The overall goal of this procedure is to induce myocardial ischemia of the neuron heart with or without ischemic preconditioning. This is accomplished by first using an optimized technique for anesthesia, positioning, ventilation, and fluid replacement. The second step of the procedure is to perform a lateral thoracotomy to facilitate access to the coronary artery.
The third step is to place an ultra thin suture under the artery and apply a hanging weight system to induce an occlusion. Finally, Evan's blue dye is injected into the carotid artery and TTC staining is performed on the heart to visualize the infarcted tissue. Ultimately, results can be obtained that show the extent of damaged myocardium in genetically modified or pharmacologically treated animals.
Generally, individuals new to this method will struggle due to the difficulty in identifying the coronary artery and the complexity of anesthesia, ventilation and fluid replacement. To begin place an anesthetized mouse in a supine position onto a temperature controlled heated table loop suture material around each extremity, and then tape the sutures to the table. Next, secure the head in place by running a suture underneath the top teeth.
Mouse movements during restraining. Using the suture indicates inappropriate anesthesia depth for the surgical procedure. If seen, the mouse should be re dosed using one third to one fourth of the initial pentobarbital dose and anesthesia confirmed via tow pinch.
Once prepared, the mouse can be covered with mineral oil to reduce exposure to allergens from the hair. Administer a saline bolus of 500 microliters and insert a rectal probe attached to a thermal feedback controller to ensure the body temperature remains at 37 degrees Celsius. To expose the trachea, make an incision along the neck and blunt.
Dissect the surrounding muscle and connective tissue. Hold the tongue with forceps and gently insert a blunt polyethylene cannula at a 15 degree angle towards the body. Confirm correct tube placement by direct visualization of the cannula within the previously exposed trachea.
Above the Corina. Connect the tube to a servo 900 C ventilator. Set the peak in inspiratory pressure at 10 millibars with a frequency of 110 breaths per minute, and a positive end expiratory pressure of three to five millibars with a fraction of inspired oxygen equal to 0.4.
Once the animal is successfully connected to the ventilator, a catheter is inserted into the carotid artery for measuring blood pressure and heart rate. A longer piece of artery will be exposed if the arm is first attached to the side of the body. Begin by blunt dissecting the para tracheal muscles to expose the carotid artery once isolated, not a suture at the proximal end of the artery, and secure it with a clamp or tape.
Place a second suture about 0.5 centimeters away from the first and tie, but do not tighten the suture around the artery. Dissect the artery to the very distal end and place a small clamp to prevent blood loss. Use micro scissors to cut a small diagonal opening into the artery below the first suture.
Hold the opening with fine forceps while inserting the catheter. Advance the catheter. Pass the second suture and tighten the knot to hold in place.
Next, loosen the clamp and advance the catheter further. Secure the catheter with several knots and tape. Once the catheter is in place, infuse normal saline at a rate of 0.1 milliliter per hour, and join it to the blood pressure measuring device.
Lastly, attach clamps to the extremities to monitor heart rate. Now that surgical preparations are complete, make an incision over the chest and expose the pectoral muscles. Cut the pectoralis muscles so that they can be pulled up vertically.
Use a cordy to create a horizontal line on the thorax wall where the incision occurs when using the cordy muscle. Contractions are normal reactions to the current applied. These movements should be distinguished from inappropriate anesthesia depth by pinching the toes with your fingers or forceps.
Hold the muscle up before cutting to ensure that the lungs drop down and outta the way. Cut the thorax wall with blunt scissors and use modified safety needles to keep the thorax open. Next, cut the thorax along the diaphragm away from the thoracic cavity.
First, trace the anticipated cutting line with a qy, and then make a vertical cut along the diaphragm towards the lower left side of the thorax. Dissect the pericardium to expose the heart and cut the phrenic nerve. To avoid diaphragmatic movements, use a moist cotton tipped applicator to turn the heart towards the right side.
Identify the left coronary artery or LCA and optimize the opening of the thorax. Those ensure a secure hold of the heart When placing the suture, keep the heart wet with a 37 degree Celsius saline soaked piece of cotton throughout the procedure. Once identified, place a nylon suture under the LCA.
Prepare the hanging weight system by threading both ends of the suture through a small piece of plastic tubing with blunt edges. Attach a small weight to each end of the suture and drape over a rod to create tension with the weights freely hanging over a rod. The LCA should immediately be occluded.Successful.
LCA occlusion can be confirmed by an immediate color change of the vessel from light red to dark violet and the myocardium supplied by the vessel, changing from bright red to white. Terminate the LCA occlusion at a predetermined time by relieving the pressure from the weights. Successful reperfusion is apparent when the color returns to determine the area at risk.
Or a r inject 1%Evans blue dye into the aorta. While the LCA is occluded. Evans blue will stain all myocardial tissue blue except the a r.
For further analysis of the infarct area, excise the heart and wash it in ice cold, 0.9%saline. Embed the heart into 2%aeros and leave at four degrees Celsius for 30 minutes. Cut the heart into slices one millimeter thick.
Using a heart matrix or microtome, incubate the slices with 1%TTC at 37 degrees Celsius for 10 minutes. Using a 15 milliliter blue cap in a water bath, the infarcted area will be white while viable tissues stain red After its development. This technique paved the way for researchers in the field of myocardial ischemia to explore cardioprotective effects using genetic and pharmacological models.
