这项工作是支持的JB马歇尔基金会。

1。准备过程的动物和设备<ol><li>手术开始之前，准备手术区和动物。使颈动脉导管插入到一端12“PE50管23隔距的钝针剪切在导管的另一端的约45°的斜角。消毒设备之前的程序。戴上头发阀盖，外科口罩和无菌手套。 </li><li>表面上放置一个加热垫，手术将进行。激活它，让它温暖。覆盖的区域的吸收性板凳垫。 </li><li>请确认你有25％甘露醇溶液中无晶体。如果晶体存在，溶解由小瓶放置在80℃的热水浴中，并定期搅拌。 </li><li>记录质量的动物。 </li><li>车型交付异丙酚稀释在0.8毫克/公斤/分钟的速率为0.1毫升/分钟。 </li><li>将大鼠在感应框3分钟。设置：5％异氟烷，2升/分钟呼吸机的流量。 </li><li>除一次性毛巾，刮颈部和两大腿内侧。 </li></ol> 2。插管的大鼠<ol><li>将在诱导动物靠背框3分钟。 </li><li>准备好您的气管内插管，静脉导管。对于这种规模和应变的老鼠，我们用的18 GA导管。管必须足够大，以便有足够的气流，但小到足以容纳进气管，不会造成损坏。拔出注射针的塑料导管和小心打破了针约。其锋利的尖1厘米。短打针将你的手写笔。将针头放回导管。 </li><li>一个倾斜的表面上，挂在老鼠的牙齿。 </li><li>闪耀的光直接对动物的脖子上，列队之间的耳朵。 </li><li>插入一个小锅铲约。到口咽部3厘米。向上抬起，轻轻拉出舌头出了口。 </li><li>开幕可视化的气管。您将EA小孔打开和关闭每一次呼吸。 </li><li>轻轻插入导管插入孔中。有时你可以感觉到塑料进气管的软骨。气管敏感。不要强行将导管。 </li><li>呼吸机设置为1升/分钟，60次和至2％异氟烷。 </li><li>导管连接呼吸机和磁带的操作表面的管。 </li><li>检查动物麻醉进行掐脚趾。 </li></ol> 3。建立丙泊酚静脉麻醉<ol><li>固定大鼠录音四肢和尾操作面。 </li><li>擦洗剃光颈部和腹股沟网站的洗必泰肥皂三次。然后擦洗洗必泰漱口液三次。 </li><li>将异丙酚的注射器IV扩展集。装入注射器，输液泵。 </li><li>使用手术刀，剃了大腿的皮肤通过削减。避免切削肌肉。 </li><li>打开切口力PS可视化的股静脉。 </li><li>扯远了组织在动脉暴露的容器。 </li><li>一个26 GA monoject兽医静脉导管插入静脉。当穿刺针的静脉壁，你会觉得它变得更容易推。收回该针头和导管推入容器。 </li><li>开始输注异丙酚在0.1毫升/分钟。 </li><li> 3-5分钟后，开始输注异丙酚，切换麻醉气体的混合物的50％O 2和50％N 2 O和关闭异氟醚。 </li></ol> 4。暴露颈总动脉分叉的<ol><li>在颈部正中切开手术刀。 </li><li>切割用剪刀或手术刀之间的两个大唾液腺。 </li><li>剖析了组织横向中线暴露颈内动脉。用钳子打开各层组织。您可以用剪刀除去的作品你的镊子公开的和确定minimallŸ血管。用纱布，棉签，和烧灼必要时控制出血。 </li><li>注入3毫升生理盐水IP，如果出现了中度到严重出血。 </li><li>使用拉钩，拉距颈动脉组织。分叉。 </li><li>公开枕动脉远端分叉。烧灼之间的钳子和一个电灼分开。 </li><li>使用镊子，充分暴露颈总动脉分叉和尽可能多的ECA。 </li><li>公开甲状腺上动脉分支枕动脉远端的ECA。烧灼之间的钳子和一个电灼分开。 </li></ol> 5。导尿颈外动脉<ol><li>将2种长度的4/0丝线在ECA。 </li><li>抓住您的止血钳缝合长度和收回ECA尾部向老鼠的脚。 </li><li>采取其他的缝合和打结，远颅向老鼠的嘴尽可能瓦特第i个外科医生的结。这形成了一个永久结扎牺牲ECA。 </li><li>删除第一个止血钳（一个，应用尾鳍紧张的），抢在对方缝合（一个是打结周围的ECA）和头部轻轻的张力，用止血钳。 </li><li>另一名外科医生的结领带松散的（没有安全 - 离开勉强并列）的分岔与永久结扎近端远端。 </li><li>灌溉用1％利多卡因放松动脉。 </li><li>请填写您的导管肝素化的盐水。 </li><li>将一个临时的血管夹尽可能接近的分歧。将血管夹在最大限度地提高空间的角度对导尿。将松散的结，向剪辑。 </li><li>做一个小的动脉切开术（切段），只是近端结扎microscissors的课外活动。如果剪辑和结扎是安全的，不应该有任何出血。 </li><li>到控制ECA墙，使用产钳，饲料坡口的导管进入血管。 </lI&gt; <li>松散的缝合打结，固定导管。领带的动脉导管周围。 </li><li>删除的片段，并用棉签清洁区。 </li><li>推进导管至约1毫米分叉远端。保持动脉近端结，以确保船舶被固定用钳子。 </li></ol> 6。管理甘露醇<ol><li>填写8“IV可拓集合和6毫升注射器用甘露醇，不产生任何气泡。存储在孵化器在37°C。注：工作迅速甘露醇 - 它很容易沉淀的溶液在室温下。 </li><li>为了形象化BBBD，您可以管理伊文思蓝染料，看着它进入领域的大脑的障碍已经被打乱了。要做到这一点，给动物2 ml / kg的2％伊文思蓝通过一个新的IV网站的腿对侧丙泊酚输注。 </li><li>安装分机设置为5微米过滤器，并填写甘露醇，以去除空气小家伙绩优。然后，将不产生气泡的活塞。 </li><li>管理甘露醇为0.09毫升/秒，持续25秒。观看甘露醇在颈总动脉的流量达到平衡，在一个点的ICA近端。它应该出现脉冲老鼠的心脏率。如果甘露醇向心流下来，你已经克服了老鼠的血压。这可能会导致危及生命的并发症和差BBBD的。 </li></ol>

