The overall goal of this procedure is to demonstrate the use of high-speed micro-computed tomography in the imaging of intrinsic cardio respiratory gating to evaluate global and regional myocardial functions in a mouse cardiac ischemia model. This method can help to answer key questions in cardiac pathophysiology such as what are the effects on pathology or of malfa treatments on global and regional myocardial function? The main advantage of this technique is that it provides a rapid, longitudinal, non-invasive evaluation of the most important cardiac parameters that is easily performed by non-experts.
The implications of this technique extend towards the therapy of cardiovascular diseases. As can be used as a preclinical tool for obtaining advanced insight into cardiac disease development. Demonstrating the procedure will be Nicole van Vliet, a technician as well as Elza van Deel who's a post-doc and Yanto Ridwan another technician from our lab.
Begin by making a small incision in the skin of a 12 week old mouse between the second and third ribs. Then using small hooks, pull aside the chest muscles to expose the intercostal muscles. Next use curved two millimeter blade spring scissors to carefully cut through the third intercostal muscle without damaging the lungs.
Then using forceps, rupture the pericardium and use a piece of wet gauze to push aside the lungs. Reposition the hooks to the inside of the thorax so that a large part of the left ventricular free wall and part of the left atrium are visible. Then using the atrium to locate the left coronary artery insert a seven zero silk surgical suture under the left coronary artery and knot the suture two millimeters below the edge of the left atrium to standardize the infarct size.
After visually confirming the success of the infarct induction by the paling of the left ventricular free wall, carefully reinflate the lung and tightly close the chest with a six zero silk surgical suture. Clean the wound with saline and use more silk sutures to close the skin followed by the application of wound spray to stimulate healing and to prevent infection. Then place the mouse in a cage on a heating pad until it is fully recovered.
Three to four hours after the surgery, turn on the microCT scanner, open the microCT control software and warm up the x-ray tube. Then treat the rubber vial stopper of an iodinate contrast agent container with 70%alcohol and use a low dead space syringe to withdraw five to 10 microliters of the agent per grams of the animal's body weight. After purging the air bubbles, swab the tail of the anesthetized mouse with 70%alcohol and heat dilate the tail veins.
Inject the contrast agent into a lateral tail vein. When the live mode button appears in the control software the x-ray tube is ready. Insert the small bore cover and the small animal bed.
Click the new database button in the database window and enter a name for the new database. When the dialogue box appears click the browse button and navigate to the drive where the database will be saved. Then click okay and observe the new database in the database window.
To set the scan conditions, select the appropriate parameters. Now place the injected, anesthetized mouse onto the animal bed in the scanner and slide the instrument door to the right to engage the safety interlock. Click the live mode button to view the subject in real time and observe the X capture window and the animal.
Pressing the stage Z-axis control back and forth buttons move the animal bed until the mouse chest is within the field of view. Then use the animal bed control left and right arrows to position the animal inside the blue bounding box. Select 90 from the rotation control drop-down list in the control software window and click the set button to rotate the gantry making sure the animal remains within the blue bounding box of the X capture window.
In the X capture window left-click the mouse to resize the cardio-respiratory region of interest. Dragging the edges so that the cardio-respiratory traces are clearly visible in the synchronization view. Press the home position and resize the region of interest for cardiorespiratory.
Make sure that the region of interest covers the diaphragm and the apical portion of the heart in all of the gantry positions. Then rotate the gantry 90 degrees to make sure that the cardiorespiratory traces are still clearly visible. When the animal is in position click the CT scan button to initialize the acquisition.
The CT scan confirmation message will appear. Click yes. The red x-ray energizing indicator will turn on the instrument and the scan will begin.
At the end of the scan observe the transaxial, coronal and sagittal views of the reconstructions in 2D viewer software. Finally, remove the animal from the scanner and monitor it until it is fully recovered. Here, representative blood pool short-axial end-diastolic and end-systolic cross sections of a mouse heart with a myocardial infarction are shown.
In these images short-axial end-diastolic and end-systolic cross sections of a mouse heart without myocardial infarction exhibit an excellent left ventricular cavity delineation with little background noise. Note how the areas of contrast rarefaction, corresponding to the myocardial infarction, were well demarcated on the short-axial images of the mouse heart subjected to the left anterior descending coronary artery ligation. But not in the sham-operated animal.
According to the 17 segment American Heart Association Model the left ventricle can be subdivided into basal, mid-cavity and apical short-axial portions. In these representative, circumferential polar plot displays a dissimilar profusion is clearly visible in the mid-anterior, mid-inferolateral, mid-anterolateral, apical anterior and apical lateral segments in the myocardial infarction mouse compared to the sham-operated animal. Once mastered, the induction of myocardial infarction can be completed in 45 minutes while the subsequent microCT imaging requires four hours.
While attempting this procedure it's important to remember that the cardiac functional performance is dependent on the type and degree of anesthesia. Thus for accurate measurements the heart rate should be kept as physiological as possible during the image acquisition. After its development, this technique paved the way for researchers in the field of pharmacology to explore intervention strategies in mice and to analyze the impact of level treatment strategies on disease progression.
After watching this video you should have a good understanding of the myocardial infarction surgery and know how to use the high-speed microCT system in conjunction with the iodine-based blood pool contrast agent. Don't forget that working with Isoflurane can be hazardous and that precautions such as good ventilation should always be taken while performing this procedure.
