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. Author manuscript; available in PMC: 2009 Apr 21.
Published in final edited form as: Mol Imaging. 2007 Jul–Aug;6(4):261–268.

CARDIAC MICRO-CT FOR MORPHOLOGICAL AND FUNCTIONAL PHENOTYPING OF MLP NULL MICE

Cristian T Badea 1, Laurence W Hedlund 1, Julie F Boslego Mackel 1, Lan Mao 2, Howard A Rockman 2, G Allan Johnson 1
PMCID: PMC2671027  NIHMSID: NIHMS98266  PMID: 17711781

Abstract

PURPOSE

Investigate the use of micro-CT for morphological and functional phenotyping of MLP null mice and compare micro-CT with M-mode echocardiography.

MATERIAL AND METHODS

MLP null mice and controls were imaged using both micro-CT and M-mode echocardiography. For Micro-CT imaging, we used a custom built scanner. Following a single intravenous injection of a blood pool contrast agent (Fenestra™ VC) and using a cardio-respiratory gating, we acquired eight phases of the cardiac cycle (every 15 ms) and reconstructed 3D datasets with 94 micron isotropic resolution. Wall thickness and volumetric measurements of left ventricle were performed and cardiac function was estimated.

RESULTS

Micro-CT and M mode echocardiography showed both morphological and functional aspects that separate MLP null mice from controls. End Diastolic and Systolic Volumes were increased significantly 3 and 5 fold respectively in the MLP null versus controls. Ejection Fraction was reduced by an average of 32% in MLP null mice. The data analysis shows that two imaging modalities provided different results partly due to the difference in anesthesia regimes. Other sources of errors for micro-CT are also analyzed.

CONCLUSION

Micro-CT can provide the 4D data (3D isotropic volumes over time) required for morphological and functional phenotyping in mice.

Keywords: micro-CT, phenotype, cardiac imaging, mouse, MLP

Introduction

Genetically modified mice are important for the study of cardiac disease and evaluation of potential treatment strategies. Imaging cardiac structure and function in mice is challenging due to the small size of their heart (left ventricle (LV) long axis is about 7 mm) and their rapid cardiac pace (up to 600 beats per minute). Thus, both high spatial and temporal resolutions are necessary for murine cardiac imaging. The two most popular imaging modalities for in vivo cardiac studies in mice are magnetic resonance (MR) microscopy and echocardiography. However, these modalities are not able to provide 3D images with isotropic resolution. Current MR microscopy based studies of myocardial function and morphology, involve acquisition of up to 8 images of 1 mm thick axial slices through the heart, with in-plane resolution between 50 and 120 microns[1–4], while 3D-echocardiographic techniques are capable of acquiring up to 18 gated short axis views of mouse heart, spaced 500 microns apart [3].

In vivo cardiac imaging with isotropic 3D resolution however is possible with micro-CT. Our recently implemented micro-CT system [5] allows in vivo characterization of cardiac structure and function in mice, based on true 4D datasets with an isotropic spatial resolution of 94 microns and a temporal resolution of 10 ms[6]. We have shown that cardiac micro-CT with a combination of high flux x-ray tube, precise physiologic gating, and the use of blood pool contrast agents results in images with voxels of ~ 1 × 10−3 mm3, roughly 10–50 times higher spatial resolution than reported for MR Microscopy[7, 8].

To determine if micro-CT imaging can accurately characterize the morphological and functional features of failing cardiac murine hearts, we applied this imaging method to MLP null mice. The muscle LIM protein (MLP) is an essential regulator of cardiac muscle development. Mice lacking MLP develop dilated cardiomyopathy with myocardial hypertrophy and heart failure starting shortly after birth[9, 10]. MLP null mice reproduce the morphological, functional biochemical, and gene expression changes seen in patients with the MLP gene mutations and dilated cardiomyopathy [11–16] and have been used to identify therapeutic targets in dilated cardiomyopathy[17]. Imaging studies using Echocardiography [11] and MR Microscopy [18] have shown that in MLP null mice, LV chamber are enlarged, the myocardium walls are thinned, and LV function is reduced.

Thus, the purpose of the present study was: i) to investigate the use of micro-CT in functional and morphological phenotype of failing murine hearts and ii) to compare the micro-CT measurements with M-mode echocardiography.

