作者要感谢丹妮拉 Pompilio 博士和罗伯塔 Ciccimarra 的技术帮助。

本文所描述的所有动物实验都是由校内动物福利委员会批准的基耶西 Farmaceutici 和伊拉斯谟 MC 的动物实验在协议编号: EMC 3349 (138-14-07) 遵守欧洲指令 2010/63 UE,意大利 d. Lgs 26/2014 和修订的 "实验室动物护理和使用指南"23。
注: 在使用前, 雌性近交 C57Bl/6 (7-8 周大) 小鼠至少7天适应当地 vivarium 条件 (室温: 20-24 °c; 相对湿度: 40-70%; 12-h 浅-暗循环), 可自由获得标准啮齿动物周和软化自来水。
1. 博莱霉素气管内治疗小鼠
<ol><li>准备气管内灌注的设备12,13,14。</li>
	<li>将小鼠放入麻醉室, 连接到2.5% 与氧混合的异氟醚蒸发器。检查麻醉深度的缺乏脚趾捏的反应。麻醉效果在3-5 分钟后发生。</li>
	<li>将麻醉鼠标放置在插管平台上, 将其挂在导线上。</li>
	<li>打开喉镜, 采取一双钝钳和使用钳或喉镜的提示, 轻轻地打开嘴。</li>
	<li>拔掉舌头, 用镊子把它抱在一边。将喉镜刀片朝后部, 直到气管的开口可视化, 并保持喉镜到位。</li>
	<li>另一方面, 将输送管连接到 PE 油管端部到气管内, 旋转三路阀门。提供50µL 的博莱霉素 (0.020 µg/小鼠)。灌注后, 迅速从气管中取出导管, 以防止窒息。按住鼠标直立几秒钟。<ol>
<li>管理博莱霉素 intratracheally 在0天, 并在4天重复 (图 1)。</li>
	</ol>
</li>
	<li>从插管平台上取出鼠标, 并监视鼠标30分钟 (从麻醉中完全恢复所需的时间)。</li>
	<li>在7、14和21天的双博莱霉素灌注后, 用裂变材料和微 CT 对小鼠肺部进行体内成像。</li>
</ol>2.体内荧光分子层析成像技术
注: 事先, 准备一个新的库存解决方案 6 nmol/毫升的基质敏感荧光基板680在盐水溶液 (0.9% 氯化钠), 并存储保护, 从光在4°c 之前使用。它是稳定的长达6月, 在4摄氏度。在注射动物之前, 允许基质成像剂平衡到室温。
<ol><li>
探头注入
	<ol>
<li>准备静脉注射基质探针溶液的设备。预热水在50摄氏度, 以加热鼠标尾部。</li>
		<li>要牢牢抓住鼠标的颈背, 取它的尾巴, 让它抓住笼盖。把老鼠抱在动物的肩膀上, 把尾巴放在温水烧杯中, 4 到最大8的热量伤害, 以防止对尾部皮肤的热损伤。 检查静脉肿胀, 以便于可视化和针插入。</li>
		<li>在注射过程中, 把小鼠放入塑料抑制剂, 保持它的稳定, 并将尾巴从后面的一个特殊的洞中拉出来。找到尾巴两侧的两条尾脉;不是尾巴底部的动脉</li>
		<li>将1毫升注射器的针 (26G) 插入静脉。如果针正确放置, 注射将是容易的, 探针溶液将流入容器。</li>
		<li>注入10毫升/千克的基质探针溶液。</li>
		<li>扔掉针。 注: 最佳成像时间点是在基质探针注入后的24小时, 因为它是探针被激活所需的时间。如果进行纵向研究, 最佳再注射时间为7天, 允许从鼠标完全清除代理。</li>
	</ol>
</li>
	<li>
裂变材料成像 注意: 在开始之前, 总是删除的皮毛上和周围的动物, 将被成像, 在这种情况下的胸部, 以避免在组织中的散射和吸光度。<ol>
<li>要麻醉鼠标, 将其放在麻醉室中, 将异氟醚拨号到2.5% 进行感应, 2% 用于维护。</li>
		<li>初始化数据采集软件, 在数据库中打开新的实验研究。</li>
		<li>在用禁裂变材料开始成像之前, 将鼠标转移到成像盒中。将麻醉鼠标放在成像盒中间, 以最佳方式使用裂变材料的视野。保持小鼠平坦, 安全, 并轻轻地压缩对两个图像盒的窗口。在卡带上用旋钮调整高度, 然后将其滑入坞站。</li>
		<li>在扫描窗口中单击主题, 然后单击预览以查看实时图像。</li>
		<li>将扫描区域绘制得足够大, 以便捕获所预期的荧光区域周围的组织。包括总共25个扫描点保证肺部区域将被完全扫描。</li>
		<li>绘制 ROI 后, 首先单击添加到重建队列, 然后单击扫描以图像鼠标。请确保选中了 680 nm 的正确激光。</li>
		<li>完成图像采集后, 将鼠标放回其笼中。确保它完全从麻醉中恢复。</li>
		<li>使用ROI 工具在分析软件的 "分析" 窗口中量化荧光的 picomoles, 并在来自肺部区域的信号上减少大约 700-800 mm3的 ROI。小心排除肝脏信号。复制其他主题的 ROI, 使它们在维度上相似, 并调整它们在每个动物图像中的位置。</li>
