Multiple-contrast X-ray micro-CT visualization of colon malformations and tumours in situ in living mice

The development of new therapeutic approaches against colorectal cancer requires preclinical studies in mice. In vivo imaging could greatly facilitate these trials, but the small size of the animals is a major limitation for the direct visualization of intestinal tissue. Here we report a method of in vivo imaging of the mouse intestine based on X-ray micro-computed tomography using multiple contrast agents. This method was validated in the model of non-cancerous polyp-like heteroplasia that spontaneously develops in the caecum area of Cdx2+/- mutant mice and in the model of colon adenocarcinoma induced by administration of the chemical carcinogen azoxymethane. As a simple and non-invasive method, multiple-contrast X-ray micro-computed tomography is appropriate for pre-clinical studies of intestinal diseases in living mice.     

Small animal micro-CT colonography

Microcomputed tomography colonography (mCTC) is a new method for detecting colonic tumors in living animals and estimating their volume, which allows investigators to determine the spontaneous fate of individually annotated tumors as well as their response to chemotherapeutics. This imaging platform was developed using the Min mouse, but is applicable to any murine model of human colorectal cancer. MicroCT is capable of 20 micron resolution, however, 100 microns is sufficient for this application. Scan quality is primarily dependent on animal preparation with the most critical parameters being proper anesthesia, bowel cleansing, and sufficient insufflation. The detection of colonic tumors is possible by both 2D and 3D rendering of image data. Tumor volume is estimated using a semi-automated five-step process which is based on three algorithms within the Amira software package. The estimates are precise, accurate and reproducible enabling changes in volume as small as 16% to be readily observed. Confirmation of mCTC observations by gross examination and histology is sometimes useful in this otherwise non-invasive protocol. Finally, mCTC is compared to other newly developed small animal imaging platforms including microMRI and microoptical colonoscopy. A major advantage of these platforms is that investigators can be perform longitudinal studies, which often have much greater statistical power than traditional cross-sectional studies; consequently, fewer animals are required for testing.     

Dual-energy micro-CT of the rodent lung

The purpose of this work is to investigate the use of dual-energy micro-computed tomography (CT) for the estimation of vascular, tissue, and air fractions in rodent lungs using a postreconstruction three material decomposition method. Using simulations, we have estimated the accuracy limits of the decomposition for realistic micro-CT noise levels. Next, we performed experiments involving ex vivo lung imaging in which intact rat lungs were carefully removed from the thorax, injected with an iodine-based contrast agent, and then inflated with different volumes of air (n = 2). Finally, we performed in vivo imaging studies in C57BL/6 mice (n = 5) using fast prospective respiratory gating in end inspiration and end expiration for three different levels of positive end expiratory pressure (PEEP). Before imaging, mice were injected with a liposomal blood pool contrast agent. The three-dimensional air, tissue, and blood fraction maps were computed and analyzed. The results indicate that separation and volume estimation of the three material components of the lungs are possible. The mean accuracy values for air, blood, and tissue were 93, 93, and 90%, respectively. The absolute accuracy in determining all fraction materials was 91.6%. The coefficient of variation was small (2.5%) indicating good repeatability. The minimum difference that we could detect in material fractions was 15%. As expected, an increase in PEEP levels for the living mouse resulted in statistically significant increases in air fractions at end expiration but no significant changes at end inspiration. Our method has applicability in preclinical pulmonary studies where changes in lung structure and gas volume as a result of lung injury, environmental exposures, or drug bioactivity would have important physiological implications.     

4-D micro-CT of the mouse heart

PURPOSE: Demonstrate noninvasive imaging methods for in vivo characterization of cardiac structure and function in mice using a micro-CT system that provides high photon fluence rate and integrated motion control. MATERIALS AND METHODS: Simultaneous cardiac- and respiratory-gated micro-CT was performed in C57BL/6 mice during constant intravenous infusion of a conventional iodinated contrast agent (Isovue-370), and after a single intravenous injection of a blood pool contrast agent (Fenestra VC). Multiple phases of the cardiac cycle were reconstructed with contrast to noise and spatial resolution sufficient for quantitative assessment of cardiac function. RESULTS: Contrast enhancement with Isovue-370 increased over time with a maximum of approximately 500 HU (aorta) and 900 HU (kidney cortex). Fenestra VC provided more constant enhancement over 3 hr, with maximum enhancement of approximately 620 HU (aorta) and approximately 90 HU (kidney cortex). The maximum enhancement difference between blood and myocardium in the heart was approximately 250 HU for Isovue-370 and approximately 500 HU for Fenestra VC. In mice with Fenestra VC, volumetric measurements of the left ventricle were performed and cardiac function was estimated by ejection fraction, stroke volume, and cardiac output. CONCLUSION: Image quality with Fenestra VC was sufficient for morphological and functional studies required for a standardized method of cardiac phenotyping of the mouse.     

