The authors would like to thank Maria Hedman, Tiina Laitinen, Tomi Laitinen, Pekka Poutiainen, Annika Viren, and Severi Sormunen for assistance and permitting 15O-PET imaging at Kuopio University Hospital; and Heikki Karhunen, Minna T&#246;rr&#246;nen, and Riikka Ven&#228;l&#228;inen from National Laboratory Animal Center for their assistance in animal work.
This study is supported by grants from Finnish Academy, ERC, and CardioReGenix EU Horizon 2020 grant.

The experiments presented here are performed using about 10-week-old female domestic pigs and are approved by the Animal Experiment Board in Finland. Animals weigh 30-40 kg at the beginning of the protocol, allowing the same procedural equipment and imaging modalities as possible for humans. Chronic ischemia is induced 14 days before the gene transfer, and the follow-up time after the gene transfer depends on the viral vector used. The study protocol is shown in Figure 1. This protocol can be used to perform adenoviral or AAV-mediated gene therapy injections. The time of sample collection has to be adjusted to the transgene expression peak, which depends on the viral vector used. For example, when performing adenoviral gene transfers, the time of the sample collection is set to 6 days after the gene transfer.
1. Medication
<ol>
	<li>Administer a daily dose of 200 mg of amiodarone and 2.5 mg of bisoprolol to prevent fatal ventricular arrhythmias. The medication begins 1 week before the ischemia operation and is continued daily until the follow-up.</li>
	<li>In addition, administer peroral doses of clopidogrel (300 mg) and acetylsalicylic acid (300 mg) to the animals 1 day before the ischemia operation to prevent acute in-stent thrombosis after the stent placement.</li>
	<li>Administer 100 mg of lidocaine and 2.5 mL of (246 mg/mL) MgSO4 intravenously to the animals at the beginning of the ischemia operation to prevent ventricular arrhythmias.</li>
	<li>Administer intramuscular injection of cefuroxime (500 mg) at the beginning of each operation for infection prophylaxis.</li>
	<li>Administer 30 mg of enoxaparin intravenously at the beginning of the ischemia operations and subcutaneously after the operation procedure for thrombosis prevention.</li>
	<li>For anesthesia and analgesia, administer 1.5 mL of atropin, 6 mL of azaperone (40 mg/mL), propofol 20 mg/mL, at a rate of 15 mg/kg/h, and fentanyl 50 &#181;g/mL at a rate of 10 &#181;g/kg/h. Drug dosages were the same for each pig. Refer to local animal use guidelines for dose administration.</li>
	<li>Anesthetize the animals during all operations. All the operations should be performed in a sterile environment using a sterile technique.</li>
</ol>
2. Transthoracic echocardiography
<ol>
	<li>Perform transthoracic echocardiography before ischemia operation, gene transfer, and euthanasia to evaluate any detectable pericardial fluid and determine the myocardial strain.</li>
	<li>Place the transducer in the third or fourth intercostal space under the armpit of the pig to access parasternal short-axis views at the mitral valve level, papillary muscle, and apical levels (Video 1). The marker of the transducer should point to the sternum of the pig. To save a clip, press Acquire.</li>
</ol>
3. Endovascular operations under fluoroscopic guidance
<ol>
	<li>Perform left ventricle cine imaging after coronary angiograms before ischemia operation, gene transfer and tissue collection.</li>
	<li>Preparation of the operation
	<ol>
		<li>Prepare for the operations by sedating the pigs with an intramuscular injection of 1.5 mL of atropine and 6 mL of azaperone.</li>
		<li>After the sedation, induce general propofol and fentanyl anesthesia for the angiographic procedures to the pigs with doses of 15 mg/kg/h and 10 &#181;g/kg/h, respectively. 
		NOTE: The pigs are anesthetized for the entire procedure.</li>
		<li>Support the ventilation by intubation and ventilator and monitor the vital physiological parameters, such as ECG and respiratory parameters.</li>
	</ol>
	</li>
	<li>Introducer sheath placement
	<ol>
		<li>Place an introducer sheath into the right femoral artery for all the operations as standard practice in cardiology. Use ultrasound to track the femoral artery and pierce it with an entry needle (18 G). 
		NOTE: Use an 8F introducer sheath for intramyocardial gene transfers and a 6F sheath for all other operations. Introduce the guidewire of the sheath through the needle to thread the artery and hold the guidewire still while removing the needle.</li>
		<li>Insert the introducer sheath along the guidewire, and when placed, remove the guidewire and administer 1.25 mg of sublingual dinitrate to the pig to induce coronary vasodilation.</li>
	</ol>
	</li>
	<li>Coronary angiography
	<ol>
		<li>Perform coronary angiography directly before ischemia operation, gene transfer, and tissue collection. The machinery needed for the angiograms is shown in Figure 2.</li>
		<li>Use a 6F catheter under fluoroscopic guidance with an iodine contrast agent to image the right coronary artery and the left ascending coronary artery (Video 2).</li>
	</ol>
	</li>
	<li>Left ventricle cine imaging under rest and dobutamine stress
	<ol>
		<li>Administer a 21 mL bolus of iodine contrast agent into the left ventricle via a 5F pigtail catheter using an auto-injector. First, set the bolus duration at 3 s and the total volume for 21 mL. Then, press&#160;Single&#160;and&#160;Yes.</li>
		<li>Calculate the ejection fraction by the measurement software of the angiographic workstation. To perform the calculation, select&#160;Ventricular Analysis&#160;of the image in question. Scroll the image to select a time frame, one in diastole and one in systole. Select a tool to draw ventricular outlines of each time frame. 
