所有工作是在公爵在体内显微镜中心，美国国立卫生研究院/ NIBIB 国家生物医学技术资源中心（P41 EB015897）和国家癌症研究所的小动物成像资源计划（U24 CA092656）。 NSF研究生研究奖学金（2003014921），提供额外的支持。

1。组装和校准超声诊断系统<ol><li>直径宽，足以覆盖的小鼠大脑和中心频率在2 MHz的范围内与单元素的超声换能器的超声系统开始。传感器是由50分贝功率的放大器，它是连接到一个信号发生器产生超声波脉冲序列。 </li><li>要校准的超声波系统的声压，使用与施加电压产生的声压水听器。传感器放置在一个以上的水听器的水箱。申请一个简单的脉冲（例如，换能器的频率在10周期与脉冲重复频率为10赫兹的正弦波）的传感器。使用一个3轴的翻译阶段找到峰值响应，这应该是在传感器的自然疫源地（约60毫米13毫米直径的2.15兆赫传感器）超声波束的中心。 </li><li>使多次测量过响E的输入电压（例如， 页 50-400毫伏），以验证系统的线性。用一个简单的线性回归估计输入电压，声压之间的关系。在我们的系统，258和167 mV峰峰值输入电压的峰值负声压为0.52和0.36兆帕。 </li><li>节目的信号发生器产生超声波脉冲序列组成的阵阵正弦脉冲换能器的频率在50000周期每爆裂和64毫秒的突发期。校准测量的基础上，设置脉冲幅度产生峰值负声压传感器的自然疫源地的中心，在0.36兆帕。 </li></ol> 2。准备试剂<ol><li>四水氯化锰（MnCl 2·4H 2 O）溶于无菌水，浓度在100毫米（300 mOsM）和过滤消毒。 </li><li>产生由“activati​​n的perflutren脂质微球G“小瓶天的实验。在45秒的制造商提供的搅拌器，一个单一的小瓶可以被激活一次在一天的开始，没有休息一天激活使用。 </li><li>紧接微管理，搅拌1分钟悬浮微手工小瓶。退出微气泡从小瓶时，不室内空气注入小瓶，因为这降低了剩余的微气泡。离开小瓶，直到最后一天的使用，然后存放在冰箱。一个单一的小瓶保持这种方式，可以持续数天。重新激活存储在搅拌器小瓶，在随后的日子里第一次使用前。 </li></ol> 3。动物的制备<ol><li>异氟醚麻醉的动物，鼻锥交付。鼻锥装置的设计应固定在同一位置，每次动物的头，准确和可靠。我们的设备15头保持在日E“头骨扁平化”的立场（即背侧颅骨表面是水平的）Paxinos脑图谱16。滴定麻醉维持85至125每分钟呼吸的呼吸频率。维持体温，用热灯或吹空调。保护润滑剂眼睛。 </li><li>取下的从鼠标头皮的头发用电动微调。 </li><li>将尾静脉导管及腹腔内（IP）的导管。为生存而研究，一定要使用适当的无菌技术; IP导管放置在这篇文章中的视频显示，仅适用于非生存实验是适当的。 </li><li>使神经刺激实验所需的任何额外的准备。为每桶领域皮质触须刺激的映射，使用解剖显微镜和显微剪刀削减触须尽可能接近皮肤表面的毛囊或周围的皮肤无刺激性。 </li><li>地方上的超声凝胶头皮，然后降低水载列头（例如，7.6微米的垃圾桶内胆）薄薄的塑料片上。达到水用棉签拭子列通过推得到任何空气被困气泡在超声凝胶。放置在其自然的焦距（58毫米）的超声换能器直接在老鼠大脑中的水柱，并用指尖擦拭传感器，以消除任何气泡。 </li></ol> 4。血脑屏障与微泡和超音波（BOMUS）开幕<ol><li>锰剂量为0.5 mmol /公斤的IP解决方案给予腹腔注射。例腹腔导管，所以锰不流了出来，并等待10分钟，以允许它来分发（ 图1）。 </li><li>要打开血脑屏障，管理30μLperflutren脂质微球（激活的DEFINITY），通过尾静脉导管，同时，启动超声脉冲序列。 Çontinue insonification 3分钟。 </li></ol> 5。神经刺激<ol><li>允许约40分钟脑水平的Mn 2 +稳定前开始神经刺激。在这种方式中，由于锰戏剧性的基线增强+跨越血脑屏障的扩散可以区别于微妙差增强，由于刺激。然后，开始与您选择的范例（ 图1）的刺激。 </li><li>触须的刺激，关闭异氟醚和清除鼻锥。在距离皮肤约2-5毫米的触须阵列通​​过手动移动软艺术家的画笔，在圆周运动（1-5赫兹）。继续刺激90分钟。锰有镇静的效果，让奔放的刺激的动物。如果动物变得焦躁不安，管理5％，约15秒钟，异氟醚经鼻锥。 </li></ol> 6。的MagnetiC谐振成像<ol><li>刺激后，通过鼻锥恢复麻醉。继续保持体温和滴定异氟醚的水平到85至125每分钟呼吸的呼吸频率。 </li><li>磁共振成像线圈放置在鼠标和转移的磁共振成像系统。采集高分辨率三维T 1加权磁共振图像。例如，使用三维扰相梯度回波（SPGR）序列的回顾与下列参数：重复时间为25 ms;回声时间为2 ms，翻转角30度; 15.63 kHz的带宽;鉴于20×20场× 12毫米; 128×128×60的矩阵。 </li></ol> 7。图像分析<ol><li>图像分析是特定于所使用的刺激范式。为触须刺激实验（ 图2），从一些动物导入到一个合适的分析环境的图像。如果某些动物的刺激，而其他人则在左侧，FL刺激右侧IP部分，所有图像都有效的“左刺激。”然后，比较每个大脑的刺激侧对侧无刺激侧，创建一个复制和镜像左，无刺激，创建图片集。注册所有图片到一个共同的空间，然后一个3×3×3像素高斯核平滑。 </li><li>数学分析环境，如MATLAB，数据导出。