This work was supported and funded under the QREN project 2013/30196, the &#34;la Caixa&#34; Banking Foundation, the Funda&#231;&#227;o para a Ci&#234;ncia e Tecnologia (FCT) project, LCF/PR/HP17/52190002. JS and EB were supported by the European Union&#39;s Horizon 2020 research and innovation program&#160;under the Marie Sklodowska-Curie grant agreement no.&#160;813716. PdCM was supported by the Stichting Life Sciences Health (LSH)-TKI project MEDIATOR (LSHM 21016).

Ascending Aortic Banding for Studying Pressure-Overload Induced Heart Failure

<ol>
	<li>Savarese, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+burden+of+heart+failure:+a+comprehensive+and+updated+review+of+epidemiology.">Global burden of heart failure: a comprehensive and updated review of epidemiology.</a> Cardiovascular Research. 118 (17), 3272-3287 (2023).</li><li>Hartley, 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=Trends+in+mortality+from+aortic+stenosis+in+Europe:+2000-2017.">Trends in mortality from aortic stenosis in Europe: 2000-2017.</a> Frontiers in Cardiovascular Medicine. 8, 748137 (2021).</li><li>Silva, K. A. S., Emter, 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=Large+Animal+models+of+heart+failure:+a+translational+bridge+to+clinical+success.">Large Animal models of heart failure: a translational bridge to clinical success.</a> Journal of the American College of Cardiology: Basic to Translational Science. 5 (8), 840-856 (2020).</li><li>Brink, C. B., Lewis, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+12+Rs+framework+as+a+comprehensive,+unifying+construct+for+principles+guiding+animal+research+ethics.">The 12 Rs framework as a comprehensive, unifying construct for principles guiding animal research ethics.</a> Animals (Basel). 13 (7), 1128 (2023).</li><li>Choy, J. S., Zhang, Z. D., Pitsillides, K., Sosa, M., Kassab, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+hemodynamic+measurements+in+swine+heart+failure+using+a+fully+implantable+telemetry+system.">Longitudinal hemodynamic measurements in swine heart failure using a fully implantable telemetry system.</a> PLoS One. 9 (8), 103331 (2014).</li><li>Ishikawa, K., 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=Increased+stiffness+is+the+major+early+abnormality+in+a+pig+model+of+severe+aortic+stenosis+and+predisposes+to+congestive+heart+failure+in+the+absence+of+systolic+dysfunction.">Increased stiffness is the major early abnormality in a pig model of severe aortic stenosis and predisposes to congestive heart failure in the absence of systolic dysfunction.</a> Journal of the American Heart Association. 4 (5), 001925 (2015).</li><li>Emter, C. A., Baines, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-intensity+aerobic+interval+training+attenuates+pathological+left+ventricular+remodeling+and+mitochondrial+dysfunction+in+aortic-banded+miniature+swine.">Low-intensity aerobic interval training attenuates pathological left ventricular remodeling and mitochondrial dysfunction in aortic-banded miniature swine.</a> American Journal of Physiology-Heart and Circulatory Physiology. 299 (5), H1348-H1356 (2010).</li><li>Brito, J., Raposo, L., Teles, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasive+assessment+of+aortic+stenosis+in+contemporary+practice.">Invasive assessment of aortic stenosis in contemporary practice.</a> Frontiers in Cardiovascular Medicine. 9, 1007139 (2022).</li><li>Tan, W., 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+Porcine+model+of+heart+failure+with+preserved+ejection+fraction+induced+by+chronic+pressure+overload+characterized+by+cardiac+fibrosis+and+remodeling.">A Porcine model of heart failure with preserved ejection fraction induced by chronic pressure overload characterized by cardiac fibrosis and remodeling.</a> Frontiers in Cardiovascular Medicine. 8, 677727 (2021).</li><li>Bikou, O., Miyashita, S., Ishikawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pig+model+of+increased+cardiac+afterload+induced+by+ascending+aortic+banding.">Pig model of increased cardiac afterload induced by ascending aortic banding.</a> Methods in Molecular Biology. 1816, 337-342 (2018).</li><li>Lelovas, P. P., Kostomitsopoulos, N. G., Xanthos, T. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+anatomic+and+physiologic+overview+of+the+porcine+heart.">A comparative anatomic and physiologic overview of the porcine heart.</a> Journal of the American Association for Laboratory Animal Science. 53 (5), 432-438 (2014).</li><li>Tian, L., 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=Supra-coronary+aortic+banding+improves+right+ventricular+function+in+experimental+pulmonary+arterial+hypertension+in+rats+by+increasing+systolic+right+coronary+artery+perfusion.">Supra-coronary aortic banding improves right ventricular function in experimental pulmonary arterial hypertension in rats by increasing systolic right coronary artery perfusion.</a> Acta Physiologica (Oxf). 229 (4), 13483 (2020).</li><li>Sorensen, M., Hasenkam, J. M., Jensen, H., Sloth, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcoronary+versus+supracoronary+aortic+stenosis.+An+experimental+evaluation.">Subcoronary versus supracoronary aortic stenosis. An experimental evaluation.</a> Journal of Cardiothoracic Surgery. 6, 100 (2011).</li><li>Lygate, C. 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=Serial+high+resolution+3D-MRI+after+aortic+banding+in+mice:+band+internalization+is+a+source+of+variability+in+the+hypertrophic+response.">Serial high resolution 3D-MRI after aortic banding in mice: band internalization is a source of variability in the hypertrophic response.</a> Basic Research in Cardiology. 101 (1), 8-16 (2006).</li><li>Jalal, Z., 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=Unexpected+Internalization+of+a+Pulmonary+Artery+Band+in+a+Porcine+Model+of+Tetralogy+of+Fallot.">Unexpected Internalization of a Pulmonary Artery Band in a Porcine Model of Tetralogy of Fallot.</a> World Journal for Pediatric and Congenital Heart Surgery. 8 (1), 48-54 (2017).</li></ol>

The authors have nothing to disclose.

