Waiting
Login processing...

Trial ends in Request Full Access Tell Your Colleague About Jove
Click here for the English version

Medicine

人体血液,脑脊液屏障的脉络丛上皮基于Cell的产品型号从基底外侧面研究细菌感染

Published: May 6, 2016 doi: 10.3791/54061

Introduction

血-脑脊髓液屏障(BCSFB)是血液和脑1之间三个屏障站点之一。其形态相关成分是脉络丛(CP)2,3-上皮细胞,内皮上皮回旋,这强烈血管和位于脑的脑室。将CP用来产生脑脊液(CSF),以及后者从血液中分离出来。为了实现屏障功能,在CP的上皮细胞显示出低的吞饮活性,表达特定转运,并且通过紧密连接(紧密连接)2,3-的连续网络密集连接。

人类脉络丛乳头状瘤(HIBCPP)细胞,从日本女子4的恶性脉络丛乳头状瘤衍生的,用于构建 BCSFB的体外模型的功能。 HIBCPP细胞显示一对夫妇的功能BCSFB的特点,为TJ形成股线,可以判断为跨上皮电阻(TEER)高跨上皮膜电位的发展,以及对大分子次要渗透率。此外,HIBCPP细胞表达的特点转运,这可能会起到调节离子微环境,并显示心尖/基底极性5,6,7。

该BCSFB已显示作为一个条目的网站的病原体(细菌,病毒和真菌)进入中枢神经系统(CNS)8。病原体,包括脑膜炎奈瑟氏球菌脑膜炎奈瑟氏球菌 ),一种革兰氏阴性细菌,入侵能引起严重的疾病如脑膜炎。 证据表明,它克服了CP的保护上皮屏障是由组织病理学观察患者支持与脑膜炎球菌性疾病表现出增加的血管和CP上皮细胞9,10-脑膜炎球菌的量。要进入宿主细胞巴cteria经常挟持内吞机制,其介导或由位于宿主细胞特异性表面受体触发。由于具有这些受体的病原体的相互作用可以是物种特异性11,动物模型只能协商,以一个受限制的程度。该HIBCPP细胞系提供了机会,研究入侵过程中,以及在人体模型系统的分子机制。采用细胞培养插入使我们能够分析病原体相互作用与宿主细胞从两个不同的细胞两侧。许多细菌,包括N。脑膜炎 ,强烈受重力的期间感染测定法的影响。对于与检测期间HIBCPP细胞病原体最佳交互,该细菌最初添加到细胞培养滤芯系统的上隔室。到从心尖或基底细胞侧,分别在体外系统的两种变化已经埃斯塔使感染blished:在标准系统HIBCPP细胞接种到过滤器插入件的上部隔室,模仿情况时微生物位于CSF的侧,并获得与所述细胞( 图1A,C)的顶面接触。与此相反,使用HIBCPP细胞在倒置的细胞培养滤芯系统反映当细菌进入血流的条件。微生物传播在从基底外侧侧( 图1B,D)的血液和遭遇的CP上皮细胞。值得注意的是,在该模型系统,已经表明,细菌侵入HIBCPP细胞极性的方式专门从基底细胞侧5,7。

随后向CP的感染,所述入侵病原体可通过结扎先天免疫系统对模式识别受体(的PRRs)识别。蓝耳的良好描述成员属于Toll样受体(TLR)家族。 Toll样受体能斌d,来感染性微生物的特性的结构,其被称为病原体相关分子模式(PAMP)。受体的结扎导致触发的细胞因子和趋化因子12,其反过来横跨BCSFB 13,14刺激免疫细胞的轮回的表达宿主细胞的活化信号级联。它已经显示HIBCPP细胞表达在mRNA水平数的TLR和感染N.脑膜炎导致多种细胞因子和趋化因子,包括CXCL1-3,IL6,IL8和TNFα15,16的分泌。

在这里,我们描述了一个模仿BCSFB倒置细胞培养插管系统种植和人细胞系HIBCPP的感染。此模型系统使得来研究与体内的相关基底细胞侧以及随后的细胞反应的病原体的相互作用。

Subscription Required. Please recommend JoVE to your librarian.

Protocol

1.准备播种HIBCPP细胞细胞培养滤芯在倒立模型系统

  1. 补充有5微克/毫升胰岛素预暖的DMEM / F12(HAM),100U / ml青霉素,100微克/毫升链霉素和10%胎牛血清(FCS)。
  2. 使用无菌镊子放置0.33平方厘米的生长区域的细胞培养滤芯为3微米的孔尺寸倒置放入一个12孔板( 图1E)。
  3. 填充介质进入细胞培养滤芯的下部隔室(约3毫升)和100微升在过滤器插入件的顶端。淹没板以及与介质的下隔室。吸在这样一种方式,滤波器插入件的下隔室保持填充( 图1E)的过度介质。
    注:建议使用血清吸管用于此步骤。
  4. 盖上盖12孔板和制备的细胞培养滤芯转移到孵化器,37℃,5%的CO 2,直到加入细胞。

2.栽培HIBCPP细胞传代

  1. 制备补充有5微克/毫升,100U / ml青霉素,100微克/毫升链霉素和10%FCS的DMEM / F12(HAM)。
  2. 预暖介质和PBS在37℃水浴中。从瓶中吸出网上平台。加入10毫升的PBS至烧瓶并乱舞。重复此步骤一次。
  3. 添加3毫升0.25%胰蛋白酶-EDTA至烧瓶并乱舞。放入培养箱,在37℃,5%的CO 2。
  4. 大约20分钟后,除去从孵化器的烧瓶中。确保细胞从烧瓶底部脱离,当用显微镜调查显示圆​​形。
    注意:细胞不彼此完全分离,并且经常在附聚物中。它建议使用细胞达通道38。
  5. 要停止胰蛋白酶加入17β-毫升培养基。通过上下吹打重悬细胞并转移该悬浮液到50毫升管中。离心,在50×g离心在室温下10分钟。
  6. 悬浮细胞培养基中的适当体积,并通过使用血球计数细胞。
    注意:悬浮HIBCPP细胞的浓度应为1×10 6个细胞/ ml。维护HIBCPP细胞,建议在10毫升培养基转移1-6×10 6细胞的量到一个T75烧瓶。更换培养基每隔一天。

3.播种倒细胞培养滤芯与HIBCPP细胞

  1. 每个反相滤波器插入件( 过滤器的底侧)的顶部添加80微升细胞悬浮液( ,8×10 4个细胞)( 图1E)的。
    注意:确保细胞均匀地播种前颠倒试管分布于停牌。
  2. 覆盖种子细胞培养滤芯与12孔板,并转移到孵化器的盖,在37℃,5%CO 2。
  3. 在第一天,填充1ml培养基到24孔板的孔中。通过使用镊子出12孔板的解除细胞培养滤波器的插入,丢弃培养基内,转动过滤器插入并放置在标准定向到准备24孔板( 图1E)。
  4. 放置细胞进入新鲜培养基每隔一天。准备24井用新鲜的介质和滤芯转移到它。填用0.5ml新鲜培养基插入。检查TEER每天都在第4节。
  5. 当在细胞培养插入HIBCPP接种细胞的TEER值超过70Ω点¯x平方厘米(约种植后4天),然后继续在含1%FCS和5微克/毫升胰岛素介质细胞培养物。准备24孔板用每孔1ml培养基。转移滤芯到准备好的井和交换介质上格。
    注:汇合后,此血清撤离导致的形成更高的膜电位。
  6. 从过滤器吸车厢中,转移到准备好,用500微升培养基填充。重复此步骤一次。把细胞过夜进入培养箱,在37℃,5%的CO 2。

4.跨膜电阻的测量(TEER)

