Name: pet and mri guided irradiation of a glioblastoma rat model using a micro-irradiator
Uploaded: 2017-12-28T14:00:00.000+00:00
Duration: 10 min 48 s
Description: Watch this Scientific Journal Video about PET and MRI Guided Irradiation of a Glioblastoma Rat Model Using a Micro-irradiator at JoVE.com

作者想感谢 Stichting Hemelaere 和职业妇女福利互助会国际协会支持这项工作。

这项研究由动物实验伦理学委员会 (09/23 和幼儿发展 12/28) 批准。所有商业细节都可以在材料表中找到。
1. F98 GB 鼠细胞模型
<ol>
	<li>培养 F98 GB 细胞, 从 ATCC 获得, 在单分子膜中使用 Dulbecco 氏改良的鹰培养基, 10% 小牛血清, 1% 青霉素, 1% 链霉素, 1% l-谷氨酰胺, 和0.1% 两性霉素 b, 并在 co2孵化器 (5% co2和37° c) 中放置。</li>
	<li>在雌性 Fischer F344 鼠脑中接种胶质瘤细胞 (体重170克)。<ol>
		<li>使用无菌器械, 随时佩戴无菌手套。</li>
		<li>通过注射74毫克/千克氯胺酮和11毫克/千克嗪 intrapertioneally (IP) 与胰岛素注射器 (1 毫升, 29 克) 的混合物, 麻醉大鼠。通过对肢体的撤退反射没有反应来证实麻醉。把老鼠固定在一个立体定位装置里, 用鼻子和耳朵的点。放置一个卡波姆眼凝胶, 以防止在麻醉下的眼睛干燥。</li>
		<li>把老鼠从眼睛的水平刮到头骨的后面, 用聚维酮碘消毒皮肤。</li>
		<li>通过2厘米的中线头皮切口露出颅骨, 并使1毫米孔 (金刚石钻头) 2 mm 后部和2.5 毫米侧向右前额半球的 bregma。</li>
		<li>插入一个 stereotactically 引导胰岛素针 (29 克) 和注入5µL 细胞悬浮 (2万 F98 GB 细胞) 3 毫米深使用微量泵控制器 (设置: 注入 (I50), 率 1 nL/秒 (001 私人))。</li>
		<li>慢慢取出注射器, 用骨蜡缝合切口。缝合皮肤, 用聚维酮碘消毒。</li>
		<li>用红灯稳定动物后的体温。监测鼠的觉醒, 直到它已经恢复足够的意识, 以维持胸骨卧床。在完全恢复之前, 不要将动物归还给其他动物的公司。将所有动物置于环境控制条件下 (12 小时正常光照/暗循环、20-24 ° c 和40-70% 相对湿度) 与食物和水的ad 随意。确保密切跟踪动物的体重, 食物, 水的摄入量, 他们的活动和正常行为。使用致命剂量戊巴比妥钠安乐动物 (160 毫克/千克), 如果下降20% 体重是观察或当正常行为严重恶化 (如, 缺乏梳理)。</li>
	</ol>
	</li>
</ol>
2. 肿瘤生长的确认
注: 使用 T2-weighted mri、动态造影增强 mri (DCE mri) 和造影增强 T1-weighted mri 对肿瘤生长8天 post-inoculation 进行评估。当肿瘤达到 2.5 x 2.5 x 2.5 mm3的大小时, 选择用于治疗的大鼠。
<ol>
	<li>首先, 将30克针连接到一个60厘米长的管子上, 在侧尾静脉中放置静脉注射。麻醉的大鼠通过鼻锥与2% 异氟醚混合氧 (0.3 升/分)。当老鼠对肢体的撤退反射没有反应时, 确认麻醉。用加热的毯子盖住老鼠, 把它们放在 MRI 床上。使用卡波姆眼凝胶防止干燥。</li>
	<li>将床放置在支架上, 用固定的鼠脑表面线圈, 并将床置于72毫米大鼠全身发射机线圈中。</li>
	<li>执行定位扫描, 然后进行 T2-weighted 自旋回波扫描, 以评估肿瘤的生长。T2-MRI 序列细节: TR/TE 3661/37.1 毫秒, 109 µm 各向同性内平面分辨率, 切片厚度600µm, 4 平均数, TA 9 分钟四十五年代。</li>
	<li>如果肿瘤在 T2-weighted 获得证实, 注射钆造影剂进入静脉置管 (mri 造影剂; 0.4 毫升/千克) 后, 三十年代开始的 DCE mri 采集。使用单片 (1 mm 切片厚度) 中的快速低角度拍摄 (FLASH) 序列在12分钟内获取 DCE-MRI。使用 (312 µm2) 的平面空间分辨率和 1.34 s 的时间分辨率。</li>
	<li>使用图像序列分析工具, 选择一个感兴趣的区域 (ROI) 在可疑的肿瘤区域, 以绘制信号强度随着时间的推移。随后, 分析产生的 DCE 曲线的形状以确认胶质母细胞瘤的存在 (图 3)。</li>
	<li>最后, 获得一个对比度增强的 T1-weighted 自旋回波序列。T1-MRI 序列细节: TR/TE 1539/9.7 毫秒, 117 µm 各向同性内平面分辨率, 切片厚度600µm, 3 平均数, TA 4 分钟十五年代. 典型的对比度增强 T1-weighted MR 图像显示在图 2中。</li>
	<li>在 T1-weighted 序列完成后, 动物可以在连续的监督下醒来, 直到它恢复完全意识。</li>
</ol>
3. 目标体积选择的多模成像
注: 为了能够执行 MRI 引导的 F98 GB 大鼠模型的3D 保形照射 PET 引导子促进, 3 成像模式需要执行。首先, 注入示, 然后在示踪剂摄取过程中进行 MRI, 随后进行静态 PET 采集和治疗计划 CT。
<ol>
	<li>麻醉的动物使用鼻子锥与2% 异氟醚混合氧 (0.3 升/分)。当老鼠对肢体的撤退反射没有反应时, 确认麻醉。使用卡波姆眼凝胶, 以防止在麻醉下干燥。</li>
	<li>将导管 (26 克) 插入尾静脉, 使 37 MBq 的 PET 放射性示踪剂在200µL 盐水中溶解。分别在 PET 采集前注入18f-FET 或18f 扎、30分钟或2小时。</li>
	<li>在 PET 获得前15分钟, 在尾静脉静脉注射 MRI 造影剂 (0.4 毫升/千克)。</li>
	<li>将大鼠放在一个 in-house 的多模床上, 用钩环紧固件固定, 在成像和微辐照时保持一定的位置 (图 1)。</li>
	<li>修复三多模态标记 (充满水的毛细血管) 在头骨的右侧。将大鼠, 仍然固定在多模床上, 在 MRI 扫描仪的动物持有人, 修复大鼠脑表面线圈和位置这一设置在一个72毫米的大鼠全身发射机线圈。执行定位扫描, 然后进行对比增强的 T1-weighted 自旋回波序列。</li>
	<li>运送动物执行18f-FET 或18f-扎 PET 捕获。在列表模式下获取30分钟静态 PET 扫描。扫描应获得30分钟后, 18f-FET 注入或2小时后, 18f 扎注入。</li></ol>将所有 PET 扫描重建为 200 x 200 x 64 矩阵, 由2D 最大似然期望最大化 (MLEM) 算法使用60次迭代和一个体素大小 0.5 x 0.5 x 1.157 mm。
	<li>将该动物放在多模床上, 固定在四轴微型器机器人定位台上的塑料支架上。使用 1 mm 和 20 x 20 cm (1024 x 1024 像素) 非晶硅平板探测器的铝滤光片进行高分辨率的 CT 扫描。重建 CT 图像的各向同性的体素大小为0.2 毫米, 分别修复 70 kV 和0.4 毫安的管电压和管电流。共获得360预测超过360°。</li>