Materials and Methods

Experimental Setup

Animal studies followed protocols approved by the Duke University Institutional Animal Care and Use Committee. MLP null mice (n=7) and controls C57BL/6 mice (n=7) were imaged with echocardiography and micro-CT on the same day. The M-mode echocardiographic imaging was described in detail in[17]. We used an HDI 5000 echocardiograph (ATL, Bothell, WA) with a 9 MHZ transducer. Animals were anesthetized with isoflurane (1.5 to 2%) delivered by a nose cone. Prior to micro-CT imaging the animals were anesthetized with a intraperitoneal (IP) injection of Ketamine (115mg/kg) and Diazepam (27 mg/kg) and then perorally intubated for mechanical ventilation using an in- house developed ventilator [19] at a rate of 90 breaths/min with a tidal volume of 0.4 ml. A solid-state pressure transducer on the breathing valve measured airway pressure and electrodes (Blue Sensor, Medicotest, UK) taped to the animal footpads acquired ECG signal. Both signals were processed with Coulbourn modules (Coulbourn Instruments, Allentown, PA) and displayed on a monitor using a custom-written LabVIEW application (National Instruments, Austin, TX). Body temperature was recorded using a rectal thermistor and maintained at 36.5°C by an infrared lamp and feedback controller (Digi-Sense®, Cole Parmer, Chicago, IL). A catheter was inserted into the tail vein and used for the injection of the contrast agent Fenestra™ VC (ART Advanced Research Technologies, Saint-Laurent, Quebec, Canada) at a dose of 0.014 ml/g body weight. Fenestra VC is a newly developed blood pool agent consisting of iodinated triglycerides formulated in a stable, sub-micron oil-in-water lipid emulsion [20, 21] containing 50 mg/ml iodine. Animals were placed on a Plexiglas cradle and scanned in a vertical position. During imaging the anesthesia was maintained with Ketamine (0.04 ml) delivered IP about every 30 mins.

Micro-CT Imaging System

The micro-CT system, described in detail elsewhere[5], acquires projected radiographs of the animal as it is rotated in vertical position in front of a high-resolution x-ray detector (X-ray ImageStar, Photonics Science, East Sussex, UK). The system has a large focal spot (1 mm) x-ray tube capable of producing high fluence rates with exposure times less than 10 ms. Motion is minimized for any single projection by synchronizing exposures to both cardiac and breathing cycles (see [5] for details). The x-ray settings for the present studies were 80 kVp, 170 mA, and 9 ms per exposure. The scanning time was approximately 7 min/dataset and on average, 8 points in the cardiac cycle (15 ms temporal resolution) were acquired. Each dataset contained 170 projections acquired over a 187° arc. The projection images were next used to reconstruct tomograms with a Feldkamp algorithm and Parker weighting [22] using the Cobra EXXIM software package (EXXIM Computing Corp, Livermore, CA). The reconstructed datasets are 3D image arrays (512 × 512 × 512) from which one can display 2D slices in any orientation. Dosimetric measurements were performed using a Wireless Dosimetry System Mobile MOSFET TN-RD-16, SN 63 B (Thomson/Nielsen, Ottawa, ON, Ca). Five MOSFET dosimeter silicon chips (1mm2 active area 0.2mm × 0.2mm) were positioned at the surface and the center of a acrylic rodent-like phantom.

Data Analysis

The resulting 4D micro-CT images (3D volume images at multiple time points in the R-R cycle) were used for wall thickness and volumetric measurements throughout the cardiac cycle. Estimation of the LV diameter dimensions in diastole and systole (LVDD and LVSD) and the wall thickness measurements i.e. inter-ventricular septum (IVSW) and posterior wall (PW) were performed with ImageJ [23] in a mid-ventricular slice.

