这项研究得到了德意志研究联合会（DFG，德国研究基金会）根据授权号FO 315/4-和欧盟第七框架计划（FP7/2007-2013）根据出让协议HEALTH-F2-2009-241778号CF。

1。播种内皮细胞孔板<ol><li>在T75培养瓶中，培养小鼠脑微血管内皮细胞（CEND）的改变培养基（DMEM中含有10％FCS，50单位/ ml青霉素/链霉素，1％L-谷氨酰胺），每周两次，直到达到合流。 （脑微血管内皮细胞生成和永生，请看到布列克等 ，2012年，福斯特等人 ，2005年21,19）。 </li><li>清洗细胞，用磷酸盐缓冲盐水（PBS）。取出PBS及胰蛋白酶消化细胞，用2毫升温暖的胰蛋白酶-EDTA溶液。 </li><li>孵育的细胞在37℃下持续5分钟或直到细胞层分散。 </li><li>进入细胞的培养基中加入8毫升。点击烧瓶数次分离细胞。 </li><li>查看细胞在显微镜下，以确保完全脱离烧瓶。 </li><li>移液器介质分离的细胞上下。摇动烧瓶混合细胞suspensi上。 </li><li>取20微升细胞悬浮液，放入血球计数器计数的细胞数。 </li><li>确定细胞密度和种子入井20,000细胞/ cm 2。 </li><li>细胞悬液转移到collagen1预包被的6孔柔性底培养板中（57.75厘米2），总体积为3ml /孔。每个孔的面积为9.62厘米2。 </li><li>细胞生长在37℃直至汇合一个星期。改变培养基每周两次。 </li></ol> 2。之前伸展损伤的细胞分化 <ol><li>改变细胞分化培养基（含1％血清简装的胎牛血清（ssFCS）的50单位/ ml青霉素/链霉素的DMEM）的培养基中。 </li><li>培养的细胞在37℃24小时。 </li></ol> 3。牵张诱导的内皮细胞损伤<ol><li>打开的细胞损伤的控制器设备。 </li><li>设置延迟为50毫秒。 </li><li>设置调节到15 psi的压力，并几次，直到按下扳机的注册高峰压力变得稳定。 </li><li>调节器的压力设定为所需的值。使用表1为指导，产生不同程度的牵拉伤。 </li><li>到托盘架灵活有底将6孔培养板（57.75厘米2）。确保该井选择设置为正确以及大小（ 即大井）。 </li><li>将适配器插头牢牢井。用一只手牢牢握住插头到位，而另一方面推动触发。 </li><li>记录产生的压力峰值。 </li><li>立即把盘子放回37℃培养箱所需的时间长度或立即使用后续实验或评估。 </li></ol> 4。染料摄取试验拉伸损伤评估<ol><li>后立即将细胞海峡蚀刻（步骤3.7）中，加入30微升的1毫克/毫升溶液中的活力染色细胞培养基中（ 注：染料溶液10μl的细胞毒性标记（请参阅的材料和设备的补充表），作为用于细胞培养液中的每毫升 ）。 </li><li>细胞的染料加入后，立即在荧光显微镜下检视。 </li></ol> 5。评估牵拉损伤乳酸脱氢酶（LDH） <ol><li>拉伸后的细胞（步骤3.7），立即取出200微升细胞培养液中，从井里。做同样的伤后各时间点你想包括在LDH释放（ 即如 30分钟，1小时，2小时， 等你的调查。） </li><li>离心细胞培养液中离心5分钟以除去任何细胞碎片在最高设置。除去上清液，并使用当前的随后的步骤。 </li><li>一旦。1AVE完成步骤5.1，并已采取了必要的样本，你想从不同的时间点，你想调查，裂解细胞裂解液中乳酸脱氢酶测定试剂盒（请参阅的材料和设备的补充表）。 </li><li>将100μl的测定培养基中，包括在套件中提供的在96 - 孔板的每一个孔中测定基托。 </li><li>成两个平行的测定试剂盒包括在96孔板的孔中，将100μl的无细胞的细胞培养基。 </li><li>在37°C下孵育30分钟。 </li><li>读取492nm处的吸光度。 </li></ol>

