作者想感谢约翰马斯顿畜牧业专家和博士马可波罗A. Passini技术援助，在这个项目的早期阶段。这项工作是由资助的​​国立卫生研究院慢性淋巴细胞白血病（R01NS41584; R01HD054413）。

1。侧脑室注射<ol><li>第一步是准备注入股市的解决方案，这些解决方案是病毒载体，质粒DNA，药物，应在无菌条件下注入。 </li><li>混合0.05％的注射部位的可视化W / V台盼蓝的PBS所需滴度的病毒载体（5-7微升总）。 </li><li>质粒DNA溶液（5升微升总）包含的D -（+） - 葡萄糖20％（W / V）（1μL），台盼蓝（0.05％），PBS（1μL），质粒（约5〜G /〜L ）（2〜L）和2.5 kDa的线性PEI均聚物（150毫米）（1〜L）。 </li><li>通过1-2分钟，低温麻醉，固定的PND 2新生儿。 </li><li>使用此注射针微升校准灭菌的玻璃微管，是通过长管连接到一个3毫升3毫升汉密尔顿注射器。 </li><li>打破（锅铲）调整为2毫米渗透入颅的针头的针尖。斜中含有的微量注射液管将针头。仔细地拉动注射器的柱塞装入针注射液。 </li><li>摄取的解决方案后，脱离管注射器;拉柱塞进一步，然后再次连接到针。 </li><li>在右手拇指和食指之间保持的针头和地方注射器柱塞摸右手手掌的中指和无名指之间。 </li><li>抓紧背后的头部皮肤使用左手和地方在光纤光照亮相关的解剖结构，可作为指导使用固定的鼠标。 </li><li>针2毫米深，垂直插入颅骨表面，在一个位置约0.25毫米，横向的矢状缝和0.50-0.75毫米，喙新生儿冠状动脉缝合（ 图 1A）。然后，用手掌推柱塞右手非常缓慢和谨慎。监测破裂的船只或面部肿胀。 </li><li>取出针15秒后停止活塞运动，以防止回流。 </li><li>要恢复，保持5-10分钟的小鼠，在加热的容器中，直到恢复运动和一般的响应。 </li></ol> 2。通过时间/面部静脉的静脉注射<ol><li>被注入的解决方案应该是准备在1:100稀释，并辅以过滤绿色食用色素和无菌条件下注入。 </li><li>将一个小塑料露儿100μL玻璃汉密尔顿注射器年底。露儿，附加33计0.25英寸的注射针。确保所有部件连接牢固。 </li><li>被注入量装入注射器，确保在注射器注射入​​血管的空气，没有空气是致命的。它往往是有益的吸管要你们一块的封口膜注射量。这允许摄取到注射器的解决明确的可视化。 </li><li>一个极小的景象透射是用来方便地可视化的颞浅静脉，或在新生儿的面部静脉（图2A）。注射前，确保新生儿使用手术磁带的透射。纱布应该放在新生儿和破坏动物的皮肤，以防止磁带的磁带。安全的动物，在其一侧的透射，确保磁带并不妨碍呼吸。新生儿的脖子上，应轻轻打开，使面部静脉是很容易看到，贴在鼻子应该是稳定的头部。 </li><li>使用2.25X头带放大镜，以便更容易可视化，慢慢地插入静脉的针。使针头应保持可见皮下的静脉是很肤浅的。 </li><li>静脉缓慢注入溶液的体积。绿色染料允许注射容易可视化。 </li><li>前等待15秒，取出针有一个完全压下注射器的柱塞和驱逐剩余的解决方案，由于极其狭窄的针孔之间的滞后。 </li><li>取出针后，用纱布注射部位施加压力，直到出血停止。 </li><li>监测新生儿窘迫的迹象。正确的注射后，没有明显的窘迫应得到遵守。新生儿应给予恢复它返回到笼前约5分钟。 </li><li>针/注射器单位之间的注射用PBS冲洗针的血液会很快堵塞由于小口径。 </li></ol> 3。代表性的成果： 侧脑室注射：大脑可以在不同的时间点注射后收获和可视化，以证明成功的注射技术。如果不添加染料注射液，将很难​​检查中的准确性jection。适当注射脑室之一，将允许在注射侧大脑的台盼蓝分布，注射后（ 图 1B）约10-15分钟。一台盼蓝均匀分布在左，右大脑半球和嗅球将可见注射后约60分钟（ 图1C）。这是由于连接将允许传播到相邻的脑室蓝色染料的脑室。精确的注射，也可以由散布在约12小时内注射后（ 图1D）延髓中央椎管蓝色染料的可视化。不准确的注射可以区分蓝色缺乏大脑半球。在这种情况下，进针深度是不足够的注射液有可能分布在皮肤下。另一种可能性是，针穿透过深在大脑中，从而超越了脑室及脑室底下掏空的解决方案是传递。 静脉注射：准确，高效的时间/面部静脉注射，可以可视化紧随注射。由于注入的解决方案，绿色染料允许可视化解决方案的进入和流经面部静脉（ 图2A）。此外，改变皮肤颜色的鼠标将遵守以下注射。新生儿正常的粉红色转换为绿色，由于绿色食品染料注射液存在（ 图2B） 。观察新生儿的绿色变化的程度取决于所使用的染料浓度。注射，并在实践期间的简单的可视化，1:50稀释建议。然而，显着地改变肤色，结果往往是小狗被母亲拒绝，因此，去除的染料或一个显著减少的浓度应在实验程序考虑。一个不准确的注入将导致绿色染料的积累，在注射部位的皮肤下。注射不完整的，它发生时没有完全插入静脉内针开幕，在静脉中流动时，会出现一些解决方案和一些积聚在皮肤下。如果发生此种情况，应插入针入血管。 <img src="/files/ftp_upload/2968/2968fig1.jpg" alt="图1"/> 图1示范成功的ICV PDN2小鼠注射。 一个利用侧脑室注射入脑室注射部位）示意图B）从ICV的大脑注入左心室与台盼蓝的PBS小狗，合影留念注射后15分钟，C左心室）侧脑室脑注射台盼蓝的PBS小狗，合影留念注射后60分钟，D）延髓中央椎管在侧脑室注射用台盼蓝的PBS小狗左心室上，采取12小时注射后的照片。 <img src="/files/ftp_upload/2968/2968fig2.jpg" alt="图2"/> 图2示范成功的第四PDN2小鼠注射。静脉注射剂使用的面部静脉）示意图 B）小狗注射绿色染料第四的PBS相比，联合国注入littermate，照片立即注射后。

