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。我们开发了一种新的小鼠肺纤维化模型, 需要双注入博莱霉素, 如图 118所述。成像技术是可视化疾病进展的有力工具, 是对治疗的药理反应。这种动物模型更好地概括了森林小组和无创技术的人类特点, 可以在临床前设置和临床医学实践之间建立一个桥梁27。
然而, 为了获得可靠和可重复的数据, 有些步骤是至关重要的。在小鼠完全麻醉的情况下, 使用标准化的程序, 必须进行气管灌注。微 ct 采集需要非常准确的麻醉监测, 因为 ct 投射是由呼吸频率封闭的。在成像前, 检查老鼠的麻醉深度是否相同。脱毛光学成像的一个非常重要的步骤。在开始之前, 一定要清除胸部周围的皮毛, 避免组织中的散射和吸收。探针注射需要通过动物的体重来矫正。
在不同时间点对同一小鼠的解剖变化进行调查和量化特定分子读出的可能性是了解纤维化发展的一大步, 对功能和药理学的明显进步。研究。
这种多模态成像方法是评估药物功效的一种智能工具, 它比终端评估提供更多的信息, 在更有效的药物发现过程中进行翻译。

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。

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		<col />
		<col />
		<col />
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">FMT 2500 Fluorescence Tomography System</td>
			<td style="width:335px;">Perkin Elmer Inc.</td>
			<td style="width:171px;">Experimental Builder</td>
			<td style="width:607px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">MMPsense 680</td>
			<td style="width:335px;">Perkin Elmer Inc.</td>
			<td style="width:171px;">NEV10126</td>
			<td>Protect from light, store the probe at 4 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">TrueQuant software</td>
			<td style="width:335px;">Perkin Elmer Inc.</td>
			<td style="width:171px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Female inbred C57BL/6</td>
			<td>San Pietro NatisoneHorst, The Netherlands (UD),&#160;</td>
			<td style="width:171px;"></td>
			<td>Prior to use, animals were acclimatized for at least 5 days to the local vivarium conditions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Isoflurane</td>
			<td style="width:335px;">ESTEVE spa</td>
			<td style="width:171px;">571329.8</td>
			<td>Do not inhale</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Automated cell counter</td>
			<td style="width:335px;">Dasit XT 1800J</td>
			<td>Experimental Builder</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Saline Solution, 0.9% Sodium Chloride (NaCl)</td>
			<td style="width:335px;">Eurospital</td>
			<td>15A2807</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Quantum FX Micro-CT scanner&#160;</td>
			<td style="width:335px;">Perkin Elmer Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:328px;">Bleomycin sulphate from Streptomyces Verticillus&#160;</td>
			<td style="width:335px;">Sigma</td>
			<td>&#160;B2434&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Automatic tissue Processor&#160;</td>
			<td style="width:335px;">ATP700 Histo-Line Laboratories</td>
			<td>ATP700&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Embedding system&#160;</td>
			<td style="width:335px;">EG 1160 Leica Biosystems</td>
			<td>EG 1160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Rotary microtome&#160;</td>
			<td style="width:335px;">Slee Cut 6062</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Digital slide scanner&#160;</td>
			<td style="width:335px;">NanoZoomer S60, Hamamatsu Photonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">NIS-AR image analysis software&#160;</td>
			<td style="width:335px;">Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Masson&#8217;s Trichrome Staining</td>
			<td style="width:335px;">Histo-Line Laboratories</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">10% neutral-buffered formalin</td>
			<td style="width:335px;">Sigma</td>
			<td>HT5012-1CS</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:328px;">Penn-century model DP-4M Dry power insufflator</td>
			<td style="width:335px;">Penn-century</td>
			<td>DPM-EXT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">PE190 micro medical tubing</td>
			<td style="width:335px;">2biological instruments snc</td>
			<td style="width:171px;">BB31695-PE/8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Syringe without needle 5 mL</td>
			<td style="width:335px;">Terumo</td>
			<td style="width:171px;">SS*05SE1</td>