4. RT 治疗计划
<ol>
	<li>使用临床前治疗计划系统 (PCTPS) 进行治疗计划。将规划 ct 导入 PCTPS, 并将 ct 图像手动分割成三不同的组织类别: 骨骼、软组织和空气。这种手工分割的基础上定义三不同的灰度值门限的规划 CT。应选择这些人工选择的灰度阈值, 使大脑中的空气消失, 颅骨的骨厚度为非零。一旦定义了这些阈值, 材料密度由 PCTPS 分配给骨骼、软组织和空气 (图 4)。</li>
	<li>如果只需要 mri 引导, 用 PCTPS 进行 mri 扫描和 co-register。<ol>
		<li>使用刚体变换 (三平移和三旋转), 多模标记, 和头骨。通过在 MRI 上用黑色信号覆盖头颅 CT 增加的信号强度, 可以实现精确的融合 (图 5)。</li>
		<li>在 T1-weighted MRI 上选择造影剂肿瘤中心的照射靶, 请参阅图 6和图 7。</li>
	</ol>
	</li>
	<li>当额外的宠物信息必须包括在内, 包括一个 CT/MRI/pet co-registration 使用生物医学图像量化软件 (BIQS)。<ol>
		<li>使用 BIQS 中的轮廓工具实现 PET/MRI 图像融合 (图 8)。在 co-registration 之后, 在 BIQS (图 9) 中, 选择增加的 PET 示踪剂摄取中心的目标, 并使用下列变换将坐标手动输入 PCTPS: x → x、Y → z 和 z → y。</li>
		<li>选择指定的剂量、弧数、弧位、圆弧的旋转范围和准直器大小 (图 10)。</li>
		<li>对于 MRI 引导 RT, 请使用以下设置: 规定剂量 20, 3 弧定位在沙发角-45 °, 0 °, 45 °与弧旋转120°, 和准直器大小 5 x 5 毫米。</li>
		<li>对于 PET MRI 引导 RT, 请使用以下设置: 使用3弧和 5 x 5 mm 准直器的规定剂量为 20, 额外的5子升压使用 3 non-coplanar 弧和 1 x 1 mm 准直仪。选择一个120°旋转的所有弧形, 同时改变沙发的位置 (-45 °, 0 °, 和45°)。</li>
	</ol>
	</li>
	<li>计算在动物体内的剂量分布和光束传递参数, 使用 PCTPS 将规定的剂量传递给目标。在实际照射之前, 在不同的沙发位置测试圆弧旋转, 以防止辐照期间发生碰撞。</li>
	<li>对于实际辐照, 选择一个0.15 毫米的铜过滤器, 设置 x 射线电压到220伏, 设置 x 射线电流为13毫安, 并在龙门位置正确的准直仪。通过将适当的光束传递参数从 PCTPS 转移到微器来执行 RT。</li>
	<li>在这些过程中, 大鼠保持连续的异氟醚麻醉 (2% 异氟醚, 混合氧0.3 升/分)。在最后一个弧线的执行之后, 动物可以在持续的监督下醒来, 直到它恢复全意识。</li>
</ol>
5. 剂量容积直方图 (DVHs)
注意: 要比较实际剂量传递到肿瘤靶体积和周围正常的脑组织, 计算 DVHs。
<ol>
	<li>绘制一个 volume-of-interest (VOI) 周围的肿瘤和正常的大脑 T1-weighted 对比增强磁共振图像计算的平均, 最大和最低剂量 (图 11)。</li>
	<li>作为对肿瘤体积和正常脑组织体积的最大、平均和最小剂量的替代物, 计算 d2、d50和 d90。D 代表由 x% 所接收的量的剂量, 用下标表示, 并且可以从产生的 DVH。</li>
</ol>
6. 八卦和假化疗
<ol>
	<li>为了模拟治疗患者的胶质母细胞瘤, 管理伴随化疗使用 IP 注射29毫克/千克在盐水中溶解与25% 砜 (亚甲基亚砜) 每天一次5天从辐照日起24,25. 使用1毫升, 29 克胰岛素注射器来管理注射。</li>
	<li>对于控制组, 管理步骤6.1 中的注入而不进行任何的八卦。</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+RECIST+to+PERCIST:+Evolving+Considerations+for+PET+response+criteria+in+solid+tumors.">From RECIST to PERCIST: Evolving Considerations for PET response criteria in solid tumors.</a> J Nucl Med. 50 (5), 122S-150S (2009).</li><li>Daisne, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+volume+in+pharyngolaryngeal+squamous+cell+carcinoma:+comparison+at+CT,+MR+imaging,+and+FDG+PET+and+validation+with+surgical+specimen.">Tumor volume in pharyngolaryngeal squamous cell carcinoma: comparison at CT, MR imaging, and FDG PET and validation with surgical specimen.</a> Radiology. 233 (1), 93-100 (2004).</li><li>Grosu, A. -. L., Weber, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PET+for+radiation+treatment+planning+of+brain+tumours.">PET for radiation treatment planning of brain tumours.</a> Radiother Oncol. 96 (3), 325-327 (2010).</li><li>Banissi, C., Ghiringhelli, F., Chen, L., Carpentier, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treg+depletion+with+a+low-dose+metronomic+temozolomide+regimen+in+a+rat+glioma+model.">Treg depletion with a low-dose metronomic temozolomide regimen in a rat glioma model.</a> Cancer Immunol Immunother. 58, 1627-1634 (2009).</li><li>Robinson, C. 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作者没有利益冲突披露

