Bio-Medical Materials and Engineering 24 (2014) 1341–1349 DOI 10.3233/BME-130937 IOS Press
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Mouse Coronary Angiography In Vivo Using Synchrotron Radiation Lijun Xua,1, Andi Zhangb,1, Guohao Duc, Honglan Xiec and Ying Chena,d,* a
Department of Biomedical Engineering, School of Medicine, Shanghai Jiao Tong University, No.280, South Chongqing RD, Shanghai, China b Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, No.197, Ruijin No.2 RD, Shanghai, China c Shanghai Synchrotron Radiation Facility, No.239, Zhangheng RD, Shanghai, China d Med-X Research Institute, Shanghai Jiao Tong University, No.1954, Huashan RD, Shanghai, China
Abstract. Purpose: To establish a method for mouse coronary angiography in vivo using synchrotron radiation, which is essential for physiological and pathological research on coronary diseases. Methods: 1) The imaging parameters (e.g., photon energy, spatial resolution of the detector, and injection rate of contrast agent) optimal for the quality of acquired images in a simulation were determined. 2) Through animal experiments, the effectiveness of these optimal parameters and the repeatability of in vivo coronary angiography were verified. 3) An algorithm for background subtraction and contrast enhancement was designed and employed to compensate for the effects of interference and the effective information extracted used for diagnosing coronary disease. Results and conclusions: An optimal set of the imaging parameters was finally determined: photon energy of 33-34 keV, detector’s spatial resolution of 30 m or higher, image capture rate of 20 f/s or more, concentration of lopamidol solution of 75% as contrast agent and a pulse injection of contrast agent at a high rate. Keywords: Coronary angiography, Synchrotron radiation, In vivo, Optimization, Background subtraction
1. Introduction Acute coronary syndrome (ACS) manifests as chest pain and other resulted symptoms when the heart receives insufficient amounts of blood. Recent studies have shown that acute coronary syndrome commonly occurs when an atherosclerotic plaque fissures or ulcerates, precipitating thrombus formation [1, 2]. This results in sudden myocardial infarction or even sudden cardiac death. Therefore, many doctors and researchers have been seeking effective methods to allay or prevent ACS [3, 4]. Gene knockout models, easily made in mice and common for science experiments [5, 6], are widely used in pathological or pharmacological research on coronary diseases. On the other hand, medical imaging, more than any other method, can directly examine the pathological state of the heart, the occurrence of rash symptoms, and the effectiveness of medicinal treatment. Therefore, coronary angiography methods for mice may become effective tools for diagnosis, treatment, and research (patho1 *
Co-author, contributed equally to this work Corresponding author. E-mail: [email protected]
0959-2989/14/$27.50 © 2014 – IOS Press and the authors. All rights reserved
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L. Xu et al. / Mouse coronary angiography in vivo using synchrotron radiation
logical and pharmacological) of heart and coronary diseases. X-ray angiography is one of the most common clinical methods for examining and diagnosing cardiovascular disease [7]. It is performed by injecting a contrast agent into the vessel of interest then imaging the inside of the vessel through X-ray-based techniques. The diameter of a murine coronary artery is about 100-200 m [8] and the heart rate is about 6-10 beats per second. It is hard for conventional imaging techniques with limited spatial resolution or temporal resolution, such as DSA, ultrasound and MRI [9], to obtain diagnostic coronary information. Synchrotron radiation, whose use has been gradually developed in recent years, features high flux, monochromaticity, and near parallel geometry [10]. Images produced by synchrotron radiation contain both high spatial resolution and temporal resolution, and may show information unavailable from conventional imaging techniques, thus presenting a high potential in the field of medical imaging [11]. Although synchrotron-based coronary angiography has made a breakthrough in routine physical examinations [12-14], the development of synchrotron angiography in mice for pathological research is still in progress. Advancements in mouse coronary angiography using synchrotron radiation were reported by Yamashita in 2002 [15] and in 2005. Matsushita established a method of coronary angiography in rats using synchrotron radiation with a Langendorff apparatus [16]. Further research by Matsushita in 2008 indicated that the minimum diameter of blood vessels is about 50 to 100 m, which can be measured by synchrotron-based coronary angiography to identify coronary spasms [17]. In recent years, Pearson performed a series of physiological and pathological research on the rat heart with the Langendorff apparatus using synchrotron radiation [18]. The purpose of our research was to establish an effective imaging technique for mouse coronary angiography in vivo using synchrotron radiation. This technique offers high spatial and temporal resolution, and further work on image analysis and recognition can help establish methods for physiological and pathological research on coronary diseases. The experiments described here were performed on BL13W1 at the Shanghai Synchrotron Radiation Facility (SSRF). 2. Materials and Methods 2.1. Imaging Conditions SSRF is a third-generation synchrotron radiation light source. The energy of the storage ring is 3.5 GeV, the highest value in the medium-energy light source. The experiment was performed in station BL13W1, which can provide monochromatic light with an energy range of 8 to 72.5 keV and beam size of 45 mm (horizontal) * 5mm (vertical). In-line attenuation contrast imaging was taken in this experiment at a photon energy range of 28 to 33 keV. The maximum flux at the sample site was about 1*1010 phs/s/mm2, the distance from the source to the sample was 30 m, and the distance from the sample to the detector was 50 cm. The detector image array is 4008×2672 pixels, with a 12 bit depth capable of imaging at 30 frames per second and 13 microns pixels. The light-path of the in-line attenuation contrast imaging was as simple (Fig. 1). White light was transmitted as monochromatic light after filtrations through double crystal monochromators and the photon energy selected by tuning the monochromators. Photons with low energy were filtered by the filters in order to reduce the thermal load of the monochromators. The light penetrated the samples held on the stage which can move within a distance of 5 cm in six different directions and then arrived at the detector. All experimental data were acquired from BL13W1 at SSRF.
