Name: isolation and cannulation of cerebral parenchymal arterioles
Uploaded: 2016-05-23T14:00:00.000+00:00
Duration: 9 min 49 s
Description: Watch this Scientific Journal Video about Isolation and Cannulation of Cerebral Parenchymal Arterioles at JoVE.com

Funded by NHLBI R01HL091905 (SE), the United Leukodystrophy Foundation CADASIL research grant (FD) and AHA 15POST247200 (PWP). The authors would like to thank Samantha P. Ahchay for providing the image on Figure 1, and Dr. Gerry Herrera, Ph.D., for providing critical comments on the manuscript.

1.插管和商会准备<ol><li>插入清洁硼硅玻璃毛细管（外径:1.9毫米;内径:1.1毫米;在长度为10毫米）浸入吸管拉出器与铂丝（直径:100微米）的凹槽。 </li><li>利用适当的设置，拉毛细管产生具有使用微量拉马一个细长尖（ 图2）的插管。使用的设置是:热火 - 700，拉 - 100，速度 -​​ 50，时间 - 10。 </li><li>插入套管插入压力肌动描记室的保持器。适当地对准套管。 </li><li>通过使用解剖显微镜至所需直径下的镊子小心打破插管的尖端。这里，我们表明，用10微米的尖端的套管（ 图2）。 </li><li>填充含1.8毫米的Ca 2+人工脑脊液（学联）两种插管;填充的 Ca 2+ -free脑脊液腔室补充有1％牛血清白蛋白（BSA）+10μM的地尔硫卓（所有解决方案应该在实验开始前新鲜制备）。根据室的尺寸，更改学联的5到20ml之间的容积。储存在4ᵒC中的腔，直到准备开始插管。 注:地尔硫卓是可逆的L-型电压门控钙抑制剂，这将引起小动脉的扩张，这有利于插管</li></ol> 2.实质小动脉的分离<ol><li>安乐死使用标准协议在由该机构的动物护理委员会批准的实验室解剖前立即给动物。使用巴比妥或二氧化碳是优选的，因为其它常用麻醉药可以具有在脑循环15血管扩张作用。这里显示的所有动物的程序已通过机构动物护理和医学内华达大学医学院使用委员会，并在股份公司reement与卫生"指导原则对动物的护理和使用"全国学院。 </li><li>用二氧化碳安乐死的动物。斩首之前，确保动物已经停止了呼吸，捏爪子时没有反应。 </li><li>斩首使用锋利的剪刀（鼠标）或断头台（鼠）动物。除去从动物的头在皮肤和沿着用细尖剪刀中线颅缝切割。使用钝钳（鼠标）或骨钳小心地取出颅骨和揭露大脑​​。慢慢地，精心为不损伤表面的软脑膜动脉中取出大脑。放置脑入填充用20ml 的 Ca 2+ -free脑脊液的补充有在冰上的1％BSA的容器。 </li><li>填充含有用冰冷的Ca2 + -free脑脊液垫补充有1％BSA的一个解剖盘和带朝上这个菜（ 图2A）腹面传送大脑。将这道菜dissec下婷显微镜（ 图3A）。针脚大脑用27号针头垫。要小心，以避免将引脚附近的大脑中动脉（MCA）。 </li><li>本地化威利斯和MCA从中分支的圈子。使用锋利对准Vannas弹簧剪刀，剪围绕MCA一个小矩形。确保矩形是通过MCA的整个长度接近5毫米宽。矩形的顶部应该是从过去Willis环（近端威利斯， 图2B，箭头的圆圈）分支点。 </li><li>使用Vannas弹簧剪刀，执行一个底切进入组织的深度为约2 - 3毫米。 注意:此部分是实质小动脉良好的隔离至关重要的，它可能需要一些试验，以得到它的权利。如果切口是单据，深足，孤立的实质小动脉会很短;如果切割太深，小动脉会在其分支点突破的时候解剖组织被去除。 </li><li>脑切片的最远端牵制与MCA的解剖盘使用昆虫针向上（从Willis环远端）。做一个切口，浅切软引脚附近，注意不要剪得深入到底层的大脑皮层（否则，引脚将不成立）。 </li><li>仔细抢用小镊子软脑膜的每一面，并开始脱皮从皮质软。如果有必要，扶住MCA，确保周围的软脑膜膜不被损坏。 </li><li>保持脱皮，直到包含MCA的PIA是皮质免费。需要注意的是对剥皮的阻力将增加接近圆的威利斯。在这一点上格外小心，因为较长的实质动脉将在这一地区。 </li><li>一旦软是免费的，仔细地将其放置在与相对的表面的解剖盘垫的顶部以实质朝下（ 图2B）动脉。 </li> &lt;利&gt;使用Vannas弹簧剪刀，切出来自MCA自由解剖动脉，确保该容器的端部直截了当地切断。 </li><li>收集动脉可用于压力肌动描记，分子分析，或天然平滑肌或内皮细胞的分离。 </li></ol> 3.压力肌动描记<ol><li>提前准备缝合。切深绿色尼龙线成5mm片，放置在一个双面胶带上的培养皿的粘性侧。根据解剖显微镜，分离每一块成用细镊子的细丝，和松散耦合通过传递一端长丝到另一个编写一个简单的半顺利结。 </li><li>使用镊子，拿起一个结关双面胶带，确保不要有太大的压力抓住它。拉缝线进入浴溶液中，滑结到插管并在从套管的前端的距离拧紧。重复此过程，对每个插管2缝线。 </li><li>涂层玻璃微量用10％BSA溶液，然后冲洗掉过量的补充有1％BSA的无Ca 2+脑脊液。 </li><li>拉50微升的溶液到玻璃微量，拉起一个实质动脉和传送到压力肌动描记室。推柱塞在一个流体运动来分配实质动脉进入腔室，以防止它粘到玻璃微的内部腔室。 </li><li>使用两个超精细镊子，打开PA的管腔，小心翼翼地只触及容器的尽头。 </li><li>使用镊子举行实质小动脉的边缘，船只小心拉出到插管。拉得足够远的套管，使得所述容器是没有插管（ 图3A）的非常锥形端。 </li><li>拧松套管的2缝线和在容器（ 图3B）将其拧紧。空间缝线拆开一点上的船只，并朝向操作者，而拉tigh10吧。确保任何分支都关闭实质动脉的相反端之前打结。 </li><li>转室周围，并关闭实质动脉的相对端为盲囊。为了实现这一目标，打开对面套管的关系，并把它传递到动脉。慢慢关闭结在于实质动脉将被捆绑到套管的侧这样的方式。避免动脉的纵向拉伸。 （ 图3C）。 注意:如果该研究的目标是通过内腔灌注药，导管插入的相反端，而不是捆扎实质动脉到套管的侧面。确保插管没有任何盐晶体或阻断腔内流内部集结。为了生理水平内保持剪切应力适当调节流量。通过Shih 等。一项研究表明流速内部大鼠8穿透动脉，并且可以作为圆周率的初始导很多研究。 </li><li>仔细腔室转移到显微镜的阶段也可以使用用于记录血管直径。腔室附着到显微镜阶段，连接测温探头和入口和出口用于灌注到正确的管中。加压动脉至40毫米汞柱小鼠实质动脉腔内压力或50毫米汞柱的大鼠动脉，使用可用一个压力系统在实验室中（水柱，耦合到压力传感器蠕动泵等 ）。 图4D示出了一个例子联接到压力换能器蠕动泵加压管状动脉。 </li><li>转用于检测在直径（ 图3E）的变化在系统上。调整诸如照明和对比度显微镜的设置，以获得最佳的检测成为可能。理想的是，小动脉的壁应该是暗和管腔半透明。一旦检测是合适的，开始录音笔他实验。 </li><li>启动灌流系统。 5毫升/分钟-用含有1.8mM的钙为15至20分钟，在3之间的流速温暖（37ᵒ℃）脑脊液洗插管，加压实质动脉。这学联不应包含地尔硫卓或BSA。 </li><li>用60毫米氯化钾脑脊液取代常规脑脊液来评价制剂的可行性。此时功率放大器应具有10 - 20％缩窄至60毫米氯化钾。如果动脉不收缩到那种程度，删除和导管插入另一个动脉。 </li><li>洗出60毫米氯化钾定期学联。等到一代自发生肌基调，这可能需要长达一个小时。如果动脉没有被那么有生肌基调，与其他动脉代替它。 注:生肌音被观察为一个渐进的，有时慢，还原在容器的内腔直径无刺激由收缩激动剂或高KCl溶液。生肌音由以下F计算ormula:（1 - （活性管腔直径/被动管腔直径））×100 5生肌色调的合适量可以根据治疗，株，种，转基因等变化。在一般情况下，生理生肌色调范围为15 - 30％5。 </li></ol> 4.示例压力肌动描记实验:激动剂诱导的收缩和肌反应<ol><li>制备一系列使用适当浓度在脑脊液选择的激动剂稀释。对于当前的实施例使用了血栓素A 2受体激动剂U46619。制备以1mM的浓度为1毫升U46619的储备溶液。转移100微升1mM的溶液到含有900微升脑脊液，以使药物的10倍稀释，得到在含有100μM的U46619的溶液的新管。重复此过程6稀释，从1毫米到100纳米，制备浓度 - 响应曲线大蟒。 </li><li>加上含有激动剂（1毫升首次剂量，随后900微升所有后续剂量的）至100毫升灌流脑脊液的溶液的总体积。这将导致该激动剂的额外稀释100倍。因此，最终浓度激动剂在浴中范围从10点到10微米。允许动脉孵育〜在每个浓度10分钟，直到管腔直径达到稳态。 </li><li>通过灌流与脑脊液功率放大器没有任何药物之前，PA的直径是回原始值洗出激动剂。孵育补充有10μM的地尔硫卓+ 2mM的EGTA中的 Ca 2+ -free脑脊液实质动脉（无BSA）中以诱导最大扩张并记录动脉的被动直径。 </li><li>小心地拉出，同时保持到关系的结束卸下套管实质小动脉。双去离子水冲洗容器室，清除杂物和多余的激动剂。填写与CA室2+ +学联BSA和导管插入另一个动脉。它不推荐执行每动脉多于一个的实验。 </li><li>为了执行生肌反应性的实验中，允许直到产生自发肌音（如上所述）的实质动脉在40毫米汞柱腔内压力平衡。降低腔内压力至5毫米汞柱，并允许PA以平衡〜5 - 10分钟，直到管腔直径达到稳定状态。 （在20毫米汞柱的增量例如从5到140毫米汞柱），使用所选择的时间间隔增加了腔内压力逐步。 </li><li>不要使动脉低于5毫米汞柱腔内压力，因为这可能导致崩溃和破坏血管内皮细胞，从而改变了实验的结果。 </li><li>在压力曲线的末端，superfuse用无Ca 2+脑脊液（无BSA）补充有10μM的地尔硫卓+ 2mM的EGTA实质动脉和重复的压力的步骤，开始于洛斯Ť压力。 注意:这将得到PA的被动直径，这是必要的，以计算％生肌音，由下式定义:％色调=（1 - （有源直径/被动直径））×100。 </li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SKCa+and+IKCa+Channels,+myogenic+tone,+and+vasodilator+responses+in+middle+cerebral+arteries+and+parenchymal+arterioles:+effect+of+ischemia+and+reperfusion.">SKCa and IKCa Channels, myogenic tone, and vasodilator responses in middle cerebral arteries and parenchymal arterioles: effect of ischemia and reperfusion.</a> Stroke. 40, 1451-1457 (2009).</li></ol>

