这项工作由中国国家自然科学基金（81573406,81373393,81273506,81221003,81473186和81402907），浙江省自然科学基金（LR15H310001）和浙江省科技创新队队长计划（2011R50014）资助。

所有实验均经浙江大学动物实验委员会道德准则批准实施，完全符合国家卫生研究院"实验动物护理与使用指南"。努力减少任何疼痛或不适，并使用最少数量的动物。 皮质硬膜下切片的OGD <ol><li> 解决方法: <ol><li>制备1000毫升的r-ACSF（124毫摩尔/ L，氯化钠，5毫摩尔/ L的KCl，1.25毫摩尔/ L KH 2 PO 4，2毫摩尔/升MgSO 4，26毫摩尔/ L的NaHCO 3，2毫摩尔/ L的CaCl 2， 10 mmol / L葡萄糖，最终pH 7.4）。在整个实验中，具有5％CO 2和95％O 2的气泡。 </li><li>如上所述制备200mL无葡萄糖的ACSF（gf-ACSF），但不包括葡萄糖，以5％CO 2和95％N 2鼓泡。气泡气体30分钟，然后应用于brain切片以减少溶液中的氧含量。继续冒泡直到OGD程序完成。 </li><li>通过用20％CO 2和80％O 2平衡200mL r-ACSF来制备酸性ACSF（a-ACSF）。气泡气体30分钟，然后应用于脑切片，将溶液的pH值降至pH6.8，继续鼓泡，直到APC程序完成。 </li><li>将三个组织架分别放入r-ACSF，gf-ACSF，a-ACSF中。 </li></ol></li><li> 脑切片准备: 注意:本研究使用8周龄的雄性C57B1 / 6J小鼠。将动物饲养在受控温度（22±2℃）下，12小时光暗循环周期，并进入沉淀的食物和水。 <ol><li>在感应室中用异氟醚过量的小鼠牺牲。放弃鼠标。用小剪刀和镊子解剖大脑。用薄的铲子取出大脑，小心地放入玻璃烧杯中用5％CO 2和95％O 2平衡的冰冷r-ACSF。 注意:这些步骤应尽可能快速但精细地进行。 </li><li>将大脑置于冰冷的r-ACSF中5分钟。调整气压以避免大脑的运动。 </li><li>将低温沉淀胶放在vibratome板上两条。将一块3％的琼脂糖放在离开刀片的胶条上，以支撑大脑。 </li><li>将10厘米培养皿倒入冰中，然后将滤纸放在盘子上。用冰冷的r-ACSF滴加湿滤纸。 </li><li>用镊子将大脑转移到滤纸上。用刀片和镊子切断额极和小脑。 注意:横截面应尽可能平滑，垂直于矢状面。 </li><li>将剩余的脑组织垂直放置在另一条胶水上，并保持大脑靠在琼脂糖上。将冰冷的r-ACSF加入切割槽淹没大脑，并向冰块区域加冰。将r-ACSF置于用5％CO 2和95％O 2鼓泡的容器中。 </li><li>提高水库并调整剃须刀的位置，使剃须刀尽可能靠近大脑，并在大脑之上。 </li><li>选择"CONT"模式，连续切片脑部。将切割厚度设置为400μm，振动频率为6 - 8，速度为3 - 4.按"启动/停止"按钮开始自动切割。 </li><li>切掉3 mL巴斯德吸管的尖端，使其与大脑切片的大小相匹配。 注意:开口应足够大，以避免对脑切片造成额外的损害。 </li><li>用tip-cut巴斯德移液器逐个抽出五个脑切片，并将其放入冰冷的r-ACSF中的组织保持器中，用5％CO 2和95％O 2鼓泡。不要收集生产的第一片。调整气压到avoi脑部切片的运动。 注意:脑切片不应相互覆盖。 </li><li>将脑切片与r-ACSF在室温下放置30分钟，然后在37℃下在水浴中放置另外10分钟以恢复突触功能。 </li></ol></li><li> OGD和APC: <ol><li>将gf-ACSF和a-ACSF放入37°C水浴中，并用上述1.1.2和1.1.3中所述的相应气体将缓冲液鼓泡30分钟。 </li><li>在r-ACSF中留下一片作为对照。将其他四个脑片转移到具有预热的gf-ACSF的组织保持器中并孵育15分钟。将切片转移回r-ACSF中的组织支架以实现再灌注。 </li><li>研究APC的时间窗口，将一片直接从gf-ACSF传输到a-ACSF。首先将其他三个切片转移到r-ACSF，然后分别在再灌注后5和15分钟内将其中两个转移到a-ACSF中的组织支架。 </li><li> Incubate-ACSF中的两个切片3分钟，然后将其转移回r-ACSF。将r-ACSF中的三个切片孵育另外1小时。将r-ACSF中的剩余切片设置为OGD组。 </li><li>对于剂量反应研究，首先在OGD后将所有四个切片转移到r-ACSF中的组织保持器，并孵育5分钟。然后在r-ACSF中留下一片作为OGD组，并将其他三个切片转移到-ACFF。将a-ACSF中的切片分别孵育1,3和5分钟，然后将其转移回r-ACSF。将r-ACSF中的三个切片孵育另外1小时。 </li></ol></li><li> 切片生存力测定: <ol><li>制备0.25％2,3,5-三苯基四唑盐酸盐（TTC）生理盐水溶液。加入1.25 g TTC粉末至500 mL生理盐水溶液。 注意:粉末完全溶解后，将溶液转移到覆盖在箔中的24孔板（500μL/孔）中并将其储存在4℃。用TTC染色的TTC和组织轻sitive。 </li><li>将切片转移到含有TTC溶液的24孔板（每孔一片）中，并在溶液中拉伸切片。将TTC溶液中的切片在37℃下在浅水浴中孵育30分钟。 </li><li>将切片转移到覆盖在箔中的干净的1.5 mL离心管中，并测量切片的干重。 </li><li>将乙醇/二甲基亚砜（1:1）加入到离心管中（v = 10:1），以提取甲。。在室温下将切片在乙醇/二甲基亚砜中孵育24小时。 </li><li>将管中的所有乙醇/二甲基亚砜加入到96孔板中，并通过读板器测量490nm处的吸光度。 </li><li>将吸光度归一化到切片的干重，并将生存力表示为对照切片的百分比。 </li></ol></li></ol> MCAO <ol><li> 制备: <ol><li>消毒工作台，激光多普勒流量计的表面y（LDF）仪器及其配件，含70％乙醇。 </li><li>准备高压灭菌仪器:两把剪刀，10厘米;两镊子，10厘米;一个眼科钳，11厘米;一只微型眼科剪刀，9厘米;一个微血管夹，1.8厘米; oneneedle驱动，r 12.5厘米;几条外科缝合线（△1/2 4×10）;棉签。 </li><li>用盐水溶液准备注射器（无针头）以保持水合作业区域。 </li><li>准备麻醉气体（100％O 2 +异氟烷）。 </li></ol></li><li> 脑血流量检测: <ol><li>设置LDF仪器。 </li><li>腹腔注射小鼠镇痛药:metamizol 200 mg / kg，carprofen 4 mg / kg，丁丙诺啡0.1 mg / kg。 </li><li>将小鼠放入感应室中，用4％异氟烷在氧气中进行麻醉，直到身体自发移动，并将其停止。 </li><li>将鼠标置于俯卧位置，其鼻子装入鼻锥并维持异氟醚为1.5％，以后的程序。 </li><li>在双眼睛上涂抹松香酚眼膏。 </li><li>用70％乙醇消毒头部。 </li><li>使用手术刀在眼睛和耳朵之间做出正确的paramedian皮肤切口，以暴露胭脂红。 </li><li>用无菌棉揉颅骨。 </li><li>在头骨表面涂一滴至两滴手术胶。 </li><li>将光纤探头的尖端5 mm尾部放置到腋下，并将中线的外侧6 mm。 </li><li>开始在计算机软件上记录血流量。等待直到发生稳定的血流基线，然后在胶上涂上20μL催化剂以固定探针。 注意:理想的基线应该超过500个通量。 </li><li>如果基线小于200通量，请精细调整位置以获得合适的基线。