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Fig. 1. Diagram of the light-path. White light was transmitted as monochromatic light after passage through the double crystal monochromators. This light then penetrated the sample and was received by the detector.
2.2. Experimental Design 2.2.1. Simulation To determine the optimal imaging parameters, a simulation was designed. A polyethylene tube with three sections was used to simulate mouse coronary artery. It was 0.26 mm in inner diameter, and 0.5mm in outer diameter. The three sections of the tube were filled with a 100%, 75% and 50% lopamidol solution, respectively, as contrast agent. The iodine concentration of the lopamidol solution was about 0.37 g/ml. These three sections of tubing were fixed on the same board, and imaged together under synchrotron radiation at a photon energy of 26, 28, 30, 32, 33, 33.7, and 34 keV. The spatial resolution of the detector was set to 13 m, 26 m, and 52 m, respectively, at each energy state. 20 images for each group of parameters were taken and then analyzed: for each image, the C (contrast) value was calculated as the index for evaluating image quality. The C value is the ratio of Ib to Ia. Ib means the average grayscale value of this image and Ia means the average grayscale value of the tube region filled with contrast agent. The formula used for this calculation was Eq. (1): C=Ib/Ia
(1)
After analysis, the optimal energy of 33 keV, the concentration of lopamidol solution of 75% as contrast agent and spatial resolution of 26 m were determined. 2.2.2. Animal experiment Normal male KM mice (25-30 g weight) were used in the experiment. A tube (0.5 mm in outer diameter, Natsume Manufactory) was introduced to the aortic valve of anesthetized mice from the right carotid artery to perfuse iodine contrast agent (Fig. 2). After the intubation operation, mice were fixed on the object stage in a standing position facing the light source. During the irradiation, diluted lopamidol solution was injected into the mouse aorta by an injection pump at a speed of 10 ml/min in pulse mode. As the contrast agent was injected, a sequence of images from the area of interest was taken under the optimal conditions determined from the above simulation. Photon energy, exposure time, and mouse position were adjusted during the capture of each sequence of images. For the adjustment of energy, a larger light size was found to be more beneficial for the location of interest but inversely proportion to the photon energy. Therefore, the position of the coronary artery was first determined at a low energy of 30keV then adjusted to 33 keV for imaging. For optimizing exposure time, the real light flux was adjusted to avoid excessive exposure. For adjusting the mouse position, the mouse was turned forward or backward in a standing position to widen the region of the mouse thoracic cavity being imaged. Image pre-processing methods were used to further enhance image quality to overcome limitations imposed by experimental conditions.
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Fig. 2 Diagram of the mouse surgery. A tube (red) was inserted into the right carotid artery until it reached the aortic valve. The right carotid artery was then ligatured to avoid bleeding.
3. Results Using images acquired from the simulation, the degree of contrast between the contrast agent and background under different photon energies was compared (Fig. 3). There was no significant difference in contrast when the photon energy ranged from 26 to 32 keV, but the contrast significantly increased when the photon energy ranged from 33-34 keV. The degree of contrast between different concentrations of contrast agent solution and background was also compared (Fig. 4). The image quality achieved using a 100% lopamidol solution was close to that achieved using a 75% lopamidol solution.
Fig. 3 The contrast at different energy. The three curves reflect changes in contrast at different photon energies when the spatial resolution of the detector was 13 m, 26 m, and 52 m. The concentration of contrast agent used was 100%.
Fig. 4 The contrast at different concentrations of contrast agent. The spatial resolution of the detector was 13 m. The three curves reflect changes in contrast at different photon energies when the concentration of the contrast agent was 100%, 75%, and 50%, respectively.
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I
II
III
IV Fig. 5 The imaging results. Images were taken of the murine thoracic cavity near the 4th and 5th rib. (I) the original image; (II) the image after background subtraction; (III) the image after contrast enhancement; (IV) the image after both background subtraction and contrast enhancement. Point A indicates the bifurcation of the left anterior descending artery and the left circumflex artery. Point B indicates the ascending aorta and the left main coronary artery (shown between the 4th and 5th rib on the left). Point C (near the 5th rib on the right) indicates the right coronary artery.
Fig. 6 The degree of contrast was compared under different conditions.
