作者要感谢体制的健康伴侣动物护理和使用委员会在地区医院。

1。驯化处理鼻内给药清醒小鼠介绍必须适应小鼠在发病前鼻内给药2至4个星期为一个周期的处理。驯化的处理是很重要的，因为它有助于确保正确的身体姿势清醒的鼻腔药物输送的最大效益。此外，服药后，小鼠没有得到很好的适应这个过程可以有一个严重的焦虑反应。小鼠应花费约2-3天在每个九个步骤进展到下一步骤之前，根据动物的舒适操作。鼠标的应力水平应作为衡量进展。这意味着，监测鼠标的移动，排尿，排便量/频率，浑身发抖，又咬。如果鼠标具有较高的应激反应，停留在这一步之前进展到下一个，直到响应减少。一个样品驯化SCHedule可以看出，在表1。驯化的小鼠的发展，应通过以下的一天一次的步骤。不止一次地执行这些步骤，在试图加快驯化，每天不推荐。 <ol><li>将鼠标在手心一段时间的两到三分钟，不超过一英尺以上的笼顶，动物经常跳在此介绍的步骤。如果鼠标试图爬出来的手和简单起来一个人的手臂，抬起的鼠标基地的尾巴，把它在一个人的手。 </li><li>将鼠标在三分钟的手的手掌，轻轻的动物和宠物。宠物鼠标定向从头部到尾部，而让动物自由走动。 </li><li>将鼠标在手心的三分钟按摩耳朵后面（轻轻地捏在一起，脖子后面的皮肤上，用拇指和食指）。 </li><li>保持/李FT鼠标，它的脖子颈背，持续30秒，让鼠标停留在笼顶30秒，再重复之前保持。 </li><li>按住鼠标采用鼻内抓地力，这是第2节，但不要颠倒的动物（完成步骤2.4）。类似这样的动物为30秒的期间保持和释放回到笼顶部。重复第二次，后一分钟的休息时间。 </li><li>按住鼠标鼻内握在第2条中所描述的，颠倒的动物，所以它的腹侧面朝上朝天花板。请确保动物的脖子上是与地面平行。保持这个姿势30秒。重复后一分钟的休息时间，这是第二次。一些小鼠抵抗鼻内的抓地力比别人多，尤其是在第一时间反演在这一步中驯化。如果鼠标释放自己的抓地力，把鼠标上笼顶部和再握。如果需要，重复进行重新把持，但监测的鼠标的应力水平，并返回到笼如有必要。在这一点上失去了抓地力，在驯化过程中可以发挥作用的另一个因素是缺乏经验的驯兽员的鼻腔给药技术。 </li><li>按住鼠标鼻腔内的抓地力，反转，简单地将一个空尖移液器在每个鼻孔，并保持30秒的动物的位置。重复此步骤后一分钟的休息时间。 </li><li>鼻腔内的抓地力，按住鼠标反效果，和6μl的生理盐水滴鼻管理第3（通过步骤3.7）中描述的左，右纳雷。 </li><li>按住鼠标鼻腔内的抓地力，转化它，并两次将动物的左，右纳雷上笼顶之间，如在第3节6μl的生理盐水滴鼻管理。 </li></ol> 2。鼻内握固定清醒小鼠<ol><li>将鼠标on的笼顶部。 </li><li>你的惯用手的拇指和食指按住鼠标的尾巴。用你的中指，无名指和小指的手指，轻轻地固定身体的下部笼顶（ 图1A）。让鼠标抓住的酒吧正如你轻轻地伸展鼠标，更好地抓住颈背。 </li><li>你非惯用的手，用拇指和中指捏住鼠标坚决的颈背后面的颈部和肩部多余的皮肤。松弛的皮肤向上拉一顶帐篷的皮肤在整个肩胛骨（ 图1B）。以尽可能的皮肤，但要小心，不要拉肌肉，因为这会引起不适感的鼠标。一定要谨慎，以免动物的肋骨或脊柱施加太大的压力。过度的压力或拉力的皮肤，可能会导致在某些情况下，呼吸抑制。 </li><li>你非惯用手的食指上快速地将鼠标的头尽可能靠近鼻子和在皮肤上（ 图1C，1D）拉回来。在同一时间，滑动朝向头部的最佳位置的拇指和中指。在理想的情况下，鼠标应该无法垂直或水平移动其头部。 </li><li>在保持抓地力的颈背，把鼠标到他的背部。鼠标的背部应该是在你的手掌。