In recent years, several studies have utilized surgical aortic banding as a model for left ventricular pressure overload and heart failure (descending9 to the ascending aorta10), allowing researchers to obtain various phenotypes tailored to their specific needs. Although using such models requires costly equipment and specialized knowledge, the information they provide is invaluable. Swine, due to its size and similarity to the human heart, serves as an ideal model11, gaining ethical acceptance as organ donors for xenotransplantation.
The main critical step in this method is the dissection of the aorta and the placement of the banding material (nylon cable or ePTFE graft) around it. During this step, several complications can occur, including laceration or rupture of the surrounding structures or the aorta itself. Controlling such complications can be achieved by placing a pursue-string suture or a mattress suture with pledgets on the hole if bleeding can be controlled to properly visualize the wound. It is strongly recommended to have the procedure performed by a cardiothoracic surgeon, which significantly reduces complication and mortality rates.
Another critical step is the constriction of the aorta, which should be done in sequential steps with stabilization periods in between. Paying close attention to systemic peripheral pressures is crucial, as sustained significant hypotension (mean arterial pressure below 60 mmHg) may result from the LV's inability to cope with the current stenosis. If not resolved, especially when ventricular pressures start to drop as well, acute heart failure will lead to the loss of the animal. The removal of the nylon cable or titanium clip is necessary when hypotension does not spontaneously resolve.
However, the main limitation of this model, and many aortic banding models, is the band's location relative to the coronary ostia. Supra-coronary banding placement does not entirely mimic aortic stenosis and may lead to increased blood pressure in the coronary circulation, which might be protective12. Limited evidence suggests no differences between sub-coronary and supra-coronary aortic banding in pigs13, indicating that the increased complications associated with sub-coronary banding surgery may not be worthwhile.
Depending on the animal strain used and the follow-up time, band internalization may become an issue. Although mainly described in rodents14, it has also been observed in the pulmonary artery of pigs15. Using ePTFE graft segments significantly increases the contact area and eliminates the occurrence of band internalization. However, ePTFE grafts are more expensive, and when using slow-growing breeds, such as the Vietnamese pot-bellied pig, band internalization is not an issue when using nylon zip ties. Researchers should choose their approach based on the animal breed used.
For fast-growing breeds, long-term follow-up might be challenging due to animal size (availability of infrastructure and equipment large enough to handle &gt;100 kg animals) and prohibitive maintenance costs.
Another limitation of this model, as well as all models requiring pericardial space access, is the presence of significant pericardial adhesions after surgery. Our experience shows no difference between closing or not closing the pericardial incision after band placement. While it does not affect function, dissecting the heart and identifying different structures becomes more time-consuming, and the epicardium is likely to be damaged if the pericardium is fully separated.
This minimally invasive method represents a significant refinement of the typical surgical procedure, leading to an uneventful and speedier recovery. The use of two high-fidelity catheters for simultaneous pressure measurement and real-time gradient measurement significantly improves the accuracy of the procedure and the reproducibility of the model, leading to a reduction in the number of animals required. The model can be applied to the study of new therapeutic interventions or devices aimed at left ventricular hypertrophy, as well as the determination of new pathophysiological mechanisms associated with left ventricular pressure overload.