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In Fig. 5(I), the image shows the left and right coronary artery, with the ribs noticeable against the background. Fig. 5(II) is the result of subtraction, and it shows that the difference in grayscale between the blood vessels and ribs has significantly increased. Fig. 5(III) and Fig. 5(IV) contrast-enhanced images of Fig. 5(I) and Fig. 5(II), respectively, were processed by histogram equalization. C´ values at sites A, B, and C were calculated (Fig. 5). The values represent the contrast from three sites against the surrounding region. The formula used to calculate these values was Eq. (2): C´=I´/Ia
(2)
Ia indicates the average grayscale of the specified region of contrast agent, and I’ indicates the average grayscale of the surrounding region. Contrast (C’) values for the three sites selected in the four images above were compared (Fig. 6). Contrast values of the image in Fig. 5(II), processed by correlation-based background subtraction, were slightly enhanced compared to the original image (Fig. 5(I)). After processing through contrast enhancement, the contrast value of the background subtracted image (Fig. 5(IV)) except site B was also better than the image without subtraction (Fig. 5(III)). Although the contrast at site B drops, it is not at a coronary region of interest and the contrast was sufficient. 4. Discussion 4.1. Effect of energy on image quality The results of our simulation indicate that the level of contrast from lopamidol solution is significantly increased when the photon energy reaches 33-34 keV. X-rays are greatly attenuated at an energy of 33.169 keV [19] due to significant photon absorption by iodine atoms (a critical energy called the K-edge of iodine atoms). However, as the photon energy increases, the available light spot size will be significantly reduced in the vertical direction, becoming only 4 mm at 33 keV while potentially reaching 5 mm at 30 keV. It is thus difficult to cover the whole coronary area if the energy is higher than 33 keV. As the smaller light size makes it harder to locate the coronary artery, the possible position of the coronary artery in animal experiments should first be identified at a low photon energy (with a large light size) then photon energy increased to acquire images with a higher contrast.
Fig. 7 DSA image of the mouse left anterior descending coronary artery (the site pointed by arrow).
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4.2. Effect of resolution on image quality The spatial resolution of the detectors is one of the most important factors affecting the quality of medical images; a higher resolution produces a higher image quality. However, for mouse coronary angiography, the temporal resolution is also an important factor that limits the image quality due to the high heart rate of mice (6-10 beats per second). With a spatial resolution of 0.1 mm and a temporal resolution of 25 f/s, Digital Subtraction Angiography (DSA) can display the left anterior descending coronary artery (Fig. 7). The DSA image, however, cannot meet the requirements of image analysis because it only contains 1-2 pixels (due to the narrow mouse coronary artery with a diameter of only 100-200 m). Therefore, a synchrotron imaging technique with high spatial resolution will produce better coronary angiograms from mice if the temporal resolution of the detectors is sufficiently high. For the same detector, the spatial resolution is inversely proportion to the temporal resolution, and a detector with both high spatial and temporal resolution requires a high light flux. The detector used in this experiment can take 10 images per second when its spatial resolution reaches its maximum of 13 m; thus only one image can be taken during one mouse cardiac cycle. Although this temporal resolution may meet the basic requirements of normal coronary angiography, it is unlikely to adapt to the complex changes in heart rate under pathological conditions. If the temporal resolution of the detector is increased to about 20 frames per second, its spatial resolution will be reduced to 26 m. Thus, at least 5 pixels can form, as the diameter of mouse left anterior descending artery is about 150 m. Further improvements in image analysis and detectors will improve detection of the edges of vessels. The successful imaging by DSA indicates that the coronary artery could be effectively dynamically imaged when the temporal resolution exceeds 25 frames per second. This technique specification has been realized at Spring-8 in Japan; thus research on coronary angiography under pathological conditions will be pursued. 4.3. Effect of contrast agent injection on image quality The murine coronary artery is too narrow to accommodate a tube, and the tube could only be positioned at the orifice of the coronary artery. Therefore the contrast agent must be injected at a high speed and in large amounts to ensure sufficient contrast agent is injected into the coronary artery. Unfortunately, it is impossible to perform a long-term injection of contrast agent at a high speed because of the high impendance caused by the viscous lopamidol solution and the narrow tube. Diluting the contrast agent will reduce the resistance during injection and help lengthen the injection time. Furthermore, in disease models of the future, drug injection will significantly dilute the contrast agent; thus it is necessary to determine how contrast dilution affects imaging. The results of a simulation (Fig. 4) indicate that a slight dilution of the contrast agent should not cause a reduction in image quality. The results also showed that the injection pump would stop working at an injecting speed of 4 ml/min for 12 s, 8 ml/min for 5 s, or 12 ml/min for 4 s due to the great resistance during injection. The longterm injection at high speed is difficult to maintain, although different pumps may have different capacities. Therefore, the concentration of the contrast agent was reduced to 75% in the experiment, and a pulse injection was performed to maintain the instant high speed and persistent injection during imaging.