对你的手掌牢牢抓住老鼠的尾巴基地附近的最后两个手指（环和小指）稳定的鼠标，或者，整个下半身（ 图1E，1F），你可以换你的手指。 </li><li>在这一点上使确定鼠标的颈部和下巴是平的和平行于地板。下巴应该是接近180度的角度与颈部尽可能地协助鼻腔给药。角度的微小变化可能会导致药物，结束了在错误的网站。确保鼠标能够通过观察腹部呼吸。鼠标翔民联无法移动，但应该是舒适的，而不是在任何疼痛。如果你看到鼠标的脖子和下巴的"V"，那么你需要重新握。 </li></ol> 3。清醒小鼠鼻腔给药的药物介绍使用鼻内的抓地力，每只小鼠被抑制两次，并持有与他们的颈部与地板平行，而20-30微升的液体的体积给药。体积小大小鼠是安全和有效的，我们通常使用24微升，并在本协议中所描述的。清醒鼻内可用于任何年龄的小鼠，例如在新生小鼠的发育神经科学的研究方法。在新生小鼠中，应使用一个较小的总体积（8微升）和液滴尺寸（1微升），以容纳更小的体积的鼻腔。药物通常是交付给两只老鼠一次，交替休息时间最有效地利用时间，并保持ØŇ时间表。 SET-UP会有一点点不同而有所不同，如果你安置给药组小鼠或单独饲养的小鼠。单独饲养的小鼠，将所有材料在防止老鼠从跨越到其他笼顶部网箱之间是有帮助的。随着组安置的小鼠，剂量只有从那个笼子。重要的是，一组安置笼中每个鼠标接收相同鼻内治疗，作为交叉污染的两个不同的治疗方法的潜在的存在由于药物到空气中或与皮肤接触的驱逐。我们强烈建议提前印刷，计量表，提醒技术人员的待遇分配，记录任何票据错过滴，要注意每个动物的健康，等步骤<ol><li>设置笼子里，他们应该是定量给料，并成立了一个计量站（ 图2）。 </li><li>拔下鼠标从笼，继续用药笼顶部。 </li><li>拿起鼠标，使用鼻内第2条中所述的抓地力。 </li><li>用你的优势手，加载移液器与6μl的药物或车辆。将鼠标的左鼻孔中，通常会在一个45度的角度（ 图1G，1H）附近的填充移液器的前端。如果你有粘性的解决方案，您可能需要增加你的角度为90度，因为该药物可能会遇到的侧吸头，而不是下降。如果很粘稠，擦拭前，用纸巾吸头排出的液体可能会有帮助。慢慢地喷射药物，其中大约一半的体积（3微升），形成一个小的液滴。 </li><li>液滴足够应放置在靠近鼠标的鼻孔，使鼠标可吸入液滴。后立即这鼠标吸入小滴，弹出的移液管尖中的其余部分的药物，以形成为鼠标的另一个小液滴，通过相同的鼻孔约2-3秒购买吸入。一定要注意口。如果我任何液体在嘴里，下拉并没有使其完全进入鼻子和应注意这一点。 </li><li>按住鼠标在这个位置上，持续15秒。我们强烈建议使用一个定时器来规范这个15秒的保持。 </li><li>右鼻孔，重复步骤3.3 - 3.6。如果有任何下降哼了一声，那滴应该被替换，并注意到。 </li><li>鼠标放置回他的笼子的顶部上，持续2分钟。开始2分钟，重复整个过程。在分娩后的首次下降。这给出了鼠标4 6各1μl总计24μl的溶液滴。 表2中可以看出，在一次给药的一个或两个小鼠的样品时间表。 </li><li>鼠标已收到所有滴后，返回鼠标移动到它的笼子。 </li></ol> 4。一般驯化和鼻腔给药的药物清醒小鼠的动物处理技术每个驯化后或鼻腔给药会议的，老鼠应该是给NA治疗，积极强化，如切里奥或去皮葵花籽。零食作为奖励，并转移他们的注意力，可能会导致伤害自己摸自己的鼻子。当工作组设小鼠，足够的对待应该被丢弃的每个鼠标。在返回到笼子里的动物，一定要恢复到原来的状态巢，以减少压力。剂量组设动物时，必须更换手套之间剂量的笼子，不转让香味从笼笼殖民地的殖民地，因为他们将成为另一个积极的努力。

