我们感谢并承认在英国 ADC 脑组织银行的彼得纳尔逊博士和索尼娅. 安德森提供了所有人脑组织样本 (NIH 赠款号: 国立老龄研究所的 P30 AG028383)。我们感谢马特哈兹达和汤姆. 杜兰, 信息技术服务, 学术技术和教师参与, 肯塔基大学的图形协助。该项目由国家神经系统疾病和中风 (1R01NS079507) 和1R01AG039621 国家老龄研究所的赠款编号 (a.m.s. H) 支持。内容完全是作者的责任, 不一定代表国家神经系统疾病和中风研究所或国家老龄研究所的官方意见。作者声明没有竞争的财政利益。

下面的信息是基于目前的安全和管理标准在肯塔基大学, 列克星敦, 肯塔基州, 美国。作为一种安全预防措施, 请参考该机构的生物安全计划和最新的法规和建议, 然后再与人体组织合作。
注意: 人体组织可以是血液传播病原体的来源, 包括人体免疫机能丧失病毒 (HIV)、乙肝病毒 (HBV)、丙型肝炎病毒 (HCV) 等。与人体组织一起工作会造成血液传播病原体感染的风险。因此, 在与人体组织合作保护实验室人员时, 一定要考虑到一些规章制度和安全问题。与美国的人体组织合作需要一个生物安全2级实验室以及安全预防措施和培训, 按照 NIH 科 IV-B-7, 1970年 5 (a) (1) 条的 OSHA 法案和用户的机构生物安全计划。一般而言, 必须在进行任何涉及人体材料 (组织、体液) 的研究之前获得机构生物安全委员会和/或机构审查委员会的批准。所有从事人体材料工作的人员都需要进行培训, 包括基本的实验室安全培训,例如化学卫生和实验室安全, 以及关于生物安全、危险废物和人类的具体培训。血液传播的病原体。所有使用人体材料的人员都强烈建议在使用人体材料之前获得乙肝疫苗接种。人员需要佩戴特定的个人防护设备, 同时使用人体材料, 例如, 袖口的实验室大衣和面罩, 并随时佩戴手套。所有工作都在生物安全柜 (2 类) 中进行。所有与人体材料接触的设备和从人体材料中产生的任何废料都应妥善处理, 以防止人员受到污染和/或感染。所有设备和表面清洗10% 漂白剂和75% 乙醇后, 每一个程序涉及人体材料。必须立即清理与人体材料的泄漏。玻璃器皿每次使用后都要蒸压。废物, 包括未固定的人体组织, 是收集在一个标签的生物危害废物袋和蒸压。锐器收集在一个穿刺和防漏容器标记为 biohazardous。所有从人体材料中产生的废料都是根据该机构的生物安全规定处置的。
注: 我们的实验室通过桑德斯-棕褐色中心 (IRB #B15-2602 米) 获得来自已故个体的新鲜额皮质样本。纳入标准是: 在英国的招生-ADC 纵向解剖队列研究和验尸间隔 (PMI) ≤4 h64。验尸遵循标准协议, 所有程序符合 NIH Biospecimen 最佳做法准则65。短的 PMI 低于4小时是最重要的, 以确保毛细血管存活后的隔离。新鲜和冰冻的组织都可以使用。如果需要冷冻, 新鲜获得的人脑组织应在液氮中休克冷冻, 储存在-80 摄氏度。新鲜或解冻的组织应存储在隔离缓冲 (见下文) 和处理迅速。我们发现, 10 克的新鲜人体组织产生约100毫克的脑毛细血管 (湿重)。
1. 设置
<ol><li>
缓冲准备 注意: 所需的缓冲量取决于组织的数量。以下协议中的所有缓冲卷都基于 10 g 的人脑皮质组织。<ol>
<li>L 隔离缓冲: 使用 Dulbecco 磷酸盐缓冲盐水的1.5 升 (DPBS; 2.7 毫米氯化钾, 1.47 毫米的2PO4, 136.9 毫米氯化钠, 8.1 毫米 Na2HPO4, 0.9 毫米 CaCl2, 0.49 毫米氯化镁2) 和补充与5毫米 d-葡萄糖 (1.35 g) 和1毫米丙酮酸钠 (0.165 克)。添加葡萄糖和丙酮酸后, 用氢氧化钠调节 pH 值7.4。冷却和存储缓冲区到4°c 之前使用。</li>
		<li>牛血清白蛋白 (bsa): 添加10克 BSA 粉到1升的隔离缓冲, 最后的 BSA 浓度为1%。搅拌缓慢, 以避免气泡, 调整到 pH 值 7.4, 并存储在4摄氏度过夜。立即使用前, 轻轻搅拌;避免形成气泡以避免白蛋白变性。</li>
		<li>密度梯度介质: 将浓度为18克的密度梯度介质放入玻璃瓶中, 加入一个磁搅拌杆。加入60毫升的隔离缓冲液, 用力摇动5分钟, 直到所有粉末悬浮。隔夜存放在4摄氏度, 允许密度梯度介质溶解。使用前先搅拌10分钟。</li>
		<li>将所有缓冲区存储在4摄氏度;在整个隔离过程中, 将所有工具和缓冲放在冰上。使用前请先搅拌所有缓冲器。</li>
	</ol>
</li>
	<li>
实验设置
	<ol>
<li>将波特-Elvehjem 组织磨床的杵装在电子架空搅拌器上。将波特-Elvehjem 组织磨床和 Dounce 均质机与杵在冰盖下。准备一个300µm 过滤网 (5 x 5 厘米2), 折叠它到一个锥, 并插入和附加到50毫升猎鹰管带胶带 (图 1A)。</li>