		NOTE: The software now calculates the ejection fraction and stroke volume by the Simpson&#39;s method. The ejection fraction measurement is performed during rest and under dobutamine-induced stress.</li>
	</ol>
	</li>
	<li>Stress imaging
	<ol>
		<li>Dose dobutamine intravenously in escalating doses from 10 &#181;g/kg/min to 20 &#181;g/kg/min for the dobutamine-induced stress imaging until the target heart rate of 160 bpm is reached. Then, perform the cine imaging.</li>
	</ol>
	</li>
	<li>Ischemia operation
	<ol>
		<li>Place a bottleneck stent into the left coronary artery (LAD) 14 days before the gene transfer to induce chronic myocardial ischemia. After the bottleneck stent placement, check whether the bottleneck stent is placed correctly, restricting the coronary blood flow. 
		NOTE: The bottleneck stent is placed on a dilation catheter and consists of a bare-metal stent covered by a polytetrafluoroethylene tube formed in a bottleneck shape to reduce coronary blood flow9.</li>
	</ol>
	</li>
	<li>Defining the stent size
	<ol>
		<li>Choose the size of the stent, either 3.0/3.5/4.0 x 8 mm, according to the size of the left ascending coronary artery in the angiogram by using the automatic measurement software in the angiographic workstation (Video 3)12.</li>
	</ol>
	</li>
	<li>Stent placement
	<ol>
		<li>Place a coil to the left coronary artery and glide the bottleneck stent to the LAD, placing it distally to the first diagonal.</li>
		<li>Inflate the stent to nominal pressure in the artery using an in-deflator with a stent-to-lumen ratio of 1.3, anchoring the bottleneck in place. After an additional 15 s, deflate the stent and retract the equipment from the artery. 
		NOTE: Confirm the correct placement of the bottleneck stent by angiogram.</li>
	</ol>
	</li>
</ol>
4. PET imaging
NOTE: One day before the gene transfer, perform rest and stress 15O-labeled radiowater PET/CT scans (requires hospital environment and radiological technicians).
<ol>
	<li>Reference imaging
	<ol>
		<li>Perform computed tomography (CT) scans before rest and stress imaging. Use the CT information for attenuation correction.</li>
	</ol>
	</li>
	<li>15O-labeled radiowater imaging
	<ol>
		<li>Perform rest and stress imaging using an 800 MBq 15H2O bolus.</li>
	</ol>
	</li>
	<li>Stress imaging
	<ol>
		<li>Perform stress imaging with a further 800 MBq 15O-water bolus after a suitable radioactive decay of 12 min. 
		NOTE: Hyperemia is induced by adenosine (200 &#181;g/kg/min intravenous), as described previously12.</li>
	</ol>
	</li>
</ol>
5. Gene transfer
<ol>
	<li>Electroanatomical mapping
	<ol>
		<li>Proceed to electroanatomical mapping after a coronary angiogram and functional measurements (echocardiography, LV cine imaging).</li>
		<li>Introduce a mapping catheter to the left ventricle via femoral sheath in fluoroscopic guidance. 
		NOTE: Register about 100-150 points around the left ventricle with the mapping catheter to create the electroanatomical map.</li>
	</ol>
	</li>
	<li>Finishing the electroanatomical map
	<ol>
		<li>Delete the outlier points to ensure a more reliable electroanatomical map of the left ventricle.</li>
		<li>Do this by selecting Clip Planes of the map and delete the points that differ from the points forming the ventricular shape. Next, select Trajectories for the map view and delete the points that have traveled horizontally during point registering. 
		NOTE: Make sure the remaining points cover the left ventricle and register more points if needed.</li>
	</ol>
	</li>
	<li>Gene transfer injections
	<ol>
		<li>Introduce an intramyocardial injection catheter to the left ventricle via the femoral sheath under fluoroscopic guidance. Set the injection needle length to 3 mm.</li>
	</ol>
	</li>
	<li>Criteria for intramyocardial injections
	<ol>
		<li>Guide the gene transfers by electroanatomical mapping system and target the injections into viable but hypokinetic areas of the left ventricle. 
		NOTE: For viability, use a unipolar voltage over 5 mV as a criterion. For hypokinesia, select a local linear shortening (LLS) as low as available, at least below 12% but preferably below 6%13.</li>
	</ol>
	</li>
	<li>Intramyocardial injections
	<ol>
		<li>During 30 s, inject the vector material into the point of selection (step 5.4) and keep the injection needle inside the myocardium for an additional 5 s before retracting to prevent backflow to the left ventricle.</li>
	</ol>
	</li>
</ol>
6. Euthanasia and sample collection
NOTE: After the coronary angiogram and ejection fraction measurements described in steps 3.4.1 and 3.5.2, respectively, administer 50 mL of saturated potassium chloride intravenously to the anesthetized pig.
<ol>
	<li>Perfusion fixation of the heart
	<ol>
		<li>Harvest the heart from the thoracic cavity. Rinse with water. Place an 18 G needle above the aortic valve and attach the needle to a perfusion pump. Perfuse the heart with 750 mL of 1% paraformaldehyde (PFA).</li>
	</ol>
	</li>
	<li>Sample collection
	<ol>
		<li>Slice the heart into 1 cm thick slices using a sharp kitchen knife. Collect the samples from the gene transfer area into 4% PFA and liquid nitrogen. 
		NOTE: To harvest negative controls, collect a control sample from the posterior wall of the left ventricle.</li>
	</ol>
	</li>
	<li>Safety tissue collection
	<ol>
		<li>Harvest samples from remote tissues, such as the lung, liver, kidney, spleen, and ovaries. Take samples into 4% PFA and liquid nitrogen.</li>
	</ol>
	</li>
</ol>
7. Sample storing
<ol>
	<li>Store the samples for staining in 4% PFA for 48 h at 4 &#176;C. 
	NOTE: Replace the PFA daily with a fresh liquid.
	<ol>
		<li>After 48 h, replace the PFA with 15% sucrose in deionized water. Store for at least 24 h before embedding the samples into paraffin blocks. Snap frozen samples are stored at -70 &#176;C.</li>
	</ol>
	</li>
</ol>