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没有利益冲突的声明。

疾病的小鼠模型的研究往往需要药物或其他物质的管理，新生儿。在这段视频中，我们展示了一步一步程序，利用侧脑室（ICV）注射CNS涉及两种类型的注射战略目标的中枢神经系统，可用于：1）直接注射;或2）静脉注射针对时间/面部静脉。这些注射的时间是非常重要的。由于ICV注射写意，头骨必须相对可塑性。这种类型的注射是唯一可能通过的第一个星期的生活，变得越来越具有挑战性的和有害...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td>绿色食用色素</td><td>麦考密克</td><td> N / A </td><td>必须过滤</td></tr><tr><td>汉密尔顿玻璃注射器（100微升） </td><td>西格玛Aldrick </td><td> 20702 </td><td></td></tr><tr><td> LuerMxF主题风格的白色尼龙</td><td>小零件， </td><td> VPLF - LC78 - 1 - 25 </td><td></td></tr><tr><td>细针距注射针头</td><td>波普尔</td><td> 7111 </td><td>尺寸：33号（SWG）× ¼“（6.35毫米） </td></tr><tr><td>凌晨视透射</td><td>呼吸</td><td> 1017920 </td><td></td></tr><tr><td> 2.25X头戴放大镜</td><td> MagEyes </td><td>型号5 </td><td>选择适合个人喜好的放大倍率</td></tr></tbody></table>

delivery of therapeutic agents through intracerebroventricular (icv) and intravenous (iv) injection in mice

尽管血脑屏障的保护作用，起着屏蔽大脑，中枢神经系统（CNS）的访问限制， 设计 1,2神经退行性疾病的潜在疗法的失败中最常见的结果。神经退行性疾病，如脊髓性肌萎缩症（SMA），下运动神经元受到影响，可以极大地受益，从引进治疗药物进入中枢神经系统。此视频的目的是为了演示两种不同的注射范式传递到新生小鼠出生后不久，治疗材料。其中一个方法是直接注射入脑侧脑室（侧脑室）材料传递到中枢神经系统通过脑脊液3,4结果。第二种方法是一个时间的静脉注射（静脉注射），可以引入不同的循环系统的疗法，导致全身交付，包括中枢神经系统5。如果一个合适的病毒载体和病毒血清型是利用广泛的中枢神经系统传导是可以实现的的。颞静脉注射的可视化和利用是可行的出生后第6天。但是，如果传递的材料是为了达到中枢神经系统，这些注射应采取血脑屏障，同时由于其不成熟的状态，更渗透，最好到产后第2天之前。充分发展的血脑屏障，极大地限制了静脉交付的成效。一次手术aptitude是实现两个输送系统是简单而有效的的。他们不需要任何广泛的手术设备，可以由一个人完成。然而，这些技术并非没有挑战。产后第2天的幼崽和随后的小目标领域的体积小，可以使注射难以执行，最初具有挑战性的复制。

Watch this Scientific Journal Video about Delivery of Therapeutic Agents Through Intracerebroventricular ICV and Intravenous IV Injection in Mice at JoVE.com

