Myocardial ischemia, reperfusion, and infarction in chronically instrumented, intact, conscious, and unrestrained mice

Abstract

In the United States alone, the National Heart, Lung, and Blood Institute (NHLBI) has invested several hundred million dollars in pursuit of myocardial infarct-sparing therapies. However, due largely to methodological limitations, this investment has not produced any notable clinical application or cardioprotective therapy. Among the major methodological limitations is the reliance on animal models that do not mimic the clinical situation. In this context, the limited use of conscious animal models is of major concern. In fact, whenever possible, studies of cardiovascular physiology and pathophysiology should be conducted in conscious, complex models to avoid the complications associated with the use of anesthesia and surgical trauma. The mouse has significant advantages over other experimental models for the investigation of infarct-sparing therapies. The mouse is inexpensive, has a high throughput, and presents the ability of one to create genetically modified models. However, successful infarct-sparing therapies in anesthetized mice or isolated mouse hearts may not be successful in more complex models, including conscious mice. Accordingly, a conscious mouse model of myocardial ischemia and reperfusion has the potential to be of major importance for advancing the concepts and methods that drive the development of infarct-sparing therapies. Therefore, we describe, for the first time, the use of an intact, conscious, and unrestrained mouse model of myocardial ischemia-reperfusion and infarction. The conscious mouse model permits occlusion and reperfusion of the left anterior descending coronary artery in an intact, complex model free of the confounding influences of anesthetics and surgical trauma. This methodology may be adopted for advancing the concepts and ideas that drive cardiovascular research.

coronary heart disease, including ischemic heart disease, angina pectoris, sudden cardiac death, and myocardial infarction, is the leading cause of death for men and women in the United States (88). Specifically, an estimated 935,000 Americans suffer a myocardial infarction each year with 610,000 new cases and 325,000 recurrent attacks (87). Prognosis after myocardial infarction is primarily dependent on the size of the infarction (25, 72, 79). Accordingly, worldwide efforts are currently underway to develop strategies to reduce infarct size. In the United States alone, the National Heart, Lung, and Blood Institute (NHLBI) has invested several hundred million dollars in pursuit of infarct-sparing therapies (54, 94). However, due largely to methodological limitations, this investment has not produced any notable clinical application or cardioprotective therapy (54, 94). In fact, to date, early coronary artery reperfusion is the only established treatment capable of consistently reducing infarct size in humans (94).

Among the major methodological limitations is the reliance on animal models that do not mimic the clinical or physiological situation. In this context, the NHLBI sponsored Consortium for preclinicAl assESsment of cARdioprotective therapies (CAESAR) has developed a strategy to focus on the use of relevant conscious animal models and models of comorbidities (54, 94) in the search for strategies to reduce infarct size. CAESAR's efforts are an attempt to assess molecular and other perturbations under physiological and functionally relevant conditions (42).

The mouse has significant advantages over other experimental models for the investigation of infarct-sparing therapies. The mouse is readily available, inexpensive, has a high throughput, and gives the investigator the ability to create genetically modified models. Thus mice can be studied for the initial screening of therapies that are promising. However, successful infarct-sparing therapies in isolated mouse hearts or anesthetized mice may not be successful in more complex models, including conscious mice. Accordingly, a conscious mouse model of myocardial ischemia and reperfusion has the potential to be of major importance for advancing the concepts and methods that drive the development of infarct-sparing therapies (105).

Therefore, we describe, for the first time, the use of an intact, conscious, and unrestrained mouse model of myocardial ischemia-reperfusion and infarction. The conscious mouse model permits occlusion and reperfusion of the left anterior descending coronary artery (LAD) in an intact, complex model free of the confounding influences of anesthetics, surgical trauma, and restraint stress (91). The myocardial ischemia and reperfusion protocol can be initiated after the resolution of the inflammation that occurs during the initial surgical preparation. This is an important consideration, because Nossuli and colleagues (73) demonstrated that the inflammation due to surgical trauma increases the background of cytokine induction and accentuates the response during myocardial ischemia and reperfusion causing significantly greater variability. The use of a chronic model eliminates these confounding effects.

MATERIALS AND METHODS

Animals.