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<li>Hammond, H. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intracoronary+gene+transfer+of+adenylyl+cyclase+6+in+patients+with+heart+failure%3A+A+randomized+clinical+trial%5BTitle%5D)+AND+%22JAMA+Cardiology%22%5BJournal%5D)">Intracoronary gene transfer of adenylyl cyclase 6 in patients with heart failure: A randomized clinical trial.</a> JAMA Cardiology. 1 (2), 163-171 (2016).</li>
<li>Hartikainen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adenoviral+intramyocardial+VEGF-DDNDC+gene+transfer+increasesmyocardial+perfusion+reserve+in+refractory+angina+patients%3A+A+phase+I%2FIIa+study+with+1-year+follow-up%5BTitle%5D)+AND+%22European+Heart+Journal%22%5BJournal%5D)">Adenoviral intramyocardial VEGF-DDNDC gene transfer increasesmyocardial perfusion reserve in refractory angina patients: A phase I/IIa study with 1-year follow-up.</a> European Heart Journal. 38 (33), 2547-2555 (2017).</li>
<li>Laakkonen, J. P., Ylä-Herttuala, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recent+advancements+in+cardiovascular+gene+therapy+and+vascular+biology%5BTitle%5D)+AND+%22Human+Gene+Therapy%22%5BJournal%5D)">Recent advancements in cardiovascular gene therapy and vascular biology.</a> Human Gene Therapy. 26 (8), 518-524 (2015).</li>
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The authors declare no conflicts of interest.

