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- Materials and Methods
X-ray computed tomography (CT) has been used for diagnoses of human pancreatic cancer. Although micro-CT is a useful approach to evaluate macromorphology of organs/tissue also in animal models, reports on pancreatic tumors are limited. In this study, the utility of micro-CT was assessed in characterizing chemically induced pancreatic tumors in Syrian hamsters. Hamsters treated with or without N-nitrosobis(2-oxopropyl)amine (BOP) were injected with the antispasmodic agent, scopolamine butylbromide, and contrast agents, 5 or 10 mL/kg body weight of iopamidol or Fenestra VC at 18–38 weeks, then examined by micro-CT scanning with a respiratory gating system. Both peristaltic and respiratory movements were substantially suppressed by the combination of scopolamine butylbromide treatment and the respiratory gating system, resulting in improvements of image qualities. Iopamidol clearly visualized the pancreatic parenchyma and contrasted the margins among the pancreas and other abdominal organs/tissue. Meanwhile Fenestra VC predominantly contrasted abdominal vascular systems, but the margins among pancreas and other organs/tissue remained obscure. Six pancreatic tumors of 4–13 mm in diameter were detected in four of 15 animals, but not the five tumors of 1–4 mm in diameter. The inner tumor images were heterogeneously or uniformly visualized by iopamidol and Fenestra VC. Overall, iopamidol could clearly contrast between pancreatic parenchyma and the tumors as compared with Fenestra VC. All tumors confirmed were histopathologically diagnosed as pancreatic ductal adenocarcinomas. Thus, micro-CT could be useful to evaluate the carcinogenic processes and preventive methods of pancreatic cancer in hamsters and to assess the novel contrast agents for detection of small pancreatic cancer in humans. (Cancer Sci 2010)
Pancreatic cancer is the fifth cause of cancer death in Japan and ranks high in mortality among developed countries.(1,2) Because of the difficulty in detecting pancreatic cancer in early operable stages, and because of the lack of any curative treatment approaches other than complete surgical removal, 5-year relative survival rate is <6%.(3,4) Therefore, for improvement of outcome, future strategies for early diagnosis of pancreatic cancer should aim at diagnosing most pancreatic cancers before they grow 20 mm in size.(5,6)
X-ray computed tomography (CT) has been widely used in detection and evaluation of pancreatic tumor progression and metastasis during clinical treatment. Recently, the CT devices have been markedly improved in their volume coverage speed, longitudinal resolution, and quality of three-dimensional reformations. In addition, dynamic CT scanning has been developed, in which CT-images of pancreas can be obtained for a short time with breath holding. This method reveals some adequate contrasts between pancreatic parenchyma and neoplastic lesions.(7) However, detectability of small pancreatic cancer, especially those <10 mm in size, has not yet been satisfactory.(8–10) Further amelioration of contrasting technologies that differentiate between tumors and pancreatic parenchyma is desirable to detect small pancreatic cancer.
Micro-CT is a useful tool for monitoring tumor development in living animal models, for example utility has been reported for detection of metastatic foci of xenograft rodent models(11,12) and periodic measurement of sizes in lung tumors and their growth overtime in a chemically induced carcinogenesis model.(13) In addition, respiratory gating systems appreciably resolve the thoracic movements in lung imaging.(11,13) However, the utility of micro-CT has been limited to several organs/tissue harboring distant X-ray adsorption ranges from surrounding tissue. The X-ray adsorption range is too close among abdominal organs/tissue to observe fine structures, and not only respiratory but also intestinal peristaltic movement interferes with CT imaging. Because of these limitations, pancreatic morphology and tumor imaging have not yet been reported in experimental animals.
In the present study, hamster pancreas and chemically induced pancreatic tumors were macromorphologically investigated by micro-CT with two characteristic contrast agents, iopamidol, which is a nonionic water-soluble iodine contrast agent typically used for clinical evaluations, and Fenestra VC, which is a polyiodinated lipid emulsion with blood pool properties developed for small animals.(14) The results obtained in the present study should provide basic information for CT-imaging of the pancreas and pancreatic tumor macromorphology and location in hamsters.
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- Materials and Methods
In the present study, pancreatic morphology and chemically induced pancreatic tumors in Syrian hamsters were shown to be detectable by micro-CT. The Syrian hamster model with BOP has been widely applied as an animal model for the study of human pancreatic ductal carcinogenesis and data showing morphological details of both the early and advanced lesions have been reported.(15–17)
In order to substantially suppress the respiratory and the peristaltic movements during CT scanning, the antispasmodic agent, scopolamine butylbromide, was administered in combination with the respiratory gating system. The respiratory gating system improved and reduced motion artifacts caused by respiratory movements on the present scanning parameter. The images of abdomen without scopolamine butylbromide treatment were not clearly visible, especially at the edge of organs/tissue, as compared with the images using scopolamine butylbromide (data not shown). The 5 mg/kg BW dose of scopolamine butylbromide is about 15 times higher than that in human cases. No toxicological data of scopolamine butylbromide in hamsters is available, but lethal dose 50% (LD50) of scopolamine butylbromide in an intravenous administration study in mice was estimated to 20 mg/kg BW (manufacturer’s in-house data), which is four times higher than the present dose. Therefore, it is likely that a single or short-term repeated intraperitoneal administration of scopolamine butylbromide at the present dose might be safe for administration. Although causes of the difference in adequate doses between hamsters and humans are unclear, application of both scopolamine butylbromide and the respiratory gating system is considered to be a useful method for abdominal imaging in animal models.
