Name: strategies for study of neuroprotection from cold-preconditioning
Uploaded: 2010-09-02T17:30:00
Duration: 16 min 27 s
Description: Watch this Scientific Journal Video about Strategies for Study of Neuroprotection from Cold-preconditioning at JoVE.com

这项工作是由美国国家神经紊乱和中风研究所（NS - 19108），偏头痛研究基金会，和白理查德P. Kraig基金会的赠款。玛西娅体育Kraig女士的协助下，准备文化传媒和维护切片文化。我们感谢她的意见，并在本文的最后版本的修订叶莲娜Grinberg。

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两个极其重要的概念，重要的是参与冷预处理神经保护作用的细胞因子信号系统的划分是在图6和7所示。首先，细胞因子的浓度极低，在正常脑组织中的信号分子。然而，生理细胞因子浓度的变化有一个巨大的可能改变大脑结构和功能（即表型），因为他们有能力改变基因的表达（图6）。此外，细胞因子是高度冗余和多效性，多种细胞因子，可以有类似的效果，和一个单一的细胞因子可以变量的影响（图7）。因此，要准确地建立先天的神经保护作用的细胞因子冷预处理（或其他生理预处理刺激），必须确定相关的信令变量的综合分析基地。这是通过复用检测策略。这将建立的“签名”冷预处理神经保护作用的细胞因子。 <img src="/files/ftp_upload/2192/2192fig6.jpg" alt="图6" /> 图6。脑细胞因子信号的电源 。插图传达巨大的信号的脑细胞因子的生理浓度的力量，相比其他公认同行的浓度。浓度表示为距离的倒数，作为参考点的单猫须开始。钠（10 -1 m的一粒胡椒说明）和钾（10 -3 M说明了一个西红柿）是在约150个和3毫米，分别的水平间质性脑空间，并有公认角色神经细胞的电生理功能。同样，pH值（即〜10 -7 m层的氢离子和780米沿芝加哥的湖畔所示）和钙（即〜10 -8看到7.8公里，从谷歌卫星图像沿着芝加哥的麦考密克湖畔看到的水平地点在海德公园附近的芝加哥大学岬点）。此外，这些浓度间质空间释放的神经递质影响当地的大脑区域的活动。与此相反，细胞因子（从地球到火星的距离显示），可以改变浓度的脑功能少十几万次。 <img src="/files/ftp_upload/2192/2192fig7.jpg" alt="图7" /> 图7。互动信号与生俱来的细胞因子的途径。细胞因子是高度冗余，多种细胞因子多效可以有类似的效果，可以有一个单一的细胞因子变量的影响。这种多样性源于复杂的交互信号配体，受体和磷酸化水平发生。事实上，大脑的不同类型的细胞，每个先天细胞特异的细胞因子，受体，和相关的磷酸化变化的能力，组成更加复杂源于。这里说明的目的，只有天生的细胞因子信号转导通路（从免疫细胞研究中派生）所示。为了简便起见，绘制大脑是作为一个单一的细胞显示出与生俱来的细胞因子的潜在的相互作用（白线）（IL -1α和IL -1β（这里​​称为IL -1α/β），肿瘤坏死因子-α，IL - 6，干扰素γ和IL - 10），受体（IL - 1R1，TNFR1，IL-6/gp130，IFNγR，和IL - 10R）和磷酸化（即，P38（P38 - MAPK）和JNK激酶ERK1 / 2）和转录因子（ATF - 2，NFκB，和STAT3）。例如，TNF -α信号通过TNFR1 JNK，P38 MAPK和ERK1 / 2，从而引发通过ATF - 2基因的表达。 TNF -α也可直接通过改变基因表达NFκB的激活。总之，这些转录因子的激活唤起增加（箭头）和下降（钝端）所示的细胞因子及其受体的表达。更重要的是，这些途径表明，在一个细胞因子的变化（如TNF -α）的影响其他细胞因子（如IL -1β）的生产。因此，建立准确的冷预处理，必须确定相关的信令变量的综合分析的细胞因子神经保护基地。这将建立先天的“签名”冷预处理的细胞因子。 （图片编制的数据参考＃25。）