The LV volumetric measurements were performed using the PSC Volume Browser (PSC-VB) developed by Pittsburgh Super Computing Center (http://www.psc.edu/biomed/research/VB/). An illustration of the LV volumetric estimation process is shown by fig. 1. In this approach, the user manually specifies a loosely defined 3D region of interest (ROI). A few axial CT images (typically 6) like that shown in fig. 1 (A), are used to draw a Region of Interest (ROI) similar to a basket that includes both the myocardium and the lumen (see fig. 1 (B)). It is important that the outer basket perimeter is within the muscle except at the top aortic valve plane. Thus, the basket contains only a two classes of voxels corresponding to blood and muscle. The voxels in the basket are next processed using a Gaussian mixture modeling approach [24] to estimate the means and variances of the two classes, blood and myocardial tissue (the two peaks in the histogram shown by fig.1C), and consequently the fraction of each of these components relative to the geometrically defined ROI volume. In addition to the direct measurement of LV blood volume the process also produces a threshold value that best reproduces the measured LV volume according to a simple threshold segmentation of the ROI voxels. This segmentation serves as a visual feedback that approximates the direct volume measurement (fig.1 D). Diastole and systole were selected as the heart phases that provided the maximum and minimum LV volume. Stroke volume (SV) and ejection fraction (EF) were calculated with the end-diastolic (EDV) and end systolic (ESV) volumes (SV= EDV−ESV; EF=SV/EDV). For cardiac output (CO), SV was multiplied by heart rate (HR). Cardiac Index was computed as the ratio between the cardiac output and the body weight.

Fig 1.

Fig 1

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The LV volumetric estimation process using PSC-VB: The axial CT images (A) are used to set a basket that includes both the myocardium and the lumen (B). An optimal threshold is computed for the bimodal histogram (C) between the two peaks of the plot (green line, see text for details) and the LV Volume of the blood (D) is visualized and estimated.

To validate the absence of distortions in the micro-CT imaging and the accuracy of the volumetric measurements performed with PSC-VB software, we imaged a phantom containing a calibration grid and two vials with 0.4ml and 0.2 ml solutions of Isovue 370 (Bracco Diagnostic) with 50 mg Iodine/ml concentration.

Statistical analysis was performed using two unpaired t-tests on the micro-CT data and M-mode Echocardiography for comparison of the MLP null and their controls. Comparison of correlation between wall measures (PW, IVSW), fractional shortening (FS) and Heart rate (HR) with the two modalities was performed as described by Bland Altman[25].

Results

Micro-CT images of the phantom containing two vials and a calibration grid are shown by fig.2. No geometric distortions were visible in the reconstructed grid (see fig.2C). The volumetric measurements performed using the PSC-VB software were 98% accurate.

Fig. 2.

Fig. 2

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The measurements calibration phantom: axial (A) and oblique slices (B, C) (cuts along lines) showing no geometric distortions (C) due to the reconstruction process. The volumetric measurements were 98% accurate.

Micro-CT with blood pool contrast agent Fenestra VC achieves high-resolution images with sufficient contrast-to-noise levels. Thus, for a contrast agent (Fenestra VC) dose of 0.014 ml/g mouse, the average signal intensity difference between the myocardium and the blood pool in the LV was approximately 310 HU and the noise level about 72 HU resulting in a contrast to noise ratio (CNR) of 4.3. The noise was measured in an air region defined just outside the mouse chest. Thus, we satisfied the limit imposed by the Rose criterion for contrast detectability with CNR that ranges between 3 and 5[26]. The radiation dose during micro-CT scanning of a mouse at 8 points on the cardiac cycle was 0.8 Gy.

Real 4D data at isotropic 943 microns spatial resolution and with 15 ms temporal resolution were visualized as seen in Fig. 1. Because the micro-CT datasets have isotropic resolution, the same image quality is possible in any imaging plane. Typical images corresponding to end-diastole (axial and coronal slices) are shown in fig. 3 for an MLP null (A, B) and C57BL/6 control mouse (C,D). Note the enlarged ventricles and the myocardial wall thinning in the MLP null mouse compared to the control mouse. A sequence of eight axial micro-CT images of an MLP mouse acquired during the cardiac cycle are shown by fig.4.

Fig. 3.

Fig. 3

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An example of images corresponding to diastole (axial and coronal cuts) for an MLP (A, B) and C57BL/6 (C, D) control mice.

Fig.4.

Fig.4

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Images of the same axial slice through an MLP mouse corresponding to 8 points i.e. every 15 ms in the cardiac cycle (RR=120ms) starting from the R peak.

The segmented LV volumes in diastole for one MLP and one C57BL/6 mice are shown by fig. 5A and fig. 5B. The time-volume curve of these LV volumes is shown by fig.5C. Note that MLP null mouse shows an approximately three times larger ventricular volume in diastole than seen in the C57BL/6 control. The weight of these representatives two mice were: 33g for the MLP null and 30.5g for the C57BL/6.

Fig. 5.