  1. 沉浸上皮组织voltohmmeter的电极尖端为在80%乙醇15分钟。引擎盖下转移voltohmmeter,让电极干一会儿。放置电极到用于所述细胞另外15分钟以达到平衡各自的培养基。
  2. 通过这样它每次触及下部隔室的底部的电极的较长臂定位执行测量,较短的臂放置到过滤器插入件隔室。
    注:细胞培养滤芯的电阻值,而不在介质中的细胞应作为空白值((测量值(Ω) - 空白值(Ω))×滤波器表面( 0.33平方厘米))。
  3. 测量代替电极回80%乙醇15分钟后。存放在干燥管中。

5.测定细胞旁渗透性

  1. 在补充有5微克/毫升胰岛素1%FCS的200毫升培养基溶解1g的FITC-菊粉。细胞的感染前应用50μg/ ml的溶液进入上部过滤器室的。
  2. 感染后收集培养基样品从下面的井,以确定多从菊糖将过滤器如何穿过细胞层。
  3. 制备的FITC菊粉标准溶液,并执行1:2稀释液,10倍(100%,50%,25%,12.5%,6.25%,3.13%,1.56%,0.78%,0.39%,0.2%和0%) 。在酶标仪所有样品的测量确定荧光。

6.细胞培养滤芯细菌的HIBCPP感染细胞的制备

  1. 有一天,在实验前,刮Ť他冷冻N.脑膜炎掀起了甘油的股票和连胜了与Vitox巧克力琼脂。在孵化生长过夜,在37℃下,用5% CO 2。
  2. 从过夜培养提取20-30殖民地和转移到装有8毫升蛋白胨培养基中添加0.042% 碳酸氢钠 ,0.01M的氯化镁和1%Polyvitex管。
  3. 摇细菌1.25小时,以220rpm,37℃。沉淀通过离心细菌在2684克在室温下10分钟。丢弃上清,悬浮用8ml无血清培养基中的细菌沉淀。涡流以确保均匀悬浮液。
  4. 以确定培养物密度,在600nm的光密度(OD)测量的1:10的稀释液。调节细菌悬浮液的OD 0.1 600。
    注:此悬浮液含有约。 1×10 8 CFU / ml的。
  5. 确认细菌细胞浓度,稀释该悬浮液逐步(1:10)到一个10 -5稀释和平板接种到巧克力琼脂平板上。
    注意:类似的系列稀释需要执行计算细菌生长曲线。

在双重免疫细胞培养滤芯和测定细菌侵入HIBCPP细胞感染7

  1. 继续在含有1%FCS和5微克/毫升胰岛素培养基的细胞培养后,使用上皮组织voltohmmeter测量细胞的每一天。如果电池已经达到了大约500Ω点¯x平方厘米TEER,进行感染。
  2. 感染与制备细菌悬浮液中的细胞在感染10商店感染的细胞在培养箱的(MOI)的多重性,在37℃,5%CO 2的时间的指示的时间。
    注意:MOI可以通过考虑在合流(1.21×10 6个细胞/ cm 2)每细胞培养滤芯细胞的数目来计算。
  3. 停止侵染离子洗涤用500μl无血清培养基含有1%牛血清白蛋白(BSA)的三倍(SFM)施加到过滤器室,将1ml到下​​部隔室。
  4. 用含有500微升的SFM 1%BSA缓冲液中的过滤器室和1ml在下部隔室20分钟,在室温下,以防止抗体的非特异性结合位点的粘附阻止。
  5. 用100μl初级抗体抗N.孵育meningtidisα-OMP(1:200),在过滤器室和500微升在在室温下20分钟的下隔室。
    注意:如果需要,抗体浓度需要被滴定。
  6. 通过吸从过滤室中的介质洗涤细胞,滤芯转移到含有1%BSA一个制备好用1ml SFM和填充用含有1%BSA的500μl的SFM的排空过滤隔室。重复此步骤两次。
  7. 固定用500μl的4%甲醛中的细胞过滤器COMPArtment,将1ml在室温10分钟的下隔室。
  8. 通过吸从过滤室中的介质洗涤细胞,滤芯转移到准备好用1ml PBS中并填充用500μlPBS中清空过滤隔室。重复此步骤一次。
    注:样品可以在4℃保存过夜于PBS中。
  9. 切割固定细胞培养筛选出插入物,用含有1%BSA的250μl的PBS洗涤。孵育15分钟,细胞在室温下与标记的250微升荧光(激发波长594纳米)的第二抗体鸡抗兔(1:500)染色细胞外细菌。
    注意:如果需要,抗体浓度需要被滴定。
  10. 透用含有1%BSA和0.5%的Triton X-100在室温下1小时,250微升PBS中的细胞。洗涤细胞三次用含有1%BSA的250μl的PBS中。
  11. 用250微升一抗抗孵育N.脑膜炎
  12. 应用荧光标记的(激发波长488纳米)的第二抗体(1:500)的250微升,荧光标记的(激发波长660纳米)鬼笔环肽(1:250)和4'-,6-二脒基-2-苯基二盐酸盐(DAPI)( 1:50,000)在室温下1小时,染色细胞内外的细菌,肌动蛋白细胞骨架和细胞核。
    注意:如果需要,抗体浓度需要被滴定。
  13. 洗涤细胞三次用含有1%BSA的250μl的PBS中。嵌入安装在4℃介质和存储,直到通过显微镜观察细胞。
  14. 确定每个预定义字段入侵细菌的数量。通过计算每滤膜20场做到这一点。计算入侵细菌的百分比。
    注意:使用面积coeffic乘以20微观领域的平均细菌数ient。结果表示存在于0.33平方厘米的细胞培养滤芯总细菌的量。感染的持续时间期间除以在培养基中生长的细菌的量该值。

Subscription Required. Please recommend JoVE to your librarian.

Representative Results

在这里,我们描述了一个倒置的细胞培养插管系统培养和HIBCPP细胞的感染。此模型使我们能够研究入侵机制和潜在的分子信号传导通路从基底细胞侧,再现细菌传播并且经由血液流进入上皮细胞( 图1)的生理状况。

该HIBCPP细胞显示一定的屏障功能,这使他们能够限制分子交换到最低水平。这是通过粘附连接(AJ)和紧密连接(TJ)蛋白质的表达来实现。的TJ相关蛋白Occludin蛋白,紧密连接蛋白和ZO-1的免疫荧光分析在细胞-细胞接触的部位显示一个不间断信号,表明细胞通过紧密连接的连续股线( 图2A,B,C)相互连接。 t作为详细的印象他TJ形态已被传输( 图2D)中获得,并冷冻断裂电子显微术( 图2E),这表明紧密连接位于小区之间并形成宽,啮合电缆状结构。

上皮细胞的紧密连接也充当围栏偏析心尖和基底外侧膜结构域,因此促进细胞的极性。之间的蛋白质,这是专门表达于HIBCPP细胞的基底细胞侧,是将AJ蛋白E-钙粘蛋白(ECAD)和如在图3中示出通过免疫荧光分析的肝细胞生长因子受体(HGF / MET),指向一个一定程度的极性。

当在合适的条件下培养HIBCPP细胞也表现出像形成的膜电位的BCSFB的典型屏障功能以及低渗透性为大分子cules。膜电位的测定可由TEER的测量与使用voltohmmeter来实现。细胞层的通透性是通过FITC标记的菊粉,传递在旁的方式层的糖的应用定量。这里,我们显示一个代表性实验,这表明这些屏障功能保持稳定,并通过标准浓度二甲亚砜(DMSO),为细胞刺激的化学品的共同溶剂的显示没有感情的结果。如可在图4A中可以看出,TEER值之前和HIBCPP细胞暴露于DMSO中后记录。

