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Cardiovascular Research 1999 42(3):680-684; doi:10.1016/S0008-6363(99)00005-X
© 1999 by European Society of Cardiology
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Copyright © 1999, European Society of Cardiology

Ischemic preconditioning limits infarct size following regional ischemia–reperfusion in in situ mouse hearts

Diane L Millera and Donna M Van Winklea,b,*

aDepartment of Anesthesiology, Oregon Health Sciences University, Portland, OR, USA
bResearch and Anesthesiology Services, P-8-ANES VA Medical Center 3710 SW US Veterans Hospital Road, Portland, OR 97201 USA

vanwinkd{at}ohsu.edu

* Corresponding author. Tel.: +1-503-220-8262, ex 57404; fax: +1-503-721-7956

Received 30 June 1998; accepted 29 October 1998


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Ischemic preconditioning has been demonstrated in a wide variety of animals, from dogs to rats. Experimentally-induced murine myocardial ischemia–reperfusion has been described, but ischemic preconditioning has not been reported in mouse hearts. To test the hypothesis that mouse hearts exhibit preconditioning-induced protection, experiments were conducted in anesthetized open chest mice subjected to regional myocardial ischemia–reperfusion. Methods: Following barbiturate anesthesia the FVB and C57BL/6J mice underwent a tracheostomy and were mechanically ventilated. The heart was exposed via a left thoracotomy performed with the aid of a dissecting microscope. A 7-0 silk suture on a curved taper needle was passed under the proximal left anterior descending coronary artery to form a snare. Mice were then randomly assigned to receive either no preconditioning or preconditioning. All mice were subjected to 60 min regional myocardial ischemia followed by 2.5 h of reperfusion. Ischemic preconditioning (IP) was induced with two (FVB mice) or three (C57BL/6J mice) cycles of 5 min coronary occlusion and 5 min reperfusion. Control animals did not receive preconditioning ischemia. Area-at-risk was assessed with fluorescent particles. Infarct size was assessed with triphenyl tetrazolium chloride, and is expressed below as a percentage of the area-at-risk. Results: In FVB mice preconditioning reduced infarct size 49%, from 36.7±4.5% to 18.9±2.8% (P<0.05). In C57BL/6J mice preconditioning reduced infarct size by 66%, from 56.4±8.3% to 18.9±4.2% (P<0.05). Conclusion: From these data we conclude that the infarct limiting effect of ischemic preconditioning is demonstrable in murine hearts.

KEYWORDS Ischemic preconditioning; Myocardial infarct size; Mice


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Ischemic preconditioning has been demonstrated in a wide variety of animal models of acute myocardial ischemia–reperfusion, including large species such as swine and smaller rodents such as rats [1–6]. Protection resulting from ischemic preconditioning is exhibited as a decrease in infarct size, decreased incidence of malignant arrhythmias, and improved post-ischemic contractile function. Experimentally-induced murine myocardial ischemia–reperfusion has been described [7–12], but ischemic preconditioning has not been reported in mouse hearts.

Despite their small size, mouse hearts offer one very significant advantage in the study of cardiac (patho)physiology: the availability of transgenic constructs. Although there are a few genetically manipulated rat models, the vast majority of genetically engineered animals are mice. However, the small size of the mouse heart mandates miniaturization, and specialized equipment and supplies. We wanted to determine if myocardial ischemic preconditioning could be demonstrated in mouse hearts. Studies were conducted in anesthetized open chest mice subjected to regional myocardial ischemia–reperfusion.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
Animals used in these studies were allowed access to food and water ad libitum until induction of anesthesia. With local Institutional Animal Care and Use Committee approval, all animals received humane treatment in compliance with the Guide for the Care and Use of Laboratory Animals published by the US National Institute for Health (NIH publication No. 85-23, revised 1985).

2.1 Surgical preparation
Two strains of mice were used: FVB and C57BL/6J. Mice were obtained from Harlan (Cincinnati, OH, USA) and studied at approximately 13–16 weeks of age (24–36 g). Mice were anesthetized with 90 µg/g i.p. sodium pentobarbital, and anesthesia was maintained with one i.p. supplement if needed. After induction of anesthesia the mice were placed in a supine position, a tracheostomy performed, and the mice mechanically ventilated. Ventilation rate was 40–50 breaths/min with a tidal volume of ~1 ml. End-tidal pCO2 was continuously monitored to guide ventilatory adjustments. In FVB mice blood pressure was monitored using a tail cuff and a cutaneous pulse oximeter probe distal to the tail cuff. Core body temperature was measured via a rectal temperature probe and maintained at 36.5–37.5°C with a miniature heating blanket. An ECG was monitored throughout the surgery and experimental protocol. Using a dissecting microscope (Leica MS5) with a 6.3x objective, a left thoracotomy was performed in the fourth intercostal space. The medial aspect of the incision was extended cranially to form a flap that was retracted to expose the heart. A small opening was made in the pericardium and a 7-0 silk suture on a curved taper needle was passed under the proximal left anterior descending coronary artery and both ends of the suture passed through a segment of flared PE10 tubing to form a snare. Mice were then randomly assigned to receive either no preconditioning or preconditioning.

