Research Article

Received: 12 May 2008, Revised: 23 January 2009, Accepted: 28 January 2009, Published online in Wiley InterScience: 25 March 2009

(www.interscience.wiley.com) DOI:10.1002/nbm.1386

In ovo non-invasive quantication of the myocardial function and mass of chick embryos using magnetic resonance imaging
William M. Holmesa *, Christopher McCabea, Jim M. Mullina, Barrie Condonb and Maureen M. Bainc
Magnetic resonance imaging (MRI) has evolved as one of the major non-invasive tools to study healthy and diseased hearts in animal models, especially rodent models. Even though, the chick embryo has long been used as a model for cardiovascular research, MRI has not yet been used for in vivo cardiac studies. Part of the reason for this is the difculty in monitoring the ECG and respiration of the chick embryo in the magnet for gating purposes. To overcome this complication, this paper presents the use of retrospective Cine MRI to measure the cardiac function of chick embryos in ovo for the rst time, without the need for respiratory or cardiac gating. The resulting left ventricular functional parameters, from six chick embryos at 20 days of incubation, were (mean W SD) EDV 69 W 15 mL, ESV 31 W 7 mL, SV 38 W 9 mL and EF 54.5 W 2%. The use of retrospective Cine MRI at earlier stages of development is also discussed and difculties have been highlighted. Copyright 2009 John Wiley & Sons, Ltd. Keywords: MRI; heart; chick; embryo; retrospective; in ovo; myocardial function

INTRODUCTION
The chick embryo is a well-known model for cardiovascular research, with regard to cardiac growth, morphogenesis and function (1,2), angiogenesis (3,4), cardiovascular mechanics (5) and the development of neurohumoral cardiac control (6). The chick embryo is an attractive model because the developing chicks requirements, with the exception of oxygen and heat, are provided by the egg contents and the surrounding eggshell. In addition, the basic mechanisms of foetal cardiovascular control have been found to be comparable between the chicken and mammals (7), with studies carried out either in ovo or ex ovo in shell-less culture (8). In chick embryos, the heart begins to develop and beat at about HamburgerHamilton (9) stage 910 (2938 h) and by stage 16 (5156 h) the circulation becomes well established. The developing myocardium subsequently undergoes simultaneous structural and functional maturation as the avian embryonic heart transforms from a straight tube to a four-chamber heart between days 3 and 5. From the fth day to the end of incubation the heart increases in size becoming almost 6 times longer and 4.5 times wider. Although the chick embryo is a well-established model species, the attempts to use non-invasive imaging modalities to monitor chick development and cardiac function have been limited. MicroCT is of limited use because of lack of tissue contrast (10). The presence of the calcitic egg shell limits the use of ultra-sound, although this can be overcome by using fenestrated eggs (11) or ex ovo culture techniques (12). Ultra-high frequency ultra-sound has been used to visualise the cardiac function of chick embryos both in fenestrated eggs and ex ovo, although without quantication (12). The use of MRI has been limited mainly to ex vivo imaging of the developing chick (13) mainly because of embryonic movement. This becomes particularly problematic beyond the 12th day of incubation for both ultra-sound (12) and

MRI (14). Recently, however, MRI has been used in combination with cooling of the egg, by placing the egg in a refrigerator for 1 h at 48C. This reduced embryonic motion signicantly, enabling high resolution imaging of developing chick embryos in ovo throughout their main growth phase (14). This methodology has provided some insight as to how the heart and other organs change in size during the extra-uterine development of chick embryos. However, it is not suitable for in vivo cardiac research per se as the cardiac output decreases considerably during cooling. With other animal models, such as rodents, it is now common to use Cine MRI to study cardiac function and development non-invasively (15). In order to produce cardiac MR movies without severe motion artefacts, the image sampling needs to be synchronised to the cardiac and respiratory cycles with high precision. Conventionally, the electrocardiogram (ECG) and respiration are monitored via sensors, and used to prospectively