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I., Remsen, L. G., Nixon, R., Neuwelt, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unexpected+neurotoxicity+of+etoposide+phosphate+administered+in+combination+with+other+chemotherapeutic+agents+after+blood-brain+barrier+modification+to+enhance+delivery,+using+propofol+for+general+anesthesia,+in+a+rat+model.">Unexpected neurotoxicity of etoposide phosphate administered in combination with other chemotherapeutic agents after blood-brain barrier modification to enhance delivery, using propofol for general anesthesia, in a rat model.</a> Neurosurgery. 47, 199 (2000).</li><li>Neuwelt, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+CNS+lymphoma+treated+with+osmotic+blood-brain+barrier+disruption:+prolonged+survival+and+preservation+of+cognitive+function.">Primary CNS lymphoma treated with osmotic blood-brain barrier disruption: prolonged survival and preservation of cognitive function.</a> Journal of clinical oncology. 9, 1580 (1991).</li><li>Remsen, L. G., Trail, P. A., Hellstr&#246;m, I., Hellstr&#246;m, K. E., Neuwelt, E. 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没有利益冲突的声明。

有几个的最大化疗效BBBD的装置。这是重要的，以尽量减少在切口向下阶段出血。血压和心脏速率可以由大出血的影响，这些因素已知影响程度BBBD 13的 。出血可减少通过使用缝合线结扎大血管，如甲状腺上和枕动脉，必须除以。此外，电，可以用来划分的血管和剖析领域有丰富的血液供应。同样重要的是，以保持空气和固体放入动物的所有行，满分 - 尤其是颈动脉导管。与甘露醇快速工?...