<ol>
	<li>Dhuria, S. V., Hanson, L. R., Frey, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intranasal+delivery+to+the+central+nervous+system:+mechanisms+and+experimental+considerations.">Intranasal delivery to the central nervous system: mechanisms and experimental considerations.</a> J. Pharm Sci. 99, 1654-1673 (2010).</li><li>Thorne, R. G., Pronk, G. J., Padmanabhan, V., Frey, W. 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没有利益冲突的声明。

此方法显示如何小鼠可以给出针对脑无麻醉使用的常规剂量的滴鼻药。滴鼻药可以提供给大组小鼠长时间以最小的努力。一个组的20只小鼠可以鼻内视为由一个人在小于一小时。了大量的分子，包括小铁螯合剂间充质干细胞的生长因子，已被证明能够迅速针对CNS，并已显示出在啮齿类动物模型的疗效。建议直接交付机制，从鼻子到大脑外运输以及嗅觉和三叉神经和散流在血管周围的空间1,10。</...

<table border="1">
 <tbody>
 <tr>
 <td>Materials</td>
 <td>Company</td>
 <td>Cat #</td>
 <td>Comment</td>
 </tr>
 <tr>
 <td>P20 pipettor set at 6 &mu;l</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Gel loading pipette tips</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Timer</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Waste receptacle for tips</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Dosing sheet for notes</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Pen</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Drug and vehicle</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Container to hold drug vials</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Animals to be dosed</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Spare gloves</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Paper towels to wipe urine</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Table 3. Materials Needed</td>
 </tr>
 </tbody>
</table>

intranasal administration of cns therapeutics to awake mice

鼻内给药是一种方法，治疗剂传递到中枢神经系统（CNS）。它是非侵入性的，不穿越血脑屏障进入中枢神经系统的允许大型分子。药物直接针对鼻腔给药的中枢神经系统，减少系统性风险，并因而不需要的全身性的副作用1。从鼻子到交货的中枢神经系统一起发生在几分钟之内的嗅觉和三叉神经通路通过细胞外的路线和，不需要药物绑定到任何受体或轴突运输2。鼻腔给药是一个广泛宣传的方法和目前正在使用的人体临床试验，3。 药物在动物模型中允许鼻腔给药的药代动力学分布性和有效性的初步评估。与小鼠，它是可能的管理定期使用斯佩恰清醒（非麻醉）的动物的药物汉化鼻内抓地力。清醒交付是有益的，因为它允许长期慢性无需麻醉剂量，花费较少的时间比麻醉，很多人可以学习和做的，所以，团队，技术人员可以剂量的小鼠在短期内大量。以这种方式给小鼠鼻内给药的治疗效能已被证明在一些包括胰岛素在糖尿病小鼠模型4-6和去铁胺在阿尔茨海默氏症小鼠模型的研究。7,8 小鼠鼻腔的抓地力可以学习的，但是是不容易的，需要实践，技巧和精确的抓地力，有效的药物输送到大脑和避免对肺，胃引流。抑制小鼠用手举行的颈部与地板平行，而药物传递用移液器使用优势手使用的改性在非优势手的浮渣。它通常需要3-4周的驯化处理前小鼠可以保持这种握法无应激反应。我们准备了这份朱庇特的视频，，鼻腔给药技术使这更容易。

有几种方法，以确定疗效的药物输送。首先是要管理的染料，如印度墨水或亚甲基蓝和鼻内的嗅觉上皮细胞内的动物实施安乐死后和后大脑被移除时，通过筛板进入中枢神经系统的可视化的染料。第二是要测量的药物交付通过使用放射性标记的标记，或使用未标记的分子的ELISA检测。 Hanson 等人分布在中枢神经系统用药后药物清醒小鼠中可以找到一个很好的例子。2012年7。我?...