		<li>将图像导出为 jpg 文件。</li>
	</ol>
</li>
</ol>3.体内微 CT 成像
注意: 在开始之前, 请始终清除图像区域附近的任何金属首饰或金属物体, 以避免 x 射线散射。
注: 辐射诱发肺纤维化是放射性肺损伤的常见发现24。在21天的四次微 ct 成像疗程中, 未出现与肺纤维化相关的微 ct 衍生指标和组织学发现, 这表明 x 射线剂量在微 ct 中传递给动物考试不足以影响调查结果。
<ol><li>通过按绿色电源按钮打开微 CT, 然后启动软件以预热 x 射线源。使用小孔和动物床的小鼠成像。</li>
	<li>通过单击新数据库新建一个数据库, 然后根据实验中的鼠标数生成浏览器, 或者单击连接到现有数据库, 将数据保存到以前创建的库中。</li>
	<li>在开始扫描之前, 选择软件控制窗口中的采集参数: x 射线管电压, 90 伏;CT X 线管电流, 160 µA;活 X 射线管电流, 80 µA;FOV, 36 毫米;无浇口技术;扫描技术, 高分辨率4分钟。</li>
	<li>麻醉小鼠吸入3% 异氟醚, 并把它们放在床上, 用鼻锥插入到钻孔中, 提供恒定的麻醉剂供应。将老鼠的爪子固定在床上, 让胸部暴露。</li>
	<li>滑动仪表门并打开实时模式按 "捕获" 按钮实时查看鼠标位置。使用直接位于 CT 仪器上的按钮, 移动动物床, 使胸部与视场 (FOV) 对齐。将扫描中心放在鼠标上;如果不调整位置与左, 右箭头位于 CT 仪器。</li>
	<li>通过选择90°并单击设置, 通过旋转龙门来确定最佳的床位置。确保图像的区域完全位于 FOV 内。</li>
	<li>请勿应用任何浇口技术, 并单击CT 扫描按钮开始扫描。单击是将打开的消息通知 x 射线源。 注: 一旦 x 光被打开, 在仪器的顶部的橙色灯将被照亮, 并且滑动门将不可能打开为操作员的安全。扫描完成后, 2D 查看器软件的一个新窗口显示了重建的 transaxial、日冕和矢状切片。</li>
	<li>检查图像质量, 并确保不具有模糊的图像从运动由于低水平的麻醉。如有必要, 请重复扫描。</li>
	<li>把动物放回笼子里, 确保它们完全从麻醉中恢复。</li>
</ol>4. 支气管肺泡灌洗
<ol><li>麻醉动物与3% 异氟醚和牺牲的腹部主动脉出血。</li>
	<li>用剪刀把胸笼和颈部暴露出来。然后暴露气管, 做一个小切口, 以允许使用21口径灌洗管连接到1毫升注射器没有针。注意不要割断气管。</li>
	<li>要执行该球, 填充1毫升注射器没有针与0.6 毫升的无菌溶液 [10x 汉克的平衡盐溶液 (HBSS); 100 毫米乙二胺四乙酸 (EDTA); 1 毫米 4-(2-羟基-乙基)-1-piperazineethansulphonic 酸 (HEPES); 蒸馏水]。</li>
	<li>将灌洗管插入气管内的切口中, 慢慢注射并取出溶液3次, 在第三次撤退时暂停, 以允许 BALF 的最佳收集以供后续分析。</li>
</ol>5. 组织学和形态计量学
<ol><li>用0.6 毫升10% 中性缓冲福尔马林的温和输液, 通过气管切开并取出肺部, 将样品在室温下贮存24小时。</li>
	<li>通过不同的通道脱水样品, 增加酒精溶液浓度 (60% 乙醇为1小时, 70% 乙醇为1小时, 90% 乙醇为 1 h, 95% 乙醇为 2 h, 100% 乙醇为 2 h) 使用自动组织处理器。</li>
	<li>将样品放置在二甲苯中2小时, 使其半透明。脱水结束时, 在60摄氏度的石蜡中渗透样品3小时, 并将其嵌入自动处理器中。</li>
	<li>使用旋转切片获得5µm 厚的串行段。</li>
	<li>Deparaffinize 和水化的下降等级的乙醇和染色与马尾松的三色使用自动组织处理器。</li>
	<li>手动聚焦肺部切片, 扫描20X 放大器使用幻灯片扫描仪和捕获的数字图像的整个肺部部分使用查看软件分辨率为 451 nm/像素。</li>
	<li>用半定量和定量参数对纤维化肺损伤进行形态学评价如下:<ol>
<li>确定阿什克罗夫特分数。根据阿什克罗夫特10修改的 Hübner et . 所定义的比例, 肺部分的半定量级形态学变化。25使用0-8 评分系统评估肺部分的所有实质。</li>
		<li>确定胶原含量。通过显微镜图像分析软件量化10、25的纤维化程度。随机选择三 ROIs 在10X 放大倍数, 每张幻灯片。通过标准化的颜色阈值设置, 检测胶原蛋白为绿色染色区域。</li>
		<li>确定肺泡空气区。将肺泡空气区量化为纤维化的间接参数10,25。通过相同的软件和 ROIs 应用于纤维化分数, 检测 ROIs 内的空气区域使用白阈值。</li>
	</ol>
</li>
</ol>