Experimental procedures and protocols were reviewed and approved by the Animal Care and Use Committee of Wayne State University and complied with The American Physiological Society's Guiding Principles in the Care and Use of Animals. Five adult, female C57BL/6 mice (20.2 ± 0.3 g before instrumentation and 21.9 ± 0.6 g on the day the hearts were harvested) and 4 adult, male C57BL/6 mice (26.0 ± 0.6 g before instrumentation and 28.9 ± 0.3 g on the day the hearts were harvested) were studied to determine infarct size following occlusion and reperfusion of the LAD coronary artery in intact conscious mice. Three additional sham-operated (occluder inserted but not used) male mice were studied to obtain histological sections from noninfarcted hearts for the determination of chamber size and comparison with the infarcted hearts. All mice were housed on a 12 h-12 h light-dark cycle with ad libitum access to food and water.

All surgical procedures were performed using aseptic surgical techniques. Twenty minutes before the surgical procedures, the mice received the nonsteroidal anti-inflammatory agent ketoprofen (5 mg/kg sq), the antibiotic cefazolin (10 mg/kg sq), and the muscarinic cholinergic receptor antagonist methyl atropine (0.05–0.1 mg/kg sq). The ketoprofen, cefazolin, and atropine were delivered in 1–3% (body weight) of physiological saline. The saline was absorbed and offset the hypotonic effects of the anesthetic.

Subsequently, the mice were anesthetized with pentobarbital sodium (50 mg/kg ip). The intraperitoneal bolus was injected into the lower lateral abdominal quadrant to reduce risk of intraluminal intestinal injection by using a standard headdown restraint. The injection site with needle was “pinched” when the needle was withdrawn. This prevents “back tracking” of the anesthetic out of the needle track. Back tracking of the anesthetic can result in delayed induction and/or failure to reach the surgical plane. The mice were immediately placed in a standard mouse cage that was free of bedding so that particulate bedding would not obscure airways or damage corneas as anesthesia was induced. Once the mice lost the righting reflex, it became possible to begin the initial preparation of the surgical site.

To initially prepare the surgical site, the fur was removed with clean, well-lubricated fine blade clippers. The clippers were periodically checked as the heat generated can cause thermal burns that delay the healing process. The loose fur was removed with a damp cloth. After the fur was removed, the animal was ready for tracheal intubation.

To intubate the mice, a custom-made intratracheal tube was required. Specifically, a 20-mm segment of polyethylene 50 (PE 50) tubing was inserted into a 5-mm segment of PE 190 tubing that was subsequently inserted into a 7-mm segment of PE 240 tubing. Subsequently, the three segments were heat sealed together making one mouse tracheal tube. The PE 50 tubing entered the trachea and the attached PE 240 tubing was connected to a Y type connector (cat. no. 6152-0125; Nalgene Labware). The Y connector was connected to the tubing of the ventilator (SAR 830 ventilator, CWE, Ardmore, PA). The mice were pressure-controlled ventilated at a respiratory rate of 140 breaths/min and an inspiratory pressure of ∼10 cmH₂O resulting in a tidal volume of ∼7.0 ml/kg. The mice were placed on a feedback-based temperature control system (model no. 40-90-8; FHC, Bowdoin, ME) for monitoring and maintaining body temperature within the physiological range. Once ventilated, the final preparation of the surgical site was conducted.

To complete the preparation of the surgical site the mice were positioned in a right lateral decubitus position and the skin was disinfected. Specifically, the surgical site was wiped in a single pass outward motion from incision site toward the fur with an iodine solution several times assuring 3–5 min of contact time. New gauze was used for each circular sweep. The same procedure was repeated using sterile alcohol-treated pads to remove the iodine solution. A sterile drape was positioned over the site and sterile petrolatum ophthalmic ointment (Puralube, E. Fougera, Melvile, NY) was placed in the eyes to prevent drying.

Thoracotomy procedures.

