The methods and imaging modalities used in this protocol constitute an excellent image of the safety and efficacy of gene therapy in ischemic heart before proceeding to clinical trials. The main advantages of this technique are the low invasiveness of the ischemia model, but also the precise targeting of the gene therapy to hypokinetic but still viable areas of the heart. Begin by performing the transthoracic echocardiography before ischemia operation, gene transfer, and euthanasia to evaluate any detectable pericardial fluid and determine the myocardial strain.
Place the transducer in the third or fourth intercostal space under the armpit of the pig with a marker pointing to the sternum of the pig for accessing parasternal short axis views at the mitral valve level, papillary muscle, and apical levels. Then press acquire to save a clip. For performing the coronary angiography directly before ischemia operation use a 6-F catheter under fluoroscopic guidance with an iodine contrast agent to image the right coronary artery, the left ascending coronary artery, and the left anterior descending artery.
For cine imaging in the autoinjector, set the bolus duration at three seconds, and the total volume for 21 milliliters, and then press single, and yes to administer iodine contrast agent into the left ventricle via a 5-F pigtail catheter. Perform cine imaging of the animal at rest and then administer dobutamine intravenously and escalating doses for the stress induction. Once the target heart rate of 160 beats per minute is achieved performs cine imaging and remove the pigtail catheter.
To calculate the ejection fraction open the measurement software, select ventricular analysis of the image in question. Scroll the image to select a timeframe, one in diastole and one in systole, and then select a tool to draw ventricular outlines of each timeframe. For inducing the chronic myocardial ischemia, 14 days before gene transfer, place a coil to the left coronary artery, and glide the bottleneck stent to the left coronary artery, and place the stent distally to the first diagonal.
Inflate the stent to nominal pressure in the artery, using an indeflator with a stent to lumen ratio of 1.3 for anchoring the bottleneck in place. After an additional 15 seconds, deflate the stent, retract the equipment from the artery, and ensure the correct placement of a bottleneck stent by angiography. After angiography and functional measurements under fluoroscopic guidance introduce a mapping catheter to the left ventricle via the femoral sheath for electro anatomical mapping, and collect at least 100 points from the left ventricle, and click accept to approve a point.
In the final map, for viability, use a unipolar voltage over five millivolts as a criterion. And for hypokinesia, select a local linear shortening as low as possible, at least as low as 12%but preferably below 6%After euthanizing the animal, harvest the heart from the thoracic cavity. Once the harvested heart is rinsed place an 18-gauge needle above the aortic valve and attach the needle to a perfusion pump.
Perfuse the heart with 750 milliliters of 1%paraformaldehyde. Use a sharp knife to slice the heart into one centimeter thick slices, and collect the samples from the gene transfer area into 4%paraformaldehyde and liquid nitrogen. Store the samples for staining in 4%paraformaldehyde for 48 hours at four degrees Celsius.
The efficacy of the gene therapy was analyzed by measuring the circumferential strain, ejection fraction, and myocardial perfusion by 15O-water PET scan. The tissue samples can be collected directly from the gene transfer area, by comparing the heart to the electro anatomical map. The transgene expression and therapeutic angiogenesis were evaluated through immune histological analysis by analyzing the number of positive cells after Beta-galactosidase staining, and by analyzing the myocardial capillary area, after platelet/endothelial cell adhesion molecule-1 staining.
It is important to confirm the placement of the bottleneck stent via angiogram and sample collection from the gene transfer area, which could be confirmed by comparing the heart to the electroanatomical map. Overall, it is crucial to make all measurements in the most reproducible way.