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A., Liu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dosimetry+in+Micro-computed+Tomography:+a+Review+of+the+Measurement+Methods,+Impacts,+and+Characterization+of+the+Quantum+GX+Imaging+System.">Dosimetry in Micro-computed Tomography: a Review of the Measurement Methods, Impacts, and Characterization of the Quantum GX Imaging System.</a> Mol Imaging Biol. , (2016).</li><li>Hubner, R. H., 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=Standardized+quantification+of+pulmonary+fibrosis+in+histological+samples).">Standardized quantification of pulmonary fibrosis in histological samples).</a> BioTechniques. 44 (4), 507-514 (2008).</li><li>King, T. E., Pardo, A., Selman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Idiopathic+pulmonary+fibrosis.">Idiopathic pulmonary fibrosis.</a> Lancet. 378 (9807), 1949-1961 (2011).</li><li>De Langhe, E., 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=Quantification+of+lung+fibrosis+and+emphysema+in+mice+using+automated+micro-computed+tomography.">Quantification of lung fibrosis and emphysema in mice using automated micro-computed tomography.</a> PloS one. 7 (8), 43123 (2012).</li></ol>

作者声明他们没有竞争的兴趣。

尽管许多研究小组专注于开发新药来治疗森林小组, 但目前只有两个患者可以使用。有迫切的医疗需要找到更有效的治疗方法7 , 因为只有肺 transplantationis 能够延长4-5 年26的生存。转化医学和新药开发的基本前提是能提供一种模仿森林小组特点的动物模型, 而介入研究对临床的成功有预测意义。然而, 现有的肺纤维化动物模型的效用仍然有争议的27</s...

特发性肺纤维化 (IPF) 是慢性肺病, 肺功能逐渐减少, 不幸的是, 在四年的诊断中往往是致命的1。森林小组的主要特点是胞外基质沉积和成纤维细胞增殖, 但尚未完全了解发病机制。最受支持的假说是, 多循环的肺损伤导致肺泡上皮细胞的破坏, 导致间充质细胞周期增殖的改变, 成纤维细胞和肌的夸大积累, 以及增加了矩阵的生成。在人类森林小组或博莱霉素诱导的动物模型中, 失调在纤维化发展中发现了基质金属蛋白酶等过程中所涉及的调解人。无控制的基质生产导致在肺间质和肺泡空间内的胶原沉积不平衡, 模仿异常伤口修复 1, 2.
药物发现和发展的主要障碍之一是可利用的老鼠模型, 模仿人类发病机制和疾病表型。不同的药物已被用来诱导动物模型中的肺纤维化: 辐照损伤, 石棉和二氧化硅的管理, fibrinogenic 细胞因子的管理和博莱霉素3,4;然而, 博莱霉素是最常用的老鼠, 老鼠, 豚鼠, 仓鼠, 兔子5或在大型动物 (非人类灵长类, 马, 狗和反刍动物)6,7。博莱霉素是一种抗生素, 由细菌链霉菌 verticillus8 , 并用作抗癌剂9。肺纤维化是药物的常见副作用, 因此, 它被用于实验动物模型中, 诱发肺纤维化。
博莱霉素诱导的肺纤维化模型, 在博莱霉素管理后 14-21 天发生纤维性病变。在所提出的工作中, 我们采用了新的协议来诱导小鼠肺纤维化的双博莱霉素气管灌注。博莱霉素小鼠模型是非常耗时的, 因为新的药物需要评估已建立的纤维化病变, 并测试, 以区分其抗纤维化的效果与抗炎作用。
对胶原含量、形态测量学和组织学分析的生化测定是基于死后分析, 限制了在同一动物中跟踪疾病发病机制的可能性。虽然这些参数被认为是纤维化评价的金标准, 但它们没有提供任何时间或空间分布的纤维性病变, 并排除了一个方法来调查疾病进展的过程。10
最近, 非侵入性影像技术已被应用于监测小鼠气道重塑、炎症和纤维化进展: 磁共振成像 (MRI), 微计算机断层扫描 (微 CT), 荧光分子层析成像 (裂变材料) 和生物发光 (BLI)11,12,13,14,15,,16,17,18 ,20,21。在博莱霉素挑战22之后, 我们提出一种非侵入性的影像学方法来监测纵向肺纤维化进展的裂变材料和微 CT 在不同的时间点。
许多途径参与了纤维化的建立和发展, 而不多的是已知的。只有对这些过程有更深的了解, 才能转化为可能转移到诊所的更多药物靶点。利用荧光分子层析成像技术对基质金属蛋白酶的活化进行纵向监测, 并通过显微 CT 检测肺实质变化, 可用于今后对治疗的临床反应。