为实现对大鼠脑胶质瘤靶向的精确照射, 微器的 on-board CT 引导是不够的。脑肿瘤是很难看到的, 由于缺乏足够的软组织对比, 即使使用对比增强。因此, 需要包括 MRI 来允许更精确的照射。利用 7 T 系统的序贯 MR 采集和微器的 ct 采集, 我们能够将剂量靶向脑内增强的肿瘤组织, 并利用规划 ct 计算剂量计划。这是可行的后, 图像融合和剂量计算使用 PCTPS17。然而, 应该牢记的是, MRI 容?...

高级别胶质瘤是最常见和最积极的恶性脑肿瘤在成人中中位数生存1年, 尽管目前的治疗方式。护理标准包括最大的手术切除, 其次是联合外照射治疗 (RT) 和莫 (八卦), 其次是维护八卦1,2,3。自从15年前引入了 "八卦" 以来, 在治疗这些肿瘤方面没有取得明显的进展。因此, 实施新的治疗策略是迫切的, 但应该首先在小动物肿瘤治疗模型 (主要是老鼠和老鼠) 中进行研究。含肿瘤的啮齿动物模型可以用来调查新的和复杂的辐射协议的功效, 可能与其他 (新的) 治疗剂结合, 以评估辐射反应或调查无线电保护剂。临床前辐射研究的一个主要优势是能够在受控实验条件下使用大的群体, 由于啮齿类动物的寿命较短而导致数据产量加快。临床前的发现应该被翻译成一个比当前实践中更快、更有效的方法4。
在过去的几十年中, 小动物辐射实验通常是通过固定辐射源实现的5,6,7,如, 137Cs 和60Co, 同位素, 或线性用于人类临床用途的加速器, 适用于单辐射场与 MV X 射线6,8,9,10,11。但是, 这些设备无法达到毫米精度, 这是小动物所需的12。此外, MV X 射线具有不适合辐照小目标的特性, 例如在光束入口区域的 air-tissue 界面上的剂量积聚, 以动物大小本身的顺序4,6 ,8,9,10,11。后者使它相当具有挑战性, 提供一个统一的剂量, 以肿瘤, 而保留周围的正常脑组织4,8,9,10,11。因此, 目前尚不清楚当前的动物研究在何种程度上仍然适用于现代 RT 实践12。在这方面, 最近开发的三维 (3D) 保形小动物微辐照有望弥合先进的3D 图像制导 RT 技术之间的技术差距, 如强度调制辐射疗法 (放射) 或用于人和当前小动物辐照的共形弧形4,13。这些平台利用千伏 (kV) X 射线源获得尖锐的 penumbras, 并避免剂量积聚。这些平台包括一个计算机控制的动物定位阶段, 一个用于成像和辐射处理的 kV X 射线源, 一个旋转龙门组件, 允许从不同角度进行辐射传递, 以及一个准直系统来形成辐射光束4. 2011年, 在根特大学的临床前成像实验室安装了微器 (图 1)。该系统类似于现代的人类放疗实践, 并使广泛的临床前实验, 如辐射与其他疗法的协同作用, 复杂的辐射方案, 和图像引导 sub-target 促进研究。
这些微辐照的治疗计划是基于 CT, 它相当于人类规划系统14,15。对于 CT 成像, on-board x 射线检测器与治疗期间使用的同一个 kV x 射线管相结合。CT 成像是使用, 因为它允许准确的动物定位, 并提供必要的信息, 个人辐射剂量计算通过分割。然而, 由于 CT 成像的软组织对比度较低, 小动物的脑部肿瘤, 如高等级胶质瘤, 不能轻易被描述。因此, 为了精确的目标体积划分, 多成像是必要的。与 CT 相比, MRI 提供了极大的软组织对比。这使得更容易可视化病变边界, 这将导致更好地划定目标体积, 有助于更好地照射病灶和避免周围组织, 如 图 24所示, 16。另外一个优点是 MRI 使用电离辐射, 与使用电离辐射的 CT 不同。MRI 的主要缺点是相对较长的采集时间和较高的操作成本。重要的是要注意, MRI 扫描不能用于剂量计算, 因为他们没有提供所需的电子密度信息, 虽然在这方面正在取得进展, 也随着最近的发展加速器先生。因此, 联合 CT/MRI 数据集是规划恶性胶质瘤照射的选择方法, 同时包含靶向 (MRI-based 体积) 和剂量计算 (ct 电子密度) 所需的信息。
为了减少小动物照射与临床常规的差距, mri 显然需要与微器的工作流程相结合, 需要在 mri 与 CT 之间进行正确的定位, 而这远非微不足道。本文讨论了 F98 脑胶质瘤的 MRI 引导3D 适形照射的方案, 该方法已在最近的17中发表。
虽然在微器的工作流程中纳入 CT 和 MRI 是小动物照射研究的一个明显的进步, 但这些解剖成像技术并不总是允许对目标体积进行完整的定义。CT 和 MRI 对脑组织的病理改变表现为增加含水量 (水肿) 和血脑屏障渗漏或造影增强。然而, 对比增强和超强区域的 T2-weighted MRI 并不总是准确的测量肿瘤程度。肿瘤细胞的检测远远超出了对比增强12的边缘。此外, 这些技术都不能识别肿瘤中最具侵袭性的部分, 这可能是治疗性抵抗和肿瘤复发的原因。因此, 从像 PET 这样的分子成像技术中获得的额外信息可能会增加 RT 目标体积定义的值, 因为这些技术能够使生物通路在体内12、18中可视化, 19。
在 2000年, 凌et al.引入了生物靶体积 (电视台) 的概念, 将解剖和功能成像集成到放疗工作流中, 导致他们所说的多维适形放疗20。这就创造了一种可能性, 通过给目标区域提供一个非均匀剂量的 PET 图像来改善剂量靶向。最广泛使用的肿瘤分期 PET 示踪和监测治疗反应是 fluor-18 (18F) 标记葡萄糖 (葡萄糖), 这可视化为葡萄糖代谢21。在头颈部癌, 以前的研究表明, 使用18f-葡萄糖 PET 导致更好的估计实际肿瘤体积, 由病理标本, 与 CT 和 MRI 相比,22。在原发性脑肿瘤, 葡萄糖是不有用的, 由于非常强烈的背景信号, 从正常的大脑, 氨基酸, 如11c-蛋氨酸和最近的18F-fluoroetthyltyrosine (FET), 已被调查的 GTV在氨基酸 PET 和 MRI-based GTVs 之间经常标记的区别的分界23。然而, 目前还没有对这一发现的意义进行调查的前瞻性试验。在本研究中, 我们选择了氨基酸示踪剂18f-FET 和缺氧示踪剂18f-fluoroazomycin-苷 (18f-扎)。18选择 f-FET 和18f-扎, 因为增加的氨基酸摄取量与 GB 肿瘤的增殖率密切相关, 而缺氧 PET 示踪剂的摄取与抗 (化疗) 放疗相关,18,23. 通过对小鼠 F98 GB 肿瘤的 PET 定义部分给予额外的辐射剂量, 优化了微器的亚容积升压。

