Journal of Pharmacological and Toxicological Methods
Original articleIn vivo imaging of rat coronary arteries using bi-plane digital subtraction angiography
Introduction
Preclinical imaging of small animal models has become an important component of biomedical, pharmaceutical, and genomic research programs. Imaging can be particularly useful for integrated (e.g. structure and function) approaches to the preclinical assessment of safety for novel therapeutics with the added benefit of functioning as a translational biomarker. To meet this need, the technology for in vivo, small animal imaging has advanced rapidly in recent years to include magnetic resonance microscopy (Johnson, Hedlund et al., 1992), micro-CT (Holdsworth & Thornton, 2002), micro-ultrasound (Hoit, 2001), micro-PET/SPECT (Herschman, 2003), and optical methods based on fluorescence and bioluminescence (Ntziachristos, Ripoll et al., 2005). We and others have recently added x-ray-based digital subtraction angiography (DSA) (Badea, Drangova, et al., 2008, Badea et al., 2007, Buhalog et al., nd, Lin et al., 2006) to this imaging armamentarium.
Drug-induced vascular injury is a particularly challenging drug safety concern for a number of reasons. Prominent among these are an incomplete understanding of the predictivity of preclinical models for clinical risk and lack of a sensitive and relevant translational biomarker strategy. Many of the best characterized preclinical models of vascular injury involve drugs that are vasoactive—i.e., induce vasoconstriction or vascular relaxation/vasodilation (Dogterom et al., 1992, Hanton et al., 1995, Kerns et al., 1989, Louden et al., 2006). In traditional preclinical safety paradigms, these effects are characterized by correlating changes in systemic blood pressure as a surrogate for vasoactivity with histologic evidence of lesions in blood vessels of various organ systems, including the heart. Paradoxically, these assessments are generally collected in separate study settings. Sensitive measures of systemic blood pressure are most often collected in single-dose telemetered animal safety pharmacology studies. Morphologic characterization of vascular injury, on the other hand, is done in repeat-dose studies from which representative tissues are collected for histologic examination. This paradigm lacks the ability to visualize tissue-specific vasoactivity where blood vessel lesions occur. Coronary arteries are a recognized site of drug-induced vascular injury in both rodents and non-rodents (Dogterom et al., 1992, Kerns et al., 1989, Louden et al., 2006).
In many vascular studies, useful information can be obtained from two-dimensional (2D) digital subtraction angiography (DSA) projection images. First suggested by Mistretta, Ort et al. (1973), DSA is now a routine clinical imaging modality and coronary artery DSA is one of its main applications (Struyven, Delcour et al., 1986). However, to date, there have been only limited studies using DSA for preclinical imaging (De Lin et al., 2008, Lin et al., 2009). Yet, much potential exists for this modality given its ease of use, throughput, potential high speed, and relatively low cost. DSA can be based on either temporal subtraction or k-edge subtraction. The latter technique is based on the nonlinear differences in the attenuation of iodine with the x-ray beam energy. A k-edge describes a sudden increase in the attenuation coefficient of x-ray photons. K-edge DSA ideally requires imaging on both sides of the k-edge of iodine with monochromatic x-rays obtained using a synchrotron source as in Schültke et al.. However, the need of a synchrotron limits the availability of such a method, since the majority of x-ray imaging systems are using polychromatic x-ray sources and are therefore, better suited for temporal subtraction. We report here methods for in vivo DSA based on temporal subtraction based on the injection of contrast agent and image acquisition before and after the contrast injection.
Coronary arteries in the rat have been previously visualized using synchrotron x-ray radiation (Matsushita et al., 2005, Matsushita et al., 2008). Although this approach provides very high resolution (13 μm), the studies were limited to ex vivo Langerdorff heart preparations (Skrzypiec-Spring, Grotthus et al., 2007). Recently, Ishikura, Hirayama et al. (2008) have shown three-dimensional (3D) real-time contrast echocardiography of rat coronary arteries. The image quality, however, is inferior compared to the x-ray-based imaging. In this work, we describe a bi-plane DSA system based on conventional x-ray tubes and CCD-based solid state detectors to image rat coronary arteries in vivo.
Our goal was to develop methods for imaging in rats that might provide translational information to assist safety assessment in drug discovery. We demonstrate that DSA can be used, not only for morphological evaluation of the coronary arteries, but also for assessing myocardial perfusion in a case study using the drug nitroprusside.
Section snippets
Dual source/detector x-ray system
We have developed a unique, dual tube/detector radiographic system that allows high-resolution imaging with high photon fluence rate, and integrated motion control, i.e. imaging is synchronized to respiratory and cardiac activity to reduce the motion artifacts. The system is also used for micro-CT, which was described previously (Badea, Johnston et al., 2008). The CT system uses two Varian A197 x-ray tubes (Varian Medical Systems, Palo Alto, CA) with dual focal spots of 0.3/0.8 mm. The tubes are
Results
Examples of cine display of DSA images are available as animations in the movie supplement to this manuscript. Each animation shows the first pass of the bolus of the contrast agent. For each injection, we generated two movies corresponding to the orthogonal bi-plane views. Static frames from such a cine-sequence are shown in Fig. 3. These sequences demonstrate the importance of catheter tip position. In the Fig. 3A,B series, the tip is very close to the aortic valve and apparently blocking the
Discussion and conclusions
Imaging coronary arteries in a rat is very challenging due to their small size and fast physiologic motion of the heart and respiration. These challenges are hard to overcome with commercial small animal x-ray or micro-CT systems that are using micro-focus x-ray tubes for which the exposures should be sufficiently long in time for an adequate signal to noise ratio. We have been able to achieve this goal by building an x-ray-based imaging system that uses x-ray tubes with large focal spot
Acknowledgments
All work was performed at the Duke Center for In Vivo Microscopy, an NIH/NCRR National Biomedical Technology Research Center (P41 RR005959), with additional support from NCI (U24 CA092656).
References (41)
- et al.
Micro-CT imaging assessment of dobutamine-induced cardiac stress in rats
Journal of Pharmacological and Toxicological Methods
(2011) - et al.
Measurement of myocardial infarct size in preclinical studies
Journal of Pharmacological and Toxicological Methods
(2010) Micro-PET imaging and small animal models of disease
Current Opinion in Immunology
(2003)- et al.
Micro-CT in small animal and specimen imaging
Trends in Biotechnology
(2002) - et al.
Brief communications: visualization of coronary arteries in rats by 3-dimensional real-time contrast echocardiography
Journal of the American Society of Echocardiography
(2008) - et al.
Coronary angiography in rats using synchrotron radiation.
Nuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment
(2005) - et al.
The minimum coronary artery diameter in which coronary spasm can be identified by synchrotron radiation coronary angiography
European Journal of Radiology
(2008) - et al.
Cardiac magnetic resonance imaging in small animal models of human heart failure
Medical Image Analysis
(2003) - et al.
Isolated heart perfusion according to Langendorff — still viable in the new millennium
Journal of Pharmacological and Toxicological Methods
(2007) - et al.
Myocardial perfusion planar imaging
Journal of Nuclear Cardiology
(2006)