The authors have nothing to disclose.

脑实质动脉是高抗动脉与灌注不同神经元的人口数吻合和分支。这些专门的血管是通过星形胶质细胞介导的血管扩张1脑血管自动调节和神经血管耦合中央的球员 ​​。这些专业血管脑血管疾病的重要性已经知道了约50年的时候米勒费希尔博士的开创性工作，在高血压患者验尸 16的大脑描述结构变化的实质动脉腔隙性梗死的领土内。这些改变，耦合到功能障碍，可引起的深?...

脑循环被唯一组织支持中枢神经元，即具有有限能量存储并因此在氧气压力和所需的营养供给的变化高度敏感的细胞的代谢需求。当执行特定任务特定的神经元亚群变得活跃，脉管促进灌注高度局部增加以防止局部缺氧和营养物1的耗竭。这是被称为神经血管耦合官能充血的一种形式，并且是依赖于血管神经单元的正确操作，活性神经元，星形胶质细胞和脑动脉2构成。脑实质动脉，一组的血管包含实质，穿透和预毛细管小动脉，是这种响应集中重要，这是再临界单独研究它们为了研究神经血管耦合3。 &lt;p系列="jove_content"&gt;实质小动脉小（20 - 70微米内径）高电阻血管灌注在大脑中不同的神经元的人群。从表面上的软脑膜血管分支出来，实质动脉渗入脑实质在近90ᵒ角喂地下微循环（ 图1）。这些动脉在维持适当的灌注压，因为它们是最前端平滑含有肌肉血管保护的毛细管的关键作用。与此相反的表面软脑膜循环，实质动脉缺乏侧支和吻合，因此是脑循环4的"瓶颈"。其结果是，实质小动脉功能障碍有助于脑血管疾病的发展，如血管性认知障碍和小缺血性中风（也称为沉默或腔隙性脑梗塞）。研究indicatË是实质小动脉功能障碍可以通过原发性高血压5，慢性应激6诱导，并且是小血管病变的基因小鼠模型7的早期事件。大鼠单穿透动脉进一步，实验诱导的阻塞足以使小缺血性中风是圆柱形的，类似那些在年龄较大的人8观察。 除了这些解剖优，调节收缩功能的机制脑膜动脉和实质动脉之间不同。生肌血管收缩是更大实质动脉9，可能是因为在细胞内Ca缺乏外在支配10，机械传导11的不同的模式，和差异2+在血管平滑肌细胞中的信号12,13。有证据表明，内皮依赖性血管舒张机制也是这些vascu之间不同与扩散因子如一氧化氮和前列环素14相比拉尔段，表现出对涉及钙机制的更大依赖实质激活的动脉血管壁内的K +通道和电紧张的沟通。因此，收集的数据使用的软脑膜动脉可能不一定适用于实质小动脉的实验，让我们的脑灌注的局部控制知识的差距。 尽管它们的重要性，实质小动脉有很大的不足的研究，主要是由于隔离和安装用于体外研究的技术挑战。在这个手稿，我们描述的方法来分离和导管插入脑实质动脉，其可以用于压力肌动描记，或为免疫标记，电生理学，分子生物学和生物化学分析隔离组织。

<table><tbody><tr><td colspan="4">&lt;strong&gt;Artificial Cerebrospinal Fluid&lt;/strong&gt;</td></tr><tr><td>NaCl</td><td>Fisher Scientific</td><td>S-640</td><td></td></tr><tr><td>KCl</td><td>Fisher Scientific</td><td>P217</td><td></td></tr><tr><td>MgCl Anhydrous</td><td>Sigma-Aldrich</td><td>M-8266</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Fisher Scientific</td><td>S233</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S9638</td><td></td></tr><tr><td>D-(+)-Glucose</td><td>Sigma-Aldrich</td><td>G2870</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>C4901</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A9647</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Isolation/Cannulation&lt;/strong&gt;</td></tr><tr><td>Stereo Microscope</td><td>Olympus</td><td>SZX7</td><td></td></tr><tr><td>Super Fine Forceps</td><td>Fine Science Tools</td><td>11252-00</td><td></td></tr><tr><td>Vannas Spring Scissors</td><td>Fine Science Tools</td><td>15000-00</td><td></td></tr><tr><td>Wiretrol 50 &amp;mu;l</td><td>VWR Scientific</td><td>5-000-1050</td><td></td></tr><tr><td>0.2 &amp;mu;m Sterile Syringe Filter</td><td>VWR Scientific</td><td>28145-477</td><td></td></tr><tr><td>Micropipette Puller</td><td>Sutter Instruments</td><td>P-97</td><td></td></tr><tr><td>Borosilicate Glass O.D.: 1.2 mm, I.D.: 0.68 mm</td><td>Sutter Instruments</td><td>B120-69-10</td><td></td></tr><tr><td>Dark Green Nylon Thread</td><td>Living Systems Instrumentation</td><td>THR-G</td><td></td></tr><tr><td>Linear Alignment Single Vessel Chamber</td><td>Living Systems Instrumentation</td><td>CH-1-LIN</td><td></td></tr><tr><td>Pressure Servo Controller with Peristaltic Pump</td><td>Living Systems Instrumentation</td><td>PS-200</td><td></td></tr><tr><td>Video Dimension Analyzer</td><td>Living Systems Instrumentation</td><td>VDA-10</td><td></td></tr><tr><td>Four Channel Recorder with LabScribe 3 Recording and Analysis Software</td><td>Living Systems Instrumentation</td><td>DAQ-IWORX-404</td><td></td></tr><tr><td>Heating Unit</td><td>Warner Instruments</td><td>64-0102</td><td></td></tr><tr><td>Automatic Temperature Controller</td><td>Warner Instruments</td><td>TC-324B</td><td></td></tr></tbody></table>