		<li>放置连接环和细胞应变过滤器 (孔隙大小:30 µm) 在50毫升猎鹰管。准备 biohazardous 垃圾袋。将所有必需的设备放在安全柜中 (见材料表)。</li>
	</ol>
</li>
</ol>2. 脑标本准备
注:图 1A显示了下面描述的整个隔离过程的工作流图表。人脑组织可以从皮层的任何部分中产生, 可以被新鲜或冰冻使用。冷冻脑组织可在室温下解冻 (无缓冲; ~ 30 分钟10克)。为了达到可比较的结果, 大脑组织应该从同一脑区获得每个实验。该协议是为新鲜的 (PMI < 4 小时) 的人大脑皮层没有被冻结的优化。
<ol><li>人脑组织的制备: 记录脑组织的重量。以下协议中的所有数字适用于10克新鲜人脑组织。将脑组织放置在100毫米培养皿中。小心地取出所有的脑膜与镊子。用手术刀切断白色物质。</li>
	<li>切碎的人脑组织: 小心地切断脑组织和碎它与手术刀。剁碎约5分钟 (2–3毫米片)。将脑组织转移到波特-Elvehjem 组织磨床上。添加30毫升的隔离缓冲。 注: 切碎的组织片很难看到, 因为大脑组织通过粉碎过程变成糊状。</li>
</ol>3. 同质化
<ol><li>波特-Elvehjem 组织磨床 (清除: 150–230µm): 融汇每个样品与100冲程在一个均质机速度 50 rpm。记录每25次笔画的时间和100笔画所需的总时间。见表 1所提议的均匀化协议;人额皮质均质10克的总时间约为22分钟。不要在空气中搅拌以防止气泡。</li>
	<li>Dounce 均质机 (游隙: 80–130µm): 将匀浆输送到冰上 Dounce 匀能机上。融汇20冲程 (~ 6 s/冲程, 总2分钟) 的悬浮。避免气泡。</li>
</ol>4. 离心
<ol><li>将脑匀浆均匀分布到四50毫升离心管中, 并记录匀浆的总容积。将50毫升的密度梯度缓冲器分配到离心管 (每管12.5 毫升)。使用10毫升的隔离缓冲液冲洗杵和均质体, 并分配到四离心管 (每管2.5 毫升)。</li>
	<li>用瓶盖紧紧地关闭离心机管。将匀浆、密度梯度介质和缓冲液混合在一起, 用力摇动管子。离心机在 5800 x g为15分钟在4°c (固定角度转子);选择中等减速速度以保持颗粒附着在管上。在2毫升的 1% BSA 中丢弃上清和并用重悬每个颗粒。</li>
</ol>5. 过滤
注意: 要将毛细血管与红细胞和其他细胞碎片分开, 需要采取一些过滤步骤。
<ol><li>300µm 网: 重新悬浮颗粒后, 通过300µm 网格过滤悬浮液。毛细血管通过网格过滤, 而较大的血管和更大的脑碎片留在网格上。仔细清洗网格, 多达50毫升 1% BSA。丢弃网格。 注: 这一过滤步骤清除了任何较大的血管或脑碎片大块的毛细管悬浮。</li>
	<li>
30µM 细胞应变过滤器 注意: 这个过滤步骤将毛细血管与红细胞和其他脑部碎片隔开。<ol>
<li>将毛细管滤液从步骤6.1 分布于五30µm 细胞应变过滤器 (每细胞应变过滤器约10毫升的毛细管滤液)。毛细血管被这种过滤器所控制, 而红细胞、其他单细胞和小的脑碎片通过过滤器并收集在滤液中。</li>
		<li>用25毫升 1% BSA 清洗每个过滤器。然后, 将所有滤液在第六过滤器上, 以增加产量。用50毫升 1% BSA 清洗每个过滤器;保持细胞应变过滤器含有毛细血管, 并丢弃滤液。</li>
	</ol>
</li>
</ol>6. 毛细管收集
<ol><li>将过滤器倒置, 用50毫升 1% BSA 清洗毛细血管, 将每个过滤器变成50毫升管。轻轻地将压力与5毫升 pipettor 的吸管尖端, 并移动它通过过滤器冲洗脑毛细血管。</li>
	<li>一定要洗掉所有的脑毛细血管, 特别是从过滤器的边缘。避免气泡, 因为这使得过滤过程更加困难, 增加了毛细血管损耗的机会。</li>
</ol>7. 洗涤
<ol><li>收集毛细血管后, 离心机所有样品在 1500 x g 3 分钟在4°c (摆动斗转子)。取出上清液并在大约3毫升的隔离缓冲器中重新悬浮颗粒。将所有悬浮颗粒从一个15毫升锥形管中的一个样品中组合, 然后用隔离缓冲器填充。离心机再在 1500 x g 3 分钟在4°c 和洗涤二次。</li>
	<li>用显微镜 (100X 放大) 和照相机 (图 1B) 记录毛细管纯度。 注:10 克人脑组织的脑毛细血管产量通常约为100毫克。分离的脑毛细血管现在可以用于实验, 处理 (e., 裂解, 膜分离), 或被闪光冷冻和储存在-80 °c 在 cryotubes 至少6–12月 (避免多个冻融循环)。</li>
</ol>

