How to produce infarction in the mouse in vivo
Yiru Guo, M.D.
Xian-Liang Tang, M.D.,
Weike Bao, M.D.,
Roberto Bolli, M.D
From the Experimental Research Laboratory
Division of Cardiology
University of Louisville and Jewish Hospital Heart and Lung Institute
Louisville, KY 40292
Tel (502) 852 1837
Fax (502) 852 6474
The mouse has emerged as a popular experimental species because of the increasing availability of transgenic and gene targeted strains that are now becoming important in the study of cardiovascular disease. Therefore, characterizing an applicable model of myocardial infarction (MI) in the mouse is important to test different hypotheses by utilizing transgenic and gene targeted mice, wherein the manipulation of specific genes may allow further mechanistic insights. For example, the finding that ischemic preconditioning cardioprotection against MI is abrogated in mice homozygous for a null iNOS allele (iNOS-/-) has furnished new insights into the pathophysiology of ischemic injury.
The mouse is likely to continue to be the species commonly used for transgenesis and gene targeting. Although transgenesis is theoretically possible in larger mammals (e.g., rabbits or pigs), developing such models would be extremely costly and time-consuming, making these models impractical. Furthermore, gene targeting has not been reported thus far in species other than the mouse. The development of a physiologically-relevant mouse model of myocardial infarction is a necessary step towards the utilization of genetically-engineered animals for interrogating the molecular basis of myocardial ischemia/reperfusion injury and preconditioning. The unique technical challenges associated with inducing MI in mice raise the concern that the results obtained in this model may not be as reliable and physiological as those obtained in larger species, where the margin for error is wider and physiologic parameters can be measured more easily. To thoroughly address these concerns, we have developed a murine model of myocardial infarction in which fundamental physiologic variables (body temperature, heart rate, arterial blood pressure, arterial blood gases and pH) are carefully measured and controlled.1,2 We have shown that measurements of infarct size in this model are both reliable and reproducible. Furthermore, we have found that both the early and the late phases of ischemic preconditioning are robustly expressed in the murine model.1,2 Utilizing this model, we have shown that the inducible isoform of NOS is essential for the late phase of preconditioning induced by ischemia or by pharmacologic stimulation with adenosine A1 agonists or opioid agonists.3We have also demonstrated that overexpression of PKCe confers protection against infarction and that targeted deletion of Lck blocks late preconditioning. Thus, this model appears to be a useful tool to interrogate the molecular basis of ischemia/reperfusion injury with a degree of specificity that is not possible with pharmacologic approaches in other species. Accordingly, the purpose of this chapter is to describe the methodological details that are employed for measuring infarct size in our murine model.
What mouse strain do we use?
In general, we prefer to use adult male mice. We think the best strains are ICR (outbred), C57BL/6J (inbred), and B6129F2/J (hybrid). These strains have good reproductive performance and also preconditionable.1,2,4
The ICR strain is a hardy outbred stock developed at the Institute for Cancer Research (Fox Chase Cancer Center) by T.S. Hauschka beginning in 1948. Good reproductive performance and fast growth rate characterize this strain. It has been used extensively in toxicology and pharmacology studies. It is often used for product safety testing and as an embryo donor and/or recipient mother in transgenic mouse labs. ICR mice can be obtained from Harlan Sprague Dawley (Houston, TX) or Taconic (Germantown, NY).
The C57BL/6J strain, one of the C57BL substrains, is now probably the most widely used of all inbred strains. It is popular in research applications of oncology and toxicology and is used as the female parents to produce the B6129F1, B6C3F1, and B6D2F1 hybrids mice. The mice can be purchased from Jackson Laboratory (Bar Harbor, ME) or Taconic (Germantown, NY).
