作者要感谢李光耀先生、梁振英先生和苏华莱士先生，感谢他们在制造光源和粉末充气器方面给予的善意协助:和动物成像援助的学院核心设施。这项工作得到了香港研究资助委员会（17300319）的支持。

本研究的实验已获香港大学活体动物教学及研究委员会（CULATR）批准。本研究使用喷雾冷冻干燥（SFD）制备的干粉配方，其中含有0.5%的红素酶信使RNA（mRNA）、5%的合成肽PEG12KL4和94.5%的曼尼托（w/w），用于演示肺16中的mRNA表达。SFD 粉末的质中空气动力学直径 （MMAD） 为 2.4μm。喷雾干燥（SD）曼尼托粉用于研究粉末分散16中使用的空气量的影响。SD 粉末的 MMAD 为 1.5μm。
1. 制造干粉窒息器和装载干粉
<ol><li>（可选）中和干粉（小瓶中）和 200 μL 非滤光圆凝胶加载移液器尖端的静电。根据制造商的指示，使用防静电枪或与除离子功能的平衡。</li>
	<li>准备一张尺寸约为 4 厘米 x 4 厘米的称重纸。将纸张对角线折叠成两半，然后展开。</li>
	<li>称重纸上重1-2毫克干粉。</li>
	<li>通过更宽的开口用粉末填充凝胶加载移液器尖端。轻轻敲击以包装粉末，直到粉末在尖端狭窄端附近形成松散的聚集物（图1A）。避免将粉末包装得太紧，因为它可能会阻碍粉末的分散。</li>
	<li>通过三向塞孔（图 1B ）将装粉尖连接到1mL 注射器。注射器的大小可以根据用于分散粉末的空气量而改变。连接期间垂直握住尖端和注射器，以防止粉末溢出。如果不立即执行管理，使用寄生胶片密封尖端的开口，并暂时在适当条件下存储，直到管理。</li>
</ol>2. 内存设备的制造
<ol><li>光源 （图2）<ol>
<li>用发光二极管 （LED） 手电筒和直径为 0.8-1 mm 的柔性光纤准备定制光源。</li>
		<li>用手钻或钻头在 LED 手电筒的透明透镜上制作一个中心孔，使光纤几乎无法通过。</li>
		<li>通过孔插入光纤。打开 LED 手电筒，调整插入的位置和深度，以获得光纤另一端的最大亮度。</li>
		<li>将光纤与透明环氧胶粘合到位。</li>
	</ol>
</li>
	<li>指导犬 （图 3）<ol>
<li>拿一个1mL塑料巴斯德移液器（图3A），把移液器保持在两端。</li>
		<li>使用酒精灯（或实验室中的其他热源，如邦森燃烧器）将酒精灯放在火焰上方 5-10 厘米处加热移液器中间（图 3B）。旋转移液器，以确保均匀加热。</li>
		<li>当塑料变软变形时，将移离火焰的移开移动器，轻轻伸展移液器。</li>
		<li>用一把剪刀将中间拉伸的移液器切成 A 部分和 B 部分（图 3C-E）。使用第 A 部分作为精细的尖端移液器，将 B 部分用作引导管。为了增加成功与引导管管交配的机会，在 B 部分（图 3F）的末尾制作一个斜面（不太锋利，这可能会增加伤害动物的风险）。当将 200 μL 凝胶加载移液器尖端（用于粉末装载）插入导管时，应将管子突出 1-2 mm。 注:具有适当尺寸（内部和外部直径）进行插管的导管（B部分）内部和外部针头内可容纳21根针头，同时也可以放入17号针内。在拉伸移液管时可能需要多次尝试才能达到适当的尺寸。</li>
		<li>（可选）:在导管的较宽端切开一个小开口，使其更加灵活，以便更容易保持光纤（图3F）。此开口还允许安装用于液体气溶胶管理的微喷雾器。</li>
	</ol>
</li>
</ol>3. 灌引
<ol><li>通过腹腔内注射将小鼠（BALB/c，7-9周）与氯胺酮（100毫克/千克）和西拉津（10毫克/千克）麻醉。</li>
	<li>准备一个用玻璃制成的平台，并将其安装到带夹子的支架上（图4A）。将麻醉鼠标放在平台上（倾斜度约为 60° 时）置于超平位置。平台的高度和倾斜角度可以通过支架上的夹子位置进行调整。</li>
	<li>将鼠标的切口钩在尼龙牙线上（图4B）。暂停鼠标。用胶带或橡皮筋固定鼠标的位置。</li>
	<li>在导引管打开时，将光纤插入导管中，然后用光纤水平尖进行插管。打开 LED 手电筒进行照明。</li>
	<li>用一对钳子轻轻伸出老鼠的舌头，露出它的气管。</li>
	<li>用另一只手用光纤将导管握住。通过口腔插入它们。通过光纤的照明，气管的开口可以可视化为声带之间的孔。</li>
	<li>将引导罐的斜面对准开口的中线（图 5A）。通过将导管的最佳尖端瞄准气管开口，用光纤轻轻地将引导管插入气管。</li>
	<li>入管后，迅速取出光纤，将导管留在气管内（图5B）。应观察正常的呼吸。</li>
	<li>在引导管的开口处握住细尖移液器（A部分），将少量的空气（约0.2 mL）注入小鼠的肺部。小鼠胸部的轻微膨胀表示适当的内分。在粉末管理之前取出细尖移液器。</li>
</ol>4. 粉末管理
<ol><li>按住与第 1.5 步中描述的注射器相连的粉末加载尖端。确保注射器和尖端之间的气流断开。</li>
	<li>向后拉注射器柱塞以提取 0.6 mL 的空气。 注:用于分散粉末的空气体积取决于粉末的特性和所装粉量。结果部分进一步描述了这一点。</li>
	<li>转动三向塞孔的阀门，将注射器和粉末加载尖端之间的气流连接起来。</li>
	<li>将装有粉末的尖端插入已放置在鼠标气管中的导管（图 5C）中。握住引导管，用力推注射器柱塞，以连续动作将粉末分散为气溶胶进入肺部。 注意:设备的任何前向运动应最小化，以避免伤害动物。</li>
	<li>取出尖端并检查尖端内的粉末是否已清空。如果没有，重复步骤 4.1 到 4.4。 注意:如果粉末因过度敲击而包装过于紧密，则可能无法正确分散。</li>
	<li>管理完成后，从气管中取出引导管。</li>
	<li>允许鼠标通过水平定位在一个超平位置，其舌头半突出，以避免气道的封锁恢复。</li>
</ol>