<table><tbody><tr><td>&lt;strong&gt;GB RAT model&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>F98 Glioblastoma cell line</td><td>ATCC</td><td>CRL-2397</td><td></td></tr><tr><td>Fischer F344/Ico crl Rats</td><td>Charles River</td><td>N/A</td><td>http://www.criver.com/products-services/basic-research/find-a-model/fischer-344-rat</td></tr><tr><td>Micropump system</td><td>World Precision Instruments</td><td>UMP3</td><td>Micro 4: https://www.wpiinc.com/products/top-products/make-selection-ump3-ultramicropump/#tabs-1</td></tr><tr><td>Stereotactic frame</td><td>Kopf</td><td>902</td><td>Model 902 Dual Small Animal Stereotaxic frame</td></tr><tr><td>diamant drill</td><td>Velleman</td><td>VTHD02</td><td>https://www.velleman.eu/products/view/?id=370450</td></tr><tr><td>Bone wax</td><td>Aesculap</td><td>1029754</td><td>https://www.aesculapusa.com/products/wound-closure/hemostatic-bone-wax</td></tr><tr><td>Insulin syringe Microfine</td><td>Beckton-Dickinson</td><td>320924</td><td>1 mL, 29G</td></tr><tr><td>InfraPhil IR lamp</td><td>Philips</td><td>HP3616/01</td><td></td></tr><tr><td>Ethilon</td><td>Ethicon</td><td>662G/662H</td><td>FS-2, 4-0, 3/8, 19 mm</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Cell culture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DMEM</td><td>Invitrogen</td><td>14040-091</td><td></td></tr><tr><td>Penicilline-streptomycine</td><td>Invitrogen</td><td>15140-148</td><td></td></tr><tr><td>L-glutamine</td><td>Invitrogen</td><td>25030-032</td><td></td></tr><tr><td>Fungizone</td><td>Invitrogen</td><td>15290-018</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Invitrogen</td><td>25300-062</td><td></td></tr><tr><td>PBS</td><td>Invitrogen</td><td>14040-224</td><td></td></tr><tr><td>Falcons</td><td>Thermo Scientific</td><td>178883</td><td>175 cm&lt;sup&gt;2&lt;/sup&gt; nunclon surface, disposables for cell culture with filter caps</td></tr><tr><td>Cell freezing medium</td><td>Sigma-aldrich</td><td>C6164</td><td>Cell Freezing Medium-DMSO, sterile-filtered, suitable for cell culture, endotoxin tested</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Animal irradiation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Micro-irradiator</td><td>X-strahl</td><td>SARRP</td><td></td></tr><tr><td>software for irradiation</td><td>X-strahl</td><td>MuriPlan</td><td>pre-clinical treatment planning system (PCTPS), version 2.0.5.</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Small animal PET&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>microPET system possibility 1</td><td>Molecubes</td><td>B-Cube</td><td>http://www.molecubes.com/b-cube/</td></tr><tr><td>microPET system possibility 2</td><td>TriFoil Imaging, Northridge CA</td><td>FLEX Triumph II</td><td>http://www.trifoilimaging.com</td></tr><tr><td>PET tracers</td><td>In-house made</td><td></td><td>18F-FDG, 18F-FET, 18F-FAZA, 18F-Choline</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Small animal MRI&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>microMRI system</td><td>Bruker Biospin</td><td>Pharmascan 