细胞显示,在试验的所有条件的开始达到高达约500Ω点¯x平方厘米高的膜电位。测得的TEER后2小时为高达0.2体积%DMSO处理的细胞没有发生变化。与此相反,TEER值以剂量依赖性的wh降低恩被应用更高的产量( 图4A)。这种减少在膜电位是伴随用于大分子的通透性增加。而加入DMSO至HIBCPP细胞达0.5%(体积)没有导致对菊粉的渗透率增加,显示1体积%和2体积%的效果,虽然受限制的程度上( 图4B)。此外,我们检查细胞松弛素D,肌动蛋白聚合的有效抑制剂,对HIBCPP细胞的膜电位和渗透性的影响。用1微克/毫升松弛素D的细胞的处理引起的TEER值的显著下降以及在FITC标记的菊粉通量的增加( 图4A,B)。这种效果可以通过引起细胞松弛素D.紧密连接的开口进行说明

所述HIBCPP细胞系作为BCSFB的模型可用于研究的病原体的相互作用和阻隔形成上皮细胞。有证据该细菌N.脑膜炎穿越BCSFB获得进入中枢神经系统。它已经显示, 奈瑟氏球菌自基底外侧细胞侧,这是在体内相关侵入HIBCPP细胞特异性。在此过程中的荚膜起着重要的作用:胶囊-缺乏的突变体展示浸润增加成HIBCPP细胞5。在下面,我们比较了MC58应变17的基底的侵袭,其等基因突变MC58siaD -缺陷的胶囊生产17 ​​和未包封载体隔离α1419,20。感染了超过在10免疫荧光分析的MOI 4小时的时程上述菌株HIBCPP细胞揭示显著侵袭两个致病菌株MC58和MC58siaD - ,而仅观察到轻微的侵袭率对于非致病性α14菌株( 图5A)。双immunoflu的形象代表感染了各自应变HIBCPP细胞orescence显微镜示于图5A,BC。

图1
图1:标准和倒立细胞培养系统与CP上皮细胞的细菌在标准的细胞培养系统 (A,C)的相互作用可以在顶端细胞侧,它是由微绒毛指示进行调查。倒立的细胞培养系统(B,D),可以让我们来研究与基底外侧细胞膜,这是通过血液进入CNS细菌入侵期间参与细菌相互作用。 对于倒置细胞培养物(E)的 HIBCPP细胞接种到过滤器插入件的底侧。下部井包括过滤器插入件的内室充斥着介质。 Subsequently,过量介质在使得滤波器插入件的内室保持填充的方式吸出。在播种后的第一天,细胞培养滤芯被翻转并充满网上平台。 请点击此处查看该图的放大版本。

图2
图2:HIBCPP细胞显示紧密连接的连续股线的TJ蛋白,ZO-1(A)中 ,Occludin蛋白(B)和紧密连接蛋白1(C)中HIBCPP细胞中检测到。面板AC显示Apotome图像。每个面板的中心:XY 细胞的正视图;顶部和右侧:通过在Z -平面几个光学片的横截面。刻度条表示10μm以下。 HIBCPP细胞的TJ结构通过输气分析锡安电子显微镜。细胞是相互连接的由紧密连接,这是由箭头(D)的指示。该比例尺为1微米。冷冻断裂电子显微术研究可视紧密啮合TJ链(E)。的比例尺= 0.5微米。在该图中,图像最初被发表在Schwerk 等人 ,PLoS一个 7:e30069,DOI:10.1371 / journal.pone.0030069(2012);重新打印许可。 请点击此处查看该图的放大版本。

图3
图3:在基底细胞侧HIBCPP细胞免疫荧光分析显示ECAD(A)Met(B)的表达参与细菌的入侵期间受体蛋白的表达 。该picturES显示Apotome图像。每个面板的中心:XY 细胞的正视图;顶部和右侧:通过在Z -平面几个光学片的横截面。所述HIBCPP细胞的顶膜(由箭头表示)被定位朝向顶部部分的顶部以及朝向右侧的,分别在右各帧的,。比例尺= 10微米。该图中的数据也被在Gründler 等人先前公布的, 微生物感染15:291-301,DOI:10.1016 / j.micinf.2012.12.005(2013);重新打印许可。 请点击此处查看该图的放大版本。

图4
图4:DMSO和细胞松弛素D对HIBCPP细胞的屏障功能影响的屏障FUNC影响化用TEER值和FITC菊粉焊剂的测量来确定。结果示一个代表性实验,这是在一式三份进行。 HIBCPP细胞在指示量的2小时用DMSO以及Cytochlalasin D处理。 请点击此处查看该图的放大版本。

当在浓度施加高达0.2体积%,DMSO不影响膜电位( 图4A)。浓度为0.1体积%到DMSO中0.5体积%也不会影响细胞( 图4B)的细胞旁渗透性。与此相反,1微克/毫升松弛素D引起TEER伴随通过细胞层的增加的FITC-菊粉焊剂下降( 图4A,B)。

图5 5:N.入侵 脑膜炎 成HIBCPP细胞。双免疫荧光显微镜显示入侵(绿色)和坚持(黄色)细菌。图像内的白框表示在右侧的放大的画面细节。没有观察到N.基底入侵球菌菌株MC58(A)和MC58siaD - ,缺陷的胶囊生产(B)中 ,而被确定为α14(C)仅轻微入侵速率。该图表明,MC58和MC58siaD的侵袭率-相比α14时是高度显著(**,P <0.01)。当MC58或MC58siaD显示极端意义- ,分别进行了比较,α14(***,P <0.001)和MC58siaD -进行了比较MC58(###,P <0.001)。比例尺籼稻德10微米。 。在D图最初发表于Borkowski 等人 ,J神经炎症 11:163,DOI:10.1186 / s12974-014-0163-X(2014);重新打印许可。 请点击此处查看该图的放大版本。

Subscription Required. Please recommend JoVE to your librarian.

Discussion

CP的上皮细胞形成分隔从血液2,3脑脊液的BCSFB。我们最近成立了HIBCPP细胞系作为BCSFB的功能人体模型。细胞显示在体外 BCSFB的重要屏障功能,包括一个高的膜电位的发展,一个低渗透性的大分子,以及紧密连接5的连续股线的存在。的TJ蛋白向细胞的顶/底外侧极性。极性是表面受体的定向定位特定的转运蛋白高的重要性,以及。我们已经证明了受体ECAD和Met以及ATP结合盒的部件(ABC)转运蛋白家族的极性本地化维持在CNS 6,7内的高度控制的微环境。

有几种微生物能够通过克服BCSFB获得进入中枢神经系统,导入区克至严重 ​​的疾病,包括脑膜炎8。之一是能够侵入的CP上皮细胞的病原体是人类特有的革兰氏阴性细菌N.球菌 9,10,这已被证明是在极性的方式专门从基底细胞侧5输入HIBCPP细胞。这是为横跨BCSFB脑感染的生理相关方向时细菌性脑膜炎的过程中散发和遭遇的CP上皮细胞从血液中。为了反映在体外这种情况下,HIBCPP细胞生长在过滤器的下侧。与此相反的标准过滤器插入件模型,其中细胞接种到上部隔室,这组倒置培养系统的最多允许从基底外侧细胞侧5,21特异性感染上皮细胞。

基底外侧侵入HIBCPP细胞已观察到这两个致病N.脑膜炎 - ,而非致病性载体隔离α14只显示边际入侵15。这说明了HIBCPP细胞模型提供扫描在入侵过程中所涉及的新的致病因子提供突变体文库的可能性。值得注意的是,它已经证明,其他病原体包括L.菌猪链球菌猪链球菌 )侵入HIBCPP细胞极性时尚5,7。

为了识别和入侵的微生物进入CNS响应,CP的上皮细胞表达的PRR 22,23。 Toll样受体代表蓝耳中的一个重要的家庭。定性的RT-PCR分析表明,HIBCPP细胞表达几种TLR受体以及相关的共同受体和衔接分子的MyD88 15。已经观察到,受体TLR2和TLR4是参与了细胞防御反应为N.悲风itidis荚膜多糖24。微阵列方法的结果也表明,感染了N. HIBCPP细胞脑膜炎显示信号转导途径的诱导,除其他因素也涉及转录调节某些靶基因包括IL6 25,26,促炎细胞因子的表达IκBζ15,强制性的。