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作者没有利益冲突要披露。

本文介绍了用于干粉窒息和体内插管的定制设备。在粉末装载步骤中，干粉末被加载到 200 μL 凝胶加载移液器尖端。重要的是轻轻敲击尖端，使粉末在尖端的狭窄端松散包装。但是，如果粉末包装太紧，它们会卡在尖端，无法正确分散。建议中和粉末和移液器尖端的静电荷，以方便粉末装载，特别是低密度和低相对湿度的粉末。引导管是设备的关键部件。它用于促进将装有粉末的移液器尖端插入小鼠气管。导管的直径不应太宽:否则将很难将其插入气管，并可能伤害鼠标。导管管的直径应足够宽，以适应光纤和粉装移液器尖端，移液器尖端应突出导管管约1-2毫米。
气管开口的可视化能力在插管过程中至关重要，从而能够正确插入导管。气管开口由白色软骨组成，喉咙后部有规律的开闭运动。通过光纤照明，气管的开口可以很容易地可视化。通过细尖塑料移液器吹气少量空气，胸部的膨胀表示适当的插管。如果未观察到胸部的通货膨胀或在插入过程中感觉到阻力，请迅速收回引导管并重复步骤。
有一个广泛使用的商用干粉充气器12，17，18（材料表:这种设备现在已停产）。干粉被装进设备的样品室，从3mL塑料空气注射器或空气泵通过空气散开。为了测量排放的剂量，设备必须在粉末管理前后进行称重，这导致不准确，因为粉末的剂量通常非常小（相对于设备的质量）。与商用充气器相比，定制设备的最大优点是透明凝胶加载移液器尖端没有粉末，可以观察到粉末分散的成功。由于移液器尖端很轻，因此在施用前后还可以准确称重以测量排放的剂量。移液器尖端插入导管，而不是暴露在动物的气管中。使用气管中的水分或分泌污染尖端的风险最小（这可能会影响发射剂量测量的准确性）。由于移液器尖端是一次性的，价格低廉，不同的干粉配方可以提前加载到不同的提示。在同一动物实验中可以评估各种配方，无需设备清洁和剂量补充，从而节省时间并消除残留粉末交叉污染的风险。此外，商业充气器产生的粉末分散模式因配方而异。多项研究报告，商业充气器分散的干粉很容易聚集，在19日、20日施用时未能到达深肺。相比之下，其他由类似我们的设备分散的配方，据报有高肺沉积15，21，22。
文献中还报告了其他类似的定制设备，用于将粉状气溶胶施用到动物的肺部。例如，Chaurasiya等人描述了使用管子进行管子的管子的灌输和粉末装载，在管内管进行粉末分散后，注射器与罐管相连。虽然他们的方法使用标准化的设备和材料（如示望镜，管和注射器）与较少的定制，这里的方法提供了一些明显的优势。首先，它允许在药物管理之前确认正确的内存。此步骤对经验不足的用户特别有帮助。其次，引导管可以起到保护盾的作用，防止气管中的任何分泌或水分污染凝胶加载移液器尖端，从而通过称重更准确地发射剂量测量。最后，更灵活的引导管与光纤一起可以更容易地进行灌导。
总之，本文准确介绍了一种定制的干粉充气器，它价格低廉、一次性、可重复且有效分散少量粉末。提到的入管过程是非侵入性的，快速的，可以安全准确地向小鼠提供粉末配方。它也可以用于管理肺分娩的液体配方。