<table border="1"><tbody><tr><td> 材料名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>只Long Evans鼠</td><td>哈伦实验室</td><td></td><td> 210-250克，男</td></tr><tr><td> PE 50管</td><td> Beckton - 迪金森</td><td></td><td></td></tr><tr><td> 18号×2.5“静脉导管</td><td>泰尔茂</td><td></td><td>对于ET管</td></tr><tr><td> 30“四分机设定</td><td>雅培</td><td></td><td></td></tr><tr><td> 26号兽医静脉导管</td><td> Monoject </td><td></td><td></td></tr><tr><td>伊文思蓝染料</td><td>西格玛</td><td> E2129 </td><td></td></tr><tr><td>双极性</td><td>科德曼</td><td></td><td></td></tr><tr><td> 5微米过滤器， </td><td>布劳恩</td><td></td><td></td></tr></tbody></table>

rat model of blood-brain barrier disruption to allow targeted neurovascular therapeutics

紧密连接的内皮细胞与基底膜和星形胶质细胞末端的脚环绕脑血管的形成血-脑屏障。选择性的阻挡排除分子根据它们的大小和电荷的血液和脑之间交叉。此功能可以阻碍治疗神经系统疾病的交付。化疗药物的一些，例如，将不会有效地穿过血-脑屏障到达肿瘤细胞2。因此，改善药物透过血 - 脑屏障的交付是感兴趣的区域。 为提高药物的交付到大脑的最普遍的方法是直接脑输注和血-脑屏障破坏3。直接脑内输液保证疗法到达大脑，然而，这种方法具有的能力是有限的药物分散4。血 - 脑屏障的中断（BBBD）人低点流直接从循环系统的药物 到大脑，从而更有效地达到分散的肿瘤细胞。三种方法的屏障破坏，渗透屏障破坏，药物屏障破坏，聚焦超声微泡。渗透破坏，开创Neuwelt，使用高渗溶液，25％甘露醇脱水的细胞血 - 脑屏障，使他们萎缩，瓦解他们的紧密连接。屏障破坏，也可以完成药理上具有血管活性化合物5，如组胺，缓激肽6。然而，该方法中，是有选择性的，主要用于脑肿瘤屏障7。此外，RMP-7，血管舒缓激肽的肽的类似物，被发现时相比，头-对-头与渗透BBBD与25％甘露糖醇8差。聚焦超声（FUS）与微泡的超声造影剂的结合，另一种方法，也被证明可逆打开的血-脑屏障。在比较FUS，不过，25％甘露醇有一个较长的历史，在人类患者中的安全性，它使一个行之有效的工具转化研究10-12。 为了实现BBBD，甘露糖醇，必须以很高的速率直接进入大脑的动脉循环交付。在人类中，血管内导管被引导到大脑，其中可以实现快速，直接流。 “本协议模型人BBBD尽可能接近。经过切口下降到常见的颈动脉分叉处，导管插入逆行进入ECA和用于提供甘露糖醇直接到内颈动脉（ICA）的循环。丙泊酚和N 2 O麻醉用于自己的能力发挥最大的效益屏障破坏13。如果执行得当，此过程有安全，有效，可逆打开血 - 脑屏障和即兴表演的能力e的药物，通常不会到达大脑8,13,14交付。

<img alt="图1" src="/files/ftp_upload/50019/50019fig1.jpg" /> 图1。可视化通过血-脑屏障破坏Evans蓝染料外渗。Evans蓝染料是与白蛋白结合，未外渗进入大脑在生理条件下的颜料。血 - 脑屏障的大脑的一侧上的中断允许Evans蓝进入和染色的打乱的半球的蓝色，而在非破坏的半球不变。因此，这是一个有用的标记血-脑屏障破坏， 图1A是一个示例的脑无血-脑...

