我们承认：托马斯·布迪曼博士在TSE Systems GmbH公司提供的技术专长和设备定制。约翰·弗莱（巴特尔公司）和鲁迪博士耶格（CH Technologies公司）为他们的有益的讨论。吴天，山姆Mboggo，四月米勒和肖恩·戴维斯（Amgen公司），用于实验的帮助。

在这些研究中使用的小鼠按照指南实验动物的管理和使用，第八版24人照顾。小鼠关在协会的评估和实验动物的认证（AAALAC）的国际认证的工厂在玉米芯床上用品消毒通风的微型隔离房集团。当测量的支气管收缩，将小鼠用100mg / kg的戊巴比妥腹膜和麻醉深度麻醉因缺乏脚趾捏反射的监测，并通过腹膜内麻醉必要维持。在实验结束时，小鼠经断颈过量巴比妥酸盐后安乐死。安乐死的验证因缺乏呼吸的证实。没有生存的手术都对小鼠进行。所有研究方案得到机构动物护理和使用委员会（IACUC）的批准。 1.的Phar的产生配方和设备选择maceutical气溶胶注：配方和设备选择依赖于单独药物的物理化学性质被雾化，因此一般方案见下文和读取器是由曾25和26奥赖尔登提到的评论。 <ol><li> 干粉气雾剂 <ol><li>微粉化药物在球磨机，喷射磨或类似的装置27的干燥粉末，并确保所述微粉化的颗粒尺寸分布（PSD）包含可吸入（0.5-5微米质量中值空气动力学直径，MMAD）的颗粒尺寸的颗粒。共混物需要稀释，微粉化乳糖有效的测试化合物。 注意：如果没有足够的微粉以确定与级联冲击的PSD中，微粉化粉末的一个小的（子毫克）样品的PSD可以通过光散射进行测量，以确认其含有小/可吸入颗粒。 </li><li>生成使用莱特灰尘进料干粉气溶胶发生器干粉气雾剂。使用手动液压机在大约1000磅每平方英寸（psi），以产生由所述粉尘莱特饲料气溶胶发生器17用作输入粉末的压实的糕点包装微粉化药物/乳糖粉末到圆柱形储器。 </li><li>螺杆圆柱形贮存到尘埃莱特进料推进贮存直至刮刀与所述药饼接触。 </li><li>莱特灰尘进料至一个旋风分离器和所述入口的出口连接到设置为15升/分钟的气流速率（最大压力90 psi）的压缩空气源。 </li><li>设定进给速度控制，以每分钟（rpm）0.7革命和打开莱特灰尘进料气溶胶发生器。 注：在0.7rpm使用小赖特灰尘进料圆筒贮存时相当于1克/小时的试验品滤饼进料速率。赖特灰尘进料刮擦压实PO的薄层通过旋转贮存器wder时，将测试物品蛋糕。空气中携带尘出莱特灰尘进料的，通过用于解附聚声速喷嘴，并进入旋流器以除去不可吸入颗粒和附聚物。 </li><li>旋流器的出口连接到所述吸入部（ 图1）的中心气雾剂增压室。 注：化合物可以由300在赖特灰尘进料的储存器被压缩到1500psi的。目的是足够的压缩颗粒，它们将在贮存器倒立时被保留，但没有这么多，赖特灰尘进料不能刮去用于重新气雾化的薄层。必须记住的是在手动液压机压力表以磅读取和对小的灰尘进料储存器中的柱塞具有约0.25平方英寸的表面积。因此，250磅的0.25平方英寸的压缩力是等效至1000psi。 </li></ol></li><li> 雾化液体的气溶胶 <ol><li>溶于100mL水或生理食盐水的药物。 </li><li>加载100毫升的注射器与药物溶液和注射器放入设定在1毫升/分钟的流速注射泵。 </li><li>连接注射器泵到喷射喷雾器，并从进料管线通向喷雾器清除空气。 </li><li>加压空气源连接到喷射喷雾器，并设置在空气流量计10升/分钟。 </li><li>插入喷射喷雾器进入预分离器。预分离器连接到喷雾器吸入单元的中心气室气溶胶（ 图1）。 注意：许多药物化合物具有有限的水溶解度和被更好地配制为干粉气雾剂。如果稳定的悬浮液可以从微粉化（MMAD &lt;5μM）化合物来制备，它可与喷射喷雾器一起使用。注意应作为暂停可能堵塞喷雾器。从1毫克/毫升为有效化合物（如支气管扩张剂喷雾器进料浓度异丙托铵），以40mg / mL的悬浮液（对于像硫酸沙丁胺醇较少有效的化合物）已被使用。注射泵进料速度设定为1毫升/分钟为一个实际的原因;以允许气溶胶浓度平衡时间和45分钟长的暴露，而无需重新加载注射器完成。 </li></ol></li></ol> 2.气溶胶暴露实验设置<ol><li> 测量药物浓度（通过在绝对过滤收集气溶胶）和粒度分布（通过使用级联冲击收集气溶胶）进入所述吸入部后预分离器/旋流器的气溶胶的。使用与动物的每分钟通气量，体重，以及曝光时间沿着这些参数来估算沉积于肺部药物的剂量。 <ol><li>称量绝对过滤，记录过滤器的重量。放置过滤器进入过滤器保持器并组装过滤器保持器。连接的绝对过滤器保持器的入口到中央气雾剂增压室样品端口和所述出口设置以1L / min的流速在实验的持续时间来采样气溶胶的真空源。 注：在过滤器上的药物采样45分钟后的质量可在亚微克范围和/或与乳糖，氯化钠盐，或其它车辆进行混合。读取到0.1微克微量天平是必要的。为了获得沉积在过滤器的药物的准确的重量，必须将过滤器平衡并在湿度受控环境中称重。药物的过滤器上的重量只能在剂量计算中使用，如果有在制剂中没有车辆或车辆是水。当在除水以外的所述制剂的车辆中，物质的过滤器上的重量只给出了通过高效液相色谱（HPLC）的药物含量进一步分析的估计的起点。 </li><li>称重并记录7级联冲击级滤波器的权重和一个最后的“尾片”;过滤。放置在每个级联冲击的七个阶段的过滤器和组装默瑟级联冲击28。级联冲击器的入口连接到一个中央气雾剂增压室样品端口和所述出口设置为样品在流速级联冲击在（通常为0.5或1升/分钟）校准气雾剂的持续时间的真空源实验。 </li><li>监测吸入单元与实时气溶胶监视器（见材料和​​试剂表）气溶胶内容，以确认该气溶胶发生器的功能，并产生在整个实验稳定气雾剂。实时气溶胶显示器的入口连接到一个中央气雾剂增压室样品端口和所述出口设置以1L / min的流速在实验的持续时间来采样气溶胶的真空源。 注：来自实时气溶胶监视器以μg/ L报告的信号，但被校准用于道路灰尘和必须重新校准为每个单独的药物气雾剂，得到正确的气溶胶浓度值。无需校准使用显示器来确认存在或不存在和气溶胶浓度的经时稳定性。 </li><li>设置过程控制参数（空气流量，真空，压力，气溶胶发生器功率）需要依赖于动物的数量将被连接到吸入单元的值。吸入部和气雾剂发生器被连续地控制/通过由制造商提供的计算机化的过程控制/数据采集系统（DACO）监测（见材料和​​试剂表）。空气流速向所述吸入单元应最低限度地在吸入单元中的所有动物的约2倍的总分钟通气率以避免CO 2的累积。 </li></ol></li><li> 负载的小鼠成鼻式暴露限位器在仅鼻吸入单元气溶胶之前。此外载荷约束应力控制动物到限制器呼吸室内空气。 注：剂量/反应实验由暴露于气溶胶​​不同的时间量的小鼠的多个组的。曝光时间被用于控制剂量/反应实验期间各组接受的剂量。 <ol><li>角度朝向天花板的限制筒了，而试图加载的动物，因为他们往往向上运行试图逃跑。指着管下来，同时装载鼓励转身逸出管的后面。确保鼠标的鼻子被定向到管的尖端并固定位置可变的柱塞进入所述限制器的后端。 注：柱塞被构造以允许小鼠的尾部为从保持件，其允许鼠标调节其体温而在限制器突出。 </li><li>调整柱塞以允许小鼠旋转，但不转头尾;确保气溶胶吸入。 </li><li>连续监测小鼠而在约束管中。定位在柱塞之后，小老鼠（&lt;20克），往往试图在管转动头对尾和采用的U位置，他们有呼吸困难。这种转向行为是最普遍的在第5分钟的克制，在这之后的小鼠很少尝试把头部到尾部。 </li></ol></li></ol> 3.气溶胶传递<ol><li>插入止动件，以堵塞吸入单元的输送口，并从过程控制软件内激活气溶胶发生器，压缩空气流量控制器，和吸入单元真空泵。 </li><li>一旦从实时气溶胶显示器读数表明气溶胶浓度必须达到平衡（〜30分钟， 图2），开始除去塞子和将含有小鼠到吸入单元鼻仅抑制管中。重复，直到暴露于药物的所有小鼠都连接到吸入单元。 注：在示例实验中，通过气溶胶发生器和稀释空气供给到吸入单元的总空气流量被设定在使用中提供一个0.5升/分钟的流速给每个动物暴露端口。例如，一个15升/分钟总空气流量足够在吸入单元，以供给每30个端口。这比所要求的小鼠的分钟通气得多的空气流动，但较大的空气流需要提供的能量（穿过气溶胶发生器压降）以产生（雾化/解附聚物）的气雾剂。 </li><li>一旦所有的动物被装载到曝光单元，打开连接到使用过程控制软件的绝对过滤器和级联冲击真空采样泵。 </li><li>当所有敞口完毕，关闭气溶胶发生器和从吸入部除去残留的小鼠。 注：动物会改变站头巾或戴口罩的人员进行处理。气溶胶传递的结束后，老鼠都是R从管emoved和管每次使用后消毒。 </li></ol> 4.计算所沉积的剂量的<ol><li>为了以μg计算所沉积的剂量29 / kg的（等式1）乘以药物在气雾剂X吸入分数x肺沉积分数的浓度（μg/ L）X分钟通气量（L /分）的曝光（分）×持续时间并且由体重（公斤）除以。 </li><li>通过乘以采样时间（分钟）的过滤器（L /分钟）除以药物的质量上由空气流量的绝对过滤器（微克）计算气溶胶浓度（μg/ L）。基于体重30异速生长公式估算鼠标的每分钟通气量。可吸入分数为1作为气溶胶通过预分离器，以便除去非可吸入颗粒，和肺沉积馏分从药物气雾剂MMAD确定使用（ 图3）由单分散气溶胶试验的校正曲线（ 图4）31。 </li></ol><img alt="方程" src="/files/ftp_upload/55454/55454eq1.jpg" />