肺病的施政途径为局部和系统行动提供治疗提供各种好处。对于肺病的治疗，可以通过肺分娩实现高局部药物浓度，从而减少所需剂量，降低全身副作用的发生率。此外，肺部相对较低的酶活性可以减少过早的药物代谢。肺部也有效地吸收药物的全身作用，由于大和良好的灌注表面积，极薄的上皮细胞层和肺毛细血管1的高血量。
吸入干粉配方已被广泛研究，用于预防和治疗各种疾病，如哮喘，慢性阻塞性肺病，糖尿病和肺疫苗接种2，3，4。固体状态下的药物通常比液体形式更稳定，干粉吸入器比雾化器5、6更便携、更方便用户。在开发吸入干粉配方时，需要在肺管理7后，在药物前动物模型中评估其安全性、药效和疗效。与能主动吸入干粉的人类不同，肺向小动物输送干粉具有挑战性。有必要建立一个有效的协议，将干粉送到动物的肺部。
老鼠被广泛用作研究动物模型，因为它们经济，繁殖良好。它们也易于处理，许多疾病模型都已建立起来。给小鼠肺部施用干粉有两种主要方法:吸入和体内管理。为了吸入，小鼠被放置在全身或鼻子专用室，其中干粉被气溶胶化，动物呼吸气溶胶没有安定8，9。设备昂贵，药品输送效率低。虽然全身腔在技术上可能不那么具有挑战性，但仅鼻腔暴露室可以最大限度地减少药物暴露在身体表面。无论如何，仍然很难精确控制和确定交付到肺部的剂量。干粉主要沉积在鼻咽部位，其中粘膜清除突出10。此外，在管理过程中，室内老鼠承受着巨大的压力，因为它们受到限制，得不到食物和水的供应。对于体内管理，它通常是指将物质直接引入气管。有两种不同的技术来实现这一点:气管切除术和骨质管内分。前者需要外科手术，使气管切口，这是侵入性的，很少用于粉末管理。这里只描述了第二种技术。与吸入法相比，体内输病是小鼠肺输配的常用方法，因为其输送效率高，药物损失最小。这是一种简单快捷的方法，可在几毫克内精确地将少量粉末输送到鼠标。虽然小鼠在解剖学和生理上与人类不同，在插管过程中需要麻醉，但内科管理绕过上呼吸道，为评估干粉配方的生物活动（如肺吸收、生物利用和治疗效果）提供了更有效的方法。
要在内经管理干粉，必须插管小鼠，这可能具有挑战性。本文介绍了定制干粉灌装器和管内装置的制造情况。演示了在小鼠肺部进行干燥粉末的灌接和窒息过程。