Heart failure (HF) is a life-threatening disease that affects millions of people worldwide, causing major social and economic impacts1. One of its significant etiologies is aortic valve disease or aortic stenosis (AS). Aortic stenosis is more prevalent in advanced age and ranks as the second most common valvular lesion in the United States. AS-related mortality has also increased in Europe, particularly in countries without access to recent interventional procedures2. Given the complexity of HF and the scarcity of therapeutic innovations, there is a pressing need for reliable animal models that can replicate the human condition and facilitate the testing of new interventions3. While rodent models outnumber large animal models, the latter offers several advantages due to their size and physiological similarities, allowing the testing of drug doses and medical devices intended for human use.
The aim of this method is to establish a reproducible model of ascending aortic banding (AAB) applicable to most large animal species used in biomedical research. In this study, the procedure is demonstrated in swine using a minimally invasive approach, adhering to the 3Rs principles (replacement, reduction, and refinement4). This approach ensures the creation of an accurate pressure gradient, resulting in high reproducibility (potentially reducing the number of required animals). Additionally, the small surgical incision (2-3 cm) minimizes surgical insult, improving animal well-being compared to more aggressive approaches like sternotomy and larger thoracotomies5 (refinement). Furthermore, providing a video demonstration of the method, along with detailed descriptions in the literature, could potentially reduce the need for animals used solely for training purposes (replacement), further decreasing animal usage. This model can be adapted for different swine strains/breeds with distinct growth rates and induces sustained pressure overload, leading to significant hypertrophy after 1 or 2 months of follow-up.
Current methods employ fixed stenosis6, disregarding animal size variability, or calculate gradient using fluid-filled pressure readings7, which are less reliable than high-fidelity pressure sensors and are susceptible to signal damping8. Another approach uses a single pressure measurement distal to the stenosis5. However, calibrating the stenosis through simultaneous proximal and distal pressure signals using percutaneously delivered high-fidelity pressure sensors represents a substantial optimization of the protocol, resulting in improved group homogeneity. By visually demonstrating this method, other researchers should be able to replicate it without significant obstacles, increasing the availability of this model while promoting the application of the 3Rs principles.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3-0 PDS II suture</td>
			<td>Ethicon</td>
			<td>Z683G</td>
			<td>Aorta banding</td>
		</tr>
		<tr>
			<td>5-0 prolene</td>
			<td>Ethicon</td>
			<td>7472H</td>
			<td>Aorta banding</td>
		</tr>
		<tr>
			<td>ACUSON NX2 Ultrasound System</td>
			<td>Siemens</td>
			<td>(240)11284381</td>
			<td>Vascular Access and Echocardiography</td>
		</tr>
		<tr>
			<td>Arterial Extension 200 cm</td>
			<td>PMH</td>
			<td>303.0666</td>
			<td>Anesthesia Maintenance</td>
		</tr>
		<tr>
			<td>Atlan A300 Ventilator</td>
			<td>Draeger</td>
			<td>8621300</td>
			<td>Ventilation</td>
		</tr>
		<tr>
			<td>Bone cutters</td>
			<td>Fehling</td>
			<td>AMP 367.00</td>
			<td>Aorta banding</td>
		</tr>
		<tr>
			<td>Cefazolin 1000 mg</td>
			<td>Labesfal</td>
			<td>100063</td>
			<td>Antibiotic</td>
		</tr>
		<tr>
			<td>Chlorhexidine 4% Wash Solution</td>
			<td>AGA</td>
			<td>19110008</td>
			<td>Cleaning</td>
		</tr>
		<tr>
			<td>Doyen Intestinal Forceps</td>
			<td>Aesculap</td>
			<td>EA121R</td>
			<td>Intubation</td>
		</tr>
		<tr>
			<td>Echogenic Introducer Needle</td>
			<td>Teleflex</td>
			<td>AN-04318</td>
			<td>Vascular Access</td>
		</tr>
		<tr>
			<td>Endotracheal tube</td>
			<td>Intersurgical</td>
			<td>8040070</td>
			<td>Intubation</td>
		</tr>
		<tr>
			<td>ePTFE vascular graft (5 mm x 40 cm)</td>
			<td>GORE-TEX</td>
			<td>S0504</td>
			<td>Aorta banding</td>
		</tr>
		<tr>
			<td>Extension line 100 cm</td>
			<td>PMH</td>
			<td>303.0394</td>
			<td>Anesthesia Induction</td>
		</tr>
		<tr>
			<td>F.O. Laryngoscope</td>
			<td>Luxamed</td>
			<td>E1.317.012</td>
			<td>Intubation</td>
		</tr>
		<tr>
			<td>F.O. Miller Blade 4 204 x 17 mm</td>
			<td>Luxamed</td>
			<td>3</td>