The hearts were approached via a left thoracotomy through the third intercostal space. In brief, a 1.5-cm incision was made in the skin over the third rib space. The muscles covering the rib were blunt dissected to expose the intercostal muscle. A 5- to 7-mm opening was made through the intercostal muscle and pleura of the third rib space using blunt dissection. The blunt tines of a Guthrie retractor (No. 17021-13, Fine Science Tools) were placed under the third and fourth rib, producing a separation of 7 mm. The pericardial sac was opened, and a coronary artery occluder made with a BV-1 tapered needle attached to 7-0 prolene suture (Ethicon 8703) was passed around the proximal segment of the LAD ∼1.0 mm from the origin by inserting the needle into the left ventricular wall and bringing it out on the other side of the coronary artery. The needle was cut from the suture, and the two ends of the suture were passed through guide tubing fashioned from a “modified” mouse thoracic carotid artery catheter (cat. no. M-CAC; Braintree Scientific). Specifically, the modification consisted of cutting the tapered end and then heat flaring the cut end. This modification prevents the guide tubing from causing tissue damage during the occlusion.

After implantation of the coronary artery occluder, the retractors were removed. The third and fourth ribs were approximated with 6-0 silk sutures. The superficial muscle covering the ribs was then apposed with 5-0 Vicryl sutures. The guide tubing with the two ends of the 7-0 suture were exteriorized at the back of the neck, filled with a sterile petrolatum ophthalmic ointment (Puralube), and sealed with a stainless steel pin to prevent a pneumothorax. Subsequently, the skin was closed with 5-0 nylon sutures. The local anesthetic bupivicaine (0.25%, 1.5 mg/kg) was injected subcutaneously at the incision site.

Subsequently, two ECG electrodes (DataSciences International, Standard Lead Coupler Kit: 276-0031-001) were sutured subcutaneously in a modified lead II configuration by placing the negative electrode slightly to the right of the manubrium and the positive electrode at the anterior axillary line along the fifth intercostal space. In addition, a third electrode was place subcutaneously, which served as the ground electrode.

All animals remained on the feedback-based temperature control system and ventilator until fully recovered from the anesthesia. Once the animal regained consciousness and was able to lift its head and maintain itself in sternal recumbency, the animal was placed in a “rodent recovery cage” (Thermocare Intensive Care Unit, Braintree Scientific, Braintree, MA). The animals were kept warm, quiet, clean, and dry and monitored every 15–30 min. Animals were returned to the housing room when fully recovered from the anesthesia and gained the ability to maintain body temperature. The nonsteroidal anti-inflammatory agent ketoprofen (5 mg/kg sq) and the antibiotic cefazolin (10 mg/kg sq) were continued for 2 days. At least 10 days were allowed for recovery. During the recovery period, the mice were handled, weighed, and acclimatized to the laboratory and investigators.

Induction of myocardial infarction.

Conscious, unrestrained mice were studied in their home cages for all experiments. The temperature within the cage was monitored and maintained near the thermoneutral zone for mice of ∼27–28°C (102) by use of a circulating water pad under the cage and a Presto HeatDish Plus Parabolic Heater. The ECG was directly recorded by taping the ECG electrodes to multistranded stainless steel Teflon-coated wires [Medwire part number 316SS7/44T, OD less than 0.25 mm, the same wire used to record lumbar sympathetic nerve activity in previous studies (16, 17, 24, 95)]. ECG signals were initially amplified (1,000 times) with a Grass P511 differential preamplifier and high-impedance probe (HIP 511GA). The low- and high-pass filters were set at 0.3 Hz and 10 kHz (37). Mice were allowed to adapt to the laboratory environment for ∼1 h to ensure stable hemodynamic conditions. During this time and throughout the entire experiment, the mouse was unrestrained and able to move about the cage albeit tethered to recording wires. After the stabilization period, beat by beat, steady-state heart rate and ECG parameters were recorded over 10–15 s. Subsequently, the LAD coronary artery was temporarily occluded by use of the prolene suture. Specifically, acute coronary artery occlusion was performed by pulling up on the suture that was around the LAD and securing it to the modified catheter with a vascular clip (Schwartz straight smooth clip). Rapid changes in the ECG (peaked T wave followed by S-T segment elevation) occur within seconds of pulling on the suture, documenting coronary artery occlusion (see Fig. 1). The tension on the suture was sufficient to record the maximum elevation in the ST segment. The coronary artery occlusion was maintained for 90 min; followed by 2 h of reperfusion. It is important to note that the mice did not display any discomfort or stress during the occlusion and did not develop sustained ventricular tachycardia either during the occlusion or reperfusion periods. After the 2 h of reperfusion, the mice were returned to the animal facility.

