The post-mortem assessment of myocardial infarction (MI) in rodents is based on quantification of the infarct on stained heart sections. We describe an accurate method to quantify the infarct size using systematic sampling of harvested rat hearts from base to apex and image analyses of trichrome-stained histological sections.
Myocardial infarction is defined as cardiomyocyte death due to prolonged ischemia; an inflammatory response and scar formation (fibrosis) follow the ischemic injury. Following the initial acute phase, chronic remodeling of the left ventricle (LV) modifies the structure and function of the heart. Permanent coronary ligation in small animals has been widely used as a reference model for a chronic model of MI. Thinning of the infarcted wall progressively develops to transmural fibrosis. Histological assessment of infarct size is commonly performed; nevertheless, a standardization of the methods for quantification is missing. Indeed, important methodological aspects, such as the number of sections analyzed and the sampling and quantification methods, are usually not described and therefore preclude comparison across investigations. Too often, quantification is performed on a single section obtained at the level of the papillary muscles. Because novel strategies aimed at reducing infarct expansion and remodeling are under investigation, there is an important need for the standardization of accurate heart sampling protocols. We describe an accurate method to quantify the infarct size using a systematic sampling of harvested rat heart and image analyses of trichromatic stained histological sections obtained from base to apex. We also provide evidence that calculating the expansion index (EI) allowed for infarct size assessment, taking into account changes of the left ventricle throughout the remodeling.
Myocardial infarction (MI) is a leading cause of death and disability worldwide. Coronary heart disease is the main cause; MI results from ischemia consecutive to coronary events such as occlusion. When reperfusion is not performed within the first 6 hr, ischemia induces irreversible myocardial necrosis. In patients, the characterization of MI relies on different diagnostic tools, including clinical signs, electrocardiography, assessment of plasma levels of biomarkers, echocardiography, MRI imaging, and histological analyses1. Acute and chronic MI are classified as two different phases of injury according to the timing of the myocardial necrosis relative to the time of the coronary occlusion. The acute phase, occurring during the first 7 days, is associated with the loss of cardiomyocytes, extensive inflammation, and the recruitment of fibroblasts. The sub-acute phase, characterized by healing of the cardiac tissue and the formation of a scar, occurs between 1 and 4 – 6 weeks. Expansion of the infarct, ventricle wall thinning, and ventricle dilatation characterize the chronic phase. Extensive remodeling of the left ventricle progressively results in severe heart failure2.
MI induced by permanent left anterior descending artery (LAD) ligation represents the standard rodent model of chronic myocardial infarction. The coronary ligature mimics the coronary occlusion. The size of the infarct depends on the site of the ligature. Characterization of myocardial ischemic injury in a rodent model is classically performed using biomarker plasma levels, such as troponin I and T3, echocardiography, MRI, and histology4,5. Biomarker levels are correlated with the extent of cardiomyocyte death. Echocardiography evaluates the left ventricular function impairment resulting from regional wall motion abnormalities. In addition, non-invasive imaging techniques, such as MRI or high-resolution echocardiography, allow the assessment of the reduction in wall motion, the volume of the scar area with reduced perfusion and viable myocardium, and the wall thinning. LV dimensions permit the accurate evaluation of infarct size. Finally, the quantification of viable and dead myocardium can be performed postmortem using specific stains of histological sections of harvested hearts and allows verification of the infarct size (IS). Another important feature is the evaluation of the infarct expansion index (EI)6. The EI is associated with the transmural infarct and starts within the first 3 days. The EI is characterized by a progressive reduction in wall thickness, an increase in the LV cavity size, and consequent changes in LV shape.
In order to evaluate the therapeutic efficacy of novel treatments–in particular, the regenerative strategies based on cells, matrices, and gene delivery-accurate assessment of MI in rodents is of paramount importance. When measured on a single cross section obtained at the papillary muscle level, the IS size may be biased due to the large variability that exists in infarct development following LAD ligation; the apex infarct might be then occulted. Importantly, more accurate methodologies to determined MI size have been described for mice7-9 or rats10. Nevertheless, IS is insufficient to accurately quantify LV remodeling or therapeutically induced reductions (or preventions) of the remodeling. Indeed, IS is commonly expressed as a percentage of total LV volume assessed on cross sections of the heart. Although this method is valid for acute MI, the thinning of the LV wall occurring during remodeling remains under-evaluated11,12. A complete morphometric quantification of infarct size and structural changes should quantify several parameters, such as endocardial and epicardial lengths and diameters, as well as infarct and healthy areas. We describe a methodological approach to accurately assess MI and remodeling in a chronic rat model.
All animals received humane care in compliance with the European Convention on Animal Care. Surgical procedures were performed in accordance with the Swiss Animal Protection Law after obtaining permission of the State Veterinary Office, Fribourg, approved by the Swiss Federal Veterinary Office, Switzerland.
1. Heart Harvesting
NOTE: All surgical interventions were performed under isoflurane anesthesia. Efforts were made to diminish animal suffering. In particular, all animals received a subcutaneous injection of 0.1 mg/kg buprenorphine pre-anesthesia. The surgical protocol for inducting myocardial infarction has been previously described elsewhere13.