另外，面膜中的数据集不相关的解剖。强度正常化等 Venot迭代法的图像。17,18。 </li><li>使用配对，单尾t检验比较每个大脑刺激时双方对每个大脑的刺激双方的每个像素对侧相应的像素。 </li><li>显示的结果p值的地图，以确定地区差活动（ 图3）。 </li></ol> 8。代表结果这里介绍的方法有两个基金<html> amental步骤：（1）血脑屏障开放与微泡和超声（BOMUS）和（2）激活诱导的锰增强磁共振成像“（AIM的MRI）。因为前者取决于后者的一步，它是重要的，以验证成功实施BOMUS的。 破坏的血脑屏障给药后的T缩短在对T的信号在脑实质增加1加权成像造影剂（如锰或钆基剂）结果相比，大脑这BOMUS未执行（ 图4）。这锰增强的分布是不完全一致，虽然它与动物之间是相当一致的。分布反映血脑屏障开放的唯一的不均匀性，而且是内在的非均匀分布锰大脑内的19。血脑屏障开放的空间和时间的动力已经进一步说明12。 耳鼻喉科“&gt;，一旦BOMUS已成功实施，下一步就是执行目的的MRI许多实验范式是可能的。然而，因为有许多潜在的困惑，控制和分析，必须精心设计的影响因子包括不均匀的血脑屏障开放，不均匀积累锰在大脑中，锰扩散的时空动态，非特异性神经元的活动。在这个演示中，触须单方面刺激神经反应映射。要考虑的不均匀性和锰通量，无刺激的侧各脑被用来作为内部控制。要占到非特异性神经元的活动可能会有所不同动物之间，分析，统计测试，以确定是一贯的动物（ 图2）之间的不同的地区，结果差异的三维地图和三维p值图（ 图3），其中显示区域的右侧对侧刺激触须更高的信号。在地图的左侧，表明该地区有显着较高的信号刺激触须同侧。 p值的地图确定的高架信号对侧刺激触须相当于初级感觉皮层，其响应触须刺激电20,21和2 -脱氧葡萄糖的研究已被广泛记录的每桶领域的广泛地区。一个更完整的讨论，这些结果已经公布以前13。 <img alt="图1" src="/files/ftp_upload/4055/4055fig1.jpg" /> 图1。协议的时间表与BOMUS和AIM MRI（，摘自Howles 等 13），脑功能成像。 <img alt="图2" src="/files/ftp_upload/4055/4055fig2.jpg" /> 图2。识别地区Ø分析计划f每个大脑的刺激和未刺激双方之间的不同强度。来比较每个大脑的刺激侧对侧无刺激侧，重复和镜像左，无刺激，创建图片集。这些图像登记，筛选，和规范化。最后，比较在测试图像左侧的刺激和左无刺激。 t检验是“配对”，使每个大脑的刺激侧相同的大脑相比，无刺激侧。 t检验是“单尾”，这样一个P-值图的边表示对大脑的刺激侧的信号明显较高，而p值映射对方表示无刺激侧的信号明显更高大脑（改编自Howles 等 。13）。 <img alt="图3" src="/files/ftp_upload/4055/4055fig3.jpg" /> 图3。汇集7只动物在两个不同的轴向位置的分析结果。 ţ<html>他的第一列显示对齐所有注册图像的均值，所有小鼠，以便有效地刺激了他们的左边触须。这些图像与彩色地图显示信号的平均水平，在每个像素相对增加对侧覆盖，彩条。彩色图像的右侧区域显示刺激对侧半球有更高的信号。彩色图像的左侧区域显示刺激同侧半球有更高的信号。第二列显示的p值指示信号增加的统计意义的地图覆盖相同的图像。第三列显示了同样的p值的地图覆盖Paxinos立体定向图谱16与阴影（，摘自Howles 等 13）的感觉皮层每桶领域的相应数字。 / 4055/4055fig4.jpg“/&gt; 图4。的Mn 2 +在大脑中的分布。图像被收购后为0.5 mmol /公斤的IP MnCl 2 170分钟，从BOMUS治疗组（n = 5）和对照组（n = 4）小鼠。正常化后，意味着和计算标准偏差图（左图）。增强更大的BOMUS治疗的小鼠。虽然这增强了整个大脑的统一，这是相当一致的，除了附近的大脑和心室的边缘。使用感兴趣区域（ROI），围绕各种结构绘制，平均信噪比（+ 1个SD）计算在每个组（右图）。处理BOMUS，动物表现出更大的信噪比，但也有更大的结构与动物之间的差异（，摘自Howles 等 13）。 <img alt="图5" src="/files/ftp_upload/4055/4055fig5.jpg" /> 图5。审查的BOMUS组织的影响，从BOMUS治疗小鼠的大脑是固定的，本身ctioned在500 - 微米的间隔，苏木精伊红染色。声压0.36兆帕（N = 3），0.52兆帕（4例），5.0兆帕（N = 1）所示的红细胞外渗看到在大脑各部分的平均数。错误条显示标准错误。第二个面板显示从暴露到5.0兆帕（改编从Howles 等 。12）大脑严重的红血细胞外渗的例子。 <img alt="图6" src="/files/ftp_upload/4055/4055fig6.jpg" /> 图6。行为的定量测试是用于麻醉前评估活动，觉醒，和响应，3小时和24小时后从麻醉中复苏。评分系统，前面所述12日 ，是基于行之有效的定量鼠标行为评估的发展在1968年22欧文。平均行为（±标准差）的得分（N = 3）为控制和BOMUS（0.36兆帕）处理（N = 8）动物所示。相对于麻醉前的基线，所有的动物行为3小时后，麻醉得分下降，但它们在很大程度上恢复的第二天。在每个时间点，两组之间无明显差异，这表明，的BOMUS没有可测量的影响动物的行为（改编自等人 Howles。，12）。