Watch this Scientific Journal Video about Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics at JoVE.com

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

血 - 脑屏障破坏，帮助某些药物输送到大脑。甘露醇动脉内交付收缩血管周围的细胞，以扰乱障碍。

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 ...

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19.3K 次观看.  Vanderbilt University School of Medicine. 血 - 脑屏障破坏，帮助某些药物输送到大脑。甘露醇动脉内交付收缩血管周围的细胞，以扰乱障碍。

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

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a significant obstacle to pharmaceutical treatments of brain disorders as it limits the passage of molecules from the vasculature into the brain tissue to molecules less than approximately 500 Da in size6. FUS induced BBB disruption (BBBD) is temporary and reversible4 and has an advantage over chemical means of inducing BBBD by being highly localized. FUS induced BBBD provides a means for investigating the effects of a wide range of therapeutic agents on the brain, which would not otherwise be deliverable to the tissue in sufficient concentration. While a wide range of ultrasound parameters have proven successful at disrupting the BBB2,5,7, there are several critical steps in the experimental procedure to ensure successful disruption with accurate targeting. This protocol outlines how to achieve MRI-guided FUS induced BBBD in a rat model, with a focus on the critical animal preparation and microbubble handling steps of the experiment.

Microbubble-mediated focused ultrasound disruption of the blood-brain barrier (BBB) is a promising technique for non-invasive targeted drug delivery in the brain1-3. This protocol outlines the experimental procedure for MRI-guided transcranial BBB disruption in a rat model.

MRI引导下的破坏血脑屏障在大鼠模型经颅聚焦超声

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a ...

科研

医学

Stretch in Brain Microvascular Endothelial Cells (cEND) as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying mechanisms involved in it are useful for treatment. There are both in vivo and in vitro methods available for this purpose. In vivo models can mimic actual head injury as it occurs during TBI. However, in vivo techniques may not be exploited for studies at the cell physiology level. Hence, in vitro methods are more advantageous for this purpose since they provide easier access to the cells and the extracellular environment for manipulation.
Our protocol presents an in vitro model of TBI using stretch injury in brain microvascular endothelial cells. It utilizes pressure applied to the cells cultured in flexible-bottomed wells. The pressure applied may easily be controlled and can produce injury that ranges from low to severe. The murine brain microvascular endothelial cells (cEND) generated in our laboratory is a well-suited model for the blood brain barrier (BBB) thus providing an advantage to other systems that employ a similar technique. In addition, due to the simplicity of the method, experimental set-ups are easily duplicated. Thus, this model can be used in studying the cellular and molecular mechanisms involved in TBI at the BBB.

Stretch in Brain Microvascular Endothelial Cells cEND as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

In vitro traumatic brain injury models are being developed to reproduce in vivo brain deformation. Stretch-induced injury has been employed for astrocytes, neurons, glial cells, aortic, and brain endothelial cells. However, our system uses a blood brain barrier (BBB) model that possesses properties constituting a legitimate model of the BBB to establish an in vitro&#160;TBI model.

作为脑微血管内皮细胞（CEND）的伸展<em&gt;体外</em创伤性脑损伤模型血脑屏障

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying ...

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm&#183;cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 &#177;&#160;0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Setting-up an In Vitro Model of Rat Blood-brain Barrier BBB: A Focus on BBB Impermeability and Receptor-mediated Transport

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

设置了一个<em&gt;体外</em大鼠血脑屏障&gt;模型（BBB）：在BBB抗渗性和受体介导的运输为中心

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and ...

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

缺氧缺糖再给氧作为<em&gt;在体外</em&gt;缺血再灌注损伤模型的研究血脑屏障功能障碍

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

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 ...