* Correspondence to: W. M. Holmes, GEMRIC, Wellcome Surgical Institute, Faculty of Medicine, University of Glasgow, Garscube Estate, Bearsden Road, Glasgow G61 1QH, Scotland, UK. E-mail: wmh5b@udcf.gla.ac.uk a W. M. Holmes, C. McCabe, J. M. Mullin GEMRIC, Wellcome Surgical Institute, Faculty of Medicine, University of Glasgow, Garscube Estate, Bearsden Road, Glasgow G61 1QH, Scotland, UK b B. Condon Clinical Physics, Institute of Neurological Sciences, Glasgow G51 4TF, UK c M. M. Bain Division of Cell Sciences, ICM, University of Glasgow Veterinary School, Garscube Estate, Bearsden Road, Glasgow G61 1QH, Scotland, UK Abbreviations used: ECG, electrocardiogram; EDV, end-diastolic volume; ESV, end-systolic volume; EF, ejection fraction; LV, left ventricular; PM, post mortem; RF, radiofrequency; SV, stroke volume.

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W. M. HOLMES ET AL. trigger the MRI scanner. Monitoring ECG and respiration of chick embryos in ovo although possible (16), would prove to be difcult in a high eld magnetic eld, where rapid switching of magnetic eld gradients and radio frequency pulses tends to corrupt the ECG signal (17). In this study, we have removed these obstacles by employing a Cine MRI protocol that incorporates a navigatorbased retrospective gating technique (18,19). Specically, the navigator signal is used to generate cardiac triggering and respiratory gating signals retrospectively, enabling reconstruction of cardiac images that are free of motion artefacts. The ability to produce cardiac MRI images without the need for peripheral ECG or respiration sensors, has allowed this rst non-invasive imaging study of cardiac function in late stage chick embryos in ovo. of view 4.60 4.60 cm2, matrix 230 230, in-plane resolution 200 200 mm2, slice thickness 1 mm, 1 acquisition and RARE factor 8. Due to cardiac motion, the heart was not visible in these anatomical scans. Only the general location and orientation of the cardiac cavity could be determined by using an appropriate reference atlas (20). Cine MRI was performed by using a modied Flash sequence with an in-slice navigator echo for retrospective gating, derived from the half-echo following the slice selective pulse, as described by Heijman et al. (18). Scout images were acquired by using this method, with the following parameters: echo time 1.9 ms; repetition time 5 ms; ip angle 158; sampling rate 100 kHz; eld of view 2.13 4.02 cm2; matrix 71 134 (read, phase); in-plane resolution 300 300 mm; slice thickness 1.5 mm; 300 acquisitions. For early stage embryos, a eld of view of 1.2 4.02 and a matrix 40 134 was used. To determine the short-axis orientation, several scout images were made in the transverse and long-axis plane of the LV. Once the short-axis orientation had been determined, cardiac Cine MRI data were acquired by using the above parameters, but with 900 acquisitions per slice. Within the short-axis slice, the orientation of the readout gradient direction was not xed with respect to the chicks orientation. The entire heart was scanned with eight contiguous short-axis slices, each with an acquisition time of 10 min. In addition to the above, a 300 mm isotropic resolution T2 scan was also acquired for egg 1, after Cine MRI scanning at 20 days incubation, to assess internal pipping. A RARE sequence was used with the following parameters: echo time 10.26 ms; repetition time 20 s; eld of view 5.40 4.50 cm2; matrix 180 150; in-plane resolution 300 300 mm2; 160 slices with thickness 300 mm; three acquisitions; RARE factor 10. Image analysis Analysis of the navigator echo and reconstruction of k space data were performed with a PavaVision Intragate (Bruker, Germany) software using Fourier ltering techniques, as described by Heijman et al. (18). This method uses the navigator echo to derive both cardiac and respiratory cycles. These are derived from the maximum of the navigator signal intensity, the phase at maximum signal intensity, the derivative of a rst-order polynomial t of the navigator signal intensity and the derivative of a rst-order polynomial t of the unwrapped navigator phase. Any corruption of the cardiac signal was excluded by manual selection. Subsequently, a maximum or minimum detection algorithm dened the trigger point in the derived cardiac or respiratory cycles. The time between successive maxima in the cardiac signal was divided into ten time intervals (cardiac phases), allowing the k-space data associated with each navigator echo to be assigned a phase in the cardiac cycle (18). It should be noted that the retrospective gating post-processing allows the choice of temporal resolution, although higher temporal resolution is achieved at the cost of an image with lower signal-to-noise ratio. The epicardial and endocardial borders were delineated by semi-automatic segmentation using CAAS MRV FARM program (Pie Medical Imaging, Maastricht, The Netherlands). Stroke volume (SV) was calculated as the difference between the end-diastolic volume (EDV) and end-systolic volume (ESV). Ejection fraction (EF) was calculated as SV/EDV. Left ventricular cavity and myocardial volumes in each slice were obtained by multiplication of the compartment area with the slice thickness (1.5 mm). The Left ventricular (LV) myocardial mass was dened as the volume within the epicardial border minus the LV cavity,