The timepoints of this protocol may be modified according to the viral vector used. Also, the immunohistological analyses may be selected according to the therapeutic gene. It is also possible to add more timepoints and endpoints to the protocol if needed.
This protocol comprises stages, which are essential to succeed and impossible to correct afterward. First, if one fails to induce appropriate ischemia, the animal must be excluded from further procedures and analyses. Standardization of the ...

Coronary artery disease is accountable for the vast proportion of mortality and disease burden worldwide1. Current treatment strategies are percutaneous interventions, pharmacological treatment, and bypass surgery2. However, despite the progression of these current therapeutics, many patients suffer from so-called refractory angina, underlining the unmet need for novel treatment approaches3. Gene therapy-mediated therapeutic angiogenesis could target this patient group.
Myocardial gene therapy is most often delivered by using different viral vectors, most commonly replication-deficient adenovirus4. As therapeutic genes, various angiogenic growth factors are used. The most substantially studied angiogenic growth factors are the vascular endothelial growth factors (VEGFs) that mediate their angiogenic signaling through vascular endothelial growth factor receptors (VEGFRs) and their co-receptors5. Several clinical trials have proved the benefit and safety of cardiac gene therapy and made this novel treatment method a realistic option for treating ischemic heart diseases6,7. However, this concept still needs enhancement of the therapeutic genes and viral vectors put to the test in large animal models before entering the clinics. The pig has been frequently used as a laboratory animal since its heart is very similar to the human heart. The size of the cardiovascular system of a pig allows the usage of similar catheter inventions as used in humans. All imaging modalities available for humans can be used in pigs8.
There are several large animal models for chronic ischemia. The most commonly used is the ameroid constrictor model9,10,11. The downside of this method is the invasiveness since thoracotomy is needed to access the coronary vasculature. Previously in our group, a mini-invasive bottleneck stent model for chronic myocardial ischemia has been developed12. This method is also used in this manuscript to induce myocardial ischemia.
The usability of ultrasound imaging has evolved substantially despite the age of the imaging modality. For example, myocardial strain is still mainly in research usage due to its novelty. Myocardial strain reflects changes in the contractile function of the heart better than the traditional M-mode ejection fraction measurement13. Thus, here in the large animal model, myocardial strain measurement is utilized. To evaluate the function of the heart, cardiac output is also measured by cine imaging of the left ventricle during angiography. Cardiac output is measured both at rest and under dobutamine-induced stress to evaluate myocardial function under stress.
In addition to the measurements of the heart function, information on myocardial perfusion is essential in gene therapy studies aiming at therapeutic angiogenesis. In this animal model, animals are imaged with 15O-labeled radiowater positron emission tomography (15H2O-PET) as this is the golden standard for measuring myocardial perfusion. 15H2O-PET has been previously validated for measuring perfusion of ischemic porcine heart14.
Thus, the methods and modalities mentioned above constitute an excellent perspective for evaluating the efficacy of gene therapy in the ischemic heart.