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manganese-enhanced+magnetic+resonance+imaging+(MEMRI):+methodological+and+practical+considerations.">Manganese-enhanced magnetic resonance imaging (MEMRI): methodological and practical considerations.</a> Nmr. Biomed. 17, 532-543 (2004).</li><li>Meiri, U., Rahamimoff, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuromuscular+transmission+-+inhibition+by+manganese+ions.">Neuromuscular transmission - inhibition by manganese ions.</a> Science. 176, 308 (1972).</li><li>Aschner, M., Guilarte, T. R., Schneider, J. S., Zheng, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manganese:+Recent+advances+in+understanding+its+transport+and+neurotoxicity.">Manganese: Recent advances in understanding its transport and neurotoxicity.</a> Toxicol. Appl. Pharm. 221, 131-147 (2007).</li><li>Watanabe, T., Frahm, J., Michaelis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manganese-enhanced+MRI+of+the+mouse+auditory+pathway.">Manganese-enhanced MRI of the mouse auditory pathway.</a> Magnet. Reson. Med. 60, 210-212 (2008).</li><li>Yu, X., Wadghiri, Y. Z., Sanes, D. H., Turnbull, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+auditory+brain+mapping+in+mice+with+Mn-enhanced+MRI.">In vivo auditory brain mapping in mice with Mn-enhanced MRI.</a> Nat. Neurosci. 8, 961-968 (2005).</li><li>Yu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistical+mapping+of+sound-evoked+activity+in+the+mouse+auditory+midbrain+using+Mn-enhanced+MRI.">Statistical mapping of sound-evoked activity in the mouse auditory midbrain using Mn-enhanced MRI.</a> Neuroimage. 39, 223-230 (2008).</li></ol>