The B6129F2/J has two stocks (B6129SF2/J and B6129PF2). In general, an F2 hybrid is defined as the second filial generation and is produced by mating two F1 hybrid mice. Matings to produce F2 mice provide the opportunity for genetic recombination between all differing loci to occur. Therefore, F2 hybrids are not genetically identical. For example, if strain X (which carries an 'a' allele at all loci) is mated with strain Y (which carries a 'b' allele at all loci), F2 progeny could be a/a, a/b, or b/b at each individual locus. An F1 hybrid is defined as the first filial generation, or the offspring of an outcross between two different strains. F1 hybrids are heterozygous at all loci at which their parents have different alleles. Similar to inbred strains, F1 hybrids are genetically and phenotypically uniform. Continuing with the example above where strain X and strain Y were mated, the F1 progeny would be a/b at every loci. Additionally, F1 hybrids exhibit 'hybrid vigor': increased disease resistance, better survival under stress, greater natural longevity, larger litters, and so on. Two other uses of F1 hybrids include:
1) accepting transplants of tissues from mice of either parental strain.
2) used as backgrounds for some deleterious mutations.
An F1 hybrid would be used in situations where genetic homogeneity is important; F2 hybrids might be used in situations where genetic homogeneity is less important, or in situations where they best approximate the mixed genetic background of experimental mice.
Recently, we have found that the genetic strain is an important determinant of the susceptibility of mice to myocardial infarction and ischemic preconditioning.4 Specifically, we have found that FVB/N mice have much smaller infarcts after a 30-min coronary occlusion than do the ICR, C57BL/6J, and B6/129SF2/J mice, and that 129SvEv mice do not develop late preconditioning.4Thus, when using gene targeted or transgenic mice, controls should consist of mice with a genetic background as close as possible to that of the gene targeted or transgenic mice (littermates are the best controls, if they are available). The important concept is that data obtained in one strain of mice cannot be extrapolated to another strain of mice.
How do we house the mice?
The mouse should be housed in microisolator cages under specific pathogen-free conditions in a room with a temperature of 24C, 55%-65% relative humidity, and a 12-h light-dark cycle. For maintaining pathogen-free transgenic or gene target mice, we generally keep mice in sterile microisolator cages under pathogen-free conditions, and food and water are autoclaved and all handling is done under a laminar flow hood following standard procedures. We allow the mice to acclimate to our facility for >2 wks before experimentation (because shipping may cause some stress). However, mice exhibiting symptoms of illness such as weight loss, decreased food intake, hair loss, loss of vigor, or wounds from fighting are excluded from the study before the beginning of the experiment.
How do we perform the open-chest procedure?
Mice are premedicated with atropine sulfate (0.04 mg/kg i.m.) and anesthetized 5 min later with sodium pentobarbital (50 mg/kg i.p.). Additional doses of pentobarbital are given during the protocol as needed to maintain anesthesia. The animals are placed in a supine position with the paws taped to the operating table. Surface leads are placed subcutaneously to obtain the ECG, which is recorded throughout the experiments on a thermal array chart recorder. Before starting surgery, mice are given gentamicin (0.7 mg/kg i.m.).
A midline cervical skin incision is performed and the muscles overlying the trachea are reflected to allow visualization of the endotracheal tube (a PE-60~90 tubing) as it is placed in the trachea. To facilitate intubation, a rubber band is placed behind the upper incisors and fastened to the operating table so that the neck is slightly extended. To place the endotracheal tube, the tongue is slightly retracted, and the beveled end of the tube (which is marked with a black marker) is inserted carefully through the larynx and into the trachea so as not to puncture the trachea or other structures in the pharyngeal region. The tube is advanced 8-10 mm from the larynx and taped in place to prevent dislodgment. The animals are ventilated with room air supplemented with oxygen (2 L/min) at a rate of 105/min and with a tidal volume of 2.1-2.5 ml using a rodent ventilator (Harvard Apparatus, Inc., South Natick, MA). The endotracheal tube is inserted loosely into the tube connected to the ventilator so as to avoid lung overexpansion. A catheter is inserted into the external jugular vein for fluid infusion. In selected studies, a catheter is inserted into the carotid artery for measurement of blood pressure (DTXTM pressure transducer, Viggo-Spectramed, Oxnard, CA) and analysis of blood gases. To replace blood losses, blood from a donor mouse is given i.v. at a dose of 40 ml/kg (~1 ml) divided into three equal boluses (first bolus, after connecting the endotracheal tube to the ventilator; second bolus, after opening the chest; third bolus, after closing the chest). Body temperature is carefully monitored with a rectal probe connected to a digital thermometer (Cole-Parmer Instrument Company, Vernon Hill, IL) and is maintained as close as possible to 37.0 C throughout the experiment using a heating pad and heat lamps.