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4.4. Improvement of background subtraction The contrast of the area of interest against the background of acquired images can be enhanced by image pre-processing techniques. It is beneficial to further image analysis on coronary images. Low grayscale regions, such as the skeleton (e.g., ribs), interfere with the region of interest in image analysis because the grayscale of the skeleton is similar to that of blood vessels filled with the contrast agent. Therefore, eliminating interference from the low grayscale background without affecting the contrast of vascular regions is beneficial to image analysis. The most conventional image subtraction involves subtracting the image taken before injecting contrast agent from the image taken after injection. However, this method will result in serious artifacts because of periodic changes of thoracic images caused by respiratory movements and the heartbeat of mice. In this experiment, “correlation-based background subtraction” was adopted to solve this problem. Images were taken as a sequence of dynamic images and divided into two groups: a pre-injection group and post-injection group. For each image in the post-injection group, its correlation to images in the pre-injection group was calculated and the pre-injection image that correlates the most was selected as the background of the post-injection image. After background subtraction, the images in the post-injection group formed a processed sequence of the coronary angiogram. According to comparisons (Fig. 6), the contrast of the region of interest will be slightly enhanced after correlation-based background subtraction, whether or not the image has been processed by contrast enhancement. Therefore, correlation-based background subtraction will not reduce the contrast of the vascular region or lower the image quality, instead successfully eliminating background. 5. Conclusion Ideal mouse coronary angiograms can be acquired by synchronous radiation, although the image quality depends on the imaging parameters. Using a photon energy of 33 keV for a blood vessel injected with lopamidol solution may produce an optimal contrast of grayscale. The images acquired would be difficult to analyze with a spatial resolution lower than 26 m or a temporal resolution lower than 20 f/s. The injection of contrast agent for mouse coronary angiography should be performed in pulse mode at high speed, and the concentration of lopamidol solution (as the contrast agent) should be diluted to 75%. An algorithm of correlation-based background subtraction and contrast enhancement was designed and employed to improve the quality of acquired images, thereby allowing the morphological coronary features to be precisely measured. The research here presents a method and optimal parameters for the effective and consistent acquisition of images of the mouse coronary artery in vivo. This research provides a methodology for physiological, pathological, and pharmacological research on the coronary arterial system and lays a foundation for further research on the early diagnosis and therapies for coronary diseases. References [1] [2]
P. Libby and P. Theroux, Pathophysiology of coronary artery disease, Circulation 111 (2005), 3481-8 G. Rioufol, G. Finet, I. Ginon, X. Andre-Fouet, R. Rossi, E. Vialle, E. Desjoyaux, G. Convert, J.F. Huret and A. Tabib, Multiple atherosclerotic plaque rupture in acute coronary syndrome - A three-vessel intravascular ultrasound study, Circulation 106 (2002), 804-8
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C.P. Cannon, W.S. Weintraub, L.A. Demopoulos, R. Vicari, M.J. Frey, N. Lakkis, F.J. Neumann, D.H. Robertson, P.T. DeLucca, P.M. DiBattiste, C.M. Gibson, E. Braunwald and T.T. Myocardial, Comparison of early invasive and conservative strategies in patients with unstable coronary syndromes treated with the glycoprotein IIb/IIIa inhibitor tirofiban, New England Journal of Medicine 344 (2001), 1879-87 D. Hasdai, S. Behar, L. Wallentin, N. Danchin, A.K. Gitt, E. Boersma, P.M. Fioretti, M.L. Simoons and A. Battler, A prospective survey of the characteristics, treatments and outcomes of patients with acute coronary syndromes in Europe and the Mediterranean basin - The Euro Heart Survey of Acute Coronary Syndromes (Euro Heart Survey ACS), Eur. Heart J. 23 (2002), 1190-201 W.E. Crusio, D. Goldowitz, A. Holmes and D. Wolfer, Standards for the publication of mouse mutant studies, Genes Brain Behav. 8 (2009), 1-4 D.P. Wolfer, W.E. Crusio and H.P. Lipp, Knockout mice: simple solutions to the problems of genetic background and flanking genes, Trends Neurosci 25 (2002), 336-40 A. Fouras, M.J. Kitchen, S. Dubsky, R.A. Lewis, S.B. Hooper and K. Hourigan, The past, present, and future of x-ray technology for in vivo imaging of function and form, J. Appl. Phys 105 (2009), 14 J.W. Thuroff, W. Hort and H. Lichti, Diameter of coronary arteries in 36 species of mammalian from mouse to giraffe, Basic Res Cardiol 79 (1984), 199-206 P. Liu, J.Q. Sun, J. Zhao, X.X. Liu, X. Gu, J. Li, T.Q. Xiao and L.X. Xu, Microvascular imaging using synchrotron radiation, J. Synchrot. Radiat 17 (2010), 517-21 R.E. Watson and M.L. Perlman, Seeing with a new light: synchrotron radiation, Science 199 (1977), 1295-302 M. Ando, E. Hashimoto, H. Hashizume, K. Hyodo, H. Inoue, T. Kunisada, A. Maksimenko, K. Mori, E. Rubenstein, J. Roberson, D. Shimao, H. Sugiyama, K. Takeda, F. Toyofuku, E. Ueno, K. Umetani, H. Wada and W. Pattanasiriwisawa, Clinical step onward with X-ray dark-field imaging and perspective view of medical applications of synchrotron radiation in Japan, Nuclear Instruments & Methods in Physics Research Section a-Accelerators Spectrometers Detectors and Associated Equipment 548 (2005), 1-16 B. Bertrand, F. Esteve, H. Elleaume, C. Nemoz, S. Fiedler, A. Bravin, G. Berruyer, T. Brochard, M. Renier, J. Machecourt, W. Thomlinson