EXPERIMENTAL METHODS
Animal preparation The fertile broiler breeder eggs used in these experiments each weighed between 50 and 55 g and were sourced from two separate batches of eggs (batch A and batch B) obtained from a commercial hatchery. Each batch of eggs was stored for 3 days, and then incubated in a Brinsea 40 digital tabletop incubator at 37.58C. On the sixth day of incubation the eggs were hand-candled to check that they were fertile and developing normally. In the rst experiment three eggs from batch A (denoted 13) were consecutively scanned on days 5, 8, 11, 13, 16 and 20. In terms of the Hamburger and Hamilton (9) stages, this equates to stages 25, 34, 37, 39, 42 and 45 of a 46-stage (21 days) incubation period. Prior to scanning, each egg was removed from the incubator and placed into a custom-built polystyrene holder to provide thermal insulation. This was suspended within the resonator/magnet in order to minimise vibrations arising in the gradient coils. Each egg was then individually scanned using a 7 T MRI system. After scanning on day 5, 8, 11, 13 and 16, the eggs were returned immediately to the incubator. On the 20th day of incubation, these eggs were scanned for the last time, then put into a refrigerator overnight to cull the embryos (schedule 1, euthanasia). The left ventricle (LV) of each embryo was then carefully excised and weighed (mg) for assessment of LV mass. Three additional eggs from batch B (denoted 46) were only scanned on the 20th day of incubation; after scanning, the eggs were again chilled overnight so that the LV could be carefully excised and weighed. Imaging The magnetic resonance imaging experiments were performed on a Bruker Biospec system, using a 30 cm bore, 7 T superconducting magnet (Bruker Biospec, Karlsruhe, Germany). A Bruker micro-imaging, actively shielded, gradient insert (BGA 12) and 200 A gradient ampliers provided strong linear magnetic eld gradient pulses of up to 400 mT/m, thus adapting the system to perform micro-imaging experiments. A Bruker 72 mm bird-cage RF volume resonator was used to excite and detect the 1H signal. Eggs were placed with their longest axis horizontal and parallel to the bore of the magnet. The chick embryos orientation within the egg was determined by a multislice RARE sequence, with the following parameters: echo time 13.9 ms, repetition time 5 s, eld

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IN OVO QUANTIFICATION OF MYOCARDIAL FUNCTION AND MASS OF CHICK EMBRYO

Figure 1. Short-axis end-diastolic images for eggs at day 8, 13, 16 and 20 of incubation. Left ventricle (LV) and right ventricle (RV) are indicated.

multiplied by the myocardial mass density. In this study, the myocardial mass density of the chick embryo was assumed to be 1.05 g/cm3, this value is commonly used for several mammalian species including mice, rats and humans (21).

