DOI: 10.1111/eci.12202
ORIGINAL ARTICLE Myocardial infarct size measurement using geometric angle calculation Michael Lichtenauer*,†, Catharina Schreiber‡, Christian Jung*, Lucian Beer†,§, Andreas Mangold¶, Mariann € ngyo € si¶, Bruno Karl Podesser** and Hendrik Jan Ankersmit†,§ Gyo * €tsherzzentrum Thu € ringen, Clinic of Internal Medicine I, Department of Cardiology, Friedrich Schiller University Universita Jena, Jena, Germany, †Christian Doppler Laboratory for Cardiac and Thoracic Diagnosis and Regeneration, Vienna, Austria, ‡ Department of Cardiac Surgery, University Clinic Salzburg, Salzburg, Austria, §Department of Thoracic Surgery, Medical University Vienna, Vienna, Austria, ¶Department of Cardiology, Medical University Vienna, Vienna, Austria, **Ludwig Boltzmann Cluster for Cardiovascular Research, Medical University Vienna, Vienna, Austria
ABSTRACT Background In basic cardiovascular research focusing on animal models of myocardial infarction (MI), the measurement of infarct size is performed by planimetry of histological sections of the heart. However, in the setting of chronic MI with ongoing changes in ventricular geometry caused by wall thinning and hypertrophy, the scar area tends to become smaller. Materials and Methods Here, in this study we compared infarct measurements in tissue sections (of rat and porcine hearts) based on three different calculation approaches, that is, infarct area, infarct lengths and infarct angles utilizing the centroid of the left ventricle using a newly developed calculation approach. Results Infarct sizes from all three measurement approaches showed significant correlation with parameters of cardiac function. However, results derived from area measurements were significantly smaller than those obtained using the other two measurement approaches due to scar thinning (infarct size area: 1481% 127 SEM, length: 2394% 204 SEM, angle: 2475% 213 SEM, P < 00001, n = 30). Moreover, results from angle measurements evidenced a much better correlation with parameters of cardiac function in a small animal model of chronic MI (e.g. ejection fraction, angle: r = 073; length: r = 064; area: r = 059, n = 30) as well as in a large animal model of acute MI (angle: r = 082; area: r = 067, n = 10). Conclusions We concluded that area-, length- and angle-based measurements can be used to determine the relative infarct size in acute MI models, although an area-based measurement might be less accurate in the setting of chronic MI. Our new method of infarct angle measurement is a reliable and simple way to calculate infarct size compared with conventional measurement approaches. Keywords Animal models, infarct size, measurement, myocardial infarction. Eur J Clin Invest 2014; 44 (2): 160–167
Introduction Heart failure accompanied with left ventricular (LV) remodelling and the loss of cardiac contractility is the leading cause of mortality and morbidity in the western world [1]. The major cause for the development of end stage heart failure in most cases is myocardial ischaemia [2]. Even though interventional strategies of early reperfusion have reduced the mortality after acute myocardial infarction, ischaemic heart disease still remains a major issue for patients and health care systems worldwide [3]. Also in basic science an extremely broad spectrum of possible therapeutic strategies to protect ischaemic myocardium or reduce the damage caused by ischaemia has been studied over the last decades [4–6].
160
Both in basic science studies and clinical trials the reduction of infarct size is one the paramount outcome parameters to define the success or failure of a potential treatment strategy. Total infarct size is the most critical parameter for the further prognosis of patients suffering from acute myocardial infarction (MI) because prognosis is inversely correlated to the total tissue mass of left ventricle myocardium that has been lost [7–9]. Previous studies showed that a direct relationship between the extent of infarct size and mortality exists and that the initial infarct size is a major predictor of long-term LV function [10]. In the field of basic cardiovascular research, scientist used animal models of MI to develop new strategies to salvage ischaemic myocardium. In these models, both early effects of myocardial ischaemia and reperfusion as well as the
ª 2013 Stichting European Society for Clinical Investigation Journal Foundation. Published by John Wiley & Sons Ltd
ANGLE-BASED INFARCT SIZE MEASUREMENT
pathophysiology of chronic heart failure have been studied extensively [11,12] and reduction of total infarct size served as a main indicator for the success in these studies. Infarct size in animal models of MI is in most cases calculated by planimetry using tissue specimens of explanted hearts. The most frequent method to determine total infarct size in histological sections is to compare the area of necrosis or scar tissue in relation to ventricular mass [13–15]. However, especially in animal models of chronic MI changes in ventricular geometry such as hypertrophy of viable myocardium and thinning of the fibrotic scar might affect these calculations and could bias the obtained data. Therefore, the measurement of the arc length of the fibrotic scar in relation to the total ventricular circumference has been suggested to reduce the influence of on-going changes in cardiac morphology [8,12,16]. In 2007, the group of Takagawa et al. [17] performed a profound comparison of both calculation methods for infarct size. They showed that the use of a length-based measurement approach is more appropriate to calculate infarct size in the setting of chronic MI. This was further substantiated by showing a better correlation of parameters of cardiac function with infarct size calculation using the length method. However, area measurement performed on tissue sections obtained several weeks after myocardial ischaemia is still used quite often in the experimental setting chronic MI [18–20]. Here in this study, we developed a new approach to calculate infarct size on histological specimens using an angle-based approach. We hypothesized that like the previously described length-based calculation, the measurement of infarct size using the angel of infarcted myocardium in relation to the full circumscribed circumference of the left ventricle could provide better results compared to the standard of area-based infarct size calculations. We further sought to compare the results obtained using our method of infarct angle measurement with the known gold standard (i.e. area and length measurement) both in a small and in a large animal model of acute and chronic MI.