2. Tissue Preparation
3. Masson-Goldner Trichrome Staining
4. Infarct Size Analysis
Six weeks post-LAD ligation, hearts were harvested from Lewis rats. 2-mm tissue sections were obtained from apex to base. A TTC staining procedure was performed to visualize the infarct area, which appears in white, and the healthy myocardium, which appears in red (Figure 2). Depending on the site of ligation of the LAD, the infarct size varies. For large MI, transmural infarcts were observed from apex to base (Figure 2A). Smaller infarcts presented white infarcted tissue visible from the apex to the mid-section of the heart (Figure 2B). For small non-transmural infarcts, fibrotic tissue was observed on only one or two sections from the apex (Figure 2C).
Thin 5-µm sections from each heart slice were cut and stained with Masson-Goldner trichrome. Pictures of the whole cross section of the heart were acquired under a stereomicroscope (Figure 3). Healthy tissues appeared in red and the connective tissue in green. Discrimination between each color can be easily performed with an image analysis software.
The thicknesses of the infarct and the septum, the LV cavity area and the LV total area were measured and computed to calculate the EI (Table 1). The EI was calculated for each section. Depending on the heart size, the number of sections varied from 5 to 7. For each heart, the EI is an average of the EI from each section. The EI varied from 0 to 0.278 and revealed a wide range of myocardial infarcts, with a CV of 58%. In comparison, the average of the percentage of infarct to the LV varied from 0 to 0.241, with a CV of 54% (Table 1). The EI and the percentage of infarct were significantly correlated; a non-parametric Spearman analysis provided a correlation coefficient r-value of 0.491 (p = 0.005) (Figure 4A). For close EI values, such as 0.11 and 0.13, the percentage of infarct varied from 5.8 to 24.1%.
Heart function was assessed by high-resolution echocardiography at 6 weeks post-LAD ligation on anesthetized animals and was performed once prior to heart harvesting. The EI calculated from histological quantification correlated significantly with the EF. A non-parametric Spearman analysis provided an r-value of -0.709 (p = 0.005) (Figure 4B).
Figure 1. Step-by-step Illustration of the Use of the Image Analysis Software. Automatic color delimitation and length measurements included the several steps. A. Calibration: The scale of the image was set up. A picture of a ruler was taken in the same condition as the heart section. The length of the ruler was marked to define the scale conversion factor. Conversion from pixel to millimeters was performed. B. LV selection: The right ventricle was cut out of the picture using the selection tool of the software. C. LV wall and septum thickness measurement: The single measurement tool was used to quantify distances. The scale bars indicate 3 mm. D. Automatic area quantification: Automatic color delimitation was performed in RGB mode. Auto-selection was performed after selecting the auto-selection mode. Color-based selection can be modified; the investigator performed visual control of the selected green tissue and increased or decreased the selected region as necessary. The scale bars indicate 3 mm. Please click here to view a larger version of this figure.
Figure 2. Heart Sections Stained with TTC. 2-mm sections of the full heart were stained with TTC. Healthy myocardium appeared in red and fibrotic tissues in white. Three hearts with different infarct sizes are presented. A: large transmural infarct, B: medium infarct, and C: small infarct. The scale bars indicate 3 mm. Please click here to view a larger version of this figure.
Figure 3. Heart Sections Stained with Masson-Goldner Trichrome. 5-µm sections were cut from each heart section obtained 6 weeks post-LAD ligation. Stained sections were placed under a stereomicroscope. Pictures of the full sections were obtained with 15X magnification. The representative picture, obtained at the level of the papillary muscles (back arrow), shows healthy myocardium in red and fibrotic tissues in green. The picture illustrates the automatic color delimitation with the image analysis software, as well as the site at which the infarct and septum thickness were measured (black lines). The scale bars indicate 3 mm. Please click here to view a larger version of this figure.
Figure 4. Correlation Between the Expansion Index (EI) and the Percentage of the Infarct Related to LV (A) or the Ejection Fraction (EF) (B). The linear regression (black line) and 95% confidence intervals (dotted lines) are represented. The non-parametric Spearman r-value is reported. A: The percentage of infarct and EI significantly correlated (r = 0.567; p = 0.003). The infarct expansion index (EI) was calculated as [LV cavity area/whole LV area]/[infarct thickness/septum thickness]. The whole LV area was measured as the combined LV cavity and LV tissue area. The percentage of the infarct was calculated as LV tissue area/infarct tissue area. B: The function assessed at 6 weeks post-LAD ligation using a high-resolution echocardiography significantly correlated with EI. Both parameters were significantly correlated (r = -0.709; p = 0.005). Please click here to view a larger version of this figure.
Table 1: Parameters Measured on Masson Goldner-stained Heart Sections.
Critical Steps within the Protocol
Fibrotic tissue can be accurately assessed in a chronic MI rat model using systematic sampling of the harvested heart and image analyses of trichromatic-stained histological sections obtained from base to apex. Two steps are particularly important for successful protocol implementation. First, the use of KCl for heart harvesting allows the cardiac muscle to be maintained in a relaxed state. This step is important for comparisons of infarct dimensions from different hearts. Second, the overnight fixation of the section in Bouin solution is critical to obtain bright trichrome staining.