Watch this Scientific Journal Video about Intranasal Administration of CNS Therapeutics to Awake Mice at JoVE.com

Intranasal Administration of CNS Therapeutics to Awake Mice

目的针对脑清醒小鼠鼻内给药的方法，进行说明。这种方法允许重复给药，长期使用鼻腔给药的药物无需麻醉，并以最小的全身暴露的鼻子到大脑传递。

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 ...

intranasal-administration-of-cns-therapeutics-to-awake-mice

Research

JoVE Journal

Medicine

58.5K 次观看.  HealthPartners Institute for Education and Research. 目的针对脑清醒小鼠鼻内给药的方法，进行说明。这种方法允许重复给药，长期使用鼻腔给药的药物无需麻醉，并以最小的全身暴露的鼻子到大脑传递。

视频: Intranasal Administration of CNS Therapeutics to Awake Mice

Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain barrier1. The barrier selectively excludes molecules from crossing between the blood and the brain based upon their size and charge. This function can impede the delivery of therapeutics for neurological disorders. A number of chemotherapeutic drugs, for example, will not effectively cross the blood-brain barrier to reach tumor cells2. Thus, improving the delivery of drugs across the blood-brain barrier is an area of interest.
The most prevalent methods for enhancing the delivery of drugs to the brain are direct cerebral infusion and blood-brain barrier disruption3. Direct intracerebral infusion guarantees that therapies reach the brain; however, this method has a limited ability to disperse the drug4. Blood-brain barrier disruption (BBBD) allows drugs to flow directly from the circulatory system into the brain and thus more effectively reach dispersed tumor cells. Three methods of barrier disruption include osmotic barrier disruption, pharmacological barrier disruption, and focused ultrasound with microbubbles. Osmotic disruption, pioneered by Neuwelt, uses a hypertonic solution of 25% mannitol that dehydrates the cells of the blood-brain barrier causing them to shrink and disrupt their tight junctions. Barrier disruption can also be accomplished pharmacologically with vasoactive compounds such as histamine5 and bradykinin6. This method, however, is selective primarily for the brain-tumor barrier7. Additionally, RMP-7, an analog of the peptide bradykinin, was found to be inferior when compared head-to-head with osmotic BBBD with 25% mannitol8. Another method, focused ultrasound (FUS) in conjunction with microbubble ultrasound contrast agents, has also been shown to reversibly open the blood-brain barrier9. In comparison to FUS, though, 25% mannitol has a longer history of safety in human patients that makes it a proven tool for translational research10-12.
In order to accomplish BBBD, mannitol must be delivered at a high rate directly into the brain's arterial circulation. In humans, an endovascular catheter is guided to the brain where rapid, direct flow can be accomplished. This protocol models human BBBD as closely as possible. Following a cut-down to the bifurcation of the common carotid artery, a catheter is inserted retrograde into the ECA and used to deliver mannitol directly into the internal carotid artery (ICA) circulation. Propofol and N2O anesthesia are used for their ability to maximize the effectiveness of barrier disruption13. If executed properly, this procedure has the ability to safely, effectively, and reversibly open the blood-brain barrier and improve the delivery of drugs that do not ordinarily reach the brain 8,13,14.

Blood-brain barrier disruption aids the delivery of certain drugs to the brain. Mannitol delivered intra-arterially shrinks cells surrounding blood vessels in order to physically disrupt the barrier.

允许有针对性的大鼠模型血 - 脑屏障破坏神经血管治疗

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain ...

科研

医学

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

一种新型的高分辨率<em&gt;在体内</em&gt;成像技术研究肿瘤生长及治疗颅内结构的动态响应

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

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 ...

Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic mechanisms that contribute to the onset and progression of intestinal diseases. The identification of new molecules that promote these pathologies has led to a flurry of activity focused on the development of potential new therapies to inhibit their function. As a result, various pre-clinical mouse models with an intact immune system and stromal microenvironment are now heavily used. Here we describe three experimental protocols to test the efficacy of new therapeutics in pre-clinical models of (1) acute mucosal damage, (2) chronic colitis and/or colitis-associated colon cancer, and (3) sporadic colorectal cancer. We also outline procedures for serial endoscopic examination that can be used to document the therapeutic response of an individual tumor and to monitor the health of individual mice. These protocols provide complementary experimental platforms to test the effectiveness of therapeutic compounds shown to be well tolerated by mice.