isolation and cannulation of cerebral parenchymal arterioles

脑实质动脉（功率放大器），其包括实质小动脉，穿透动脉和预毛细管小动脉，是高电阻的血管从脑膜动脉和小动脉和潜水分支出来进入脑实质。个人PA灌注实质的离散圆柱形领土和其中所包含的神经元。这些小动脉脑血流量在全球（脑血管自动调节），并在本地（功能性充血）的调节中的球员。功率放大器是神经血管单元，一个相匹配的大脑内局部血流到代谢活性结构的一部分，并且还包括神经元，的interneurons，和星形胶质细胞。通过功率放大器灌注直接链接到该特定领土神经元和神经元代谢铅增加的活性，造成进料的PA的扩张局部灌注的增大。 PA的调控从更好的特点，不同软脑膜动脉。压力诱导的血管收缩更大的功率放大器和血管舒张机制有所不同。此外，功率放大器不会从血管周围神经受到外在的支配 - 神经支配是通过与星形胶质细胞endfeet内在联系和本质上是间接的。因此，通过使用软脑膜血管研究积累关于收缩调节数据并不直接转化为理解的PA的功能。此外，病理状态，如高血压，糖尿病，如何影响PA结构和反应性仍然未定。这方面的知识缺口部分的有关PA隔离和插管技术困难的结果。在这个手稿中，我们提出了隔离和啮齿动物功率放大器插管的协议。此外，我们显示，可以与这些小动脉，包括激动剂诱导收缩和肌原反应进行的实验的例子。虽然这个手稿的重点是PA插管和压力肌动描记，PA隔离s时，可以也可以用于生物化学，生物物理，分子，和成像研究。

图5A示出鼠标PA的60毫米氯化钾脑脊液一个代表收缩来评价制剂的完整性。在60毫米氯化钾存在30％ - 功率放大器应15之间收缩。如果收缩低于15％，则丢弃该PA和导管插入另一个之一，因为它表明，在分离和插管过程中动脉被损坏。 图5B说明了增加血栓素的浓度2类似物U-46619（晚上10时至1μM）到?...

Watch this Scientific Journal Video about Isolation and Cannulation of Cerebral Parenchymal Arterioles at JoVE.com

Isolation and Cannulation of Cerebral Parenchymal Arterioles

This manuscript describes a simple and reproducible protocol for isolation of intracerebral arterioles (a group of blood vessels encompassing parenchymal arterioles, penetrating arterioles and pre-capillary arterioles) from mice, to be used in pressure myography, immunofluorescence, biochemistry, and molecular studies.

分离及脑实质小动脉插管

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance ...

isolation-and-cannulation-of-cerebral-parenchymal-arterioles

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

11.6K 次观看.  University of Nevada School of Medicine. This manuscript describes a simple and reproducible protocol for isolation of intracerebral arterioles (a group of blood vessels encompassing parenchymal arterioles, penetrating arterioles and pre-capillary arterioles) from mice, to be used in pressure myography, immunofluorescence, biochemistry, and molecular studies.