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没有利益冲突的声明。

在体外的机械性损伤的影响进行了研究和方法已被建立为星形胶质细胞，神经元，神经胶质细胞和血管内皮细胞8，9，22。有，然而，迄今为止，仍然没有已知的伸展培养的脑损伤的内皮细胞的体外模型。细胞模型的创伤提供了有价值的动物模型的优势，因为可以精确地控制6的细胞的机械环境。因此，通过牵拉伤，用培养的脑血管内皮细胞在体外创伤模型，行为模型血脑屏障（BBB），如我们的协议是什么礼物可能被证明是有用的。 该协议利用CEND细胞，在我们的实验室建立BBB模型。由于BBB击穿往往是记录在颅脑损伤患者脑外伤往往与破坏的血脑屏障，这可能会导致水肿形成23，24，PRES的方法这里ented具体可以TBI进行BBB研究中使用。 在此模型中，重要的是照顾多少牵拉损伤程度被施加到细胞。在尽可能多的在任何情况下，无论施加的压力量受伤细胞，损伤的程度，可以影响内皮细胞多大差别很大的其他细胞类型。主动脉内皮细胞更耐牵拉伤比星形胶质细胞的或混合神经胶质细胞的9。此外，他们更迅速地修复损伤后，相比于其他类型的细胞。因此，对于脑血管内皮细胞，特别是CEND细胞，更大量的牵拉损伤是必要的，以产生一个高程度的损伤。人们可以通过施加相应的压力示于表1中达到所需程度的损伤。然而，对于CEND细胞，严重伤害是首选，由于其电阻应变。 LDH进行的分析表明作为程度伸展的增加，更多的乳酸脱氢酶分泌到上清液中。与此相反，分别获得了低的数量牵拉损伤的细胞生产的乳酸脱氢酶的量控制细胞相似。如上文在协议中，必须注意，使用适量的介质，因为介质的量的增加或减少，可能会导致施加到各孔中的峰值压力的差异。例如，一个含有5毫升的流体寄存器中的平均为4.0 psi的峰值压力而空以及寄存器45 psi的压力被施加时，平均为3.8磅。因此，最好是推多次触发以及一个控制，以确保产生的峰值压力，对应到所需的量。 在我们的实验中，我们使用的可行性染色伸展，以确定影响细胞膜的通透性。光学系统使我们能够灵活底培养板，我们用vi的EW染色细胞在显微镜下直接。然而，当你想进行免疫标记研究，直接拉伸损伤后，可能会出现困难。首先，板的尺寸和厚度，可能会引起问题与一些显微镜观察平台。第二，以及柔性膜的光学系统可能是一个障碍清除观看。 然而，尽管有上述的限制，所描述的过程可以用作体外血脑屏障的机械性损伤的模型。创伤性脑损伤（TBI）包括两部分，即，局部缺血和创伤。为脑外伤后继发性损伤的情况时，存在着严重的失血导致血压低，或由于脑肿胀限制氧气供应到大脑缺血发生。它被认为是延迟的，非机械的损伤，较上面的那一刻开始，损伤25的连续的病理过程。一个缺氧的发生FTER重型颅脑损伤是常见的26。氧糖剥夺（OGD）是目前正在使用的方法， 在体外缺血模型。因此，受到细胞OGD后作为辅助侮辱细胞弹力，可以模仿缺血脑外伤后的发病率。因此，要改善我们目前的体外模型TBI和模式，它尽可能接近实际TBI，因为它发生在体内 ，在未来，我们也将聘请OGD结合牵拉伤。