RESULTS
Using eggs from Batch A (denoted 13), an attempt was made to study the cardiac function of chick embryos in ovo at 5, 8, 11, 13, 16 and 20 days of incubation. At the earlier stages of incubation

this study was hindered by difculty in locating the small embryonic heart and bulk embryonic motion during scanning. Nevertheless, we were able to obtain examples of Cine MRI at 8, 13, 16 and 20 days incubation and examples of these are shown in Fig. 1. Examples of T2 weighted images of eggs at 8 and 20 days incubation are shown in Fig. 2. Even with repeated attempts, it was not possible to successfully acquire data on each day for each egg in batch A, due to the problem of embryonic movement during scanning. However, it was found that by day 20 of incubation, there was no bulk motion of the embryo during

Figure 2. RARE T2 weighted sagittal (A) and coronal (B) images of egg 2 at day 8 of incubation. RARE T2 weighted sagittal(C) and coronal (D) images of egg 1 at day 20 of incubation.

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Figure 3. Short-axis MR images of a fully developed chick embryo heart (day 20, Stage 46) from base to apex (AH) taken during diastole. The in-plane image resolution is 300 300 mm, with a slice thickness of 1.5 mm. Scalebar 5 mm.

scanning, and excellent cardiac images were obtained for each egg in Batch A. Hence, three more eggs from batch B were scanned at this stage, giving a total group size of six. Up to eight short-axis slices covering the entire heart were acquired for each egg (n 6), as shown in Fig. 3. Figure 4 shows examples of

short-axis end-diastolic images taken from a central slice through the heart of all six chick embryos (Batch A and B) at day 20 of incubation. These images are free of respiration and cardiac movement artefacts, with clear denition of endo- and epicardial borders and high contrast between blood and myocardium.

Figure 4. Short-axis end-diastolic images taken from the central slice through the heart of embryos 16 at day 20, as shown in Table 1.

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IN OVO QUANTIFICATION OF MYOCARDIAL FUNCTION AND MASS OF CHICK EMBRYO

Figure 5. Short-axis images showing the temporal evolution of the cardiac cycle taken from the central slice through the heart at day 20 (Slice C, in Figure 3). Image 1 shows end-diastole and image 6 end-systole. Slice thickness 1.5 mm and in-plane resolution 300 300 mm. Scalebar 5 mm.

Figure 5 shows an example of the temporal evolution of the central short-axis slice through the LV. Analysis of the navigator echoes allowed the cardiac and respiratory cycles to be derived; examples of these are shown in Fig. 6. The average cardiac and respiration rates of all six eggs imaged at day 20 of incubation are provided in Table 1. The egg surface temperature following scanning ranged from 35 to 378C, with the temperature maintained by thermal insulation and slight heating effect of the sequence. LV mass as determined at post mortem, was found to be similar to the estimated LV mass calculated from MRI images (99 11 and 97 10 mg respectively). From the Cine MRI images we were also able to calculate LV functional parameters such as the end diastolic volume (69 15 mL), end systolic volume (31 7 mL), SV (38 9 mL) and EF (54.5 2%) from each of the eggs at day 20 (see Table 1). Analysis of the Cine MRI data also allowed consideration of the temporal changes in cardiac parameters; for example Fig. 7 shows the evolution in the LV blood volume with the cardiac phase.

DISCUSSION
Serial cine MRI of cardiac development In this paper, Cine MRI with retrospective gating was used to non-invasively image the cardiac function of chick embryos in ovo. This technique uses the signal from a navigator echo to derive the cardiac and respiratory cycles (18), which are then used for retrospective cardiac and respiratory gating. This eliminates the need for both ECG and respiratory gating, both of which are very difcult to monitor in ovo inside the MR magnet. With the eggs from batch A an attempt was made to serially image the developmental changes in cardiac function in ovo, on 5, 8, 11, 13, 16 and 20 days of incubation (HH stages 25, 34, 37, 39, 42 and 45). Although the embryos used in this study were killed at day 20 to measure the LV mass, previous MRI studies by Bain et al. (14) found no adverse effects of serial MRI scanning, where all eggs hatched successfully and grew to adulthood. There were three main difculties encountered in Cine MRI of the earlier

Figure 6. Examples of respiration and cardiac signals at 20 days incubation derived from navigator echoes, taken from (A) egg 3, showing rapid regular respiration, (B) egg 6, showing slow regular respiration and (C) egg 1, showing no regular respiration.