没有利益冲突的声明。

在这里，一个方法是为无创性开放血脑屏障贯穿整个鼠脑超声和微泡（BOMUS）。随着BBB开放，锰2 +管理和激活诱导锰增强磁共振成像（MRI检查目的）被用来形象短时间刺激在轻轻镇静小鼠的神经元反应。 取得足够的血脑屏障开放与负峰值声压为0.36兆帕。请注意，这是在超声束的中心在头皮表面的压力。单元素的传感器的光束轮廓的测量表明，在光束边缘的声压​​是...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>水听器</td><td>索诺拉州医疗系统，朗蒙特，CA </td><td>四至-251的SN </td><td></td></tr><tr><td>翻译阶段</td><td> Newport公司，加利福尼亚州欧文</td><td></td><td></td></tr><tr><td>超声换能器</td><td>奥林巴斯公司，无损检测，沃尔瑟姆马</td><td> A306S-SU </td><td>回顾伴随传感器找到确切的中心频率，特别是传感器，对目录中所列的标称频率可能不同于制造商的测试表。 （例如，我们的传感器的标称频率为2.25 MHz的，但实际的中心频率为2.15 MHz的。） </td></tr><tr><td> VEVO影像站</td><td> VisualSonics，Inc。加拿大多伦多</td><td>; </td><td></td></tr><tr><td> 50 dB的功率放大器</td><td> E和我，纽约州罗切斯特市</td><td>模型240L </td><td></td></tr><tr><td>信号发生器</td><td>安捷伦科技公司，加利福尼亚州圣克拉拉</td><td>型号33220A </td><td></td></tr><tr><td> MnCl 2 - （H2O）4 </td><td>西格玛</td><td></td><td>分子量不同批次，要求制造商精确测量</td></tr><tr><td> perflutren脂微</td><td>马北比尔里卡，医学影像，lantheus </td><td> DEFINITY的</td><td></td></tr><tr><td>微搅拌器</td><td>马北比尔里卡，医学影像，lantheus </td><td> VIALMIX </td><td></td></tr><tr><td>磁共振成像线圈</td><td> M2M影像公司，Hillcrest的，OH </td><td></td><td> 35毫米直径的正交传输/接收量线圈</td></tr><tr><td>磁共振成像系统</td><td> GE医疗，MilwaukeE，WI </td><td></td><td> GE EXCITE控制台的操作7-T的水平孔磁铁</td></tr><tr><td>图像分析环境</td><td>面相影像，加利福尼亚州圣迭戈，MathWorks公司，Natic​​k市马</td><td>阿米拉的MATLAB </td><td></td></tr></tbody></table>

functional neuroimaging using ultrasonic blood-brain barrier disruption and manganese-enhanced mri

