Characterization of Transgenic NSG-SGM3 Mouse Model of Precision Radiation-Induced Chronic Hyposalivation.

Regenerative medicine holds promise to cure radiation-induced salivary hypofunction, a chronic side effect in patients with head and neck cancers, therefore reliable preclinical models for salivary regenerative outcome will promote progress towards therapies. In this study, our objective was to develop a cone beam computed tomography-guided precision ionizing radiation-induced preclinical model of chronic hyposalivation using immunodeficient NSGSGM3 mice. Using a Schirmer's test based sialagogue-stimulated saliva flow kinetic measurement method, we demonstrated significant differences in hyposalivation specific to age, sex, precision-radiation dose over a chronic (6 months) timeline. NSG-SMG3 mice tolerated doses from 2.5 Gy up to 7.5 Gy. Interestingly, 5-7.5 Gy had similar effects on stimulated-saliva flow (∼50% reduction in young female at 6 months after precision irradiation over sham-treated controls), however, >5 Gy led to chronic alopecia. Different groups demonstrated characteristic saliva fluctuations early on, but after 5 months all groups nearly stabilized stimulated-saliva flow with low-inter-mouse variation within each group. Further characterization revealed precision-radiation-induced glandular shrinkage, hypocellularization, gland-specific loss of functional acinar and glandular cells in all major salivary glands replicating features of human salivary hypofunction. This model will aid investigation of human cell-based salivary regenerative therapies.

Imaging and Dosimetry Study of Inter-fraction Setup Error in a Murine Xenograft Flank Tumor Radiation Model.

Modern small animal irradiation platforms provide for accurate delivery of radiation under 3D image guidance. However, leveraging these improvements currently comes at the cost of lower-throughput experimentation. Herein, we characterized setup accuracy and dosimetric robustness for mock/sham irradiation of a murine xenograft flank tumor model using the X-RAD SmART+ with the vendor-supplied Monte Carlo (MC) treatment planning system (SmART ATP). The chosen beam arrangement was parallel-opposing using a 20 mm square collimator, aligned off-axis for ipsilateral lung sparing. Using a cohort of five mice imaged with cone beam computed tomography (CBCT) over five consecutive mock-irradiation fractions, we compared inter-fraction setup variability resulting from a vendor-supplied multi-purpose bed with anesthesia nose cone with a more complicated immobilization solution with an integrated bite block with nose cone and Styrofoam platform. A hypothetical "high-throughput" image-guidance scenario was investigated, wherein the day 1 stage coordinates (resulting from CBCT guidance) were applied on days 2-5. Daily inter-fraction setup errors were evaluated per specimen (days 2-5) using CBCT-derived offsets from day 1 stage coordinates. Using the CBCT images and Monte Carlo dose calculation, 3D dosimetric plan robustness was evaluated for the vendor-supplied immobilization solution, for both the high-throughput guidance scenario as well as for use of daily CBCT-based alignment. Inter-fraction setup offset magnitude was 3.6 (±1.5) mm for the vendor-supplied immobilization compared to 3.3 (±1.8) mm for the more complicated solution. For the vendor-supplied immobilization, we found that daily CBCT was needed to adequately cover the flank tumors dosimetrically, given our chosen treatment approach.

Quantifying the setup uncertainty of a stereotactic murine micro-image guided radiation therapy system.

OBJECTIVE:: Investigate the reproducibility of murine cranial positioning using solely a stereotactic stage, and quantify the potential improvements from the on-board image guidance of the X-RAD SmART irradiator. METHODS:: For intermouse reproducibility, athymic nude mice (N = 5, ×4 groups) were cranially fixed on a stereotactic stage. Each mouse was imaged via cone-beam CT (CBCT). A virtual isocenter target was placed in the brain, the stage shifted to that target, and the couch positions recorded. The mouse was removed from the stage and this process repeated twice (N=60 measurements). The first acquired CBCT coordinates (within each group of five mice) were used to define "stereotactic couch coordinates." CBCT shifts were calculated to quantify the accuracy of setup based on couch coordinates alone. For intramouse reproducibility, C57BL/6 mice (N=4) were imaged daily for 7 days. Each mouse had individual stereotactic coordinates defined from their first day of CBCT localization, and positional shifts required on the six subsequent days of imaging were quantified (N = 24 measurements). RESULTS:: The mean vector shift between stereotactic setup and CBCT alignment for inter and intramouse analysis was 0.78 ± 0.27 mm and 0.82 ± 0.34 mm, respectively. CONCLUSION:: Cranial irradiation that can permit positional uncertainties on the order of a millimeter can rely solely on stereotactic coordinates derived from a single daily CBCT. Irradiations of subregions requiring submillimeter accuracy require daily image guidance for each mouse. ADVANCES IN KNOWLEDGE:: This is the first investigation of stereotactic reproducibility using the X-RAD SmART and it suggests a method for increased efficiency in high-throughput experiments.

Impact of breathing 100% oxygen on radiation-induced cognitive impairment.

Future space missions are expected to include increased extravehicular activities (EVAs) during which astronauts are exposed to high-energy space radiation while breathing 100% oxygen. Given that brain irradiation can lead to cognitive impairment, and that oxygen is a potent radiosensitizer, there is a concern that astronauts may be at greater risk of developing cognitive impairment when exposed to space radiation while breathing 100% O(2) during an EVA. To address this concern, unanesthetized, unrestrained, young adult male Fischer 344 × Brown Norway rats were allowed to breathe 100% O(2) for 30 min prior to, during and 2 h after whole-body irradiation with 0, 1, 3, 5 or 7 Gy doses of 18 MV X rays delivered from a medical linear accelerator at a dose rate of ~425 mGy/min. Irradiated and unirradiated rats breathing air (~21% O(2)) served as controls. Cognitive function was assessed 9 months postirradiation using the perirhinal cortex-dependent novel object recognition task. Cognitive function was not impaired until the rats breathing either air or 100% O(2) received a whole-body dose of 7 Gy. However, at all doses, cognitive function of the irradiated rats breathing 100% O(2) was improved over that of the irradiated rats breathing air. These data suggest that astronauts are not at greater risk of developing cognitive impairment when exposed to space radiation while breathing 100% O(2) during an EVA.