Materials and methods Rat model of chronic myocardial infarction Animal experiments were approved by the committee for animal research, Medical University of Vienna (vote:BMBWK66.009/0278-BrGT/2005). Myocardial infarction was induced in adult male Sprague–Dawley rats (weight 300–350 g) by ligating the left anterior descending artery (LAD). Animals were anaesthetized intraperitoneally with a mixture of xylazine (1 mg/ 100 g bodyweight) and ketamine (10 mg/100 g bodyweight) and ventilated mechanically. A left lateral thoracotomy was performed in the fourth intercostal space and the pericardium was opened. The heart was then delivered to the surface of the
thoracic incision and a ligature using 6–0 prolene was placed around the LAD beneath the left atrium. After the onset of ischaemia, the thoracic incision was closed again. The perioperative mortality in the first 48 h ranged around 25%.
Assessment of cardiac function by echocardiography Six weeks after induction of MI, rats were anaesthetized with 100 mg/kg ketamine and 20 mg/kg xylazine. Transthoracic echocardiography was performed on a Vivid seven system (General Electric Medical Systems, Fairfield, CT, USA). Analyses were performed by an experienced observer in rodent imaging blinded to the treatment groups to which the animals were allocated. The chest was shaved, animals were placed on their left side and sufficient quantities of transducer gel were applied to the animals’ thorax. Parasternal long-axis and parasternal short axis view B-mode images were acquired. Mmode images were recorded for measurements of LV dimensions at end-systole and end-diastole. Average LV volumes were calculated from three representative measurements over multiple heart cycles.
Histology Animals were sacrificed 6 weeks after experimental infarction. Hearts were sliced at three layers at the level of the largest extension of infarcted area (n = 30). Slices were fixed in 10% neutral buffered formalin and embedded in paraffin. Tissue samples were stained according to an Elastica van Gieson (EVG) staining protocol. Specimens were evaluated on an Olympus AX70 microscope (Olympus Optical Co. Ltd., Shinjuku, Tokyo, Japan) and captured digitally. For further morphological analyses, specimens were digitalised at a definition of 9600 dpi on a high performance office scanner (Hewlett Packard, Palo Alto, CA, USA).
Infarct area measurements Planimetric evaluation after 6 weeks was carried out on tissue samples stained with EVG. IMAGE J software (Rasband, W.S., Image J, U.S. National Institutes of Health, Bethesda, MD, USA) was utilized to determine the area extent of myocardial infarction after 6 weeks. The extent of infarcted myocardial tissue (% of left ventricle) was calculated as follows: the infarct scar area and the total area of the LV tissue were traced manually in the digital images and measured automatically by the software. Infarct size, expressed as a percentage, was calculated by dividing the area of infarct areas by total LV area.
Infarct length measurements For the infarct length measurement, four length measurements were performed on histological specimens obtained 6 weeks after MI. These included epicardial and endocardial infarct lengths and total epicardial and endocardial circumferences.
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The epicardial infarct length ratio was obtained by dividing the epicardial infarct lengths by the total of epicardial circumference. Endocardial infarct ratio was calculated similarly. Infarct size derived from this approach was calculated as [(epicardial infarct ratio + endocardial infarct ratio)/2] 9 100.