			<td>Intubation</td>
		</tr>
		<tr>
			<td>Fenestrated Sterile Drape</td>
			<td>Bastos Viegas</td>
			<td>4882-256</td>
			<td>Aseptic Technique</td>
		</tr>
		<tr>
			<td>Fentanyl 0.5 mg/10 mL</td>
			<td>B.Braun</td>
			<td>5758883</td>
			<td>Anesthesia / Analgesia</td>
		</tr>
		<tr>
			<td>Guidewire 260 cm J-tip</td>
			<td>B.Braun</td>
			<td>J3 FC-FS 260-035</td>
			<td>Left Ventricle catheterization</td>
		</tr>
		<tr>
			<td>Infusomat Space Infusion Pump</td>
			<td>B.Braun</td>
			<td>24101800</td>
			<td>Fluids / Drug administration</td>
		</tr>
		<tr>
			<td>Intercostal retractor</td>
			<td>Fehling Surgical</td>
			<td>MRP-1</td>
			<td>Thoracotomy</td>
		</tr>
		<tr>
			<td>Introcan Certo IV Catheter 20G</td>
			<td>B.Braun</td>
			<td>4251326</td>
			<td>Fluids / Drug administration</td>
		</tr>
		<tr>
			<td>Isotonic Saline Solution 0.9%</td>
			<td>B.Braun</td>
			<td>5/44929/1/0918</td>
			<td>Fluids / Drug administration</td>
		</tr>
		<tr>
			<td>Ketamidor 100 mg/mL</td>
			<td>Richter pharma</td>
			<td>1121908AB</td>
			<td>Anesthesia Induction</td>
		</tr>
		<tr>
			<td>L10-5v Linear Transducer</td>
			<td>Siemens</td>
			<td>11284481</td>
			<td>Vascular Access</td>
		</tr>
		<tr>
			<td>Midazolam 15 mg/3 mL</td>
			<td>Labesfal</td>
			<td>PLB762-POR/2</td>
			<td>Anesthesia Induction</td>
		</tr>
		<tr>
			<td>Mikro-cath</td>
			<td>Millar</td>
			<td>63405(1)</td>
			<td>Pressure recording</td>
		</tr>
		<tr>
			<td>MP1 guide catheter 6 Fr</td>
			<td>Cordis</td>
			<td>67027000</td>
			<td>Left Ventricle catheterization</td>
		</tr>
		<tr>
			<td>Needle Holder</td>
			<td>Fehling Surgical</td>
			<td>ZYY-5</td>
			<td>Aorta banding</td>
		</tr>
		<tr>
			<td>Non-woven adhesive</td>
			<td>Bastos Viegas</td>
			<td>442-002</td>
			<td>Fluids / Drug administration</td>
		</tr>
		<tr>
			<td>P4-2 Phased Array Transducer</td>
			<td>Siemens</td>
			<td>11284467</td>
			<td>Echocardiography</td>
		</tr>
		<tr>
			<td>Perfusor Compact Syringe Perfusion Pump</td>
			<td>B.Braun</td>
			<td>8717030</td>
			<td>Fluids / Drug administration</td>
		</tr>
		<tr>
			<td>Pressure Signal Conditioner</td>
			<td>ADinstruments</td>
			<td>PCU-2000</td>
			<td>Pressure recording</td>
		</tr>
		<tr>
			<td>Propofol Lipuro 2%</td>
			<td>B.Braun</td>
			<td>357410&#160;</td>
			<td>Anesthesia Maintenance</td>
		</tr>
		<tr>
			<td>Radifocus Introducer II Standard Kit B - Introducer Sheath</td>
			<td>Terumo</td>
			<td>RS+B60K10MQ</td>
			<td>Vascular Access</td>
		</tr>
		<tr>
			<td>Radiopaque marker</td>
			<td>Scanlan</td>
			<td>1001-83</td>
			<td>Aorta banding</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fehling Surgical</td>
			<td></td>
			<td>Thoracotomy</td>
		</tr>
		<tr>
			<td>Skinprep (Chlorhexidine 2% / 70% Isopropyl alcohol)</td>
			<td>Vygon</td>
			<td>SKPC015ES</td>
			<td>Disinfection</td>
		</tr>
		<tr>
			<td>Stopcock manifold (3 ports)</td>
			<td>PMH</td>
			<td>310.0489</td>
			<td>Fluids / Drug administration</td>
		</tr>
		<tr>
			<td>Straight forceps</td>
			<td>Fehling Surgical</td>
			<td>ZYY-1</td>
			<td>Thoracotomy</td>
		</tr>
		<tr>
			<td>Stresnil 40 mg/mL</td>
			<td>ecuphar</td>
			<td>572184.2</td>
			<td>Anesthesia Induction</td>
		</tr>
		<tr>
			<td>Syringe Luer Lock 20 cc</td>
			<td>Omnifix B.Braun</td>
			<td>4617207V</td>
			<td>Anesthesia Induction</td>
		</tr>
		<tr>
			<td>Syringe Luer Lock 50 cc</td>
			<td>Omnifix B.Braun</td>
			<td>4617509F</td>
			<td>Anesthesia Maintenance</td>
		</tr>
		<tr>
			<td>Transdermal fentanyl Patch 50 mcg/h</td>
			<td>Mylan</td>
			<td>5022153</td>
			<td>Analgesia</td>
		</tr>
		<tr>
			<td>Ultravist</td>
			<td>Bayer</td>
			<td>KT0B019</td>
			<td>Angiography</td>
		</tr>
		<tr>
			<td>Universal Hemostasis Valve Adapter</td>
			<td>Merit Medical</td>
			<td>UHVA08</td>
			<td>Left Ventricle catheterization</td>
		</tr>
		<tr>
			<td>Velcro Limb Immobilizer</td>
			<td>PMH</td>
			<td>SU-211</td>
			<td>Animal stabilization</td>
		</tr>
		<tr>
			<td>Venofix A, 21 G</td>
			<td>B.Braun</td>
			<td>4056337</td>
			<td>Anesthesia Induction</td>
		</tr>
		<tr>
			<td>Vista 120S Patient Monitor</td>
			<td>Draeger</td>
			<td>MS32997</td>
			<td>Monitoring</td>
		</tr>
		<tr>
			<td>Weck titanium clip</td>
			<td>Teleflex</td>
			<td>523760</td>
			<td>Aorta banding</td>
		</tr>
		<tr>
			<td>Weck titanium clip applier</td>
			<td>Teleflex</td>
			<td>523166</td>
			<td>Aorta banding</td>
		</tr>
		<tr>
			<td>Zhiem Vision</td>
			<td>Iberdata</td>
			<td>N/A</td>
			<td>Fluoroscopy</td>
		</tr>
	</tbody>
</table>