Vascular imaging in small rodents using micro-CT

In vivo animal models of neoplasm, stroke, subarachnoid hemorrhage, and other diseases involving alterations in vessel anatomy and diameter, require a fast and easy-to-use imaging tool that captures anatomical structure and biologic function data. Micro-computed tomography angiography (μCTA) offers high spatial and temporal resolution and is suitable to perform this task. However, conducting μCTA in small rodents, especially in mice, requires a high degree of accuracy and precision. This article describes a setup for in vivo μCTA in mice using both a bolus technique with a conventional contrast agent, as well as, angiography with a blood-pool contrast agent. Our setup in mice is at isotropic resolutions up to 16 μm with scanning times less than 1 min. The described protocol also addresses some of the technical challenges associated with the imaging of vascular structures in mice models.     

Application of micro-CT in small animal imaging

Over the past decade, the number of publications using micro-computed tomography (μCT) imaging in preclinical in vivo studies has risen exponentially. Higher spatial and temporal resolution are the key technical advancements that have allowed researchers to capture increasingly detailed anatomical images of small animals and to monitor the progression of disease in small animal models. The purpose of this review is to present the technical aspects of μCT, as well as current research applications. Our objectives are threefold: to familiarize the reader with the basics of μCT techniques; to present the type of experimental designs currently used; and to highlight limitations, future directions, in μCT-scanner research applications, and experimental methods. As a first step we present different μCT setups and components, as well as image contrast generation principles. We then present experimental approaches in order of the evaluated organ system. Finally, we provide a short summary of some of the technical limitations of μCT imaging and discuss potential future developments in μCT-scanner techniques and experimental setups.     

Small-animal imaging using clinical positron emission tomography/computed tomography and super-resolution

Considering the high cost of dedicated small-animal positron emission tomography/computed tomography (PET/CT), an acceptable alternative in many situations might be clinical PET/CT. However, spatial resolution and image quality are of concern. The utility of clinical PET/CT for small-animal research and image quality improvements from super-resolution (spatial subsampling) were investigated. National Electrical Manufacturers Association (NEMA) NU 4 phantom and mouse data were acquired with a clinical PET/CT scanner, as both conventional static and stepped scans. Static scans were reconstructed with and without point spread function (PSF) modeling. Stepped images were postprocessed with iterative deconvolution to produce super-resolution images. Image quality was markedly improved using the super-resolution technique, avoiding certain artifacts produced by PSF modeling. The 2 mm rod of the NU 4 phantom was visualized with high contrast, and the major structures of the mouse were well resolved. Although not a perfect substitute for a state-of-the-art small-animal PET/CT scanner, a clinical PET/CT scanner with super-resolution produces acceptable small-animal image quality for many preclinical research studies.     

Induction of radio-adaptive response in colony formation by low dose X-ray irradiation.

In this study, we examined the induction of a radio-adaptive response to cell death using a colony formation test in m5S, G401.2/6TG.1 and HeLa cells. When m5S cells were subjected to priming irradiation of 0.05 to approximately 0.15 Gy 4 hr before being irradiated with 4.5 Gy, the survival ratios increased significantly to 39 to approximately 42%. The priming irradiation effect was also observed when G401.2/6TG.1 cells were subjected to priming irradiation of 0.025 to approximately 0.1 Gy 4 hr before being irradiated with 0.8 Gy. This effect showed a two-phasic characteristic, where the first peak was reached at 0.025 Gy, and the second peak was reached at 0.075 Gy. The first peak showed a survival ratio of 56%, while the second peak was at 55%. However, in HeLa cells, this priming irradiation effect was not observed. These results indicated that induction of the radio-adaptive response did not depend on whether cells are normal or cancerous. One of the differences in these cells is that m5S and G401.2/6TG.1 cells have gap-junctional intercellular communication, but HeLa cells do not. Induction of the radio-adaptive response may be related to gap-junctional intercellular communication.     

A new boring bryozoan from the Middle Jurassic of the Moscow Region and its micro-CT research

A new boring ctenostome bryozoan without calcareous skeleton, Orbignyopora opulenta sp. nov. (Vesiculariida, Eurystomata), is described from deposits of the Middle Callovian of the Moscow Region. A pioneering micro-ct examination of a colony of this species reveals its diagnostic features hidden in the shell substratum: shape, size, and distinctive features in the budding of zooids and stolons, and also tubular processes (tubulets) developed on the frontal surface of autozooids. The diagnosis of the genus Orbignyopora is added by dimorphism of autozooids (A-zooids and B-zooids) and spindly kenozooids.     