在实验期间，将探头连接到颅骨。 </li><li>用碘伏消毒头连接光纤探头。 </li></ol></li><li> MCAO: <ol><li>将麻醉的小鼠（用LDF探针连接到其头骨）放置并固定，仰卧位置，并在整个手术过程中使用加热灯保持其体温。用70％乙醇消毒颈部。 </li><li>使用手术刀在颈部做一个paramedian皮肤切口;用镊子钝化软组织以暴露血管。向暴露的组织中加入一滴盐水以保持水分。 </li><li>使用眼科钳将周围组织和迷走神经解剖颈总动脉。小心不要损伤迷走神经。颈总动脉暴露后再次用碘伏消毒颈部皮肤。 </li><li>在颈总动脉的远端放置微血管夹，然后在近端用6-0丝线缝合死结。将夹具尽可能靠近以进行后续操作。确保长度钳夹与死结之间的血管尽可能长，以便后续操作。 </li><li>通过捆扎一个松散的结将临时缝合线靠近夹具。确保单丝插入之间有足够的空间。 </li><li>用微型眼科剪刀在两节之间进行一个小的纵向切口。确保切口尽可能靠近死结，以便后续操作。小心不要切断船只。 </li><li>通过切口插入12毫米尖端钝的单丝进入动脉内腔，并将其推进几毫米。拧紧单丝尖端的松散结，然后取下夹具。 </li><li>使用眼科钳将长丝推进颈内动脉（10mm，以阻塞大脑中动脉），直到LDF软件显示血流量急剧下降（基线血流减少80％以上）。记录遮挡的开始时间。 注意:LDF仪器设置为在2.2节中描述。 </li><li>大脑中动脉闭塞1 h。切断LDF探针，并将其放入30℃的培养箱中，以保持闭塞状态。 </li></ol></li><li> 再灌注和APC: <ol><li>在开始闭塞后55分钟，用异氟醚再次麻醉动物。将小鼠放置并固定在仰卧位。打开颈部切口并重新暴露颈总动脉。 </li><li>在闭塞期后用眼科钳子轻轻拉出长丝，以达到再灌注，并通过拧紧结而将临时缝合线变成永久缝合线。 </li><li>为酸中毒的治疗，再灌注后5，50，或100分钟改变由鼻锥吸入到含有20％CO 2，20％O 2和60％N 2 5分钟混合气体的气体。用中断的手术缝合缝合切口。 </li><li>将小鼠放在30°C的保温笼中，直到小鼠恢复意识然后将老鼠送回清洁的个别笼子里。为小鼠提供水培养和滋润的食物。手术后密切观察小鼠的过度疼痛和死亡。 </li></ol></li><li> 脑梗塞体积测量: <ol><li>再灌注24 h后采用TTC染色测量脑梗死体积。 </li><li>在指定的牺牲时间之前，按步骤1.4.1中所述准备0.25％的TTC溶液。将溶液转移到覆盖在箔中的24孔板（1mL /孔）中并将其储存在4℃。 注意:TTC和TTC染色的组织对光敏感。 </li><li>在再灌注后24小时，在诱导室中用异氟醚过量处死动物。解脱老鼠用小剪刀和镊子解剖大脑。检查大脑上的流血点，以排除在威利斯圈发生蛛网膜下腔出血的小鼠。 </li><li>将大脑放在-20°的干净的玻璃片上<html> ; C冰包。将大脑和玻片放在-20°C冰箱中5分钟，使大脑更容易切片。 </li><li>将大脑和玻璃片从-20°C取出，放回-20°C冰袋。用刀片和镊子解剖额极和小脑。 </li><li>用刀片将脑切片水平切割成1mm厚度以产生5片。将切片转移到含有TTC溶液的24孔板（每孔1片）中并用镊子拉伸，并拉伸溶液中的切片。将TTC溶液中的切片在37℃下在浅水浴中孵育30分钟。 </li><li>吸出TTC溶液。加入10％甲醛（1mL /孔），并在室温下孵育30分钟。将切片按切片顺序放置在玻璃纸上，并将片段扫描到照相板成像仪中。 </li><li>使用ImageJ分析软件分析梗塞面积占整个脑切片的百分比lass ="xref"&gt; 8基于视觉识别; 见图2 （左图）。 </li></ol></li></ol>