作者没有什么可透露的。

本议定书描述了从新鲜组织中分离完整和可行的人脑毛细血管。在本节中, 我们将详细讨论以下内容: 1) 对协议的修改, 2) 常见错误的疑难解答, 3) 技术的局限性, 4) 该模型对于现有的和替代的血脑屏障模型的意义, 以及 5)用于隔离人脑毛细血管的潜在应用。
这里描述的协议是优化 10 g 的新鲜人类额叶皮质组织。然而, 相对简单的修改这个程序为: 1) 或多或少超过10克的组织, 2) 冷冻脑组织, 或 3) 脑组织从大脑区域以外的额叶皮质。首先, 有或多或少超过10克的脑组织, 缓冲的必要体积可以简单地放大或下降到可用数量的组织。因此, 如果只有5克的脑组织可用, 缓冲的体积应该减少一半。第二, 我们描述了一种使用新鲜脑组织的毛细管隔离, 但是如果新鲜的组织不可用, 可以使用冷冻组织38。第三, 我们使用新鲜的脑组织从额叶皮质, 但毛细血管可能是孤立的其他皮质脑区, 如果有足够的可用组织。也可以分离非皮质脑区毛细血管 (e., 白质), 但这些区域有不同的细胞组成和毛细血管密度66,67,68。因此, 使用来自不同大脑区域的组织可能需要调整协议 (例如缓冲体积、梯度介质密度、离心速度和/或过滤步骤数)。
毛细管隔离程序, 虽然不复杂本身, 是敏感的小扰动或修改的协议。修改可能导致毛细血管产量减少或毛细血管活力降低。表2概述隔离过程中遇到的最常见的错误和问题, 并列出提示, 以避免这些错误和解决方案在发生故障时进行疑难解答。与该程序有关的最常见问题是低毛细管产量。毛细血管的损耗通常是每个步骤中小损失的累计总和, 是由于整个过程中的小偏差造成的。一个关键的步骤, 其中大量的毛细血管可能会失去的是密度离心。缓冲区中不正确的浓度会导致不正确的密度, 从而使毛细血管与细胞碎片分离, 从而减少毛细管颗粒的体积。图 3显示了离心步骤中相对于脑匀浆的密度梯度介质过少或太多的后果。调整到正确的浓度可以解决这个问题。请注意, 离心机的加速度和减速速度也会影响脑毛细颗粒的形成。毛细血管可能也会丢失在步骤6–7如果部分毛细血管材料粘在吸管的提示。这个问题可以通过彻底冲洗每个吸管提示, 然后再改变它。在步骤7中, 从细胞应变过滤器中冲洗毛细血管可能不完整, 毛细血管可能粘在过滤器的边缘。这可以通过检查显微镜下的过滤器和其他洗涤步骤来避免。在隔离过程的每一个步骤中, 失去毛细血管会导致一个微不足道的毛细管颗粒或没有足够的毛细管材料进行进一步的加工和实验。
从新鲜的人体组织中分离出脑毛细血管是一种独特的血脑屏障模型, 它与体内的情况相似。但是, 该技术存在一些局限性。一个挑战是是否有新鲜的人体组织。由于最佳 pmi 是≤4 h, 在一个显着的更长的 pmi 收集的大脑组织将不足够新鲜的一些下游应用。在某些情况下, 可能很难获得足够大的组织数量, 足以为多个实验组, 从而限制下游应用。因此, 从啮齿目动物19、犬69、牛42或猪70脑组织中分离出新鲜毛细血管可能更适合于模拟血脑屏障。在解释和发布数据时, 应考虑到影响人脑组织变异性的因素, 如年龄、性别、种族、疾病状态、药物史、样本脑区和 PMI 等。在实验水平上, 重要的是要注意, 孤立的毛细血管仍然包括毛细血管, 但胶质 endfeet 被删除的程序44。需要考虑的是, 这里提出的模型作为血脑屏障 (即毛细血管内皮细胞) 的前体模型, 而不是作为神经血管单元的模型。
与任何人类组织一起工作总是带来固有的安全风险, 研究人员必须在隔离过程中采取适当的预防措施, 以避免感染。具体地说, 在美国, 与人体组织的工作需要指定的实验室空间, 是 BSL 2 认证, 包括一个生物安全柜 (类 A2)。此外, 工作人员还必须使用个人防护设备 (如, 实验室大衣, 手套和面罩), 以及与人体组织合作的指定设备, 并实施 biohazardous 废物处理。实施这些安全措施是费时费力的, 而且增加了程序的难度, 特别是对经验不足的实验室人员来说。
血液脑屏障是高度保守的有机体之间与一个明确定义的中枢神经系统71。人血脑屏障的建模很难, 因为神经血管单元细胞间存在复杂的神经血管耦合。据估计, 成年人脑平均大约有860亿个神经元, 并且认为几乎每个神经元都有自己的毛细血管在附近, 以确保适当的供应与氧气和营养素72,73。毛细血管内皮细胞构成血脑界面的最大表面积 (12–18 m2为健康的成年人类)。紧的连接点是阻碍旁细胞扩散溶质的广泛 pharmacotherapeutics 的障碍。此外, 许多研究描述神经退行性疾病的屏障功能障碍, e., 阿尔茨海默病74, 中风75, 癫痫38,76, 多发性硬化77, 和创伤性脑损伤28,78。因此, 必须建立密切代表人血脑屏障的模型, 以便更好地了解健康和疾病中的屏障功能。
血脑屏障的多体外细胞培养模型存在;为专家审查在题目看见41,49,51,69,79,80。简单地, 两个新的孤立的脑毛细血管内皮细胞的原代培养和永生化脑毛细血管内皮细胞线可用。脑微血管内皮细胞的原代培养主要用于小鼠、大鼠、猪和奶牛。然而, 由于细胞必须新鲜地被隔绝, 主要细胞培养是劳力密集的。永生化的脑毛细血管内皮细胞系可从鼠, 鼠和人, 并减少劳动密集型, 因为它们可以传代长期使用。然而, 即使是永生化的细胞系也限制了它们在失去内皮特性之前传代的频率。主要细胞和永生化细胞系通常培养在板上, 以模型的脑毛细血管内皮和测量屏障通透性和药物转运跨细胞单层, 从而模拟血到脑运输41 ,81,82。这些模型中的培养基也可以用星形胶质细胞、毛细血管或其他生理相关因素 (如41、83、84) 来修改或补充。
永生化细胞系的优势在于它们相对容易获得和可用性。当培养的内皮细胞可以达到汇合, 他们失去内皮细胞的性质, 因为他们并排生长在单层。例如, 培养的 hCMECs 显示减少了转运体的表达, 如 P gp, 紧密连接蛋白, 并显示可变渗透率为外来化合物41。图 4显示了与新鲜隔绝的人毛细血管相比, hCMEC/D3 细胞中 P gp 蛋白表达的西方印迹。尽管总蛋白含量降低了10倍, 但与 hCMEC/D3 细胞相比, 对 P gp 的信号更强。这表明 hCMEC/D3 细胞在培养中丢失了大量的 p-gp 蛋白表达。此外, 在不同的文化媒体, 环境和设备影响屏障完整性的关键措施,即志愿者测量 Transwell 板检测。这些问题中的一些可以通过利用孤立的脑毛细管模型来克服, 这种模式更密切地代表人体血脑屏障。
孤立的脑毛细血管被用于广泛的研究, 包括基因组学, 蛋白质组学, 功能蛋白质组学和 cellomics 研究 (图 5)。此外, 许多技术和方法都存在, 以分析孤立的脑毛细血管在每个领域。值得注意的是,图 5中所示的实验技术可以用于从许多来源 (包括人、牛、啮齿动物和猪组织) 中分离出来的脑毛细血管, 这可能促进翻译研究。例如, 李等。85通过纯化大鼠脑毛细血管分离 mRNA, 研究了血脑屏障的基因组学。另外,等等。86采用 rt-pcr 和 qRT pcr 方法研究 PXR 对 p-gp 的调节。许多蛋白质组学研究利用西方印迹3, 斑点杂交分析87, 简单的西方化验3,38, ELISA88,89, 免疫沉淀3, 和染色3,90,91。为了辨别转运者在脑血管内皮的贩运, 麦卡弗里等。92使用分离的脑毛细血管的亚细胞分馏。皮诺和艾尔。93采用分离的牛内皮细胞膜囊泡, 辨别转运体的位置和跨血脑屏障的转运方向。在其他蛋白质组研究中, 研究人员使用液相色谱和串联质谱定量分析转运蛋白94,95,96。功能蛋白质组学研究利用转运体和渗漏化验2,38。Hartz等。2使用酶谱法测定分离的脑毛细血管中的酶活性。此外, 大量的 cellomic 研究使用细胞培养产生了许多内皮细胞线和模型的血脑屏障82,83,84。使用这些模型, 常用的化验方法包括迁移化验97、98、可行性和毒性化验99和血管生成化验98。
孤立的脑毛细血管可以精确地描述蛋白质的表达和活性, 并描述血脑屏障的信号通路。这在某种程度上归结于脑子的毛细血管含量, 仅大约 1% (v/v)。因此, 用全脑匀浆或脑切片代替纯化的毛细血管内皮细胞, 最有可能导致信噪比差24。此外, 隔离后, 脑毛细血管是可行的至少6小时 (未发表的数据从老鼠和老鼠), 这使得研究, 以辨别特定的信号通路。建议从同一准备中包括一个控制组。
需要对血脑屏障的代表性和平移模型, 如本报告中所讨论的孤立毛细管模型, 来研究健康和疾病中的屏障功能。在这里, 我们提出了一个协议, 以获得一个良好的产量和高质量的孤立的人脑毛细血管, 可以作为一个前体内模型的血脑屏障。孤立毛细血管保留其原有的结构和功能, 允许使用它们进行一些分离后的分子, 生物化学和生理学的化验。在处理人体组织样本时应注意, 最好是在 BSL 2 或更高的环境中进行。