and J.F. Le Bas, Comparison of synchrotron radiation angiography with conventional angiography for the diagnosis of in-stent restenosis after percutaneous transluminal coronary angioplasty, Eur. Heart J. 26 (2005), 1284-91 S. Ohtsuka, Y. Sugishita, T. Takeda, Y. Itai, J. Tada, K. Hyodo and M. Ando, Dynamic intravenous coronary angiography using 2D monochromatic synchrotron radiation, Br. J. Radiol 72 (1999), 24-8 S. Ohtsuka, I. Yamaguchi, W. Jin, T. Takeda, K. Hyodo and M. Ando, Clinical program of intravenous coronary angiography using synchrotron radiation, Med. Imaging Technol 23(2005), 292-6 T. Yamashita, S. Kawashima, M. Ozaki, M. Namiki, T. Hirase, N. Inoue, K. Hirata, K. Umetani, K. Sugimura and M. Yokoyama, Mouse coronary angiograph using synchrotron radiation microangiography, Circulation 105 (2002), E3-E4 S. Matsushita, K. Hyodo, S. Akishima, F. Sato, T. Imazuru, M. Noma, Y. Hiramatsu, O. Shigeta and Y. Sakakibara, Coronary angiography in rats using synchrotron radiation, Proceedings 4th Workshop on Medical Applications of Synchrotron Radiation (MARS2004), pp 94-8 S. Matsushita, K. Hyodo, T. Imazuru, C. Tokunaga, F. Sato, Y. Enomoto, Y. Hiramatsu and Y. Sakakibara, The minimum coronary artery diameter in which coronary spasm can be identified by synchrotron radiation coronary angiography, Proceedings Conference on Medical Applications of Synchrotron Radiation (2008), pp S84-S8 J.T. Pearson, M.J. Jenkins, Y. Okura, D. Tani, D.O. Schwenke, K. Umetani, K. Kangawa and M. Shirai, Dynamic synchrotron imaging of the coronary vasculature in vivo in rat models of heart failure and diabetes, J. Physiol. Sci. 59 (2009), 326 R. Carr, Exotic sources of x-rays fro iodine k-edge angiography, Nuclear Instruments & Methods in Physics Research Section a-Accelerators Spectrometers Detectors and Associated Equipment 347 (1994),510-4
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Mouse Coronary Angiography In Vivo Using Synchrotron Radiation Lijun Xua,1, Andi Zhangb,1, Guohao Duc, Honglan Xiec and Ying Chena,d,* a
Department of Biomedical Engineering, School of Medicine, Shanghai Jiao Tong University, No.280, South Chongqing RD, Shanghai, China b Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, No.197, Ruijin No.2 RD, Shanghai, China c Shanghai Synchrotron Radiation Facility, No.239, Zhangheng RD, Shanghai, China d Med-X Research Institute, Shanghai Jiao Tong University, No.1954, Huashan RD, Shanghai, China
Abstract. Purpose: To establish a method for mouse coronary angiography in vivo using synchrotron radiation, which is essential for physiological and pathological research on coronary diseases. Methods: 1) The imaging parameters (e.g., photon energy, spatial resolution of the detector, and injection rate of contrast agent) optimal for the quality of acquired images in a simulation were determined. 2) Through animal experiments, the effectiveness of these optimal parameters and the repeatability of in vivo coronary angiography were verified. 3) An algorithm for background subtraction and contrast enhancement was designed and employed to compensate for the effects of interference and the effective information extracted used for diagnosing coronary disease. Results and conclusions: An optimal set of the imaging parameters was finally determined: photon energy of 33-34 keV, detector’s spatial resolution of 30 m or higher, image capture rate of 20 f/s or more, concentration of lopamidol solution of 75% as contrast agent and a pulse injection of contrast agent at a high rate. Keywords: Coronary angiography, Synchrotron radiation, In vivo, Optimization, Background subtraction
1. Introduction Acute coronary syndrome (ACS) manifests as chest pain and other resulted symptoms when the heart receives insufficient amounts of blood. Recent studies have shown that acute coronary syndrome commonly occurs when an atherosclerotic plaque fissures or ulcerates, precipitating thrombus formation [1, 2]. This results in sudden myocardial infarction or even sudden cardiac death. Therefore, many doctors and researchers have been seeking effective methods to allay or prevent ACS [3, 4]. Gene knockout models, easily made in mice and common for science experiments [5, 6], are widely used in pathological or pharmacological research on coronary diseases. On the other hand, medical imaging, more than any other method, can directly examine the pathological state of the heart, the occurrence of rash symptoms, and the effectiveness of medicinal treatment. Therefore, coronary angiography methods for mice may become effective tools for diagnosis, treatment, and research (patho1 *
Co-author, contributed equally to this work Corresponding author. E-mail: [email protected]
0959-2989/14/$27.50 © 2014 – IOS Press and the authors. All rights reserved
1342
L. Xu et al. / Mouse coronary angiography in vivo using synchrotron radiation
logical and pharmacological) of heart and coronary diseases. X-ray angiography is one of the most common clinical methods for examining and diagnosing cardiovascular disease [7]. It is performed by injecting a contrast agent into the vessel of interest then imaging the inside of the vessel through X-ray-based techniques. The diameter of a murine coronary artery is about 100-200 m [8] and the heart rate is about 6-10 beats per second. It is hard for conventional imaging techniques with limited spatial resolution or temporal resolution, such as DSA, ultrasound and MRI [9], to obtain diagnostic coronary information. Synchrotron radiation, whose use has been gradually developed in recent years, features high flux, monochromaticity, and near parallel geometry [10]. Images produced by synchrotron radiation contain both high spatial resolution and temporal resolution, and may show information unavailable from conventional imaging