Fig. 1.

An analog recording of the electrocardiogram (ECG) at the onset of occlusion of the left anterior descending coronary artery (LAD) in an intact, conscious, and unrestrained male mouse is shown. The onset of occlusion is indicated by the dotted line. Rapid changes in the ECG (peaked T wave and S-T segment elevation) occurred within a second of pulling on the suture, documenting coronary artery occlusion.

Two weeks later for the 4 male mice and 4 wk later for the 5 female mice, the mice were brought to the laboratory and the ECG was recorded for 90 min. Subsequently, the mice were deeply anesthetized and the hearts were harvested, sectioned, and processed with Masson Trichrome Stain kit (Sigma: HT15) to visualize the infarct scar. Masson Trichrome stain is useful to examine connective tissue and muscle characterized by fibrotic changes. Specifically, the cytoplasm and muscle fibers stain red, whereas the collagen displays blue (see Figs. 3–11). Using this staining protocol, infarct size scores were calculated using the length measurement approaches for infarct size described below.

Fig. 3.

Top: 1-s analog recordings of the ECG before occlusion of the LAD (control), following 45 min of occlusion of the LAD and following 2 h of reperfusion of the LAD in a chronically instrumented, intact, conscious, and unrestrained mouse. Note the ST segment elevation at 45 min of occlusion, which remained elevated for the entire 90-min occlusion period. After 2 h of reperfusion, note the prominent Q wave, which was absent in the control condition. Bottom: photomicrographs of tissue sections taken from the infarcted heart after 2 wk of reperfusion. The sections were taken from the apex through the base of the left ventricle at 300-μm intervals. Note the intense blue staining indicating the abundance of collagen, the large left ventricular chamber area, and the thin left ventricular wall.

Fig. 4.

ECG before, during, and after occlusion of the LAD in a chronically instrumented, intact, conscious, unrestrained mouse (top) and photomicrographs of histological sections taken from the infarcted heart 2 wk postocclusion (bottom). See Fig. 3 legend for more details.

Fig. 5.

ECG before, during, and after occlusion of the LAD in a chronically instrumented, intact, conscious, unrestrained mouse (top) and photomicrographs of histological sections taken from the infarcted heart 2 wk postocclusion (bottom). See Fig. 3 legend for more details.

Fig. 6.

ECG before, during, and after occlusion of the LAD in a chronically instrumented, intact, conscious, unrestrained mouse (top) and photomicrographs of histological sections taken from the infarcted heart 2 wk postocclusion (bottom). See Fig. 3 legend for more details.

Fig. 7.

ECG before, during, and after occlusion of the LAD in a chronically instrumented, intact, conscious, unrestrained mouse (top) and photomicrographs of histological sections taken from the infarcted heart 4 wk postocclusion (bottom). See Fig. 3 legend for more details.

Fig. 8.

ECG before, during, and after occlusion of the LAD in a chronically instrumented, intact, conscious, unrestrained mouse (top) and photomicrographs of histological sections taken from the infarcted heart 4 wk postocclusion (bottom). See Fig. 3 legend for more details.

Fig. 9.

ECG before, during, and after occlusion of the LAD in a chronically instrumented, intact, conscious, unrestrained mouse (top) and photomicrographs of histological sections taken from the infarcted heart 4 wk postocclusion (bottom). See Fig. 3 legend for more details.

Fig. 10.

ECG before, during, and after occlusion of the LAD in a chronically instrumented, intact, conscious, unrestrained mouse (top) and photomicrographs of histological sections taken from the infarcted heart 4 wk postocclusion (bottom). See Fig. 3 legend for more details.

Fig. 11.

ECG before, during, and after occlusion of the LAD in a chronically instrumented, intact, conscious, unrestrained mouse (top) and photomicrographs of histological sections taken from the infarcted heart 4 wk postocclusion (bottom). See Fig. 3 legend for more details.

Preparation of heart sections.