虽然老鼠是主导的模型系统，为研究神经科学，脑功能成像在小鼠遗传和分子基础仍然是技术上的挑战。一种方法，激活诱导锰增强磁共振成像（MRI检查目的），已成功地用于映射在啮齿类动物的神经元活动1-5。在AIM MRI，锰2 +作用钙模拟，并在6,7去极化神经元累积。由于锰2 +缩短的T 1组织的财产，升高的神经元活动的地区，将加强在MRI。此外，锰2 +清除慢慢从活化地区，因此，可以进行刺激之前成像磁铁外，从而提高实验的灵活性。然而，因为锰2 +不容易穿过血脑屏障（BBB）的，需要打开血脑屏障限制使用的AIM MRI，尤其是在小鼠。 开放血脑屏障的工具之一是ULTrasound。虽然潜在的破坏性，如果超声结合管理与充气微泡（即超声造影剂），血脑屏障开放所需的声压是相当低的。可以使用这种超声和微泡的结合，可靠地打开，而不会造成组织损伤8-11血脑屏障。 在这里，一种方法是执行旨在利用微气泡和超声波打开血脑屏障的MRI。静脉注射perflutren微泡后，重点不突出的脉冲超声波束被剃光鼠标头3分钟。为简单起见，我们称这BBB的开放的技术与微泡和超声BOMUS 12。使用BOMUS打开整个血脑屏障的两个大脑半球，锰是管理整个鼠脑。轻轻镇静小鼠的实验刺激后，AIM MRI是用来映射神经反应。 至证明这种方法，本BOMUS和AIM MRI用于映射单方面的触须轻轻镇静小鼠13机械刺激。因为BOMUS可以打开整个两半球血脑屏障，无刺激大脑的一侧是用来控制背景非特异性刺激。由此产生的3D激活地图同意的桶领域皮质14的触须地区出版的申述。在超声开放血脑屏障的是快速，非侵入性的，可逆的，因此这种方法是适用于高吞吐量和/或纵向研究在清醒小鼠。

Watch this Scientific Journal Video about Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI at JoVE.com

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

一个被描述为广泛的开放血脑屏障在鼠标使用微泡和超声技术。使用这种技术，锰，可管理的鼠脑。因为锰是一个磁共振造影剂，在去极化神经元的积累，这种方法能够使神经元的活动影像。

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

functional-neuroimaging-using-ultrasonic-blood-brain-barrier

Research

JoVE Journal

Neuroscience

14.9K 次观看.  Stanford University. 一个被描述为广泛的开放血脑屏障在鼠标使用微泡和超声技术。使用这种技术，锰，可管理的鼠脑。因为锰是一个磁共振造影剂，在去极化神经元的积累，这种方法能够使神经元的活动影像。

视频: Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Measuring the Subjective Value of Risky and Ambiguous Options using Experimental Economics and Functional MRI Methods

Most of the choices we make have uncertain consequences. In some cases the probabilities for different possible outcomes are precisely known, a condition termed "risky". In other cases when probabilities cannot be estimated, this is a condition described as "ambiguous". While most people are averse to both risk and ambiguity1,2, the degree of those aversions vary substantially across individuals, such that the subjective value of the same risky or ambiguous option can be very different for different individuals. We combine functional MRI (fMRI) with an experimental economics-based method3 to assess the neural representation of the subjective values of risky and ambiguous options4. This technique can be now used to study these neural representations in different populations, such as different age groups and different patient populations.
In our experiment, subjects make consequential choices between two alternatives while their neural activation is tracked using fMRI. On each trial subjects choose between lotteries that vary in their monetary amount and in either the probability of winning that amount or the ambiguity level associated with winning. Our parametric design allows us to use each individual's choice behavior to estimate their attitudes towards risk and ambiguity, and thus to estimate the subjective values that each option held for them. Another important feature of the design is that the outcome of the chosen lottery is not revealed during the experiment, so that no learning can take place, and thus the ambiguous options remain ambiguous and risk attitudes are stable. Instead, at the end of the scanning session one or few trials are randomly selected and played for real money. Since subjects do not know beforehand which trials will be selected, they must treat each and every trial as if it and it alone was the one trial on which they will be paid. This design ensures that we can estimate the true subjective value of each option to each subject. We then look for areas in the brain whose activation is correlated with the subjective value of risky options and for areas whose activation is correlated with the subjective value of ambiguous options.

Using functional MRI and behavioral methods to determine the neural representation of the subjective value of risky and ambiguous options in the human brain.

，测量主观价值风险和不明确的选项，使用实验经济学和功能磁共振成像的方法

Most of the choices we make have uncertain consequences. In some cases the probabilities for different possible outcomes are precisely known, a condition ...