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Z., Jiang, S., Zeng, Y. M., Tang, Q. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+study+of+the+neuroprotective+effect+of+propofol+on+hypoxic+hippocampal+slice.">An in vitro study of the neuroprotective effect of propofol on hypoxic hippocampal slice.</a> Brain Inj. 28 (13-14), 1758-1765 (2014).</li><li>Niu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+profiling+with+HPLC-FTMS+of+exogenous+and+endogenous+chemicals+susceptible+to+the+administration+of+chotosan+in+an+animal+model+of+type+2+diabetes-induced+dementia.">Chemical profiling with HPLC-FTMS of exogenous and endogenous chemicals susceptible to the administration of chotosan in an animal model of type 2 diabetes-induced dementia.</a> J Pharm Biomed Anal. 104, 21-30 (2015).</li></ol>

作者没有什么可以披露的。

在这里我们提出两个实验模型来研究APC对脑缺血的神经保护作用。在脑切片中，APC通过在再灌注发生后用20％CO 2鼓泡的酸性缓冲液中孵育小鼠皮质纹状体切片来实现，而在MCAO模型中，APC是通过在再灌注后向小鼠吸入20％CO 2而实现的。这两种模型反映了APC对脑缺血的神经保护作用。保护与通过缺血后处理实现的保护相当，但是具有更宽的时间窗口。在MCAO模型，再灌注后相比10分钟?...

中风是全球死亡和残疾的主要原因之一。在过去几十年中，为了找到有效的卒中治疗，已经做出了巨大的努力，但是成绩还不尽如人意。后处理是在缺血发作后由亚毒性应激操纵的过程。后处理，包括缺血性，缺氧，低血糖和远程缺血后处理，触发内源性适应机制，并已被证明是有前途的治疗对脑缺血1，2，3，4。然而，缺血后处理可能引起额外的损伤。肢体远程缺血后处理通常需要5的几个周期-在同侧或双侧后肢5，6，7 20分钟闭塞和再灌注。钍因此，这些后处理操作在临床实践中是危险的或不切实际的。为了克服这些缺点，我们开发了APC为小鼠8局灶性脑缺血的疗法。通过吸入20％CO 2诱导，APC以更可行和更安全的方式显着减少缺血性脑损伤。最近我们已经证明，APC延长了再灌注窗口，突出了APC对卒中治疗的重要性9 。 在这里我们提出两个实验模型来研究APC对脑缺血的神经保护作用。第一个是小鼠皮质纹状体切片中的氧 - 葡萄糖剥夺（OGD）模型。通过人造脑脊液（ASCF）将脑切片快速制备和转移到人造环境中，可以维持细胞活力和神经元电路，这样可以在体外研究脑功能10sup&gt;， 11 。在ASCF模仿脑缺血缺氧缺糖，诱导缺血性损伤12，13，14。在OGD之后，将脑切片用常规ASCF（r-ASCF）刷新以提供再灌注，然后使用用20％CO 2鼓泡的酸性ASCF处理APC。与原代培养细胞相比，皮质硬膜下切片维持完整的组织学表征。 为了研究体内脑功能，采用小鼠中脑动脉闭塞（MCAO）模型。通过颈总动脉插入火焰钝的单丝阻止大脑中动脉。作为最广泛使用的中风模型之一，MCAO模型显示临床相关性，单丝的应用使得更容易实现再灌注。简单地通过reperfusio发病后吸入含有20％CO 2含氧量正常的混合气体n，APC对脑梗死体积减少所表明的脑缺血有显着的保护作用。

<table border="0" cellpadding="0" cellspacing="0" width="723">
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</table>

experimental models to study the neuroprotection of acidic postconditioning against cerebral ischemia