The heart was excised under deep anesthesia. The left and right atria and large vessels were removed, and the heart was quickly rinsed in 10 mM Tris, 0.9% NaCl, 0.05% thimerosal in 10 mM phosphate buffer, pH 7.4 (TPBS) and then immersion fixed in formaldehyde-zinc fixative for 60 min, washed in TPBS (3 × 10 min) then cryoprotected overnight in 30% sucrose (prepared in half strength TPBS). Subsequently, the heart was embedded in OCT compound and sliced transversely from the apex to the base at 10-μm intervals with the use of a cryostat. An interval of 300 μm was maintained between each section. All sections were thaw mounted on Superfrost Plus slides and stained with Masson Trichrome for quantitative analysis of infarct size.

Measurement of infarct size.

After an experimental acute myocardial infarction, infarct size is typically measured as the area of the infarcted region in relation to the area at risk (70, 76, 81, 103, 115). However, after a chronic myocardial infarction, cardiac structural remodeling consisting of chamber dilatation and wall thinning in the infarcted region with hypertrophy in the viable region may confound the area-based approaches (68, 77, 78). Specifically, the opposing changes of wall thickness in the necrotic and viable myocardium may result in spurious measurements of the infarcted area. Despite these potentially confounding variables (progressive thinning of the infarcted wall with reduction in the volume of the infarcted region) area measurements are also used in the chronic infarct setting (51, 52, 104). However, to account for the cardiac structural remodeling in the chronic model of myocardial infarction, histological measurement of the arc length of the infarct scar is commonly used (46, 75, 78, 80, 103).

Length measurement.

All histological sections were examined with an Olympus BH-2 microscope using a ×1 objective. Our primary end point was infarct size. To achieve this end point, every section from apex to base (300 μm apart) totaling 14–15 sections per heart was quantified from the digital photomicrographs using image analysis software (Image J 1.45s). Four lengths, including epicardial and endocardial infarct lengths and epicardial and endocardial circumferences, were traced manually. Endocardial infarct length was taken as the length of the endocardial infarct scar surface that included greater than 50% of the whole thickness of myocardium (103). Epicardial infarct length was taken as the length of the transmural infarct region (see Figs. 3–11 and Ref. 103).Epicardial infarct ratio was obtained by dividing the sum of the epicardial infarct lengths from all sections by the sum of epicardial circumferences from all sections. Endocardial infarct ratio was calculated similarly. Infarct size derived from this approach was calculated as [(epicardial infarct ratio + endocardial infarct ratio)/2] × 100 (103).

Data analysis.

All physiological recordings were sampled at 4 kHz, and the data were expressed as means ± SE. An unpaired t-test was used to determine differences in infarct size following 2 and 4 wk of reperfusion. A one-way ANOVA was used to determine differences in left ventricular chamber size in noninfarcted hearts and infarcted hearts following 2 and 4 wk of reperfusion.

RESULTS

Figure 1 presents an analog recording of the ECG at the onset of occlusion of the LAD (gray dotted line) in an intact, conscious, and unrestrained male mouse. Rapid changes in the ECG (peaked T wave and S-T segment elevation) occurred within a second of pulling on the suture, documenting coronary artery occlusion.

Photomicrographs of myocardial tissue sections taken from one noninfarcted male mouse heart are presented in Fig. 2. The sections were taken from the apex through the base of the left ventricle at 300-μm intervals. Note the absence of collagen (i.e., no blue stain), the small left ventricular chamber area, and the thick left ventricular wall.

Fig. 2.

Photomicrographs of myocardial sections from one noninfarcted male mouse heart are shown. The sections were taken from the apex through the base of the left ventricle at 300-μm intervals and processed with Masson Trichrome stain. Note the absence of collagen (i.e., no blue stain), small left ventricular chamber area, and thick left ventricular wall.

Analog recordings of the ECG before occlusion of the LAD (control), following 45 min of occlusion of the LAD and following 2 h of reperfusion of the LAD in each of the intact, conscious mice are presented in the top panels of Figs. 3–11. Coronary artery occlusion produced rapid changes in the ECG (peaked T wave and S-T segment elevation; Fig. 1). The ST segment was elevated at 45 min of occlusion and remained elevated during the entire 90 min occlusion. After 2 h of reperfusion, note the prominent Q wave, which was absent in the control condition. These changes in the ECG were observed in all mice.

Photomicrographs of myocardial sections taken from each of the infarcted hearts after 2 wk of reperfusion (Figs. 3–6) or 4 wk of reperfusion (Figs. 7–11) are presented in the bottom panel. The sections were taken from the apex through the base of the left ventricle at 300-μm intervals. Note the intense blue staining indicating the abundance of collagen, the large left ventricular chamber area, and the thin left ventricular wall.