a minimally invasive model of aortic stenosis in swine

Large animal models of heart failure play an essential role in the development of new therapeutic interventions due to their size and physiological similarities to humans. Efforts have been dedicated to creating a model of pressure-overload induced heart failure, and ascending aortic banding while still supra-coronary and not a perfect mimic of aortic stenosis in humans, closely resembling the human condition.
The purpose of this study is to demonstrate a minimally invasive approach to induce left ventricular pressure overload by placing an aortic band, precisely calibrated with percutaneously introduced high-fidelity pressure sensors. This method represents a refinement of the surgical procedure (3Rs), resulting in homogenous trans-stenotic gradients and reduced intragroup variability. Additionally, it enables swift and uneventful animal recovery, leading to minimal mortality rates. Throughout the study, animals were followed for up to 2 months after surgery, employing transthoracic echocardiography and pressure-volume loop analysis. However, longer follow-up periods can be achieved if desired. This large animal model proves valuable for testing new drugs, particularly those targeting hypertrophy and the structural and functional alterations associated with left ventricular pressure overload.

During the initial development of the model, the mortality rate was approximately 30%, with animals dying from acute heart failure after banding and surgical complications. However, after the model was established, surgical complications became less common, and the mortality rate dropped to around 15%. The two deaths that occurred were due to aortic rupture during dissection.
The use of high-fidelity pressure sensors enables obtaining high-quality pressure signals (Figure 2), allowing real-time and accurate calibration of the stenosis. This ensures that all operated animals experience a similar degree of left ventricular pressure overload, reducing variability within the group. Moreover, the catheter itself has a 2.3 F shaft, which has minimal impact on flow obstruction compared to larger fluid-filled catheters. After an initial investment, the catheters can be reused multiple times, and if sterilization is needed, ethylene oxide can be used (usually available through collaboration with surgical departments at the hospital).
The trans-stenotic gradient can be calculated in real-time by the software, which measures the pressure difference between the left ventricle (proximal pressure) and the distal aorta (distal pressure). A few minutes of stabilization between each constriction step ensures that the left ventricle has time to adapt. After determining the desired constriction degree, a 15 min stabilization period should be applied to ensure that the banding degree remains stable and the animal is compensated (Figure 2A).
This approach is superior to other methodologies that do not measure trans-stenotic gradient in real-time and lack both the homogeneity of having a similar gradient between all animals (92.3 &#177; 2.3 mmHg, mean and standard error of the mean, respectively, for 7 operated animals) and tight monitoring of left ventricular pressures. Additionally, this approach avoids the difficulties associated with performing transthoracic echocardiography in swine, particularly in certain breeds like the Vietnamese potbellied pig, which has a more significantly protruding sternum.
Transthoracic echocardiography can confirm aortic banding both immediately after the surgery and during follow-up time points (Figure 3). The banding surgery results in significant stenosis of the aorta with turbulent flow, which can be qualitatively evaluated or quantified using continuous wave Doppler. Figure 2 displays representative images of 2-month follow-up echocardiography, showing significant aortic stenosis (upper row) and left ventricular concentric hypertrophy (middle and bottom rows). Two months after banding, the animals develop significant cardiac hypertrophy. The macroscopic evaluation revealed larger hearts and a thicker left ventricular wall (Figure 4). The two-month follow-up period was determined based on the growth rate of the used animals, as a longer follow-up period would result in animals too large to be handled by our infrastructures.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65780/65780fig01.jpg" /> 
Figure 1: Schematics of the aortic banding protocol. After receiving 20-25 kg male pigs, the animals are submitted to a 1 week quarantine period. On the day of the procedure, the animals are anesthetized, the LV and aorta are catheterized, and high-fidelity pressure sensors are placed, followed by aortic banding and animal recovery. The whole procedure, once mastered, lasts around 2 h. Two months after surgery, the animals are submitted to a terminal evaluation, including collection of samples and measurement of physiological variables. AB-aortic banding, Ao-aorta, LV-left ventricle, PV-pressure-volume, RHC-right heart catheterization, US-ultrasound. <a href="https://www.jove.com/files/ftp_upload/65780/65780fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65780/65780fig02.jpg" /> 
Figure 2: Pressure measurements during aortic banding. (A) Representative traces of LV and aortic (distal to the banding) pressures during aortic banding. Zoom in on LV and aortic pressure before (B) and after (C) constriction, showing the gradient creation (difference between peak systolic LV and aortic pressure). (D) Pull-off of the ventricular pressure sensor, transitioning from the aorta proximal to the banding to the aorta distal to the banding. AP-arterial pressure, LVP-left ventricle pressure, MC-high-fidelity pressure sensor. <a href="https://www.jove.com/files/ftp_upload/65780/65780fig02large.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/65780/65780fig03.jpg" /> 
Figure 3: Transthoracic echocardiography. Follow-up at 2 months after surgery reveals significant stenosis of the aorta (black arrow, upper row). LV hypertrophy is apparent, both in 2D (white arrows, middle row), as well as in M-mode, which also demonstrates concentric hypertrophy (white arrows, bottom row). The vertical bar corresponds to 3 cm, and 2D PSAX images were acquired at a depth of 15 cm. <a href="https://www.jove.com/files/ftp_upload/65780/65780fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65780/65780fig04.jpg" /> 
Figure 4: Post-mortem macroscopic analysis of the heart. Aortic banding leads to cardiomegaly, with clear hypertrophy of the LV wall. Heart slices are base, mid-cavity, and apex from left to right. Pericardial adhesions can be seen throughout the epicardium. Scale bars represent 1 cm (upper row) and 4 cm (lower row). <a href="https://www.jove.com/files/ftp_upload/65780/65780fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Development of a Minimally Invasive Large-Animal Model for Reliable and Reproducible Cardiovascular Research at JoVE.com

Author Spotlight: Development of a Minimally Invasive Large-Animal Model for Reliable and Reproducible Cardiovascular Research 

This protocol describes a minimally invasive surgical procedure for ascending aortic banding in swine.