<table>
	<tbody>
		<tr>
			<td>BALB/c mouse</td>
			<td></td>
			<td></td>
			<td>Female; 7-9 weeks old; Body weight 20-25 g</td>
		</tr>
		<tr>
			<td>CleanCap Firefly Luciferase mRNA</td>
			<td>TriLink Biotechnology</td>
			<td>L-7602</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry Powder Insufflator</td>
			<td>PennCentury</td>
			<td>Model DP-4M</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine 10%</td>
			<td>Alfasan International B.V.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Light emitting diode (LED) torch</td>
			<td>Unilite Internation</td>
			<td>PS-K1</td>
			<td></td>
		</tr>
		<tr>
			<td>Mannitol (Pearlitol 160C)</td>
			<td>Roquette</td>
			<td>450001</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-filter round gel loading pipette tip (200 &#181;L)</td>
			<td>Labcon</td>
			<td>1034-800-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon floss</td>
			<td>Reach</td>
			<td>30017050</td>
			<td></td>
		</tr>
		<tr>
			<td>One milliliter syringe without needle</td>
			<td>Terumo</td>
			<td>SS-01T</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical fibre</td>
			<td>Fibre Data</td>
			<td>OMPF1000</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG12KL4 peptide</td>
			<td>EZ Biolab</td>
			<td></td>
			<td>(PEG12)-KLLLLKLLLLKLLLLKLLLLK-NH2</td>
		</tr>
		<tr>
			<td>Plastic Pasteur fine tip pipette</td>
			<td>Alpha Labotatories</td>
			<td>LW4061</td>
			<td></td>
		</tr>
		<tr>
			<td>Three-way stopcock</td>
			<td>Braun</td>
			<td>D201</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine 2%</td>
			<td>Alfasan International B.V.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Zerostat 3 anti-static gun</td>
			<td>MILTY</td>
			<td>5036694022153</td>
			<td></td>
		</tr>
	</tbody>
</table>

intratracheal administration of dry powder formulation in mice

在开发可吸入干粉配方时，必须评估其在前层动物模型中的生物活动。本文介绍了一种在小鼠体内输送干粉制剂的非侵入性方法。提供由 200 μL 凝胶装载移液器尖端组成的干粉装装置，通过三向塞孔连接到 1 mL 注射器。少量干粉（1-2毫克）被装入移液器尖端，并在注射器中通过0.6毫升的空气分散。由于移液器尖端是一次性的，价格低廉，因此可以提前将不同的干粉配方加载到不同的提示中。在同一动物实验中，无需设备清洁和剂量补充，即可评估各种配方，从而节省时间并消除残留粉末交叉污染的风险。粉末分散的程度可以通过移液器尖端残留的粉末量来检查。包括一个带有定制光源和导引管的鼠标内导管协议。适当的插管是影响干粉配方在健康内输送到小鼠深肺区域的关键因素之一。

当使用干粉充气器将粉末气溶胶输送到动物的肺部时，所使用的空气量至关重要，因为它会影响粉末的安全性以及粉末分散效率。为了优化该方法，使用不同体积的空气（0.3 mL、0.6 mL 和 1.0 mL）来分散干粉（1 毫克喷雾干曼尼托），并在给药后监测小鼠的重量 48 小时（图 6）。使用0.3 mL和0.6 mL的空气不会导致小鼠在施用后体重减轻至48小时。将粉末与 1 mL 空气分散，在 24 小时内导致超过 5% 的体重减轻，48 小时后仍未完全恢复。在此协议中，使用了7-9周大的BALB/c小鼠。根据物种、菌株和动物年龄、粉末特性（如颗粒大小分布、凝聚力和密度）以及要施用的粉末质量，用于高效粉末分散和动物耐受性的空气量可能需要调查人员对不同动物模型进行优化。
使用上述方法将喷雾冷冻干燥 （SFD） 制备的干粉配方交付给小鼠。SFD配方含有0.5%的mRNA表达红素酶蛋白，5%的合成肽作为输送载体，94.5%的曼尼托16。BALB/c 小鼠在体外施用 1 毫克含有 5μg mRNA 的 SFD 粉末，使用体内成像系统 （IVIS） （图 7）在 24 h 时评估肺部的红素酶表达。SFD粉末分散在深肺中，并观察到红道酶的表达。作为比较，SFD粉末在PBS中重组（最终体积为75μL），并以相同的灌引程序作为液体给小鼠施用，但使用微喷雾器代替产生液体气溶胶16。重组配方的红素酶表达明显高于干粉配方，这可能是由于粉末溶解问题或粉末和液体形式之间的药理动力学特征不同。用mRNA干粉气溶胶治疗的肺的组织学特征与未经治疗的控制和脂糖（LPS）治疗组（图8）进行了比较。没有任何治疗的肺部显示出健康的呈现，而肺内用10微克LPS治疗，显示空气空间分布不规则，炎症细胞渗透到间歇和肺间空间。用SFD粉末治疗的肺部没有出现任何炎症迹象。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61469/61469fig01.jpg"> 图1:定制干粉充气器。 （A） 粉末在尖端的狭窄端附近包装。（B） 凝胶加载移液器尖端通过三向塞锁连接到 1 mL 注射器。该图改编自廖等人21。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61469/61469fig01large.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/61469/61469fig02.jpg"> 图2:定制的光源进行灌引。 柔性光纤通过在镜头上创建一个小孔连接到 LED 手电筒。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61469/61469fig02large.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/61469/61469fig03.jpg"> 图3:指导性坎努拉。 （A） 1 mL 塑料巴斯德移液器用于制作导管。（B） 移液器通过加热软化。（C） 加热的移液器被拉伸和切割。（D） 移液器的 A 部分用作细尖移液器。（E&F）移液器的 B 部分用作导管。创建斜面以方便灌引程序。可以进行一个小的开口（可选的），以增加管子的灵活性。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61469/61469fig03large.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/61469/61469fig04.jpg"> 图4:灌引平台。 （A） 受管平台由安装在支架上的玻璃板组成。（B） 麻醉鼠标被放置在平台上，处于超平位置，通过用尼龙牙线钩住切口而悬浮。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61469/61469fig04large.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/61469/61469fig05.jpg"> 图5:说明插管程序的示意图图。 （A） 导管斜面与气管开口的中线对齐。（B） 引导管插入气管，并准备进行粉末管理。（C） 粉末加载尖端（通过三向塞子连接到注射器）插入已放置在鼠标气管中的导管中。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61469/61469fig05large.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/61469/61469fig06.jpg"> 图6:不同空气量的干粉的体内管理。 BALB/c 小鼠在体内使用喷雾干燥 （SD） 曼尼托粉进行静脉注射，这些粉末被分散在 0.3 mL、0.6 mL 和 1.0 mL 的空气中。在施用前和施用后18小时、24小时和48小时对小鼠的体重进行了监测。数据以体重变化百分比的平均值（n=2）的形式呈现。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61469/61469fig06large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61469/61469fig07.jpg"> 图7:MRNA制剂作为干粉和重组液体气溶胶的体内管理。 BALB/c 小鼠使用定制的干粉充气器或重组液体气溶胶（75 μL PBS 中的 1 毫克）配方，在体内使用喷雾冷冻干燥 （SFD） 0.5% mRNA （luciferase） 配方作为粉末气溶胶 （1 毫克） 进行静脉注射。每只小鼠接受5微克的mRNA剂量。PBS （75μL） 用作控制。在24小时后（A）肺被隔离用于生物发光成像:（B） 测量肺组织的红斑狼疮蛋白表达。这些数据被表示为每毫克蛋白质的相对光单位 （RLU） 的平均值，由单向 ANOVA 分析，然后是 Tukey 的临时测试***p < 0.001 （n=4）。这个数字改编自邱等人16。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61469/61469fig07large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/61469/61469fig08.jpg"> 图8:mRNA干粉配方的体内管理后，小鼠肺部的病理学。 （A） 未经处理的控制:小鼠在体内使用（B） LPS （10 毫克在 25μL PBS 中） 和（C）喷雾冷冻干 mRNA 粉 （1 毫克）。使用直立显微镜以 20 倍放大（比例杆 = 100 mm）查看幻灯片。这个数字改编自邱等人16。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61469/61469fig08large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Intratracheal Administration of Dry Powder Formulation in Mice at JoVE.com

Intratracheal Administration of Dry Powder Formulation in Mice

吸入干粉配方在治疗呼吸系统疾病方面有很大的潜力。在进入人类研究之前，有必要在前科研究中评估干粉配方的功效。提出了一种简单而非侵入性的方法，通过体外路线对小鼠的干粉进行管理。

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 ...

intratracheal-administration-of-dry-powder-formulation-in-mice

Nanyang Technological University

Research

JoVE Journal

Medicine

9.8K 次观看.  The University of Hong Kong. 吸入干粉配方在治疗呼吸系统疾病方面有很大的潜力。在进入人类研究之前，有必要在前科研究中评估干粉配方的功效。提出了一种简单而非侵入性的方法，通过体外路线对小鼠的干粉进行管理。

视频: Intratracheal Administration of Dry Powder Formulation in Mice

Improving IV Insulin Administration in a Community Hospital

Diabetes mellitus is a major independent risk factor for increased morbidity and mortality in the hospitalized patient, and elevated blood glucose concentrations, even in non-diabetic patients, predicts poor outcomes.1-4 The 2008 consensus statement by the American Association of Clinical Endocrinologists (AACE) and the American Diabetes Association (ADA) states that "hyperglycemia in hospitalized patients, irrespective of its cause, is unequivocally associated with adverse outcomes."5 It is important to recognize that hyperglycemia occurs in patients with known or undiagnosed diabetes as well as during acute illness in those with previously normal glucose tolerance.
The Normoglycemia in Intensive Care Evaluation-Survival Using Glucose Algorithm Regulation (NICE-SUGAR) study involved over six thousand adult intensive care unit (ICU) patients who were randomized to intensive glucose control or conventional glucose control.6 Surprisingly, this trial found that intensive glucose control increased the risk of mortality by 14% (odds ratio, 1.14; p=0.02). In addition, there was an increased prevalence of severe hypoglycemia in the intensive control group compared with the conventional control group (6.8% vs. 0.5%, respectively; p&lt;0.001). From this pivotal trial and two others,7,8 Wyoming Medical Center (WMC) realized the importance of controlling hyperglycemia in the hospitalized patient while avoiding the negative impact of resultant hypoglycemia. 
Despite multiple revisions of an IV insulin paper protocol, analysis of data from usage of the paper protocol at WMC shows that in terms of achieving normoglycemia while minimizing hypoglycemia, results were suboptimal. Therefore, through a systematical implementation plan, monitoring of patient blood glucose levels was switched from using a paper IV insulin protocol to a computerized glucose management system. By comparing blood glucose levels using the paper protocol to that of the computerized system, it was determined, that overall, the computerized glucose management system resulted in more rapid and tighter glucose control than the traditional paper protocol. Specifically, a substantial increase in the time spent within the target blood glucose concentration range, as well as a decrease in the prevalence of severe hypoglycemia (BG &lt; 40 mg/dL), clinical hypoglycemia (BG &lt; 70 mg/dL), and hyperglycemia (BG &gt; 180 mg/dL), was witnessed in the first five months after implementation of the computerized glucose management system. The computerized system achieved target concentrations in greater than 75% of all readings while minimizing the risk of hypoglycemia. The prevalence of hypoglycemia (BG &lt; 70 mg/dL) with the use of the computer glucose management system was well under 1%.

When compared to the previous paper protocol, implementation of a computerized glucose management system results in a substantial increase in blood glucose concentration measurements within the target range. Using a computerized glucose management system to monitor blood glucose levels, decreases in severe hypoglycemia (&lt;40 mg/dL), clinical hypoglycemia (&lt;70 mg/dL), and hyperglycemia (&gt;180 mg/dL) also can be observed.

提高在社区医院静脉注射胰岛素政府

Diabetes mellitus is a major independent risk factor for increased morbidity and mortality in the hospitalized patient, and elevated blood glucose ...

科研

医学

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

一种新的手术方法气管内注射生物活性剂在胎鼠模型

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Intranasal Administration of CNS Therapeutics to Awake Mice

Intranasal administration is a method of delivering therapeutic agents to the central nervous system (CNS). It is non-invasive and allows large molecules that do not cross the blood-brain barrier access to the CNS. Drugs are directly targeted to the CNS with intranasal delivery, reducing systemic exposure and thus unwanted systemic side effects1. Delivery from the nose to the CNS occurs within minutes along both the olfactory and trigeminal neural pathways via an extracellular route and does not require drug to bind to any receptor or axonal transport2. Intranasal delivery is a widely publicized method and is currently being used in human clinical trials3.
Intranasal delivery of drugs in animal models allows for initial evaluation of pharmacokinetic distribution and efficacy. With mice, it is possible to administer drugs to awake (non-anesthetized) animals on a regular basis using a specialized intranasal grip. Awake delivery is beneficial because it allows for long-term chronic dosing without anesthesia, it takes less time than with anesthesia, and can be learned and done by many people so that teams of technicians can dose large numbers of mice in short periods. Efficacy of therapeutics administered intranasally in this way to mice has been demonstrated in a number of studies including insulin in diabetic mouse models 4-6 and deferoxamine in Alzheimer's mouse models. 7,8
The intranasal grip for mice can be learned, but is not easy and requires practice, skill, and a precise grip to effectively deliver drug to the brain and avoid drainage to the lung and stomach. Mice are restrained by hand using a modified scruff in the non-dominant hand with the neck held parallel to the floor, while drug is delivered with a pipettor using the dominant hand. It usually takes 3-4 weeks of acclimating to handling before mice can be held with this grip without a stress response. We have prepared this JoVE video to make this intranasal delivery technique more accessible.

A method to intranasally administer drugs to awake mice for the purpose of targeting the brain is described. This method allows for repeat dosing over long periods using intranasal administration of drug without anesthesia, and nose-to-brain delivery with minimal systemic exposure.

清醒小鼠滴鼻的CNS药物治疗管理

Intranasal administration is a method of delivering  therapeutic agents to the central nervous system (CNS). It is  non-invasive and allows large ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

颈动脉输液的紫杉烷类化合物在小鼠体内的药代动力学和药效学分析

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Intubation-mediated Intratracheal (IMIT) Instillation: A Noninvasive, Lung-specific Delivery System

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences between the murine and human respiratory systems, especially in their upper respiratory tracts (URT). In some models, these differences in the murine nasal cavity can have a significant impact on disease progression and presentation in the lower respiratory tract (LRT) when using intranasal instillation techniques, potentially limiting the usefulness of the mouse model to study these diseases. For these reasons, it would be advantageous to develop a technique to instill bacteria directly into the mouse lungs in order to study LRT disease in the absence of involvement of the URT. We have termed this lung specific delivery technique intubation-mediated intratracheal (IMIT) instillation. This noninvasive technique minimizes the potential for instillation into the bloodstream, which can occur during more invasive traditional surgical intratracheal infection approaches, and limits the possibility of incidental digestive tract delivery. IMIT is a two-step process in which mice are first intubated, with an intermediate step to ensure correct catheter placement into the trachea, followed by insertion of a blunt needle into the catheter to mediate direct delivery of bacteria into the lung. This approach facilitates a &#62;98% efficacy of delivery into the lungs with excellent distribution of reagent throughout the lung. Thus, IMIT represents a novel approach to study LRT disease and therapeutic delivery directly into the lung, improving upon the ability to use mice as surrogates to study human respiratory disease. Furthermore, the accuracy and reproducibility of this delivery system also makes it amenable to Good Laboratory Practice Standards (GLPS), as well as delivery of a wide range of reagents which require high efficiency delivery to the lung.

Intubation-mediated Intratracheal IMIT Instillation: A Noninvasive, Lung-specific Delivery System

Intubation-mediated intratracheal (IMIT) instillation of reagents is an excellent, noninvasive method for studying respiratory disease, as well as a method for instilling therapeutic reagents directly into the lung. It is a rapid and highly reproducible method which is suitable for preclinical testing.

插管介导气管内（IMIT）灌输:一种无创，龙专用输送系统

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences ...

Transarterial Administration of Oncolytic Viruses for Locoregional Therapy of Orthotopic HCC in Rats

Hepatocellular carcinoma (HCC) is a disease with limited treatment options and poor prognosis. In recent years, oncolytic virotherapies have proven themselves to be potentially powerful tools to fight malignancy. Due to the unique dual blood supply in the liver, it is possible to apply therapies locally to orthotopic liver tumors, which are predominantly fed by arterial blood flow. We have previously demonstrated that hepatic arterial delivery of oncolytic viruses results in safe and efficient transduction efficiency of multifocal HCC lesions, resulting in significant prolongation of survival in immune competent rats. This procedure closely mimics the application of transarterial embolization in patients, which is the standard palliative care provided to many HCC patients. The ability to administer tumor therapies through the hepatic artery in rats allows for a highly sophisticated preclinical model for evaluating novel viral vectors under development. Here we describe the detailed protocol for microdissection of the hepatic artery for infusion of oncolytic virus vectors to treat orthotopic HCC.

Oncolytic virotherapies are under development as novel therapeutics for the treatment of hepatocellular carcinoma (HCC). Here we describe a method for locoregional therapy of HCC via hepatic arterial administration of oncolytic virus.

溶瘤病毒的Transarterial管理局大鼠原位肝癌的局部治疗

Hepatocellular carcinoma (HCC) is a disease with limited treatment options and poor prognosis.  In recent years, oncolytic virotherapies have proven ...