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只有A鼻吸入系统及其操作提供药用气雾剂啮齿动物的肺部进行了描述。在鼻式保持器抑制动物是用于暴露动物空降材料通常使用的方法。进行实验证明了抗胆碱能支气管扩张药异丙托溴铵34能够有效地逆转醋甲胆碱诱导的支气管收缩时通过鼻式吸入用的沉积剂量为0.1μg/ kg的ED 50递送至小鼠。在异丙托的有效剂量大于10倍的增加被要求支气管扩张以下气管内递送（ED 50气管内= 1.3微克/千克，数据未示出）输送。这是由于在通过气管内给药3，5，10产生的肺部药物的非均匀沉积图案。 10倍和更高的有效剂量吸入气管和给药技术之间的差异对其他药物已被他人4先前观察到的。 在气管内给药所产生的肺药物的非均匀沉积图案也由35肺减慢药物吸收，减少在其中药物进入全身循环的速率和减小看到的全身性副作用的可能性。因此，为了优化安全性/功效（治疗指数）的新吸入药物36时，应使用鼻只吸入递送。气管内沉积将通过产生错误的高有效剂量在肺中和低剂量全身循环得到TI的不准确估计。 如吸入途径提供新的药物的开发，它从临床前药效学研究的有效药物剂量适当地转换为预测的EF是必不可少的ficacious人用剂量的临床试验。 Tusko的臭名昭著的死亡大象37文献中常常被引用来提醒我们使用异速生长尺度预测物种间的药物剂量和种间药物剂量不应线性外推的身体群众的一个简单比较的基础上。这是通常使用的异速生长方式与0.67的异速生长指数b。从临床前药效学研究38预测人类药物剂量。使用鼠标吸入的0.9微克/千克为异丙托铵，0.67的异速指数，0.03千克小鼠体重，和60公斤人体重ED 90; 0.07微克的估计人类ED 90 /公斤（（0.9 *（0.03 / 60）（1-0.67）= 0.07）。该预测值可以被计算为从我们的鼠标数据的预测的人类剂量沉积比得上实际人类的0.26微克/千克有效沉积剂量，这可以从所传送的执行来计算的40微克39由60公斤乘以16的0.4（（40/60）* 0.4 = 0.26）人口腔吸入肺部沉积分数人体质量除以本身。有效的人吸入沉积量的估计也有助于计划在临床试验需要40吸入毒理学研究中使用的递送剂量。 显影唯一鼻吸入技术当专门的设备和大量的用于鼻吸入仅药物递送所需的试验化合物（克数量）可以是显著限制。如果需要特定的剂量（通常是毒理学和不药效剂量/应答）的研究，并且该校准运行将需要更多的药物可用，而不存在动物甲校准运行是必要的。此校准运行是必要的，因为每一种药物/制剂的物理化学性质可以变化足以极大地影响所沉积的剂量在肺部药物。空气和药物的进料速率，以在校准运行期间气雾剂发生器的优化是必需的，以实现所需的特定剂量的气溶胶颗粒大小和浓度。而在所述方法中所建议的气溶胶发生器的空气和药物的进料速率是一个合理的起点，有潜在的对特定的药物配制剂的非可吸入（粒度&gt; 5mm）的气雾剂将生成。在临床前研究阶段，往往存在足够的可用药物做校准运行，不可能事先知道什么剂量（如果有的话）进入肺部。另外，静电在气雾剂产生过程中产生的并影响了气溶胶浓度达到平衡所需的时间。重要的是要正确接地设备，以尽量减少静电荷是很重要的。以最小化静电另一种选择是湿度增加供给到吸入单元的空气，以增加导电性和消散上的颗粒25的静电荷。加湿供给到气溶胶发生器的空气期间短（&lt;1个小时）给药会话不需要用于动物的舒适性，但如果使用较长的给药时间应该被考虑。 药物可以释放到动物的被动鼻式吸入或绕过鼻咽沉积直接气管内给药方法肺部。鼻式吸入递送在吸入毒理学41的领域中常用的，但微在药物发现过程的早期使用。是需要进行，由于鼻式吸入研究的多学科研究小组：需要大量的药物，制定，产生和表征气溶胶所需的专门知识;操作复杂的设备，并测量呼吸疾病的动物模型药物疗效。本文所描述的递送技术被用来开发SmaI位升分子吸入药物，但在将来可应用于开发吸入生物制剂42,43。但愿这份手稿记录的程序和技巧，将有利于新的临床前药物发现和毒理学研究人员获得必要通过雾化吸入，以药物输送到啮齿动物的技能。