The myocardial ischemia and reperfusion protocol and a myocardial infarction was induced in 4 male and 5 female intact, conscious, and unrestrained mice. Fourteen days (4 male mice) or twenty-eight days (5 female mice) after the ischemia and reperfusion protocol, the hearts were harvested, sectioned, and stained with Masson trichrome to visualize the infarct scar. Subsequently, the infarct size scores were calculated using the length measurement approach based on epicardial and endocardial scar arc lengths. The average infarct size (open circles) and individual infarct sizes (closed circles) for males after 2 wk of reperfusion and females after 4 wk of reperfusion are presented in Fig. 12A. Although infarct size following 4 wk of reperfusion was greater than infarct size following 2 wk of reperfusion, it must be noted that sex differences may have accounted for this effect. Figure 12B presents the average (open circles) and individual (closed circles) left ventricular chamber sizes from the three noninfarcted hearts as well as the hearts from males after 2 wk of reperfusion and females after 4 wk of reperfusion. Left ventricular chamber size after 4 wk of reperfusion was significantly larger compared with the chamber size of the noninfarcted hearts but not different from the chamber size after 2 wk of reperfusion.

Fig. 12.

A: average infarct size (open circles) and individual infarct sizes (closed circles) for males after 2 wk of reperfusion and females after 4 wk of reperfusion. B: average (open circles) and individual (closed circles) left ventricular chamber sizes for the 3 noninfarcted hearts as well as the hearts from males after 2 wk of reperfusion and females after 4 wk of reperfusion. *P < 0.05, 4 wk reperfusion vs. 2 wk reperfusion; #P < 0.05, 4 wk reperfusion vs. noninfarcted.

DISCUSSION

Worldwide efforts are currently underway using mice to identify infarct sparing strategies. Often, the therapeutic effects of infarct sparing strategies of mice are not fully appreciated, due in part to the difficulty of measuring physiological responses in complex, conscious models free of the confounding influences of anesthetics, surgical trauma, and the use of crystalloid perfused hearts that are devoid of many of the vital components of blood. To address this concern, as well as provide a resource for investigators using wild-type mice or available spontaneous or engineered mouse mutants, we used existing technology, surgical techniques, and experimental protocols for the study of myocardial ischemia-reperfusion and infarction in the intact, conscious, and unrestrained mouse.

Specifically, we describe, for the first time, the use of an intact, conscious, and unrestrained mouse model of myocardial ischemia-reperfusion and infarction. The conscious mouse model permits occlusion and reperfusion of the LAD after recovery from the anesthesia and surgical trauma. This is an important consideration since it allows for the resolution of the inflammation that occurs during the initial surgical preparation (13, 39, 74). Furthermore, many anesthetic agents confer protection against ischemia-reperfusion injury across species (20, 34, 98, 105) as well as in a variety of tissues, including heart (20, 22, 58, 82, 100), brain (47, 106, 114), kidney (43), lung (56), and liver (8). Anesthesia also significantly alters the autonomic nervous system (36), and disturbances in cardiac autonomic balance play a critical role in the determination of infarct size. Specifically, reductions in parasympathetic activity or increases in sympathetic activity increase the morbidity and mortality associated with myocardial infarction (11). Accordingly, avoiding these complications associated with the use of anesthesia has the potential to improve the validity and clinical significance of the results.

Successful infarct-sparing therapies in isolated hearts may not be successful in more complex models, including conscious mice, because perfusion solutions that are devoid of many of the vital components of blood may confound the response to myocardial ischemia and reperfusion. Specifically, isolated perfused heart preparations, which are devoid of humoral influences and neuronal regulation, most often use a hemoglobin-free perfusate that requires an unphysiologically high arterial oxygen tension (28) and coronary perfusion rate (9). Furthermore, because the perfusate is devoid of many of the vital components of blood, the crystalloid-perfused hearts exhibit significantly more edema than blood-perfused hearts after ischemia and reperfusion (23, 83). In addition, the increased edema alters myocardial function and contributes to ultrastructural damage (7). Thus, although precise control of the determents of myocardial oxygen consumption, e.g., preload, afterload, heart rate, and contractility are possible with this model, results should be confirmed in complex, conscious systems.