中风是全球死亡和残疾的主要原因之一，治疗方法有限。作为神经保护的内源性策略，后处理已被证明是有希望的针对脑缺血的治疗方法。然而，复杂的程序和潜在的安全问题限制了其临床应用。为了克服这些缺点，我们开发了酸性后处理（APC）作为实验性局灶性脑缺血的治疗方法。 APC是指温和酸中毒治疗由局部缺血再灌注期间吸入的CO 2。在这里，我们分别提出了两种模型来在体外和体内执行APC。使用小鼠的氧 - 葡萄糖剥夺（OGD）治疗和小鼠的皮质静脉闭塞和中脑动脉闭塞（MCAO）来模拟脑缺血。 APC可以通过将脑切片转移到用20％CO 2鼓泡的酸性缓冲液中来实现r通过吸入20％CO 2的小鼠。 APC显示出对脑缺血的显着保护作用，如组织存活力和脑梗死体积所反映的。

在上述皮质纹状体切片模型中，在再灌注后1小时通过TTC测定定量皮质硬膜下切片存活力。通过将490nm处的吸收归一化到对照切片来计算TTC转化。根据TTC转换，APC在起始时间和持续时间依赖的方式保护OGD诱导的再灌注损伤。详细地，1和3分钟的酸中毒治疗在OGD 15分钟后5分钟显着改善了活力，而5分钟没有（OGD:0.609±0.029,5 / 1:0.758±0.034,5 / 3:0.821±0.041， 5/5:0.672±0.053，数据报告为?...

Watch this Scientific Journal Video about Experimental Models to Study the Neuroprotection of Acidic Postconditioning Against Cerebral Ischemia at JoVE.com

Experimental Models to Study the Neuroprotection of Acidic Postconditioning Against Cerebral Ischemia

酸性后处理可防止脑缺血。在这里我们提出两个模型来执行APC。分别通过体外氧葡萄糖剥夺后将皮质硬膜下切片转移到酸性缓冲液中，并在体内大脑中动脉闭塞后吸入20％CO 2 。

实验模型研究酸性后处理对脑缺血的神经保护作用

Stroke is one of the leading causes of mortality and disability worldwide, with limited therapeutic approaches. As an endogenous strategy for ...

experimental-models-to-study-neuroprotection-acidic-postconditioning

Research

JoVE Journal

Neuroscience

7.4K 次观看.  Zhejiang University. 酸性后处理可防止脑缺血。在这里我们提出两个模型来执行APC。分别通过体外氧葡萄糖剥夺后将皮质硬膜下切片转移到酸性缓冲液中，并在体内大脑中动脉闭塞后吸入20％CO 2 。 

视频: Experimental Models to Study the Neuroprotection of Acidic Postconditioning Against Cerebral Ischemia

An Experimental Platform to Study the Closed-loop Performance of Brain-machine Interfaces

The non-stationary nature and variability of neuronal signals is a fundamental problem in brain-machine interfacing. We developed a brain-machine interface to assess the robustness of different control-laws applied to a closed-loop image stabilization task. Taking advantage of the well-characterized fly visuomotor pathway we record the electrical activity from an identified, motion-sensitive neuron, H1, to control the yaw rotation of a two-wheeled robot. The robot is equipped with 2 high-speed video cameras providing visual motion input to a fly placed in front of 2 CRT computer monitors. The activity of the H1 neuron indicates the direction and relative speed of the robot's rotation. The neural activity is filtered and fed back into the steering system of the robot by means of proportional and proportional/adaptive control. Our goal is to test and optimize the performance of various control laws under closed-loop conditions for a broader application also in other brain machine interfaces.

We use a closed-loop fly-machine interface to investigate general principles in neuronal control.

研究脑机接口的闭环性能的实验平台

The non-stationary nature and variability of neuronal signals is a fundamental problem in brain-machine interfacing. We developed a brain-machine ...

科研

神经科学

Cerebral Blood Oxygenation Measurement Based on Oxygen-dependent Quenching of Phosphorescence