有许多优点的药物呼吸道疾病治疗吸入给药。吸入给药直接将治疗剂作用的部位，肺。在肺中药物的局部浓度高提供了减少剂量和全身暴露显著的优势，并最大限度地提高疗效。这是一个重要优点，即能大大提高治疗指数（TI，药物引起过度的药物剂量，提供功效的副作用的剂量比）的药物。吸入β2 -肾上腺素能激动剂，皮质类固醇和抗胆碱能药物已被证明在提高患者的哮喘和COPD肺功能有效，同时最小化的全身副作用（心动过速，免疫抑制，和便秘）当这些药物口服观察到。新的药物类（例如，PDE4抑制剂1和BTK抑制剂2）最近证明改善在临床前的动物疾病模型，但是，类似于β2 -激动剂，皮质甾类和抗胆碱能药物肺功能有效，遭受全身副作用，可能通过吸入输送最小化。由于吸入与口服药物开发所增加的成本，吸入制剂只应考虑呼吸迹象成功后口服/全身给药剂量揭示限制基于机制的全身性的副作用。 临床前，吸入化合物经优化以增加TI，这需要在体内效力和副作用的测量。最初，这些测量可以在分开的测定中，通常是一个局部递送功效测量和全身递送副作用测量，但要真正比较化合物制成，疗效及副作用应在吸入给药后的相同的动物进行测定。这需要剂量/响应的研究，交流hieve给予肺足够化合物以诱导可测量的副作用。的唯一方法目前大剂量药物的均匀地分配到多个小动物的肺同时仅鼻吸入3，4，5。的优势和不同的吸入暴露弱点的技术最近已经审查6，7，8。专门的设备和大量的测试化合物（克数量）的所需要的鼻吸入仅药物递送，但验证的概念研究可以通过其他手段是可能的。 当药物的量是有限的（毫克的量），直接施用方法是一种选择，但所有从非均匀沉积遭受与更多的药物沿着中央气道浓缩和较差表示在实质/肺泡区域3，4，5。直接滴入提供有效剂量是始终较高，不能直接进行比较，以吸入剂量4。直接滴入方法包括鼻内9，气管内液体10，11和喷雾滴注12，或干粉末吹入13，14可被用作筛选工具来确定购买仅鼻吸入研究的近似剂量范围内，或确定效力/毒性的排名的一系列结构相似的药物15。由于中心气道沉积图案，直接给药方法可能更有用，以确定化合物的，关于中央气道比I（支气管扩张药或肥大细胞抑制剂）作用的影响n中的外周肺（抗炎药）。 与人类不同，谁可以从吸入器在一个单一的深呼吸吸入气溶胶浓缩的相当大的剂量，连续产生可呼吸的（0.5-5微米质量中值空气动力学直径，MMAD）气雾剂，长达一个小时，则需要沉积一种有效的药物剂量成一个仅鼻吸入系统16自主呼吸啮齿动物的肺。气溶胶发生器（喷射雾化器或赖特灰尘进料17），其可以连续地产生用于仅鼻吸入研究所需的气溶胶颗粒大小和浓度是不是在产生高质量（吸入）气溶胶非常有效的。所述药物进料速率为有效的这些气溶胶发生器（IC 50 pM至nM的功能性的基于细胞的测定）的化合物在1〜10毫克/分范围通常是和药物气雾剂通常小于1％会向呼吸区动物（图1）。许多产生的粒子的过大（&gt; 5微米），以进入肺并通过气溶胶分类器（预分离器或旋风分离器具有5μm切口的点）被除去以避免在鼻子大剂量的药物。添加到仅鼻吸入系统的低效率是可吸入颗粒的小颗粒尺寸范围（0.5至5微米MMAD）。许多小于0.5微米的呼出（如香烟烟雾），而不是沉积于肺部18中的气溶胶粒子的。此外，许多“较大”的气溶胶颗粒的（〜5微米）在鼻子存款被吸收或通过粘膜纤毛清除朝向在那里它们被吞咽到胃19中的咽喉后部输送。当仅使用鼻吸入，敷在鼻剂量总是比沉积于肺部剂量和鼻剂量可以向全身暴露和副作用20大。本质上，吸入DRU克剂量的小（在微克范围）最小化由鼻，肺，或胃肠道组织吸收药物的任何全身副作用的潜力。甚至当提供给动物的气溶胶的颗粒大小是在吸入范围，平均只有4％的气溶胶颗粒，它使在肺部动物沉积物的呼吸区。更有效的气溶胶发生器是可用的，但喷射式喷雾器和Wright灰尘进料是无与伦比它们对来自不同液体和干粉制剂，分别产生连续气溶胶能力。 从预分离器吸入气雾剂传递成是基于流过鼻子设计22中的鼻式暴露吸入部21。吸入单元具有3层（仅两层在图1中示出），并且每个层包含对动物和气溶胶取样10个曝光端口。这些端口位于外围周围的CEntral气溶胶全会。有意识的小鼠被放置在玻璃抑制保持器（6英寸长英寸直径的1.2）和呼吸吸入单元的气溶胶成分。小鼠未驯化的约束装置23。先前的经验表明，小鼠耐受管克制不到一个小时的持续时间类似地，具有或不具有适配2。 吸入单元设计为直接递送药物气溶胶的动物的肺，同时避免暴露于运营商。这些药物的效力/毒性通常是未知和多个工程安全控制来避免暴露给运营商。运营商必须始终佩戴个人防护装备（手套，实验服，呼吸器，安全眼镜）。吸入单元的外气室是下在操作期间的任何时候都负压，从而允许安全移除的单个或动物组无覆盖UT切断气雾发生器。吸入单元也包含在通过排气口在天花板维持在负压力，以防止气雾剂的任何逃逸进入室内在故障的情况下的二次外壳。从吸入系统中的所有流出物的空气被释放到环境中之前，一个HEPA过滤器过滤。在这个手稿中使用的鼻式暴露系统是从单一供应商（参见材料的补充表 ）购得。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Nose-only exposure inhalation unit</td>
			<td>TSE systems</td>
			<td>700100-KNES-040-ss</td>
			<td>Custom configurations available</td>
		</tr>
		<tr>
			<td>DACO data acquisition system</td>
			<td>TSE systems</td>
			<td>700400-PRO-C-D/1</td>
			<td></td>
		</tr>
		<tr>
			<td>MC One Jet Mill</td>
			<td>Jetpharma</td>
			<td>DEC MicroJet 10</td>
			<td></td>
		</tr>
		<tr>
			<td>Turbula Mixer</td>
			<td>GlenMills Inc</td>
			<td>T2F</td>
			<td></td>
		</tr>
		<tr>
			<td>Micronized Lactose</td>
			<td>DFE Pharma</td>
			<td>Lactohale 200</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydraulic press</td>
			<td>Specac</td>
			<td>GS15011</td>
			<td></td>
		</tr>
		<tr>
			<td>Cascade impactor filters</td>
			<td>Pall Life Sciences</td>
			<td>7219</td>
			<td>Emfab filter</td>
		</tr>
		<tr>
			<td>Absolute filters</td>
			<td>Whatman</td>
			<td>10370302</td>
			<td>5 cm diameter</td>
		</tr>
		<tr>
			<td>Real time aerosol monitor 
			Microdust Pro Monitor</td>
			<td>Casella</td>
			<td>CEL-712</td>
			<td></td>
		</tr>
		<tr>
			<td>Ipratropium bromide</td>
			<td>Spectrum Chemical</td>
			<td>I1178</td>
			<td>pre-micronized</td>
		</tr>
		<tr>
			<td>flexiVent FX1 system</td>
			<td>scireq</td>
			<td>FV-FXCS</td>
			<td></td>
		</tr>
	</tbody>
</table>