<table><tbody><tr><td>FMT 2500 Fluorescence Tomography System</td><td>Perkin Elmer Inc.</td><td>Experimental Builder</td><td></td></tr><tr><td>MMPsense 680</td><td>Perkin Elmer Inc.</td><td>NEV10126</td><td>Protect from light, store the probe at 4 &amp;deg;C</td></tr><tr><td>TrueQuant software</td><td>Perkin Elmer Inc.</td><td></td><td></td></tr><tr><td>Female inbred C57BL/6</td><td>San Pietro NatisoneHorst, The Netherlands (UD),&amp;nbsp;</td><td></td><td>Prior to use, animals were acclimatized for at least 5 days to the local vivarium conditions</td></tr><tr><td>Isoflurane</td><td>ESTEVE spa</td><td>571329.8</td><td>Do not inhale</td></tr><tr><td>Automated cell counter</td><td>Dasit XT 1800J</td><td>Experimental Builder</td><td></td></tr><tr><td>Saline Solution, 0.9% Sodium Chloride (NaCl)</td><td>Eurospital</td><td>15A2807</td><td></td></tr><tr><td>Quantum FX Micro-CT scanner&amp;nbsp;</td><td>Perkin Elmer Inc.</td><td></td><td></td></tr><tr><td>Bleomycin sulphate from Streptomyces Verticillus&amp;nbsp;</td><td>Sigma</td><td>&amp;nbsp;B2434&amp;nbsp;</td><td></td></tr><tr><td>Automatic tissue Processor&amp;nbsp;</td><td>ATP700 Histo-Line Laboratories</td><td>ATP700&amp;nbsp;</td><td></td></tr><tr><td>Embedding system&amp;nbsp;</td><td>EG 1160 Leica Biosystems</td><td>EG 1160</td><td></td></tr><tr><td>Rotary microtome&amp;nbsp;</td><td>Slee Cut 6062</td><td></td><td></td></tr><tr><td>Digital slide scanner&amp;nbsp;</td><td>NanoZoomer S60, Hamamatsu Photonics</td><td></td><td></td></tr><tr><td>NIS-AR image analysis software&amp;nbsp;</td><td>Nikon</td><td></td><td></td></tr><tr><td>Masson&amp;rsquo;s Trichrome Staining</td><td>Histo-Line Laboratories</td><td></td><td></td></tr><tr><td>10% neutral-buffered formalin</td><td>Sigma</td><td>HT5012-1CS</td><td></td></tr><tr><td>Penn-century model DP-4M Dry power insufflator</td><td>Penn-century</td><td>DPM-EXT</td><td></td></tr><tr><td>PE190 micro medical tubing</td><td>2biological instruments snc</td><td>BB31695-PE/8</td><td></td></tr><tr><td>Syringe without needle 5 mL</td><td>Terumo</td><td>SS*05SE1</td><td>Cut the boards of the piston by scissors</td></tr><tr><td>Hamilton 0.10 mL (model 1710)</td><td>Gastight</td><td>81022</td><td></td></tr><tr><td>Discofix 3-way Stopcock</td><td>Braun</td><td>4095111</td><td></td></tr><tr><td>Syringe with needle 1 mL</td><td>Pic solution</td><td>3,071,260,300,320</td><td>Use without needle</td></tr><tr><td>Plastic feeding tubes 18 ga x 50 mm</td><td>2biological instruments snc</td><td>FTP-18-50</td><td>Cut obliquely the tip&amp;nbsp;</td></tr></tbody></table>

a multimodal imaging approach based on micro-ct and fluorescence molecular tomography for longitudinal assessment of bleomycin-induced lung fibrosis in mice