制备切片文化<table border="1"><tbody><tr><td> 试剂/设备名称 </td><td> 公司 </td><td> 目录/型号 </td><td> 评论 </td></tr><tr><td> Millicell小CM组织培养插入（30毫米） </td><td>赛默飞世尔</td><td> PICM0 - 3050 </td><td></td></tr><tr><td>基础培养基鹰</td><td> Invitrogen公司</td><td> 21010 </td><td></td></tr><tr><td>厄尔s平衡盐溶液</td><td>西格玛</td><td> E2888 </td><td></td></tr><tr><td>马血清</td><td> Invitrogen公司</td><td> 26050-088 </td><td></td></tr><tr><td> Glutamax </td><td> Invitrogen公司</td><td> 35050 </td><td></td></tr><tr><td>庆大霉素</td><td> Invitrogen公司</td><td> 15710-064 </td><td></td></tr><tr><td> Fungizone </td><td> Invitrogen公司</td><td> 15295 </td><td></td></tr><tr><td> D -葡萄糖</td><td>西格玛</td><td> G8769 </td><td></td></tr><tr><td> BSL - 1通风柜</td><td>面包师傅</td><td></td><td></td></tr><tr><td> McIlwain组织菜刀</td><td>布林克曼</td><td></td><td></td></tr><tr><td>特氟隆磁盘</td><td>布林克曼</td><td> 023-49-310-1 </td><td></td></tr><tr><td>抹刀</td><td>赛默飞世尔</td><td> 14 - 373 - 25A </td><td></td></tr><tr><td>弧形光圈剪刀</td><td>精细科学的工具</td><td> 14091-09 </td><td></td></tr><tr><td>长直剪</td><td>精细科学的工具</td><td> 14002-14 </td><td></td></tr><tr><td>长钳</td><td>赛默飞世尔</td><td> 13-812-40 </td><td></td></tr><tr><td>斜角钳</td><td>精细科学的工具</td><td> 11808-02 </td><td></td></tr><tr><td>短钳</td><td>赛默飞世尔</td><td> 13-812-38 </td><td></td></tr><tr><td> ＃5 Inox公司钳</td><td>精细科学的工具</td><td> 11254-20 </td><td></td></tr><tr><td> 2虹膜铲刀</td><td>精细科学的工具</td><td> 10094-13 </td><td></td></tr><tr><td> 150 × 15毫米培养皿</td><td>赛默飞世尔</td><td> 08-757-148 </td><td></td></tr><tr><td> 60 × 15毫米培养皿</td><td>赛默飞世尔</td><td> 08 - 757 - 100B </td><td></td></tr><tr><td>野生M8的体视显微镜</td><td>野生Heerbrugg </td><td></td><td></td></tr><tr><td> Gey的平衡盐溶液</td><td>西格玛</td><td> G9779 </td><td></td></tr><tr><td> Blak -射线短波紫外线测量仪</td><td> UVP </td><td> J - 225 </td><td></td></tr><tr><td> 6猎鹰培养皿</td><td>赛默飞世尔</td><td> 08 - 772 - 1B </td><td></td></tr><tr><td> Neurobasal </td><td> Invitrogen公司</td><td> 21103</td><td></td></tr><tr><td> B27的补充</td><td> Invitrogen公司</td><td> 17504 </td><td></td></tr><tr><td> Gem21 Neuroplex </td><td>双子座生物制品</td><td> 400-160-010 </td><td></td></tr><tr><td>抗坏血酸</td><td>西格玛</td><td> A4544 </td><td></td></tr><tr><td> CO 2分析仪</td><td>巴哈拉赫</td><td> ＃2820 </td><td></td></tr></tbody></table>预检片文化的活力<table border="1"><tbody><tr><td> 试剂/设备名称 </td><td> 公司 </td><td> 目录/型号 </td><td> 评论 </td></tr><tr><td> Sytox绿</td><td> Invitrogen公司</td><td> S7020 </td><td></td></tr><tr><td> DMIBRE倒置显微镜</td><td>徕卡</td><td></td><td></td></tr></tbody></table>冷预处理<table border="1"><tbody><tr><td> 试剂/设备名称 </td><td> 公司 </td><td> 目录/型号 </td><td> 评论 </td></tr><tr><td> BSL - 2通风柜</td><td> NuAire </td><td></td><td></td></tr></tbody></table>兴奋毒性损伤<table