Infarct angle calculations Digitalized images of cardiac specimens were imported into image processing software (Adobe Photoshop CS5, San Jose, CA, USA). The left ventricle was designated using the marquee tool and copied into a new image file. The image section of the left ventricle was adjusted to a 1 : 1 ratio in order to increase the accuracy of angle calculations. The newly aligned digital images were loaded into IMAGE J software (Rasband, W.S., Image J, U.S. National Institutes of Health) and the circumference of the left ventricle was traced manually using the freehand selection tool. The centroid of the left ventricle was calculated automatically (Set Measurement options). Starting from the centroid, angle calculations were performed in relation to endo-, myo- and epicardial border zones between vital myocardium and scar tissue. Infarct size (expressed as a percentage of the left ventricle) was calculated as follows: mean of all three angle measurements divided by 360° 9 100.
Porcine closed chest reperfused myocardial infarction model Large animal experiments utilizing a porcine closed chest reperfused MI model were carried out at the Institute of Diagnostics and Oncoradiology, University of Kaposvar, Hungary as previously described [21]. Animal experiments were approved by the University of Kaposvar (vote:246/002/ SOM2006,MAB-28-2005). Female Large White pigs used for these experiments weighing approximately 30 kg were sedated with 12 mg/kg ketamine hydrochloride, 1 mg/kg xylazine and 004 mg/kg atropine. After the administration of 200 IU/kg of heparin, a 6F guiding catheter was introduced into the left coronary ostium and angiography of the left coronary arteries was performed. A balloon catheter was inserted into the LAD after the origin of the second major diagonal branch. The LAD was then occluded by inflating the balloon slowly at 4–6 atm. After 90 min of occlusion, the balloon was deflated and reperfusion was established. After 24 h, echocardiography was performed as described above to obtain parameters of cardiac function. Hereafter, euthanasia was performed by the administration of saturated potassium chloride. Double-staining with 1% Evans blue dye and a 4% solution of 2,3,5-triphenyltetrazolium chloride (TTC) was performed to delineate areas at risk for ischaemia and infarcted (necrotic) areas. After explantation of the heart, the LAD was occluded again at same position where the balloon was situated before and both coronary arteries were perfused with Evans blue solution to delineate the
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area at risk and non-risk regions. The hearts were cut in into 7 mm thick slices starting from the apex towards the level of the occlusion (seven layers per heart). The slices were incubated in 500 mL of TTC solution at 37 °C in a shaking water bath for 20 min. All slices were photographed using a digital camera (Panasonic HDC-HS700; Kadoma, Osaka, Japan) mounted on a fixed stand. Infarct size measurements were performed using IMAGE J software (Rasband, W.S., Image J, U.S. National Institutes of Health).
Statistical methods All data are expressed as means plus standard deviation (SD). Statistical analysis was performed using GRAPHPAD Prism software (GraphPad Software, La Jolla, CA, USA). The Wilcoxon matched pairs test was performed to calculate significances between the three methods of measurement. P-values < 005 were considered statistically significant. Correlations between infarct size and LVEF, SF, LVEDD and LVESD were tested by linear regression analysis.
Results Calculations of infarction size using an angle-based approach in a small animal model Myocardial infarction was induced in 30 Sprague–Dawley rats by permanent ligation of the LAD. The angle of total infarcted myocardium was calculated as described in the materials in methods section. Figure 1 shows an overview of the calculation approach. In the first step, the histological specimen is digitalised using a commercially available office scanner (Fig. 1a). In the next step, the obtained picture needs to be adapted into a 1 : 1 ratio using digital image editing software. Then the centroid of the left ventricle is calculated (Fig. 1b). Based on the centroid, three angle measurements are performed (to the endo-, mid- and epicardial limits of the infarct scar tissue (Fig. 1c). Three tissue sections per animal were evaluated using this approach (total number of animals n = 30).
Calculations of infarct size using infarct area, length and angle In this small animal model of chronic MI, we performed an evaluation of parameters of cardiac function (LV ejection fraction, shortening fraction, left ventricular systolic and diastolic diameters) by means of echocardiography. Thereafter rats were euthanized, hearts were explanted and stained with EVG staining. Digital images of the obtained histological specimens were obtained and infarct size calculations were performed using the previously described planimetric area and length measurement approach, and also using an infarction size score based on the angle of the myocardial scar tissue compared to the full circumference of the left ventricle.