特发性肺纤维化 (IPF) 是一种致命的肺部疾病的特点是渐进和不可逆转的破坏肺结构, 这导致严重恶化的肺功能和随后死亡的呼吸衰竭。
博莱霉素在实验动物模型中的发病机制已被注射。本文研究了双气管内注射博莱霉素诱导的森林小组样小鼠模型。用于研究肺纤维化的标准组织学评估是侵入性终端程序。这项工作的目的是通过非侵入性成像技术, 如荧光分子层析和微 CT 监测肺纤维化。通过组织学研究证实的这两种技术可以代表一种革命性的功能性方法, 用于对森林小组疾病的严重程度和进展进行实时无创监测。不同方法的融合是进一步了解森林小组疾病的一步, 在这种情况下, 在病理条件下发生的分子事件可以用裂变材料观察到, 随后的解剖变化可以通过微 CT 监测。

肺纤维化病变的自发分辨率观察三周后, 单一的博莱霉素管理和适度的结构变化突出了这个模型的局限性。只有预防性治疗才能执行, 因为狭窄的治疗窗口不代表临床实践17。
在这里, 我们证明, 我们的双博莱霉素气管灌注的协议能够发展长期持久的肺纤维化在小鼠18。实验设计显示在<strong cla...

Watch this Scientific Journal Video about A Multimodal Imaging Approach Based on Micro-CT and Fluorescence Molecular Tomography for Longitudinal Assessment of Bleomycin-Induced Lung Fibrosis in Mice a

A Multimodal Imaging Approach Based on Micro-CT and Fluorescence Molecular Tomography for Longitudinal Assessment of Bleomycin-Induced Lung Fibrosis in Mice

基于显微 CT 和荧光分子层析成像的多模态成像方法对博莱霉素诱导的小鼠肺纤维化的纵向评价

本文描述了一种基于微 CT 和荧光分子层析成像的无创多模成像方法, 用于对博莱霉素双气管灌注诱导的小鼠肺纤维化模型进行纵向评价。

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by the progressive and irreversible destruction of lung architecture, which ...

a-multimodal-imaging-approach-based-on-micro-ct-fluorescence

Research

JoVE Journal

Immunology and Infection

11.4K 次观看.  Chiesi Farmaceutici S.p.A.. 本文描述了一种基于微 CT 和荧光分子层析成像的无创多模成像方法, 用于对博莱霉素双气管灌注诱导的小鼠肺纤维化模型进行纵向评价。

视频: A Multimodal Imaging Approach Based on Micro-CT and Fluorescence Molecular Tomography for Longitudinal Assessment of Bleomycin-Induced Lung Fibrosis in Mice

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography (&mu;CT).
High resolution &mu;CT is a very versatile yet accurate (1-2 microns of resolution) technique for 3D examination of ex-vivo biological samples1, 2. As opposed to electron tomography, the &mu;CT allows the examination of up to 4 cm thick samples. This technique requires only few hours of measurement as compared to weeks in histology. In addition, &mu;CT does not rely on 2D stereologic models, thus it may complement and in some cases can even replace histological methods3, 4, which are both time consuming and destructive. Sample conditioning and positioning in &mu;CT is straightforward and does not require high vacuum or low temperatures, which may adversely affect the structure. The sample is positioned and rotated 180&deg; or 360&deg;between a microfocused x-ray source and a detector, which includes a scintillator and an accurate CCD camera, For each angle a 2D image is taken, and then the entire volume is reconstructed using one of the different available algorithms5-7. The 3D resolution increases with the decrease of the rotation step. The present video protocol shows the main steps in preparation, immobilization and positioning of the sample followed by imaging at high resolution.

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography ( CT).

高分辨率三维成像 EX - VIVO生物样品显微CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized ...

科研

生物工程

Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence imaging using IVIS Spectrum was employed to monitor the distribution and development of the tumor cells following the delivery procedure including DLIT reconstruction to measure the tumor signal and its location.
Development of metastatic lesions to the bone tissues triggers osteolytic activity and lesions to tibia and femur were evaluated longitudinally using micro CT. Imaging was performed using a Quantum FX micro CT system with fast imaging and low X-ray dose. The low radiation dose allows multiple imaging sessions to be performed with a cumulative X-ray dosage far below LD50. A mouse imaging shuttle device was used to sequentially image the mice with both IVIS Spectrum and Quantum FX achieving accurate animal positioning in both the bioluminescence and CT images. The optical and CT data sets were co-registered in 3-dimentions using the Living Image 4.1 software. This multi-mode approach allows close monitoring of tumor growth and development simultaneously with osteolytic activity.