血脑屏障是血液和大脑之间的紧密控制的界面, 它决定了大脑中的什么进入和出来。解剖学上, 内皮细胞构成血脑屏障, 形成一个复杂的连续毛细血管网络。从生理上来说, 这种毛细管网络为大脑提供氧气和营养, 同时处理二氧化碳和代谢废物。重要的是, 证据支持对屏障的改变会导致许多病症, 包括阿尔茨海默病、癫痫和中风1、2、3、4、5,6,7. 脑血管内皮细胞也可以阻断药物对大脑的吸收, 例如肿瘤切除术后化疗多形性胶质瘤,8,9, 10. 在这方面, 孤立的人脑毛细血管代表了一种独特的前体血脑屏障模型, 它与体内的屏障特性密切相关,可以对健康的屏障功能和功能障碍进行研究。和疾病。在这篇文章中, 我们提供了一个协议, 以保持良好的毛细血管质量和产量, 以研究血脑屏障的人脑毛细血管从人脑。
在 1969年, Siakotos 。11是第一个报告使用密度梯度离心和玻璃珠柱分离的牛和人脑组织的脑毛细血管分离。后来,等等。12通过添加多个过滤步骤, 减少了研究大鼠脑毛细血管所需的组织量, 同时保持葡萄糖转运的代谢活动, 改进了这种方法。从那时起, 研究人员对毛细血管隔离程序进行了多次优化, 改进了每次迭代13、14、15的方法和脑毛细管模型。例如, Pardridge等。16分离的牛毛细血管使用酶消化而不是机械均匀化, 然后通过一个毛细管悬浮通过一个210µm 网格过滤器和一个玻璃珠柱。这些修改改善了孤立脑毛细血管的台盼蓝排斥染色, 从而提高了内皮细胞的生存能力。二十世纪九十年代代初, 达赖尔. 17 glutamyl transpeptidase (γ-GTase) 和碱性磷酸酶的分离的牛和大鼠毛细血管, 其神经元污染明显, 维持代谢活性。在 2000年, 米勒和al。18、用分离的大鼠和猪脑毛细血管与共焦显微镜相结合, 显示输送基质在毛细血管腔内的积聚。随后, 我们的实验室继续优化脑毛细血管隔离程序, 我们已经建立了运输化验, 以确定 p-糖蛋白 (p gp)19,20,21, 乳腺癌抗性蛋白 (BCRP)22,23, 多药耐药性蛋白 2 (Mrp2)24运输活动。在 2004年, 我们发表了两份报告, 我们使用孤立的大鼠脑毛细血管调查各种信号通路。在 Hartz等。21, 我们发现肽 endothelin-1 通过内皮素受体 b (ETb) 受体、一氧化氮合酶 (NOS) 和蛋白激酶 C (PKC), 快速、可逆地减少脑毛细血管中的 P gp 转运功能。在鲍尔等。19, 我们显示核受体 pregnane X 受体 (PXR) 的表达, 并显示 PXR 调节的 P gp 表达和传输功能的脑毛细血管。在转基因人性化 PXR 小鼠实验中, 我们扩大了这一研究方向, 并通过 hPXR 活化25对植入 P gp在体内收紧屏障。在 2010年, Hartz 。26使用这种方法来恢复过度表达 tshr 开心的转基因人淀粉样蛋白 (开心) 小鼠的 p-gp 蛋白表达和转运活性。此外, 恢复开心小鼠 p-gp 显著降低淀粉样β (Aβ)40和 Aβ42的大脑水平。
除了研究信号通路, 孤立的脑毛细血管可以用来确定毛细血管通透性的变化, 我们称之为毛细血管渗漏。特别是, 德州红色泄漏检测是用来评估从毛细管腔内的荧光染料的泄漏, 从毛细血管流明, 然后这些数据被用来分析泄漏率。与控制毛细血管相比, 毛细血管渗漏率的增加表明血脑屏障2的物理完整性发生了变化。这是有价值的, 因为有许多疾病状态与障碍干扰, e., 癫痫, 多发性硬化症, 阿尔茨海默病, 创伤性脑损伤27,28,29, 30。其他组也利用孤立的毛细血管来辨别信号通路, 调节蛋白质表达和蛋白质31,32,33,34的传输活动, 35,36,37。最后, 我们继续优化这种方法, 以隔离人脑毛细血管和, 最近, 我们显示增加 P gp 表达在人血脑屏障的癫痫患者相比无癫痫控制个人38.结合起来, 这些发展表明, 孤立的脑毛细血管可以作为一个多功能模型来研究屏障功能。
各种体内、体外、内外血脑屏障模型已应用于基础研究和工业药物筛选, 主要目的是检测药物向大脑的传递39、40、41 ,42,43,44。除了离体脑毛细血管外, 目前的血脑屏障模型包括在硅模型中, 分离的脑毛细血管内皮细胞的离体细胞培养或永生化细胞系从各种人多潜能干细胞 (hPSC) 的体外培养, 分化为脑毛细血管内皮细胞和微流控芯片模型。
在以预测吸收、分布、新陈代谢和排泄 (ADME) 性质为基础的药物开发中, 在硅模型中最常用的方法是选择药物候选者。定量结构-属性关系 (QSPR) 模型和定量结构-活动关系模型是高吞吐量筛选的常用方法, 用于预测药物候选者的脑内渗透率。45,46. 这些模型对于屏蔽渗透特性的分子是有用的。
Betz等。47建立培养的脑毛细血管内皮细胞作为体外血脑屏障模型体系。体外细胞培养模型使用新鲜组织或永生化内皮细胞系如人脑微血管内皮细胞 (hCMECs) 可以是另一个高通量筛选工具的大脑渗透或机械研究。然而, 脑毛细血管内皮细胞培养模型缺乏毛细血管腔内血流的生理剪应力, 在整体生物学复杂性上受到限制, 并经历了重要屏障成分的表达和定位变化。如紧密连接蛋白, 表面受体, 转运体, 酶, 离子通道48,49,50。反之, 从 hPSCs 中提取的内皮细胞, 与 hCMEC/D3 培养相比具有低的蔗糖渗透性, 并含有某些血脑屏障转运体、黏附分子和紧密连接点51的极化表达,52. 然而, 这些细胞也受文化中变化的特性的改变, 必须对系统进行验证, 以确认其在体内屏障性质52中的重述。
血脑屏障研究的新趋势包括利用3D 组织培养系统制造人造毛细血管, 利用芯片上的器官技术产生微流控装置, 或利用中空纤维技术53,54,55. 然而, 人造毛细血管的直径 (100–200µm) 明显大于脑毛细血管 (3–7µm)。因此,体外剪切力不完全类似于体内的情况。这是在 "血脑屏障芯片" 微流控装置, 其中人工膜形成 "血液" 和 "大脑" 车厢和流体泵浦通过这些装置产生微流控剪切力。同样, 不同组合的内皮细胞与星形胶质细胞和血管平滑肌干细胞的联合培养也被应用于中空纤维技术, 以重现体内条件下的流变参数56 。,57,58. 然而, 目前还不清楚这个模型是如何反映血脑屏障的其他特性的, 如运输、新陈代谢、信号传递和其他。这些人造毛细管和切片模型适用于高通量筛选药物, 但用于生成这些模型的细胞在培养过程中也会发生变化。
冷冻和固定脑切片或原发性脑毛细血管内皮细胞培养是额外的模型, 可用于研究人类微血管5,59,60,61。例如, 固定脑组织的免疫组化被用来确定蛋白质的定位和健康的表达与患病的组织相比。
除了以上所述的组织切片和体外模型之外, 还可以利用新鲜的脑毛细血管来研究血脑屏障功能。这种孤立的毛细管模型的局限性包括难以获得新鲜的人脑组织, 缺乏星形胶质细胞和神经元, 以及一个相对耗时的隔离过程。分离的脑毛细管模型的优点是, 该模型与体内情况相似, 因此可以用来表征屏障功能和功能障碍。重要的是, 它也可以用来辨别信号机制使用大量的化验和分子技术3,19,62,63。
我们的实验室可以通过桑德斯-棕褐色中心 (IRB #B15-2602 米)64获得新鲜和冰冻的人脑组织。在这种情况下, 解剖遵循标准的协议, 大脑在 < 4 小时内获得, 所有程序符合 NIH Biospecimen 最佳做法准则65。考虑到人类大脑组织的这种独特的接触, 我们建立并优化了一项协议, 以隔离人脑组织中的毛细血管, 从而导致完整、可行的人脑毛细血管的高产。两个常见的端点的兴趣是确定的蛋白质表达和活动。在这方面, 我们和其他人已经建立了不同的化验方法, 可用于分离的脑毛细血管, 以研究蛋白质表达和活动水平。这些分析包括: 西方印迹, 简单的西方检测, 酶联免疫吸附试验 (ELISA), 反向转录聚合酶链反应 (rt-pcr), 定量聚合酶链反应 (qPCR), 酶谱法, 运输活动的检测, 和毛细管渗漏化验。这些化验可以让研究人员研究人体病理状况中屏障功能的变化, 确定控制蛋白表达和活性的途径, 并确定治疗血脑屏障的药理靶点。疾病。
结合起来, 新鲜的孤立的脑毛细血管可以作为一个强健的和可再生的血脑屏障模型。特别是, 该模型可以结合多种不同的检测方法, 确定广泛的端点来研究屏障功能。

