessed and confirmed with echocardiography, and the reproducibility of the model is reported.&#160;&#160;

A rodent model of left heart volume overload from mitral regurgitation is reported. Mitral regurgitation of controlled severity is induced by advancing a needle of defined dimensions into the anterior leaflet of the mitral valve, in a beating heart, with ultrasound guidance.&#160;

Mitral regurgitation (MR) is a widely prevalent heart valve lesion, which causes cardiac remodeling and leads to congestive heart failure. Though the ...

Real-Time Monitoring and Modulation of Blood Pressure in a Rabbit Model of Ischemic Stroke

Control of blood pressure, in terms of both absolute values and its variability, affects outcomes in ischemic stroke patients. However, it remains challenging to identify the mechanisms that lead to poor outcomes or evaluate measures by which these effects can be mitigated because of the prohibitive limitations inherent to human data. In such cases, animal models can be utilized to conduct rigorous and reproducible evaluations of diseases. Here we report refinement of a previously described model of ischemic stroke in rabbits that is augmented with continuous blood pressure recording to assess the impacts of modulation on blood pressure. Under general anesthesia, femoral arteries are exposed through surgical cutdowns to place arterial sheaths bilaterally. Under fluoroscopic visualization and roadmap guidance, a microcatheter is advanced into an artery of the posterior circulation of the brain. An angiogram is performed by injecting the contralateral vertebral artery to confirm occlusion of the target artery. With the occlusive catheter remaining in position for a fixed duration, blood pressure is continuously recorded to allow for tight titration of blood pressure manipulations, whether through mechanical or pharmacological means. At the completion of the occlusion interval, the microcatheter is removed, and the animal is maintained under general anesthesia for a prescribed length of reperfusion. For acute studies, the animal is then euthanized and decapitated. The brain is harvested and processed to measure the infarct volume under light microscopy and further assessed with various histopathological stains or spatial transcriptomic analysis. This protocol provides a reproducible model that can be utilized for more thorough preclinical studies on the effects of blood pressure parameters during ischemic stroke. It also facilitates effective preclinical evaluation of novel neuroprotective interventions that might improve care for ischemic stroke patients.

Continuous arterial blood pressure recording allows the investigation of impacts of various hemodynamic parameters. This report demonstrates the application of continuous arterial blood pressure monitoring in a large animal model of ischemic stroke for determination of stroke pathophysiology, impact of different hemodynamic factors, and the assessment of novel treatment approaches.

A Rabbit Aortic Valve Stenosis Model Induced by Direct Balloon Injury

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