70/16</td><td>https://www.bruker.com/products/mr/preclinical-mri/pharmascan/overview.html</td></tr><tr><td>Dotarem contrast agent</td><td>Guerbet</td><td></td><td>MRI contrast agent, Dotarem 0,5 mmol/ml</td></tr><tr><td>rat whole body transmitter coil</td><td>Rapid Biomedical</td><td>V-HLS-070</td><td></td></tr><tr><td>rat brain surface coil</td><td>Rapid Biomedical</td><td>P-H02LE-070</td><td></td></tr><tr><td>Water-based heating unit</td><td>Bruker Biospin</td><td>MT0125</td><td></td></tr><tr><td>30 G Needle for IV injection</td><td>Beckton-Dickinson</td><td>305128</td><td>30 G</td></tr><tr><td>PE 10 tubing (60 cm/injection)</td><td>Instech laboratories, Inc</td><td>BTPE-10</td><td>BTPE-10, polyethylene tubing 0.011 x .024 in (0.28 x 60 mm), non sterile, 30 m (98 ft) spool, Instech laboratories, Inc Plymouth meeting PA USA- (800) 443-4227- http://www.instechlabs.com</td></tr><tr><td>non-heparinised micro haematocrit capillaries</td><td>GMBH</td><td>7493 21</td><td>these capillaries are filled with water to create markers visible on MRI and CT</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Consumables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>isoflurane: Isoflo</td><td>Zoetis</td><td>B506</td><td>Anaesthesia</td></tr><tr><td>ketamine: Ketamidor</td><td>Ecuphar</td><td></td><td>Anaesthesia</td></tr><tr><td>xylazine: Sedaxyl</td><td>Codifar NV</td><td></td><td>Anaesthesia</td></tr><tr><td>catheter</td><td>Terumo</td><td>Versatus-W</td><td>26G</td></tr><tr><td>Temozolomide</td><td>Sigma-aldrich</td><td>T2577-100MG</td><td>chemotherapy</td></tr><tr><td>DMSO</td><td>Sigma-aldrich</td><td>276855-100ML</td><td></td></tr><tr><td>Insulin syringe Microfine</td><td>Beckton-Dickinson</td><td>320924</td><td>1 mL, 29G</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Image analysis&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PMOD software</td><td>PMOD technologies LLC</td><td>PFUS (fusion tool)</td><td>biomedical image quantification software (BIQS), version 3.405, https://www.pmod.com/web/?portfolio=22-image-processing-pfus</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Anesthesia-equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anesthetic movabe unit</td><td>ASA LTD</td><td>ASA 0039</td><td>ASA LTD, 5 valley road, Keighley, BD21 4LZ</td></tr><tr><td>Oxygen generator</td><td>Veterinary technics Int.</td><td>7F-3</td><td>BDO-Medipass, Ijmuiden</td></tr></tbody></table>

pet and mri guided irradiation of a glioblastoma rat model using a micro-irradiator

几十年来, 小动物的辐射研究大多是使用相当粗糙的实验设置, 应用简单的单技术, 而没有能力针对特定或界定肿瘤体积。辐射的交付使用固定的辐射源或线性加速器生产 megavoltage (MV) x-射线。这些设备无法达到小动物所需的毫米精度。此外, 高剂量传递到健康的周围组织阻碍反应评估。为了增加小动物研究和人类之间的翻译, 我们的目标是模仿在老鼠模型中对人脑胶质瘤的治疗。为了在临床前设置更精确的照射, 最近开发了精确的图像引导小动物辐射研究平台。类似于人类规划系统, 这些微辐照的治疗计划是基于计算机断层扫描 (CT)。然而, 低软组织对比 CT, 使它非常具有挑战性的定位目标在某些组织, 如大脑。因此, 与 CT 相比, 磁共振成像 (MRI) 具有优异的软组织对比度, 能够更精确地描述靶点的照射。在过去的十年中, 生物成像技术, 如正电子发射断层扫描 (PET) 获得了辐射治疗治疗指导的兴趣。PET 使如, 葡萄糖消耗, 氨基酸运输, 或缺氧, 目前在肿瘤的可视化。以更高的剂量靶向肿瘤的高度增殖或耐药部位, 可以获得生存的好处。这个假说导致了生物肿瘤容量 (电视台) 的介绍, 除常规总目标容量 (GTV)、临床目标容量 (中视) 和计划的目标容量 (PTV) 之外。
在根特大学的前临床影像实验室, 有一个微型器, 一个小动物宠物, 和一个 7 T 小动物的 MRI 是可用的。其目的是将 MRI 引导的照射和 PET 引导子在胶质瘤大鼠模型中的增强。

