Chronic Model of Ischemia/Reperfusion

Myocardial ischemia and reperfusion: a murine model [Michael et al, 1995]

Male and female C57BL/6 mice 8-16 weeks of age (20 - 30 g body wt) are used for most studies. The following brief description is excerpted from the more detailed reference. Anesthesia will be produced by an intraperitoneal injection of sodium pentobarbital (4 mg/ml; 10 ml/g). Mice are placed in a supine position with paws taped to the operating table. With direct visualization of the trachea, an endotracheal tube is inserted and connected to a Harvard rodent volume-cycled ventilator cycling at 100/minute with volume sufficient to adequately expand the lungs, but not over expand. The inflow valve is supplied with 100 percent oxygen.

For studies of the myocardial response to permanent occlusion, ligation of the anterior descending branch of the left coronary artery is achieved by tying 8-0 silk suture around the artery. The suture is passed under the artery at a position approximately 1mm from the tip of the normally positioned left auricle. If inflammatory factors are key components please see below.

For studies of the effect of reperfusion after coronary artery occlusion, the ligature is tied at the same location on the coronary artery used for the permanent occlusion. However, to allow subsequent re-establishment of blood flow, occlusion is produced by placing a 1 mm length of polyethylene tubing (OD = 0.61 mm, Intramedic PE-10, Clay Adams, Inc., Parsippany, NJ) on the artery and fixing it in place with the ligature. The artery is then compressed by tightening the ligature producing myocardial blanching and electrocardiographic ST-segment elevation as observed in permanent ligations [Michael et al, 1995]. After occlusion for the desired time, blood flow is restored by removing the ligature and PE tubing. The chest wall is then closed by a 5-0 TiCron suture with one layer through the chest wall and muscle and a second layer through the skin and subcutaneous layer.

After surgical closing of the chest, the endotracheal tube is removed, warmth provided by a heat lamp and 100 percent oxygen via nasal cone. The animal is given 0.01 mg/kg buprenorphine as an analgesic, and becomes sternally recumbent within one hour. After surviving the experimental infarct the mice recover and are followed post-operatively with physiological measurements. Sham operated mice undergo the identical procedure with placement of the ligature but not coronary artery occlusion. This model is widely used and quoted; we have trained personnel from more than 20 laboratories.

Chronic Closed-Chest Model of Coronary Artery Occlusion-Reperfusion

Closed Chest Ischemia and Reperfusion Model
Figure 1. Closed chest ischemia and reperfusion model

After thoracotomy (approximate 5 mm windows), the pericardium is dissected and an 8-0 Surgipro monofilament polypropylene suture with the U-shaped tapered needle is passed under the LAD. The needle is then cut from the suture, and the 2 ends of the 8-0 suture are threaded through a 0.5 mm piece of PE-10 tubing, forming a loose snare around the LAD. Each end of the suture is then threaded through the end of a size 3 Kalt suture needle (Fine Science Tools, Inc.) and exteriorized through each side of the chest wall. The chest is closed with 4 interrupted stitches utilizing 6-0 suture, carefully avoiding pneumothorax.

The ends of the exteriorized 8-0 suture are tucked under the skin, which is then also closed with 6-0 suture. After removal from the respirator and with the endotracheal tube withdrawn, the animal is kept warm with a heat lamp and allowed to breathe 100 percent oxygen via nasal cone until full sternally recumbent.

At 7 to 10 days post-instrumentation, allowing recovery from the surgical insult, the animals are re-anesthetized with 1.5 percent isofluorane and buprenorphine given I.P. for analgesia and cefazolin as a topical antibiotic. The extremities are taped to a Lead II ECG board, and the skin above the chest wall is re-opened. The 8-0 suture, which had been previously exteriorized outside the chest wall, is cleared of debris and carefully taped to heavy metal picks as shown in Figure 1. Occlusion of the LAD for a specified time (15 minutes to 2 hours or permanent) is accomplished by gently pulling the heavy metal picks apart until an S-T elevation appears on the ECG. The ECG is constantly monitored throughout the entire ischemic interval to insure persistent ischemia.

If reperfusion is desired at the end of the occlusion period, reperfusion is accomplished by pushing the metal picks towards the animal, and cutting the suture close to the chest wall. Reperfusion is confirmed by return of the S-T elevation to preocclusion state which usually occurs within a few minutes. The skin is closed with 6-0 suture, and the animal is allowed to recover in a warm cage. During this procedure, the animal is neither re-intubated, nor put on oxygen, but rather breathes room air under normal ventilation.

For repetitive occlusion studies (I/RC), a 15-minute LAD occlusion followed by 24h reperfusion is repeated for 3.5 and 7 days. Multiple LAD occlusions can be performed without obvious local injury [Dewald et al, 2003]. The model is actively studied under support of HL-089792A and became an intriguing model of non-infarctive adverse remodeling and fibrosis [Haudek et al, 2006; Frangogiannis et al, 2007].

Aortic Constriction/Coarctation and Cardiac Stress

Effects of Transverse Aortic Banding on Carotid Blood Flow Patterns in Mice
Figure 2. Effects of transverse aortic banding on carotid blood flow patterns in mice

Because baseline cardiac function can be insufficiently revealing, we have developed a calibrated method of inducing increased left ventricular work in mice that has allowed us to evaluate the effects of hemodynamic stress on various transgenic and gene deletion models [Kurrelmeyer et al, 2000]. Mice are taken at 8-12 weeks of age 20-25 g body weight and are subjected to partial occlusion of the aorta utilizing a technique developed in the laboratory. To create the occlusion, a 6-0 suture is tied twice around a blunt 3mm segment of a 27 gauge needle, which is positioned adjacent to the aorta between the r. innominate and left carotid arteries, then the needle is removed after placement of the ligature. The degree of constriction is controlled by measuring peak flow velocity in each of the carotid arteries before and after constriction.

Depending on the experiment, animals are subjected to degrees of constriction resulting in right to left peak velocity of 5-10. Mice are monitored again at two days to assure that the load is remaining constant. Using this method, we have had considerable success in developing constant amounts of left ventricular hypertrophy in a cohort of mice. This method has been successful in showing stress related changes [Kurrelmeyer et al, 2000] (Figure 2*). Sham animals are generated by following the entire surgical procedure without constricting the ligature. The constrictors are placed on the aorta at 14 days which corresponds to the formation of mature scar in the mouse [Michael et al, 1995; Michael et al, 1999].

*Figure 2. Carotid artery velocity signals from a normal mouse before and after placement of a constricting band around aortic arch. The band creates pressure overload cardiac hypertrophy, and analysis of the differences in the carotid flow pulsatility (PI) index taken at surgery or at one day post-op can be used to predict the degree of hypertrophy after one week. It is interesting that the mean velocities (M) change very little after banding.