1.

Introduction

The rat is a classic mammalian animal model for a wide range of embryonic studies aimed to address congenital abnormalities. Rat physiology is considered to be more complex and informative than in the mouse, providing a closer animal model to humans for neurobehavioral, toxicological, and organ manipulation studies. Until recently, the use of rats was hampered by the lack of genetic manipulation techniques, but derivation of rat embryonic stem (ES) cells by Buehr ¹ and Li ² provides a basis for a wide range of direct genetic alterations to create disease models with more relevance to human disorders, including better models of congenital cardiovascular defects. Together with advances in genetic manipulation, higher resolution imaging methods such as optical coherence tomography (OCT) have emerged as improved phenotyping tools. OCT is a 3-D optical imaging technique with a spatial resolution of 2 to 20 μm and an imaging depth of 2 to 3 mm in tissue. It does not require the application of contrast agents and can be combined with Doppler OCT detection of velocity at the same spatial resolution.

OCT has been successfully used by several groups for live embryonic imaging of Xenopus laevis,^{3, 4} quail,⁵ and chick.⁶ Recently, our group optimized protocols for static mouse embryo culture for OCT imaging, demonstrating that OCT and Doppler OCT can be successfully applied to live embryonic 3-D structural imaging and cardiovascular and hemodynamic analysis⁷ at early, postimplantation embryonic stages E7.5 to E10.5. Our recent work has shown that this method is a highly sensitive means to image blood flow as it is possible to make velocity measurements from individual blood cells during early stages of circulation in mouse embryos.⁸ While OCT has proven to be an extremely useful modality for live embryonic imaging in different species, its capabilities have never been tested in the rat model.

In this study, we have used OCT to image dynamic cardiovascular development in the rat model at the embryonic stages E10.5 to E11.5, equivalent to E8.5 to E9.5 in mouse embryos. We performed 3-D structural imaging of live rat embryos, dynamic heart imaging, and Doppler OCT analysis of the beating heart. Here, we report the first live dynamic imaging of the rat embryo at cellular resolution, opening the door for a wide range of embryonic physiological studies in the rat model.

2.

Materials and Methods

3.

Results and Discussion

3.2.

Structural 3-D SS-OCT Imaging

Figure 1 shows 3-D reconstructions of the embryos with extraembryonic yolk sacs at E11.5. All major structures are clearly outlined on the reconstructions. Even though the rat and the mouse are both rodents, their embryonic development differs noticeably. Rat embryos are about 30% larger than the mouse embryos at the corresponding developmental stage, they develop slower, and the gestation period is longer than in the mouse. Despite the larger size, the rat embryos at the studied stages are within the imaging depth of the SS-OCT system and the embryonic structures are resolved better than the ones of the mouse at the corresponding developmental stages due to their larger size. At later time points, high-resolution OCT imaging can be complimented with high-frequency ultrasound imaging, which enables deeper tissue imaging with the expense of lower resolution (30 to 50 μm).

Fig. 1

Live 3-D imaging of early rat embryos with SS-OCT. (a) 3-D rendering view of a live E10.5 embryo cultured on the imaging stage. The clipping plane on the reconstruction is at a 60-deg angle to the direction of the OCT laser beam. (b) A cross-section view of a typical 3-D reconstruction of an E10.5 embryo. Major embryonic structures are clearly distinguishable on the reconstructions. Both data sets consist of 512 × 512 A-scans.

3.3.

Dynamic Structural Imaging

To test the potential of SS-OCT to analyze dynamic structural changes in rat embryos, we acquired 2-D time-lapse sequences. Figure 2a shows a representative frame taken from a time lapse acquired of the beating embryonic heart tube. The time lapse was acquired at 512 A-scans per frame and 28 frames/s. The walls of the beating heart are nicely visualized, providing an opportunity to observe heart dynamics during the heartbeat. Figure 2b is a frame from a time lapse taken through the embryo along the dorsal aorta. Individual circulating erythrocytes are clearly detectable within the heart tube [Fig. 2a] and the dorsal aorta [Fig. 2b].

Fig. 2

Dynamic imaging of live rat embryos. Representative frames out of time lapse (a) through embryonic heart and (b) along the embryonic body. The time lapses are acquired at about 28 frames/s. Individual circulating blood cells and small groups of cells are distinguishable in the heart tube (a) and dorsal aorta (b).

3.4.

Doppler SS-OCT Imaging of Cardiodynamics and Hemodynamics

We performed Doppler SS-OCT imaging to visualize hemodynamics within rat embryos. Figure 3 shows a structural SS-OCT image through the embryonic heart and a series of color-coded Doppler images of the same area taken from a time lapse and representing different phases of the heartbeat cycle. The detected Doppler shift signal was generated by the velocity component, which is parallel to the OCT laser beam. The blue shift corresponds to movement toward the detector, while the red shift corresponds to movement away from the detector. As one can see from the figure, the Doppler shift was detected from the circulating blood cells inside the heart (labeled as b) as well as from the moving heart wall (labeled as hw). Even though the heart at the studied stages is within the imaging depth of the SS-OCT system and the structure is well resolved, hemodynamic analysis in the heart is complicated by the Doppler shift phase wrapping caused by the high flow velocities and the difficulty of mapping of the flow direction required for the velocity calculation. While several groups are developing and optimizing algorithms to overcome these limitations,^{6, 10} this paper demonstrates that Doppler shift maps can be acquired from the beating rat embryonic heart for live hemodynamic analysis.

Fig. 3

Doppler SS-OCT velocity imaging in the live embryonic heart. (a) Structural image acquired from the primitive ventricle of the beating embryonic heart at E10.5 and (b) to (g) the corresponding representative Doppler color-coded maps from the same area taken out of a time lapse. Doppler imaging was performed at about 26 frames/s at 512 A-lines per frame. (Color online only.)

4.

Conclusions

For the first time we demonstrated live imaging of rat embryos with resolution at the single cell level. We performed (1) 3-D imaging of live embryos cultured on the imaging stage, which provides a tool for structural analysis of embryo morphology; (2) dynamic 2-D imaging; and (3) dynamic Doppler shift velocity imaging of a beating heart. Our results show that OCT imaging of cultured live rat embryos is a useful tool for studying morphological changes during development as well as for analyzing dynamic physiological processes, such as heartbeat and blood flow. Comparative studies between rat and mouse embryos indicate differences between these rodent species, potentially highlighting processes that may benefit from the generation of new mutant rat models.