<table>
	<tbody>
		<tr>
			<td>Personal Protective Equipment (PPE)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Diamond Grip Plus Latex Gloves, Microflex Medium</td>
			<td>VWR, Radnor, PA, USA</td>
			<td>32916-636</td>
			<td>PPE</td>
		</tr>
		<tr>
			<td>Disposable Protective Labcoats</td>
			<td>VWR, Radnor, PA, USA</td>
			<td>470146-214</td>
			<td>PPE; due to the nature of the human source material, the use of a disposable lab coat is recommended</td>
		</tr>
		<tr>
			<td>Face Shield, disposable</td>
			<td>Thermo Fisher Scientific, Pittsburgh, PA, USA</td>
			<td>19460102</td>
			<td>PPE; due to the nature of the human source material, the use of a disposable face shield is recommended</td>
		</tr>
		<tr>
			<td>Safety Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clavies High-Temperature Autoclave Bags 8X12</td>
			<td>Thermo Fisher Scientific, Pittsburgh, PA, USA</td>
			<td>01-815-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Versi Dry Bench Paper 18&#34; x 20&#34;</td>
			<td>Thermo Fisher Scientific, Pittsburgh, PA, USA</td>
			<td>14-206-32</td>
			<td>to cover working areas</td>
		</tr>
		<tr>
			<td>VWR Sharps Container Systems</td>
			<td>Thermo Fisher Scientific, Pittsburgh, PA, USA</td>
			<td>75800-272</td>
			<td>for used scalpels</td>
		</tr>
		<tr>
			<td>Bleach 8.2% Clorox Germicidal 64 oz</td>
			<td>UK Supply Center, Lexington, KY, USA</td>
			<td>323775</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4&#176;C Refrigerator</td>
			<td>Thermo Fisher Scientific, Pittsburgh, PA, USA</td>
			<td>13-986-148</td>
			<td></td>
		</tr>
		<tr>
			<td>Accume BASIC AB15 pH Meter</td>
			<td>Thermo Fisher Scientific, Pittsburgh, PA, USA</td>
			<td>AB15</td>
			<td></td>
		</tr>
		<tr>
			<td>Heidolph RZR 2102 Control</td>
			<td>Heidolph, Elk Grove Village, IL, USA</td>
			<td>501-21024-01-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall LEGEND XTR Centrifuge</td>
			<td>Thermo Fisher Scientific, Pittsburgh, PA, USA</td>
			<td>75004521</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica L2 Dissecting Microscope</td>
			<td>Leica Microsystems Inc, Buffalo Grove IL, USA</td>
			<td></td>
			<td>used to remove meninges</td>
		</tr>
		<tr>
			<td>POLYTRON PT2500 Homogenizer</td>
			<td>Kinematica AG, Luzern, Switzerland</td>
			<td>9158168</td>
			<td></td>
		</tr>
		<tr>
			<td>Scale P-403</td>
			<td>Denver Instrument, Bohemia, NY, USA</td>
			<td>0191392</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard mini Stir</td>
			<td>Thermo Fisher Scientific, Pittsburgh, PA, USA</td>
			<td>1151050</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo-Flasks Liquid Nitrogen Dewar</td>
			<td>Thermal Scientific, Mansfiled, TX, USA</td>
			<td>11-670-4C</td>
			<td>used to freeze the tissue?</td>
		</tr>
		<tr>
			<td>Voyager Pro Analytical Balance</td>
			<td>OHAUS, Parsippany, NJ, USA</td>
			<td>VP214CN</td>
			<td></td>
		</tr>
		<tr>
			<td>ZEISS Axiovert Microcope</td>
			<td>Carl Zeiss, Inc Thornwood, NY, USA</td>
			<td></td>
			<td>used to check isolated capillaries</td>
		</tr>
		<tr>
			<td>Tools and Glassware</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Finnpipette II Pipette 1-5mL</td>
			<td>Thermo Fisher Scientific, Pittsburgh, PA, USA</td>
			<td>21377823T1</td>
			<td>wash capillaries off filter</td>
		</tr>
		<tr>
			<td>Finnpipette II Pipette 100-1000 &#181;L</td>
			<td>Thermo Fisher Scientific, Pittsburgh, PA, USA</td>
			<td>21377821T1</td>
			<td>resuspend pellet in BSA</td>
		</tr>
		<tr>
			<td>Pipet Boy</td>
			<td>Integra, Hudson, NH, USA</td>
			<td>739658</td>
			<td></td>
		</tr>
		<tr>
			<td>50mL Falcon tubes 25/rack - 500/cs</td>
			<td>VWR, Radnor, PA, USA</td>
			<td>21008-951</td>
			<td></td>
		</tr>
		<tr>
			<td>EISCO Scalpel Blades</td>
			<td>Thermo Fisher Scientific, Pittsburgh, PA, USA</td>
			<td>S95938C</td>
			<td>to mince brain tissue</td>
		</tr>
		<tr>
			<td>PARAFILM</td>
			<td>VWR, Radnor, PA, USA</td>
			<td>52858-000</td>
			<td>to cover beaker and volumetric flask</td>
		</tr>
		<tr>
			<td>Thermo Scientific Finntip Pipet Tips 5 ml</td>
			<td>Thermo Fisher Scientific, Pittsburgh, PA, USA</td>
			<td>21-377-304</td>
			<td>to wash capillaries off filter</td>
		</tr>
		<tr>
			<td>60 ml syringe with Luer-Lok</td>
			<td>Thermo Fisher Scientific, Pittsburgh, PA, USA</td>
			<td>BD309653</td>
			<td>used with connector ring to filter capillaries</td>
		</tr>
		<tr>
			<td>Scalpel Handle #4</td>
			<td>Fine Science Tools, Foster City, CA, USA</td>
			<td>10060-13</td>
			<td>used for mincing</td>
		</tr>
		<tr>
			<td>Dumont Forceps #5</td>
			<td>Fine Science Tools, Foster City, CA, USA</td>
			<td>11251-10</td>
			<td>used to remove meninges</td>
		</tr>
		<tr>
			<td>Potter-Elvehjem Tissue Grinder</td>
			<td>Thomas Scientific, Swedesboro, NJ, USA</td>
			<td>3431E25</td>
			<td>50 ml volume, clearance: 150-230 &#956;m</td>
		</tr>
		<tr>
			<td>Dounce Homogenizer</td>
			<td>VWR, Radnor PA USA</td>
			<td>62400-642</td>
			<td>15 ml volume, clearance: 80-130 &#956;m</td>
		</tr>
		<tr>
			<td>Spectra/Mesh Woven Filters (300 &#181;m)</td>
			<td>Spectrum Laboratories, Rancho Dominguez, CA, USA</td>
			<td>146424</td>
			<td>Used to filter capillary suspension to remove any meninges that may be left</td>
		</tr>
		<tr>
			<td>pluriStrainers (pore size: 30 &#181;m)</td>
			<td>pluriSelect Life Science, Leipzig, Germany</td>
			<td>43-50030-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Connector Ring</td>
			<td>pluriSelect Life Science, Leipzig, Germany</td>
			<td>41-50000-03</td>
			<td>reuse multiple time</td>
		</tr>
		<tr>
			<td>1 l Volumetric Flask</td>
			<td></td>
			<td></td>
			<td>for preparation of Isolation Buffer</td>
		</tr>
		<tr>
			<td>1 l Beaker</td>
			<td></td>
			<td></td>
			<td>for preparation of 1% BSA</td>
		</tr>
		<tr>
			<td>Stir Bar</td>
			<td></td>
			<td></td>
			<td>for preparation of 1% BSA and Ficoll&#174;</td>
		</tr>
		<tr>
			<td>Schott Bottle (60 ml)</td>
			<td></td>
			<td></td>
			<td>for preparation of Ficoll&#174;</td>
		</tr>
		<tr>
			<td>Ice Bucket</td>
			<td></td>
			<td></td>
			<td>to keep everything cold</td>
		</tr>
		<tr>
			<td>100 mm Petri Dish</td>
			<td></td>
			<td></td>
			<td>for mincing of brain tissue</td>
		</tr>
		<tr>
			<td>Tissue Culture Cell Scraper</td>
			<td>VWR, Radnor, PA, USA</td>
			<td>89260-222</td>
			<td>to remove supernatant after centrifugation</td>
		</tr>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BSA Fraction V, A-9647</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>A9647-500g</td>
			<td>prepare in DPBS with Ca2+ &#38; Mg2+ the day before. Avoid bubbles during preparation. Store in the refrigerator. Slowly stir for 10 min before use.</td>
		</tr>
		<tr>
			<td>DPBS with Ca2+ &#38; Mg2+</td>
			<td>Hyclone</td>
			<td>SH30264.FS</td>
			<td>DPBS - part of the Isolation Buffer</td>
		</tr>
		<tr>
			<td>Ficoll PM400</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>F4375</td>
			<td>Exact measurement is important here. Weigh out in bottle with stir bar. Shake vigurously after adding DPBS. Keep in the fridge O/N. It will be clear in the morning. Stir gently for 10-15 min before use. Keep on ice until use.</td>
		</tr>
		<tr>
			<td>Glucose (D-(+) Dextrose)</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>G7528</td>
			<td>Glucose (D-(+) Dextrose) Concentration: 5 mM</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide Standard Solution</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>71474</td>
			<td>to adjust pH of the DPBS</td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>P2256</td>
			<td>Concentration: 1 mM</td>
		</tr>
	</tbody>
</table>