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Table 1. Post mortem LV mass and MRI derived LV functional parameters Heart Rate (bpm) 217 208 297 265 234 250 245 33 Respiration (bpm) 54 89 18 9 11 PM Mass (mg) 88 89 102 114 91 108 99 11 Calculated Mass (mg) 96 93 84 91 97 119 97 10 EDV (mL) 84 60 55 91 69 57 69 15 ESV (mL) 36 24 23 41 34 26 31 7 Stroke Volume (mL) 48 32 32 50 35 31 38 9 Ejection Fraction (%) 56.9 54.1 57.1 54.6 50.4 53.9 54.5 2

Egg 1 Batch A 2 Batch A 3 Batch A 4 Batch B 5 Batch B 6 Batch B Mean SD

See text for abbreviations.

stage chick embryos: rstly, the embryonic motion; secondly, the small size of the heart; and lastly, the myocardial contrast. The problem of embryonic motion can be best appreciated from Fig. 2a,b which shows a sagittal and coronal slice through an egg at day 8. The embryo is surrounded by albumen and yolk and oats relatively freely on these media; hence, bulk motion of the embryo during Cine MRI scanning was a major problem. Even with repeated attempts, it was not always possible to acquire images unaffected by motion. The second problem was cardiac size. At day 5 of development, the heart could not be localised. At day 8 the size of a chick embryos heart ($1.6 mm LV outer diameter) is close to the scanning resolution (0.3 mm) and slice thickness (1.5 mm). However, it was possible at day 8 to obtain a clear cardiac signal from the navigator echoes and reconstruct Cine MRI showing a beating heart (22). As would be expected, no regular respiration signals were seen on days 8, 13 and 16 as gas exchange is performed entirely via the chorioallantoic membrane (CAM). However, irregular respiration signals were detected, similar to those shown in Fig. 6c, which was probably associated with embryonic motion rather than respiration. A further confound at earlier stages of development was the lack of myocardial contrast, see Fig. 1. The white blood contrast of these images allowed easy identication of the inner myocardial

boundary, but the outer boundary was harder to identify. The myocardial contrast appeared to improve with development, but was best dened at day 20 of incubation.

Cine MRI of late stage embryos Although it was difcult to scan early stage embryos using Cine MRI, it was easy to scan embryos at day 20 of incubation. At this late stage, the hearts were $9 mm wide and 11 mm long and showed good myocardial contrast. In addition, the embryo had grown to almost completely to ll the egg (Fig. 2c,d), which severely restricted its motion. As a result, high quality multislice Cine MRI data could be obtained and used for quantitative measurements of cardiac function at day 20. The egg surface temperature after scanning ranged from 35 to 378C, with the temperature maintained by the thermal insulation of the polystyrene egg holder and the slight heating effect of the MRI sequence. Tazawa et al. (23) have shown that such small temperature variations have a negligible inuence on the cardiac rate ($10 beats/min) of the individual 20 day old embryos, which is much less than the natural variation between chicks. The heart rate values derived from the navigator echo for the six chick embryos at day 20 were in the range of 208297 beats/min (Table 1). This is comparable with the range of 200300 beats/min reported by Aubert et al. (16) for chick embryos at 19.5 days incubation. The respiration rates of the six embryos, however, varied widely, which can be understood in terms of an asynchrony in internal pipping that occurs on average 12.3 h before hatching (20). During the main phase of development, the lungs are non-functional and there is no respiration. Gas exchange occurs indirectly via the chorioallantoic membrane (CAM) which ts tightly against the inner surface of the eggshell. The porosity of the egg shell permits the diffusion of oxygen and carbon dioxide between the environment and the blood. This function can be paralleled to that of the placenta for the mammalian foetus. At Stage 45, which corresponds to day 2021 of incubation, the embryo pierces the air cell (internal pipping), allowing aeration of the air sacs and lungs, which establishes respiration. Following internal pipping the lungs in combination with the CAM contribute to the embryos total gas exchange. Full transition from CAM to lungs in providing gas exchange can last several hours to days (24). This is in sharp contrast to the mammalian case where pulmonary ventilation and placental

Figure 7. Temporal evolution of total LV blood volume during the cardiac cycle for all six eggs at day 20 of incubation. Phase 1 represents an end-diastole.