为了证实这些发现,感染了N. HIBCPP细胞在一个倒置培养系统脑膜炎已被用于研究的细胞因子和趋化因子的释放。已知的是这个过程的目的是指使由先天免疫系统13,14的活化的炎症反应。 HIBCPP细胞 产生和分泌的细胞因子和趋化因子,包括CXCL1-3,IL6,IL8和TNFα15,16,这已经描述吸引嗜中性粒细胞和单核细胞27。通过采用我们的模型系统,我们发现polymorphnuclear中性粒细胞,单核细胞和也的T淋巴细胞通过HIBCPP细胞迁移16,28,强调HIBCPP细胞是适合破译免疫细胞的迁移跨上机制。

本文中所提出的模型系统还提供修改的机会。 HIBCPP细胞可以用抗体或化学抑制剂来识别受体和信号感染期间参与转导通路进行预处理。 DMSO中,对于细胞刺激试剂的共同溶剂可以合适的量被施加到HIBCPP细胞,其显示不受整个研究时间过程的化学稳定屏障功能。这个属性也相应于药物研究模型。

虽然我们已经证明,HIBCPP细胞显示极性的一定程度,就不会自然地反映在体内模型的每一个细节。 <EM>例如ABC转运蛋白家族成员的定位只是部分匹配的预期模式6,为什么制药应用可以大概只适用于一个有限的范围内。另外,HIBCPP细胞似乎没有产生CSF(数据未显示)。然而,我们已发现,HIBCPP细胞表达对RNA水平5蛋白质甲状腺素,胰岛素样生长因子2(IGF2)和叉头框J1(FOXJ1),这是用于CP上皮细胞29,30,31,32特性标志物蛋白。这一观察结果支持HIBCPP细胞保留了CP上皮细胞的典型特性。重要的是,它应当指出,HIBCPP细胞不存在接触抑制。因此,至关重要的使用细胞进行实验时,达到适当的TEER值。此外,HIBCPP细胞不应超出通道38使用,因为潜在的开发阻挡功能似乎具有更高的通道下降。最后,研究小鬼在BCSFB感染的共培养模型组成的CP上皮细胞和内皮的期间细胞 - 细胞相互作用的行为将是高的兴趣,但这样的模式仍需要开发。

总之,HIBCPP细胞培养物模型可用于揭示重要侵入途径和底层信号转导途径,特别是在BCSFB特定人类病原体。因为它是已知的CP的上皮细胞也参与神经变性疾病,包括阿尔茨海默病以及多发性硬化症2,以及在肿瘤细胞33的脑转移,一般种类繁多的机会该系统提供对疾病相关机制调查,提供寻找新的治疗目标的潜力。

Subscription Required. Please recommend JoVE to your librarian.

Materials

Name Company Catalog Number Comments
0.25% Trypsin-EDTA Gibco 25200-056
4´,6 diamidino-2-phenylindole (DAPI) Life Technologies D1306
12-well plates Starlab CC7682-7512
24-well plates Starlab CC7682-7524
Anti Neisseria meningitidis α-OMP This antibody was a gift from Drs. H. Claus and U. Vogel (University of Würzburg, Germany)
Alexa Fluor 488 (chicken anti rabbit) Invitrogen A21441
Alexa Fluor 594 (chicken anti rabbit) Invitrogen A21442
Alexa Fluor 660 Phalloidin Invitrogen A22285
Bovine serum albumine (BSA) Calbiochem 12659
Chocolate agar plates Biomerieux 43109
Cytochalasin D Sigma C8273
DMEM/F12 + L-Glut + 15 mM HEPES Gibco 31330-095
DMEM/F12 + L-Glut + 15 mM HEPES w/o Phenolred Gibco 11039-047
Dimethyl sulfoxide Sigma D2650
Fetal calf serum (FCS) Life Technologies 10270106
FITC-Inulin Sigma F3272
Insulin Sigma 19278
MgCl2 Sigma 2393
NaHCO3 Sigma 55761
PBS + Mg + Ca Gibco 14040-174
Penicillin/Streptomycin MP Biomedicals 1670049
Polyvitex Biomerieux 55651
Proteose peptone BD 211684
Serum-free medium Gibco 10902-096
Thincert cell culture inserts for 24-well plates, pore size 3 µm Greiner 662630
Tissue culture flask 75 cm² red cap sterile Greiner 658175
Triton X-100 Sigma T8787
Volt-Ohm Meter Millicell-ERS2 with MERSSTX01 electrode Millipore MERSSTX00