isolation of cerebral capillaries from fresh human brain tissue

在生理和病理生理学条件下了解血脑屏障功能对于制定新的治疗策略具有重要的作用, 这些战略有望加强脑药物的传递, 改善脑保护, 并治疗脑障碍。然而, 研究人体血脑屏障功能具有挑战性。因此, 迫切需要适当的模型。在这方面, 从人脑组织中分离出来的脑毛细血管代表了一种独特的工具, 可以在尽可能接近人类体内的情况下研究屏障功能。在这里, 我们描述了一个优化的协议, 以隔离毛细血管从人的大脑组织高产和一致的质量和纯度。毛细血管从新鲜的人脑组织中分离出来, 采用机械均匀化、密度梯度离心和过滤。隔离后, 人脑毛细血管可用于各种应用, 包括渗漏化验, 活细胞成像, 免疫分析, 以研究蛋白质的表达和功能, 酶活性, 或细胞内信号。孤立的人脑毛细血管是阐明人体血脑屏障功能调节的独特模型。该模型可为中枢神经系统 (cns) 发病机制提供深入的见解, 有助于发展治疗中枢性疾病的治疗策略。

隔离从人脑组织中产生的悬浮液富含人脑毛细血管 (图 1B), 少量较大的血管, 红细胞, 其他单细胞, 和一些细胞碎片。有些毛细血管是分枝的, 在某些情况下, 红血球被包裹在毛细血管流明。典型毛细管有3–7µm 直径和大约100-200 µm 长以开放流明;大多数毛细管末端都是折叠的。使用共焦显微镜, 孤立的人脑毛细血管揭示一个管状, 完整的结构和形态学。图 2A显示了一个具有代表性的透射光图像的人脑毛细血管与附周细胞和红细胞在腔内。所有关于直径、大小和形态学的研究结果都与以前关于隔离脑毛细血管结构12、17、18的报告一致。以 C219 为主要抗体 (1 µg/毫升), 以 immunostained 为 P gp (绿色), 在图 2B中分离人脑毛细血管;核复染与 DAPI (1 µg/毫升)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57346/57346fig1.jpg"> 图 1: 毛细管隔离的流程图.(A) 象形文字说明了将脑毛细血管与新鲜人体组织隔离的程序的主要步骤。(B) 图片显示隔离后直接在光显微镜下的人脑毛细血管 (100X 倍放大)。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57346/57346fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57346/57346fig2.jpg"> 图 2: 孤立的人脑毛细血管.(a) 一种孤立的人脑毛细血管的透射光图像。(B) 共焦显微镜图像显示了一个单独的人脑毛细血管 immunostained 对 P gp (绿色;C219 1 µg/毫升);细胞核复染 DAPI (蓝色; 1 µg/毫升)。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57346/57346fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57346/57346fig3.jpg"> 图 3: 密度离心故障排除.象形文字显示了密度梯度离心后的制备方法。它突出了太多和太少的密度梯度介质的影响, 以及如何影响分离和产生的毛细管颗粒。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57346/57346fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57346/57346fig4.jpg"> 图 4: 人脑毛细血管中的 p-gp 蛋白表达.与 hCMEC\D3 细胞相比, 在孤立的人毛细血管中, 西方印迹显示了对 P gp (1 µg/毫升) 的强频带。β肌动蛋白被用作负荷控制 (1 µg/µL)。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57346/57346fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57346/57346fig5.jpg"> 图 5: 用于隔离人脑毛细血管的应用.文献中发表的关于孤立脑毛细血管的最常见应用的概述。隔离毛细血管已用于: 1) 基因组学85,86, 2) 蛋白质组学3,38,87,88,89,90, 91、92、94、95、96、3) 功能蛋白质组学2、38和 4) Cellomics82, 83,84,97,98,99。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57346/57346fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>中风</td>
			<td>时间 [分钟]</td>
		</tr>
<tr>
<td>1–25</td>
			<td>7–7。5</td>
		</tr>
<tr>
<td>26–50</td>
			<td>5–5。5</td>
		</tr>
<tr>
<td>51–75</td>
			<td>5–5。5</td>
		</tr>
<tr>
<td>76–100</td>
			<td>5–5。5</td>
		</tr>
<tr>
<td>总时间:</td>
			<td>22–24分钟</td>
		</tr>
</tbody></table>表 1: 均匀化协议.波特-Elvehjem 组织磨床的均匀化协议, 融汇10克的人额皮质, 均匀速度为 50 rpm。请注意, 头几个笔画需要额外的时间来融汇切碎的组织。在这最初的均匀化以后, 每冲程是十二年代在期间 (6 s 为向下运动, 6 s 为向上运动)。因此, 在初始均匀化后, 5 笔画可以在1分钟内完成, 或在5分钟内25笔画。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>问题</td>
			<td>潜在原因</td>
			<td>解决 方案</td>
		</tr>
<tr>
<td rowspan="3">无毛细管颗粒</td>
			<td>1) 不正确的 Ficoll 浓度</td>
			<td>1) 调整 Ficoll 浓度</td>
		</tr>
<tr>
<td>2) 离心速度不正确</td>
			<td>2) 调整离心速度</td>
		</tr>
<tr>
<td>3) 不正确的加速度和/或减速速度</td>
			<td>3) 调整加速度和/或减速速度</td>
		</tr>
<tr>
<td rowspan="4">低毛细管产量</td>
			<td>1) 脑膜阻断过滤步骤</td>
			<td>1) 过滤前取出所有脑膜</td>
		</tr>
<tr>
<td>2) 隔离过程中失去的毛细血管过多</td>
			<td>2) 正确计算缓冲浓度, 冲洗吸管提示</td>
		</tr>
<tr>
<td>3) PluriStrainer 过滤器清洗毛细血管不足</td>
			<td>3) 打开过滤器, 仔细检查毛细血管 (使用显微镜)</td>
		</tr>
<tr>
<td>4) resuspensions 期间的过剩气泡</td>
			<td>4) 吸管慢慢避免气泡</td>
		</tr>
<tr>
<td rowspan="4">无活力毛细血管</td>
			<td>1) 延长的验尸间隔</td>
			<td>1) 减少时间间隔, 如果可能, 或使用管理冻结大脑</td>
		</tr>
<tr>
<td>2) 使用冷冻组织进行隔离</td>
			<td>2) 使用新鲜纸巾</td>
		</tr>
<tr>
<td>3) 隔离过程时间过长</td>
			<td>3) 优化工作流程</td>
		</tr>
<tr>
<td>4) 在隔离过程中没有将设备/缓冲器放在冰上</td>
			<td>4) 隔离时将设备和缓冲放在冰上</td>
		</tr>
</tbody></table>表 2: 常见问题的疑难解答.在隔离过程中发生的最常见错误和问题的列表, 以及如何解决它们。