创伤性脑损伤（TBI）是领先全球的死亡原因之一。脑外伤的一个主要的健康和医疗问题1，每年约有10万人受到影响。由于这个原因，各种在体内和体外模型TBI已建立和发展，研究其机 ​​制2,3,4更好地了解脑外伤可以帮助改善病人的治疗和减少相关的死亡率，发病率和成本。 利用在体内和体外方法存在许多脑损伤模型， 在体内模型可以模仿实际发生的事件，头部受伤。然而，由于体内情况的复杂性，无障碍组织的利益变得有限2。在理解的生理反应的单个细胞的伤害，作为结果，它是重要的细胞分离的全身效应可能会抑制或改变他们的个人响应5。出于这个原因，提供了有价值的细胞模型的创伤在动物模型的优点，因为可以精确地控制6的细胞的机械环境。 在体外系统中，采用机械负载的细胞或组织损伤引起的这样的方法来确定改变的使用已经开发出来。例如，已建立了一种用于研究细胞的机械性损伤的效果为星形胶质细胞，神经元，神经胶质细胞和血管内皮细胞7,8,9。 在体外创伤模型研究啮齿动物和人类的星形胶质细胞反应10的压力控制装置相同，我们用什么为我们的模型建立。同样的方法适用于诱导损伤小鼠脑微血管内皮细胞（bEnd3）11和皮层神经元，脑endoth 12,13以及通过伸展新生仔猪14。的elial细胞培养装置的底部变形以及由此产生机械牵拉伤10。伤害时培养的细胞中细胞的空气压力，由应用程序。此压力可偏转膜细胞的生长，从而拉伸细胞。通过设置相应的空气压力脉冲的持续时间和强度，可以实现不同程度的伸展（ 即 “低，”温和的“，或”严重“）拉伸损伤此方法已在体内 7与外伤，而且损伤此方法允许细胞外环境的精确控制，可以很容易地被再现。 虽然已被用于许多其他的脑细胞类型，包括bEnd3类似的方法，我们的模型中使用的小鼠脑微血管内皮细胞（CEND）产生一个优点，即它使在我们的实验室。该细胞系是一个非常适合用作BBB模型在体外细胞培养模型血脑屏障（BBB）。应具备的特点，这将使他们能够作为渗透屏幕。 体外细胞模型的预测血脑屏障通透性的重要标准之一是，它应该具有生理现实细胞结构15。即使bEnd3细胞显示独特的主轴，如鳞状形态文化16，他们表现出体外形态不规则行为，使他们形成囊肿样腔，而不是常规的管状结构中纤维蛋白凝胶17。此外，当细胞注入胚胎和新生小鼠，诱导快速增长的肿瘤，但不是在新生儿和年轻小鼠致死小鼠胚胎。因此，建议管正常的内皮细胞生长，迁移和分化的一个或多个进程已经改变或消除在此细胞株18。另一方面，形态学，免疫细胞化学评价内皮细胞和BBB标志物的表达，生物电，和细胞旁通量测量表明，我们的BBB模型CEND确实是一个合适的模型是BBB 19。 脑血管内皮细胞在体内具有极其严密的渗透与跨内皮电阻（TEER）从2,000-5,000欧姆厘米2。对于研究脑微血管系统的阻隔性能，医药品，细胞旁限制性和气密性应予以考虑。在大多数脑微血管内皮细胞（BCEC），这是不保存细胞展览TEER从50-100欧姆厘米2 20。永生化的脑内皮细胞株bEnd3生成TEER值不大于60欧姆厘米2 15。 CEND细胞的培养基中含有相比之下分化降低血清显示屏TEER值范围从300-500Ωcm的2 19,21。 至目前为止， 在体外培养的脑血管内皮细胞牵拉损伤模型是稀缺的。因此，通过牵拉伤，用培养的脑血管内皮细胞作为模型是BBB创伤体外模型可能会被证明是有用的。在这个协议中，我们提出的体外模型，可以模仿的脑细胞，特别是大脑的血脑屏障的微血管内皮细胞，在TBI收到的实际影响。该模型的主要优点是，受伤的量施加的细胞，以及细胞外的环境中，可以很容易地控制，以精确的方式，能够容易重现性实验设置。