In addition, inflammation contributes significantly to the development and progression of cardiovascular diseases. Indicators of systemic inflammation, such as C-reactive protein, a marker of low-grade inflammation (1, 2, 86), and serum levels of myeloperoxidase are indicative of the risk for adverse cardiovascular events (5, 14). Furthermore, it is well recognized that myocardial ischemia elicits an acute inflammatory response. Importantly, the inflammatory response is markedly augmented by reperfusion. Specifically, reperfusion elevates proinflammatory cytokines and infiltration of neutrophils in the tissue (27, 55, 108). Accordingly, a significant part of myocardial injury after ischemia and reperfusion is attributable to the inflammatory processes (13, 39, 74). In this context, the surgical trauma associated with open-chest preparations is also associated with inflammatory responses. Specifically, acute surgical trauma associated with open-chest preparations in sham-operated animals results in highly variable background levels of inflammation that are indistinguishable from those of ischemic-reperfused animals (15, 71). Since the background levels of inflammation associated with surgical trauma in open-chest preparations compromised the ability to assess inflammation directly due to myocardial ischemia and reperfusion, Michael and colleagues (71) developed a chronic model of myocardial ischemia and reperfusion in dogs that allowed for the resolution of the surgical trauma and inflammation that occur during the initial surgical preparations of the animals before the induction of the ischemia and reperfusion protocol. This is an important consideration because acute surgical trauma increases background levels of inflammatory markers and accentuates and primes the response causing significantly greater variability (73). Similarly, environmental stimuli that induces inflammation predisposes individuals to more severe cardiovascular injury in response to an ischemic events (21, 38, 44, 85, 109, 113). In this context, Nossuli and colleagues (73) demonstrated that the inflammation due to the surgical trauma increases the background of cytokine induction and accentuates the response causing significantly greater variability. The use of a chronic model eliminates these limitations.

Current American College of Cardiology/American Heart Association guidelines recommend reperfusion therapy (thrombolytic therapy, primary percutaneous transluminal coronary angioplasty, or coronary artery bypass grafting) be administered to all patients regardless of age, sex, or race who have symptoms suggestive of a myocardial infarction and who present to the hospital within 12 h of symptom onset and have diagnostic changes on their 12-lead ECG (ST segment elevation or bundle-branch block) (3, 89). Accordingly, most patients experiencing an acute myocardial infarction receive reperfusion therapy (6). Furthermore, the open-artery hypothesis suggests that survival after a myocardial infarction depends more on improved left ventricular remodeling and healing, electrical stability, and myocardial perfusion than on reduction in infarct size (12, 40, 49, 53, 79, 110). Thus survival may improve even when reperfusion therapy is administered late, after irreversible necrosis has occurred. Thus early or late reperfusion is recommended for individuals with myocardial infarction. However, many studies investing infarct-sparing strategies in animal models use permanent rather than temporary coronary artery occlusion. The model described in this communication permits occlusion and reperfusion of the LAD.

An important consideration is that most myocardial infarctions in humans occur in the absence of anesthetic agents and surgical trauma and the hearts are perfused with blood. Furthermore, most myocardial ischemia and reperfusion protocols cannot be conducted in humans. Thus the study of relevant complex, conscious models are essential (54, 94) and animals must be considered. In this context, the mice did not exhibit signs of pain, distress, or agitation during or after the ischemia and reperfusion protocol. Accordingly, we do not have evidence that the mice experience pain during myocardial ischemia. This may be due, in part, to the fact that myocardial ischemic episodes are asymptomatic (silent) in as many as 80% of cases (4, 18, 69). Similarly, myocardial infarction is silent in up to 68% of cases (4, 18, 45, 69). Thus the majority of myocardial ischemic episodes are silent, indicating an inability or failure to sense ischemic damage or stress on the heart (32). In fact, chest pain has a low specificity as an indicator of myocardial ischemia and twice as many people die from a silent heart attack compared with those that experienced a myocardial infarction with chest pain (30, 50).