We describe methods for longitudinal monitoring of the efficacy of therapeutics for the treatment of colonic pathologies in mice using a rigid endoscope. This protocol can be readily used for the characterization of the therapeutic response of an individual tumor in live mice and also for monitoring potential disease relapse.

新的治疗肠道病变的疗效，使用活体小鼠的串行内镜成像的非侵入性评估

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic ...

A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of treatments in vivo1. The traditional, established protocol has been further fine-tuned to minimize the damage to the animal. Notably, the pectoral muscle layers are teased away rather than simply cut, and the thoracotomy is approached intercostally as opposed to breaking the ribs in a sternotomy, preserving the integrity of the ribcage. With these changes, the overall stress on the animal is decreased. 
Stem cell therapies aimed to alleviate the damage caused by MIs have shown promise over the years for their pro-angiogenic and anti-apoptotic benefits. Current approaches of delivering cells to the heart surface typically involve the injection of the cells either near the damaged site, within a coronary artery, or into the peripheral blood stream2-4. While the cells have proven to home to the damaged myocardium, functionality is limited by their poor engraftment at the site of injury, resulting in diffusion into the blood stream5. This manuscript highlights a procedure that overcomes this obstacle with the use of a cell-encapsulated hydrogel patch. The patch is fabricated prior to the surgical procedure and is placed on the injured myocardium immediately following the occlusion of the left coronary artery. To adhere the patch in place, biocompatible external fibrin glue is placed directly on top of the patch, allowing for it to dry to both the patch and the heart surface. This approach provides a novel adhesion method for the application of a delicate cell-encapsulating therapeutic construct.

This protocol aims to alleviate the limitation of poor cell engraftment for stem cell treatment of myocardial infarctions through the use of a hydrogel system and a fibrin-based glue. With this approach, cell-to-tissue contact post-infarction can be maintained, increasing the therapeutic potential of beneficial agents at the site of injury.

水凝胶构造和纤维蛋白基胶方法来提供治疗的小鼠心肌梗死模型。

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of ...

A Rat Carotid Balloon Injury Model to Test Anti-vascular Remodeling Therapeutics

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is also a valuable model system to test, and to evaluate therapeutics and drugs that negate maladaptive remodeling in the vessel. The injury, or barotrauma, in the vessel lumen caused by an inflated balloon via an inserted catheter induces subsequent neointimal growth, often leading to hyperplasia or thickening of the vessel wall that narrows, or obstructs the lumen. The method described here is sufficiently sensitive, and the results can be obtained in relatively short time (2 weeks after the surgery). The efficacy of the drug or therapeutic against the induced-remodeling can be evaluated either by the post-mortem pathological and histomorphological analysis, or by ultrasound sonography in live animals. In addition, this model system has also been used to determine the therapeutic window or the time course of the administered drug. These studies can leadto the development of a better administrative strategy and a better therapeutic outcome. The procedure described here provides a tool for translational studies that bring drug and therapeutic candidates from bench research to clinical applications.

The rat carotid balloon injury model described below allows researchers to evaluate drugs or therapeutics that negate injury-induced arterial hyperplasia. Detailed pre-surgical preparation, surgical procedure, and post-surgical cares of the animal are described.

大鼠颈动脉球囊损伤模型试验抗血管重建治疗

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is ...

Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing factor in the overall outcome. Immunoblotting, flow cytometry and fixed cell experiments are often used to study how cells respond to anti-cancer drugs. These methods are important, but they have several shortcomings. Variability in drug responses between cancer and normal cells, and between cells of different cancer origin, and transient and rare responses are difficult to understand using population averaging assays and without being able to directly track and analyze them longitudinally. The microscope is particularly well suited to image live cells. Advancements in technology enable us to routinely image cells at a resolution that enables not only cell tracking, but also the observation of a variety of cellular responses. We describe an approach in detail that allows for the continuous time-lapse imaging of cells during the drug response for essentially as long as desired, typically up to 96 hr. Using variations of the approach, cells can be monitored for weeks. With the employment of genetically encoded fluorescent biosensors numerous processes, pathways and responses can be followed. We show examples that include tracking and quantification of cell growth and cell cycle progression, chromosome dynamics, DNA damage, and cell death. We also discuss variations of the technique and its flexibility, and highlight some common pitfalls.