DOWNLOAD MATERIALS LIST

References

  1. Abott, N. J., Patabendige, A. A. K., Dolman, D. E. M., Yusof, S. R., Begley, D. J. Structure and function of the blood-brain barrier. Neurobiol Dis. 37, 13-25 (2009).
  2. Wolburg, H., Paulus, W. Choroid plexus: biology and pathology. Acta Neuropathol. 119, 75-88 (2010).
  3. Engelhardt, B., Sorokin, L. The blood-brain and the blood-cerebrospinal fluid barriers: function and dysfunction. Semin Imunopathol. 31, 497-511 (2009).
  4. Ishiwata, I., Ishiwata, C., Ishiwata, E., Sato, Y., Kiguchi, K., Tachibana, T., et al. Establishment and characterization of a human malignant choroid plexus papilloma cell line (HIBCPP). Hum Cell. 18, 67-72 (2005).
  5. Schwerk, C., Papandreou, T., Schuhmann, D., Nickol, L., Borkowski, J., Steinmann, U., et al. Polar invasion and translocation of Neisseria meningitidis and Streptococcus suis in anovel human model of the blood-cerebrospinal fluid barrier. PloS One. 7, e30069 (2012).
  6. Bernd, A., Ott, M., Ishikawa, H., Schroten, H., Schwerk, C., Fricker, G. Characterization of efflux transport proteins of the human choroid plecus papilloma cell line HIBCPP, a functional in vitro model of the blood-cerebrospinal fluid barrier. Pharm Res. , (2014).
  7. Gründler, T., Quednau, N., Stump, C., Orian-Rousseau, V., Ishikawa, H., Wolburg, H., et al. The surface proteins InlA and InlB are interdependently required for polar basolateral invasion by Listeria monocytogenes in a human model of the blood-cerebrospinal fluid barrier. Microbes Infect. 15, 291-301 (2013).
  8. Schwerk, C., Tenenbaum, T., Kwang, S. K., Schroten, H. The choroid plexus - a multi-role player during infectious diseases of the CNS. Front Cell Neurosci. 9, 80 (2015).
  9. Pron, B., Taha, M. K., Rambaud, C., Fournet, J. C., Pattey, N., Monnet, J. P., et al. Interaction of Neisseria meningtidis with the components of the blood-brain barrier correlates with increased expression of PilC. J Infect Dis. 176, 1285-1292 (1997).
  10. Guarner, J., Greer, P. W., Whitney, A., Shieh, W. J., Fischer, M., White, E. H., Carlone, G. M., et al. Pathogenesis and diagnosis of human meningococcal disease using immunohistochemical and PCR assays assays. Am J Clin Pathol. 122, 754-764 (2004).
  11. Pizarro-Cerda, J., Kuhbacher, A., Cossart, P. Entry of Listeria monocytogenes in mammalian epithelial cells: an updated view. Cold Spring Harb Perspect Med. 2, (2012).
  12. Beutler, B. Microbe sensing, positive feedback loops and the pathogenesis of inflammatory diseases. Immunol. Rev. 227, 248-263 (2009).
  13. Wilson, E. H., Weninger, W., Hunter, C. A. Trafficking of immune cells in the central nervous system. J Clin Invest. 120, 1368-1379 (2010).
  14. Meeker, R. B., Williams, K., Killebrew, D. A., Hudson, L. C. Cell trafficking through the choroid plexus. Cell Adh Migr. 6, 390-396 (2012).
  15. Borkowski, J., Li, L., Steinmann, U., Quednau, N., Stump-Guthier, C., Weiss, C., et al. Neisseria meningitidis elicits a pro-inflammatory response involving I kappa B zeta in a human blood-cerebrospinal fluid barrier model. J Neuroinflammation. 11, 163 (2014).
  16. Steinmann, U., Borkowski, J., Wolburg, H., Schroppel, B., Findeisen, P., Weiss, C., et al. Transmigration of polymorphnuclear neutrophils and monocytes through the human blood-cerebrospinal fluid barrier after bacterial infection in vitro. J Neuroinflammation. 10, 30 (2013).
  17. McGuiness, B. T., Clarke, I. N., Lambden, P. R., Barlow, A. K., Poolman, J. T., Heckels, J. E. Point mutation in meningococcal por A gene associated with increased endemic disease. Lancet. 337, 514-517 (1991).
  18. Ram, S., Cox, A. D., Wright, J. C., Vogel, U., Getzlaff, S., Boden, R. Neisserial lipopolysaccharide is a target for complement component C4b. inner core phosphoethanolamine residues define C4b linkage specificity. J Biol Chem. 278, 50853-50862 (2003).
  19. Claus, H., Maiden, M. C., Maag, R., Frosch, M., Vogel, U. Many carried meningococci lack the genes required for capsule synthesis and transport. Microbiology. 148, 1813-1819 (2002).
  20. Claus, H., Maiden, M. C., Wilson, D. J., Mccarthy, N. D., Jolley, K. A., Urwin, R., et al. Genetic analysis of meningococci carried by children and young adults. J Infect Dis. 191, 1263-1271 (2005).
  21. Tenenbaum, T., Papandreou, T., Gellrich, D., Friedrichs, U., Seibt, A., Adam, R., et al. Polar bacterial invasion and translocation of Streptococcus suis across the blood-cerebrospinal fluid barrier in vitro. Cell Microbiol. 11, 323-336 (2009).
  22. Laflamme, N., Echchannaoui, H., Landmann, R., Rivest, S. Cooperation between toll-like receptor 2 and 4 in the brain of mice challenged with cell wall components derived from gram-negative and gram-positive bacteria. Eur J Immunol. 33, 1127-1138 (2003).
  23. Laflamme, S., Rivest, S. Toll-like receptor 4: the missing link of the cerebral innate immune response triggered by circulating gram-negative bacterial cell wall components. FASEB J. 15, 155-163 (2001).
  24. Zughaier, S. M. Neisseria meningitidis capsular polysaccharides indice inflammatory responses via TLR2 and TLR4-MD-2. J Leukoc Biol. 89, 469-480 (2011).
  25. Yamamoto, M., Yamazaki, S., Uematsu, S., Sato, S., Hemmi, M., Hoshino, K., et al. Regulation of Toll/IL-1-receptor -mediated gene expression by the inducible nuclear protein IkappaBzeta. Nature. 430, 218-222 (2004).
  26. Lorenz, J., Zahlten, J., Pollok, I., Lippmann, J., Scharf, S., N'Guessan, P. D., et al. Legionella pheumophila-induced IkappaBzeta-dependent expression of interleukin-6 in lung epithelium. Eur Respir J. 37, 648-657 (2011).
  27. Jaerve, A., Muller, H. W. Chemokines in CNS injury and repair. Cell Tissue Res. 349, 229-248 (2012).
  28. Schneider, H., Weber, C. E., Schoeller, J., Steinmann, U., Borkowski, J., Ishikawa, H., et al. Chemotaxis of T-cells after infection of human choroid plexus papilloma cells with Echovirus 30 in an in vitro model of the blood-cerebrospinal fluid barrier. Virus Res. 170, 66-74 (2012).
  29. Chodobski, A., Szmydynger-Chodobska, J. Choroid plexus: Target for polypeptides and site of their synthesis. Microsc. Res. Tech. 52, 65-82 (2001).
  30. Dickson, P. W., Schreiber, G. High levels of messenger RNA for transthyretin (prealbumin) in human choroid plexus. Neurosci. Lett. 66, 311-315 (1986).
  31. Stylianopoulou, F., Herbert, J., Soares, M. B., Efstratiadis, A. Expression of the insulin-like growth factor II gene in the choroid plexus and the leptomeninges of the adult rat central nervous system. Proc Natl Acad Sci USA. 85, 141-145 (1988).
  32. Lim, L., Zhou, H., Costa, R. H. The winged helix transcription factor HFH-4 is expressed during choroid plexus epithelial development in the mouse embryo. Proc. Natl Acad. Sci. USA. 94, 3094-3099 (1997).
  33. Vandenhaute, E., Stump-Guthier, C., Lasierra Losada, M., Tenenbaum, T., Rudolph, H., Ishikawa, H., et al. The choroid plexus may be an underestimated site of tumor invasion to the brain: an in vitro study using neuroblastoma cell lines. Cancer Cell Int. , 15-102 (2015).

Tags

医药,第111,血 - 脑脊液屏障,细胞培养滤芯,细胞系,脉络丛,HIBCPP,宿主 - 病原体相互作用,人,
人体血液,脑脊液屏障的脉络丛上皮基于Cell的产品型号从基底外侧面研究细菌感染
Play Video
PDF DOI DOWNLOAD MATERIALS LIST

Cite this Article

Dinner, S., Borkowski, J.,More

Dinner, S., Borkowski, J., Stump-Guthier, C., Ishikawa, H., Tenenbaum, T., Schroten, H., Schwerk, C. A Choroid Plexus Epithelial Cell-based Model of the Human Blood-Cerebrospinal Fluid Barrier to Study Bacterial Infection from the Basolateral Side. J. Vis. Exp. (111), e54061, doi:10.3791/54061 (2016).