Fig. 5

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A comparison of the LV volumes for an MLP null (A) and C57BL/6 (B) mouse in diastole. The time volume curves (C) for the LV in MLP null and C57BL/6 mice: y-axis is the LV volume, x-axis is time from the R peak. Note the larger LV volumes of the MLP null mouse indicating an enlarged ventricle.

Micro-CT based measurements (mean and standard errors) are given the plot in fig. 6(A) while fig. 6(B) shows the results for the measurements obtained with M mode echocardiography both for MLP null and C57BL/6. With the exception of wall thickness (PW, IVSW) and cardiac index (CI) all micro-CT measured values for MLP null are significantly different than for the C57BL/6. The same situation with one exception (PW) is shown by fig.6 (B) but with M mode echocardiography data.

Fig. 6.

Fig. 6

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Mean and Standard Error plots for Micro-CT (A) and M Mode echocardiography (B). The abbreviations stand for: BW= body weight, LVD(S)D: left ventricle diameter in end diastole (systole), PW=posterior wall thickness, IVSW=intraventricular septum wall, HR =heart rate, ED(S)V=end diastolic(systolic) volume, EF=Ejection Fraction, CO=cardiac output, FS=fractional shortening. Statistical significance (p<0.05, unpaired t-test) is visually marked by * symbol on the graphs.

The Bland Altman plots for common measures between the two imaging modalities are shown by fig. 7 for PW (A), IVSW(B), FS (C) and HR (D). The graphs plot the differences versus the average values between the measurements with the two imaging modalities for each animal. In all graphs mean differences are different than zero suggesting the existence of a bias but there is no apparent trend in the distribution of the differences and the values are in the limits of agreement of two standard deviation.

Fig. 7.

Fig. 7

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Bland Altman plots using both Micro-CT and Echocardiography data for PW (A), IVSW (B), FS (C) and HR (D). The heavy line is the mean difference between the two imaging methods and the stroked lines represent +- 2SD. The square symbols represent MLP null data while the triangle symbols show the C57BL/6 data.

Discussion and Conclusions

This study demonstrates our unique ability to obtain 4D cardiac data from mice using micro-CT. As shown here, both morphological (LV dilatation) and functional parameters values such as ejection fraction (EF), stroke volume (SV), cardiac output (CO) and fractional shorthening (FS) were found to be significantly different in the MLPs null versus controls (see fig. 6A). Compared to control mice, in MLP null, EDV was three times greater and ESV was increased five fold. However EF is reduced by an average of 32% in MLP null mice versus the C57BL/6 indicating dilated cardiomyopathy with hypertrophy. Some of our results compare well with those provided by Wilding et al using MR Microscopy[18]. For example, the EF for C57BL/6 mice was 59. 6% with micro-CT and 66 % with MR while for MLP null mice the EF was 27.8% with micro-CT and 25% with MR Microscopy. However, in our case the cardiac output and stroke volume were larger for the MLP null mice. This could be due to the larger body size of the MLP mice. Indeed, after normalization to the body weight the cardiac index becomes similar (0.39 for the MLP null versus 0.31 for C57BL/6 mice) and are not significantly different (see fig. 6(A)). As shown by Bland Altman plots (fig. 7), the two modalities i.e. micro-CT and M mode echocardiography provided different results for the same measures (mean of differences is not zero) but within the limits of agreement of two standard deviations. The reasons for such differences can be attributed partly to the different heart rates (see fig. 7D) and anesthesia regimes (Ketamine for micro-CT and isoflurane for M mode echocardiography). We notice an average difference of about 0.3 mm in the wall thickness (fig. 7A, B) and of about 15% in FS. Micro-CT allows in vivo estimation of functional measures based on LV that are not possible with M-Mode echocardiography (compare Fig. 6A and B). Typically, the long axis measurements are very difficult and inaccurate using the M-Mode echocardiography. Some current M mode methods to measure LV volumes in murine hearts are invasive as they use piezoelectric crystals surgically attached to the wall of the heart. This approach was used by Esposito et al. [11] to obtain LV volumes in MLP null mice. They found, similar to our results, that MLP null mice had almost equal and even slightly larger cardiac index than their controls in this open chest study.