border="1"><tbody><tr><td> 试剂/设备名称 </td><td> 公司 </td><td> 目录/型号 </td><td> 评论 </td></tr><tr><td> CoolSnap FX CCD相机</td><td> Photometrics </td><td></td><td></td></tr><tr><td> Fluoroscein </td><td> Invitrogen公司</td><td> F36915 </td><td></td></tr><tr><td> MetaMorph软件</td><td>分子器件</td><td>五，04年7月5日</td><td></td></tr><tr><td> 100微米深hemacytometer </td><td> Hausser科学</td><td></td><td></td></tr><tr><td> N -甲基- D -天冬氨酸</td><td> Calbiochem </td><td> 454575 </td><td></td></tr><tr><td> SigmaStat软件</td><td> SYSTAT软件公司</td><td>诉3.5 </td><td></td></tr></tbody></table>冷预处理应用的联合治疗<table border="1"><tbody><tr><td> 试剂/设备名称 </td><td> 公司 </td><td> 目录/型号 </td><td> 评论 </td></tr><tr><td> sTNFR1 </td><td> R＆D系统</td><td> 425 - R1 - 050 </td><td></td></tr><tr><td>牛血清白蛋白</td><td>西格玛</td><td> A - 4503 </td><td></td></tr></tbody></table> RNA分离<table border="1"><tbody><tr><td> 试剂/设备名称 </td><td> 公司 </td><td> 目录/型号 </td><td> 评论 </td></tr><tr><td> RNAL ATER </td><td> Ambion公司</td><td> AM7021 </td><td></td></tr><tr><td>微的RNeasy试剂​​盒</td><td> Qiagen公司</td><td> 74004 </td><td></td></tr><tr><td> β-巯基乙醇</td><td>费舍尔</td><td> 03446-100 </td><td></td></tr><tr><td> Diposable 1.5 RNase的免费杵</td><td>南泽西精密工具和模具公司</td><td> 749521-1590 </td><td></td></tr><tr><td> RNasin </td><td> Promega公司</td><td> N261A </td><td></td></tr><tr><td>三羟甲基]氨基甲烷</td><td>西格玛</td><td> T3069 </td><td></td></tr><tr><td> EDTA（乙二胺四乙酸二钠水合物</td><td>西格玛</td><td> ＃E7889 </td><td></td></tr></tbody></table> RNA定量<table border="1"><tbody><tr><td> 试剂/设备名称 </td><td> 公司 </td><td> 目录/型号 </td><td> 评论 </td></tr><tr><td> RiboGreen含量</td><td> Invitrogen公司</td><td> R11491 </td><td></td></tr><tr><td>酵母tRNA </td><td> Invitrogen公司</td><td> 15401-011 </td><td></td></tr><tr><td> 1.5 mL离心管</td><td>美国科学</td><td> 16155500 </td><td></td></tr><tr><td> 96荧光检测板</td><td> NUNC </td><td> DK - 4000 </td><td></td></tr><tr><td> 96孔荧光酶标仪</td><td> TECAN </td><td>沙费尔二</td><td></td></tr></tbody></table>定量PCR <table border="1"><tbody><tr><td> 试剂/设备名称 </td><td> 公司 </td><td> 目录/型号 </td><td> 评论 </td></tr><tr><td> iScript反向转录试剂盒</td><td> Bio - Rad公司</td><td> 170-8891 </td><td></td></tr><tr><td> SYBR GREEN </td><td> Invitrogen公司</td><td> S7563 </td><td></td></tr><tr><td>白金Taq聚合酶</td><td> Invitrogen公司</td><td> RNA的Ultrasense 11732-927 </td><td></td></tr><tr><td> iCycler热循环仪</td><td> Bio - Rad公司</td><td> iCycler光模块</td><td></td></tr><tr><td> iCycler系统软件</td><td> Bio - Rad公司</td><td>诉3.1 </td><td></td></tr></tbody></table>定量PCR微阵列<table border="1"><tbody><tr><td> 试剂/设备名称 </td><td> 公司 </td><td> 目录/型号 </td><td> 评论 </td></tr><tr><td>核糖核酸酶客队 </td><td>分子生物产品</td><td> ＃7000 </td><td></td></tr><tr><td> RT 2第一链套件</td><td> SABiosciences </td><td> C - 03 </td><td></td></tr><tr><td>基于SYBR GREEN - PCR混合物</td><td> SABiosciences </td><td> PA - 011 </td><td>特定iCycler </td></tr><tr><td> RT 2探查PCR阵列</td><td> SABiosciences </td><td> PARN - 011A </td><td></td></tr></tbody></table>蛋白质组学的含量<table border="1"><tbody><tr><td> 试剂/设备名称 </td><td> 公司 </td><td> 目录/型号</td><td> 评论 </td></tr><tr><td>细胞裂解试剂盒</td><td> Bio - Rad公司</td><td> 171-304011 </td><td></td></tr><tr><td>离心</td><td> Fisher Scientific则</td><td>微型7 </td><td></td></tr><tr><td> BSA的蛋白质股票</td><td> Bio - Rad公司</td><td> 500-0007 </td><td></td></tr><tr><td> BCA蛋白检测试剂盒</td><td> Thermo Scientific的</td><td> 23225 </td><td></td></tr><tr><td>免疫96微孔板</td><td> NUNC </td><td> 449824 </td><td></td></tr><tr><td>密封胶带</td><td> Bio - Rad公司</td><td> 2239444 </td><td></td></tr><tr><td> 96孔板读者</td><td> Perkin Elmer公司</td><td> 1420 - mulilabel柜台</td><td></td></tr><tr><td> Bioplex大鼠肿瘤坏死因子-α细胞因子的测定</td><td> Bio - Rad公司</td><td> X0000001T </td><td></td></tr></tbody></table>免疫染色<table border="1"><tbody><tr><td> 试剂/设备名称 </td><td> 公司 </td><td> 目录/型号 </td><td> 评论 </td></tr><tr><td> 16％多聚甲醛</td><td>阿法埃莎</td><td> 43368 </td><td></td></tr><tr><td>赖氨酸</td><td>西格玛</td><td> L - 6001 </td><td></td></tr><tr><td>磷酸钠</td><td> Fisher Scientific则</td><td> S374 - 1 </td><td></td></tr><tr><td>高碘酸钠</td><td>西格玛</td><td> S - 1878年</td><td></td></tr><tr><td>叠氮化钠</td><td>西格玛</td><td> S - 2002 </td><td></td></tr><tr><td>过氧化氢（30％） </td><td>西格玛</td><td> H1009 </td><td></td></tr><tr><td>山羊血清</td><td>科罗拉多州的血清公司</td><td> CS 0920 </td><td></td></tr><tr><td> TRITON X - 100 </td><td>西格玛</td><td> T - 8787 </td><td></td></tr><tr><td>染色菜</td><td>特德佩拉公司</td><td> 14511 </td><td></td></tr><tr><td>板振动筛</td><td>实验室线仪器公司</td><td> 3520 </td><td></td></tr><tr><td> ＃1滤纸（185毫米） </td><td>瓦特曼</td><td> 1001-185 </td><td></td></tr><tr><td> 3,3 - 二氨基</td><td>西格玛</td><td> D8001 </td><td></td></tr><tr><td>二甲基亚砜</td><td>西格玛</td><td> D8418 </td><td></td></tr><tr><td>乙醇</td><td> Pharmco - AAPER </td><td> 111000200 </td><td></td></tr><tr><td>二甲苯</td><td> Fisher Scientific则</td><td> X3S - 4 </td><td></td></tr><tr><td> Permount安装媒体</td><td> Fisher Scientific则</td><td> SP15 - 100 </td><td></td></tr><tr><td>低温恒温器</td><td>徕卡</td><td>厘米3 050小号</td><td></td></tr><tr><td>组织Tek的</td><td>特德佩拉公司</td><td> 2705​​0 </td><td></td></tr><tr><td>细尖的油漆刷</td><td>特德佩拉</td><td> 11806 </td><td></td></tr><tr><td>人民行动党堵笔</td><td>特德佩拉公司</td><td> 22309 </td><td></td></tr><tr><td> SFX信号增强器</td><td> Invitrogen公司</td><td> A31631 </td><td></td></tr><tr><td>延长抗淬灭</td><td> Invitrogen公司</td><td> P7481 </td><td></td></tr></tbody></table>