How do we perform the coronary artery occlusion procedure?
With the aid of a dissecting microscope (Fisher Scientific, Pittsburgh, PA) and a microcoagulator (ASSI Polar-Mate Isolator, San Diego, CA), the chest is opened through a midline sternotomy. An 8-0 nylon suture (Ethicon, Inc. Johnson & Johnson Co. Somerville, NJ) is passed with a tapered needle under the left anterior descending coronary artery 2-3 mm from the tip of the left auricle, and a nontraumatic balloon occluder is applied on the artery. Coronary occlusion is induced by inflating the balloon occluder. Successful performance of coronary occlusion and reperfusion is verified by visual inspection (i.e., by noting the development of a pale color in the distal myocardium upon inflation of the balloon and the return of a bright red color due to hyperemia after deflation) and by observing ST-segment elevation and widening of the QRS on the ECG during ischemia and their resolution after reperfusion. After the coronary occlusion/reperfusion protocol, the chest is closed in layers, and a small catheter is left in the thorax for 10-20 min to evacuate air and fluids. The mice are removed from the ventilator, kept warm with heat lamps, given fluids (1.0-1.5 ml of 5% dextrose in water i.p.), and allowed 100% oxygen via nasal cone.
How do we perform postmortem analysis?
Figure 1. Representative example of a heart from a control group (ICR subjected to a 30-min coronary occlusion and 24 h of reperfusion). The infarcted region was delineated by perfusing the aortic root with TTC; the region at risk was delineated by perfusing the aortic root with Phthalo blue after tying the previously occluded artery (see text for details). As a result of this procedure, the nonischemic portion of the left ventricle was stained dark blue, the viable tissue within the region at risk was stained bright red, whereas the infarcted tissue was light yellow. Note the large, confluent areas of infarction spanning most of the thickness of the LV wall, with thin rims of viable subendocardial tissue. This pattern was characteristic of all nonpreconditioned groups (including AMI and sham groups). The scale at the bottom is in mm.
At the conclusion of the study,
the mice are given heparin (1 U/g i.p.), after which they are
anesthetized with sodium pentobarbital (35 mg/kg i.p.) and
euthanized with an i.v. bolus of potassium chloride
(KCl). The heart is excised and perfused with
Krebs-Henseleit solution through an aortic cannula (22- or
23-gauge needle) using a Langendorff apparatus. To
delineate infarcted from viable myocardium, the heart is then
perfused with a 1% solution of triphenyltetrazolium chloride
(TTC) in phosphate buffer (pH 7.4, 37 C) at a pressure of 60 mmHg
(approximately 3 ml over 3 min). To delineate the
occluded/reperfused coronary vascular bed (the region at risk),
the coronary artery is then tied at the site of the previous
occlusion and the aortic root is perfused with a 5-10% solution
of Phthalo blue dye (Heucotech Ltd., Fairless Hill, Pa) in normal saline (2 ml over
3 min). As a result of this procedure, the portion of the
left ventricle (LV) supplied by the previously occluded coronary
artery (region at risk) is identified by the absence of blue
dye, whereas the rest of the LV is stained dark blue. The
heart is frozen, after which all atrial and right ventricular
tissues are excised. The LV is cut into 5-7 transverse
slices, which are fixed in 10% neutral buffered formaldehyde and,
24 h later, weighed and photographed (Nikon AF N6006). The
transparencies are projected onto a paper screen at a 30-fold
magnification, and the borders of the infarcted,
ischemic-reperfused, and nonischemic regions are traced.