ª 2013 Stichting European Society for Clinical Investigation Journal Foundation. Published by John Wiley & Sons Ltd
ANGLE-BASED INFARCT SIZE MEASUREMENT
(a)
(b)
Infarct size (% of LV)
(d)
(c)
P
ORIGINAL ARTICLE Myocardial infarct size measurement using geometric angle calculation Michael Lichtenauer*,†, Catharina Schreiber‡, Christian Jung*, Lucian Beer†,§, Andreas Mangold¶, Mariann € ngyo € si¶, Bruno Karl Podesser** and Hendrik Jan Ankersmit†,§ Gyo * €tsherzzentrum Thu € ringen, Clinic of Internal Medicine I, Department of Cardiology, Friedrich Schiller University Universita Jena, Jena, Germany, †Christian Doppler Laboratory for Cardiac and Thoracic Diagnosis and Regeneration, Vienna, Austria, ‡ Department of Cardiac Surgery, University Clinic Salzburg, Salzburg, Austria, §Department of Thoracic Surgery, Medical University Vienna, Vienna, Austria, ¶Department of Cardiology, Medical University Vienna, Vienna, Austria, **Ludwig Boltzmann Cluster for Cardiovascular Research, Medical University Vienna, Vienna, Austria
ABSTRACT Background In basic cardiovascular research focusing on animal models of myocardial infarction (MI), the measurement of infarct size is performed by planimetry of histological sections of the heart. However, in the setting of chronic MI with ongoing changes in ventricular geometry caused by wall thinning and hypertrophy, the scar area tends to become smaller. Materials and Methods Here, in this study we compared infarct measurements in tissue sections (of rat and porcine hearts) based on three different calculation approaches, that is, infarct area, infarct lengths and infarct angles utilizing the centroid of the left ventricle using a newly developed calculation approach. Results Infarct sizes from all three measurement approaches showed significant correlation with parameters of cardiac function. However, results derived from area measurements were significantly smaller than those obtained using the other two measurement approaches due to scar thinning (infarct size area: 1481% 127 SEM, length: 2394% 204 SEM, angle: 2475% 213 SEM, P < 00001, n = 30). Moreover, results from angle measurements evidenced a much better correlation with parameters of cardiac function in a small animal model of chronic MI (e.g. ejection fraction, angle: r = 073; length: r = 064; area: r = 059, n = 30) as well as in a large animal model of acute MI (angle: r = 082; area: r = 067, n = 10). Conclusions We concluded that area-, length- and angle-based measurements can be used to determine the relative infarct size in acute MI models, although an area-based measurement might be less accurate in the setting of chronic MI. Our new method of infarct angle measurement is a reliable and simple way to calculate infarct size compared with conventional measurement approaches. Keywords Animal models, infarct size, measurement, myocardial infarction. Eur J Clin Invest 2014; 44 (2): 160–167
Introduction Heart failure accompanied with left ventricular (LV) remodelling and the loss of cardiac contractility is the leading cause of mortality and morbidity in the western world [1]. The major cause for the development of end stage heart failure in most cases is myocardial ischaemia [2]. Even though interventional strategies of early reperfusion have reduced the mortality after acute myocardial infarction, ischaemic heart disease still remains a major issue for patients and health care systems worldwide [3]. Also in basic science an extremely broad spectrum of possible therapeutic strategies to protect ischaemic myocardium or reduce the damage caused by ischaemia has been studied over the last decades [4–6].
160
Both in basic science studies and clinical trials the reduction of infarct size is one the paramount outcome parameters to define the success or failure of a potential treatment strategy. Total infarct size is the most critical parameter for the further prognosis of patients suffering from acute myocardial infarction (MI) because prognosis is inversely correlated to the total tissue mass of left ventricle myocardium that has been lost [7–9]. Previous studies showed that a direct relationship between the extent of infarct size and mortality exists and that the initial infarct size is a major predictor of long-term LV function [10]. In the field of basic cardiovascular research, scientist used animal models of MI to develop new strategies to salvage ischaemic myocardium. In these models, both early effects of myocardial ischaemia and reperfusion as well as the
ª 2013 Stichting European Society for Clinical Investigation Journal Foundation. Published by John Wiley & Sons Ltd
ANGLE-BASED INFARCT SIZE MEASUREMENT
pathophysiology of chronic heart failure have been studied extensively [11,12] and reduction of total infarct size served as a main indicator for the success in these studies. Infarct size in animal models of MI is in most cases calculated by planimetry using tissue specimens of explanted hearts. The most frequent method to determine total infarct size in histological sections is to compare the area of necrosis or scar tissue in relation to ventricular mass [13–15]. However, especially in animal models of chronic MI changes in ventricular geometry such as hypertrophy of viable myocardium and thinning of the fibrotic scar might affect these calculations and could bias the obtained data. Therefore, the measurement of the arc length of the fibrotic scar in relation to the total ventricular circumference has been suggested to reduce the influence of on-going changes in cardiac morphology [8,12,16]. In 2007, the group of Takagawa et al. [17] performed a profound comparison of both calculation methods for infarct size. They showed that the use of a length-based measurement approach is more appropriate to calculate infarct size in the setting of chronic MI. This was further substantiated by showing a better correlation of parameters of cardiac function with infarct size calculation using the length method. However, area measurement performed on tissue sections obtained several weeks after myocardial ischaemia is still used quite often in the experimental setting chronic MI [18–20]. Here in this study, we developed a new approach to calculate infarct size on histological specimens using an angle-based approach. We hypothesized that like the previously described length-based calculation, the measurement of infarct size using the angel of infarcted myocardium in relation to the full circumscribed circumference of the left ventricle could provide better results compared to the standard of area-based infarct size calculations. We further sought to compare the results obtained using our method of infarct angle measurement with the known gold standard (i.e. area and length measurement) both in a small and in a large animal model of acute and chronic MI.