为了模拟在临床前模型中对胶质母细胞瘤照射的人类治疗方法, 必须纳入 MRI 引导放射治疗。使用 PCTPS 和微器界面, 我们能够在 T1-weighted MRI17上用多形 non-coplanar 弧照射大鼠 F98 胶质母细胞瘤。将刚体变换与多床结合, 用于 MRI 与规划 CT 的图像配准。在 T1-weighted MRI (图 7) 中, 在造影增强肿瘤区的中心选择了等照射。
<p class="jove_content...

Watch this Scientific Journal Video about PET and MRI Guided Irradiation of a Glioblastoma Rat Model Using a Micro-irradiator at JoVE.com

PET and MRI Guided Irradiation of a Glioblastoma Rat Model Using a Micro-irradiator

PET 与 MRI 引导下微器对大鼠胶质瘤模型的照射

在过去, 小动物照射通常是没有能力靶界定肿瘤体积。其目的是模仿大鼠的人脑胶质瘤的治疗。使用小动物辐照平台, 我们进行了 MRI 引导3D 共形照射与 PET-based 子促进在临床前设置。

For decades, small animal radiation research was mostly performed using fairly crude experimental setups applying simple single-beam techniques without ...

pet-mri-guided-irradiation-glioblastoma-rat-model-using-micro

Research

JoVE Journal

Cancer Research

9.4K 次观看.  Ghent University Hospital. 在过去, 小动物照射通常是没有能力靶界定肿瘤体积。其目的是模仿大鼠的人脑胶质瘤的治疗。使用小动物辐照平台, 我们进行了 MRI 引导3D 共形照射与 PET-based 子促进在临床前设置。

视频: PET and MRI Guided Irradiation of a Glioblastoma Rat Model Using a Micro-irradiator

Stem Cell Transplantation Strategies for the Restoration of Cognitive Dysfunction Caused by Cranial Radiotherapy

Radiotherapy often provides the only clinical recourse for those afflicted with primary or metastatic brain tumors. While beneficial, cranial irradiation can induce a progressive and debilitating decline in cognition that may, in part, be caused by the depletion of neural stem cells. Given the increased survival of patients diagnosed with brain cancer, quality of life in terms of cognitive health has become an increasing concern, especially in the absence of any satisfactory long-term treatments.
To address this serious health concern we have used stem cell replacement as a strategy to combat radiation-induced cognitive decline. Our model utilizes athymic nude rats subjected to cranial irradiation. The ionizing radiation is delivered as either whole brain or as a highly focused beam to the hippocampus via linear accelerator (LINAC) based stereotaxic radiosurgery. Two days following irradiation, human neural stem cells (hNSCs) were stereotaxically transplanted into the hippocampus. Rats were then assessed for changes in cognition, grafted cell survival and for the expression of differentiation-specific markers 1 and 4-months after irradiation. Our cognitive testing paradigms have demonstrated that animals engrafted with hNSCs exhibit significant improvements in cognitive function. Unbiased stereology reveals significant survival (10-40%) of the engrafted cells at 1 and 4-months after transplantation, dependent on the amount and type of cells grafted. Engrafted cells migrate extensively, differentiate along glial and neuronal lineages, and express a range of immature and mature phenotypic markers.
Our data demonstrate direct cognitive benefits derived from engrafted human stem cells, suggesting that this procedure may one day afford a promising strategy for the long-term functional restoration of cognition in individuals subjected to cranial radiotherapy. To promote the dissemination of the critical procedures necessary to replicate and extend our studies, we have provided written and visual documentation of several key steps in our experimental plan, with an emphasis on stereotaxic radiosurgey and transplantation.

Brain tumor patients routinely undergo cranial radiotherapy, and while beneficial, this treatment often results in debilitating cognitive dysfunction. This serious unresolved problem has at present, no clinical recourse, and has driven our efforts to devise stem cell based therapies for the recovery of radiation-induced cognitive decrements.

颅放疗引起的认知功能障碍恢复干细胞移植策略

Radiotherapy often provides the only clinical recourse for those afflicted with primary or metastatic brain tumors. While beneficial, cranial irradiation ...

科研

医学

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the understanding of the mechanics of tumor invasion, and direct intracerebral inoculation of tumor provides the opportunity of observing the invasive process in a physiologically appropriate environment2. As far as human brain tumors are concerned, the orthotopic models currently available are established either by stereotaxic injection of cell suspensions or implantation of a solid piece of tumor through a complicated craniotomy procedure3. In our technique we harvest cells from tissue culture to create a cell suspension used to implant directly into the brain. The duration of the surgery is approximately 30 minutes, and as the mouse needs to be in a constant surgical plane, an injectable anesthetic is used. The mouse is placed in a stereotaxic jig made by Stoetling (figure 1). After the surgical area is cleaned and prepared, an incision is made; and the bregma is located to determine the location of the craniotomy. The location of the craniotomy is 2 mm to the right and 1 mm rostral to the bregma. The depth is 3 mm from the surface of the skull, and cells are injected at a rate of 2 &mu;l every 2 minutes. The skin is sutured with 5-0 PDS, and the mouse is allowed to wake up on a heating pad. From our experience, depending on the cell line, treatment can take place from 7-10 days after surgery. Drug delivery is dependent on the drug composition. For radiation treatment the mice are anesthetized, and put into a custom made jig. Lead covers the mouse's body and exposes only the brain of the mouse. The study of tumorigenesis and the evaluation of new therapies for GBM require accurate and reproducible brain tumor animal models. Thus we use this orthotopic brain model to study the interaction of the microenvironment of the brain and the tumor, to test the effectiveness of different therapeutic agents with and without radiation.