A Minimally Invasive Model of Aortic Stenosis in Swine

Large animal models of heart failure play an essential role in the development of new therapeutic interventions due to their size and physiological ...

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922 Views.  University of Porto. This protocol describes a minimally invasive surgical procedure for ascending aortic banding in swine.

Video: Author Spotlight: Development of a Minimally Invasive Large-Animal Model for Reliable and Reproducible Cardiovascular Research 

A Swine Model of Neonatal Asphyxia

Annually more than 1 million neonates die worldwide as related to asphyxia. Asphyxiated neonates commonly have multi-organ failure including hypotension, perfusion deficit, hypoxic-ischemic encephalopathy, pulmonary hypertension, vasculopathic enterocolitis, renal failure and thrombo-embolic complications. Animal models are developed to help us understand the patho-physiology and pharmacology of neonatal asphyxia. In comparison to rodents and newborn lambs, the newborn piglet has been proven to be a valuable model. The newborn piglet has several advantages including similar development as that of 36-38 weeks human fetus with comparable body systems, large body size (&tilde;1.5-2 kg at birth) that allows the instrumentation and monitoring of the animal and controls the confounding variables of hypoxia and hemodynamic derangements.
We here describe an experimental protocol to simulate neonatal asphyxia and allow us to examine the systemic and regional hemodynamic changes during the asphyxiating and reoxygenation process as well as the respective effects of interventions. Further, the model has the advantage of studying multi-organ failure or dysfunction simultaneously and the interaction with various body systems. The experimental model is a non-survival procedure that involves the surgical instrumentation of newborn piglets (1-3 day-old and 1.5-2.5 kg weight, mixed breed) to allow the establishment of mechanical ventilation, vascular (arterial and central venous) access and the placement of catheters and flow probes (Transonic Inc.) for the continuously monitoring of intra-vascular pressure and blood flow across different arteries including main pulmonary, common carotid, superior mesenteric and left renal arteries. Using these surgically instrumented piglets, after stabilization for 30-60 minutes as defined by Z&lt;10% variation in hemodynamic parameters and normal blood gases, we commence an experimental protocol of severe hypoxemia which is induced via normocapnic alveolar hypoxia. The piglet is ventilated with 10-15% oxygen by increasing the inhaled concentration of nitrogen gas for 2h, aiming for arterial oxygen saturations of 30-40%. This degree of hypoxemia will produce clinical asphyxia with severe metabolic acidosis, systemic hypotension and cardiogenic shock with hypoperfusion to vital organs. The hypoxia is followed by reoxygenation with 100% oxygen for 0.5h and then 21% oxygen for 3.5h. Pharmacologic interventions can be introduced in due course and their effects investigated in a blinded, block-randomized fashion.

Large animal models have good translational values in the examination of physiology and pharmacology of neonatal asphyxia. Using newborn piglets, we develop an experimental protocol to simulate neonatal asphyxia which has advantages of studying the systemic and regional hemodynamics, oxygen transport with biochemical and pathologic pathways and correlations.

Annually more than 1 million neonates die worldwide as related to asphyxia. Asphyxiated neonates commonly have multi-organ failure including hypotension, ...

Photothrombotic Ischemia: A Minimally Invasive and Reproducible Photochemical Cortical Lesion Model for Mouse Stroke Studies

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected light-sensitive dye. Following illumination, the dye is activated and produces singlet oxygen that damages components of endothelial cell membranes, with subsequent platelet aggregation and thrombi formation, which eventually determines the interruption of local blood flow. This approach, initially proposed by Rosenblum and El-Sabban in 1977, was later improved by Watson in 1985 in rat brain and set the basis of the current model. Also, the increased availability of transgenic mouse lines further contributed to raise the interest on the photothrombosis model. Briefly, a photosensitive dye (Rose Bengal) is injected intraperitoneally and enters the blood stream. When illuminated by a cold light source, the dye becomes activated and induces endothelial damage with platelet activation and thrombosis, resulting in local blood flow interruption. The light source can be applied on the intact skull with no need of craniotomy, which allows targeting of any cortical area of interest in a reproducible and non-invasive way. The mouse is then sutured and allowed to wake up. The evaluation of ischemic damage can be quickly accomplished by triphenyl-tetrazolium chloride or cresyl violet staining. This technique produces infarction of small size and well-delimited boundaries, which is highly advantageous for precise cell characterization or functional studies. Furthermore, it is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

Photothrombosis is a quick, minimally-invasive technique for inducing small and well-delimited infarction in areas of interest in highly reproducible manner. It is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected ...