<table><tbody><tr><td>1% PFA</td><td>VWR</td><td>VWRC28794.295</td><td>Prepared from paraformaldehyde powder</td></tr><tr><td>15 % sucrose</td><td>VWR</td><td>VWRC27480.294</td><td>Prepared from solid sucrose</td></tr><tr><td>4% PFA</td><td>VWR</td><td>VWRC28794.295</td><td>Prepared from paraformaldehyde powder</td></tr><tr><td>5 F pigtail catheter</td><td>Cordis</td><td>534-550S</td><td></td></tr><tr><td>6 F catheter AR2</td><td>Cordis</td><td>670-112-00</td><td></td></tr><tr><td>6 F introducer sheath</td><td>Cordis</td><td>504-606X</td><td></td></tr><tr><td>8 F introducer sheath</td><td>Cordis</td><td>504-608X</td><td></td></tr><tr><td>Acetylsalicylic acid</td><td></td><td></td><td>Varying producer</td></tr><tr><td>Amiodarone</td><td></td><td></td><td>Varying producer</td></tr><tr><td>Angiographic station</td><td>GE Healthcare</td><td></td><td></td></tr><tr><td>Angiolaboratory set</td><td>M&amp;ouml;lnlycke</td><td></td><td>designed for the needs of our angiolaboratory, contains sterile drapes, cups and swabs</td></tr><tr><td>Bisoprolol</td><td></td><td></td><td>Varying producer</td></tr><tr><td>Cefuroxime</td><td></td><td></td><td>Varying producer</td></tr><tr><td>Clopidogrel</td><td></td><td></td><td>Varying producer</td></tr><tr><td>Coroflex Blue stent</td><td>B.Braun Medical</td><td>5029012</td><td>Catalog number depends on stent size</td></tr><tr><td>Crile forceps</td><td></td><td></td><td></td></tr><tr><td>Cyclotron</td><td>GE Healthcare</td><td></td><td></td></tr><tr><td>Dobutamine</td><td></td><td></td><td>Varying producer</td></tr><tr><td>Electroanatomical mapping system</td><td>Biologics Delivery Systems, Johnson &amp;amp; Johnson company</td><td></td><td></td></tr><tr><td>Enoxaparin</td><td></td><td></td><td>Varying producer</td></tr><tr><td>Fentanyl</td><td></td><td></td><td>Varying producer</td></tr><tr><td>Intramyocardial injection catheter</td><td>Johnson &amp;amp; Johnson</td><td></td><td></td></tr><tr><td>Iodine contrast agent</td><td>Iomeron</td><td></td><td></td></tr><tr><td>Kitchen knife</td><td></td><td></td><td>Varying producer</td></tr><tr><td>Lidocaine</td><td></td><td></td><td>Varying producer</td></tr><tr><td>Liquid nitrogen</td><td></td><td></td><td>Varying producer</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td></td><td></td><td>Varying producer</td></tr><tr><td>Needle 18 G</td><td>Cordis</td><td>12-004943</td><td></td></tr><tr><td>Perfusion pump</td><td></td><td></td><td></td></tr><tr><td>PET-CT scanner</td><td>Siemens Healthcare</td><td></td><td></td></tr><tr><td>Polytetrafluoroethylene tube</td><td></td><td></td><td></td></tr><tr><td>Propofol</td><td></td><td></td><td>Varying producer</td></tr><tr><td>Scalpel no 11</td><td>VWR</td><td>SWAN0503</td><td></td></tr><tr><td>Sublingual dinitrate</td><td>Takeda</td><td></td><td></td></tr><tr><td>Ultrasound machine</td><td>Philips</td><td></td><td></td></tr></tbody></table>

large animal model for evaluating the efficacy of the gene therapy in ischemic heart

Coronary artery disease is one of the significant causes of mortality and morbidity worldwide. Despite the progression of current therapeutics, a considerable proportion of coronary artery disease patients remain symptomatic. Gene therapy-mediated therapeutic angiogenesis offers a novel therapeutic method for improving myocardial perfusion and relieving symptoms. Gene therapy with different angiogenic factors has been studied in few clinical trials. Due to the novelty of the method, the progress of myocardial gene therapy is a continuous path from bench to bedside. Therefore, large animal models are needed for evaluating the safety and efficacy. The more the large animal model identifies the original disease and the endpoints used in clinics, the more predictable outcomes are from clinical trials. Here, we introduce a large animal model for evaluating the efficacy of the gene therapy in the ischemic porcine heart. We use clinically relevant imaging methods such as ultrasound imaging and 15H2O-PET. For targeting the gene transfers into the desired area, electroanatomical mapping is used. The aim of this method is: (1) to mimic chronic coronary artery disease, (2) to induce therapeutic angiogenesis at hypoxic areas of the heart, and (3) to evaluate the safety and efficacy of the gene therapy by using relevant endpoints.

The success of the ischemia operation can be confirmed with this protocol by coronary angiogram and by determining the hypokinetic area by transthoracic ultrasound (Figure 1) before proceeding to the gene delivery. The state of the coronary occlusion can be evaluated by coronary angiogram, and the electroanatomical mapping ensures the ischemic and hibernating areas.
The efficacy of the gene therapy can be analyzed by measuring the circumferential strain, ejection ...

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Large Animal Model for Evaluating the Efficacy of the Gene Therapy in Ischemic Heart

Myocardial gene therapy for ischemic heart disease holds great promise for future therapeutics. Here, we introduce a large animal model for evaluating the efficacy of gene therapy in the ischemic heart.