			<td>Cut the boards of the piston by scissors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Hamilton 0.10 mL (model 1710)</td>
			<td style="width:335px;">Gastight</td>
			<td>81022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Discofix 3-way Stopcock</td>
			<td style="width:335px;">Braun</td>
			<td>4095111</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Syringe with needle 1 mL</td>
			<td style="width:335px;">Pic solution</td>
			<td>3,071,260,300,320</td>
			<td>Use without needle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Plastic feeding tubes 18 ga x 50 mm</td>
			<td style="width:335px;">2biological instruments snc</td>
			<td>FTP-18-50</td>
			<td>Cut obliquely the tip&#160;</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。实验设计显示在图 119,20,21中。本研究的目的是观察无创成像技术的小鼠肺纤维化进展。博莱霉素是 intratracheally 管理两次 (在0和4天, 1 毫克/千克的博莱霉, 在50µL 每一次)。对于每组十二只小鼠, intratracheally 在与博莱霉素组相同的时间内对同一体积的车辆进行了挑战。为评价纤维化, 在阿什克罗夫特评分系统的基础上进行了半定量组织学分析。10
本研究采用显微 CT 和裂变材料技术结合经典组织学方法, 对博莱霉素诱导小鼠模型肺纤维化的发展进行了评价。影像学技术的无创性是肺纤维化进展的临床前评价的真实价值, 与组织学吻合的结果是一个很好的步骤。微 CT 对纤维化病变纵向肺实质变化的量化有重要作用。
博莱霉素治疗组的组织学图片显示, 从7天开始, 主要作为单纤维化肿块, 并在14天进展到融合的替代胶原蛋白, 保持不变直到21天 (图 2 a-2 c)。博莱霉素治疗引起的肺部炎症 (图 3A), 其中白细胞的数量显著高于 BALF 治疗小鼠 7, 14, 21 天相比, 汽车组。有趣的是, 每次取样时淋巴细胞和单核细胞分数也会增加 (图 3 b-3 c);相比之下, 在7天 (图 3D) 中, 已观察到中性粒细胞分数的显著增加。
本研究采用显微 CT 对肺实质变化进行纵向监测。从基线观察的不同时间点的肺结构的渐进解剖学变化清楚地看到在微 CT 预测 (图 4 a-4 b)。支气管树远端的气道半径 (图5A 和 5C)19、20、21和总肺纤维化百分比 (图5B 和 5D) 可量化纤维化进展。与组织学评分相比, 博莱霉素治疗组7天的纤维化百分率 (图 5D) 略有高估。这可以解释为炎症和纤维化发病的双重反应, 使得很难区分这两种症状。从显微 CT 图像处理中选择肺实质变化的定量 (图 5 c-5 d)19,20,21.这些显微 CT 参数很好地与组织学结果一致, 如图 2 a/2 c所示。
微 CT 成像直接反映了肺实质的病理和治疗变化, 裂变材料技术提供了与森林小组喜欢的蛋白质表达有关的定量信息。在这项研究中, 我们选择了一个基于它与森林小组相关的基质探针, 我们在博莱霉素治疗小鼠 (图 6)18中发现了特异的基质金属蛋白酶活化。用 activable 荧光探针在选定的时间点注射载体或博莱霉素治疗小鼠, 研究了基质金属蛋白酶的作用。注射二十四小时后, 小鼠通过裂变材料的图像显示, 纤维化小鼠可以激活特定的基质荧光探针体内(图 6 a-6 d)18和前体 (图 6E)18.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56443/56443fig1.jpg"> 图 1: 实验性地建立了博莱霉素诱导的小鼠肺纤维化.C57BL/6 雌性小鼠有生理盐水或博莱霉素 intratracheally 两次, 0 和4天。小鼠在基线 (0 天)、7、14和21天的微 CT 扫描器中成像。12只小鼠被牺牲 7, 14, 21 天, 他们的肺部评估胶原沉积, 以关联组织学结果与图像获得的µCT。此数字已从已发布的文章21中进行了修改。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56443/56443fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56443/56443fig2.jpg"> 图 2:博莱霉素诱导小鼠肺纤维化的组织学分析时间过程.  (A)通过阿什克罗夫特评分在不同时间点的车辆或博莱霉素治疗小鼠肺纤维化的量化。实验重复了三次, 每个点代表了12只动物的平均扫描电镜。统计分析是由方差计算, 其次是 Tukey 的测试。* p < 0.05;** < 0.01。 (B)阿什克罗夫特在轻度、中等和严重子类别中分配的评分频率分布。 (C) intratracheally 双注入博莱霉素或生理盐水治疗小鼠7、14、21天后处理 (放大10X、鳞条200µm) 对马尾松腺染色小鼠肺切片的代表性组织学。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56443/56443fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56443/56443fig3.jpg"> 图 3:细胞浸润时间过程进入博莱霉素诱导的小鼠肺纤维化 BALF.  (A)的白细胞数、 (B)单核细胞、 (C)淋巴细胞和(D)嗜中性粒度。在 BALF 中找到的单元格表示为 cells*103/µL 的数目。实验重复了三次, 每个点代表了9只动物的平均扫描电镜。统计分析是由方差计算, 其次是 Dunnett 的测试。* p < 0.05;** < 0.01。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56443/56443fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56443/56443fig4.jpg"> 图 4: 对博莱霉素诱导的肺纤维化和车辆治疗小鼠的纵向微 CT 成像预测。(A)微 ct、博莱霉素治疗小鼠和(B)微 ct、盐水治疗小鼠<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56443/56443fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56443/56443fig5.jpg"> 图 5: 呼吸道, 纤维化肺裂片的定量和分割基于重复的微 CT 成像. (A)气道被分为中央和远端部分。(B)呼吸道的远端是用于识别和分裂成肺裂片的肺内道, 如: 右颅叶 (RCrL)、右中叶 (RMdL)、右尾叶 (RCdL)、右副瓣 (RAcL) 和左肺 (LL)。(C)气道半径和(D)全肺纤维化量化无论是车辆或博莱霉素治疗小鼠在不同的时间点。每一点都代表5只动物的平均扫描电镜, 总共有30只老鼠。统计分析是由方差计算, 其次是 Dunnett 的测试。* p < 0.05;** < 0.01。此数字已从已发布的文章21中进行了修改。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56443/56443fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56443/56443fig6.jpg"> 图。6. 用裂变材料 inbleomycin 诱导的肺纤维化小鼠荧光信号的时间过程。在体内 (A 和 B)和前体(C 和 D)用车辆或博莱霉素探针治疗的小鼠注射的典型图像。(E)用裂变材料图像软件自动计算肺荧光信号的总量。实验重复了三次, 每个点代表了9种动物的平均标准偏差。统计分析是由方差计算, 其次是 Dunnett 的测试。* p < 0.05;** < 0.01。此数字已从已发布的文章18中进行了修改。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56443/56443fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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