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

Minimally Invasive Transverse Aortic Constriction in Mice

Minimally invasive transverse aortic constriction (MTAC) is a more desirable method for the constriction of the transverse aorta in mice than standard open-chest transverse aortic constriction (TAC). Although transverse aortic constriction is a highly functional method for the induction of high pressure in the left ventricle, it is a more difficult and lengthy procedure due to its use of artificial ventilation with tracheal intubation. TAC is oftentimes also less survivable, as the newer method, MTAC, neither requires the cutting of the ribs and intercostal muscles nor tracheal intubation with a ventilation setup. In MTAC, as opposed to a thoracotomy to access to the chest cavity, the aortic arch is reached through a midline incision in the anterior neck. The thyroid is pulled back to reveal the sternal notch. The sternum is subsequently cut down to the second rib level, and the aortic arch is reached simply by separating the connective tissues and thymus. From there, a suture can be wrapped around the arch and tied with a spacer, and then the sternal cut and skin can be closed. MTAC is a much faster and less invasive way to induce left ventricular hypertension and enables the possibility for high-throughput studies. The success of the constriction can be verified using high-frequency trans-thoracic echocardiography, particularly color Doppler and pulsed-wave Doppler, to determine the flow velocities of the aortic arch and left and right carotid arteries, the dimension of the blood vessels, and the left ventricular function and morphology. A successful constriction will also trigger significant histopathological changes, such as cardiac muscle cell hypertrophy with interstitial and perivascular fibrosis. Here, the procedure of MTAC is described, demonstrating how the resulting flow changes in the carotid arteries can be examined with echocardiography, gross morphology, and histopathological changes in the heart.

Minimally invasive transverse aortic constriction (MTAC) conserves the essentials of regular transverse aortic constriction (TAC) while eliminating the use of a ventilator with tracheal intubation. It proves to be a highly desirable method for high-throughput studies on left ventricular overload, particularly in translational studies.

Minimally invasive transverse aortic constriction (MTAC) is a more desirable method for the constriction of the transverse aorta in mice than standard ...

Technique of Minimally Invasive Transverse Aortic Constriction in Mice for Induction of Left Ventricular Hypertrophy

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure overload-induced left ventricular hypertrophy (LVH) and its progression to heart failure. In the majority of the reported investigations, this procedure is performed with intubation and ventilation of the animal which renders it demanding and time-consuming and adds to the surgical burden to the animal. The aim of this protocol is to describe a simplified technique of minimally invasive TAC without intubation and ventilation of mice. Critical steps of the technique are emphasized in order to achieve low mortality and high efficiency in inducing LVH.
Male C57BL/6 mice (10-week-old, 25-30 g, n=60) were anesthetized with a single intraperitoneal injection of a mixture of ketamine and xylazine. In a spontaneously breathing animal following a 3-4 mm upper partial sternotomy, a segment of 6/0 silk suture threaded through the eye of a ligation aid was passed under the aortic arch and tied over a blunted 27-gauge needle. Sham-operated animals underwent the same surgical preparation but without aortic constriction. The efficacy of the procedure in inducing LVH is attested by a significant increase in the heart/body weight ratio. This ratio is obtained at days 3, 7, 14 and 28 after surgery (n = 6 - 10 in each group and each time point). Using our technique, LVH is observed in TAC compared to sham animals from day 7 through day 28. Operative and late (over 28 days) mortalities are both very low at 1.7%.
In conclusion, our cost-effective technique of minimally invasive TAC in mice carries very low operative and post-operative mortalities and is highly efficient in inducing LVH. It simplifies the operative procedure and reduces the strain put on the animal. It can be easily performed by following the critical steps described in this protocol.

The aim of this protocol is to describe step-by-step the technique of minimally invasive transverse aortic constriction (TAC) in mice. By elimination of intubation and ventilation which are mandatory for the commonly used standard procedure, minimally invasive TAC simplifies the operative procedure and reduces the strain put on the animal.

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure ...

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an increased use of mouse models in orthopedic research was noted, most probably because mouse models offer a large number of genetically-modified strains and special antibodies for the analysis of molecular mechanisms of fracture healing. To control the biomechanical conditions, well-characterized osteosynthesis techniques are mandatory, also in mice. Here, we report on the design and use of a closed bone healing model to stabilize femur fractures in mice. The intramedullary screw, made of medical-grade stainless steel, provides through fracture compression an axial and rotational stability compared to the mostly used simple intramedullary pins, which show a complete lack of axial and rotational stability. The stability achieved by the intramedullary screw allows the analysis of endochondral healing. A large amount of callus tissue, received after stabilization with the screw, offers ideal conditions to harvest tissue for biochemical and molecular analyses. A further advantage of the use of the screw is the fact that the screw can be inserted into the femur with a minimally invasive technique without inducing damage to the soft tissue. In conclusion, the screw is a unique implant that can ideally be used in closed fracture healing models offering standardized biomechanical conditions.

This protocol describes a minimally invasive osteosynthesis technique using an intramedullary screw for standardized stabilization of femur fractures, which can be used to analyze endochondral bone healing in mice.

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an ...