Coronary artery disease is one of the significant causes of mortality and morbidity worldwide. Despite the progression of current therapeutics, a ...

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Research

JoVE Journal

Medicine

1.8K Views.  University of Eastern Finland. De methoden en beeldvormingsmodaliteiten die in dit protocol worden gebruikt, vormen een uitstekend beeld van de veiligheid en werkzaamheid van gentherapie in ischemisch hart voordat wordt overgegaan tot klinische onderzoeken. De belangrijkste voordelen van deze techniek zijn de lage invasiviteit van het ischemiemodel, maar ook de precieze afstemming van de gentherapie op hypokinetische maar nog steeds levensvatbare delen van het hart. Begin met het uitvoeren van de transthoracale echocardiog...

Video: Large Animal Model for Evaluating the Efficacy of the Gene Therapy in Ischemic Heart

Gene Transfer for Ischemic Heart Failure in a Preclinical Model

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine their efficacy and safety.
Gene therapy using viral vectors is one of the most promising approaches for treating cardiac diseases. Viral delivery of various different genes by changing the carrier gene has immeasurable therapeutic potential.
In this video, the full process of an animal model of heart failure creation followed by gene transfer is presented using a swine model. First, myocardial infarction is created by occluding the proximal left anterior descending coronary artery. Heart remodeling results in chronic heart failure. Unique to our model is a fairly large scar which truly reflects patients with severe heart failure who require aggressive therapy for positive outcomes. After myocardial infarct creation and development of scar tissue, an intracoronary injection of virus is demonstrated with simultaneous nitroglycerine infusion. Our injection method provides simple and efficient gene transfer with enhanced gene expression. This combination of a myocardial infarct swine model with intracoronary virus delivery has proven to be a consistent and reproducible methodology, which helps not only to test the effect of individual gene, but also compare the efficacy of many genes as therapeutic candidates.

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine ...

Evaluation of Exercise Tolerance of Pigs Subjected to Exercise

Exercise Test for Evaluation of the Functional Efficacy of the Pig Cardiovascular System

Despite the progress in treatments, cardiovascular diseases are still one of the biggest causes of mortality and morbidity worldwide. Gene therapy-based therapeutic angiogenesis is a promising approach for treating patients with significant symptoms, despite optimal pharmacological therapy and invasive procedures. However, many promising cardiovascular gene therapy techniques have failed to accomplish expectations in clinical trials. One explanation is a mismatch between preclinical and clinical endpoints used to measure efficacy. In animal models, the emphasis has usually been on easily quantifiable endpoints, such as the number and area of the capillary vessels calculated from histological sections. Apart from mortality and morbidity, endpoints in clinical trials are subjective, such as exercise tolerance and quality of life. However, the preclinical and clinical endpoints likely measure different aspects of the applied therapy. Nevertheless, both types of endpoints are required to develop successful therapeutic approaches. In clinics, the main goal is always to alleviate patients' symptoms and improve their prognosis and quality of life. To achieve better predictive data from preclinical studies, endpoint measurements must be better matched to those in clinical studies. Here, we introduce a protocol for a clinically relevant treadmill exercise test in pigs. This study aims to: (1) provide a reliable exercise test in pigs that can be used to evaluate the safety and functional efficacy of gene therapy and other novel therapies, and (2) better match the endpoints between preclinical and clinical studies.

Author Spotlight: Exercise Test for Evaluation of the Functional Efficacy of the Pig Cardiovascular System  

The present protocol describes a large animal exercise test model to assess the functional capacity of the cardiovascular system for evaluating the efficiency of new therapies in the preclinical setting. It is comparable to a clinical exercise test.

Despite the progress in treatments, cardiovascular diseases are still one of the biggest causes of mortality and morbidity worldwide. Gene therapy-based ...

Genetics

Myocardial Infarction and Functional Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ligation is a challenging procedure and has a high mortality rate due to its nature. We developed a method to consistently transplant bioprinted patches of cells and hydrogels onto the epicardium of an infarcted mouse heart to test their regenerative properties in a robust and feasible way. First, a deeply anesthetized mouse is carefully intubated and ventilated. Following left lateral thoracotomy (surgical opening of the chest), the exposed LAD is permanently ligated and the bioprinted patch transplanted onto the epicardium. The mouse quickly recovers from the procedure after chest closure. The advantages of this robust and quick approach include a predicted 28-day mortality rate of up to 30% (lower than the 44% reported by other studies using a similar model of permanent LAD ligation in mice). Moreover, the approach described in this protocol is versatile and could be adapted to test bioprinted patches using different cell types or hydrogels where high numbers of animals are needed to optimally power studies. Overall, we present this as an advantageous approach which may change preclinical testing in future studies for the field of cardiac regeneration and tissue engineering.