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IN OVO QUANTIFICATION OF MYOCARDIAL FUNCTION AND MASS OF CHICK EMBRYO diffusion overlap for only a few minutes at birth. For chick embryos following internal pipping, there are a number of stages involved in this respiratory transition, viz.: (A) initial irregular breathing, (B) regular respiration with low rate and (C) transition to regular rapid respiration (25). Figure 6 shows examples of different respiration signals derived from the navigator echoes, it is clear that different eggs are at different stages in this respiratory transition. These results are also consistent with other studies which have reported respiration rates of 72.7 breaths per minute at 19.9 days of incubation (26) and 51 breaths per minute at 20 days of incubation (24). A subsequent T2 scan of egg 1, shown in Fig. 2c,d conrmed that the chick in this egg had not yet undergone internal pipping, i.e. the beak had not pierced the air cell, thus explaining the lack of respiration recorded for this particular egg (Table 1). We are therefore condent that the asynchrony in internal pipping at the time of scanning is responsible for the variation in respiration rates presented in Table 1. The retrospective gating technique allowed Cine MRI images to be recorded free of respiration and cardiac movement artefacts thus allowing measurement of EDV and ESV. From these data SV and EF were derived, see Table 1. Among the parameters determined, the estimated EFs were the most comparable between chicks, viz. 54.5 2%. As this is the rst study of its type, there is no other data available upon which to make comparisons. However, it is interesting to note that in similar studies using rodents, typical values for EF were 68.6 6.6% in mice (27) and 69 1% in rat (28). Inaccuracies in measuring the post mortem LV mass (99 11 mg) and in vivo MRI measurement of LV mass (97 10 mg) are to be expected in a study of this type. Even with great caution, dissection of the LV of a chick embryo heart is impossible without inducing an inaccuracy of several milligrams. For this measurement, our results are therefore comparable to similar cardiac studies performed on mice, which gave a post mortem LV mass of 72.8 10.0 mg and in vivo MRI measurement of 76.1 6.5 mg (27). The accuracy of the MRI measurement of LV mass could be improved by using thinner slices and higher in-plane resolution, although this would require an improvement in signal detection sensitivity. The present study used a volume resonator (72 mm diameter) to image the eggs of approximately 42 mm diameter, however sensitivity could be improved by a better match of the volume resonator diameter to the egg diameter. In rodent cardiac studies, sensitivity has been increased by using parallel imaging techniques with an appropriate coil array (29). In a similar way, a custom-designed array of coils surrounding the egg would greatly improve the sensitivity for in ovo cardiac studies. This would allow not only increased resolution but also much reduced scanning time, increasing the throughput. Currently, the rat is the mainstay of basic cardiovascular research. MRI is routinely used to assess the structure and function in rat models (28). Important advances in transgenic (30) and mutagenesis techniques (31) in rats has increased the need for high throughput screening. Currently, the rapid throughput of rodent models is limited by the time required to prepare, anaesthetise and monitor the animal (29). In this regard, the use of late stage chick embryos may have an advantage, as they require no preparation before scanning. Indeed, avian transgenic technology is now progressing rapidly (32). Thus, late stage chick embryos may potentially be better suited than rodent models for high throughput screening experiments of this type.

CONCLUSION
We conclude that Cine-MRI with retrospective gating offers a novel approach for future cardiovascular research, in which late stage (day 20) chick embryos in ovo could be used as the model species. Currently, the high throughput of rodent models is limited by the time taken to prepare, anaesthetise and physiologically monitor the animals. In contrast, late stage chick embryos in ovo require no such preparation, thus leading to potentially higher throughput. However, MRI proved impractical for routine developmental studies of earlier stage embryos.

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