Semi-Minimal Invasive Method to Induce Myocardial Infarction in Rats and the Assessment of Cardiac Function by an Isolated Working Heart System

Despite the significant improvements in therapy, numbers of patients that develop heart failure following acute myocardial infarction is still increasing. To observe the transition from acute MI to heart failure, we need reproducible animal models. We demonstrate a model of MI which is relatively easy, reproducible and causes minimal discomfort to the animals.

In addition, the isolated heart apparatus provides a highly reproducible preparation that can be studied in a time and cost-efficient manner. In collaboration with other research groups worldwide, we are using this technique to clarify the safety and efficacy of novel drugs and therapeutic approaches that improve myocardial repair and function following MI.Visual demonstration of the most critical steps of this protocol such as intubation or the level of LAD occlusion can help to prevent common pitfalls. The step-by-step description permits reproducible results even if performed by untrained researchers.

Milat Inci, lab technician, is performing the working heart measurements, and Michael Koch, mathematician and electronic engineer, is contributing in computational analysis. After anesthetizing the rat, intubate it with a 14 gauge tube and volume-controlled ventilation with a mixture of oxygen, air, and isoflurane. Place the rat on a heated operating table in a supine position and fix the forelimbs with tape.

Then place the ECG probes subcutaneously in the extremities of the animal. Use a scalpel to make an incision starting two millimeters parasternal on the left thorax at the level of the third intercostal space and continuing to the anterior axillary line at the level of the fifth intercostal space. Gently move the superficial muscles so that the ribs can be seen.

Perform the thoracotomy at the level of the fourth intercostal space and insert a retractor to gain visibility of the heart and lung. Then carefully open the pericardium to avoid bleeding. To induce ischemia reperfusion over a defined time, occlude the left anterior descending artery with a tourniquet.

After rupturing the myocardium, identify the left artery and place the suture two to three millimeters underneath its border at the ventral margin of the heart. Reopen the artery by removing the tourniquet after 30 minutes. Alternatively, the artery can be permanently occluded by making six to seven knots with a 6-0 suture to close the ligation.

Use a 4-0 single monofilament suture to close the thorax with three single button sutures. Prior to tightening the last suture, remove any residual air from the thorax with a 10 millimeter syringe to prevent a pneumothorax. Replace the muscles and turn off the volatile anesthesia.

Then suture the skin with a continuous 4-0 suture. Administer an antiseptic spray to protect against infection and biting of the suture. For deep anesthesia, administer a mixture of xylazine four milligrams per kilogram body weight and ketamine 100 milligrams per kilogram body weight intraperitoneally prior to echocardiography or working heart measurements.

For echocardiography, place the rat in a supine position on the heating tray and obtain parasternal short axis views of the left ventricular cavity at the level of the papillary muscle. Then perform M-mode echocardiography in order to measure left ventricular ejection fraction and morphology. To harvest the organs, perform an intravenous heparin injection.

Then use a scalpel to make a skin incision under the xyphoid and extended parallel to the ribs on both sides with scissors. Cut the ribs in the front axillary line and grab the xiphoid to lift up the chest. Remove anatomical or fibrotic tissue adhesions by carefully rupturing the tissue with two pairs of forceps.

Take blood samples from the vena cava with a five milliliter syringe. Then perform the excision of the whole heart at the inlet and outlet level. Immerse the heart in ice cold Krebs-Henseleit buffer and mount it on the erythrocyte-perfused isolated heart system by cannulating the aorta.

Start with the Langendorff mode. And after 15 minutes, switch to working heart mode. Cannulate the left atrium via a pulmonary vein.

Then change the flow direction by opening the clip that occludes the atrial cannula which results in a perfusion to the left atrium and a physiological blood flow in the left heart. Record hemodynamic measurements for 20 minutes in the working heart mode, collecting blood drops of the coronaries with a two milliliter syringe to measure coronary flow every five minutes. Perform continuous measurements of left atrial flow and aortic flow with a probe.

If needed, insert a high fidelity catheter retrogradely via the aortic valve into the left ventricle and measure the left ventricular systolic pressure. Conclude the analysis by calculating the stroke volume and external heart work as described in the text manuscript. Histological cuts and stains were performed to prove the reproducibility of the procedure.

The fibrotic scar in MIR and treated animals was comparable with the scar formation of the sham animals while the difference in fibrosis between sham and MIR groups was significant. Additionally, MIR and remote ischemic per-conditioning treated animals showed significantly reduced fibrosis compared to MIR-treated animals. In vivo echography was used to measure ejection fraction, LV end-diastolic and end-systolic diameters.

The analysis showed significantly reduced cardiac function due to MIR treatment, but hemodynamic parameters were preserved by RIPerc. Ex vivo hemodynamic data exhibited the effectiveness of the procedure as the MIR group showed significant decreases in LV systolic pressure, cardiac output, stroke volume, and external heart work. When attempting this procedure, it is important to remember that proper positioning of the occluding suture and mounting of the heart to the isolated heart apparatus significantly increase the chance of gaining reproducible results.

There are many ways of assessing cardiac function in the intact animal through acute or chronic instrumentation or even noninvasively by echocardiography or MRI. However, the isolated reperfused heart remains a prominent investigative tool and offers a whole array of unique advantages.