Less
Copy Citation Download Citation Reprints and Permissions
View Video

PLAYLIST

  • Research • Medicine
    Estimation of Urinary Nanocrystals in Humans using Calcium Fluorophore Labeling and Nanoparticle Tracking Analysis
  • Research • Medicine
    Development and Evaluation of 3D-Printed Cardiovascular Phantoms for Interventional Planning and Training
  • Research • Medicine
    Human Fetal Blood Flow Quantification with Magnetic Resonance Imaging and Motion Compensation
  • Research • Medicine
    Digital Handwriting Analysis of Characters in Chinese Patients with Mild Cognitive Impairment
  • Research • Medicine
    Segmentation and Linear Measurement for Body Composition Analysis using Slice-O-Matic and Horos
  • Research • Medicine
    Magnetic Resonance Imaging of Multiple Sclerosis at 7.0 Tesla
  • Research • Medicine
    Real-Time Magnetic Resonance Guided Focused Ultrasound for Painful Bone Metastases
  • Research • Medicine
    Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping
  • Research • Medicine
    Obtaining Quality Extended Field-of-View Ultrasound Images of Skeletal Muscle to Measure Muscle Fascicle Length
  • Research • Medicine
    Lung CT Segmentation to Identify Consolidations and Ground Glass Areas for Quantitative Assesment of SARS-CoV Pneumonia
  • Research • Medicine
    Electroretinogram Recording for Infants and Children under Anesthesia to Achieve Optimal Dark Adaptation and International Standards
  • Research • Medicine
    Measurement of Tissue Oxygenation Using Near-Infrared Spectroscopy in Patients Undergoing Hemodialysis
  • Research • Medicine
    Evaluation of Capnography Sampling Line Compatibility and Accuracy when Used with a Portable Capnography Monitor
  • Research • Medicine
    Simultaneous Laryngopharyngeal and Conventional Esophageal pH Monitoring
  • Research • Medicine
    Real-Time Monitoring of Neurocritical Patients with Diffuse Optical Spectroscopies
  • Research • Neuroscience
    Evaluating Postural Control and Lower-extremity Muscle Activation in Individuals with Chronic Ankle Instability
  • Research • Medicine
    Assessment of Dependence in Activities of Daily Living Among Older Patients in an Acute Care Unit
  • Research • Medicine
    Validated LC-MS/MS Panel for Quantifying 11 Drug-Resistant TB Medications in Small Hair Samples
  • Research • Medicine
    International Expert Consensus and Recommendations for Neonatal Pneumothorax Ultrasound Diagnosis and Ultrasound-guided Thoracentesis Procedure
  • Research • Biology
    A Finite Element Approach for Locating the Center of Resistance of Maxillary Teeth
  • Research • Medicine
    Lower Limb Biomechanical Analysis of Healthy Participants
  • Research • Neuroscience
    Assessing Early Stage Open-Angle Glaucoma in Patients by Isolated-Check Visual Evoked Potential
  • Research • Medicine
    Oral Health Assessment by Lay Personnel for Older Adults
  • Research • Medicine
    Determining and Controlling External Power Output During Regular Handrim Wheelchair Propulsion
  • Research • Medicine
    A Whole Body Dosimetry Protocol for Peptide-Receptor Radionuclide Therapy (PRRT): 2D Planar Image and Hybrid 2D+3D SPECT/CT Image Methods
  • Research • Medicine
    Measurement of Carotenoids in Perifovea using the Macular Pigment Reflectometer
  • Research • Medicine
    Assessment of Static Graviceptive Perception in the Roll-Plane using the Subjective Visual Vertical Paradigm
  • Research • Medicine
    Learning Modern Laryngeal Surgery in a Dissection Laboratory
  • Research • Medicine
    DIPLOMA Approach for Standardized Pathology Assessment of Distal Pancreatectomy Specimens
  • Research • Medicine
    A Computerized Functional Skills Assessment and Training Program Targeting Technology Based Everyday Functional Skills
  • Research • Medicine
    Imaging Features of Systemic Sclerosis-Associated Interstitial Lung Disease
  • Research • Medicine
    Integrating Augmented Reality Tools in Breast Cancer Related Lymphedema Prognostication and Diagnosis
  • Research • Medicine
    Ultrasonographic Assessment During Cardiopulmonary Resuscitation
  • Research • Medicine
    Measurement of the Hepatic Venous Pressure Gradient and Transjugular Liver Biopsy
  • Research • Medicine
    Patient Directed Recording of a Bipolar Three-Lead Electrocardiogram using a Smartwatch with ECG Function
  • Research • Medicine
    Traditional Trail Making Test Modified into Brand-new Assessment Tools: Digital and Walking Trail Making Test
  • Research • Medicine
    Use of Magnetic Resonance Imaging and Biopsy Data to Guide Sampling Procedures for Prostate Cancer Biobanking
  • Research • Medicine
    A Fluorescence-based Assay for Characterization and Quantification of Lipid Droplet Formation in Human Intestinal Organoids
  • Research • Medicine
    A Novel Non-invasive Method for the Detection of Elevated Intra-compartmental Pressures of the Leg
  • Research • Medicine
    Quantitative Mapping of Specific Ventilation in the Human Lung using Proton Magnetic Resonance Imaging and Oxygen as a Contrast Agent
  • Research • Neuroscience
    Portable Thermographic Screening for Detection of Acute Wallenberg's Syndrome
  • Research • Medicine
    Use of MRI-ultrasound Fusion to Achieve Targeted Prostate Biopsy
  • Research • Medicine
    Testing of all Six Semicircular Canals with Video Head Impulse Test Systems
  • Research • Medicine
    Protocol and Guidelines for Point-of-Care Lung Ultrasound in Diagnosing Neonatal Pulmonary Diseases Based on International Expert Consensus
  • Research • Neuroscience
    Bilateral Assessment of the Corticospinal Pathways of the Ankle Muscles Using Navigated Transcranial Magnetic Stimulation
  • Research • Medicine
    Targeting Gray Rami Communicantes in Selective Chemical Lumbar Sympathectomy
  • Research • Medicine
    Multi-Modal Home Sleep Monitoring in Older Adults
  • Research • Medicine
    Cardiac Magnetic Resonance for the Evaluation of Suspected Cardiac Thrombus: Conventional and Emerging Techniques
  • Research • Medicine
    Observational Study Protocol for Repeated Clinical Examination and Critical Care Ultrasonography Within the Simple Intensive Care Studies
  • Research • Medicine
    Measurements of Motor Function and Other Clinical Outcome Parameters in Ambulant Children with Duchenne Muscular Dystrophy
  • Research • Medicine
    Assessment of the Efficacy of An Osteopathic Treatment in Infants with Biomechanical Impairments to Suckling
  • Research • Medicine
    Quantification of Levator Ani Hiatus Enlargement by Magnetic Resonance Imaging in Males and Females with Pelvic Organ Prolapse
  • Research • Medicine
    Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain
  • Research • Medicine
    Generation of Human 3D Lung Tissue Cultures (3D-LTCs) for Disease Modeling
  • Research • Medicine
    Proton Therapy Delivery and Its Clinical Application in Select Solid Tumor Malignancies
  • Research • Medicine
    Combining Volumetric Capnography And Barometric Plethysmography To Measure The Lung Structure-function Relationship
  • Research • Medicine
    Two-Dimensional X-Ray Angiography to Examine Fine Vascular Structure Using a Silicone Rubber Injection Compound
  • Research • Medicine
    Preparation, Procedures and Evaluation of Platelet-Rich Plasma Injection in the Treatment of Knee Osteoarthritis
  • Research • Medicine
    Cardiac Magnetic Resonance Imaging at 7 Tesla
  • Research • Medicine
    Semi-quantitative Assessment Using [18F]FDG Tracer in Patients with Severe Brain Injury
  • Research • Medicine
    Handheld Metal Detector Screening for Metallic Foreign Body Ingestion in Children
  • Research • Medicine
    Conducting Maximal and Submaximal Endurance Exercise Testing to Measure Physiological and Biological Responses to Acute Exercise in Humans
  • Research • Medicine
    A Metadata Extraction Approach for Clinical Case Reports to Enable Advanced Understanding of Biomedical Concepts
  • Research • Medicine
    Autonomic Function Following Concussion in Youth Athletes: An Exploration of Heart Rate Variability Using 24-hour Recording Methodology
  • Research • Medicine
    Hydra, a Computer-Based Platform for Aiding Clinicians in Cardiovascular Analysis and Diagnosis
  • Research • Medicine
    Objective Nociceptive Assessment in Ventilated ICU Patients: A Feasibility Study Using Pupillometry and the Nociceptive Flexion Reflex
  • Research • Medicine
    'Boden Food Plate': Novel Interactive Web-based Method for the Assessment of Dietary Intake
  • Research • Medicine
    Anogenital Distance and Perineal Measurements of the Pelvic Organ Prolapse (POP) Quantification System