An experimental mouse model of bone metastasis was established following intracardiac delivery of luciferase expressing mammary tumor cells. Tumor development and resulted osteolytic lesion were monitored longitudinally with bioluminescence and micro CT imaging.

生物发光和微型CT成像监测肿瘤转移和溶骨性病变

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence ...

医学

Multimodal Bioluminescent and Positronic-emission Tomography/Computational Tomography Imaging of Multiple Myeloma Bone Marrow Xenografts in NOG Mice

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular interactions that exist within this microenvironment. Yet the BM microenvironment cannot be easily replicated in vitro, which potentially limits the physiologic relevance of many in vitro and ex vivo experimental models. These issues can be overcome by utilizing a xenograft model in which luciferase (LUC)-transfected 8226 MM cells will specifically engraft in the mouse skeleton. When these mice are given the appropriate substrate, D-luciferin, the effects of therapy on tumor growth and survival can be analyzed by measuring changes in the bioluminescent images (BLI) produced by the tumors in vivo. This BLI data combined with positronic-emission tomography/computational tomography (PET/CT) analysis using the metabolic marker 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) is used to monitor changes in tumor metabolism over time. These imaging platforms allow for multiple noninvasive measurements within the tumor/BM microenvironment.

Here we use bioluminescent, X-ray, and positron-emission tomography/computed tomography imaging to study how inhibiting mTOR activity impacts bone marrow-engrafted myeloma tumors in a xenograft model. This allows for physiologically relevant, non-invasive, and multimodal analyses of the anti-myeloma effect of therapies targeting bone marrow-engrafted myeloma tumors in vivo.

nog 小鼠多发性骨髓瘤异种移植的多模态生物发光和正电子发射成形断层扫描成像

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular ...

癌症研究

Computed Tomography-guided Time-domain Diffuse Fluorescence Tomography in Small Animals for Localization of Cancer Biomarkers

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light absorption by tissue chromophores (e.g., hemoglobin, water, lipids, melanin) typically limits optical signal propagation through thicknesses larger than a few millimeters2. Compared to other visible wavelengths, tissue absorption for red and near-infrared (near-IR) light absorption dramatically decreases and non-elastic scattering becomes the dominant light-tissue interaction mechanism. The relatively recent development of fluorescent agents that absorb and emit light in the near-IR range (600-1000 nm), has driven the development of imaging systems and light propagation models that can achieve whole body three-dimensional imaging in small animals3.
Despite great strides in this area, the ill-posed nature of diffuse fluorescence tomography remains a significant problem for the stability, contrast recovery and spatial resolution of image reconstruction techniques and the optimal approach to FMI in small animals has yet to be agreed on. The majority of research groups have invested in charge-coupled device (CCD)-based systems that provide abundant tissue-sampling but suboptimal sensitivity4-9, while our group and a few others10-13 have pursued systems based on very high sensitivity detectors, that at this time allow dense tissue sampling to be achieved only at the cost of low imaging throughput. Here we demonstrate the methodology for applying single-photon detection technology in a fluorescence tomography system to localize a cancerous brain lesion in a mouse model.
The fluorescence tomography (FT) system employed single photon counting using photomultiplier tubes (PMT) and information-rich time-domain light detection in a non-contact conformation11. This provides a simultaneous collection of transmitted excitation and emission light, and includes automatic fluorescence excitation exposure control14, laser referencing, and co-registration with a small animal computed tomography (microCT) system15. A nude mouse model was used for imaging. The animal was inoculated orthotopically with a human glioma cell line (U251) in the left cerebral hemisphere and imaged 2 weeks later. The tumor was made to fluoresce by injecting a fluorescent tracer, IRDye 800CW-EGF (LI-COR Biosciences, Lincoln, NE) targeted to epidermal growth factor receptor, a cell membrane protein known to be overexpressed in the U251 tumor line and many other cancers18. A second, untargeted fluorescent tracer, Alexa Fluor 647 (Life Technologies, Grand Island, NY) was also injected to account for non-receptor mediated effects on the uptake of the targeted tracers to provide a means of quantifying tracer binding and receptor availability/density27. A CT-guided, time-domain algorithm was used to reconstruct the location of both fluorescent tracers (i.e., the location of the tumor) in the mouse brain and their ability to localize the tumor was verified by contrast-enhanced magnetic resonance imaging.
Though demonstrated for fluorescence imaging in a glioma mouse model, the methodology presented in this video can be extended to different tumor models in various small animal models potentially up to the size of a rat17.