视频: Isolation and Cannulation of Cerebral Parenchymal Arterioles

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow (ischemia) 1. Middle cerebral artery (MCA) delivers a great majority of blood to the lateral surface of the cortex 2, is the most common site of human stroke 3, and ischemia within its territory can result in extensive dysfunction or death 1,4,5. Survivors of ischemic stroke often suffer loss or disruption of motor capabilities, sensory deficits, and infarct. In an effort to capture these key characteristics of stroke, and thereby develop effective treatment, a great deal of emphasis is placed upon animal models of ischemia in MCA.
Here we present a method of permanently occluding a cortical surface blood vessel. We will present this method using an example of a relevant vessel occlusion that models the most common type, location, and outcome of human stroke, permanent middle cerebral artery occlusion (pMCAO). In this model, we surgically expose MCA in the adult rat and subsequently occlude via double ligature and transection of the vessel. This pMCAO blocks the proximal cortical branch of MCA, causing ischemia in all of MCA cortical territory, a large portion of the cortex. This method of occlusion can also be used to occlude more distal portions of cortical vessels in order to achieve more focal ischemia targeting a smaller region of cortex. The primary disadvantages of pMCAO are that the surgical procedure is somewhat invasive as a small craniotomy is required to access MCA, though this results in minimal tissue damage. The primary advantages of this model, however, are: the site of occlusion is well defined, the degree of blood flow reduction is consistent, functional and neurological impairment occurs rapidly, infarct size is consistent, and the high rate of survival allows for long-term chronic assessment.

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

We describe a highly reproducible method for the permanent occlusion of a rodent major cerebral blood vessel. This technique can be accomplished with very little peripheral damage, minimal blood loss, a high rate of long-term survival, and consistent infarct volume commensurate with the human clinical population.

永久性脑血管闭塞<em&gt;通过</em&gt;双人结扎横断

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow ...

科研

医学

Comprehensive Endovascular and Open Surgical Management of Cerebral Arteriovenous Malformations

Arteriovenious malformations (AVMs) are associated with significant morbidity and mortality, and have a rupture risk of ~3% per year. Treatment of AVMs must be tailored specifically to the lesion, with surgical resection being the gold standard for small, accessible lesions. Pre-operative embolization of AVMs can reduce nidal blood flow and remove high-risk AVM features such as intranidal or venous aneurysms, thereby simplifying a challenging neurosurgical procedure. Herein, we describe our approach for the staged endovascular embolization and open resection of AVMs, and highlight the advantages of having a comprehensively trained neurovascular surgeon leading a multi-disciplinary clinical team. This includes planning the craniotomy and resection to immediately follow the final embolization stage, thereby using a single session of anesthesia for aggressive embolization, and rapid resection. Finally, we provide a representative case of a 22-year-old female with an unruptured right frontal AVM diagnosed during a seizure workup, who was successfully treated via staged embolizations followed by open surgical resection.

Surgery is the gold standard for accessible arteriovenious malformations (AVMs), and pre-operative embolization can simplify this procedure. We describe our approach for staged endovascular embolization and open resection of AVMs, and provide a representative clinical example highlighting the advantages of a comprehensively trained neurovascular surgeon leading a multi-disciplinary clinical team.

脑动静脉畸形的综合血管内和开放性手术治疗

Arteriovenious malformations (AVMs) are associated with significant morbidity and mortality, and have a rupture risk of ~3% per year. Treatment of AVMs ...

神经科学

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier (BBB). Rats with induced ischemic stroke were established and used as in vivo models to investigate the functional integrity of the BBB. Spectrophotometric detection of Evans blue (EB) in the brain samples with ischemic injury could provide reliable justification for the research and development of novel therapeutic modalities. This method generates reproducible results, and is applicable in any laboratory without a need for special equipment. Here, we present a visualized and technical guideline on the detection of the extravasation of EB following induction of ischemic stroke in rats.

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

The overall goal of this procedure is to provide a highly reproducible technique for in vivo assessment of the blood-brain barrier disruption in rat models of ischemic stroke.

一种体内评价大鼠缺血性中风模型中血脑屏障的破坏

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier ...

Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other vessels), and describes techniques for evaluating tissue perfusion using Laser Doppler Flowmetry (LDF) and microvessel density utilizing fluorescently labeled Griffonia simplicifolia (GS1) lectin. Current methods for studying isolated resistance arteries at transmural pressures encountered in vivo and in the absence of parenchymal cell influences provide a critical link between in vivo studies and information gained from molecular reductionist approaches that provide limited insight into integrative responses at the whole animal level. LDF and techniques to selectively identify arterioles and capillaries with fluorescently-labeled GS1 lectin provide practical solutions to enable investigators to extend the knowledge gained from studies of isolated resistance arteries. This paper describes the application of these techniques to gain fundamental knowledge of vascular physiology and pathology in the rat as a general experimental model, and in a variety of specialized genetically engineered &#34;designer&#34; rat strains that can provide important insight into the influence of specific genes on important vascular phenotypes. Utilizing these valuable experimental approaches in rat strains developed by selective breeding strategies and new technologies for producing gene knockout models in the rat, will expand the rigor of scientific premises developed in knockout mouse models and extend that knowledge to a more relevant animal model, with a well understood physiological background and suitability for physiological studies because of its larger size.

This manuscript describes in vitro video microscopy protocols for evaluating vascular function in rat cerebral resistance arteries. The manuscript also describes techniques for evaluating microvessel density with fluorescently labeled lectin and tissue perfusion using Laser Doppler Flowmetry.

利用大鼠离体阻力动脉的视频显微镜对血管控制机制的评价

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other ...

Ex Vivo Pressurized Hippocampal Capillary-Parenchymal Arteriole Preparation for Functional Study

From subtle behavioral alterations to late-stage dementia, vascular cognitive impairment typically develops following cerebral ischemia. Stroke and cardiac arrest are remarkably sexually dimorphic diseases, and both induce cerebral ischemia. However, progress in understanding the vascular cognitive impairment, and then developing sex-specific treatments, has been partly limited by challenges in investigating the brain microcirculation from mouse models in functional studies. Here, we present an approach to examine the capillary-to-arteriole signaling in an ex vivo hippocampal capillary-parenchymal arteriole (HiCaPA) preparation from mouse brain. We describe how to isolate, cannulate, and pressurize the microcirculation to measure arteriolar diameter in response to capillary stimulation. We show which appropriate functional controls can be used to validate the HiCaPA preparation integrity and display typical results, including testing potassium as a neurovascular coupling agent and the effect of the recently characterized inhibitor of the Kir2 inward rectifying potassium channel family, ML133. Further, we compare the responses in preparations obtained from male and female mice. While these data reflect functional investigations, our approach can also be used in molecular biology, immunochemistry, and electrophysiology studies.

The present manuscript details how to isolate hippocampal arterioles and capillaries from the mouse brain and how to pressurize them for pressure myography, immunofluorescence, biochemistry, and molecular studies.

Ex Vivo 加压希马卡毛细管- 功能研究准备

From subtle behavioral alterations to late-stage dementia, vascular cognitive impairment typically develops following cerebral ischemia. Stroke and ...

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the choroidal vessels supply the photoreceptors. Alterations in retinal perfusion contribute to numerous sight-threatening disorders, including diabetic retinopathy, glaucoma and retinal branch vein occlusions. Understanding the molecular mechanisms involved in the control of blood flow through the retina and how these are altered during ocular disease could lead to the identification of new targets for the treatment of these conditions. Retinal arterioles are the main resistance vessels of the retina, and consequently, play a key role in regulating retinal hemodynamics through changes in luminal diameter. In recent years, we have developed methods for isolating arterioles from the rat retina which are suitable for a wide range of applications including cell physiology studies. This preparation has already begun to yield new insights into how blood flow is controlled in the retina and has allowed us to identify some of the key changes that occur during ocular disease. In this article, we describe methods for the isolation of rat retinal arterioles and include protocols for their use in patch-clamp electrophysiology, calcium imaging and pressure myography studies. These vessels are also amenable for use in PCR-, western blotting- and immunohistochemistry-based studies.

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

This manuscript describes a straightforward protocol for the isolation of arterioles from the rat retina that can be used in electrophysiological, calcium imaging and pressure myography studies.

体外细胞生理学研究中视网膜微动脉的分离

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the ...

生物学

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.
Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 &#176;C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial &#34;tubes&#34; are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

新鲜分离和内联小鼠脑内皮细胞内钙和膜电位的同时测量

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...