科研

神经科学

Optogenetic Functional MRI

The investigation of the functional connectivity of precise neural circuits across the entire intact brain can be achieved through optogenetic functional magnetic resonance imaging (ofMRI), which is a novel technique that combines the relatively high spatial resolution of high-field fMRI with the precision of optogenetic stimulation. Fiber optics that enable delivery of specific wavelengths of light deep into the brain in vivo are implanted into regions of interest in order to specifically stimulate targeted cell types that have been genetically induced to express light-sensitive trans-membrane conductance channels, called opsins. fMRI is used to provide a non-invasive method of determining the brain's global dynamic response to optogenetic stimulation of specific neural circuits through measurement of the blood-oxygen-level-dependent (BOLD) signal, which provides an indirect measurement of neuronal activity. This protocol describes the construction of fiber optic implants, the implantation surgeries, the imaging with photostimulation and the data analysis required to successfully perform ofMRI. In summary, the precise stimulation and whole-brain monitoring ability of ofMRI are crucial factors in making ofMRI a powerful tool for the study of the connectomics of the brain in both healthy and diseased states.

This protocol describes the steps and data analysis required to successfully perform optogenetic functional magnetic resonance imaging (ofMRI). ofMRI is a novel technique that combines high-field fMRI readout with optogenetic stimulation, allowing for cell type-specific mapping of functional neural circuits and their dynamics across the whole living brain.

光遗传学功能磁共振成像

The investigation of the functional connectivity of precise neural circuits across the entire intact brain can be achieved through optogenetic functional ...

High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. Transcytosis across the BBB regulates the delivery of hormones, metabolites, and therapeutic antibodies to the brain parenchyma. Here, we present a protocol that combines immunofluorescence of free-floating sections with laser scanning confocal microscopy and image analysis to visualize subcellular organelles within endothelial cells at the BBB. Combining this data-set with 3D image analysis software allows for the semi-automated segmentation and quantification of capillary volume and surface area, as well as the number and intensity of intracellular organelles at the BBB. The detection of mouse endogenous immunoglobulin (IgG) within intracellular vesicles and their quantification at the BBB is used to illustrate the method. This protocol can potentially be applied to the investigation of the mechanisms controlling BBB transcytosis of different molecules in vivo.

Here we present a microscopy-based protocol for high-resolution imaging and a three-dimensional reconstruction of the mouse neurovascular unit and blood-brain barrier using brain free-floating sections. This method allows for the visualization, analysis, and quantification of intracellular organelles at the BBB.

血脑屏障的高分辨率共聚焦成像: 成像、3D 重建和 Transcytosis 的量化

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. ...

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains brain homeostasis. The principal sites of the barrier are endothelial cells of the brain capillaries whose barrier function results from tight intercellular junctions and efflux transporters expressed on the plasma membrane. This function is regulated by pericytes and astrocytes that together form the neurovascular unit (NVU). Several neurological diseases such as stroke, Alzheimer&#39;s disease (AD), brain tumors are associated with an impaired BBB function. Assessment of the BBB permeability is therefore crucial in evaluating the severity of the neurological disease and the success of the treatment strategies employed.
We present here a simple yet robust permeability assay that have been successfully applied to several mouse models both, genetic and experimental. The method is highly quantitative and objective in comparison to the tracer fluorescence analysis by microscopy that is commonly applied. In this method, mice are injected intraperitoneally with a mix of aqueous inert fluorescent tracers followed by anesthetizing the mice. Cardiac perfusion of the animals is performed prior to harvesting brain, kidneys or other organs. Organs are homogenized and centrifuged followed by fluorescence measurement from the supernatant. Blood drawn from the cardiac puncture just before perfusion serves for normalization purpose to the vascular compartment. The tissue fluorescence is normalized to the wet weight and serum fluorescence to obtain a quantitative tracer permeability index. For additional confirmation, the contralateral hemi-brain preserved for immunohistochemistry can be utilized for tracer fluorescence visualization purposes.

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Here we present a mouse brain vascular permeability assay using intraperitoneal injection of fluorescent tracers followed by perfusion that is applicable to animal models of blood-brain barrier dysfunction. One hemi-brain is used for assessing permeability quantitatively and the other for tracer visualization/immunostaining. The procedure takes 5 - 6 h for 10 mice.

用荧光标记示踪剂对小鼠血脑屏障通透性的体内检测

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains ...

