Medicine
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Confirmation of Myocardial Ischemia and Reperfusion Injury in Mice Using Surface Pad Electrocardiography
Chapters
Summary November 24th, 2016
During murine myocardial ischemia/reperfusion surgery, correct placement of the occluding ligature is typically confirmed by visible observation of myocardial pallor. Herein, a method of electrocardiographically confirming ischemia and reperfusion, to supplement observed myocardial pallor, is demonstrated in male C57Bl/6 mice.
Transcript
The overall goal of this procedure is to use electrocardiography to confirm a successful myocardial ischemia and reperfusion in mice. This method can help increase the consistency and reproducibility of the ischemia reperfusion surgery. The main advantage of this technique is that it allows the success of the ischemia and reperfusion to be assessed during the procedure, as opposed to necessitating time-consuming post-surgical analysis.
After preparing the ECG pad, place a smaller than dime-sized amount of highly conductive electrolyte gel onto each of the four metallic electrodes on the ECG pad. Then press each paw of an anesthetized supine mouse to a piece of tape, and attach the tape to an ECG electrode, taking care that each paw is in contact with the gel and the electrode. Next, configure the machine so that the ECG waveform can be visualized in real time, adjusting the resolution to account for the differences in amplitude as necessary.
If the peak of the R wave or the trough of the Q wave is out of the visual frame, adjust the resolution until the entire height of the waveform can be observed. Then clear the ECG pad of tools, taking care that the mouse is still and untouched and obtain a baseline waveform of the animal's normal, healthy ECG. To perform the ischemia reperfusion, begin by removing the hair around the surgical site and disinfecting the area with betadine and alcohol solutions.
Next, use a scalpel to make a vertical incision parallel to the esophagus and trachea, gently moving the lymph nodes to each side of the incision until the thin tissue covering the trachea is exposed. Using forceps, gently separate the tissue until the white cartilage rings of the trachea are visible. Then quickly insert the ventilation tubing into the mouth towards the throat.
When the tip of the tubing is visible through the exposed neck area, align the tube with the start of the trachea. Gently wiggle the tube side to side while applying upward pressure until the tubing slides into the trachea, as confirmed visually through the translucent tissue. When the tube is in place, use a loop of string to hook the mouse's two front teeth and tape the string ends to the ECG pad to steady the head of the animal and to ensure the ventilation tubing does not move during surgery.
Then quickly attach the ventilation tubing to the rodent ventilator and adjust the ventilation settings according to the weight of the animal. Cover the exposed trachea with a gauze soaked in warm saline to keep the tissue from drying. Then use a scalpel to make a vertical incision along the left side of the sternum.
Using forceps, gently separate the fascia layer from the muscle layer, followed by careful cutting of the underlying muscles layers without damaging any visible blood vessels. Use the forceps to gently pull the third rib upwards. Then, maintaining the grip on the rib with one hand, use surgical scissors to carefully cut the intercostal tissue between the third and fourth rib.
When the lungs have retracted, use the forceps to gently separate the thin layer of pericardium surrounding the heart. Then move the ribs into a position where the heart is visible between the ribs. To position the heart so that the left atrial auricle is easily visible, move the mouse's lower left paw so that it overlaps the lower right paw.
Locate the left anterior descending coronary artery, or LAD, beneath the left auricle, and swiftly insert a 7-0 silk tapered suturing needle into the myocardium deeply enough to pass under the LAD, but not so deeply as to penetrate the left ventricle cavity. Hold the suturing ligature until there is about four centimeters of suturing silk left on the free end, and begin to tie a simple suture knot. Once the free end of the suturing silk has been pulled through the loops to form the knot, grasp both the free and needle ends of the suturing silk with forceps and insert an approximately one-centimeter section of PE10 tubing under the forming knot atop the epicardial surface.
When the tubing is in place, return the lower left paw to its original electrode and tighten the knot so that the PE10 tubing is sutured to the heart. Release all physical contact with the mouse to allow the ECG waveform to stabilize for 10 seconds, visually checking the waveform and recording it as the time of occlusion. Then, if the ECG and myocardial color changes indicate ischemia, double-knot the suture around the PE10 tubing and cover the open chest cavity with warm saline gauze.
At the end of the ischemic period, remove the gauze covering the chest cavity and visualize the heart. Cut the suturing silk atop the PE10 tubing and remove the tubing, followed by the gentle removal of the ligature from the myocardium. Release all physical contact with the mouse and allow the ECG waveform to cycle for 10 seconds, recording the waveform as the time of reperfusion.
At the experimental endpoint, adjust the resolution for any changes in the amplitude, as necessary. If the T wave changes and the myocardium returns to red, indicating a successful reperfusion, suture the intercostal space with a 5-0 silk suture, while applying gentle pressure to the mouse's chest to expel any excess air that entered the cavity during surgery to close the chest cavity. Then suture the muscle layers and skin, allowing the mouse to fully recover in a constant temperature-controlled environment with monitoring.
This figure follow the progression of one mouse from a baseline rating all the way through reperfusion, with the first waveform displaying a normal sinus rhythm at the recorded baseline. The second waveform displays the ECG one minute after the ligature was tied and the artery became occluded, with an elevated, hyperacute T wave peak observed, compared to the baseline, preligature waveform. The third waveform shows a complete ST segment elevation at the five-minute time point, with the S portion of the complex not reaching as deeply negatively as in the baseline waveform, before progressing into the J and T waves.
At 20 minutes of ischemia, the QT interval has widened, and the ST segment is still elevated, with both sections remaining as such up to the 45-minute mark. After five minutes of reperfusion, evidence of deep, significant Q waves begin to appear, and the T wave returns to the isoelectric line. After 30 minutes, however, the negative Q waves still remain, and are likely indicative of both permanent damage and that the dying heart tissue is deflecting the electrical currents around the damaged area.
Further, continuous ischemia and the progression to injury and infarction leads to an enhanced negative T wave projection that is usually permanent. While attempting this procedure, it's important to remember to obtain a baseline ECG before making any marks on the animal. ECG analysis can be applied to detect any electrical changes or abnormalities.
The heart can be further processed for protein analysis to investigate proteomic changes or tissue staining to investigate proteomic or morphometric changes. After watching this video, you should have a good understanding of how to use electrocardiography to confirm successful myocardial ischemia and reperfusion in a mouse.
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