techniques, thus presenting a high potential in the field of medical imaging [11]. Although synchrotron-based coronary angiography has made a breakthrough in routine physical examinations [12-14], the development of synchrotron angiography in mice for pathological research is still in progress. Advancements in mouse coronary angiography using synchrotron radiation were reported by Yamashita in 2002 [15] and in 2005. Matsushita established a method of coronary angiography in rats using synchrotron radiation with a Langendorff apparatus [16]. Further research by Matsushita in 2008 indicated that the minimum diameter of blood vessels is about 50 to 100 m, which can be measured by synchrotron-based coronary angiography to identify coronary spasms [17]. In recent years, Pearson performed a series of physiological and pathological research on the rat heart with the Langendorff apparatus using synchrotron radiation [18]. The purpose of our research was to establish an effective imaging technique for mouse coronary angiography in vivo using synchrotron radiation. This technique offers high spatial and temporal resolution, and further work on image analysis and recognition can help establish methods for physiological and pathological research on coronary diseases. The experiments described here were performed on BL13W1 at the Shanghai Synchrotron Radiation Facility (SSRF). 2. Materials and Methods 2.1. Imaging Conditions SSRF is a third-generation synchrotron radiation light source. The energy of the storage ring is 3.5 GeV, the highest value in the medium-energy light source. The experiment was performed in station BL13W1, which can provide monochromatic light with an energy range of 8 to 72.5 keV and beam size of 45 mm (horizontal) * 5mm (vertical). In-line attenuation contrast imaging was taken in this experiment at a photon energy range of 28 to 33 keV. The maximum flux at the sample site was about 1*1010 phs/s/mm2, the distance from the source to the sample was 30 m, and the distance from the sample to the detector was 50 cm. The detector image array is 4008×2672 pixels, with a 12 bit depth capable of imaging at 30 frames per second and 13 microns pixels. The light-path of the in-line attenuation contrast imaging was as simple (Fig. 1). White light was transmitted as monochromatic light after filtrations through double crystal monochromators and the photon energy selected by tuning the monochromators. Photons with low energy were filtered by the filters in order to reduce the thermal load of the monochromators. The light penetrated the samples held on the stage which can move within a distance of 5 cm in six different directions and then arrived at the detector. All experimental data were acquired from BL13W1 at SSRF.
L. Xu et al. / Mouse coronary angiography in vivo using synchrotron radiation
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Fig. 1. Diagram of the light-path. White light was transmitted as monochromatic light after passage through the double crystal monochromators. This light then penetrated the sample and was received by the detector.
2.2. Experimental Design 2.2.1. Simulation To determine the optimal imaging parameters, a simulation was designed. A polyethylene tube with three sections was used to simulate mouse coronary artery. It was 0.26 mm in inner diameter, and 0.5mm in outer diameter. The three sections of the tube were filled with a 100%, 75% and 50% lopamidol solution, respectively, as contrast agent. The iodine concentration of the lopamidol solution was about 0.37 g/ml. These three sections of tubing were fixed on the same board, and imaged together under synchrotron radiation at a photon energy of 26, 28, 30, 32, 33, 33.7, and 34 keV. The spatial resolution of the detector was set to 13 m, 26 m, and 52 m, respectively, at each energy state. 20 images for each group of parameters were taken and then analyzed: for each image, the C (contrast) value was calculated as the index for evaluating image quality. The C value is the ratio of Ib to Ia. Ib means the average grayscale value of this image and Ia means the average grayscale value of the tube region filled with contrast agent. The formula used for this calculation was Eq. (1): C=Ib/Ia
(1)
After analysis, the optimal energy of 33 keV, the concentration of lopamidol solution of 75% as contrast agent and spatial resolution of 26 m were determined. 2.2.2. Animal experiment Normal male KM mice (25-30 g weight) were used in the experiment. A tube (0.5 mm in outer diameter, Natsume Manufactory) was introduced to the aortic valve of anesthetized mice from the right carotid artery to perfuse iodine contrast agent (Fig. 2). After the intubation operation, mice were fixed on the object stage in a standing position facing the light source. During the irradiation, diluted lopamidol solution was injected into the mouse aorta by an injection pump at a speed of 10 ml/min in pulse mode. As the contrast agent was injected, a sequence of images from the area of interest was taken under the optimal conditions determined from the above simulation. Photon energy, exposure time, and mouse position were adjusted during the capture of each sequence of images. For the adjustment of energy, a larger light size was found to be more beneficial for the location of interest but inversely proportion to the photon energy. Therefore, the position of the coronary artery was first determined at a low energy of 30keV then adjusted to 33 keV for imaging. For optimizing exposure time, the real light flux was adjusted to avoid excessive exposure. For adjusting the mouse position, the mouse was turned forward or backward in a standing position to widen the region of the mouse thoracic cavity being imaged. Image pre-processing methods were used to further enhance image quality to overcome limitations imposed by experimental conditions.