Diffuse fluorescence tomography offers a relatively low-cost and potentially high-throughout approach to preclinical in vivo tumor imaging. The methodology of optical data collection, calibration, and image reconstruction is presented for a computed tomography-guided non-contact time-domain system using fluorescent targeting of the tumor biomarker epidermal growth factor receptor in a mouse glioma model.

电脑断层导引的时域漫荧光体层摄影术在小动物的肿瘤生物标志物的本土化

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light ...

Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. With the increasing availability of knockout and transgenic derivatives, together with a vast amount of genetic information, mice provide one of the best models to study the molecular mechanisms underlying the pathology and physiology of lung diseases. Inhalation, intranasal instillation, intratracheal instillation, and intratracheal intubation are the most widely used techniques by a number of investigators to administer materials of interest to mouse lungs. There are pros and cons for each technique depending on the goals of a study. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented. This technique was applied to administer bleomycin to mouse lungs as a model to study pulmonary fibrosis.

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented.

无创性气管内插管，以研究小鼠肺的病理生理

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular ...

Hybrid &#181;CT-FMT imaging and image analysis

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to assess the biodistribution of novel probes and to assess disease progression using established molecular probes or reporter genes. The combination with an anatomical modality, e.g., micro computed tomography (&#181;CT), is beneficial for image analysis and for fluorescence reconstruction. We describe a protocol for multimodal &#181;CT-FMT imaging including the image processing steps necessary to extract quantitative measurements. After preparing the mice and performing the imaging, the multimodal data sets are registered. Subsequently, an improved fluorescence reconstruction is performed, which takes into account the shape of the mouse. For quantitative analysis, organ segmentations are generated based on the anatomical data using our interactive segmentation tool. Finally, the biodistribution curves are generated using a batch-processing feature. We show the applicability of the method by assessing the biodistribution of a well-known probe that binds to bones and joints.

Hybrid  &#181;CT-FMT imaging and image analysis

We describe a protocol for hybrid imaging, combining fluorescence-mediated tomography (FMT) with micro computed tomography (&#181;CT). After fusion and reconstruction, we perform interactive organ segmentation to extract quantitative measurements of the fluorescence distribution.

混合μCT-FMT成像和图像分析

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to ...

Using Micro-computed Tomography for the Assessment of Tumor Development and Follow-up of Response to Treatment in a Mouse Model of Lung Cancer

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse models of lung cancer are being used to study the mechanism of cancer development and to experiment with various therapeutic strategies. However, the absence of a real-time technique to identify the development of tumor nodules in mice lungs and to monitor the changes in their size in response to various experimental and therapeutic interventions hampers the ability to obtain an accurate description of the course of the disease and its timely response to treatments. In this study, a method using a micro-computed tomography (CT) scanner for the detection of the development of lung tumors in a mouse model of lung adenocarcinoma is described. Next, we show that monthly follow-up with micro-CT can identify dynamic changes in the lung tumor, such as the appearance of additional nodules, increase in the size of previously detected nodules, and decrease in the size or complete resolution of nodules in response to treatment. Finally, the accuracy of this real-time assessment method was confirmed with end-point histological quantification. This technique paves the way for planning and conducting more complex experiments on lung cancer animal models, and it enables us to better understand the mechanisms of carcinogenesis and the effects of different treatment modalities while saving time and resources.

We describe a method for the detection of tumor nodule development in the lungs of an adenocarcinoma mouse model using micro-computed tomography and its use for monitoring changes in nodule size over time and in response to treatment. The accuracy of the assessment was confirmed with end-point histological quantification.

采用微计算机断层扫描对肿瘤发展的评估和后续反应来治疗肺癌的小鼠模型

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse ...

Induction of Mouse Lung Injury by Endotracheal Injection of Bleomycin

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models continue to be an essential tool for developing new antifibrotic strategies. Here we provide an effective method to investigate in vivo antifibrotic activity of human mesenchymal stromal cells obtained from whole umbilical cord (hUC-MSC) in attenuating bleomycin-induced lung injury. C57BL/6 mice receive a single endotracheal injection of bleomycin (1.5 U/kg body weight) followed by a double infusion of hUC-MSC (2.5 x 105) into the tail vein, 24 h and 7 days after the bleomycin administration. Upon sacrifice at days 8, 14, or 21, inflammatory and fibrotic changes, collagen content, and hUC-MSC presence in explanted lung tissue are analyzed. The injection of bleomycin into the mouse&#160;trachea allows the direct targeting of the lungs, leading to extensive pulmonary inflammation and fibrosis. The systemic administration of a double dose of hUC-MSC results in the early blunting of the bleomycin-induced lung injury. Intravenously infused hUC-MSC are transiently engrafted into the mouse lungs, where they exert their anti-inflammatory and antifibrotic activity. In conclusion, this protocol has been successfully applied for the preclinical testing of hUC-MSC in an experimental mouse model of human pulmonary fibrosis. However, this technique can be easily extended both to study the effect of different endotracheally administered substances on the pathophysiology of the lungs and to validate new anti-inflammatory and antifibrotic systemic therapies.