dry powder and nebulized aerosol inhalation of pharmaceuticals delivered to mice using a nose-only exposure system

阻塞性呼吸系统疾病，如哮喘和慢性阻塞性肺疾病（COPD）目前由吸入抗炎和支气管扩张药治疗。尽管多次治疗的可用性，这两种疾病都是不断增长的公共健康问题。大部分哮喘患者都很好地控制电流吸入疗法但重症哮喘患者有相当数量都没有。哮喘影响全世界估计有3亿人，大约20％有疾病是一种严重的。相较于哮喘，很少有有效的治疗慢性阻塞性肺病。人口估计有10％的人COPD并同时减少其他主要疾病死亡率的趋势是COPD增加。虽然开发药物的吸入给药是具有挑战性的，只有鼻子吸入单元使啮齿动物的预临床疗效和安全性/毒理学研究肺直接交付的新型药物。吸入给药具有用于呼吸系统疾病，其中肺中的高浓度提高疗效和减少全身性副作用的多个优点。吸入糖皮质激素和支气管扩张从这些优势中获益，并吸入输送还可以适用于未来的生物疗法的潜力。本文所描述的吸入单元可以产生，样品表征和在啮齿类动物的肺中均匀地沉积的药物气雾剂。这使得临床前确定疗效并存放于啮齿动物肺部药物剂量的安全性，启动临床开发之前所需的关键数据。