  • Research • Medicine
    Bedside Ultrasound for Guiding Fluid Removal in Patients with Pulmonary Edema: The Reverse-FALLS Protocol
  • Research • Medicine
    Muscle Imbalances: Testing and Training Functional Eccentric Hamstring Strength in Athletic Populations
  • Research • Medicine
    Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae
  • Research • Medicine
    Skeletal Muscle Neurovascular Coupling, Oxidative Capacity, and Microvascular Function with 'One Stop Shop' Near-infrared Spectroscopy
  • Research • Medicine
    Collecting Hair Samples for Hair Cortisol Analysis in African Americans
  • Research • Medicine
    In Vivo Morphometric Analysis of Human Cranial Nerves Using Magnetic Resonance Imaging in Menière's Disease Ears and Normal Hearing Ears
  • Research • Medicine
    Measuring the Carotid to Femoral Pulse Wave Velocity (Cf-PWV) to Evaluate Arterial Stiffness
  • Research • Medicine
    Standardized Measurement of Nasal Membrane Transepithelial Potential Difference (NPD)
  • Research • Medicine
    Taste Exam: A Brief and Validated Test
  • Research • Medicine
    Absorption of Nasal and Bronchial Fluids: Precision Sampling of the Human Respiratory Mucosa and Laboratory Processing of Samples
  • Research • Medicine
    Methodology for Sputum Induction and Laboratory Processing
  • Research • Medicine
    Electrophysiological Measurement of Noxious-evoked Brain Activity in Neonates Using a Flat-tip Probe Coupled to Electroencephalography
  • Research • Medicine
    A Detailed Protocol for Physiological Parameters Acquisition and Analysis in Neurosurgical Critical Patients
  • Research • Medicine
    Oral Biofilm Sampling for Microbiome Analysis in Healthy Children
  • Research • Medicine
    Using Retinal Imaging to Study Dementia
  • Research • Medicine
    Application of an Amplitude-integrated EEG Monitor (Cerebral Function Monitor) to Neonates
  • Research • Medicine
    3D Ultrasound Imaging: Fast and Cost-effective Morphometry of Musculoskeletal Tissue
  • Research • Medicine
    The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo
  • Research • Medicine
    A Component-resolved Diagnostic Approach for a Study on Grass Pollen Allergens in Chinese Southerners with Allergic Rhinitis and/or Asthma
  • Research • Medicine
    A Novel Method: Super-selective Adrenal Venous Sampling
  • Research • Medicine
    A Method for Quantifying Upper Limb Performance in Daily Life Using Accelerometers
  • Research • Medicine
    Non-invasive Assessments of Subjective and Objective Recovery Characteristics Following an Exhaustive Jump Protocol
  • Research • Medicine
    Experimental Protocol of a Three-minute, All-out Arm Crank Exercise Test in Spinal-cord Injured and Able-bodied Individuals
  • Research • Medicine
    Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle
  • Research • Medicine
    Assessment of Pulmonary Capillary Blood Volume, Membrane Diffusing Capacity, and Intrapulmonary Arteriovenous Anastomoses During Exercise
  • Research • Medicine
    Assessment of Child Anthropometry in a Large Epidemiologic Study
  • Research • Medicine
    Video Movement Analysis Using Smartphones (ViMAS): A Pilot Study
  • Research • Medicine
    Network Analysis of Foramen Ovale Electrode Recordings in Drug-resistant Temporal Lobe Epilepsy Patients
  • Research • Medicine
    A Model to Simulate Clinically Relevant Hypoxia in Humans
  • Research • Medicine
    Interictal High Frequency Oscillations Detected with Simultaneous Magnetoencephalography and Electroencephalography as Biomarker of Pediatric Epilepsy
  • Research • Medicine
    Induction and Assessment of Exertional Skeletal Muscle Damage in Humans
  • Research • Medicine
    A Detailed Protocol for Perspiration Monitoring Using a Novel, Small, Wireless Device
  • Research • Medicine
    Drug-Induced Sleep Endoscopy (DISE) with Target Controlled Infusion (TCI) and Bispectral Analysis in Obstructive Sleep Apnea
  • Research • Medicine
    Integrated Compensatory Responses in a Human Model of Hemorrhage
  • Research • Medicine
    Transthoracic Speckle Tracking Echocardiography for the Quantitative Assessment of Left Ventricular Myocardial Deformation
  • Research • Medicine
    Impression Cytology of the Lid Wiper Area
  • Research • Behavior
    A Protocol of Manual Tests to Measure Sensation and Pain in Humans
  • Research • Medicine
    Unbiased Deep Sequencing of RNA Viruses from Clinical Samples
  • Research • Medicine
    A Choroid Plexus Epithelial Cell-based Model of the Human Blood-Cerebrospinal Fluid Barrier to Study Bacterial Infection from the Basolateral Side
  • Research • Medicine
    Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile
  • Research • Medicine
    Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing
  • Research • Medicine
    Ultrasound Assessment of Endothelial Function: A Technical Guideline of the Flow-mediated Dilation Test
  • Research • Medicine
    Using a Laminating Technique to Perform Confocal Microscopy of the Human Sclera
  • Research • Medicine
    Intravenous Endotoxin Challenge in Healthy Humans: An Experimental Platform to Investigate and Modulate Systemic Inflammation
  • Research • Medicine
    Modeling and Simulations of Olfactory Drug Delivery with Passive and Active Controls of Nasally Inhaled Pharmaceutical Aerosols
  • Research • Medicine
    Exosomal miRNA Analysis in Non-small Cell Lung Cancer (NSCLC) Patients' Plasma Through qPCR: A Feasible Liquid Biopsy Tool
  • Research • Medicine
    A Multimodal Imaging- and Stimulation-based Method of Evaluating Connectivity-related Brain Excitability in Patients with Epilepsy
  • Research • Medicine
    Measuring Cardiac Autonomic Nervous System (ANS) Activity in Toddlers - Resting and Developmental Challenges
  • Research • Medicine
    Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function
  • Research • Medicine
    Quantitative Fundus Autofluorescence for the Evaluation of Retinal Diseases
  • Research • Medicine
    Diagnosis of Musculus Gastrocnemius Tightness - Key Factors for the Clinical Examination
  • Research • Medicine
    Stereo-Electro-Encephalo-Graphy (SEEG) With Robotic Assistance in the Presurgical Evaluation of Medical Refractory Epilepsy: A Technical Note
  • Research • Medicine
    Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease
  • Research • Medicine
    Transcutaneous Microcirculatory Imaging in Preterm Neonates
  • Research • Medicine
    Using an Ingestible Telemetric Temperature Pill to Assess Gastrointestinal Temperature During Exercise
  • Research • Medicine
    Design, Fabrication, and Administration of the Hand Active Sensation Test (HASTe)
  • Research • Medicine
    MRI-guided dmPFC-rTMS as a Treatment for Treatment-resistant Major Depressive Disorder
  • Research • Medicine
    Functional Human Liver Preservation and Recovery by Means of Subnormothermic Machine Perfusion
  • Research • Medicine
    A Multicenter MRI Protocol for the Evaluation and Quantification of Deep Vein Thrombosis
  • Research • Medicine
    Determining The Electromyographic Fatigue Threshold Following a Single Visit Exercise Test
  • Research • Medicine
    Use of Electromagnetic Navigational Transthoracic Needle Aspiration (E-TTNA) for Sampling of Lung Nodules
  • Research • Medicine
    Trabecular Meshwork Response to Pressure Elevation in the Living Human Eye
  • Research • Medicine
    In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses
  • Research • Medicine
    Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy
  • Research • Medicine
    Human Brown Adipose Tissue Depots Automatically Segmented by Positron Emission Tomography/Computed Tomography and Registered Magnetic Resonance Images
  • Research • Medicine
    Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy
  • Research • Medicine
    A Methodological Approach to Non-invasive Assessments of Vascular Function and Morphology
  • Research • Medicine
    Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling
  • Research • Medicine
    State of the Art Cranial Ultrasound Imaging in Neonates
  • Research • Medicine
    Measurement of Dynamic Scapular Kinematics Using an Acromion Marker Cluster to Minimize Skin Movement Artifact
  • Research • Medicine
    The Supraclavicular Fossa Ultrasound View for Central Venous Catheter Placement and Catheter Change Over Guidewire
  • Research • Medicine
    Ultrasound Assessment of Endothelial-Dependent Flow-Mediated Vasodilation of the Brachial Artery in Clinical Research
  • Research • Medicine
    Tracking the Mammary Architectural Features and Detecting Breast Cancer with Magnetic Resonance Diffusion Tensor Imaging