A micro-CT approach for determination of insect respiratory volume

Variation in the morphology of the insect tracheal system can strongly affect respiratory physiology, with implications for everything from pest control to evolution of insect body size. However, the small size of most insects has made measuring the morphology of their tracheal systems difficult. Historical approaches including light microscopy and scanning and transmission electron microscopy (SEM, TEM) are technically difficult, labor intensive, and can introduce preparation artifacts. More recently, synchrotron X-ray microtomography (SR-μCT) has allowed for detailed analysis of tracheal morphology of diverse insects. However, linear accelerators required for SR-μCT are not readily available, making the approach unavailable for most labs. Recent advancements in microcomputed tomography (μCT) have made possible fine resolution of internal morphology of very small insects. However, μCT has never been used to quantify insect tracheal system dimensions. We measured respiratory volume of a grasshopper (Schistocerca americana) by analysis of high resolution μCT scans. Volume estimates from μCT closely matched volume estimates by water displacement as well as literature estimates for this species. The μCT approach may thus provide a widely available, cost-effective, and straightforward approach to characterizing the internal morphology of insect respiratory systems.     

In vivo respiratory-gated micro-CT imaging in small-animal oncology models

Micro-computed tomography(micro-CT) is becoming an accepted research tool for the noninvasive examination of laboratory animals such as mice and rats, but to date, in vivo scanning has largely been limited to the evaluation of skeletal tissues. We use a commercially available micro-CT device to perform respiratory gated in vivo acquisitions suitable for thoracic imaging. The instrument is described, along with the scan protocol and animal preparation techniques. Preliminary results confirm that lung tumors as small as 1 mm in diameter are visible in vivo with these methods. Radiation dose was evaluated using several approaches, and was found to be approximately 0.15 Gy for this respiratory-gated micro-CT imaging protocol. The combination of high-resolution CT imaging and respiratory-gated acquisitions appears well-suited to serial in vivo scanning.     

A novel 3D mouse embryo atlas based on micro-CT

The goal of the International Mouse Phenotyping Consortium (IMPC) is to phenotype targeted knockout mouse strains throughout the whole mouse genome (23,000 genes) by 2021. A significant percentage of the generated mice will be embryonic lethal; therefore, phenotyping methods tuned to the mouse embryo are needed. Methods that are robust, quantitative, automated and high-throughput are attractive owing to the numbers of mice involved. Three-dimensional (3D) imaging is a useful method for characterizing morphological phenotypes. However, tools to automatically quantify morphological information of mouse embryos from 3D imaging have not been fully developed. We present a representative mouse embryo average 3D atlas comprising micro-CT images of 35 individual C57BL/6J mouse embryos at 15.5 days post-coitum. The 35 micro-CT images were registered into a consensus average image with our automated image registration software and 48 anatomical structures were segmented manually. We report the mean and variation in volumes for each of the 48 segmented structures. Mouse organ volumes vary by 2.6-4.2% on a linear scale when normalized to whole body volume. A power analysis of the volume data reports that a 9-14% volume difference can be detected between two classes of mice with sample sizes of eight. This resource will be crucial in establishing baseline anatomical phenotypic measurements for the assessment of mutant mouse phenotypes, as any future mutant embryo image can be registered to the atlas and subsequent organ volumes calculated automatically.     

荧光素酶标的人肝癌细胞裸鼠异种移植瘤模型的生物发光成像和PET-CT成像比较

目的利用荧光素酶基因标记的人肝癌细胞株BEL-7402建立裸鼠肝原位移植模型,及小鼠肝原位移植模型的生物发光和小动物PET-CT成像的比较。方法构建表达荧光素酶基因的真核表达载体并将其转入人肝癌细胞BEL-7402,经梯度浓度G418筛选获得稳定表达荧光素酶基因的细胞克隆并扩大培养。BALB/cA-nu裸鼠肝门静脉接种5×105个发光细胞使其成瘤,活体荧光成像和小动物PET-CT成像系统观察肿瘤的生长情况。结果获得了稳定表达Luc的人肝癌细胞株,将其接种到裸鼠体内,活体荧光成像系统观察发现能够成瘤,小动物PET-CT影像观察发现小鼠肝脏边缘对18 F-FDG有高摄取区域。结论利用荧光素酶基因标记的人肝癌细胞BEL-7402成功建立了原位肝癌裸鼠模型,小动物活体成像结合小动物PET-CT技术为原位肿瘤模型的建立提供了一种新的可靠的技术,为进一步研究肝癌生长转移机制和药物开发提供了新的有用工具。