Monitoring of the spatiotemporal characteristics of cerebral blood and tissue oxygenation is crucial for better understanding of the 
neuro-metabolic-vascular relationship. Development of new pO2 measurement modalities with simultaneous monitoring of pO2 in larger fields of view with higher 
spatial and/or temporal resolution will enable greater insight into the functioning of the normal brain and will also have significant impact on diagnosis 
and treatment of neurovascular diseases such as stroke, Alzheimer's disease, and head injury.
Optical imaging modalities have shown a great potential to provide high spatiotemporal resolution and quantitative imaging of pO2 based on hemoglobin absorption in visible and near infrared range of optical spectrum. However, multispectral measurement of cerebral blood oxygenation relies on photon migration through the highly scattering brain tissue. Estimation and modeling of tissue optical parameters, which may undergo dynamic changes during the experiment, is typically required for accurate estimation of blood oxygenation. On the other hand, estimation of the partial pressure of oxygen (pO2) based on oxygen-dependent quenching of phosphorescence should not be significantly affected by the changes in the optical parameters of the tissue and provides an absolute measure of pO2. Experimental systems that utilize oxygen-sensitive dyes have been demonstrated in in vivo studies of the perfused tissue as well as for monitoring the oxygen content in tissue cultures, showing that phosphorescence quenching is a potent technology capable of accurate oxygen imaging in the physiological pO2 range. 
Here we demonstrate with two different imaging modalities how to perform measurement of pO2 in cortical vasculature based on phosphorescence lifetime imaging. In first demonstration we present wide field of view imaging of pO2 at the cortical surface of a rat. This imaging modality has relatively simple experimental setup based on a CCD camera and a pulsed green laser. An example of monitoring the cortical spreading depression based on phosphorescence lifetime of Oxyphor R3 dye was presented. In second demonstration we present a high resolution two-photon pO2 imaging in cortical micro vasculature of a mouse. The experimental setup includes a custom built 2-photon microscope with femtosecond laser, electro-optic modulator, and photon-counting photo multiplier tube. We present an example of imaging the pO2 heterogeneity in the cortical microvasculature including capillaries, using a novel PtP-C343 dye with enhanced 2-photon excitation cross section.
Click here to view the related article <a href="http://www.jove.com/Details.php?ID=1731">Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems.</a>

We present an experimental procedure for measuring the partial pressure of oxygen (pO2) in cerebral vasculature based on oxygen-dependent quenching of phosphorescence. Animal preparation and imaging procedures were outlined for both large field of view CCD-based imaging of pO2 in rats and 2-photon excitation based imaging of pO2 in mice.

脑血氧测量基于磷光氧依赖淬火

Monitoring of the spatiotemporal characteristics of cerebral blood and tissue oxygenation is crucial for better understanding of the 
...

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable clinical state; in the development of animal models of focal ischemia, however, achieving reproducibility of experimentally induced infarct volume is essential. The rat is a widely used animal model for stroke due to its relatively low animal husbandry costs and to the similarity of its cranial circulation to that of humans2,3. In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple methods of MCA occlusion (MCAO) have been described to mimic this clinical syndrome in animal models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. In this video, we demonstrate the transient endovascular suture MCAO model in the spontaneously hypertensive rat (SHR). A filament with a silicon tip coating is placed intraluminally at the MCA origin for 60 minutes, followed by reperfusion. Note that the optimal occlusion period may vary in other rat strains, such as Wistar or Sprague-Dawley. Several behavioral indicators of stroke in the rat are shown. Focal ischemia is confirmed using T2-weighted magnetic resonance images and by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO.

Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.

焦距脑缺血模型大鼠大脑中动脉的血管内缝合闭塞

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable ...

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium (hfRPE) cultures with morphological, physiological and genetic characteristics of native human RPE. These hfRPE cell cultures exhibit heavy pigmentation, and electron microscopy show extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied mammalian models of native RPE, including human. These results were extended by the development of therapeutic interventions in several animal models of human eye disease. We have focused on strategies for the removal of abnormal fluid accumulation in the retina or subretinal space. The extracellular subretinal space separates the photoreceptor outer segments and the apical membrane of the RPE and is critical for maintenance of retinal attachments and a whole host of RPE/retina interactions.

We provide a reproducible method for culturing confluent monolayers of human fetal retinal pigment epithelial cells (hfRPE) cells that exhibit morphology, physiology, polarity, and protein and gene expression patterns of adult native tissue. This work has been extended to an animal model of several eye diseases.

视网膜色素上皮的生理与病理生理学研究的实验模型

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium ...