Retroductal Submandibular Gland Instillation and Localized Fractionated Irradiation in a Rat Model of Salivary Hypofunction

Normal tissues that lie within the portals of radiation are inadvertently damaged. Salivary glands are often injured during head and neck radiotherapy. Irreparable cell damage results in a chronic loss of salivary function that impairs basic oral activities, and increases the risk of oral infections and dental caries. Salivary hypofunction and its complications gravely impact a patient's comfort. Current symptomatic management of the condition is ineffective, and newer therapies to assuage the condition are needed. 
Salivary glands are exocrine glands, which expel their secretions into the mouth via excretory ducts. Cannulation of these ducts provides direct access to the glands. Retroductal delivery of a contrast agent to major salivary glands is a routine out-patient procedure for diagnostic imaging. Using a similar procedure, localized treatment of the glands is feasible. However, performing this technique in preclinical studies with small animals poses unique challenges. In this study we describe the technique of retroductal administration in rat submandibular glands, a procedure that was refined in Dr. Bruce Baum's laboratory (NIH)1, and lay out a procedure for local gland irradiation.

Salivary gland hypofunction, a major adverse effect of head and neck radiotherapy diminishes a patient's quality of life. The demonstration of efficacy of new therapies in animal models is a prerequisite before clinical transition. This protocol describes retroductal administration and local irradiation of rat submandibular glands.

Retroductal颌下腺灌输和本地化分割照射在涎腺功能减退症大鼠模型

Normal tissues that lie within the portals of radiation are inadvertently damaged. Salivary glands are often injured during head and neck radiotherapy. ...

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by astrocytes, pericytes and brain endothelial cells (BECs). Here, we detail methods to assess BBB coverage using single cultures of immortalized human BECs, single cultures of primary mouse BECs, and a humanized triple culture model (BECs, astrocytes and pericytes) of the BBB. To highlight the applicability of the assays to disease states, we describe the effect of oligomeric amyloid-&#946; (oA&#946;), which is an important contributor to Alzheimer&#39;s disease (AD) progression, on BBB coverage. Further, we utilize the epidermal growth factor (EGF) to illuminate the drug screening potential of the techniques. Our results show that single and triple cultured BECs form meshwork-like structures under basal conditions, and that oA&#946; disrupts this cell meshwork formation and degenerates the preformed mesh structures, but EGF blocks this disruption. Thus, the techniques described are important for dissecting fundamental and disease-relevant processes that modulate BBB coverage.

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Maintaining blood-brain barrier coverage is key for the homeostasis of the central nervous system. This protocol describes in vitro techniques to delineate the fundamental and pathological processes that modulate blood-brain barrier coverage.

评估血脑屏障网状血管形成和破坏的体外测定

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

一种基于猪脑内皮细胞的体外血脑屏障模型的改进方法

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Delivery of Cardioactive Therapeutics in a Porcine Myocardial Infarction Model

Myocardial infarction is one of the leading causes of death and disability worldwide, and there is an urgent need for novel cardioprotective or regenerative strategies. An essential component of drug development is determining how a novel therapeutic is to be administered. Physiologically relevant large animal models are of critical importance in assessing the feasibility and efficacy of various therapeutic delivery strategies. Due to their similarities to humans in cardiovascular physiology, coronary vascular anatomy, and heart weight to body weight ratio, swine is one of the preferred species in the preclinical evaluation of new therapies for myocardial infarction. The present protocol describes three methods of administering cardioactive therapeutic agents in a porcine model. After percutaneously induced myocardial infarction, female landrace swine received treatment with novel agents through either: (1) thoracotomy and transepicardial injection, (2) catheter-based transendocardial injection, or (3) intravenous infusion via jugular vein osmotic minipump. The procedures employed for each technique are reproducible, resulting in reliable cardioactive drug delivery. These models can be easily adapted to suit individual study designs, and each of these delivery techniques can be used to investigate a variety of possible interventions. Therefore, these methods are a useful tool for translational scientists pursuing novel biological approaches in cardiac repair following myocardial infarction.

The present protocol describes three methods of administering cardioactive therapeutic agents in a porcine model. Female landrace swine received treatment through either: (1) thoracotomy and transepicardial injection, (2) catheter-based transendocardial injection, or (3) intravenous infusion via jugular vein osmotic minipump.