支气管扩张异丙托溴铵以1毫克/毫升的浓度溶解于0.9％的生理盐水。 100毫升的注射器中填充的异丙托铵配制剂溶液和将注射器插入到以1mL / min的流速注射泵设定喂喷射喷雾器（ 图1）。激活控制系统，用于吸入单元发起10L /分钟的空气流的喷射喷雾器，5L /分钟的稀释空气流向稀释室，和15.5 L /真空气流的分钟来绘制药物气雾剂出的吸入单元一旦在它通过小鼠的鼻子。注射泵和实时监测气溶胶采样泵被激活。二十四只小鼠插入到吸入单元的实时气溶胶监视器确认在吸入部的气溶胶浓度已达到平衡（15-30分钟， 图2）之后。样品泵的绝对过滤器（1升/分钟）和级联冲击器（0.5L /分钟）被开启一次所有小鼠连接到吸入单元。 8只小鼠的第一组5分钟，15分钟之后的第二组，和45分钟后的第三组后从吸入装置中取出。支气管收缩32被测量为在呼吸系统阻力（Rrs）的增加对乙酰甲胆碱的单一雾化剂量（30毫克/毫升）使用啮齿动物呼吸器33给药后异丙托品输送2个小时。在Rrs的增加的百分比对于每个动物，作为最大的Rrs过雾化乙酰甲胆碱（Rmax）为后3分钟间隔计算减去基线测量的乙酰甲胆碱（RBASE）之前的Rrs值由RBASE（在Rrs的=％增加（RMAX划分-Rbase）/ RBASE），被用于定量的支气管收缩。 气溶胶暴露的45分钟期间沉积在级联冲击过滤器和绝对过滤气溶胶溶解在50％乙腈和异丙托铵的通过HPLC（表1）定量的质量。的异丙托铵气溶胶的MMAD的x / GSD经计算为1.7×/ 1.5微米（ 图3）和用于使用小鼠用于气雾剂为1.7微米（ 图4）的MMAD的0.037的沉积分数（DF）。可吸入分数（IF）为1作为气溶胶通过预分离器，以便除去不可吸入颗粒。乘以采样时间穿过过滤器异丙托铵在气溶胶（0.5微克/升）的平均浓度从异丙托铵的质量计算上通过空气的流率除以绝对过滤器（22微克）拉（1L /分钟） （45分钟）。的平均体重的小鼠的为0.019 kg和它们的预测分钟通气计算为0.021升/分钟。使用等式1，所沉积的剂量为5，15和45个分钟暴露组分别为0.1，0.3和0.9微克/千克;分别。 =“1”&gt;异丙托8周龄的C57BL / 6小鼠抑制雾化醋甲胆碱诱导的支气管收缩。乙酰甲胆碱雾化给药（ 图5，上图）的秒内增大的Rrs。对照组0.62±0.03 CMH 2 O * S / mL的基线值增加到1.66±0.12 CMH 2 O * S / mL的最大值的RR（暴露于室内空气代替异丙托气溶胶），较168±9％增加RRS，70秒乙酰甲胆碱气雾剂给药之后。在呼吸系统阻力的增加％是由吸入剂量异丙托铵以剂量依赖性方式抑制。在Rrs的增加的百分比由51±9％，79±14％，和89±2％在沉积吸入剂量0.1，0.3异丙托铵的分别抑制（ 图5，下图），和0.9微克/千克，。 克” /&gt; 图1：吸入单元与干粉气溶胶发生器附接。用来产生从液体制剂气雾剂喷雾器被示在右边。 1）莱特灰尘进料; 2）旋流器，3）注射泵，4）喷射式喷雾器插入预分离器，5）稀释空气混合器。当递送雾化气溶胶，尘埃进料和气旋由注射器泵，喷射式喷雾器/预分离器，和稀释空气混合器代替。放大图（从奥尔德姆21修改）示出了在由插入所述约束管向所述吸入单元创建围绕动物的鼻子呼吸区气溶胶流动路径。气溶胶填充吸入单元的中心气雾剂（灰色）气室，流出到动物的鼻子在那里由在外部（白色）气室并进入废物收集过滤器在轻微真空拉回（未示出）。 <a href="http://ecsource.jove.com/files/ftp_upload/55454/55454fig1large.jpg" target="_blank">PL</a>轻松点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/55454/55454fig2.jpg" /> 图2：实时气溶胶监视器测量证实在吸入部的气溶胶浓度达到平衡〜接通气雾剂发生器在30分钟后。 <a href="http://ecsource.jove.com/files/ftp_upload/55454/55454fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55454/55454fig3.jpg" /> 图3：收集在级联冲击（蓝色条线图）的每个阶段异丙托质量覆盖有用来计算气溶胶颗粒大小分布的MMAD和GSD曲线拟合（黑色曲线）。 <a href="http://ecsource.jove.com/files/ftp_upload/55454/55454fig3large.jpg" target="_blank">请</a>点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/55454/55454fig4.jpg" /> 图4：小鼠肺作为MMAD的用于通过鼻式吸入递送气雾剂的功能沉积分数（从谢31修改）。对于粒径小于0.5μm的大多数气雾剂的呼出和肺不会沉积（如香烟烟雾）。对于颗粒尺寸大于5μm，最气溶胶是由鼻子过滤掉。 <a href="http://ecsource.jove.com/files/ftp_upload/55454/55454fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/55454/55454fig5.jpg" /> 图5：在呼吸系统阻力迅速增加通过雾化乙酰甲胆碱诱导administe红色到小鼠是通过鼻式吸入递送至小鼠（n = 8每组，上图）异丙托铵阻断。异丙托抑制在呼吸系统阻力醋甲胆碱诱导的增加为0.1微克/千克的ED 50剂量沉积（* P &lt;0.05vs。对照，下图）。 <a href="http://ecsource.jove.com/files/ftp_upload/55454/55454fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td colspan="3">级联冲击数据用来计算MMAD和GSD </td></tr><tr><td>级过滤器</td><td>异丙托溴铵的过滤器 通过HPLC（毫克） </td><td>各级馏份 直径（mm） </td></tr><tr><td> 1 </td><td> 0.002 </td><td> 6 </td></tr><tr><td> 2 </td><td> 0.19 </td><td> 4.5 </td></tr><tr><td> 3 </td><td> 1.43 </td><td> 2.5 </td></tr><tr><td> 4</td><td> 2.55 </td><td> 1.5 </td></tr><tr><td>五</td><td> 2.95 </td><td> 1.2 </td></tr><tr><td> 6 </td><td> 1.03 </td><td> 0.7 </td></tr><tr><td> 7 </td><td> 0.37 </td><td> 0.5 </td></tr></tbody></table> 表1：使用的级联冲击器的数据来计算MMAD和GSD

Watch this Scientific Journal Video about Dry Powder and Nebulized Aerosol Inhalation of Pharmaceuticals Delivered to Mice Using a Nose-only Exposure System at JoVE.com

Dry Powder and Nebulized Aerosol Inhalation of Pharmaceuticals Delivered to Mice Using a Nose-only Exposure System

本文所描述的吸入单元可以产生，样品表征和在啮齿类动物的肺中均匀地沉积的药物气雾剂。这使得临床前确定的有效性和沉积于肺部药物剂量的安全性的;关键数据，使临床吸入药物的发展。

干粉和药品的雾化吸入气雾剂递送小鼠使用鼻只曝光系统

Obstructive respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD) are currently treated by inhaled anti-inflammatory and ...

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40.3K 次观看.  Amgen. 本文所描述的吸入单元可以产生，样品表征和在啮齿类动物的肺中均匀地沉积的药物气雾剂。这使得临床前确定的有效性和沉积于肺部药物剂量的安全性的;关键数据，使临床吸入药物的发展。 

视频: Dry Powder and Nebulized Aerosol Inhalation of Pharmaceuticals Delivered to Mice Using a Nose-only Exposure System

Endotracheal Intubation in Mice via Direct Laryngoscopy Using an Otoscope

Mice, both wildtype and transgenic, are the principal mammalian model in biomedical research currently. Intubation and mechanical ventilation are necessary for whole animal experiments that require surgery under deep anesthesia or measurements of lung function. Tracheostomy has been the standard for intubating the airway in these mice to allow mechanical ventilation. Orotracheal intubation has been reported but has not been successfully used in many studies because of the substantial technical difficulty or a requirement for highly specialized and expensive equipment. Here we report a technique of direct laryngoscopy using an otoscope fitted with a 2.0 mm speculum and using a 20 G&#160;intravenous catheter as an endotracheal tube. We have used this technique extensively and reliably to intubate and conduct accurate assessments of lung function in mice. This technique has proven safe, with essentially no animal loss in experienced hands. Moreover, this technique can be used for repeated studies of mice in chronic models.

Endotracheal Intubation in Mice via Direct Laryngoscopy Using an Otoscope

We have developed a simple, reliable, and relatively inexpensive method for endotracheal intubation in mice via direct laryngoscopy using an otoscope with a 2.0 mm speculum. This technique is atraumatic and can be used for repeated measurements in chronic experiments. We find it superior to tracheostomy or previously reported nonsurgical techniques.

气管插管小鼠<em&gt;通过</em&gt;直接喉镜使用耳镜

Mice, both wildtype and transgenic, are the principal mammalian model in biomedical research currently. Intubation and mechanical ventilation are ...

科研

医学

Flexible Colonoscopy in Mice to Evaluate the Severity of Colitis and Colorectal Tumors Using a Validated Endoscopic Scoring System

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental endoscopy has been highly useful for studies that require repeated assessments in a single laboratory animal, such as those evaluating mechanisms of chronic inflammatory bowel disease and the progression of colorectal cancer. However, the methods used across studies are highly variable. At least three endoscopic scoring systems have been published for murine colitis and published protocols for the assessment of colorectal tumors fail to address the presence of concomitant colonic inflammation. This study develops and validates a reproducible endoscopic scoring system that integrates evaluation of both inflammation and tumors simultaneously. This novel scoring system has three major components: 1) assessment of the extent and severity of colorectal inflammation (based on perianal findings, transparency of the wall, mucosal bleeding, and focal lesions), 2) quantitative recording of tumor lesions (grid map and bar graph), and 3) numerical sorting of clinical cases by their pathological and research relevance based on decimal units with assigned categories of observed lesions and endoscopic complications (decimal identifiers). The video and manuscript presented herein were prepared, following IACUC-approved protocols, to allow investigators to score their own experimental mice using a well-validated and highly reproducible endoscopic methodology, with the system option to differentiate distal from proximal endoscopic colitis (D-PECS).

Over the past few years, new generation endoscopes have emerged as important diagnostic research aids for evaluating murine colitis and colorectal tumors. We present herein a detailed protocol for endoscopic assessment of inflammation and colorectal tumors in mice, as well as a novel scoring system that uses decimal identifiers to document the endoscopic severity of colitis and colorectal tumors.

灵活的结肠镜检查在小鼠使用经过验证的内镜评分系统来评估结肠炎和结肠直肠肿瘤的严重程度

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental ...

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an otherwise dynamic system, and successive phases are easily overlooked or misinterpreted. Live-cell imaging and time-lapse microscopy, in which living cells can be followed for hours or even days in a more or less continuous fashion, are therefore very informative. The protocol described here allows for the investigation of the fate of chemotherapeutic nanoparticles after the delivery of doxorubicin (dox) in living cells. Dox is an intercalating agent that must be released from its nanocarrier to become biologically active. In spite of its clinical registration for more than two decades, its uptake, breakdown, and drug release are still not fully understood. This article explores the hypothesis that lipid-based nanoparticles are taken up by the tumor cells and are slowly degraded. Released dox is then translocated to the nucleus. To prevent fixation artifacts, live-cell imaging and time-lapse microscopy, described in this experimental procedure, can be applied.