Watch this Scientific Journal Video about Isolation of Cerebral Capillaries from Fresh Human Brain Tissue at JoVE.com

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

新鲜人脑组织中毛细血管的分离

从人脑组织中分离出的脑毛细血管可作为研究生理和病理生理学条件下屏障功能的临床前模型。在这里, 我们提出了一个优化的协议, 以隔离脑毛细血管与新鲜的人脑组织。

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

isolation-of-cerebral-capillaries-from-fresh-human-brain-tissue

Nanyang Technological University

Research

JoVE Journal

Neuroscience

12.5K 次观看.  University of Kentucky. 从人脑组织中分离出的脑毛细血管可作为研究生理和病理生理学条件下屏障功能的临床前模型。在这里, 我们提出了一个优化的协议, 以隔离脑毛细血管与新鲜的人脑组织。

视频: Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure.

Double Fluorescence in situ Hybridization in Fresh Brain Sections

This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.

双荧光原位杂交食脑切片

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

科研

神经科学

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

神经元形态的研究方法:<em&gt;体外</em&gt; RNAi技术在胚胎小鼠大脑皮质的电穿孔

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

Fiber-optic Implantation for Chronic Optogenetic Stimulation of Brain Tissue

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard for analyzing patterns of synaptic connectivity, but paired electrophysiological recordings can be both cumbersome and experimentally limiting. The development of optogenetics has introduced an elegant method to stimulate neurons and circuits, both in vitro1 and in vivo2,3. By exploiting cell-type specific promoter activity to drive opsin expression in discrete neuronal populations, one can precisely stimulate genetically defined neuronal subtypes in distinct circuits4-6. Well described methods to stimulate neurons, including electrical stimulation and/or pharmacological manipulations, are often cell-type indiscriminate, invasive, and can damage surrounding tissues. These limitations could alter normal synaptic function and/or circuit behavior. In addition, due to the nature of the manipulation, the current methods are often acute and terminal. Optogenetics affords the ability to stimulate neurons in a relatively innocuous manner, and in genetically targeted neurons. The majority of studies involving in vivo optogenetics currently use a optical fiber guided through an implanted cannula6,7; however, limitations of this method include damaged brain tissue with repeated insertion of an optical fiber, and potential breakage of the fiber inside the cannula. Given the burgeoning field of optogenetics, a more reliable method of chronic stimulation is necessary to facilitate long-term studies with minimal collateral tissue damage. Here we provide our modified protocol as a video article to complement the method effectively and elegantly described in Sparta et al.8 for the fabrication of a fiber optic implant and its permanent fixation onto the cranium of anesthetized mice, as well as the assembly of the fiber optic coupler connecting the implant to a light source. The implant, connected with optical fibers to a solid-state laser, allows for an efficient method to chronically photostimulate functional neuronal circuitry with less tissue damage9 using small, detachable, tethers. Permanent fixation of the fiber optic implants provides consistent, long-term in vivo optogenetic studies of neuronal circuits in awake, behaving mice10 with minimal tissue damage.

The development of optogenetics now provides the means to precisely stimulate genetically defined neurons and circuits, both in vitro and in vivo. Here we describe the assembly and implantation of a fiber optic for chronic photostimulation of brain tissue.

光纤植入慢性Optogenetic刺激脑组织

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard ...

Isolation of Cerebrospinal Fluid from Rodent Embryos for use with Dissected Cerebral Cortical Explants

The CSF is a complex fluid with a dynamically varying proteome throughout development and in adulthood. During embryonic development, the nascent CSF differentiates from the amniotic fluid upon closure of the anterior neural tube. CSF volume then increases over subsequent days as the neuroepithelial progenitor cells lining the ventricles and the choroid plexus generate CSF. The embryonic CSF contacts the apical, ventricular surface of the neural stem cells of the developing brain and spinal cord. CSF provides crucial fluid pressure for the expansion of the developing brain and distributes important growth promoting factors to neural progenitor cells in a temporally-specific manner. To investigate the function of the CSF, it is important to isolate pure samples of embryonic CSF without contamination from blood or the developing telencephalic tissue. Here, we describe a technique to isolate relatively pure samples of ventricular embryonic CSF that can be used for a wide range of experimental assays including mass spectrometry, protein electrophoresis, and cell and primary explant culture. We demonstrate how to dissect and culture cortical explants on porous polycarbonate membranes in order to grow developing cortical tissue with reduced volumes of media or CSF. With this method, experiments can be performed using CSF from varying ages or conditions to investigate the biological activity of the CSF proteome on target cells.