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Fig. 2 Diagram of the mouse surgery. A tube (red) was inserted into the right carotid artery until it reached the aortic valve. The right carotid artery was then ligatured to avoid bleeding.
3. Results Using images acquired from the simulation, the degree of contrast between the contrast agent and background under different photon energies was compared (Fig. 3). There was no significant difference in contrast when the photon energy ranged from 26 to 32 keV, but the contrast significantly increased when the photon energy ranged from 33-34 keV. The degree of contrast between different concentrations of contrast agent solution and background was also compared (Fig. 4). The image quality achieved using a 100% lopamidol solution was close to that achieved using a 75% lopamidol solution.
Fig. 3 The contrast at different energy. The three curves reflect changes in contrast at different photon energies when the spatial resolution of the detector was 13 m, 26 m, and 52 m. The concentration of contrast agent used was 100%.
Fig. 4 The contrast at different concentrations of contrast agent. The spatial resolution of the detector was 13 m. The three curves reflect changes in contrast at different photon energies when the concentration of the contrast agent was 100%, 75%, and 50%, respectively.
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I
II
III
IV Fig. 5 The imaging results. Images were taken of the murine thoracic cavity near the 4th and 5th rib. (I) the original image; (II) the image after background subtraction; (III) the image after contrast enhancement; (IV) the image after both background subtraction and contrast enhancement. Point A indicates the bifurcation of the left anterior descending artery and the left circumflex artery. Point B indicates the ascending aorta and the left main coronary artery (shown between the 4th and 5th rib on the left). Point C (near the 5th rib on the right) indicates the right coronary artery.
Fig. 6 The degree of contrast was compared under different conditions.
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In Fig. 5(I), the image shows the left and right coronary artery, with the ribs noticeable against the background. Fig. 5(II) is the result of subtraction, and it shows that the difference in grayscale between the blood vessels and ribs has significantly increased. Fig. 5(III) and Fig. 5(IV) contrast-enhanced images of Fig. 5(I) and Fig. 5(II), respectively, were processed by histogram equalization. C´ values at sites A, B, and C were calculated (Fig. 5). The values represent the contrast from three sites against the surrounding region. The formula used to calculate these values was Eq. (2): C´=I´/Ia
(2)
Ia indicates the average grayscale of the specified region of contrast agent, and I’ indicates the average grayscale of the surrounding region. Contrast (C’) values for the three sites selected in the four images above were compared (Fig. 6). Contrast values of the image in Fig. 5(II), processed by correlation-based background subtraction, were slightly enhanced compared to the original image (Fig. 5(I)). After processing through contrast enhancement, the contrast value of the background subtracted image (Fig. 5(IV)) except site B was also better than the image without subtraction (Fig. 5(III)). Although the contrast at site B drops, it is not at a coronary region of interest and the contrast was sufficient. 4. Discussion 4.1. Effect of energy on image quality The results of our simulation indicate that the level of contrast from lopamidol solution is significantly increased when the photon energy reaches 33-34 keV. X-rays are greatly attenuated at an energy of 33.169 keV [19] due to significant photon absorption by iodine atoms (a critical energy called the K-edge of iodine atoms). However, as the photon energy increases, the available light spot size will be significantly reduced in the vertical direction, becoming only 4 mm at 33 keV while potentially reaching 5 mm at 30 keV. It is thus difficult to cover the whole coronary area if the energy is higher than 33 keV. As the smaller light size makes it harder to locate the coronary artery, the possible position of the coronary artery in animal experiments should first be identified at a low photon energy (with a large light size) then photon energy increased to acquire images with a higher contrast.
Fig. 7 DSA image of the mouse left anterior descending coronary artery (the site pointed by arrow).