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

Obstructive respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD) are currently treated by inhaled anti-inflammatory and bronchodilator drugs. Despite the availability of multiple treatments, both diseases are growing public health concerns. The majority of asthma patients are well controlled on current inhaled therapies but a substantial number of patients with severe asthma are not. Asthma affects an estimated 300 million people worldwide and approximately 20 percent have a severe form of the disease. In contrast to asthma, there are few effective therapies for COPD. An estimated 10% of the population has COPD and the trend in death rates is increasing for COPD while decreasing for other major diseases. Although developing drugs for inhaled delivery is challenging, the nose-only inhalation unit enables direct delivery of novel drugs to the lung of rodents for pre-clinical efficacy and safety/toxicology studies. Inhaled drug delivery has multiple advantages for respiratory diseases, where high concentration in the lung improves efficacy and minimizes systemic side effects. Inhaled corticosteroids and bronchodilators benefit from these advantages and inhaled delivery may also hold potential for future biologic therapies. The inhalation unit described herein can generate, sample for characterization, and uniformly deposit a drug aerosol in the lungs of rodents. This enables the pre-clinical determination of the efficacy and safety of drug doses deposited in the lungs of rodents, key data required before initiating clinical development.

The inhalation unit described herein can generate, sample for characterization, and uniformly deposit a drug aerosol in the lungs of rodents. This enables the pre-clinical determination of the efficacy and safety of drug doses deposited in the lungs; key data enabling clinical inhaled drug development.

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 ...

A Pleural Effusion Model in Rats by Intratracheal Instillation of Polyacrylate/Nanosilica

Pleural effusion is a prevalent clinical finding of many pulmonary diseases. Having a useful animal pleural effusion model is very important to study these pulmonary diseases. Previous pleural effusion models paid more attention to biological factors rather than nanoparticles in the environment. Here, we introduce a model to make pleural effusion in rats by intratracheal instillation of polyacrylate/nanosilica, and a method of nanoparticle isolation in the pleural effusion. By intratracheal instillation of polyacrylate/nanosilica with concentrations of 3.125, 6.25 and 12.5 mg/kg&#8729;mL, the pleural effusion in rats presented on day 3, peaked at days 7-10 in 6.25 and 12.5 mg/kg&#8729;mL groups, then slowly decreased and disappeared on day 14. When the concentration of polyacrylate/nanosilica increased, the pleural effusion is produced more and faster. This pleural fluid was detected by ultrasound examination or CT chest scanning and confirmed by dissection of rats. Silica nanoparticles were observed in the rats' pleural effusion by transmission electron microscope. These results showed that the exposure to polyacrylate/nanosilica leads to the induction of pleural effusion, which was consistent with our previous report in humans. Additionally, this model is beneficial for the further study of nanotoxicology and the pleural effusion diseases.

Here, we present a protocol to construct a pleural effusion model in rats by intratracheal instillation of polyacrylate/nanosilica.

聚丙烯酸/纳米水的气管内灌注积液模型

Pleural effusion is a prevalent clinical finding of many pulmonary diseases. Having a useful animal pleural effusion model is very important to study ...

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. Various approaches have been described, such as the inhalation of aerosolized LPS as well as nasal or intratracheal instillation. The presented protocol describes a detailed step-by-step procedure to induce ALI in mice by direct intratracheal LPS instillation and perform FACS analysis of blood samples, bronchoalveolar lavage (BAL) fluid, and lung tissue. After intraperitoneal sedation, the trachea is exposed and LPS is administered via a 22 G venous catheter. A robust and reproducible inflammatory reaction with leukocyte invasion, upregulation of proinflammatory cytokines, and disruption of the alveolo-capillary barrier is induced within hours to days, depending on the LPS dosage used. Collection of blood samples, BAL fluid, and lung harvesting, as well as the processing for FACS analysis, are described in detail in the protocol. Although the use of the sterile LPS is not suitable to study pharmacologic interventions in infectious diseases, the described approach offers minimal invasiveness, simple handling, and good reproducibility to answer mechanistic immunological questions. Furthermore, dose titration as well as the use of alternative LPS preparations or mouse strains allow modulation of the clinical effects, which can exhibit different degrees of ALI severity or early vs. late onset of disease symptoms.

Presented here is a step-by-step procedure to induce acute lung injury in mice by direct intratracheal lipopolysaccharide instillation and to perform FACS analysis of blood samples, bronchoalveolar lavage fluid, and lung tissue. Minimal invasiveness, simple handling, good reproducibility, and titration of disease severity are advantages of this approach.

通过直接内切内脂多糖灌输诱导小鼠急性肺损伤

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. ...

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 ...