Assessment of Cerebral Lateralization in Children using Functional Transcranial Doppler Ultrasound (fTCD)

There are many unanswered questions about cerebral lateralization. In particular, it remains unclear which aspects of language and nonverbal ability are lateralized, whether there are any disadvantages associated with atypical patterns of cerebral lateralization, and whether cerebral lateralization develops with age. In the past, researchers interested in these questions tended to use handedness as a proxy measure for cerebral lateralization, but this is unsatisfactory because handedness is only a weak and indirect indicator of laterality of cognitive functions1. Other methods, such as fMRI, are expensive for large-scale studies, and not always feasible with children2.
Here we will describe the use of functional transcranial Doppler ultrasound (fTCD) as a cost-effective, non-invasive and reliable method for assessing cerebral lateralization. The procedure involves measuring blood flow in the middle cerebral artery via an ultrasound probe placed just in front of the ear. Our work builds on work by Rune Aaslid, who co-introduced TCD in 1982, and Stefan Knecht, Michael Deppe and their colleagues at the University of M&uuml;nster, who pioneered the use of simultaneous measurements of left- and right middle cerebral artery blood flow, and devised a method of correcting for heart beat activity. This made it possible to see a clear increase in left-sided blood flow during language generation, with lateralization agreeing well with that obtained using other methods3.
The middle cerebral artery has a very wide vascular territory (see Figure 1) and the method does not provide useful information about localization within a hemisphere. Our experience suggests it is particularly sensitive to tasks that involve explicit or implicit speech production. The 'gold standard' task is a word generation task (e.g. think of as many words as you can that begin with the letter 'B') 4, but this is not suitable for young children and others with limited literacy skills. Compared with other brain imaging methods, fTCD is relatively unaffected by movement artefacts from speaking, and so we are able to get a reliable result from tasks that involve describing pictures aloud5,6. Accordingly, we have developed a child-friendly task that involves looking at video-clips that tell a story, and then describing what was seen.

Assessment of Cerebral Lateralization in Children using Functional Transcranial Doppler Ultrasound fTCD

Functional transcranial Doppler sonography (fTCD) is a simple and non-invasive ultrasound technique which can be used to assess the lateralization of cognitive functions, especially language, and is suitable for use with children.

儿童使用功能经颅多普勒超声评估大脑偏侧（fTCD）

There are many unanswered questions about cerebral lateralization.  In particular, it remains unclear which aspects of language and nonverbal ability are ...

Strategies for Study of Neuroprotection from Cold-preconditioning

Neurological injury is a frequent cause of morbidity and mortality from general anesthesia and related surgical procedures that could be alleviated by development of effective, easy to administer and safe preconditioning treatments. We seek to define the neural immune signaling responsible for cold-preconditioning as means to identify novel targets for therapeutics development to protect brain before injury onset. Low-level pro-inflammatory mediator signaling changes over time are essential for cold-preconditioning neuroprotection. This signaling is consistent with the basic tenets of physiological conditioning hormesis, which require that irritative stimuli reach a threshold magnitude with sufficient time for adaptation to the stimuli for protection to become evident.
Accordingly, delineation of the immune signaling involved in cold-preconditioning neuroprotection requires that biological systems and experimental manipulations plus technical capacities are highly reproducible and sensitive. Our approach is to use hippocampal slice cultures as an in vitro model that closely reflects their in vivo counterparts with multi-synaptic neural networks influenced by mature and quiescent macroglia / microglia. This glial state is particularly important for microglia since they are the principal source of cytokines, which are operative in the femtomolar range. Also, slice cultures can be maintained in vitro for several weeks, which is sufficient time to evoke activating stimuli and assess adaptive responses. Finally, environmental conditions can be accurately controlled using slice cultures so that cytokine signaling of cold-preconditioning can be measured, mimicked, and modulated to dissect the critical node aspects. Cytokine signaling system analyses require the use of sensitive and reproducible multiplexed techniques. We use quantitative PCR for TNF-&alpha; to screen for microglial activation followed by quantitative real-time qPCR array screening to assess tissue-wide cytokine changes. The latter is a most sensitive and reproducible means to measure multiple cytokine system signaling changes simultaneously. Significant changes are confirmed with targeted qPCR and then protein detection. We probe for tissue-based cytokine protein changes using multiplexed microsphere flow cytometric assays using Luminex technology. Cell-specific cytokine production is determined with double-label immunohistochemistry. Taken together, this brain tissue preparation and style of use, coupled to the suggested investigative strategies, may be an optimal approach for identifying potential targets for the development of novel therapeutics that could mimic the advantages of cold-preconditioning.