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4.2. Effect of resolution on image quality The spatial resolution of the detectors is one of the most important factors affecting the quality of medical images; a higher resolution produces a higher image quality. However, for mouse coronary angiography, the temporal resolution is also an important factor that limits the image quality due to the high heart rate of mice (6-10 beats per second). With a spatial resolution of 0.1 mm and a temporal resolution of 25 f/s, Digital Subtraction Angiography (DSA) can display the left anterior descending coronary artery (Fig. 7). The DSA image, however, cannot meet the requirements of image analysis because it only contains 1-2 pixels (due to the narrow mouse coronary artery with a diameter of only 100-200 m). Therefore, a synchrotron imaging technique with high spatial resolution will produce better coronary angiograms from mice if the temporal resolution of the detectors is sufficiently high. For the same detector, the spatial resolution is inversely proportion to the temporal resolution, and a detector with both high spatial and temporal resolution requires a high light flux. The detector used in this experiment can take 10 images per second when its spatial resolution reaches its maximum of 13 m; thus only one image can be taken during one mouse cardiac cycle. Although this temporal resolution may meet the basic requirements of normal coronary angiography, it is unlikely to adapt to the complex changes in heart rate under pathological conditions. If the temporal resolution of the detector is increased to about 20 frames per second, its spatial resolution will be reduced to 26 m. Thus, at least 5 pixels can form, as the diameter of mouse left anterior descending artery is about 150 m. Further improvements in image analysis and detectors will improve detection of the edges of vessels. The successful imaging by DSA indicates that the coronary artery could be effectively dynamically imaged when the temporal resolution exceeds 25 frames per second. This technique specification has been realized at Spring-8 in Japan; thus research on coronary angiography under pathological conditions will be pursued. 4.3. Effect of contrast agent injection on image quality The murine coronary artery is too narrow to accommodate a tube, and the tube could only be positioned at the orifice of the coronary artery. Therefore the contrast agent must be injected at a high speed and in large amounts to ensure sufficient contrast agent is injected into the coronary artery. Unfortunately, it is impossible to perform a long-term injection of contrast agent at a high speed because of the high impendance caused by the viscous lopamidol solution and the narrow tube. Diluting the contrast agent will reduce the resistance during injection and help lengthen the injection time. Furthermore, in disease models of the future, drug injection will significantly dilute the contrast agent; thus it is necessary to determine how contrast dilution affects imaging. The results of a simulation (Fig. 4) indicate that a slight dilution of the contrast agent should not cause a reduction in image quality. The results also showed that the injection pump would stop working at an injecting speed of 4 ml/min for 12 s, 8 ml/min for 5 s, or 12 ml/min for 4 s due to the great resistance during injection. The longterm injection at high speed is difficult to maintain, although different pumps may have different capacities. Therefore, the concentration of the contrast agent was reduced to 75% in the experiment, and a pulse injection was performed to maintain the instant high speed and persistent injection during imaging.
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4.4. Improvement of background subtraction The contrast of the area of interest against the background of acquired images can be enhanced by image pre-processing techniques. It is beneficial to further image analysis on coronary images. Low grayscale regions, such as the skeleton (e.g., ribs), interfere with the region of interest in image analysis because the grayscale of the skeleton is similar to that of blood vessels filled with the contrast agent. Therefore, eliminating interference from the low grayscale background without affecting the contrast of vascular regions is beneficial to image analysis. The most conventional image subtraction involves subtracting the image taken before injecting contrast agent from the image taken after injection. However, this method will result in serious artifacts because of periodic changes of thoracic images caused by respiratory movements and the heartbeat of mice. In this experiment, “correlation-based background subtraction” was adopted to solve this problem. Images were taken as a sequence of dynamic images and divided into two groups: a pre-injection group and post-injection group. For each image in the post-injection group, its correlation to images in the pre-injection group was calculated and the pre-injection image that correlates the most was selected as the background of the post-injection image. After background subtraction, the images in the post-injection group formed a processed sequence of the coronary angiogram. According to comparisons (Fig. 6), the contrast of the region of interest will be slightly enhanced after correlation-based background subtraction, whether or not the image has been processed by contrast enhancement. Therefore, correlation-based background subtraction will not reduce the contrast of the vascular region or lower the image quality, instead successfully eliminating background. 5. Conclusion Ideal mouse coronary angiograms can be acquired by synchronous radiation, although the image quality depends on the imaging parameters. Using a photon energy of 33 keV for a blood vessel injected with lopamidol solution may produce an optimal contrast of grayscale. The images acquired would be difficult to analyze with a spatial resolution lower than 26 m or a temporal resolution lower than 20 f/s. The injection of contrast agent for mouse coronary angiography should be performed in pulse mode at high speed, and the concentration of lopamidol solution (as the contrast agent) should be diluted to 75%. An algorithm of correlation-based background subtraction and contrast enhancement was designed and employed to improve the quality of acquired images, thereby allowing the morphological coronary features to be precisely measured. The research here presents a method and optimal parameters for the effective and consistent acquisition of images of the mouse coronary artery in vivo. This research provides a methodology for physiological, pathological, and pharmacological research on the coronary arterial system and lays a foundation for further research on the early diagnosis and therapies for coronary diseases. References [1] [2]
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