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			<td height="20" style="height:20px;width:355px;">Cell Injury Controller II</td>
			<td style="width:351px;">Custom Design and Fabrication, Virginia, USA</td>
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			<td style="width:435px;">http://www.radiology.vcu.edu/research/customdesign/cic.html</td>
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			<td height="20" style="height:20px;">Bioflex Culture Plate - Collagen Type I</td>
			<td>Flexcell, Dunn Labortechnik</td>
			<td>BF - 3001C</td>
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			<td height="20" style="height:20px;width:355px;">Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)</td>
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			<td style="width:143px;">D5796</td>
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			<td style="width:351px;">PAA Laboratories</td>
			<td style="width:143px;">A15110-1333</td>
			<td style="width:431px;">final concentration 10%, heat-inactivated (30 min at 56 &#176;C)</td>
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			<td height="20" style="height:20px;width:355px;">L-glutamine</td>
			<td style="width:351px;">Biochrom AG</td>
			<td style="width:143px;">K0282</td>
			<td style="width:431px;">Storage: &#8804; -15 &#176;C</td>
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			<td height="20" style="height:20px;width:355px;">MEM Vitamin</td>
			<td style="width:351px;">Biochrom AG</td>
			<td style="width:143px;">K0373</td>
			<td style="width:431px;">Storage: &#8804; -15 &#176;C</td>
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			<td height="20" style="height:20px;width:355px;">Na-pyruvate</td>
			<td style="width:351px;">Biochrom AG</td>
			<td style="width:143px;">L0473</td>
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			<td style="width:351px;">Biochrom AG</td>
			<td style="width:143px;">K0293</td>
			<td style="width:431px;">Storage at 4 &#176;C</td>
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			<td height="20" style="height:20px;width:355px;">Penicilin/Streptomycin</td>
			<td style="width:351px;">Biochrom AG</td>
			<td style="width:143px;">A2212</td>
			<td style="width:431px;">Storage: &#8804; -15 &#176;C</td>
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			<td height="20" style="height:20px;width:355px;">Fetal Calf Serum, charcoal stripped (ssFCS)</td>
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			<td>12676-011</td>
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			<td height="20" style="height:20px;width:355px;">Trypsin-EDTA solution</td>
			<td style="width:351px;">PAA Laboratories</td>
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			<td style="width:351px;">Life Technologies</td>
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			<td style="width:431px;">Storage: &#8804; -15 &#176;C, protected from light</td>
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			<td height="20" style="height:20px;">Cytotoxicity Detection Kit PLUS (LDH)</td>
			<td>Roche</td>
			<td>4744926001</td>
			<td style="width:431px;">Storage: &#8804; -15 &#176;C</td>
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stretch in brain microvascular endothelial cells (cend) as an in vitro traumatic brain injury model of the blood brain barrier

可用于治疗由于死亡率高的事件带来的创伤性脑损伤（TBI）的方法，使一个更好的了解相关机制参与。有在体内和体外方法可用于这一目的。 体内模型可以模拟实际的头部受伤，因为它发生在脑外伤。然而， 在体内的技术可能无法被利用在细胞的生理水平的研究。因此， 在体外培养的方法是更有利的用于此目的，因为它们提供了更容易获得的细胞和细胞外环境进行操纵。 我们的协议提出了一个TBI使用牵拉损伤脑微血管内皮细胞的体外模型。它利用压力细胞培养灵活底孔。可以很容易地控制和施加的压力可以产生药害，范围从低到严重。鼠脑微血管内皮细胞（CEND）的在我们的实验室中产生的，是一个非常适合的模型血脑屏障（BBB），从而提供了一个到其他系统，采用了类似的技术优势。此外，由于简单的方法，实验装置很容易被复制。因此，这种模式可以用来研究细胞和分子机制参与TBI在BBB。