Modifications and Troubleshooting
Absence of staining may be due to difficulties during the fixation of the section in Bouin solution, such as reduced incubation time or expired Bouin solutions. The TTC staining is optional and could be used to visualize the infarct size in a rapid but non-quantitative way. It is important to note that TTC can be performed for the same heart prior to the inclusion in either OCT or paraffin.
Limitations of the Technique
The present approach permits one to choose between paraffin-embedded and cryo-preserved heart sections and can be used for immunostaining. Nevertheless, the full heart has to be sectioned, and the tissue cannot be used for further analysis, such as western blot or RT-PCR for protein and gene expression analyses.
Significance of the Technique with Respect to Existing/Alternative Methods
Because the quantification procedures and, in particular, the number of analyzed sections vary widely between investigations, defining the minimal number of sections necessary to obtain a reliable quantification is paramount. Takagawa et al.9 demonstrated that reliability is maximized with a minimal heart slice number of 6 – 8 sections of the whole heart of a mouse using 1-mm intervals. In the present protocol, 5 – 7 sections of the entire rat heart were obtained when performing 2-mm thick sectioning. Furthermore, 2-mm sections are a standard and frequently used size for TTC staining. In addition, the systemic sampling is simple to apply and presents a periodic aspect that allows characterization of the entire heart.
From each of the 2-mm slices, 5-µm sections were cut and stained. For each 5-µm section, quantification of the septum thickness, scar thickness and length, total scar area, total LV area, and LV cavity area were performed, and the average of each parameter was calculated per animal. We used Masson-Goldner staining of thin sections rather than TTC staining of 2-mm sections to improve the accuracy of both the automatic color detection and the assessment of lengths. Indeed, TTC staining is mostly interesting for early-phase or ischemic reperfusion models for the detection of the area at risk8,14, rather than for the chronic stage.
MI induced from LAD ligation may vary depending on the ligature site. In the presented chronic infarction model, the ligation size was intentionally modified for each animal, and results for various conditions of infarct size and remodeling were present after 6 weeks. Accordingly, the calculated EI revealed a wide range of myocardial infarcts and supported the difference observed in heart function correlated with EF. When extensive thinning of the infarcted segment occurred, the area of the infarct was greatly reduced in the case of transmural fibrosis. In this condition, defining the infarct size by calculating the area of the infarct tissue in relation to the entire LV (expressed as a percentage of the LV) would weakly evaluate the extent of the infarct. The lack of consideration for remodeling-induced LV dilation and extreme thinning of the infarct LV wall may underestimate the infarct size, while an infarct present in the absence of wall thinning would over-estimate the infarct size, as represented by the values outside of the 95% confidence interval. This shortcoming could be eliminated through the calculation of the EI.
It has been shown that the LV shape change is positively correlated with the EI and wall thinning15, 16. Although EI can be a predictor of LV function, it is important to emphasize that echocardiography and histological analyses are complementary methods to assess the myocardial infarct at the functional and tissue level, respectively. Echocardiography allows longitudinal study, and histological analyses provide fundamental end-point assays that allow additional quantification of LV morphology, such as wall thickness.
Future Applications or Directions after Mastering This Technique
The systemic sampling of the entire heart and the calculation of the EI are of particular interest when assessing chronic MI. In addition, this method will be suitable for the evaluation of treatment efficacy, in particular for novel treatments, such as cell- and matrix-based treatments that aim at reducing infarct size and remodeling. Reliable quantification of infarct expansion is of paramount importance for comparisons between non-treated and treated animals, as end-point histological analyses preclude the comparison of infarct size pre- and post-treatment.
The authors have nothing to disclose.
The study was supported by the Swiss National Foundation [SNF 310030-149986 to MNG], the University of Fribourg, and Fribourg Hospital.
Acrylic rat heart matrix 2mm | 72-5015 | Harvard Appartus | |
INSPIRA ADVANCED VOLUME CONTROLLED VENTILATOR | HARVARD APPARATUS | 557058 | |
CATHETER INSYTE 14G | BD | 381267 | |
O.C.T | BDHA361603E | VWR | |
TTC | T8877-10G | Sigma Aldrich | |
Mayer hematoxylin | MHS32-1L | Sigma Aldrich | |
Acid Fuchsin CI 42685 |
F8129-50G | Sigma Aldrich | |
Ponceau Xylidin CI 16150 |
P2395-25G | Sigma Aldrich | |
Orange G CI 16230 |
O3756-100G | Sigma Aldrich | |
Light green CI 42095 |
L5382-25G | Sigma Aldrich | |
KCl | P9333-500G | Sigma Aldrich | |
Xylol | 10315083 | HoneyWell | |
Ethanol absolute | 10303990 | HoneyWell | |
2-methylbutane | M32631-1L | Sigma Aldrich | |
Stereogical microscope | SM2800 | Nikon | |
Formaldehyde | 99340 | Reactolab | |
Embedding cassette | K113.1 | Carl Roth | |
Bersoft Image measurement Software | Bersoft.com | Licensed software |