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Live-cell imaging is a powerful tool to visualize dynamic processes. The examination of fixed cells provides only static pictures, which can lead to misinterpretation and confusion about the process. This work presents a method to study uptake, drug release, and intracellular localization of liposomal nanoparticles in living cells.

使用体外活体细胞成像技术研究脂纳米粒子化疗

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an ...

Comparing the Effects of Electronic Cigarette Vapor and Cigarette Smoke in a Novel In Vivo Exposure System

Electronic cigarettes (E-cigarettes) are being widely used, and growing in popularity. It is estimated that more than 9 million adults use them regularly. The potential adverse health effects of electronic cigarette vapor (E-vapor) exposure are poorly defined. While several animal models of E-vapor exposure have been developed, few models expose rodents to clinically relevant quantities of nicotine and make direct comparisons to cigarette smoke within the same exposure system. Here, we present a method for constructing and operating an E-vapor chamber and cigarette smoke chamber. The chambers are constructed by outfitting anesthesia chambers with a computer controlled pumping system that delivers consistent amounts of E-vapor or cigarette smoke to rodents. Nicotine exposure is measured indirectly by quantifying pre and post-exposure serum cotinine levels. This exposure system can be modified to accommodate various types of E-cigarettes&#160;and tobacco cigarettes, and can be used to compare the effects of E-vapor&#160;and cigarette smoke in vivo.

Comparing the Effects of Electronic Cigarette Vapor and Cigarette Smoke in a Novel In Vivo Exposure System

This protocol describes a method for exposing rodents to electronic cigarette vapor (E-vapor) and cigarette smoke. Exposure chambers are constructed by modifying anesthesia chambers with an automated pumping system that delivers E-vapor or cigarette smoke to rodents. This system can easily be modified to accommodate many experimental endpoints.

比较电子烟和烟雾在小说中的影响<em&gt;体内</em&gt;曝光系统

Electronic cigarettes (E-cigarettes) are being widely used, and growing in popularity. It is estimated that more than 9 million adults use them regularly. ...

Generation of a Chronic Obstructive Pulmonary Disease Model in Mice by Repeated Ozone Exposure

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high incidence in aging populations. The current conventional therapies for COPD focus mainly on symptom-modifying drugs; thus, the development of new therapies is urgently needed. Qualified animal models of COPD could help to characterize the underlying mechanisms and can be used for new drug screening. Current COPD models, such as lipopolysaccharide (LPS) or the porcine pancreatic elastase (PPE)-induced emphysema model, generate COPD-like lesions in the lungs and airways but do not otherwise resemble the pathogenesis of human COPD. A cigarette smoke (CS)-induced model remains one of the most popular because it not only simulates COPD-like lesions in the respiratory system, but it is also based on one of the main hazardous materials that causes COPD in humans. However, the time-consuming and labor-intensive aspects of the CS-induced model dramatically limit its application in new drug screening. In this study, we successfully generated a new COPD model by exposing mice to high levels of ozone. This model demonstrated the following: 1) decreased forced expiratory volume 25, 50, and 75/forced vital capacity (FEV25/FVC, FEV50/FVC, and FEV75/FVC), indicating the deterioration of lung function; 2) enlarged lung alveoli, with lung parenchymal destruction; 3) reduced fatigue time and distance; and 4) increased inflammation. Taken together, these data demonstrate that the ozone exposure (OE) model is a reliable animal model that is similar to humans because ozone overexposure is one of the etiological factors of COPD. Additionally, it only took 6 - 8 weeks, based on our previous work, to create an OE model, whereas it requires 3 - 12 months to induce the cigarette smoke model, indicating that the OE model might be a good choice for COPD research.

This study describes the successful generation of a new chronic obstructive pulmonary disease (COPD) animal model by repeatedly exposing mice to high concentrations of ozone.

反复臭氧暴露对小鼠慢性阻塞性肺疾病模型的产生

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high ...

Contractions of Human-iPSC-derived Cardiomyocyte Syncytia Measured with a Ca-sensitive Fluorescent Dye in Temperature-controlled 384-well Plates

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CM) are a useful model of human cardiac physiology and pharmacology. Various methods have been proposed to record this spontaneous activity and to evaluate drug effects, but many of these methods suffer from limited throughput and/or physiological relevance. We developed a high-throughput screening system to quantify the effects of exogenous compounds on hiPSC-CM's beating frequency, using a Ca-sensitive fluorescent dye and a temperature-controlled imaging multi-well plate reader. We describe how to prepare the cell plates and the compound plates and how to run the automated assay to achieve high sensitivity and reproducibility. We also describe how to transform and analyze the fluorescence data to provide reliable measures of drug effects on spontaneous rhythm. This assay can be used in drug discovery programs to guide chemical optimization away from, or toward, compounds affecting human cardiac function.

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells are useful models of human cardiac physiology and pharmacology. Here we present a high-throughput screening system to quantify the effects of exogenous compounds on beating frequency, using a Ca-sensitive fluorescent dye and a temperature-controlled imaging multi-well plate reader.

温度控制384孔板中用 Ca 敏感荧光染料测定人 iPSC 的心肌细胞较多的收缩

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CM) are a useful model of human cardiac ...

A Rabbit Model of Aqueous-Deficient Dry Eye Disease Induced by Concanavalin A Injection into the Lacrimal Glands: Application to Drug Efficacy Studies

Dry eye disease (DED), a multifactorial inflammatory disease of the ocular surface, affects 1 in 6 humans worldwide with staggering implications for quality of life and health care costs. The lack of informative animal models that recapitulate its key features impedes the search for new therapeutic agents for DED. Available DED animal models have limited reproducibility and efficacy. A model is presented here in which DED is induced by injecting the mitogen concanavalin A (Con A) into the orbital lacrimal glands of rabbits. Innovative aspects of this model are the use of ultrasound (US) guidance to ensure optimal and reproducible injection of Con A into the inferior lacrimal gland; injection of Con A into all orbital lacrimal glands that limits compensatory production of tears; and use of periodic repeat injections of Con A that prolong the state of DED at will. DED and its response to test agents are monitored with a panel of parameters that assess tear production, the stability of the tear film, and the status of the corneal and conjunctival mucosa. They include tear osmolarity, tear break-up time, Schirmer&#39;s tear test, rose bengal staining, and tear lactoferrin levels. The induction of DED and the monitoring of its parameters are described in detail. This model is simple, robust, reproducible, and informative. This animal model is suitable for the study of tear physiology and of the pathophysiology of DED as well as for the assessment of the efficacy and safety of candidate agents for the treatment of DED.