This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart.

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ...

Bioengineering

A Chronic Cardiac Ischemia Model in Swine Using an Ameroid Constrictor

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but regardless of the approach, an in vivo model is needed to test each treatment. The pig is one of the most used large animal models for cardiovascular disease. Its heart is very similar in anatomy and function to that of a human. The ameroid placement technique creates an ischemic area of the heart, which has many useful applications in studying myocardial infarction. This model has been used for surgical research, pharmaceutical studies, imaging techniques, and cell therapies.
There are several ways of inducing an ischemic area in the heart. Each has its advantages and disadvantages, but the placement of an ameroid constrictor remains the most widely used technique. The main advantages to using the ameroid are its prevalence in existing research, its availability in various sizes to accommodate the anatomy and size of the vessel to be constricted, the surgery is a relatively simple procedure, and the post-operative monitoring is minimal, since there are no external devices to maintain. This paper provides a detailed overview of the proper technique for the placement of the ameroid constrictor.

The purpose of this protocol is to demonstrate the placement of a delayed constricting device (an ameroid constrictor) around a coronary artery in a swine model. This device creates an ischemic area of the heart that is useful for studying new diagnostic imaging techniques and new methods of treatment.

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but ...

Percutaneous Contrast Echocardiography-guided Intramyocardial Injection and Cell Delivery in a Large Preclinical Model

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection fraction (HFrEF). Before they can be considered for use, and implemented in humans, extensive preclinical studies are required in large animal models to evaluate the safety, efficacy, and fate of the injectate (e.g., stem cells) once delivered into the myocardium. Small rodent models offer advantages (e.g., cost effectiveness, amenability for genetic manipulation); however, given inherent limitations of these models, the findings in these rarely translate into the clinic. Conversely, large animal models such as rabbits, have advantages (e.g., similar cardiac electrophysiology compared to humans and other large animals), whilst retaining a good cost-effective balance. Here, we demonstrate how to perform a percutaneous contrast echocardiography-guided intramyocardial injection (IMI) technique, which is minimally invasive, safe, well tolerated, and very effective in the targeted delivery of injectates, including cells, into several locations within the myocardium of a rabbit model. For the implementation of this technique, we also have taken advantage of a widely available clinical echocardiography system. After putting in practice the protocol described here, a researcher with basic ultrasound knowledge will become competent in the performance of this versatile and minimally invasive technique for routine use in experiments, aimed at hypothesis testing of the capabilities of cardiac regenerative therapeutics in the rabbit model. Once competency is achieved, the whole procedure can be performed within 25 min after anaesthetizing the rabbit.

Novel therapeutic strategies in cardiac regenerative medicine require extensive and detailed studies in large preclinical animal models before they can be considered for use in humans. Here, we demonstrate a percutaneous contrast echocardiography-guided intramyocardial injection technique in rabbits, which is valuable for hypothesis testing the efficacy of such novel therapies.

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection ...

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves focal endothelial-selective transgenesis, thereby allowing an investigator to determine the biological roles of endothelial-expressed transgenes and to quantify the in vivo transcriptional activity of DNA sequences in large artery endothelial cells. The method uses surgical isolation of rabbit common carotid arteries and an arteriotomy to deliver a transgene-expressing viral vector into the arterial lumen. A short incubation period of the vector in the lumen, with subsequent aspiration of the lumen contents, is sufficient to achieve efficient and durable expression of the transgene in the endothelium, with no detectable transduction or expression outside of the isolated arterial segment. The method allows assessment of the biological activities of transgene products both in normal arteries and in models of human vascular disease, while avoiding systemic effects that could be caused either by targeting gene delivery to other sites (e.g. the liver) or by the alternative approach of delivering genetic constructs to the endothelium by germ line transgenesis. Application of the method is limited by the need for a skilled surgeon and anesthetist, a well-equipped operating room, the costs of purchasing and housing rabbits, and the need for expertise in gene-transfer vector construction and use. Results obtained with this method include: transgene-related alterations in arterial structure, cellularity, extracellular matrix, or vasomotor function; increases or reductions in arterial inflammation; alterations in vascular cell apoptosis; and progression, retardation, or regression of diseases such as intimal hyperplasia or atherosclerosis. The method also allows measurement of the ability of native and synthetic DNA regulatory sequences to alter transgene expression in endothelial cells, providing results that include: levels of transgene mRNA, levels of transgene protein, and levels of transgene enzymatic activity.