strategies for study of neuroprotection from cold-preconditioning

神经损伤的发病率和死亡率的常见原因，从一般的麻醉和相关手术过程中，可以有效的发展缓解，易于管理和安全的预处理治疗。我们寻求定义冷预处理手段确定疗法的发展新目标，以保护脑损伤发病前的神经免疫信号。低级亲炎症介质，信号随时间变化的冷预处理神经保护作用是必不可少的。这个信号是一致的生理调节兴奋效应的基本原理，这就需要刺激性的刺激达到足够的时间适应保护的刺激阈值的大小，才能显现。 因此，在冷预处理神经保护作用的免疫信号划分要求，生物系统和实验操作，再加上技术能力的高度重复性和敏感。我们的做法是使用作为海马脑片培养，在体外模型，密切反映他们在与多突触的神经网络，成熟的和静态的胶质细胞/小胶质细胞的影响体内同行。小胶质细胞胶质状态尤为重要，因为它们的细胞因子的主要来源，这是femtomolar范围执行。此外，切片文化，可以在体外保持几个星期，这是足够的时间来唤起激活刺激和评估的适应性反应。最后，环境条件，可以准确地控制使用切片文化，使细胞因子信号寒冷的预处理可以进行测量，模仿，并调制，剖析关键的节点方面。细胞因子信号系统的分析需要使用的敏感和可重复性复用的技术。我们使用肿瘤坏死因子-α定量PCR，实时定量PCR定量阵列筛选，评估组织范围内的细胞因子变化的小胶质细胞激活屏幕。后者是一个最敏感的和可重复性的手段来衡量多个细胞因子系统，同时信号的变化。重大变化，然后有针对性的qPCR蛋白质检测证实。我们探头组织为基础的细胞因子蛋白复微流式细胞仪检测使用Luminex公司的技术变化。确定双标免疫组化细胞特异性细胞因子的产生。两者合计，这个脑组织编制和使用的样式，再加上建议的调查战略可能是一个确定发展的新型疗法，可以模仿冷预处理的优势的潜在目标的最佳途径。

Watch this Scientific Journal Video about Strategies for Study of Neuroprotection from Cold-preconditioning at JoVE.com

Strategies for Study of Neuroprotection from Cold-preconditioning

我们寻求定义冷预处理手段确定疗法的发展新目标，以保护脑损伤发病前的神经免疫信号。我们目前所做的这些工作需要的生物系统，实验操作，加上技术能力的高度重复性和敏感的战略。

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 ...

Research

JoVE Journal

Neuroscience

Laser Capture Microdissection of Drosophila Peripheral Neurons

Laser Capture Microdissection of Drosophila Peripheral Neurons

激光捕获显微切割技术的果蝇外周神经元

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

科研

神经科学

Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

突触改变老化过程中淀粉样蛋白病理研究从大鼠和转基因小鼠海马急性片的制备

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The ...

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Vibrodissociation从啮齿动物脑切片的神经元，研究突触传递和图像突触前的码头

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

连续光漂白划定单雪旺细胞在神经肌肉接头处

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

膜下丘脑腹内侧神经元的电位染料成像从成年小鼠为研究葡萄糖传感

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins

从海马CA1区片核富含分的分离，研究突触核信使蛋白质的活性依赖进口核

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular ...

Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

作为一种工具来描绘分子途径参与神经毒性和神经保护神经元细胞在线实时阻抗为基础的细胞分析仪

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness 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. ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

时间依赖性增加对微电极阵列刺激神经元细胞培养物的网络响应

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

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

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 ...