我预涂胶原细胞培养6以及灵活的平底培养板（57.75厘米2）受到不同程度的牵拉伤，使用电池担架设备。细胞损伤后，在显微镜下检查细胞形态的伸展引起的损伤的影响。有人指出，为更大程度的弹力施加的细胞（ 图1），也可以观察到细胞有更大程度的失真。 图1A中所示，控制不受到损伤的细胞，这些细胞没有任何指示细胞肿胀或失真为规则形状的脑血管内皮细胞（CEND）的出现。当牵拉伤（ 图1B-D），变形可以在光学显微镜下观察。拉伸的细胞后，严重的峰值压力3.5-4.5磅之间，CEND细胞出现明显回缩，肿胀，变形，不<html>能够间隙。此外，吸收的可行性污垢（100nM的最终浓度）也增加了牵拉损伤的程度的显着升高（ 图2）。的可行性染色使用的是染料不能渗透对健康细胞的细胞质膜完整性受到损害时，成为透性。的染料排除在大部分的控制单元，因此，只有少数的细胞进行染色（ 图2A）相比，拉伸的细胞（ 图2B-D）。更多的绿色荧光蛋白细胞牵拉损伤的程度增加。 由于损伤，乳酸脱氢酶（LDH）释放酶的生化指标也根据制造商的指导检查。 图3可以看出 ，增加细胞牵拉损伤引起乳酸脱氢酶（LDH）释放增加。 928/50928fig1.jpg“宽度=”500px的“/&gt; 图1。光镜检查正常与受伤的细胞（A）普通拉伸融合细胞单层istightly包装和拉长。当细胞被拉伸通过施加峰值压力脉冲1.8-2.0磅（ 即低拉伸），他们显得不那么紧凑，空间的箭头（B）所示。当细胞与2.5-3.0压力峰值脉冲中度受伤，其中一些出现肿胀和变形（C）。细胞变得严重3.5-4.5磅，如箭头（D）表示的伸展与缩回。 100X的放大倍率。 <a href="https://www.jove.com/files/ftp_upload/50928/50928fig1highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/50928/50928fig2.jpg" width="500px" /> 图2。萤石escence镜检正常与受伤的细胞，细胞可行性染色2小时，伤后处理。 答：控制拉伸细胞BD：拉伸细胞（B -低-中度，C，D中-重度）。 100X的放大倍率。 <a href="https://www.jove.com/files/ftp_upload/50928/50928fig2highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/50928/50928fig3.jpg" width="500px" /> 图3。伸展损伤后在不同的时间间隔，测定乳酸脱氢酶（LDH）酶的释放到上清液后伸展损伤。释放到培养基中的乳酸脱氢酶。 LDH表达的总释放LDH乳酸脱氢酶（LDH媒体加细胞）的百分比。值是±SEM。各时间点的n是5，除0 HR V值受到严重拉伸，其中n = 3。 LDH释放低，适中弹力细胞没有差异拉伸控制，并从对方明显。造成严重的拉伸的细胞，它们被释放的LDH量，作为一个显着更大的比其他所有样品，除了适度拉伸的样品在1小时。 （P &lt;0.05，单因素方差分析，霍尔姆-通富法）。 <a href="https://www.jove.com/files/ftp_upload/50928/50928fig3highres.jpg" target="_blank">点击这里查看大图</a> 。 表1中。引导产生不同程度的牵拉伤。 <table border="1"><tbody><tr><th>调压阀</th><th>峰值压力</th><th>损伤程度</th></tr><tr><td> 15磅</td><td> 1.2-1.5磅</td><td> &lt;低</td></tr><tr><td> 20-25磅</td><td> 1.8-2.0磅</td><td>低</td></tr><tr><td> 30-35磅</td><td> 2.5-3.0磅</td><td>中度</tD&gt; </tr><tr><td> 40-50磅</td><td> 3.5-4.5磅</td><td>严重</td></tr><tr><td> 60psi的</td><td> 4.8-6.0磅</td><td> &gt;严重</td></tr></tbody></table>

Watch this Scientific Journal Video about Stretch in Brain Microvascular Endothelial Cells cEND as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier at JoVE.com

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

在体外创伤性脑损伤的车型正在研发中再现体内脑变形。伸展损伤已受聘为星形胶质细胞，神经元，神经胶质细胞，主动脉，脑血管内皮细胞。然而，我们的系统采用了血脑屏障（BBB）模型，该模型具有BBB构成一个合法的模型建立在体外 TBI模型的属性。

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 ...