Chemoprevention by N-acetylcysteine of low-dose CT-induced murine lung tumorigenesis.

Data from the National Lung Screening Trial suggested that annual computed tomography (CT) screening of at-risk patients decreases lung cancer mortality by 20%. We assessed the effects of low-dose CT radiation in mice exposed to 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) to mimic the effects of annual CT screening in heavy smokers and ex-smokers. A/J mice were treated at 8 weeks with NNK followed 1 week later by 4 weekly doses of 0, 10, 30 or 50 mGy of whole-body CT and euthanized 8 months later. Irradiated mice exhibited significant 1.8- to 2-fold increases in tumor multiplicity in males (16.1 ± 0.8 versus 9.1 ± 1.5 tumors per mouse; P < 0.0001) and females (21.6 ± 0.8 versus 10.5 ± 1.4 tumors per mouse; P < 0.0001), respectively, compared with unirradiated mice with no dose effect observed; female mice exhibited higher sensitivity to radiation exposure than did males (P < 0.0001). Similar results were obtained when tumor area was determined. To assess if the deleterious effects of radiation could be prevented by antioxidants, female mice were fed a diet containing 0.7% N-acetylcysteine (NAC) starting 3 days prior to the first CT exposure and continuing for a total of 5 weeks. NAC prevented CT induced increases in tumor multiplicity (10.5 ± 1.2 versus 20.7 ± 1.5 tumors per mouse; P < 0.0001) back to levels seen in NNK/unirradiated mice (10.5 ± 1.2). Our data suggest that exposure of sensitive populations to CT radiation increases the risk of tumorigenesis, and that antioxidants may prevent the long-term carcinogenic effects of low-dose radiation exposure. This would allow annual screening with CT while preventing the potential long-term toxicity of radiation exposure.

A gated-7T MRI technique for tracking lung tumor development and progression in mice after exposure to low doses of ionizing radiation.

A gated-7T magnetic resonance imaging (MRI) application is described that can accurately and efficiently measure the size of in vivo mouse lung tumors from ∼0.1 mm(3) to >4 mm(3). This MRI approach fills a void in radiation research because the technique can be used to noninvasively measure the growth rate of lung tumors in large numbers of mice that have been irradiated with low doses (<50 mGy) without the additional radiation exposure associated with planar X ray, CT or PET imaging. High quality, high resolution, reproducible images of the mouse thorax were obtained in ∼20 min using: (1) a Bruker 7T micro-MRI scanner equipped with a 60 mm inner diameter gradient insert capable of generating a maximum gradient of 1000 mT/m; (2) a 35 mm inner diameter quadrature radiofrequency volume coil; and (3) an electrocardiogram and respiratory gated Fast Low Angle Shot (FLASH) pulse sequence. The images had an in-plane image resolution of 98 µm and a 0.5 mm slice thickness. Tumor diameter measured by MRI was highly correlated (R(2) = 0.97) with the tumor diameter measured by electronic calipers. Data generated with an initiation/promotion mouse model of lung carcinogenesis and this MRI technique demonstrated that mice exposed to 4 weekly fractions of 10, 30 or 50 mGy of CT radiation had the same lung tumor growth rate as that measured in sham-irradiated mice. In summary, this high-field, double-gated MRI approach is an efficient way of quantitatively tracking lung tumor development and progression after exposure to low doses of ionizing radiation.

Cancer-prone mice expressing the Ki-rasG12C gene show increased lung carcinogenesis after CT screening exposures.

A >20-fold increase in X-ray computed tomography (CT) use during the last 30 years has caused considerable concern because of the potential carcinogenic risk from these CT exposures. Estimating the carcinogenic risk from high-energy, single high-dose exposures obtained from atomic bomb survivors and extrapolating these data to multiple low-energy, low-dose CT exposures using the Linear No-Threshold (LNT) model may not give an accurate assessment of actual cancer risk. Recently, the National Lung Cancer Screening Trial (NLST) reported that annual CT scans of current and former heavy smokers reduced lung cancer mortality by 20%, highlighting the need to better define the carcinogenic risk associated with these annual CT screening exposures. In this study, we used the bitransgenic CCSP-rtTA/Ki-ras mouse model that conditionally expresses the human mutant Ki-ras(G12C) gene in a doxycycline-inducible and lung-specific manner to measure the carcinogenic risk of exposure to multiple whole-body CT doses that approximate the annual NLST screening protocol. Irradiated mice expressing the Ki-ras(G12C) gene in their lungs had a significant (P = 0.01) 43% increase in the number of tumors/mouse (24.1 ± 1.9) compared to unirradiated mice (16.8 ± 1.3). Irradiated females had significantly (P < 0.005) more excess tumors than irradiated males. No tumor size difference or dose response was observed over the total dose range of 80-160 mGy for either sex. Irradiated bitransgenic mice that did not express the Ki-ras(G12C) gene had a low tumor incidence (≤ 0.1/mouse) that was not affected by exposure to CT radiation. These results suggest that (i) estimating the carcinogenic risk of multiple CT exposures from high-dose carcinogenesis data using the LNT model may be inappropriate for current and former smokers and (ii) any increased carcinogenic risk after exposure to fractionated low-dose CT-radiation may be restricted to only those individuals expressing cancer susceptibility genes in their tissues at the time of exposure.