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

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

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

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

一种体内评价大鼠缺血性中风模型中血脑屏障的破坏

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

Neuroimaging Field Methods Using Functional Near Infrared Spectroscopy (NIRS) Neuroimaging to Study Global Child Development: Rural Sub-Saharan Africa

Portable neuroimaging approaches provide new advances to the study of brain function and brain development with previously inaccessible populations and in remote locations. This paper shows the development of field functional Near Infrared Spectroscopy (fNIRS) imaging to the study of child language, reading, and cognitive development in a rural village setting of C&#244;te d&#39;Ivoire. Innovation in methods and the development of culturally appropriate neuroimaging protocols allow&#160;a first-time look into the brain&#39;s development and children&#39;s learning outcomes in understudied environments. This paper demonstrates protocols for transporting and setting up a mobile laboratory, discusses considerations for field versus laboratory neuroimaging, and presents a guide for developing neuroimaging consent procedures and building meaningful long-term collaborations with local government and science partners. Portable neuroimaging methods can be used to study complex child development contexts, including the impact of significant poverty and adversity on brain development. The protocol presented here has been developed for use in C&#244;te d&#39;Ivoire, the world&#39;s primary source of cocoa, and where reports of child labor in the cocoa sector are common. Yet, little is known about the impact of child labor on brain development and learning. Field neuroimaging methods have the potential to yield new insights into such urgent issues, and the development of children globally.

Neuroimaging Field Methods Using Functional Near Infrared Spectroscopy NIRS Neuroimaging to Study Global Child Development: Rural Sub-Saharan Africa

Portable neuroimaging approaches (functional Near Infrared Spectroscopy) provide advances to the study of the brain in previously inaccessible regions; here, rural C&#244;te d&#39;Ivoire. Innovation in methods and development of culturally-appropriate neuroimaging protocols permits novel study of the brain&#39;s development and children&#39;s learning outcomes in environments with significant poverty and adversity.

利用功能近红外光谱成像技术研究全球儿童发展: 农村撒哈拉以南非洲地区的神经影像场方法

Portable neuroimaging approaches provide new advances to the study of brain function and brain development with previously inaccessible populations and in ...

A Benchtop Approach to the Location Specific Blood Brain Barrier Opening using Focused Ultrasound in a Rat Model

Stereotaxic surgery is the gold standard for localized drug and gene delivery to the rodent brain. This technique has many advantages over systemic delivery including precise localization to a target brain region and reduction of off target side effects. However, stereotaxic surgery is highly invasive which limits its translational efficacy, requires long recovery times, and provides challenges when targeting multiple brain regions. Focused ultrasound (FUS) can be used in combination with circulating microbubbles to transiently open the blood brain barrier (BBB) in millimeter sized regions. This allows intracranial localization of systemically delivered agents that cannot normally cross the BBB. This technique provides a noninvasive alternative to stereotaxic surgery. However, to date this technique has yet to be widely adopted in neuroscience laboratories due to the limited access to equipment and standardized methods. The overall goal of this protocol is to provide a benchtop approach to FUS BBB opening (BBBO) that is affordable and reproducible and can therefore be easily adopted by any laboratory.

Focused ultrasound with microbubble agents can open the blood brain barrier focally and transiently. This technique has been used to deliver a wide range of agents across the blood brain barrier. This article provides a detailed protocol for the localized delivery to the rodent brain with or without MRI guidance.

在鼠模型中使用聚焦超声波打开位置特定血脑屏障开口的台面方法

Stereotaxic surgery is the gold standard for localized drug and gene delivery to the rodent brain. This technique has many advantages over systemic ...

Focused Ultrasound Induced Blood-Brain Barrier Opening for Targeting Brain Structures and Evaluating Chemogenetic Neuromodulation