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

14.8K 次观看.  Zentrum für operative Medizin der Universität Würzburg.  在体外创伤性脑损伤的车型正在研发中再现体内脑变形。伸展损伤已受聘为星形胶质细胞，神经元，神经胶质细胞，主动脉，脑血管内皮细胞。然而，我们的系统采用了血脑屏障（BBB）模型，该模型具有BBB构成一个合法的模型建立在体外 TBI模型的属性。 

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

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

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 ...

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 ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

脑Microslices的兴奋性刺激作为<em&gt;在体外</em&gt;中风模型

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

控制皮层影响模型创伤性脑损伤

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

A Novel Model of Mild Traumatic Brain Injury for Juvenile Rats

Despite growing evidence that childhood represents a major risk period for mild traumatic brain injury (mTBI) from sports-related concussions, motor vehicle accidents, and falls, a reliable animal model of mTBI had previously not been developed for this important aspect of development. The modified weight-drop technique employs a glancing impact to the head of a freely moving rodent transmitting acceleration, deceleration, and rotational forces upon the brain. When applied to juvenile rats, this modified weight-drop technique induced clinically relevant behavioural outcomes that were representative of post-concussion symptomology. The technique is a rapidly applied procedure with an extremely low mortality rate, rendering it ideal for high-throughput studies of therapeutics. In addition, because the procedure involves a mild injury to a closed head, it can easily be used for studies of repetitive brain injury. Owing to the simplistic nature of this technique, and the clinically relevant biomechanics of the injury pathophysiology, the modified weight-drop technique provides researchers with a reliable model of mTBI that can be used in a wide variety of behavioural, molecular, and genetic studies.

The modified weight-drop technique is an easy, cost-effective procedure used for the induction of mild traumatic brain injury in juvenile rats. This novel technique produces clinically relevant symptomology that will advance the study of mild traumatic brain injury (mTBI) and concussion.

轻度脑外伤的幼年大鼠一种新模式

Despite growing evidence that childhood represents a major risk period for mild traumatic brain injury (mTBI) from sports-related concussions, motor ...

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 Mouse Model of Single and Repetitive Mild Traumatic Brain Injury

Mild Traumatic Brain Injury (mTBI) can result in the acute loss of brain function, including a period of confusion, a loss of consciousness (LOC), focal neurological deficits and even amnesia. Athletes participating in contact sports are at high risk of exposure to large number of mTBIs. In terms of the level of injury in a sporting athlete, a mTBI is defined as a mild injury that does not cause gross pathological changes, but does cause short-term neurological deficits that are spontaneously resolved. Despite previous attempts to model mTBI in mice and rats, many have reported gross adverse effects including skull fractures, intracerebral bleeding, axonal injury and neuronal cell death. Herein, we describe our highly reproducible animal model of mTBI that reproduces clinically relevant symptoms. This model uses a custom made pneumatic impactor device to deliver a closed-head trauma. This impact is made under precise velocity and deformation parameters, creating a reliable and reproducible model to examine the mechanisms that contribute to effects of single or repetitive concussive mTBI.

Athletes absorb several hundred mild traumatic brain injuries (mTBI)/concussions every year; however, the consequence of these on the brain is poorly understood. Therefore, an animal model of single and repetitive mTBI that consistently replicates clinically relevant symptoms provides the means to advance the study of mTBI and concussion.

单一和重复轻度创伤性脑损伤的小鼠模型

Mild Traumatic Brain Injury (mTBI) can result in the acute loss of brain function, including a period of confusion, a loss of consciousness (LOC), focal ...

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) ...