In the present study, two different types of abdominal organs/tissue and the pancreatic tumor images, which reflected the characteristics of iopamidol and Fenestra VC, were obtained by micro-CT scanning. In living animals, iopamidol clearly enhanced the visibility of major abdominal organs/tissue parenchyma, and simultaneously the kidneys and urinary tract were prominently contrasted and the radial white linear artifacts were seen in the regions. However, the contrasts among organs/tissue were thought to be attenuated by renal excretion during micro-CT scanning. In a sacrificed animal, the parenchyma and the edge of pancreas and other organs/tissue were clearly visible and Ao and IVC were also visualized as compared with living animals. The artifacts by iopamidol which appeared in living animals were not observed in the sacrificed animals. Assessments of such contrast agents, which are excreted rapidly, by using sacrificed animals may also be useful in addition to using living animals. On the other hand, Fenestra VC predominantly increased the visibility of major abdominal vascular systems and was likely retained during micro-CT scanning for 40 min. Ford et al.(18) reported that contrasts in blood vessels and several abdominal organs in mice were retained for 24 h after injection of Fenestra VC. The present study also supported the benefits of Fenestra VC to vascular systems imaging when scanning for longer periods of time.
Most pancreatic tumor imaging with contrast agents such as nonionic iodine materials including iopamidol typically appeared as a hypoattenuating mass relative to the pancreatic parenchyma in humans.(19) The same is true with the case of BOP-treated hamsters. However the contrast between tumors and parenchyma in hamster pancreas was slightly inferior to that in humans, despite the higher dose of iopamidol than the manufacturer’s recommendations for humans. It is implied that the difference in effects of nonionic contrast agents on humans and hamsters is the rapid clearance of iopamidol during image scanning, resulting in attenuation of the contrast of pancreatic parenchyma. From the viewpoint of toxicological effects of such contrast agents, repeated intravenous administrations of iopamidol for 5 weeks in rats at a dose of 2 g/kg BW iodine equivalent, which is almost similar to the present dose (1.85 g/kg BW iodine equivalent), did not show remarkable influences on body weights, food intakes, and hematological parameters (manufacturer’s in-house data). Therefore, it is likely that the present doses of each agent under the Isoflurane anesthesia with the use of respiratory gating systems might be safe in single or short-term repeated administration studies. However, the influence of repeated treatment with iopamidol on general toxicities and pancreatic carcinogenesis in hamsters is still unclear. Further detailed analyses are be required to elucidate these points. Baron reported that differences of at least 10–15 CT value (HU; Hounsfield unit) are required for visual detection of the tumor.(20) In other words, higher contrasts between tumor and parenchyma images by micro-CT are required for differentiating histomorphological changes in the tumors as compared with pancreatic parenchyma. In the present study, all the tumors observed by micro-CT were histopathologically diagnosed as pancreatic ductal carcinomas, which are also the frequently observed pancreatic carcinoma type in humans.
Micro-CT with contrast agents enhanced the imaging of BOP-induced pancreatic tumors over 4 mm in diameter. However, carcinoma-localized intralobules were not fully detected by micro-CT. The reason for this might also be due to insufficient contrast between the pancreatic tumor and parenchyma. Recently, Hainfeld et al.(21) reported that gold nanoparticles with higher X-ray absorptive power than iodine-based agents may be useful as a novel X-ray contrast agent. Further studies using powerful X-ray absorptive agents may shed light on the limitations of imaging with smaller sized pancreatic tumors. For evaluations of novel contrast agents, X-ray micro-CT imaging systems with BOP-induced pancreatic carcinogenesis models could be adequate.
In the present study, we focused on the imaging of both pancreatic morphology and BOP-induced pancreatic tumors at one time point using two types of contrast agents. Time-dependent imaging of pancreatic tumors might also be useful for evaluations of the tumor development and preventives for pancreatic carcinogenesis. Overall, iopamidol could clearly contrast between pancreatic parenchyma and the tumors as compared with Fenestra VC.
In conclusion, our results provided evidence that respiratory-gated micro-CT scanning of hamsters has potential as a method for evaluating changes in the growth of pancreatic tumors. These studies on scanning and pancreatic carcinogenesis model systems may also be useful for evaluation of newly developing contrast agents for pancreatic tumors. Development of novel contrast agents in combination with the improvement of devices may lead to detection of smaller pancreatic tumors and precancerous lesions.