Acoustically Targeted Chemogenetics (ATAC) allows for the noninvasive control of specific neural circuits. ATAC achieves such control through a combination of focused ultrasound (FUS) induced blood-brain barrier opening (FUS-BBBO), gene delivery with adeno-associated viral (AAV) vectors, and activation of cellular signaling with engineered, chemogenetic, protein receptors and their cognate ligands. With ATAC, it is possible to transduce both large and small brain regions with millimeter precision using a single noninvasive ultrasound application. This transduction can later allow for a long-term, noninvasive, device-free neuromodulation in freely moving animals using a drug. Since FUS-BBBO, AAVs, and chemogenetics have been used in multiple animals, ATAC should also be scalable for the use in other animal species. This paper expands upon a previously published protocol and outlines how to optimize the gene delivery with FUS-BBBO to small brain regions with MRI-guidance but without a need for a complicated MRI-compatible FUS device. The protocol, also, describes the design of mouse targeting and restraint components that can be 3D-printed by any lab and can be easily modified for different species or custom equipment. To aid reproducibility, the protocol describes in detail how the microbubbles, AAVs, and venipuncture were used in ATAC development. Finally, an example data is shown to guide the preliminary investigations of studies utilizing ATAC.

This protocol delineates steps necessary for the gene delivery through focused ultrasound blood brain barrier (BBB) opening, evaluation of the resulting gene expression, and measurement of neuromodulation activity of chemogenetic receptors through histological tests.

聚焦超声诱导血脑屏障打开，用于靶向大脑结构和评估化学遗传神经调控

Acoustically Targeted Chemogenetics (ATAC) allows for the noninvasive control of specific neural circuits. ATAC achieves such control through a ...

Role of Diffusion MRI Tractography in Endoscopic Endonasal Skull Base Surgery

Endoscopic endonasal surgery has gained a prominent role in the management of complex skull base tumors. It allows the resection of a large group of benign and malignant lesions through a natural anatomical extra-cranial pathway, represented by the nasal cavities, avoiding brain retraction and neurovascular manipulation. This is reflected by the patients' prompt clinical recovery and the low risk of permanent neurological sequelae, representing the main caveat of conventional skull base surgery. This surgery must be tailored to each specific case, considering its features and relationship with surrounding neural structures, mostly based on preoperative neuroimaging. Advanced MRI techniques, such as tractography, have been rarely adopted in skull base surgery due to technical issues: lengthy and complicated processes to generate reliable reconstructions for inclusion in the neuronavigation system. 
This paper aims to present the protocol implemented in the institution and highlights the synergistic collaboration and teamwork between neurosurgeons and the neuroimaging team (neurologists, neuroradiologists, neuropsychologists, physicists, and bioengineers) with the final goal of selecting the optimal treatment for each patient, improving the surgical results and pursuing the advancement of personalized medicine in this field.

We present a protocol to integrate diffusion MRI tractography in patient work-up to endoscopic endonasal surgery for a skull base tumor. The methods for adopting these neuroimaging studies in the pre- and intra-operative phases are described.

扩散MRI造影术在内窥镜内窥镜颅骨基础外科中的作用

Endoscopic endonasal surgery has gained a prominent role in the management of complex skull base tumors. It allows the resection of a large group of ...

跨内皮抗性测量以评估细胞屏障完整性

Barrier Functional Integrity Recording on bEnd.3 Vascular Endothelial Cells via Transendothelial Electrical Resistance Detection

The blood-brain barrier (BBB) is a dynamic physiological structure composed of microvascular endothelial cells, astrocytes, and pericytes. By coordinating the interaction between restricted transit of harmful substances, nutrient absorption, and metabolite clearance in the brain, the BBB is essential in preserving central nervous system homeostasis. Building in vitro models of the BBB is a valuable tool for exploring the pathophysiology of neurological disorders and creating pharmacological treatments. This study describes a procedure for creating an in vitro monolayer BBB cell model by seeding bEnd.3 cells into the upper chamber of a 24-well plate. To assess the integrity of cell barrier function, the conventional epithelial cell voltmeter was used to record the transmembrane electrical resistance of normal cells and CoCl2-induced hypoxic cells in real-time. We anticipate that the above experiments will provide effective ideas for the creation of in vitro models of BBB and drugs to treat disorders of central nervous system diseases.

作者聚焦：TEER 检测在评估细胞屏障完整性中的应用 

This protocol describes a dependable and efficient in vitro model of the brain blood barrier. The method uses mouse cerebral vascular endothelial cells bEnd.3 and measures transmembrane electrical resistance.

通过跨内皮电阻检测对bEnd.3血管内皮细胞进行屏障功能完整性记录

The blood-brain barrier (BBB) is a dynamic physiological structure composed of microvascular endothelial cells, astrocytes, and pericytes. By coordinating ...