However, cardiac micro-CT in mice is not without problems. A temporal resolution of 15 ms in sampling could cause inaccurate determination of the end-systolic and end-diastolic volumes. In a different experiment we acquired data using retrospective gating and reconstructed with projection images binned post acquisition in various number of time points in the cardiac cycle. We noticed a reduction of 3% in EF estimation when the time points were reduced from ten to eight in the cardiac cycle. Increased temporal resolution would require a larger number of datasets requiring more sampling time and an increased radiation dose. We were able to achieve a 10 ms temporal resolution in other studies[27, 28]. However, in the present study we compromised on the temporal resolution versus duration of our imaging session to keep the scanning time to less than one hour.

Another concern with the present study is the vertical positioning of the animal during micro-CT. It is well known [29] that such a position in larger mammals and humans results in an orthostasis effect, i.e. reduction of venous return, LV volumes and cardiac output. However this effect is not observed in rodents. The influence of a vertical body position on murine systemic blood pressure and left ventricular (LV) hemodynamics over time has been investigated with MR Microscopy[4, 30]. These studies showed that tilting to vertical position had no significant changes in murine LV hemodynamics. Similar results were seen in rats[31]. An acceptable explanation could be that the column of fluid in a rodent is only a few centimeters and therefore the small gravitational effect is negated by the autoregulatory system. Thus, the vertical position was not considered to be an issue in our study.

Additionally, cardiac micro-CT involves the use of an injected blood pool contrast agent which could affect the hemodynamics due to hypervolemia. Considering that mice have a blood volume that is about 6% of the body weight, by injecting Fenestra VC in dose of 0.014 ml/g, we added ~ 20% to the blood volume. But according to one study [32] an increase in hypervolemia of ~30% in the total blood volume by adding saline did not significantly affect cardiac index in mice. We note however that the contrast agent used here is a lipid emulsion and we do not know its influence on the hemodynamics.

MR Microscopy is considered to be the gold standard for in vivo measurement of cardiac morphology function and new methods such as 3D echocardiography are often compared to MR Microscopy[3]. But micro-CT has the potential advantage over MR Microscopy by providing isotropic resolution. This is not the case with MR Microscopy where typically a number of axial slices are scanned and the LV volume is estimated by interpolation between axial slices acquired every 1 mm[33].

When comparing MR Microscopy and micro-CT, isotropic resolution should not be however considered isolated. The inferior spatial resolution of MR Microscopy is counterbalanced by a higher temporal resolution, higher tissue contrast, and its versatile character i.e. the ability to measure perfusion, tagging, targeted molecular imaging contrast studies, or spectroscopic metabolic studies. Further studies will be needed to establish the role of cardiac micro-CT versus MR Microscopy. Other mouse models of cardiac disease such as for myocardial infarction involving regional variation will be more suitable to show the advantage in isotropy of micro-CT imaging versus MR Microscopy.

More recently, new echocardiographic systems (VisualSonics Inc, Toronto, Canada) are available for small animals and are able to provide B-mode measurements in small animals. We add, however, that although echocardiography seems to be the method of choice to estimate cardiac function, it still uses some model-based estimations that rely on geometric assumptions that are appropriate only for normal hearts[34, 35].

Another invasive method to measure LV volume in mice is based on conductance catheters but a recent study[36] compared the conductance catheter measurements with those from MR Microscopy and found that that absolute volumetric values are strikingly underestimated by conductance catheter measurements.

In conclusion, we show how micro-CT can characterize cardiac anatomy and function in a genetic model of dilated cardiomyopathy. The use of truly isotropic voxels with volume resolution more than 10 times of previous methods provides significant increase in both precision and accuracy over both echocardiography and MR Microscopy. This technique has a great potential to become an important tool in cardiac phentoyping and this paper reports on our first attempts towards such a goal.

Acknowledgments

All work was performed at the Duke Center for In Vivo Microscopy, an NIH/NCRR National Biomedical Technology Resource (NCRR P41 RR005959), with additional support from NCI R24 CA 092656, and NHLBI 5R01-HL055348. We thank to Art Wetzel and Stu Pomerantz from Pittsburg Supercomputing Center for their help with the PSC-VB software and to Sally Zimney for editorial assistance.

Abbreviations

MLP

muscle LIM protein

LV

left ventricle

MR

Magnetic Resonance

LVD(S)D

left ventricle end diastolic (systolic) dimension

PW

posterior wall thickness

IVSW

interventricular septum wall

ED(S)V

end diastolic (systolic) volume

EF

ejection fraction

SV

stroke volume

CO

cardiac output

HR

heart rate

FS

fractional shortening

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