  • Research • Medicine
    A Neuroscientific Approach to the Examination of Concussions in Student-Athletes
  • Research • Medicine
    DTI of the Visual Pathway - White Matter Tracts and Cerebral Lesions
  • Research • Medicine
    Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples
  • Research • Medicine
    Fundus Photography as a Convenient Tool to Study Microvascular Responses to Cardiovascular Disease Risk Factors in Epidemiological Studies
  • Research • Medicine
    A Multi-Modal Approach to Assessing Recovery in Youth Athletes Following Concussion
  • Research • Medicine
    Clinical Assessment of Spatiotemporal Gait Parameters in Patients and Older Adults
  • Research • Medicine
    Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue
  • Research • Medicine
    Collection and Extraction of Saliva DNA for Next Generation Sequencing
  • Research • Medicine
    Fast and Accurate Exhaled Breath Ammonia Measurement
  • Research • Medicine
    Developing Neuroimaging Phenotypes of the Default Mode Network in PTSD: Integrating the Resting State, Working Memory, and Structural Connectivity
  • Research • Medicine
    Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens
  • Research • Medicine
    Assessment of Vascular Function in Patients With Chronic Kidney Disease
  • Research • Medicine
    Coordinate Mapping of Hyolaryngeal Mechanics in Swallowing
  • Research • Medicine
    Network Analysis of the Default Mode Network Using Functional Connectivity MRI in Temporal Lobe Epilepsy
  • Research • Medicine
    EEG Mu Rhythm in Typical and Atypical Development
  • Research • Medicine
    The Multiple Sclerosis Performance Test (MSPT): An iPad-Based Disability Assessment Tool
  • Research • Medicine
    Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples
  • Research • Medicine
    Dynamic Visual Tests to Identify and Quantify Visual Damage and Repair Following Demyelination in Optic Neuritis Patients
  • Research • Medicine
    Primary Culture of Human Vestibular Schwannomas
  • Research • Medicine
    Utility of Dissociated Intrinsic Hand Muscle Atrophy in the Diagnosis of Amyotrophic Lateral Sclerosis
  • Research • Medicine
    Lesion Explorer: A Video-guided, Standardized Protocol for Accurate and Reliable MRI-derived Volumetrics in Alzheimer's Disease and Normal Elderly
  • Research • Medicine
    Pulse Wave Velocity Testing in the Baltimore Longitudinal Study of Aging
  • Research • Medicine
    Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters
  • Research • Medicine
    3D-Neuronavigation In Vivo Through a Patient's Brain During a Spontaneous Migraine Headache
  • Research • Medicine
    A Novel Application of Musculoskeletal Ultrasound Imaging
  • Research • Medicine
    Computerized Dynamic Posturography for Postural Control Assessment in Patients with Intermittent Claudication
  • Research • Medicine
    Collecting Saliva and Measuring Salivary Cortisol and Alpha-amylase in Frail Community Residing Older Adults via Family Caregivers
  • Research • Medicine
    Diffusion Tensor Magnetic Resonance Imaging in the Analysis of Neurodegenerative Diseases
  • Research • Medicine
    Transcriptomic Analysis of Human Retinal Surgical Specimens Using jouRNAl
  • Research • Medicine
    Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens
  • Research • Medicine
    Evaluation of Respiratory Muscle Activation Using Respiratory Motor Control Assessment (RMCA) in Individuals with Chronic Spinal Cord Injury
  • Research • Medicine
    Minimal Erythema Dose (MED) Testing
  • Research • Medicine
    Measuring Cardiac Autonomic Nervous System (ANS) Activity in Children
  • Research • Medicine
    Collecting And Measuring Wound Exudate Biochemical Mediators In Surgical Wounds
  • Research • Medicine
    A Research Method For Detecting Transient Myocardial Ischemia In Patients With Suspected Acute Coronary Syndrome Using Continuous ST-segment Analysis
  • Research • Medicine
    Using a Chemical Biopsy for Graft Quality Assessment
  • Research • Medicine
    Characterizing Exon Skipping Efficiency in DMD Patient Samples in Clinical Trials of Antisense Oligonucleotides
  • Research • Medicine
    In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes
  • Research • Medicine
    Normothermic Ex Situ Heart Perfusion in Working Mode: Assessment of Cardiac Function and Metabolism
  • Research • Medicine
    Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats
  • Research • Medicine
    Bronchoalveolar Lavage (BAL) for Research; Obtaining Adequate Sample Yield
  • Research • Medicine
    Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants
  • Research • Medicine
    Tilt Testing with Combined Lower Body Negative Pressure: a "Gold Standard" for Measuring Orthostatic Tolerance
  • Research • Medicine
    Driving Simulation in the Clinic: Testing Visual Exploratory Behavior in Daily Life Activities in Patients with Visual Field Defects
  • Research • Medicine
    Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods
  • Research • Medicine
    Portable Intermodal Preferential Looking (IPL): Investigating Language Comprehension in Typically Developing Toddlers and Young Children with Autism
  • Research • Medicine
    Intraoperative Detection of Subtle Endometriosis: A Novel Paradigm for Detection and Treatment of Pelvic Pain Associated with the Loss of Peritoneal Integrity
  • Research • Medicine
    The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease
  • Research • Medicine
    Collection Protocol for Human Pancreas
  • Research • Medicine
    The α-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva
  • Research • Medicine
    The Measurement and Treatment of Suppression in Amblyopia
  • Research • Medicine
    Corneal Donor Tissue Preparation for Endothelial Keratoplasty
  • Research • Medicine
    Quantification of Atherosclerotic Plaque Activity and Vascular Inflammation using [18-F] Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography (FDG-PET/CT)
  • Research • Medicine
    Eye Tracking Young Children with Autism
  • Research • Medicine
    Doppler Optical Coherence Tomography of Retinal Circulation
  • Research • Medicine
    Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System
  • Research • Medicine
    Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR
  • Research • Medicine
    Method to Measure Tone of Axial and Proximal Muscle
  • Research • Medicine
    The Trier Social Stress Test Protocol for Inducing Psychological Stress
  • Research • Medicine
    Probing the Brain in Autism Using fMRI and Diffusion Tensor Imaging
  • Research • Medicine
    Multifocal Electroretinograms
  • Research • Medicine
    Isolation of Human Islets from Partially Pancreatectomized Patients
  • Research • Medicine
    Examining the Characteristics of Episodic Memory using Event-related Potentials in Patients with Alzheimer's Disease
  • Research • Medicine
    Magnetic Resonance Imaging Quantification of Pulmonary Perfusion using Calibrated Arterial Spin Labeling
  • Research • Medicine
    Manual Muscle Testing: A Method of Measuring Extremity Muscle Strength Applied to Critically Ill Patients
  • Research • Medicine
    Expired CO2 Measurement in Intubated or Spontaneously Breathing Patients from the Emergency Department
  • Research • Medicine
    A Protocol for Comprehensive Assessment of Bulbar Dysfunction in Amyotrophic Lateral Sclerosis (ALS)
  • Research • Medicine
    An Investigation of the Effects of Sports-related Concussion in Youth Using Functional Magnetic Resonance Imaging and the Head Impact Telemetry System
  • Research • Medicine
    Corneal Confocal Microscopy: A Novel Non-invasive Technique to Quantify Small Fibre Pathology in Peripheral Neuropathies
  • Research • Medicine
    Methods to Quantify Pharmacologically Induced Alterations in Motor Function in Human Incomplete SCI
  • Research • Medicine
    Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology
  • Research • Medicine
    Technique to Collect Fungiform (Taste) Papillae from Human Tongue
  • Research • Medicine
    Assessing Endothelial Vasodilator Function with the Endo-PAT 2000
  • Research • Medicine
    Making Sense of Listening: The IMAP Test Battery
  • Research • Medicine
    An Experimental Paradigm for the Prediction of Post-Operative Pain (PPOP)
  • Research • Biology
    Bioelectric Analyses of an Osseointegrated Intelligent Implant Design System for Amputees
  • Research • Biology
    Demonstration of Cutaneous Allodynia in Association with Chronic Pelvic Pain
  • Get cutting-edge science videos from JoVE sent straight to your inbox every month.

    Waiting X
    Simple Hit Counter