The ventricular cerebrospinal fluid (CSF) bathes the neuroepithelial and cerebral cortical progenitor cells during early brain development in the embryo. Here we describe the method developed to isolate ventricular CSF from rodent embryos of different ages in order to investigate its biological function. In addition, we demonstrate our cerebral cortical explant dissection and culture technique that allows for explant growth with minimal volumes of culture medium or CSF.

脑脊液分离解剖大脑皮质外植体使用的啮齿动物胚胎

The CSF is a complex fluid with a dynamically varying proteome throughout development and in adulthood. During embryonic development, the nascent CSF ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

从胚胎前脑内皮细胞的分离和培养

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

Isolation and Cannulation of Cerebral Parenchymal Arterioles

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance blood vessels branching out from pial arteries and arterioles and diving into the brain parenchyma. Individual PA perfuse a discrete cylindrical territory of the parenchyma and the neurons contained within. These arterioles are a central player in the regulation of cerebral blood flow both globally (cerebrovascular autoregulation) and locally (functional hyperemia). PAs are part of the neurovascular unit, a structure that matches regional blood flow to metabolic activity within the brain and also includes neurons, interneurons, and astrocytes. Perfusion through PAs is directly linked to the activity of neurons in that particular territory and increases in neuronal metabolism lead to an augmentation in local perfusion caused by dilation of the feed PA. Regulation of PAs differs from that of better-characterized pial arteries. Pressure-induced vasoconstriction is greater in PAs and vasodilatory mechanisms vary. In addition, PAs do not receive extrinsic innervation from perivascular nerves &#8212; innervation is intrinsic and indirect in nature through contact with astrocytic endfeet. Thus, data regarding contractile regulation accumulated by studies using pial arteries does not directly translate to understanding PA function. Further, it remains undetermined how pathological states, such as hypertension and diabetes, affect PA structure and reactivity. This knowledge gap is in part a consequence of the technical difficulties pertaining to PA isolation and cannulation. In this manuscript we present a protocol for isolation and cannulation of rodent PAs. Further, we show examples of experiments that can be performed with these arterioles, including agonist-induced constriction and myogenic reactivity. Although the focus of this manuscript is on PA cannulation and pressure myography, isolated PAs can also be used for biochemical, biophysical, molecular, and imaging studies.

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 ...

Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific sets of sparsely distributed activated neurons called neuronal ensembles that mediate learned associations. Fluorescence Activated Cell Sorting (FACS) has recently been optimized for adult rat brain tissue and allowed isolation of activated neurons using antibodies against the neuronal marker NeuN and Fos protein, a marker of strongly activated neurons. Until now, Fos-expressing neurons and other cell types were isolated from fresh tissue, which entailed long processing days and allowed very limited numbers of brain samples to be assessed after lengthy and complex behavioral procedures. Here we found that yields of Fos-expressing neurons and Fos mRNA from dorsal striatum were similar between freshly dissected tissue and tissue frozen at -80 &#186;C for 3 - 21 days. In addition, we confirmed the phenotype of the NeuN-positive and NeuN-negative sorted cells by assessing gene expression of neuronal (NeuN), astrocytic (GFAP), oligodendrocytic (Oligo2) and microgial (Iba1) markers, which indicates that frozen tissue can also be used for FACS isolation of glial cell types. Overall, it is possible to collect, dissect and freeze brain tissue for multiple FACS sessions. This maximizes the amount of data obtained from valuable animal subjects that have often undergone long and complex behavioral procedures.

Fluorescence Activated Cell Sorting FACS and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

Here we present a Fluorescence Activated Cell Sorting (FACS) protocol to study molecular alterations in Fos-expressing neuronal ensembles from both fresh and frozen brain tissue. The use of frozen tissue allows FACS isolation of many brain areas over multiple sessions to maximize the use of valuable animal subjects.

荧光活化细胞分选（FACS）和新鲜和冷冻大鼠脑组织的Fos表达神经元基因表达分析

The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific ...

A Rat Model of Mild Intrauterine Hypoperfusion with Microcoil Stenosis

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies of this phenomenon have been performed in either models with severe intrauterine ischemia or models with gradient degree of intrauterine hypoperfusion. No study has been performed in a model on uniform mild intrauterine hypoperfusion (MIUH). Two models have been used for studies of MIUH: a model based on suture ligation of either side of the arterial arcade formed with the uterine and ovarian arteries, and a transient model based on clipping the bilateral ovarian arteries and aorta having patency. Those two rodent models of MIUH have some limitations, e.g., not all fetuses are subjected to MIUH, depending on their position in the uterine horn. In our MIUH model, all fetuses are subjected to a comparable level of intrauterine hypoperfusion. MIUH was achieved by mild stenosis of all four arteries feeding the uterus, i.e., the bilateral uterine and ovarian arteries.
Arterial stenosis was induced by metal microcoils wrapped around the feeding arteries. Producing arterial stenosis with microcoils allowed us to control, optimize, and reproduce decreased blood flow with very little inter-animal variability and a low mortality rate, thus enabling accurate evaluation. When microcoils with an inner diameter of 0.24 mm were used, the blood flow in both the placenta and fetus was mildly decreased (approximately 30% from the pre-stenosis level in the placenta). The offspring of our MIUH model clearly demonstrates long-lasting alterations in neurological, neuroanatomical and behavioral test results.

Mild intrauterine hypoperfusion was produced by artery stenosis with metal microcoils wrapped around the uterine and ovarian arteries in rats at embryonic day 17. This procedure produced prenatal hypoperfusion and intrauterine growth restriction.

轻度宫内灌注圈狭窄的大鼠模型

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.
Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 &#176;C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 &#176;C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) &#62; 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

斑马鱼脑幼虫神经元、巨噬细胞和小胶质的分离和 RNA 提取

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...

Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). We have identified five steps in the workflow that are crucial for obtaining RNA isolates from enteric ganglia with sufficiently high quality and quantity for RNA-seq. First, when preparing intestinal tissue, each sample must have all excess liquid removed by blotting prior to flattening the serosa as much as possible across the bottom of large base molds. Samples are then quickly frozen atop a slurry of dry ice and 2-methylbutane. Second, when sectioning the tissue, it is important to position cryomolds so that intestinal sections parallel the full plane of the myenteric plexus, thereby yielding the greatest surface area of enteric ganglia per slide. Third, during LCM, polyethylene napthalate (PEN)-membrane slides offer the greatest speed and flexibility in outlining the non-uniform shapes of enteric ganglia when collecting enteric ganglia. Fourth, for distinct visualization of enteric ganglia within sections, ethanol-compatible dyes, like Cresyl Violet, offer excellent preservation of RNA integrity relative to aqueous dyes. Finally, for the extraction of RNA from captured ganglia, we observed differences between commercial RNA extraction kits that yielded superior RNA quantity and quality, while eliminating DNA contamination. Optimization of these factors in the current protocol greatly accelerates the workflow and yields enteric ganglia samples with exceptional RNA quality and quantity.

The goal of this protocol is to obtain high-integrity RNA samples from enteric ganglia isolated from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). This protocol involves preparing flash-frozen samples of human intestinal tissue, cryosectioning, ethanolic staining and dehydration, LCM, and RNA extraction.

激光捕获显微切割分离新鲜冷冻人体肠神经节的优化

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue ...