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1 Department of Gene Therapy, Faculty of Medicine at the National Heart & Lung Institute, Imperial College, London, UK and2 Angiogenesis Research Center, Beth Israel Deaconess Medical Center, Harvard University, Boston, MA, USA3 Angiogenesis Research Center, Beth Israel Deaconess Medical Center, Harvard University, Boston, MA, USA
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Abstract |
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(Received 16 January 2004;
accepted after revision 6 May 2004; first published online 6 May 2004)
Corresponding author U. Griesenbach: Department of Gene Therapy, Faculty of Medicine at the National Heart & Lung Institute, Imperial College, London, UK. Email: u.griesenbach{at}ic.ac.uk
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Introduction |
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Preclinical models of myocardial ischaemia have been reported in several large animal species, including dogs (Schaper et al. 1970; Shou et al. 1997) and goats (Mannion et al. 1996). The model that most closely resembles the response seen in humans is the pig ameroid model (Ware & Simons, 1999), which has been used in a variety of therapeutic studies (Giordano et al. 1996; Hariawala et al. 1996; Lopez et al. 1998; Mack et al. 1998; Tio et al. 1999; Patel et al. 1999; Laham et al. 2000; Sato et al. 2000, 2001). However, the expense and practical demands of porcine surgical facilities severely limit the extent of such studies, precluding the use of this model for larger scale screening studies of novel therapeutic approaches.
A rodent model of acute myocardial infarction (MI) was first developed in the rat (Pfeffer et al. 1979). More recently a murine equivalent has been described (Michael et al. 1995), providing the means to exploit the increasing availability of many useful transgenic and knockout mouse strains. Complete occlusion of the left anterior descending (LAD) coronary artery induces an acute MI. The resulting ischaemia in the left ventricular wall has been visualized with Evans blue/TTC perfusion assays (Du et al. 1998; Lutgens et al. 1999; Michael et al. 1999; Scherrer-Crosbie et al. 1999), which allows quantification of the infarct area. Further work by Guo et al. (1998) has demonstrated ischaemic preconditioning after short-term LAD occlusion in mouse, thus validating the physiologic relevance of this infarct model. Previous studies show that the size of the infarct varies between 20–65% (Michael et al. 1995), 8–54% (Du et al. 2000), 14–65% (Patten et al. 1998) and 15–55% (Gao et al. 2000) in individual studies. Because of this, some studies divide animals into subgroups based on infarct size (e.g. Large Infarct versus Moderate Infarct) for further functional analysis (Patten et al. 1998; Du et al. 2000). Clearly this would not be appropriate, however, for studies aimed at assessing the effect of novel therapies.
Several assays of cardiac function have also been developed in this model. Haemodynamic measurements, such as heart rate, blood pressure and contractility (maximum change in pressure over time, dP/dTmax), have been carried out following catheterization of the left ventricle (Gao et al. 2000). Further functional assessments have been made using echocardiography (Scherrer-Crosbie et al. 1999). Although variability was fairly high, these end-points have been sufficient to distinguish the MI mouse from sham-operated or wild-type control animals (Patten et al. 1998; Lutgens et al. 1999; Du et al. 2000; Gao et al. 2000). However, in order to discern possible treatment effects, the intrinsic variability of the model must be kept to a minimum.
Before embarking on interventional studies, it is important first to evaluate the model's baseline characteristics. Here we attempted to critically appraise the usefulness of the model for future therapeutic studies. Since the placement of the occluding suture is likely to be a key factor in the extent of the resulting cardiac insult, we used three distinct occlusion sites along the LAD coronary artery. We then selected end-points suitable for relatively high-throughput preclinical screening studies. We assessed the effects of suture placement on survival and disease phenotype. We further evaluated end-points of haemodynamic function, lesion size in different regions of the left ventricle, and the correlation between infarct size and cardiac function.
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Methods |
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All procedures were approved by the British Home Office, in accordance with the Animals (Scientific Procedures) Act 1986, and by the Imperial College Ethical Review Panel. Pain and distress were limited by regular administration of analgesia in accordance with advice from Imperial College Named Veterinary Surgeons. C57B6/J mice (male, 20–25 g, 6–10 weeks old) were used. Animals were anaesthetized with 250 mg kg–1 Avertin (Papaioannou & Fox, 1993; 0.1 ml 2.5% Avertin per 10 g body weight injected intraperitoneally). The analgesic buprenorphine was given preoperatively (10–20 µg (kg body weight)–1). Occlusion of the left anterior descending coronary artery (LAD) was performed as previously reported (Michael et al. 1999), under sterile conditions, with minor alterations. Briefly, the anaesthetized animal was intubated endotracheally in a supine position, and ventilated with a Harvard Mouse Mini-Vent (Harvard Apparatus, March-Hugstetten, Germany), which supplied 0.2–0.25 ml room air 120 times per minute. The animal was moved onto its right side, and a left thoracotomy provided access to the beating heart. After removing the pericardium, the LAD was visualized with a stereomicroscope (Leica MZ6, Heerbrugg, Switzerland), and occluded with 8/0 (0.4) Prolene suture (Ethicon, Johnson & Johnson, Brussels, Belgium). Occlusion was confirmed by observation of left ventricular pallor immediately post ligation. The chest was closed, the lungs re-inflated and the animal moved to a prone position until spontaneous breathing occurred. Open-chest surgery itself has been shown to cause haemodynamic changes observable 7 days after surgery (Lutgens et al. 1999). Thus, control (sham) animals underwent a thoracotomy with removal of the pericardium, but no LAD occlusion. No suture was placed in the sham animals' hearts, in order to avoid unintended vessel damage or occlusion. Animals were monitored closely for signs of infection at the surgical site; none were observed in any animal.
Suture placement
After experience of approximately 100 procedures had been gained, and the operator was able to locate the LAD in every animal, it became possible to choose finely distinguished suture target sites. A total of 46 animals were divided into three different groups: High, Middle and Low Suture (n= 10–15 per group). In the High Suture group, the occluding suture was placed at the atrioventricular junction. This was located at the edge of the atrial appendage. In the Middle Suture group, the suture was placed 0.3 mm distal to the atrioventricular junction. Accurate measurement of this distance was made possible by use of the stereomicrocope, using the suture needle (width 0.3 mm) as a guide. In the Low Suture group, the suture was placed 0.6 mm distal to the atrioventricular junction. In all groups, this resulted in the suture being placed ‘upstream’ of the first major branching of the artery. Figure 1 shows a schematic representation of suture placement sites. Previous studies have reported suture placement 1–3 mm from the atrial appendage (Michael et al. 1999; Du et al. 2000; Gao et al. 2000), whereas here the most distal suture was placed 0.6 mm from the atrial appendage. Apart from suture placement, all other aspects of the procedure were identical for each group. Mice were randomised to each group in order to avoid the effects of any minor variation in age or size.
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Seven days after surgery, the phenotype of each animal was scored semiquantitatively as follows: 3 = phenotypically indistinguishable from a wild-type animal; 2 = at least one sign of illness (hunched posture or ruffled coat), but otherwise as active as a wild-type animal; 1 = hunched posture, ruffled coat and less active than a wild-type animal; 0 = dead. All surviving animals were anaesthetized with Avertin, intubated and ventilated as above for haemodynamic measurements. Access to the apex of the heart was gained via the abdominal cavity after removal of the diaphragm. A 23 gauge needle filled with heparinized saline (200 IU ml–1 heparin in 0.9% saline) was inserted through the apex into the left ventricle. The needle was connected to a Digi-Medical Heart Performance Analyser (Micro-Med Inc., Louisville, KY, USA), which recorded maximum left ventricular pressure (LVP) and maximum change in pressure over time (dP/dTmax). All measurements and analysis were performed in a blinded manner.
Histologic analysis
After haemodynamic assessment, animals were killed by exsanguination caused by removal of the transducer-attached catheter. The heart was removed and fixed in fresh 4% paraformaldehyde, pH 7.3. The tissue was processed and embedded in paraffin using routine histological procedures. Five micrometre transverse step sections were collected every 200 µm through the entire ventricle (approximately 10–12 sections per animal), and stained with Haematoxylin and Eosin (H&E). At 7 days after occlusion, it is too early to differentiate the final results of the cardiac insult and subsequent repair process. Therefore the total lesion area, expressed as a percentage of the total left ventricular wall, was calculated using MetaMorph software (Universal Imaging Corporation, West Chester, PA, USA), and a mean value calculated for each heart. Mid-papillary sections were then divided into anterior, lateral, posterior and septal regions, and lesion size in each of these regions was scored semiquantitatively. Each region was divided into four equal quadrants, and each quadrant scored 1 (lesion present) or 0 (no lesion). The sum of these quadrant scores was used to give each region an overall score from 0 to 4. All analysis was done in a blinded manner.
Statistical analysis
All values are expressed as the mean ±S.E.M. or the median (range), as appropriate. Except for survival analysis, data between groups were compared using the Kruskal–Wallis analysis of variance for multiple unpaired, non-parametric groups of variables, followed (where permitted by the Kruskal–Wallis analysis) by the Mann–Whitney U test, with a Bonferroni correction for multiple comparisons. Survival data were analysed by constructing a Kaplan–Meier table to derive a 
2 value. Correlation between selected variables was analysed using the least-squares method for linear regression. The null hypothesis was rejected at P < 0.05.
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Results |
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Figure 2 shows survival for sham, Low, Middle and High Suture groups, up to 7 days after the procedure. There was no difference in survival among the sham, Low Suture and Middle Suture groups (100, 90 and 73% survival at day 7, respectively). However, in the High Suture group survival was only 33%, which was significantly (P < 0.05) lower than in the sham-operated control group. Thus, the number of animals that completed the protocol was n= 9, n= 8 and n= 5 in the Low, Middle and High Suture groups, respectively. Phenotype was assessed 7 days after the procedure using a semiquantitative scoring system (Fig. 3). There was no difference among the sham, Low Suture and Middle Suture groups, but the High Suture group had a significantly (P < 0.01) lower phenotypic score compared to sham-operated controls.
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The effect of occlusion on cardiac function was determined by measuring maximum LVP and dP/dTmax 7 days after the procedure (Fig. 4). Although both the Low and Middle Suture groups had low intragroup variability (coefficient of variance (%c.v.): 20 and 17%, respectively, for maximum LVP; 27 and 23%, respectively, for dP/dTmax), only the Middle Suture group showed a significant reduction in maximum LVP (Fig. 4A) and dP/dTmax (Fig. 4B) compared to the sham control group (maximum LVP: sham group 73.9 mmHg (45.9–101.3 mmHg), Middle Suture group 58.5 mmHg (40.2–65.6 mmHg); dP/dTmax: sham group 4337 (2016–6129), Middle Suture group 2045 (1294–2803); n= 8–10, P < 0.05 and P < 0.01 for maximum LVP and dP/dTmax, respectively). The High Suture group, with low surviving n numbers and high variability (%c.v.: 43 and 53% for maximum LVP and dP/dTmax, respectively), was not different from any other group. For all groups, heart rate during cardiac function measurement was 400–500 beats min–1.
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A representative H&E-stained cross-section from a Middle Suture group animal 7 days after myocardial infarction is shown in Fig. 5. Among the Suture groups, there were generally no histologic changes proximal to the suture. Lesion size as a proportion of the left ventricular wall varied considerably through the myocardium; however, there was no consistent pattern of lesion size change in any Suture group. Thus, in 43% of animals the lesion increased from base to apex; in 31% of animals the lesion increased and then decreased; in 22% of animals the lesion remained approximately the same; and in 4% of animals the lesion reduced from base to apex. The septum and right ventricular wall were rarely affected, and if affected, less than 1% of the total septal or right ventricular area was diseased. No histologic changes were seen in any sham-operated animal.
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There was no correlation between lesion area and maximum LVP or dP/dTmax (data not shown). However, the effect of MI on cardiac function in humans depends greatly on which region of the left ventricular wall is affected. We therefore analysed lesion area semiquantitatively in each of four regions: the anterior, lateral and posterior walls and the septum. There was no difference between the Suture groups in regional distribution of injury. In all groups, the anterior wall was most affected, followed by the lateral wall, while the posterior wall and septum were minimally affected, if at all. Both anterior (Fig. 7A) and lateral wall (Fig. 7B) lesion scores showed a significant, albeit weak, negative correlation with dP/dTmax (r=–0.46 for both, P < 0.05). There was also a similar correlation between maximum LVP and anterior wall infarct (r=–0.44, P < 0.05), but not with lateral wall infarct. Neither the posterior wall nor the septum showed any correlation with cardiac function.