Materials and methods Rat model of chronic myocardial infarction Animal experiments were approved by the committee for animal research, Medical University of Vienna (vote:BMBWK66.009/0278-BrGT/2005). Myocardial infarction was induced in adult male Sprague–Dawley rats (weight 300–350 g) by ligating the left anterior descending artery (LAD). Animals were anaesthetized intraperitoneally with a mixture of xylazine (1 mg/ 100 g bodyweight) and ketamine (10 mg/100 g bodyweight) and ventilated mechanically. A left lateral thoracotomy was performed in the fourth intercostal space and the pericardium was opened. The heart was then delivered to the surface of the
thoracic incision and a ligature using 6–0 prolene was placed around the LAD beneath the left atrium. After the onset of ischaemia, the thoracic incision was closed again. The perioperative mortality in the first 48 h ranged around 25%.
Assessment of cardiac function by echocardiography Six weeks after induction of MI, rats were anaesthetized with 100 mg/kg ketamine and 20 mg/kg xylazine. Transthoracic echocardiography was performed on a Vivid seven system (General Electric Medical Systems, Fairfield, CT, USA). Analyses were performed by an experienced observer in rodent imaging blinded to the treatment groups to which the animals were allocated. The chest was shaved, animals were placed on their left side and sufficient quantities of transducer gel were applied to the animals’ thorax. Parasternal long-axis and parasternal short axis view B-mode images were acquired. Mmode images were recorded for measurements of LV dimensions at end-systole and end-diastole. Average LV volumes were calculated from three representative measurements over multiple heart cycles.
Histology Animals were sacrificed 6 weeks after experimental infarction. Hearts were sliced at three layers at the level of the largest extension of infarcted area (n = 30). Slices were fixed in 10% neutral buffered formalin and embedded in paraffin. Tissue samples were stained according to an Elastica van Gieson (EVG) staining protocol. Specimens were evaluated on an Olympus AX70 microscope (Olympus Optical Co. Ltd., Shinjuku, Tokyo, Japan) and captured digitally. For further morphological analyses, specimens were digitalised at a definition of 9600 dpi on a high performance office scanner (Hewlett Packard, Palo Alto, CA, USA).
Infarct area measurements Planimetric evaluation after 6 weeks was carried out on tissue samples stained with EVG. IMAGE J software (Rasband, W.S., Image J, U.S. National Institutes of Health, Bethesda, MD, USA) was utilized to determine the area extent of myocardial infarction after 6 weeks. The extent of infarcted myocardial tissue (% of left ventricle) was calculated as follows: the infarct scar area and the total area of the LV tissue were traced manually in the digital images and measured automatically by the software. Infarct size, expressed as a percentage, was calculated by dividing the area of infarct areas by total LV area.
Infarct length measurements For the infarct length measurement, four length measurements were performed on histological specimens obtained 6 weeks after MI. These included epicardial and endocardial infarct lengths and total epicardial and endocardial circumferences.
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The epicardial infarct length ratio was obtained by dividing the epicardial infarct lengths by the total of epicardial circumference. Endocardial infarct ratio was calculated similarly. Infarct size derived from this approach was calculated as [(epicardial infarct ratio + endocardial infarct ratio)/2] 9 100.