Here we present an effective method to investigate the antifibrotic activity of intravenously infused human mesenchymal stromal cells obtained from the whole umbilical cord following the induction of lung injury by an endotracheal injection of bleomycin in C57BL/6 mice. This protocol can be easily extended to the preclinical testing of other therapeutics.

小鼠肺损伤的诱导,通过博霉素内切注射

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models ...

免疫与感染

Pre-Conditioning the Airways of Mice with Bleomycin Increases the Efficiency of Orthotopic Lung Cancer Cell Engraftment

Lung cancer is a deadly treatment refractory disease that is biologically heterogeneous. To understand and effectively treat the full clinical spectrum of thoracic malignancies, additional animal models that can recapitulate diverse human lung cancer subtypes and stages are needed. Allograft or xenograft models are versatile and enable the quantification of tumorigenic capacity in vivo, using malignant cells of either murine or human origin. However, previously described methods of lung cancer cell engraftment have been performed in non-physiological sites, such as the flank of mice, due to the inefficiency of orthotopic transplantation of cells into the lungs. In this study, we describe a method to enhance orthotopic lung cancer cell engraftment by pre-conditioning the airways of mice with the fibrosis inducing agent bleomycin. As a proof-of-concept experiment, we applied this approach to engraft tumor cells of the lung adenocarcinoma subtype, obtained from either mouse or human sources, into various strains of mice. We demonstrate that injuring the airways with bleomycin prior to tumor cell injection increases the engraftment of tumor cells from 0-17% to 71-100%. Significantly, this method enhanced lung tumor incidence and subsequent outgrowth using different models and mouse strains. In addition, engrafted lung cancer cells disseminate from the lungs into relevant distant organs. Thus, we provide a protocol that can be used to establish and maintain new orthotopic models of lung cancer with limiting amounts of cells or biospecimen and to quantitatively assess the tumorigenic capacity of lung cancer cells in physiologically relevant settings.

We describe a method to significantly enhance orthotopic engraftment of lung cancer cells into the murine lungs by pre-conditioning the airways with injury. This approach may also be applied to study stromal interactions within the lung microenvironment, metastatic dissemination, lung cancer co-morbidities, and to more efficiently generate patient derived xenografts.

博莱霉素对小鼠气管的预处理提高原位肺癌细胞植入的效率

Lung cancer is a deadly treatment refractory disease that is biologically heterogeneous. To understand and effectively treat the full clinical spectrum of ...

Implantation and Monitoring by PET/CT of an Orthotopic Model of Human Pleural Mesothelioma in Athymic Mice

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. MPM development is mainly associated with asbestos. Treatments provide only modest survival since the median survival average is 9&#8211;18 months from the time of diagnosis. Therefore, more effective treatments must be identified. Most data describing new therapeutic targets were obtained from in vitro experiments and need to be validated in reliable in vivo preclinical models. This article describes one such reliable MPM orthotopic model obtained after injection of a human MPM cell line H2052/484 into the pleural cavity of immunodeficient athymic mice. Transplantation in the orthotopic site allows studying the progression of tumor in the natural in vivo environment. Positron emission tomography/computed tomography (PET/CT) molecular imaging using the clinical [18F]-2-fluoro-2-deoxy-D-glucose ([18F]FDG) radiotracer is the diagnosis method of choice for examining patients with MPM. Accordingly, [18F]FDG-PET/CT was used to longitudinally monitor the disease progression of the H2052/484 orthotopic model. This technique has a high 3R potential (Reduce the number of animals, Refine to lessen pain and discomfort, and Replace animal experimentation with alternatives) since the tumor development can be monitored non-invasively and the number of animals required could be significantly reduced.
This model displays a high development rate, a rapid tumor growth, is cost-efficient and allows for rapid clinical translation. By using this orthotopic xenograft MPM model, researchers can assess biological responses of a reliable MPM model following therapeutic interventions.

This article describes the generation of an orthotopic mouse model of human pleural mesothelioma by implantation of H2052/484 mesothelioma cells into the pleural cavity of immunocompromised athymic mice. The longitudinal monitoring of the development of intrapleural tumors was assessed by non-invasive multimodal [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography imaging.

小鼠中人类胸膜皮质瘤的PET/CT的植入与监测

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. ...