The purpose of this article is to describe the use of an orthotopic glioblastoma model for chemoradiation studies. This article will go though cell processing, implanting, and radiotherapy of the mouse using an intracranial model.

在原位小鼠脑肿瘤模型结合放疗

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the ...

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality treatment consisting of surgical resection, post-operative radiation therapy and temozolomide chemotherapy 1. New therapeutic approaches are clearly needed to improve patient survival and quality of life. The development of more effective treatment strategies would be aided by animal models of GBM that recapitulate human disease yet allow serial imaging to monitor tumor growth and treatment response. In this paper, we describe our technique for the precise stereotactic implantation of bio-imageable GBM cancer cells into the brains of nude mice resulting in tumor xenografts that recapitulate key clinical features of GBM 2. This method yields tumors that are reproducible and are located in precise anatomic locations while allowing in vivo bioluminescent imaging to serially monitor intracranial xenograft growth and response to treatments 3-5. This method is also well-tolerated by the animals with low perioperative morbidity and mortality.

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

We describe an integrated method for the precise, stereotactic implantation of human glioblastoma multiforme cells into the brains of nude mice and subsequent serial in vivo imaging to monitor growth and response to treatment of the resultant xenografts.

立体定向颅内植入和<em在体内</em生物发光成像的肿瘤异种移植的小鼠模型系统的多形性胶质母细胞瘤

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality ...

Positron Emission Tomography-based Dose Painting Radiation Therapy in a Glioblastoma Rat Model using the Small Animal Radiation Research Platform

A rat glioblastoma model to mimic chemo-radiation treatment of human glioblastoma in the clinic was previously established. Similar to the clinical treatment, computed tomography (CT) and magnetic resonance imaging (MRI) were combined during the treatment-planning process. Positron emission tomography (PET) imaging was subsequently added to implement sub-volume boosting using a micro-irradiation system. However, combining three imaging modalities (CT, MRI, and PET) using a micro-irradiation system proved to be labor-intensive because multimodal imaging, treatment planning, and dose delivery have to be completed sequentially in the preclinical setting. This also results in a workflow that is more prone to human error. Therefore, a user-friendly algorithm to further optimize preclinical multimodal imaging-based radiation treatment planning was implemented. This software tool was used to evaluate the accuracy and efficiency of dose painting radiation therapy with micro-irradiation by using an in silico study design. The new methodology for dose painting radiation therapy is superior to the previously described method in terms of accuracy, time efficiency, and intra- and inter-user variability. It is also an important step towards the implementation of inverse treatment planning on micro-irradiators, where forward planning is still commonly used, in contrast to clinical systems.

Here we present a protocol to perform preclinical positron emission tomography-based radiotherapy in a rat glioblastoma model using algorithms developed in-house to optimize the accuracy and efficiency.

使用小动物辐射研究平台在胶质母细胞瘤大鼠模型中基于正电子发射断层扫描的剂量绘画放射治疗

A rat glioblastoma model to mimic chemo-radiation treatment of human glioblastoma in the clinic was previously established. Similar to the clinical ...

癌症研究

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology rely on the injection of a suspension of glioma cells into the brain parenchyma following incision of the dura-mater. Whereas the cells have to be injected superficially to be accessible to intravital two-photon microscopy, superficial injections fail to recapitulate the physiopathological conditions. Indeed, escaping through the injection tract most tumor cells reach the extra-dural space where they expand abnormally fast in absence of mechanical constraints from the parenchyma.
Our improvements consist not only in focally implanting a glioma spheroid rather than injecting a suspension of glioma cells in the superficial layers of the cerebral cortex but also in clogging the injection site by a cross-linked dextran gel hemi-bead that is glued to the surrounding parenchyma and sealed to dura-mater with cyanoacrylate. Altogether these measures enforce the physiological expansion and infiltration of the tumor cells inside the brain parenchyma. Craniotomy was finally closed with a glass window cemented to the skull to allow chronic imaging over weeks in absence of scar tissue development.
Taking advantage of fluorescent transgenic animals grafted with fluorescent tumor cells we have shown that the dynamics of interactions occurring between glioma cells, neurons (e.g. Thy1-CFP mice) and vasculature (highlighted by an intravenous injection of a fluorescent dye) can be visualized by intravital two-photon microscopy during the progression of the disease.
The possibility to image a tumor at microscopic resolution in a minimally compromised cerebral environment represents an improvement of current GBM animal models which should benefit the field of neuro-oncology and drug testing.

We have established a cortical orthotopic glioblastoma model in mice for intravital two-photon microscopy that recapitulates the biophysical constraints normally at play during the growth of the tumor. A chronic glass window replacing the skull above the tumor enables the follow-up of the tumor progression over time by two-photon microscopy.

原位胶质母细胞瘤小鼠模型的维护脑实质的物理约束，并适用于活体双光子显微镜

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology ...