The corresponding areas are measured by computerized
videoplanimetry (Adobe Photoshop, version 4.0, NIH Image, or
Image tool), and from these measurements infarct size is
calculated as a percentage of the region at risk.1,2
What kind of anesthetic do we use?
Initially, we induced anesthesia with xylazine (7.5 mg/kg i.m.) and ketamine (55 mg/kg i.m.); however, we found that the heart rate was quite low (280-330 bpm), not within the physiological range. We therefore chose pentobarbital anesthesia. After selecting the anesthetic, a series of pilot studies was performed in 47 mice.2 First, we sought to establish physiological parameters to be used as a reference for subsequent experiments. In eight mice, ECG leads were placed subcutaneously and the animals were allowed to recover. Heart rate was monitored in the conscious state on the following days, and was found to average 668±31 bpm (range, 490 to 760 bpm). In 16 pentobarbital-anesthetized mice, mean arterial blood pressure prior to thoracotomy was found to average 97.2±4.4 mmHg. In 39 pentobarbital-anesthetized mice, rectal temperature prior to thoracotomy was found to average 37.0±0.2°C. Therefore, under pentobarbital anesthesia, heart rate and arterial pressure are within physiological limits. We have found that mice recover fairly quickly after closing the thoracotomy, and believe that pentobarbital provides a satisfactory means to induce anesthesia.
What ventilatory parameters do we use?
Because of the obvious importance of adequate oxygenation and acid-base balance, we recommend that investigators identify the optimal ventilatory parameters in their particular model. In our studies, since the endotracheal tube used is without a cuff, we adjust the tidal volume by observing the inflation of the lungs after the chest is opened. We found that the average tidal volume of 2.2 +/- 0.1 ml results in adequate inflation of the lungs without over-expansion. (Over-expansion is dangerous because it can cause rupture of the lungs) Using this tidal volume, we tested different ventilatory rates in 23 open-chest mice and analyzed arterial blood gases in each animal (Table 1).2 We found that even small changes in ventilatory rate resulted in significant changes in arterial blood gases (Table 1), emphasizing the importance of the ventilatory parameters. A ventilatory rate of 105/min was found to produce optimal values of pO2, pCO2, and pH, as detailed in Table 1. This rate is within the range observed in spontaneously breathing mice.5,6 The ventilatory rate and the tidal volume that produce the optimal blood gas values will vary from one laboratory to another. What is important is that each laboratory determines these values before starting large-scale studies of myocardial infarction in mice.
How can arterial blood pressure be kept in the normal range during the occlusion/reperfusion procedure?
We measured arterial blood pressure in mice subjected to open-chest surgery.2 The results are summarized in Fig 2. In five mice, one dose of blood (13.3 ml/kg; ~0.4 ml) was given immediately after the thoracotomy in an effort to prevent hypotension. Despite this, mean arterial blood pressure fell to 63.4±3.7 mmHg after opening the chest (probably due to the loss of negative intrathoracic pressure and to the positive end-expiratory pressure) (Fig. 2). Furthermore, after closing the chest, another drop in blood pressure was noted, to a nadir of 63.0±9.7 mmHg (Fig. 2). Because these hypotensive episodes could induce ischemic preconditioning, we decided to administer three doses of blood (instead of one): the first was given before opening the chest, the second immediately after opening the chest, and the third after closing the chest. Each dose consisted of 13.3 ml/kg (~0.4 ml). Using this protocol, mean arterial pressure remained at or above 80 mmHg throughout a 1-h period of open-chest state (Fig. 2). Next, we tested whether this protocol of fluid replacement was sufficient to prevent severe hypotension in mice undergoing a sequence of six coronary occlusion/reperfusion cycles, in which myocardial ischemia would be expected to cause a further fall in arterial pressure. Although each coronary occlusion caused a drop in arterial pressure, the three doses of blood resulted in mean arterial pressure being maintained at or above 80 mmHg throughout the six occlusion/reperfusion cycles (Fig. 2). Thus, using the fluid supplementation protocol detailed above and careful precautions to minimize blood loss, arterial blood pressure could be kept at adequate levels throughout the six occlusion/reperfusion cycles.