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

This method is to introduce a transgene into the endothelium of rabbit carotid arteries. Introduction of the transgene allows the assessment of the biological role of the transgene product either in normal arteries or disease models. The method is also useful for measuring activity of DNA regulatory sequences.

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves ...

A Pre-Clinical Porcine Model of Orthotopic Heart Transplantation

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart failure. Multiple small-animal models of heart transplantation have been used to study the acute and long-term effects of novel therapies. However, few are tested and demonstrated success in clinical trials. It is of critical importance to evaluate new therapies in a clinically relevant large-animal model for efficient and reliable translation of basic studies&#39;&#160;findings. Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and previously used to investigate novel cardioprotective strategies. This procedure focuses on acute ischemia-reperfusion injury and is a reliable method to investigate novel interventions which have been tested and validated in smaller experimental models, such as the murine model. We demonstrate its usefulness in assessing cardiac performance during the early post-transplantation period and other potential possibilities enabled by the model.

Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and utilized to investigate novel cardioprotective strategies.

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart ...

Establishing a Swine Model of Post-myocardial Infarction Heart Failure for Stem Cell Treatment

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major causes of mortality and morbidity around the world. Cell-based therapies for cardiac repair and improvement of left ventricular function after MI have attracted considerable attention. Accordingly, the safety and efficacy of these cell transplantations should be tested in a preclinical large animal model of HF prior to clinical use. Pigs are widely used for cardiovascular disease research due to their similarity to humans in terms of heart size and coronary anatomy. Therefore, we sought to present an effective protocol for the establishment of a porcine chronic HF model using closed-chest coronary balloon occlusion of the left circumflex artery (LCX), followed by rapid ventricular pacing induced with pacemaker implantation. Eight weeks later, the stem cells were administered by intramyocardial injection in the peri-infarct area. Then the infarct size, cell survival, and left ventricular function (including echocardiography, hemodynamic parameters, and electrophysiology) were evaluated. This study helps establish a stable preclinical large animal HF model for stem cell treatment.

We sought to establish a swine model of heart failure induced by left circumflex artery blockage and rapid pacing to test the effect and safety of intramyocardial administration of stem cells for cell-based therapies.

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major ...

Delivery of Cardioactive Therapeutics in a Porcine Myocardial Infarction Model

Myocardial infarction is one of the leading causes of death and disability worldwide, and there is an urgent need for novel cardioprotective or regenerative strategies. An essential component of drug development is determining how a novel therapeutic is to be administered. Physiologically relevant large animal models are of critical importance in assessing the feasibility and efficacy of various therapeutic delivery strategies. Due to their similarities to humans in cardiovascular physiology, coronary vascular anatomy, and heart weight to body weight ratio, swine is one of the preferred species in the preclinical evaluation of new therapies for myocardial infarction. The present protocol describes three methods of administering cardioactive therapeutic agents in a porcine model. After percutaneously induced myocardial infarction, female landrace swine received treatment with novel agents through either: (1) thoracotomy and transepicardial injection, (2) catheter-based transendocardial injection, or (3) intravenous infusion via jugular vein osmotic minipump. The procedures employed for each technique are reproducible, resulting in reliable cardioactive drug delivery. These models can be easily adapted to suit individual study designs, and each of these delivery techniques can be used to investigate a variety of possible interventions. Therefore, these methods are a useful tool for translational scientists pursuing novel biological approaches in cardiac repair following myocardial infarction.

The present protocol describes three methods of administering cardioactive therapeutic agents in a porcine model. Female landrace swine received treatment through either: (1) thoracotomy and transepicardial injection, (2) catheter-based transendocardial injection, or (3) intravenous infusion via jugular vein osmotic minipump.

Large Animal Model for Evaluating the Efficacy of the Gene Therapy in Ischemic Heart

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