Magnetic Resonance-Guided High Intensity Focused Ultrasound Generated Hyperthermia: A Feasible Treatment Method in a Murine Rhabdomyosarcoma Model

Magnetic resonance-guided high intensity focused ultrasound (MRgHIFU) is an established method for producing localized hyperthermia. Given the real-time imaging and acoustic energy modulation, this modality enables precise temperature control within a defined area. Many thermal applications are being explored with this noninvasive, nonionizing technology, such as hyperthermia generation, to release drugs from thermosensitive liposomal carriers. These drugs can include chemotherapies such as doxorubicin, for which targeted release is desired due to the dose-limiting systemic side effects, namely cardiotoxicity. Doxorubicin is a mainstay for treating a variety of malignant tumors and is commonly used in relapsed or recurrent rhabdomyosarcoma (RMS). RMS is the most common solid soft tissue extracranial tumor in children and young adults. Despite aggressive, multimodal therapy, RMS survival rates have remained the same for the past 30 years. To explore a solution for addressing this unmet need, an experimental protocol was developed to evaluate the release of thermosensitive liposomal doxorubicin (TLD) in an immunocompetent, syngeneic RMS mouse model using MRgHIFU as the source of hyperthermia for drug release.

Presented here is a protocol to use controlled hyperthermia, generated by magnetic resonance-guided high intensity focused ultrasound, to trigger drug release from temperature-sensitive liposomes in a rhabdomyosarcoma mouse model.

磁共振引导的高强度聚焦超声产生热疗:小鼠横纹肌肉瘤模型中的可行治疗方法

Magnetic resonance-guided high intensity focused ultrasound (MRgHIFU) is an established method for producing localized hyperthermia. Given the real-time ...

在小鼠中使用 2P-IVM 实时跟踪脑肿瘤中的纳米颗粒

Two-Photon Intravital Microscopy of Glioblastoma in a Murine Model

The delivery of intravenously administered cancer therapeutics to brain tumors is limited by the blood-brain barrier. A method to directly image the accumulation and distribution of macromolecules in brain tumors in vivo would greatly enhance our ability to understand and optimize drug delivery in preclinical models. This protocol describes a method for real-time in vivo tracking of intravenously administered fluorescent-labeled nanoparticles with two-photon intravital microscopy (2P-IVM) in a mouse model of glioblastoma (GBM).
The protocol contains a multi-step description of the procedure, including anesthesia and analgesia of experimental animals, creating a cranial window, GBM cell implantation, placing a head bar, conducting 2P-IVM studies, and post-surgical care for long-term follow-up studies. We show representative 2P-IVM imaging sessions and image analysis, examine the advantages and disadvantages of this technology, and discuss potential applications.
This method can be easily modified and adapted for different research questions in the field of in vivo preclinical brain imaging.

作者聚焦：优化胶质母细胞瘤治疗中药物递送和早期检测的多模式成像策略

We present a novel approach for two-photon microscopy of the tumor delivery of fluorescent-labeled iron oxide nanoparticles to glioblastoma in a mouse model.

小鼠模型中胶质母细胞瘤的双光子活体显微镜

The delivery of intravenously administered cancer therapeutics to brain tumors is limited by the blood-brain barrier. A method to directly image the ...

<meta charset="utf8"></head><body>氧化铁纳米颗粒用于小鼠胶质母细胞瘤的图像引导治疗递送

Nanoparticle Delivery of an Oligonucleotide Payload in a Glioblastoma Multiforme Animal Model

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a significant hurdle in the delivery of therapies to GBM. Reported here is an image-guided, iron oxide-based therapeutic delivery nano platform capable of bypassing this physiological barrier by virtue of size and accumulating in the tumor region, delivering its payload. This 25 nm nano platform consists of crosslinked dextran-coated iron oxide nanoparticles labeled with Cy5.5 fluorescent dye and containing antisense oligonucleotide as a payload. The magnetic iron oxide core enables tracking of the nanoparticles through in vivo magnetic resonance imaging, while Cy5.5 dye allows tracking by optical imaging. This report details the monitoring of the accumulation of this nanoparticle platform (termed MN-anti-miR10b) in orthotopically implanted glioblastoma tumors following intravenous injection. In addition, it provides insight into the in vivo delivery of RNA oligonucleotides, a problem that has hampered the translation of RNA therapeutics into the clinic.

<meta charset="utf8"></head><body>作者聚焦:使用氧化铁纳米颗粒治疗胶质母细胞瘤的创新癌症疗法

The blood-brain barrier is a significant hurdle in the delivery of therapies for glioblastoma, a disease for which there is no cure. Here, we report an in vivo image-guided iron oxide therapeutic nano platform that can bypass this physiological barrier by virtue of size and accumulate in the tumor.

多形性胶质母细胞瘤动物模型中寡核苷酸有效载荷的纳米颗粒递送

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a ...

PET 与 MRI 引导下微器对大鼠胶质瘤模型的照射

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此视频中的章节

颅放疗引起的认知功能障碍恢复干细胞移植策略

在原位小鼠脑肿瘤模型结合放疗

立体定向颅内植入和

使用小动物辐射研究平台在胶质母细胞瘤大鼠模型中基于正电子发射断层扫描的剂量绘画放射治疗

原位胶质母细胞瘤小鼠模型的维护脑实质的物理约束，并适用于活体双光子显微镜

磁共振引导的高强度聚焦超声产生热疗:小鼠横纹肌肉瘤模型中的可行治疗方法

小鼠模型中胶质母细胞瘤的双光子活体显微镜

小鼠模型中胶质母细胞瘤的双光子活体显微镜

小鼠模型中胶质母细胞瘤的双光子活体显微镜

多形性胶质母细胞瘤动物模型中寡核苷酸有效载荷的纳米颗粒递送