Of all the determinants of infarct size, temperature is probably the most important. Accordingly, temperature should be strictly controlled throughout the experiment. We control temperature by adjusting the heating pad and the heat lamps during the surgical procedures. We allow a minimal drift in temperature, from 36.7° to 37.3°C. As a result of these compulsive measures, rectal temperature remains within the physiological range in our studies (Table 2). With regard to heart rate, as mentioned earlier, pentobarbital anesthesia results in heart rates those are within the physiologic range for conscious mice (this range is from 500 to 760 beats/min) (Table 2).
How to select the duration of reperfusion?
In view of the added complexity inherent in following mice for 24 h after reperfusion, we investigated whether extending the reflow period beyond 4 h was important for assessing the final extent of infarct size. Birnbaum et al.7 have reported that at least 3 h of reperfusion are necessary to assess the final extent of infarction in rabbits. Accordingly, we allowed a minimum of 4 h of reperfusion. When infarct size was compared after 4 h and 24 h of reperfusion, the results were similar, both in nonpreconditioned hearts (group II vs. group I; group VII vs. group V) and in preconditioned hearts (group VIII vs. group VI) (Fig. 3 and Table 3), supporting the conclusion that a 4-h reperfusion interval is sufficient to evaluate ischemic preconditioning in the mouse.2 This information should be useful in designing future studies, particularly studies of early preconditioning, which could be done acutely without the need for survival surgery.2 Although the results obtained with 4 h and 24 h of reperfusion are similar, we think it is preferable, whenever possible, to allow 24 h of reperfusion, particularly when using mice with genetic mutations of proteins that may be important in modulating the inflammatory response after reperfusion. However, when mice are particularly frail, a 4-h reperfusion interval is preferable because it reduces mortality.
We have developed a reliable and physiologically-relevant model of ischemic preconditioning that can be used in genetically-engineered animals. Our results can be summarized as follows (i) despite the small size of the mouse, it is possible to study MI in vivo in this model under conditions in which basic physiologic variables are kept within normal limits (body temperature, arterial oxygenation, acid-base balance, heart rate, and arterial blood pressure); and (ii) both the quality of the postmortem staining for region at risk and infarction and the reproducibility of the measurements of infarct size are excellent and compare favorably with those in larger species.
This murine model of MI should be useful for investigating the impact of genetic manipulations upon physiological end-points in vivo. By applying this model to mice with overexpression or targeted disruption of individual genes implicated in the cellular pathways underlying ischemic heart diseases, it should be possible to conclusively establish the role of a specific gene product in the genesis of ischemic heart diseases in the intact animal.
A major concern in the design of our model was to ensure that the results would be physiologically relevant. The minuscule size of the murine heart necessitates miniaturization of the procedures used in larger species and therefore poses a unique challenge in terms of maintaining general experimental conditions within normal values and avoiding artifacts. Thus, when starting a mouse model, a considerable amount of preliminary work should be performed prior to the actual studies.