In support of this concept, mice produce a variety of vocalizations, including vocalizations audible to humans, as well as ultrasonic vocalizations. Ultrasonic vocalizations utilize frequencies higher than 30 kHz (90), and therefore cannot be detected directly by human ears. A number of studies have shown that mice produce ultrasonic vocalizations during stress (31, 33, 97, 101, 111). We used an ultrasonic frequency detector and have not heard vocalizations during coronary artery occlusion and reperfusion in the mouse suggesting that the mice are not stressed. Furthermore, visual inspection of the animal's appearance, posture, and spontaneous behavior suggest the absence of pain or discomfort. Specifically, there is an absence of the, well-known symptoms of pain; e.g., piloerection, unkempt coat/rough hair, hunched posture, apathy, aggression, or self-mutilation.

Limitations.

Genetically modified mice are increasingly used in cardiovascular research (92). Importantly, recent reports on the sequencing of both the human and mice genomes revealed that, to date, only 300 or so genes appear to be unique to one species or the other (107). Furthermore, human and mouse proteins show 80% homology (10). Yet, despite this conservation there are significant differences between mice and humans in cardiovascular function. Such differences are not surprising since the two species differ markedly in heart size, body mass, oxygen consumption, heart rate, and lifespan. Accordingly, a word of caution is required as it is increasingly important to understand the potential limitations of extrapolating data from mice to humans (10, 92, 107).

Specifically, caution should be used when comparing data obtained in species with high heart rates (mice, rats) to data obtained in species with low heart rates (rabbits, dogs, humans) because electrophysiology properties, autonomic regulation, and hemodynamic function are species dependent as a result of differences in heart size, body mass, oxygen consumption, and heart rate (10). Thus mice may not reflect human cardiovascular physiology as closely as larger mammals. Accordingly, extrapolation of mouse data to the human physiological condition must be made with caution; and the murine model may best be used to establish “proof of concepts” that will need to be confirmed in other models that more closely approximate human physiology.

While caution in interpreting preclinical data obtained in mice is clearly warranted, mice will continue to be an important model for human cardiovascular research and will be essential for progress in understanding cardiovascular function in health and disease. For example, numerous studies highlight the relevancy of the mouse as a model of human disease (10, 26, 29, 35, 48, 57, 84, 93, 96, 112).

An additional consideration is that even within small animals with high heart rates (rats and mice) responses to coronary artery occlusion are markedly different. Specifically, we preformed virtually identical procedures in conscious rats many times (19, 59–67). Every rat developed sustained ventricular tachycardia within 10 min of coronary artery occlusion. We expected the mouse to develop sustained ventricular arrhythmia as well, however, this never happened. These data are consistent with previous reports, documenting that it is nearly impossible to elicit ischemia- or reperfusion-induced sustained ventricular arrhythmias in the mouse (99). Understanding the differences between species may advance the concepts and ideas that drive cardiovascular research.

Perspectives and Significance

This paper describes the application of existing technology, surgical techniques, and experimental protocols for the study of myocardial ischemia-reperfusion and infarction in the chronically instrumented, intact, conscious, and unrestrained mouse. The methodology allows for the accurate documentation of infarct size following coronary artery occlusion and reperfusion in conscious mice. Investigators may be encouraged to adopt these existing procedures to their investigations of myocardial infarction since highly reliable data can be obtained in mice under physiological conditions.

It is important to acknowledge that the data gathered from experiments performed at many levels, from molecules to humans, have been and will be critical in developing infarct sparing strategies. Accordingly, a wide range of investigations, rather than a single model or experimental technique, is required to develop novel infarct sparing strategies (41). In this context, the conscious mouse provides an additional tool in the prevention, treatment, and rehabilitation from cardiovascular disease.

GRANTS

This study was supported by National Heart, Lung, and Blood Institute Grants HL-88615 (S. E. DiCarlo) and HL-98945 (J-P. Jin).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: H.L.L., H.-Z.F., J.-P.J., and S.E.D. conception and design of research; H.L.L., H.J., and S.E.D. performed experiments; H.L.L., H.J., and S.E.D. analyzed data; H.L.L. and S.E.D. interpreted results of experiments; H.L.L., H.J., and S.E.D. prepared figures; H.L.L. and S.E.D. drafted manuscript; H.L.L. and S.E.D. edited and revised manuscript; H.L.L., H.J., H.-Z.F., J.-P.J., and S.E.D. approved final version of manuscript.