2.2 Induction of ischemia
Ischemia was induced by gently snugging the snare tight, and clamping the PE10 tubing with a vascular bulldog clamp which was supported on a sling. Ischemia was verified by epicardial cyanosis followed by blanching, and ECG changes. Reperfusion was elicited by loosening the snare, and was verified by epicardial hyperemia. All mice were subjected to 60 min regional myocardial ischemia followed by 2.5 h of reperfusion. Ischemic preconditioning (IP) was induced with two (FVB mice) or three (C57BL/6J mice) cycles of 5 min coronary occlusion and 5 min reperfusion. Control (C) animals did not receive preconditioning ischemia.

2.3 Measurement of risk and infarct sizes
At the end of reperfusion, the coronary snare was retightened, and the risk area was delineated by infusing zinc–cadmium sulfide particles (through an 8-µm syringe filter) into the left ventricular lumen via the apex. The zinc cadmium sulfide particles fluoresce bright yellow under UV light and demarcate the area-at-risk as a negative image. Hearts were then weighed, sliced transversely into ~5 slices of ~1-mm thickness, and incubated in triphenyl tetrazolium chloride (TTC, 1% w/v in sodium phosphate buffer, pH 7.4, 37°C) for 20 min. Contrast between stained and unstained tissue was enhanced by incubation of the heart slices in 10% formalin for 10 min. Heart slices arranged cephalad surface facing upward on a microscope slide and then compressed slightly to a uniform thickness of 0.62 mm, using another microscope slide with four cover slips (one at each corner) as shims. The slices were photographed under UV and full spectrum light and the images projected to fill an 8.5''x11'' paper. The outlines of the risk area (zinc cadmium negative) and the infarct area (TTC negative) were traced for each slice, and then digitized using computer-assisted planimetry. The resultant areas were multiplied by slice thickness to calculate volume per slice, then summed. The same individual traced all images without knowledge of the experimental group. Each image was traced three times and averaged. For each heart, infarct size is normalized to area-at-risk.

2.4 Data analysis
ECG was recorded continuously on a strip chart recorder (Model 3400, Gould Instruments, Valley View, OH, USA). Measurements of systolic pressure was made at baseline, immediately prior to the long CAO, immediately before reperfusion, and at the end of reperfusion.

The primary dependent variable analyzed to assess presence or absence of cardioprotection was infarct size. Because the amount of left ventricular myocardium that progresses to infarction depends upon the size of the risk zone, infarct size was normalized as a percentage of the risk zone. Because the risk versus infarct relationship displays a non-zero intercept, we also examined the risk versus infarct relationship for each experimental group. Data analysis was performed with a PC-based statistical software package (CRUNCH 4, Crunch Software, Oakland, CA, USA). Differences between groups and within time points are assessed with analysis of variance. Post-hoc testing between groups was performed using the Newman–Keuls test for multiple comparisons. Post-hoc testing within groups is performed with the Dunnett test for multiple comparisons.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
Twenty-eight FVB and fourteen C57BL/6J mice were randomized into the study; twenty (ten C and ten IP) and fourteen (seven C and seven IP) contributed to the final dataset, respectively. Premature deaths resulting from progressive and irreversible bradycardia and hypotension occurred in two FVB mice (one C and one IP). Six additional FVB hearts were excluded from analysis due to technical failures in delineating the risk zone. Ventricular fibrillation did not occur in any mice.

Hemodynamic data for the mice are shown in Table 1 There were no differences between groups in heart rate or arterial pressure at baseline or any other timepoint. Table 2 and Figs. 1 and 2Go show infarct data. Non-preconditioned mice displayed marked infarction following 60 min coronary occlusion and 2.5 h reperfusion. In FVB mice preconditioning reduced infarct size 49%, from 36.7±4.5% to 18.9±2.8% (P≤0.01, Fig. 1). In C57BL/6J mice preconditioning reduced infarct size by 66%, from 56.5±8.3% to 18.9±4.2% (P≤0.01, Fig. 2.). Because there was a positive relationship between absolute infarct volume and absolute risk volume, and this relationship exhibited a positive x-intercept, we also performed ANCOVA analysis of absolute infarct volume with absolute risk volume as the covariate. This analysis also showed that preconditioning reduced infarct size in both FVB and C57BL/6J mice (P<0.01 both strains).


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  		Table 1 Haemodynamic valuesa

 

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  		Table 2 Weights and volumesa

 

Figure 1
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  		Fig. 1 Infarct size was smaller in preconditioned FVB mice, whether expressed as infarct as percentage of area-at-risk (top panel) or absolute infarct volume (bottom panel). Empty symbols represent data from individual hearts, and filled symbols indicate the group mean±S.E.M. C, control; IP, preconditioned. Levels of statistical significance are indicated as follows: *, P=0.004 (ANOVA); {dagger}, P=0.016 (ANOVA); §, P=0.007 (ANCOVA, risk volume as co-variate).