Invasive Hemodynamic Monitoring of Aortic and Pulmonary Artery Hemodynamics in a Large Animal Model of ARDS

One of the leading causes of morbidity and mortality in patients with heart failure is right ventricular (RV) dysfunction, especially if it is due to pulmonary hypertension. For a better understanding and treatment of this disease, precise hemodynamic monitoring of left and right ventricular parameters is important. For this reason, it is essential to establish experimental pig models of cardiac hemodynamics and measurements for research purpose. 
This article shows the induction of ARDS by using oleic acid (OA) and consequent right ventricular dysfunction, as well as the instrumentation of the pigs and the data acquisition process that is needed to assess hemodynamic parameters. To achieve right ventricular dysfunction, we used oleic acid (OA) to cause ARDS and accompanied this with pulmonary artery hypertension (PAH). With this model of PAH and consecutive right ventricular dysfunction, many hemodynamic parameters can be measured, and right ventricular volume load can be detected. 
All vital parameters, including respiratory rate (RR), heart rate (HR) and body temperature were recorded throughout the whole experiment. Hemodynamic parameters including femoral artery pressure (FAP), aortic pressure (AP), right ventricular pressure (peak systolic, end systolic and end diastolic right ventricular pressure), central venous pressure (CVP), pulmonary artery pressure (PAP) and left arterial pressure (LAP) were measured as well as perfusion parameters including ascending aortic flow (AAF) and pulmonary artery flow (PAF). Hemodynamic measurements were performed using transcardiopulmonary thermodilution to provide cardiac output (CO). Furthermore, the PiCCO2 system (Pulse Contour Cardiac Output System 2) was used to receive parameters such as stroke volume variance (SVV), pulse pressure variance (PPV), as well as extravascular lung water (EVLW) and global end-diastolic volume (GEDV). Our monitoring procedure is suitable for detecting right ventricular dysfunction and monitoring hemodynamic findings before and after volume administration.

We present a protocol of creating right ventricular dysfunction in a pig model by inducing ARDS. We demonstrate invasive monitoring of left and right ventricular cardiac output using flow probes around the aorta and the pulmonary artery, as well as blood pressure measurements in the aorta and pulmonary artery.

One of the leading causes of morbidity and mortality in patients with heart failure is right ventricular (RV) dysfunction, especially if it is due to ...

Electromagnetic Navigation Transthoracic Nodule Localization for Minimally Invasive Thoracic Surgery

The increased use of chest computed tomography (CT) has led to an increased detection of pulmonary nodules requiring diagnostic evaluation and/or excision. Many of these nodules are identified and excised via minimally invasive thoracic surgery; however, subcentimeter and subsolid nodules are frequently difficult to identify intra-operatively. This can be mitigated by the use of electromagnetic transthoracic needle localization. This protocol delineates the step-by-step process of electromagnetic localization from the pre-operative period to the postoperative period and is an adaptation of the electromagnetically guided percutaneous biopsy previously described by Arias et al. Pre-operative steps include obtaining a same day CT followed by the generation of a three-dimensional virtual map of the lung. From this map, the target lesion(s) and an entry site are chosen. In the operating room, the virtual reconstruction of the lung is then calibrated with the patient and the electromagnetic navigation platform. The patient is then sedated, intubated, and placed in the lateral decubitus position. Using a sterile technique and visualization from multiple views, the needle is inserted into the chest wall at the prechosen skin entry site and driven down to the target lesion. Dye is then injected into the lesion and, then, continuously during needle withdrawal, creating a tract for visualization intra-operatively. This method has many potential benefits when compared to the CT-guided localization, including a decreased radiation exposure and decreased time between the dye injection and the surgery. Dye diffusion from the pathway occurs over time, thereby limiting intra-operative nodule identification. By decreasing the time to surgery, there is a decrease in wait time for the patient, and less time for dye diffusion to occur, resulting in an improvement in nodule localization. When compared to electromagnetic bronchoscopy, airway architecture is no longer a limitation as the target nodule is accessed via a transparenchymal approach. Details of this procedure are described in a step-by-step fashion.

Presented here is a protocol for lung nodule localization using dye marking via electromagnetically navigated transthoracic needle access. The technique described here can be accomplished in the peri-operative period to optimize nodule localization and to successful resection when performing minimally invasive thoracic surgery.

The increased use of chest computed tomography (CT) has led to an increased detection of pulmonary nodules requiring diagnostic evaluation and/or ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...

Novel Percutaneous Approach for Deployment of 3D Printed Coronary Stenosis Implants in Swine Models of Ischemic Heart Disease

Minimally invasive methods for creating models of focal coronary narrowing in large animals are challenging. Rapid prototyping using three-dimensionally (3D) printed coronary implants can be employed to percutaneously create a focal coronary stenosis. However, reliable delivery of the implants can be difficult without the use of ancillary equipment. We describe the use of a mother-and-child coronary guide catheter for stabilization of the implant and for effective delivery of the 3D printed implant to any desired location along the length of the coronary vessel. The focal coronary narrowing was confirmed under coronary cineangiography and the functional significance of the coronary stenosis was assessed using gadolinium-enhanced first-pass cardiac perfusion MRI. We showed that reliable delivery of 3D printed coronary implants in swine models (n = 11) of ischemic heart disease can be achieved through repurposing mother-and-child coronary guide catheters. Our technique simplifies the percutaneous delivery of coronary implants to create closed-chest swine models of focal coronary artery stenosis and can be performed expeditiously, with a low procedural failure rate.

We describe a novel, cost-effective, and efficient technique for percutaneous delivery of three-dimensionally printed coronary implants to create closed-chest swine models of ischemic heart disease. The implants were fixed in place using a mother-and-child extension catheter with high success rate.

Minimally invasive methods for creating models of focal coronary narrowing in large animals are challenging. Rapid prototyping using three-dimensionally ...