This article describes the development of a method to induce acute or chronic dry eye disease in rabbits by injecting concanavalin A to all portions of the orbital lacrimal gland system. This method, superior to those already reported, generates a reproducible, stable model of dry eye suitable for the study of pharmacological agents.

由康卡纳林注射到乳原区引起的水性干眼病的兔子模型：药物功效研究的应用

Dry eye disease (DED), a multifactorial inflammatory disease of the ocular surface, affects 1 in 6 humans worldwide with staggering implications for ...

Establishment of a Severe Dry Eye Model Using Complete Dacryoadenectomy in Rabbits

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic step. Despite advances in our understanding of DED, significant knowledge gaps remain. Advances are limited in part due to the lack of informative animal models. The authors recently reported on a method of DED induced by injecting all orbital lacrimal gland (LG) tissues with the lectin concanavalin A. Here, we report a novel model of aqueous-deficient DED based on the surgical resection of all orbital LG (dacryoadenectomy) tissues. Both methods use rabbits because of their similarity to human eyes in terms of the size and structure of the ocular surface. One week after removal of the nictitating membrane, the orbital superior LG was surgically removed under anesthesia, followed by removal of the palpebral superior LG, and finally removal of the inferior LG. Dacryoadenectomy induced severe DED, evidenced by a marked reduction in the tear break up time test and the Schirmer&#39;s tear test, and significantly increased tear osmolarity and rose bengal staining. Dacryoadenectomy-induced DED lasted at least eight weeks. There were no complications and animals tolerated the procedure well. The technique can be mastered relatively easily by those with adequate surgical experience and appreciation of the relevant rabbit anatomy. Since this model recapitulates the features of human aqueous-deficient DED, it is suitable for studies of ocular surface homeostasis, DED, and candidate therapeutics.

A novel approach is presented to induce chronic dry eye disease in rabbits by surgically removing all orbital lacrimal glands. This method, distinct from those previously reported, produces a stable, reproducible model of aqueous deficient dry eye well suited to study tear physiology and pathophysiology and ocular therapeutics.

在兔子中使用完全的白眼切除术建立严重干眼模型

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic ...

Intratracheal Administration of Dry Powder Formulation in Mice

In the development of inhalable dry powder formulations, it is essential to evaluate their biological activities in preclinical animal models. This paper introduces a noninvasive method of intratracheal delivery of dry powder formulation in mice. A dry powder loading device that consists of a 200 &#181;L gel loading pipette tip connected to an 1 mL syringe via a three-way stopcock is presented. A small amount of dry powder (1-2 mg) is loaded into the pipette tip and dispersed by 0.6 mL of air in the syringe. Because pipette tips are disposable and inexpensive, different dry powder formulations can be loaded into different tips in advance. Various formulations can be evaluated in the same animal experiment without device cleaning and dose refilling, thereby saving time and eliminating the risk of cross-contamination from residual powder. The extent of powder dispersion can be inspected by the amount of powder remaining in the pipette tip. A protocol of intubation in mouse with a custom-made light source and a guiding cannula is included. Proper intubation is one of the key factors that influences the intratracheal delivery of dry powder formulation to the deep lung region of the mouse.

Dry powder formulations for inhalation have great potential in treating respiratory diseases. Before entering human studies, it is necessary to evaluate the efficacy of the dry powder formulation in preclinical studies. A simple and noninvasive method of the administration of dry powder in mice through the intratracheal route is presented.

小鼠干粉配方的内经管理

In the development of inhalable dry powder formulations, it is essential to evaluate their biological activities in preclinical animal models. This paper ...

A Novel Inhalation Mask System to Deliver High Concentrations of Nitric Oxide Gas in Spontaneously Breathing Subjects

Nitric Oxide (NO) is administered as gas for inhalation to induce selective pulmonary vasodilation. It is a safe therapy, with few potential risks even if administered at high concentration. Inhaled NO gas is routinely used to increase systemic oxygenation in different disease conditions. The administration of high concentrations of NO also exerts a virucidal effect in vitro. Owing to its favorable pharmacodynamic and safety profiles, the familiarity in its use by critical care providers, and the potential for a direct virucidal effect, NO is clinically used in patients with coronavirus disease-2019 (COVID-19). Nevertheless, no device is currently available to easily administer inhaled NO at concentrations higher than 80 parts per million (ppm) at various inspired oxygen fractions, without the need for dedicated, heavy, and costly equipment. The development of a reliable, safe, inexpensive, lightweight, and ventilator-free solution is crucial, particularly for the early treatment of non-intubated patients outside of the intensive care unit (ICU) and in a limited-resource scenario. To overcome such a barrier, a simple system for the non-invasive NO gas administration up to 250 ppm was developed using standard consumables and a scavenging chamber. The method has been proven safe and reliable in delivering a specified NO concentration while limiting nitrogen dioxide levels. This paper aims to provide clinicians and researchers with the necessary information on how to assemble or adapt such a system for research purposes or clinical use in COVID-19 or other diseases in which NO administration might be beneficial.

This simple and highly adaptable system device for the inhalation of high-concentration nitric oxide (NO) gas does not require mechanical ventilators, positive pressure, or high gas flows. Standard medical consumables and a snug-fitting mask are used to safely deliver NO gas to spontaneously breathing subjects.

一种新型吸入面膜系统，可在自发呼吸受试者中提供高浓度的一氧化氮气体

Nitric Oxide (NO) is administered as gas for inhalation to induce selective pulmonary vasodilation. It is a safe therapy, with few potential risks even if ...