Optimization of the Longa Middle Cerebral Artery Occlusion Method for Complete Reperfusion

The middle cerebral artery occlusion model serves as the primary animal model for studying ischemic stroke. Despite being used in research for over three decades, its standardization remains inadequate. Predominantly conducted on rats and mice, the procedure poses challenges due to mice's smaller and more fragile nature. Unlike the Koizumi common carotid artery method, the Longa external carotid artery is the sole intraluminal filament stroke model ensuring complete reperfusion post-ischemia. This aspect holds critical significance for studies investigating reperfusion phenomena. The surgical modifications demonstrated in this article ensure continuous blood flow from the common carotid artery throughout the ischemic phase and after the reperfusion onset. The goal of these modifications is to selectively occlude the middle cerebral artery by keeping the perfusion uninterrupted in branches proximal to the middle cerebral artery during the ischemia period. Furthermore, the onset of reperfusion is sudden and can be precisely controlled, thereby modeling endovascular thrombectomy in human medicine more accurately. Our aim in presenting this comprehensive video article is to ease the training of new surgeons and promote the standardization of surgical procedures within the scientific community.

This protocol presents a step-by-step guide for researchers to perform the middle cerebral artery occlusion procedure on mice using the modified Longa external carotid artery method. Modifications presented in this article aim to increase the accuracy of middle cerebral artery occlusion and ensure complete reperfusion.

Longa 大脑中动脉闭塞方法的优化

The middle cerebral artery occlusion model serves as the primary animal model for studying ischemic stroke. Despite being used in research for over three ...

Isolation and Functional Analysis of Arteriolar Endothelium of Mouse Brain Parenchyma

Cerebral blood flow is conveyed by vascular resistance arteries and downstream parenchymal arterioles. Steady-state vascular resistance to blood flow increases with decreasing diameter from arteries to arterioles that ultimately feed into capillaries. Due to their smaller size and location in the parenchyma, arterioles have been relatively understudied and with less reproducibility in findings than surface pial arteries. Regardless, arteriolar endothelial cell structure and function&#8212;integral to the physiology and etiology of chronic degenerative diseases&#8212;requires extensive investigation. In particular, emerging evidence demonstrates that compromised endothelial function precedes and exacerbates cognitive impairment and dementia.
In the parenchymal microcirculation, endothelial K+ channel function is the most robust stimulus to finely control the spread of vasodilation to promote increases in blood flow to areas of neuronal activity. This paper illustrates a refined method for freshly isolating intact and electrically coupled endothelial &#34;tubes&#34; (diameter, ~25 &#181;m) from mouse brain parenchymal arterioles. Arteriolar endothelial tubes are secured during physiological conditions (37 &#176;C, pH 7.4) to resolve experimental variables that encompass K+ channel function and their regulation, including intracellular Ca2+ dynamics, changes in membrane potential, and membrane lipid regulation. A distinct technical advantage versus arterial endothelium is the enhanced morphological resolution of cell and organelle (e.g., mitochondria) dimensions, which expands the usefulness of this technique. Healthy cerebral perfusion throughout life entails robust endothelial function in parenchymal arterioles, directly linking blood flow to the fueling of neuronal and glial activity throughout precise anatomical regions of the brain. Thus, it is expected that this method will significantly advance the general knowledge of vascular physiology and neuroscience concerning the healthy and diseased brain.

Intensive preparation of intact mouse cerebral endothelial &#34;tubes&#34; from cerebral parenchymal arterioles is illustrated for studying cerebral blood flow regulation. Further, we demonstrate the experimental strengths of this endothelial study model for fluorescence imaging and electrophysiology measurement of key cellular signaling pathways, including changes in intracellular [Ca2+] and membrane potential.

小鼠脑实质小动脉内皮的分离及功能分析

Cerebral blood flow is conveyed by vascular resistance arteries and downstream parenchymal arterioles. Steady-state vascular resistance to blood flow ...

Preparing Cerebral Endothelial Tubes from Mouse Parenchymal Arterioles

Source: Hakim, M. A., et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/63463">Isolation and Functional Analysis of Arteriolar Endothelium of Mouse Brain Parenchyma.</a> J. Vis. Exp. (2022)
This video demonstrates a method for isolating the endothelial tubes from mouse cerebral parenchyma. Through a combination of enzymatic digestion and mechanical dissociation, the process yields an intact tube with endothelial cells.

从小鼠实质小动脉制备脑内皮管

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 
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