Here, we describe a method of long-term time-lapse microscopy to longitudinally track single cells in response to anti-cancer therapeutics.

透过玻璃看:时间推移显微镜和单细胞的纵向跟踪研究抗癌治疗

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing ...

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 &#956;m of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer&#39;s disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer&#39;s disease.

纵<em&gt;在体内</em&gt;的Cerebrovasculature影像:相关性疾病CNS

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

Intranasal Delivery of Therapeutic Stem Cells to Glioblastoma in a Mouse Model

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of therapeutic stem cells via the intranasal route is a recently discovered alternative strategy, with strong potential for clinical translation, due to its non-invasive nature compared to intracranial implantation or delivery via systemic routes. The lack of blood brain barrier further strengthens the therapeutic potential of stem cells undergoing intranasal brain entry. This article summarizes the essential techniques utilized in our studies and outlines the basic principles of intranasal strategy for stem cell delivery using a mouse model of intracranial glioma xenografts. We demonstrate the optimized procedures that generate consistent and reproducible results with specific predetermined experimental parameters and offer guidelines for streamlined work flow that ensure efficient execution and reliable experimental outcome. The article is designed to serve as a baseline for further experimental customization based on hypothesis, stem cell types, or tumor specifics.

Stem cells are promising therapeutic carriers to treat brain tumors due to their intrinsic tumor tropism. Non-invasive intranasal stem cell delivery bypasses the blood brain barrier and demonstrates strong potential for clinical translation. This article summarizes the basic principles of intranasal stem cell delivery in a mouse model of glioma.

在小鼠模型中将胶质母细胞瘤治疗干细胞的鼻内递送

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of ...

Novel Methods for Intranasal Administration Under Inhalation Anesthesia to Evaluate Nose-to-Brain Drug Delivery

Intranasal administration has been reported to be a potential pathway for nose-to-brain delivery of therapeutic agents that circumvents the blood-brain barrier. However, there have been few reports regarding not only the quantitative analysis but also optimal administration conditions and dosing regimens for investigations of nose-to-brain delivery. The limited progress in research on nose-to-brain pathway mechanisms using rodents represents a significant impediment in terms of designing nose-to-brain delivery systems for candidate drugs.
To gain some headway in this regard, we developed and evaluated two novel methods of stable intranasal administration under inhalation anesthesia for experimental animals. We also describe a method for the evaluation of drug distribution levels in the brain via the nose-to-brain pathway using radio-labeled [14C]-inulin (molecular weight: 5,000) as a model substrate of water-soluble macromolecules.
Initially, we developed a pipette-based intranasal administration protocol using temporarily openable masks, which enabled us to perform reliable administration to animals under stable anesthesia. Using this system, [14C]-inulin could be delivered to the brain with little experimental error.
We subsequently developed an intranasal administration protocol entailing reverse cannulation from the airway side through the esophagus, which was developed to minimize the effects of mucociliary clearance (MC). This technique led to significantly higher levels of [14C]-inulin, which was quantitatively detected in the olfactory bulb, cerebrum, and medulla oblongata, than the pipette method. This appears to be because retention of the drug solution in the nasal cavity was substantially increased by active administration using a syringe pump in a direction opposite to the MC into the nasal cavity.
In conclusion, the two methods of intranasal administration developed in this study can be expected to be extremely useful techniques for evaluating pharmacokinetics in rodents. The reverse cannulation method, in particular, could be useful for evaluating the full potential of nose-to-brain delivery of drug candidates.

Here, we describe two novel methods of stable intranasal administration under inhalation anesthesia with minimal physical stress for experimental animals. We also describe a method for quantitative evaluation of drug distribution levels in the brain via the nose-to-brain pathway using radiolabeled [14C]-inulin as a model substrate of water-soluble macromolecules.

吸入麻醉下鼻内给药评价鼻腔给脑药物的新方法

Intranasal administration has been reported to be a potential pathway for nose-to-brain delivery of therapeutic agents that circumvents the blood-brain ...