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Discussion |
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In this study, survival was significantly lower in the High Suture group; this has previously been observed. Du et al. (1998) reported that a ‘too-high’ occlusion site generated an ischaemic zone comprising up to 70% of the left ventricle, which the mice did not survive. Here, similarly, the High Suture group showed low survival and poor phenotype. Assessment of overall phenotype in this way does not necessarily indicate a specific aetiology; however, as suture placement was the only experimental variable, these results suggest that phenotypic changes were related to the High suture placement, and that this resulted in too severe an infarction. With 67% mortality and high morbidity, the surviving animals are not likely to be a representative sample. This is shown by the highly variable cardiac function among the survivors. Although low surviving n numbers made reliable statistical analysis difficult, the High Suture group was not different from sham-operated control animals. Thus the surviving animals in the High Suture group are likely to be a selected population with better endogenous collateralization or greater reliance on other coronary arteries. In addition, it is ethically difficult to justify the use of a model with such severe outcomes, especially when the Middle Suture group provides a reproducible model without such severe effects on morbidity and mortality.
Survival in the Middle and Low Suture groups was similar to previously published studies, which have reported 50–76% survival 7 days after induction of MI (Lutgens et al. 1999). Intra-group variability was lowest in the Middle and Low Suture groups. However, the Low Suture group was not different from sham-operated control animals in haemodynamic end-point assays. Due to the ‘low’ occlusion, a substantial proportion of the left ventricle remained perfused and therefore functional. In contrast, in the Middle Suture group a reduction of 20 mmHg in maximum LVP and 2400 in dP/dTmax compared favourably with previous published studies (Lutgens et al. 1999; Du et al. 2000; Gao et al. 2000). Moreover, the use of a catheter-based system provided global cardiac functional data in a practical, accessible and timely form, which is desirable for relatively high-throughput screening studies.
Specific measurements of intragroup variability have not yet been reported. In this study we determined the coefficients of variance (%c.v.) to characterize the variability of haemodynamic and histologic measurements within each group. Variability was also used to evaluate the usefulness of each end-point for future therapeutic experiments. Low variability (17–23%) in maximum LVP and dP/dTmax in the Middle Suture group, in conjunction with significant reductions compared to sham-operated controls, suggests that these end-points may be robust enough to assess potential treatments. Power calculations, based on intragroup variability, predict that n= 12 mice per group would be required in order to measure accurately a 20% improvement in maximum LVP and a 30% improvement in dP/dTmax. In contrast, higher variability in our histologic data means that n= 14 mice per group would be required to demonstrate a 40% reduction in overall lesion size. The increased experimental ‘noise’ in this end-point may also contribute to the fact that we did not observe a significant correlation between total left ventricular lesion area and cardiac function. This is in contrast to a previous study which showed a significant negative correlation between overall infarct size and echocardiographic parameters (Gao et al. 2000).
When we analysed four different regions (anterior, lateral and posterior wall and the septum) separately, however, cardiac function correlated most strongly with lesion size in the anterior and lateral regions. These are the regions whose supply is most dependent on the LAD coronary artery, and which have the largest influence on myocardial contractions. Not surprisingly therefore, as in humans, injury to these regions has the greatest negative effect on cardiac function. However, the strength of the correlations suggests that other factors also contribute, which probably include endogenous collateralization and/or reliance on coronary vessels other than the LAD coronary artery. It therefore appears that lesion size may be a useful secondary end-point for future treatment studies, as long as the limitations of the assay are acknowledged.
In summary, we have shown that occlusion of the LAD coronary artery 0.3 mm distal to the atrioventricular junction produces a significant and reproducible reduction in cardiac function, and a significant increase in histologic changes in the left ventricle. Although some, more sophisticated, measurements, such as functional assessment of collateralization using coloured microspheres, are not yet technically feasible in the mouse, the model as described here may provide a useful preclinical tool. Further, we provide power calculations which may facilitate the development of studies to assess potential new treatments in this model.
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