Delivery of Therapeutic Agents Through Intracerebroventricular ICV and Intravenous IV Injection in Mice

本文演示了两种非常不同的方法：注射入脑（脑室）和2）全身（静脉注射）1）引入到中央的新生小鼠的中枢神经系统治疗药物。

交付的治疗药物通过侧脑室（ICV）和静脉（IV）注射液在小鼠

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most ...

delivery-therapeutic-agents-through-intracerebroventricular-icv

Research

JoVE Journal

Medicine

Delivery of Therapeutic Agents Through Intracerebroventricular (ICV) and Intravenous (IV) Injection in Mice

60.2K 次观看.  University of Missouri. 本文演示了两种非常不同的方法：注射入脑（脑室）和2）全身（静脉注射）1）引入到中央的新生小鼠的中枢神经系统治疗药物。

视频: Delivery of Therapeutic Agents Through Intracerebroventricular ICV and Intravenous IV Injection in Mice

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 ...

科研

医学

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

啮齿动物的中枢神经系统药物直接脑室交货

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

神经科学

<meta charset="utf8"></head><body>小鼠模型中注射后的颈总动脉修复

Facilitating Repeat Intracarotid Injections in Mouse Models by a Novel Injection Site Repair Technique

Given recent advances in the delivery of novel antitumor therapeutics using endovascular selective intraarterial delivery methods in neuro-oncology, there is an urgent need to develop methods for intracarotid injections in mouse models, including methods to repair the carotid artery in mice after injection to allow for subsequent injections. We developed a method of intracarotid injection in a mouse model to deliver therapeutics into the internal carotid artery (ICA) with two alternative procedures. 
During injection, the needle is inserted into the common carotid artery (CCA) after tying a suture around the external carotid artery (ECA) and injected therapeutics are delivered into the ICA. Following injection, the common carotid artery (CCA) can be ligated, which limits the number of intracarotid injections to one. The alternative procedure described in this article includes a modification where intracarotid artery injection is followed by injection site repair of the CCA, which restores blood flow within the CCA and avoids the complication of cerebral ischemia seen in some mouse models. 
We also compared the delivery of bone marrow-derived human mesenchymal stem cells (BM-hMSCs) to intracranial tumors when delivered through intracarotid injection with and without injection site repair following the injection. Delivery of BM-hMSCs does not differ significantly between the methods. Our results demonstrate that injection site repair of the CCA allows for repeat injections through the same artery and does not impair the delivery and distribution of injected material, thus providing a model with greater flexibility that more closely emulates intracarotid injection in humans.

<meta charset="utf8"></head><body>作者聚焦:通过溶瘤病毒和 MicroRNA 的动脉内递送策略推进胶质母细胞瘤治疗

Repair of the intracarotid artery in a mouse model after injection returns blood flow to the artery without negatively impacting the distribution of the injected material. Injection site repair facilitates subsequent injections through the same artery and prevents cerebral ischemia in mouse strains that lack a complete Circle of Willis.

通过一种新颖的注射部位修复技术促进小鼠模型中的重复颈内注射

Given recent advances in the delivery of novel antitumor therapeutics using endovascular selective intraarterial delivery methods in neuro-oncology, there ...

癌症研究

Intracerebroventricular and Intravascular Injection of Viral Particles and Fluorescent Microbeads into the Neonatal Brain

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the hematogenous route, which involves infection of the endothelial cells and pericytes of the brain. The second is the intracerebroventricular (ICV) route. Once within the central nervous system (CNS), viruses may spread to the subarachnoid space, meninges, and choroid plexus via the cerebrospinal fluid. In experimental models, the earliest stages of CNS viral distribution are not well characterized, and it is unclear whether only certain cells are initially infected. Here, we have analyzed the distribution of cytomegalovirus (CMV) particles during the acute phase of infection, termed primary viremia, following ICV or intravascular (IV) injection into the neonatal mouse brain. In the ICV injection model, 5 &#181;l of murine CMV (MCMV) or fluorescent microbeads were injected into the lateral ventricle at the midpoint between the ear and eye using a 10-&#181;l syringe with a 27 G needle. In the IV injection model, a 1-ml syringe with a 35 G needle was used. A transilluminator was used to visualize the superficial temporal (facial) vein of the neonatal mouse. We infused 50 &#181;l of MCMV or fluorescent microbeads into the superficial temporal vein. Brains were harvested at different time points post-injection. MCMV genomes were detected using the in situ hybridization method. Fluorescent microbeads or green fluorescent protein expressing recombinant MCMV particles were observed by fluorescent microscopy. These techniques can be applied to many other pathogens to investigate the pathogenesis of encephalitis.

