The overall goal of this procedure is to perform a murn closed chest model for time, delayed myocardial ischemia and reperfusion to reduce modulation of reperfusion injury and inflammatory response by the surgical trauma itself. This is accomplished by first performing a blunt thoracotomy in the left fourth intercostal space. The second step is to place a ligation with an occlusion device around the left anterior descending artery or LAD.
Next, the ligation ends are knotted to form a loop and placed into the subcutaneous tissue after closing the chest and letting the mouse recover for five days. Hanging weights are used to produce controlled ischemia and reperfusion while ECGs are recorded. Ultimately, TC staining or histology are used to show infarct size.
The main advantage of this technique over existing methods like open chest models of myocardial ischemia and reperfusion, is that with this technique, the inflammatory response by the surgical trauma itself is blunted. After confirming sedation, begin the procedure by applying depilatory cream to the neck and left chest of the mouse. After one minute, wipe off the cream and apply povidone iodine for local skin disinfection.
Then inject buprenorphine subcutaneously for pain relief. Next, use a small pair of scissors to make a midline neck skin incision, and then blunt, dissect the glands and muscles that cover the trachea. Then turn on the ventilator, setting the respiratory rate to 105 per minute and the tidal volume to 200 microliters.
Now using a pair of forceps, pull the animal's tongue and then gently insert a 22 gauge metal tube into the animal's mouth. Confirm intubation by direct visualization of the tube inside the trachea and by chest movement. Finally, bypass the induction box by switching to waste gas tubing to prevent contamination of laboratory space.
Begin by making a skin incision in the left midclavicular line of the intubated mouse, and then by blunt, dissecting the subcutaneous tissue towards the axilla. Now identify the border of the major pectoralis muscle, and then blunt, dissect it from the minor pectoralis muscle underneath. Pull the minor pectoralis muscle to the right to gain a direct view of the rib cage.
Identify the fourth intercostal space, and then use the tips of a pair of forceps to bluntly penetrate and span the intercostal space, allowing insertion of retractors. Adjust the retractors with rubber bands attached to the operation table until there is a clear view of the heart, including the left oracle. Then gently pull off the pericardium without injuring the heart.
Finally, identify the LAD by lifting off the left atrial oracle from the anterior wall of the left ventricle. Begin by bending the tapered needle tip of an eight T proline suture into a U-shape. Then pass the needle through the myocardium deep enough underneath the LAD that the needle is not visible through the tissue.
Now cut off the needle end of the suture to have one centimeter of suture visible at each side. Soak a length of PE 10 tubing in alcoholic disinfection such as the two propanol for at least two minutes. Then caught off a one millimeter section of tubing to create an udder.
Finally, thread both suture ends through the udder. Then using a size three cult suture needle guide, both suture ends out of the upper intercostal space. Begin by using a six aut prolene suture to tie together both the upper and lower ribs in the opened intercostal space of the animal.
Then before closing the chest clamp the expiratory tube to open up ectasis to hyperinflate the lungs for a few respiratory cycles. Set the ventilator tidal volume back to 200 microliters. Now tie both ends of the eight TLAD ligation suture to have them form a next hook up.
ECG leads to the mouse. Hook weights onto the eight OTT loop, letting them hang carefully. There should be a significant ST elevation within a few heartbeats.
Then release the weights and place the loop in a subcutaneous pocket. Closing the skin with six aught single knot sutures. Finally, let the mouse recover after extubation underneath a warming lamp.
After five days recovery, perform the myocardial ischemia protocol as described in the written text at the end of the desired reperfusion time, cut the chest skin of an anesthetized intubated animal in the midline to the xiphoid. Then open the abdomen and cut the diaphragm below the rib cage, cutting the chest open on both sides of the midclavicular line. Now, fix the flapped anterior chest wall with the suture to gain unobstructed access to the heart Ligating.
The LAD is the trickiest part of this procedure. It requires fine motor skills and a steady hand. Carefully prepare the eight aut suture loop.
Cut the loop and tie knot to ligate the LAD. Then inject 10%fallow blue, slowly into the left atrium, aspirating from time to time. The surface of the area at risk also turned blue here, which happens on rare occasions.
Next, inject potassium chloride into the left atrium, arresting the heart in diastole for equal infarct size assessment, and then cut out the heart leaving as much extra cardio tissue as possible to ease the dissection of the organ. Wash the excised heart in PBS and then freeze it in isop pentane and liquid nitrogen. Alternatively, hearts can be placed into a freezer until lightly frozen.
When the heart is fully frozen, align the tissue so that it will be cut perpendicular to the long axis of the heart, and then cut the tissue into one millimeter slices. Place each slice in an individual well of a 96 well plate, and then incubate the slices in 1.5%TTC at 37 degrees Celsius for 20 minutes. Then fix the slices with 4%formaldehyde overnight.
The next day, place the slices on a microscope slide, and then cover the slices with another slide. Use one millimeter metal spacers at the end of each slide holding the two slides together with paperclips. Finally, take a digital image from both sides of each slice.
Use a software program like Image J for P telemetry after opening the chest of an anesthetized intubated animal to both sides of the midclavicular line as just shown. Remove any extra cardio tissue and blunt, dissect the thymus covering the aortic root. Next, use forceps to grab the ascending aorta and cut out the heart with as little extra cardiac tissue as possible.
Next, wash and squeeze the heart gently in Cardioplegic solution and place the heart into a P 35 dish filled with more cardioplegic solution. Fill a 24 gauge IV line with zinc formula and fixative now cannulate the ascending aorta and then cut a hole between the left oracle and left atrium. Insert a 26 gauge catheter into the left atrium with a 16 centimeter long tubing attached to it.
Finally, perfuse the heart with a zinc formal and fixative. After 10 minutes, place the heart into a tube filled with fixative for a maximum of 24 hours at seven degrees Celsius. In this figure, the infarct size and percentage of area at risk represented along the Y axis is compared for mice that underwent 30 minutes of ischemia, followed by 120 minutes of reperfusion represented by the black bar to mice that underwent 30 minutes of ischemia, followed by three cycles of 20 seconds.
Each of reperfusion and occlusion or ischemic post-condition represented by the red bar, note that there is a significantly higher percentage in infarct size in the ischemia reperfusion group than for the IPC mice. This figure demonstrates how the white infarct areas are distinguishable from the red at-risk areas and from the blue non occluded areas. Here a representative slice of a white infarcted area with red TTC staining for viable myo card is shown.
Note that due to the conical shape of the left ventricle close to the apex, the epi myocardi in the pink and blue outer area denoted with the asterisk appears as a plain area and should not be considered for planimetric measurement. Most importantly, compared to animals that underwent myocardial infarction, sham operated animals that underwent all surgical procedures except ischemia and reperfusion exhibited low expression of cytokine RNA. For example, as seen in this bar, graph animals that underwent 30 minutes of ischemia and 120 minutes of reperfusion express significant levels of IL one beta.
But animals in control and sham operated animal groups did not. Similarly, animals that underwent ischemic reperfusion also expressed significant levels of IL six compared to animals in control and sham operated groups. Finally, no significant production of myocardial TNF Alpha mRNA expression was noted for control sham operated or ischemia reperfused animals After its development.
This technique paved the way for researchers in the field of cardiovascular immunology to explore the innate immune response in cardiovascular diseases in mice.
