Abstract：
OBJECTIVE: The aim of this study was to develop an enhanced intestinal toxicity assay with three outputs assessing proliferation, villi morphology and DNA damage after irradiation.
METHODS: Whole 5 cm jejunal lengths were collected from mice following total body x-ray irradiation (0-15 Gy) at 0-84 h. Tissues were wrapped into swirls for cryopreservation and immunohistochemically stained for EdU, CD31, and γH2AX. A semi-automated image analysis was developed for the proliferation, villi morphology, and DNA damage models.
RESULTS: Proliferation assessed via EdU staining varied with cycles of damage repair, hyperproliferation, and homeostasis after radiation, with the time to onset of each cycle variable based on radiation dose. An analysis model evaluating the amount of proliferation per unit length of jejunum analyzed was developed, with a dose-response curve identified at 48 h post treatment. The villi length model measured the length of intact and healthy CD31-stained capillary beds between the crypts and villi tips at 3.5 days post treatment within a 0-10 Gy dose range. The DNA damage model evaluated the intensity of γH2AX staining within cellular nuclei, with a useful dose-response identified at 1 h post-radiation treatment.
CONCLUSIONS: This assay demonstrates flexibility for assessing radiation-induced damage, with analysis of proliferation, villi length, or direct DNA damage achievable at defined time points and within useful radiation dose curves. The software-assisted image analysis allows for rapid, comprehensive, and objective data generation with an assay turnover time of days instead of weeks on samples that are representative of most of the treated jejunum.
METHODS: Whole 5 cm jejunal lengths were collected from mice following total body x-ray irradiation (0-15 Gy) at 0-84 h. Tissues were wrapped into swirls for cryopreservation and immunohistochemically stained for EdU, CD31, and γH2AX. A semi-automated image analysis was developed for the proliferation, villi morphology, and DNA damage models.
RESULTS: Proliferation assessed via EdU staining varied with cycles of damage repair, hyperproliferation, and homeostasis after radiation, with the time to onset of each cycle variable based on radiation dose. An analysis model evaluating the amount of proliferation per unit length of jejunum analyzed was developed, with a dose-response curve identified at 48 h post treatment. The villi length model measured the length of intact and healthy CD31-stained capillary beds between the crypts and villi tips at 3.5 days post treatment within a 0-10 Gy dose range. The DNA damage model evaluated the intensity of γH2AX staining within cellular nuclei, with a useful dose-response identified at 1 h post-radiation treatment.
CONCLUSIONS: This assay demonstrates flexibility for assessing radiation-induced damage, with analysis of proliferation, villi length, or direct DNA damage achievable at defined time points and within useful radiation dose curves. The software-assisted image analysis allows for rapid, comprehensive, and objective data generation with an assay turnover time of days instead of weeks on samples that are representative of most of the treated jejunum.
摘要：
目的：本研究的目的是开发一种增强的肠毒性试验，有三个输出评估增殖，照射后绒毛形态和DNA损伤。
方法：在0-84小时进行全身X射线照射（0-15Gy）后，从小鼠中收集整个5cm空肠长度。将组织包裹在漩涡中进行冷冻保存，并对EdU进行免疫组织化学染色，CD31和γH2AX。为增殖开发了半自动图像分析，绒毛形态,和DNA损伤模型。
结果：通过EdU染色评估的增殖随损伤修复周期而变化，过度增殖,辐射后的体内平衡，每个周期的开始时间取决于辐射剂量。建立了一个分析模型，该模型评估了每单位长度空肠的增殖量，在治疗后48小时确定剂量反应曲线。绒毛长度模型在0-10Gy剂量范围内治疗后3.5天测量隐窝和绒毛尖端之间的完整和健康的CD31染色毛细血管床的长度。DNA损伤模型评估了细胞核内γH2AX染色的强度，在放射治疗后1小时确定有用的剂量反应。
结论：该试验证明了评估辐射诱导损伤的灵活性，通过对增殖的分析，绒毛长度,或在定义的时间点和有用的辐射剂量曲线内可实现的直接DNA损伤。软件辅助的图像分析允许快速，全面,和客观的数据生成，其中分析周转时间为几天而不是几周，样品代表了大多数处理的空肠。
方法：在0-84小时进行全身X射线照射（0-15Gy）后，从小鼠中收集整个5cm空肠长度。将组织包裹在漩涡中进行冷冻保存，并对EdU进行免疫组织化学染色，CD31和γH2AX。为增殖开发了半自动图像分析，绒毛形态,和DNA损伤模型。
结果：通过EdU染色评估的增殖随损伤修复周期而变化，过度增殖,辐射后的体内平衡，每个周期的开始时间取决于辐射剂量。建立了一个分析模型，该模型评估了每单位长度空肠的增殖量，在治疗后48小时确定剂量反应曲线。绒毛长度模型在0-10Gy剂量范围内治疗后3.5天测量隐窝和绒毛尖端之间的完整和健康的CD31染色毛细血管床的长度。DNA损伤模型评估了细胞核内γH2AX染色的强度，在放射治疗后1小时确定有用的剂量反应。
结论：该试验证明了评估辐射诱导损伤的灵活性，通过对增殖的分析，绒毛长度,或在定义的时间点和有用的辐射剂量曲线内可实现的直接DNA损伤。软件辅助的图像分析允许快速，全面,和客观的数据生成，其中分析周转时间为几天而不是几周，样品代表了大多数处理的空肠。