We seek to define the neural immune signaling responsible for cold-preconditioning as means to identify novel targets for therapeutics development to protect brain before injury onset. We present strategies for such work that require biological systems, experimental manipulations plus technical capacities that are highly reproducible and sensitive.

冷预处理神经保护作用的研究策略

Neurological injury is a frequent cause of morbidity and mortality from general anesthesia and related surgical procedures that could be alleviated by ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Evaluation of Bioenergetic Function in Cerebral Vascular Endothelial Cells

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types that comprise the BBB; these cells have a very high-energy demand, which requires optimal mitochondrial function. In the case of disease or injury, the mitochondrial function in these cells can be altered, resulting in disease or&#160;the opening of the BBB. In this manuscript, we introduce a method to measure mitochondrial function in CVE cells by using whole, intact cells and a bioanalyzer. A mito-stress assay is used to challenge the cells that have been perturbed, either physically or chemically, and evaluate their bioenergetic function. Additionally, this method also provides a useful way to screen new therapeutics that have direct effects on mitochondrial function. We have optimized the cell density necessary to yield oxygen consumption rates that allow for the calculation of a variety of mitochondrial parameters, including ATP production, maximal respiration, and spare capacity. We also show the sensitivity of the assay by demonstrating that the introduction of the&#160;microRNA, miR-34a, leads to a pronounced and detectable decrease in mitochondrial activity. While the data shown in this paper is optimized for the bEnd.3 cell line, we have also optimized the protocol for primary CVE cells, further suggesting the utility in preclinical and clinical models.

Endothelial cell mitochondria are critical to maintain blood-brain-barrier integrity. We introduce a protocol to measure bioenergetic function in cerebral vascular endothelial cells.

在脑血管内皮细胞生物能量功能的评价

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types ...

A Magnetic Resonance Imaging Protocol for Stroke Onset Time Estimation in Permanent Cerebral Ischemia

MRI provides a sensitive and specific imaging tool to detect acute ischemic stroke by means of a reduced diffusion coefficient of brain water. In a rat model of ischemic stroke, differences in quantitative T1 and T2 MRI relaxation times (qT1 and qT2) between the ischemic lesion (delineated by low diffusion) and the contralateral non-ischemic hemisphere increase with time from stroke onset. The time dependency of MRI relaxation time differences is heuristically described by a linear function and thus provides a simple estimate of stroke onset time. Additionally, the volumes of abnormal qT1 and qT2 within the ischemic lesion increase linearly with time providing a complementary method for stroke timing. A (semi)automated computer routine based on the quantified diffusion coefficient is presented to delineate acute ischemic stroke tissue in rat ischemia. This routine also determines hemispheric differences in qT1 and qT2 relaxation times and the location and volume of abnormal qT1 and qT2 voxels within the lesion. Uncertainties associated with onset time estimates of qT1 and qT2 MRI data vary from &plusmn; 25 min to &plusmn; 47 min for the first 5 hours of stroke. The most accurate onset time estimates can be obtained by quantifying the volume of overlapping abnormal qT1 and qT2 lesion volumes, termed 'Voverlap' (&plusmn; 25 min) or by quantifying hemispheric differences in qT2 relaxation times only (&plusmn; 28 min). Overall, qT2 derived parameters outperform those from qT1. The current MRI protocol is tested in the hyperacute phase of a permanent focal ischemia model, which may not be applicable to transient focal brain ischemia.

A protocol for stroke onset time estimation in a rat model of stroke exploiting quantitative magnetic resonance imaging (qMRI) parameters is described. The procedure exploits diffusion MRI for delineation of the acute stroke lesion and quantitative T1 and T2 (qT1 and qT2) relaxation times for timing of stroke.

脑缺血中风发作时间估计的磁共振成像协议

MRI provides a sensitive and specific imaging tool to detect acute ischemic stroke by means of a reduced diffusion coefficient of brain water. In a rat ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

器官海马切片培养模型研究肿瘤细胞的神经保护和侵袭性

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...