Infarct angle calculations Digitalized images of cardiac specimens were imported into image processing software (Adobe Photoshop CS5, San Jose, CA, USA). The left ventricle was designated using the marquee tool and copied into a new image file. The image section of the left ventricle was adjusted to a 1 : 1 ratio in order to increase the accuracy of angle calculations. The newly aligned digital images were loaded into IMAGE J software (Rasband, W.S., Image J, U.S. National Institutes of Health) and the circumference of the left ventricle was traced manually using the freehand selection tool. The centroid of the left ventricle was calculated automatically (Set Measurement options). Starting from the centroid, angle calculations were performed in relation to endo-, myo- and epicardial border zones between vital myocardium and scar tissue. Infarct size (expressed as a percentage of the left ventricle) was calculated as follows: mean of all three angle measurements divided by 360° 9 100.
Porcine closed chest reperfused myocardial infarction model Large animal experiments utilizing a porcine closed chest reperfused MI model were carried out at the Institute of Diagnostics and Oncoradiology, University of Kaposvar, Hungary as previously described [21]. Animal experiments were approved by the University of Kaposvar (vote:246/002/ SOM2006,MAB-28-2005). Female Large White pigs used for these experiments weighing approximately 30 kg were sedated with 12 mg/kg ketamine hydrochloride, 1 mg/kg xylazine and 004 mg/kg atropine. After the administration of 200 IU/kg of heparin, a 6F guiding catheter was introduced into the left coronary ostium and angiography of the left coronary arteries was performed. A balloon catheter was inserted into the LAD after the origin of the second major diagonal branch. The LAD was then occluded by inflating the balloon slowly at 4–6 atm. After 90 min of occlusion, the balloon was deflated and reperfusion was established. After 24 h, echocardiography was performed as described above to obtain parameters of cardiac function. Hereafter, euthanasia was performed by the administration of saturated potassium chloride. Double-staining with 1% Evans blue dye and a 4% solution of 2,3,5-triphenyltetrazolium chloride (TTC) was performed to delineate areas at risk for ischaemia and infarcted (necrotic) areas. After explantation of the heart, the LAD was occluded again at same position where the balloon was situated before and both coronary arteries were perfused with Evans blue solution to delineate the
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area at risk and non-risk regions. The hearts were cut in into 7 mm thick slices starting from the apex towards the level of the occlusion (seven layers per heart). The slices were incubated in 500 mL of TTC solution at 37 °C in a shaking water bath for 20 min. All slices were photographed using a digital camera (Panasonic HDC-HS700; Kadoma, Osaka, Japan) mounted on a fixed stand. Infarct size measurements were performed using IMAGE J software (Rasband, W.S., Image J, U.S. National Institutes of Health).
Statistical methods All data are expressed as means plus standard deviation (SD). Statistical analysis was performed using GRAPHPAD Prism software (GraphPad Software, La Jolla, CA, USA). The Wilcoxon matched pairs test was performed to calculate significances between the three methods of measurement. P-values < 005 were considered statistically significant. Correlations between infarct size and LVEF, SF, LVEDD and LVESD were tested by linear regression analysis.
Results Calculations of infarction size using an angle-based approach in a small animal model Myocardial infarction was induced in 30 Sprague–Dawley rats by permanent ligation of the LAD. The angle of total infarcted myocardium was calculated as described in the materials in methods section. Figure 1 shows an overview of the calculation approach. In the first step, the histological specimen is digitalised using a commercially available office scanner (Fig. 1a). In the next step, the obtained picture needs to be adapted into a 1 : 1 ratio using digital image editing software. Then the centroid of the left ventricle is calculated (Fig. 1b). Based on the centroid, three angle measurements are performed (to the endo-, mid- and epicardial limits of the infarct scar tissue (Fig. 1c). Three tissue sections per animal were evaluated using this approach (total number of animals n = 30).
Calculations of infarct size using infarct area, length and angle In this small animal model of chronic MI, we performed an evaluation of parameters of cardiac function (LV ejection fraction, shortening fraction, left ventricular systolic and diastolic diameters) by means of echocardiography. Thereafter rats were euthanized, hearts were explanted and stained with EVG staining. Digital images of the obtained histological specimens were obtained and infarct size calculations were performed using the previously described planimetric area and length measurement approach, and also using an infarction size score based on the angle of the myocardial scar tissue compared to the full circumference of the left ventricle.
ª 2013 Stichting European Society for Clinical Investigation Journal Foundation. Published by John Wiley & Sons Ltd
ANGLE-BASED INFARCT SIZE MEASUREMENT
(a)
(b)
Infarct size (% of LV)
(d)
(c)
P