Here, we describe a simple method of intracerebroventricular and intravascular injection of viral particles or fluorescent microbeads into the neonatal mouse brain. The localization pattern of the virus and nanoparticles could be detected by microscopic evaluation or by in situ hybridization.

病毒颗粒和荧光微球侧脑室和血管内注入新生儿脑

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the ...

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous substances into the brain. These exogenous substances may induce injurious impact or constitute therapeutic strategy. The most common delivery methods of exogenous substances into the brain are based on systemic deliveries, such as subcutaneous or intravenous routes. Although commonly used, these approaches have several limitations, including low delivery efficacy into the brain. In contrast, surgical methods that locally deliver substances into the CNS are more specific and prevent the uptake of the exogenous substances by other organs. Several surgical methods for CNS delivery are available; however, they tend to be very traumatic. Here, we describe a mouse infusion microsurgery technique, which effectively delivers substances into the brain via the internal carotid artery, with minimal trauma and no interference with normal CNS functionality.

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

The present protocol describes a mouse microsurgery infusion technique, which effectively delivers substances directly into the brain via the internal carotid artery.

鼠标显微注水技术进行有针对性的物质传递到中枢神经系统<i&gt;通过</i&gt;的颈内动脉

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous ...

Intravenous Injections in Neonatal Mice

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are technically feasible and routine. However, some mouse models can have early onset of disease with a rapid progression that makes administration of potential therapies difficult. The temporal (or facial) vein is just anterior to the ear bud in mice and is clearly visible for the first two days after birth on either side of the head using a dissecting microscope. During this window, the temporal vein can be injected with volumes up to 50 &#956;l. The injection is safe and well tolerated by both the pups and the dams. A typical injection procedure is completed within 1-2 min, after which the pup is returned to the home cage. By the third postnatal day the vein is difficult to visualize and the injection procedure becomes technically unreliable. This technique has been used for delivery of adeno-associated virus (AAV) vectors, which in turn can provide almost body-wide, stable transgene expression for the life of the animal depending on the viral serotype chosen.

Animal models of pediatric disease can experience early onset and aggressive disease progression. Clinically relevant therapy delivery to young mouse models can be technically difficult. This protocol describes a non-invasive intravenous injection method for newborn mice within the first two postnatal days of life.

在新生小鼠静脉注射

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are ...

生物学

Intraventricular Drug Delivery and Sampling for Pharmacokinetics and Pharmacodynamics Study

Although the blood-brain barrier (BBB) protects the brain from foreign entities, it also prevents some therapeutics from crossing into the central nervous system (CNS) to ameliorate diseases or infections. Drugs are administered directly into the CNS in animals and humans to circumvent the BBB. The present protocol describes a unique way of treating brain infections through intraventricular delivery of antibiotics, i.e., polymyxins, the last-line antibiotics to treat multi-drug resistant Gram-negative bacteria. A straightforward stereotaxic surgery protocol was developed to implant a guide cannula reaching into the lateral ventricle in rats. After a recovery period of 24 h, rats can be injected consciously and repeatedly through a cannula that is fitted to the guide. Injections can be delivered manually as a bolus or infusion using a microinjection pump to obtain a slow and controlled flow rate. The intraventricular injection was successfully confirmed with Evans Blue dye. Cerebrospinal fluid (CSF) can be drained, and the brain and other organs can be collected. This approach is highly amenable for studies involving drug delivery to the CNS and subsequent assessment of pharmacokinetic and pharmacodynamic activity.

<meta charset="utf8"></head><body>用于药代动力学和药效学研究的脑室内药物输送和取样

Delivery of therapeutics directly into the central nervous system is one way of circumventing the blood-brain barrier. The present protocol demonstrates intracerebroventricular injection for subsequent collection of cerebrospinal fluid and bodily organs. This facilitates the investigation of drug pharmacokinetics and pharmacodynamics in animal models for developing new treatments.

用于药代动力学和药效学研究的脑室内药物输送和取样

Although the blood-brain barrier (BBB) protects the brain from foreign entities, it also prevents some therapeutics from crossing into the central nervous ...

交付的治疗药物通过侧脑室（ICV）和静脉（IV）注射液在小鼠

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清醒小鼠滴鼻的CNS药物治疗管理

啮齿动物的中枢神经系统药物直接脑室交货

通过一种新颖的注射部位修复技术促进小鼠模型中的重复颈内注射

通过一种新颖的注射部位修复技术促进小鼠模型中的重复颈内注射

通过一种新颖的注射部位修复技术促进小鼠模型中的重复颈内注射

病毒颗粒和荧光微球侧脑室和血管内注入新生儿脑

鼠标显微注水技术进行有针对性的物质传递到中枢神经系统

在新生小鼠静脉注射

用于药代动力学和药效学研究的脑室内药物输送和取样

用于药代动力学和药效学研究的脑室内药物输送和取样