Minimal Invasive Surgical Procedure of Inducing Myocardial Infarction in Mice

The overall goal of this procedure is to induce a highly reproducible myocardial infarction in mice with minimally invasive manipulations. This is achieved by intubation under a stereo microscope to control the intubation cannula position during the procedure. In the second step, a discreet lateral left incision is made between the ribs to open the thoracic cavity and to facilitate quick healing.

The ligature of the left descending coronary artery is then placed according to the appropriate downstream manipulation. Ultimately, the size and shape of the infarction can be assessed by standard immunohisto morphometric analysis. The main advantages of this technique of existing methods are that is minimally invasive, highly reproducible, and easily perform, enabling researcher with even minimal experience with a method to apply it in small animal models.

Generally, individuals new to this method will struggle because the size of the mouse heart requires high precision of manipulation. During microsurgery. Begin by placing an anesthetized eight to 10 week old male, C 57 BL six wild type mouse in the supine position on a heated surgery table.

After confirming the appropriate level of sedation by toe pinch, apply eye ointment to the animal and use a small razor to remove the hair from the ventral neck area and the left half of the thorax disinfect the skin with 70%ethanol and brood derm. Then make a small 0.5 centimeter median incision in the center of the neck. Once under the skin, pass a pair of sterile curved forceps through the two fatty bodies to facilitate the visualization of the trachea through the covering muscle.

With a stereo microscope, introduce the intubation cannula orally into the trachea. The cannula should be observed through the transparent tissue. After confirming the proper placement of the instrument, connect the cannula to the small animal ventilator and adjust the ventilation settings to a tidal volume between 100 and 150 microliters and a respiration rate between 100 and 150 aspirations per minute to induce a myocardial infarction.

Next, make an incision less than 0.5 centimeters long in the skin between the xiphoid process and the left Xi.Then using forceps, separate the muscle layer from the underlying ribs for a chronic infarction. Make an incision in the fifth intercostal space for an ischemia reperfusion model. Make the incision in the fourth intercostal space.

Next place retractors into the incision to open the thoracic cavity and to visualize the heart. Then carefully remove the pericardium to prevent excessive fibrotic processes and visualize the left descending coronary artery as the deeply positioned light red vessel. For the chronic infarction model, place a ligature in the middle of the ventral side of the heart between the oracle and apex using the left descending coronary artery as a reference.

Then to obtain the transmural anterior and posterior infarction, use a zero seven silk suture to bind both branches of the artery. For the ischemia reperfusion model, place the ligature under the oracle and over a silicone tube and the main body of the left descending coronary artery. During the ischemia period, place temporal sutures on the ribs, moistening the sutures with a compress to avoid drying the tissue.

Then after the ischemia, remove the silicone tube and cut the suture with small scissors to visualize the reperfusion. After inducing the infarction, fill the thorax with warm isotonic salt solution to eliminate the residual air. Then close the thorax with 3 0 6 sutures, positioning the medial sutures at a 90 degree angle to ensure a sealed closure of the ribs.

Close the muscle layer with two sutures, followed by closure of the skin with three to four sutures as necessary. Then disconnect the intubation cannula from the ventilator to allow spontaneous breath marking the mouse for later identification, and place the animal under a red lamp until it wakes. To analyze the myocardial infarction data, regularly monitor the ejection fraction fractional shortening cardiac output and heart dimensions by echocardio.

At the end point of the experiment, open the thoracic cavity and excise the heart. Place the tissue in sterile PBS for extensive washing. Then once all the blood has been removed, stop the heart in diastole with a saturated potassium chloride solution and fix the tissue in 10%formalin overnight.

Embed the thin section of the heart tissue in paraffin positioning the tip of the heart at the bottom of the mounting block to allow transverse sectioning of the tissue. Then collect 20 serial sections of five microns, discarding the next 300 microns. Continue to collect 25 micron sections every 300 microns until the mitral valve level is reached.

Then use mori's one step staining to measure the infarction size of the tissue to perform TTC staining. Rebuild the ligature and use an aortic cannula to perfuse the heart with 200 microliters of 1%Evans blue solution and freeze the heart in a small plastic bag at negative 20 degrees Celsius without washing. After two hours, use a sharp scalpel to harvest five transverse sections from the tissue and incubate the sections for 10 to 15 minutes.

In TTC solution at 37 degrees Celsius according to the manufacturer's instructions. After another 10 minute fixation in 10%formalin, transfer the sections to glass microscope slides. For histological analysis.

Depending on the model used, the infarction size will differ. The chronic infarction model induces a circular transmural infarction of the apex, while the ischemia reperfusion model induces a thin middle wall throughout all of the heart. To analyze the direct effect of the infarction on cardiac viability, Evans blue TTC staining can be used to reveal the healthy myocardium not affected by the ischemia in blue, the viable at risk myocardium inside the ischemic area in red and the dead tissue in white.

Usually the infarction size is expressed as a percentage of the risk area go Maori's. One step staining allows the measurement of the mature scar that results from the remodeling process with the blue stained infarcted and red stained healthy ventricular areas analyzed in the first section from each level of the heart tissue acquired before the mitral valve. The infarction from all of the sections is then expressed as a percentage of the total left ventricular volume.

Conventional staining procedures also can be performed, for example, in these images. Staining for CD 31 and smooth muscle actin was used to reveal the presence of angiogenesis and myofibroblasts in the infarcted tissue respectively. Use double fluorescent staining to identify the different target molecules in the infarction area as no autofluorescence is observed in the absence of cardiomyocytes.

Once mastered, this technique can be completed in 25, 30 minutes if it is performed properly. Sufficient pain management is necessary to ensure high reproducibility Following this procedure. Other functional analysis can be carried out such as intraventricular, pressure measurements, ultrasound, or isolated heart lung.

End of perfusion.