 

Figure 2
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  		Fig. 2 As was seen for FVB mice, infarct size was also smaller in preconditioned C57BL/6J mice, whether expressed as infarct as percentage of area-at-risk (top panel) or absolute infarct volume (bottom panel). Empty symbols represent data from individual hearts, and filled symbols indicate the group mean±S.E.M. The data point indicated by the arrow corresponded to a heart with a large risk zone (41 mm [3], average risk zone size=25 mm3). C, control; IP, preconditioned. Levels of statistical significance are indicated as follows: *, P=0.004 (ANOVA); {dagger}, P=0.016 (ANOVA); §, P=0.007 (ANCOVA, risk volume as co-variate).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The major finding of the present data is that in situ mouse hearts display infarct limitation as a consequence of myocardial ischemic preconditioning. There have been no previous studies examining myocardial ischemic preconditioning in mice. Recently, measurement of infarct size following experimentally-induced myocardial ischemia and reperfusion has been described in murine in situ and isolated hearts [7–13]. In the in situ studies, mice were subjected to permanent or reversible coronary artery ligation and infarct size was assessed with triphenyl tetrazolium. Hemodynamics in these studies were not reported [8,10,12,13]. In the isolated heart studies, hearts were subjected to global ischemia–reperfusion, and protection assessed by changes in post-ischemic contractile function and/or infarct limitation.

As noted by Kass et al., many murine experimental preparations deviate markedly from the normal conscious physiologic values of heart rate and mean arterial pressure (550–620 beats/min and 110–115 mmHg, respectively) [14]. This is certainly true in isolated murine hearts which are not paced, in which the intrinsic heart rate is reported to be approximately ~300 beats/min [7,11]. Only one of the isolated murine heart studies reported utilized pacing, with heart rate set at 360 beats/min [11]. As noted above, the in situ studies of murine myocardial infarct size have not reported hemodynamic values. Although we used anesthetized mice, our presently reported hemodynamic values suggest that the experimental preparation we employed approximates normal physiologic parameters fairly well. Heart rate was 509±14 and 528±13 beats/min in the FVB and C57BL/6J mice, respectively, and arterial pressure was 93±1 mmHg (FVB).

It is not currently known whether the cellular triggers and mediators of the infarct limiting effect of preconditioning in the mice are the same as those described for other species (e.g., adenosine, bradykinin, protein kinase C, ATP-sensitive potassium channels). However, several studies have examined endogenous cardioprotection in mice by using trangenically induced over-expression of specific substances. Matherne et al. have shown that cardiac specific over-expression of the A1 adenosine receptor results in increased time to ischemic contracture and improved functional recovery following 30 min of global ischemia–reperfusion in isolated buffer perfused mouse hearts. These data suggest that adenosine functions as an endogenous protectant during ischemia in mice [11]. Similarly, over-expression of extracellular superoxide dismutase (EC-SOD) results in improved cardiac function following brief (7 min) global ischemia in working isolated buffer perfused mouse hearts [7]. Limitation of infarct size following 30 min global myocardial ischemia has also been reported in murine hearts over-expressing heat shock protein 72 [8]. Therefore, several endogenous substances that are known to confer cardioprotection in other species (e.g., rabbit, rat, dog) are also cardioprotective in mouse hearts. It is likely that ischemic preconditioning in mice is mediated in a manner similar to that used by hearts of other species.

Our initial studies utilized FVB mice because of their larger size and calmer temperament. Once we demonstrated that preconditioning did limit infarct size in this strain, we examined preconditioning in the black C57 strain; this strain was chosen because it is commonly used for genetic manipulation. The experiments with FVB mice used two cycles of preconditioning prior to myocardial ischemia and showed that infarct size could be reduced by 50%. In the second set of experiments with C57BL/6J mice, we hoped increase the difference between control infarct size and preconditioned infarct size by increasing the preconditioning stimulus to three cycles. Our data confirmed an increased myocardial protection with three cycles of preconditioning, with infarct size reduced by 66%. We feel that the greater difference in infarct size found with three cycles of preconditioning increases the usefulness of the model. We did note a difference in control infarct size between the two strains employed, with control infarct size being larger in the C57BL/6J mice. The reason for this difference in not known. Inter-strain physiologic differences have been reported, but none readily explain a variation in infarct susceptibility [15].

In summary, the present data demonstrate that in situ mouse hearts can be preconditioned. The ability to precondition mouse hearts permits the application of transgenic technology to studies examining the cellular mediation of ischemic preconditioning.

Time for primary review 22 days.


   Acknowledgements
 
This work was supported by a VA Merit Review grant (D.V.W.).


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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