Nanyang Technological University

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

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

使用人工痰介质测试抗生素药效试验<em&gt;绿脓杆菌</em&gt;更多相关的肺囊肿性纤维化的条件

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

科研

免疫与感染

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated &mu;CT (DLIT-&mu;CT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-&mu;CT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-&mu;CT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and &mu;CT scans using a single imaging platform. The low dose &mu;CT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with &mu;CT imaging to create a 4D movie of C. rodentium infection.

4D多模态成像<em&gt;柠檬酸rodentium</em在小鼠感染

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

长期慢性<em&gt;铜绿假单胞菌</em&gt;在呼吸道感染小鼠

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

一种简单有效的方法构建突变牛痘病毒载体

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. 
Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. 
Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Here, we describe the purification of Legionella pneumophila (L. pneumophila) outer membrane vesicles (OMVs) from liquid cultures. These purified vesicles are then used for the treatment of macrophages to analyze their pro-inflammatory potential.

嗜肺军团菌外膜囊泡:隔离及对巨噬细胞促炎潜力分析

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a &#34;target hypothesis&#34;, which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

基于荧光的淋巴细胞检测适用于小分子的高通量筛选

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

荧光为基础的神经氨酸酶抑制测定流感病毒的易感性评估抗病毒药的神经氨酸酶抑制剂类

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

报告酶荧光法检测小鼠结核分枝杆菌的影像学

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-&#945;), and interleukin 1 alpha (IL-1&#945;) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances.

人精切肺切片中物质的细胞毒性及免疫调节作用的评价

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new ...

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. ...