Because temperature is a major determinant of infarct size,8-11 this variable is tightly controlled throughout the experiment by using heating pads and heat lamps while continuously monitoring rectal temperature. Our results demonstrate that by using these procedures, temperature can be kept within a narrow range (36.4-37.6°C) (Table 2), which represents the normal range for the mouse,5,12 as confirmed by our pilot studies in which rectal temperature averaged 37.0°±0.4°C. Hypoxemia, acidosis, and alkalosis may also have a major influence upon animal survival, infarct size, and/or ischemic preconditioning. Accordingly, we measured arterial pH, pO2, pCO2, and bicarbonate levels in mice subjected to open-chest surgery (Table 1). These measurements demonstrated that, with a ventilatory rate of 105/min and an average tidal volume of 2.2 ml, all parameters were within the physiologic range for the mouse13; in particular, arterial pH was kept at ~7.40 and adequate oxygenation was maintained throughout the open-chest state (Table 1). Careful control of blood gases is important in the mouse, since small variations in ventilatory rate result in marked variations in arterial blood gases (Table 1).
Heart rate and arterial pressure are important indices of normal cardiovascular homeostasis and are also important determinants of the severity of myocardial ischemia. As elaborated earlier, we avoided anesthesia with ketamine/xylazine because these agents resulted in unacceptably low heart rates (280 to 330 bpm), clearly outside of the physiological range, which in our pilot studies in conscious mice was found to be 490 to 760 bpm (average, 688±31 bpm). With pentobarbital anesthesia, the heart rates recorded in the present experiments (Table 2) were reasonably close to those measured in conscious mice in our pilot studies and in previous studies.6,12,14,15 The blood volume of a 25-g mouse has been estimated to range between 1.5 and 2.3 ml.5 To avoid hypotension, surgery was performed with a microcoagulator, and every effort was made to minimize blood losses. Pilot studies, however, showed that opening the chest caused a significant drop in arterial blood pressure, so that the mice became severely hypotensive despite the administration of 13.3 ml/kg (~0.4 ml) of blood (Fig. 2). Besides causing mortality, severe hypotension could lead to myocardial hypoperfusion and, possibly, induce preconditioning as a result of myocardial ischemia and/or reflex adrenergic activation. We therefore modified our protocol by administering three doses of blood (total of 40 ml/kg or ~1.2 ml), as detailed earlier, which resulted in values of mean arterial blood pressure >80 mmHg throughout the sequence of six coronary occlusion/reperfusion cycles (Fig. 2). These values of arterial pressure are within the range reported by others in normal mice.5,14-22
Using this model, the average infarct size in nonpreconditioned mice (groups I, II, III, V and VII combined, see Fig. 3) was found to be 52% of the region at risk, which, surprisingly, is similar to the average infarct size measured after the same duration of coronary occlusion (30 min) in conscious rabbits (56.9±5.9% 23and 56.8±5.3% 24of the region at risk) and in open-chest rabbits (52.0±5.2% 25, 53.6±5.7% 26, 48.1±3.9% 27, and 49.1±4.3% 28 of the region at risk). The ranges of individual infarct sizes (Fig. 3 and Table 3) and the slopes and x-intercepts of the infarct size-risk region relationships (Fig. 4) were also similar to those previously observed in conscious rabbits after a 30-min occlusion.23,24
With the development of genetically-engineered mice, there is increasing interest in using transgenic or knockout mice as a tool to interrogate the cellular mechanisms of cardiovascular disease. By overexpression or targeted disruption of specific genes, these murine models provide a unique approach to understanding the role of specific gene products in abnormal cardiovascular function. In the case of ischemic preconditioning, however, the exploitation of genetic manipulations has been hindered by the lack of in vivo physiologic correlates. We have developed a mouse model of myocardial infarction in which several fundamental physiological variables are carefully controlled and kept within normal limits. Mortality is relatively low (<20%). Measurements of infarct size are accurate and reproducible. Our results also demonstrate that a robust infarct-sparing effect occurs during the early and the late phases of preconditioning in the mouse and that the quantitative aspects of this effect are consistent with previous experience in other species. This model has been useful to elucidate the molecular basis of ischemic preconditioning by making it possible to apply molecular biology techniques to intact animal preparations in order to dissect the precise roles of individual proteins.
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