我们要感谢美国国家卫生赠款R01NS057476，P50NS010828，P01NS055104，R01EB000790，K99NS0670​​50，R01HL081273和R01EB007279和美国心脏协会授予0855772D研究院的支持。

1。宽视场成像PO2查看老鼠的皮质血管<ol><li>最初麻醉与异氟醚组（250克-350克），股动脉和静脉插管监测心率，血压，血气，以及静脉滴注。体温保持在37 ± 0.1 ° C。进行气管切开术，大鼠和通风与空气和氧气的混合物。 </li><li>用于测量血液气体的血液样本每次30-45分钟，通风和麻醉的调整，以保持正常的生理范围内的血气。 </li><li>颅顶骨4毫米x 4毫米大小的窗口，是一个封闭的准备成像。骨和硬脑膜和颅窗口充满了1.5％琼脂糖，密封，用显微镜的盖玻片。 1毫米2毛刺额骨上的孔是用来诱导intracortical显微注射氯化钾（〜10μL，1米）惩教署。应采取特别小心，以避免任何损害的皮质血管。多余的热量，机械压力，或缺氧的短暂时期，可能会危及从血管进入间质空间血脑屏障的染料，并导致泄漏。 </li><li>从光学不透明材料的面具是周围放置颅窗口。面膜的目的是吸收磷光信号，从泄露组织的染料，从硬脑膜问题边缘未来。染料的间隙是非常明亮的磷光，可以破坏测量的来源。间质空间中的染料的亮度增加，是导致低氧分压和猝灭速率低的环境中建立的染料。实验血管的损伤，因此容易识别的外观明亮的磷光点，在这种情况下的实验数据将被拒绝。 </li><li>手术完成后，异氟醚停止麻醉切换到alphachloralose（50毫克/公斤静脉注射40毫克/（千克H）输液）。 </li><li>动物转移上车，呼吸，血压和体温监测。动物呼吸室内空气的同时，迅速转移到成像设置适当的空气和氧气的混合物，并重新连接流量计。 </li><li>持有的动物脑立体定位框架，是摆下的目标。颅窗口集中在视野下的目标和定位焦平面平行。 </li><li>脉冲激光开启，并设置为最小的脉冲能量。脉冲电能表和脉冲能量传递到样品的激光束指向调整为不超过10兆焦耳/ 厘米 2 。束与斜入射角度〜60度和激光ISS国际空间站上的颅窗口为本关闭。 </li><li>血液通风和麻醉气体测量和调整执行，直到所有的动物生理参数正常生理范围内。 </li><li>假设血容量是体重约7％，达到4 × 10 -5米的探头血药浓度的1毫升生理盐水溶解适量的磷光探针Oxyphor R3 。探头解决方案是通过股静脉注射。 </li><li>一个1米氯化钾解决方案的编制和〜1 UL是通过额骨上的毛刺小孔，用注射器注入诱导惩教署。 </li><li>打开激光成像KCl溶液注射后立即开始。磷光成像是在〜10分钟。 </li><li>另一种血液中的气体进行测量，以确认动物生理参数仍然在正常范围内。 </li><li>磷光寿命得到所有的像素，通过拟合单指数衰减，使用Matlab中的统计加权非线性平方米装修。磷光寿命转换为使用一个实证斯特恩Volmer关系（见下文）的PO2值。 </li></ol> 2。高分辨率双光子PO2在皮质鼠标的微型血管成像<ol><li>小鼠麻醉isofluorene和股动脉插管监测心率，血压，血气，以及染料的管理。体温保持在37 ± 0.1 ° C和动物自主呼吸的空气和氧气的混合物。 </li><li>颅窗口是根据最初是由大卫柯菲德和温弗里德Denk开发的技术准备。应采取一个良好的护理，以避免任何损害血管间质空间，防止可能泄漏的染料。 </li><li>在整个筹备过程和一个用于测量血液中气体的少量血液样本采取通风和麻醉的调整，以保持血气ES在正常生理范围。 </li><li>动物移动到成像装置。一个修改后的立体框架，认为动物是摆下的目标。颅窗口集中在视野下的奥林巴斯4X目标和定位焦平面平行。 </li><li>颅窗口的图像与数码相机通过目镜，4X的目标是与奥林巴斯20X物镜（NA = 0.95）所取代。 </li><li>血液通风和麻醉气体测量和调整，直到所有的动物生理参数，是一种正常的生理范围内执行。 </li><li>假设血液量的约7％的体重，适量的磷光探针PTP - C343是0.2毫升生理盐水达到〜15微米的探针的血药浓度溶解。探头解决方案是经股动脉注射。 </li><li>开始实验，通过设置在每个像素的排放染料激发和收集所需的时间间隔。 </li><li>取得的一项调查显示扫描，这是一个缓慢的二维光栅扫描显示在当前深入的血管结构的磷光强度衰减。目的是颅窗口垂直移动（Z轴）和磷光强度缓慢2D调查扫描使用840纳米的最小激光功率，发现在成像平面上的微血管。 </li><li>在每个成像深度，在这个深度，基于磷光调查扫描，血管内的点被选中，并记录在每个点上的磷光衰减的平均预定的号码重复。 </li><li>设置所需的平均量，测量时间间隔，和实验的持续时间，并开始测量。在实验期间，PO 2测量是在选定的地点在指定的测量间隔收购。 </li><li>在测量过程中，该软件重新激发激光指示选定的地点，通过改变振镜扫描镜的位置。控制振镜扫描仪，光电调制器，和所有其他的设备进行定制LabVIEW软件。 </li><li>磷光寿命得到所有选中的点，通过拟合单指数衰减，使用Matlab中的非线性平方米装修。磷光寿命转换为使用一个实证斯特恩Volmer关系（见下文）的PO2值。 </li><li>收集各种皮层深处的数据后，注入血管成像微血管结构右旋糖酐标记的荧光染料。 </li><li>执行FITC荧光双光子成像，使用绿色通道，四通道探测器获取血管的结构图像的堆栈。 </li><li>动物实验是由美国马萨诸塞州总医院研究动物保健小组委员会提出的准则和法规的规定执行。 </li></ol> 3。代表性的成果: <img src="/files/ftp_upload/1694/1694fig1.jpg" alt="图1" /> 图1（一）对这个数字的左侧面板显示惩教署波到来前查看形象氧气压力的一个广泛的领域。 （b）显示面板右侧平均氧气压力随时间的演化（一）在惩教署面板上标记的利益区域内传播。 图2（AVI电影）:这部电影显示惩教署波的传播过程中的氧气压力在整个颅窗口的时间演化。比例尺表示氧气毫米汞柱的压力。 <img src="/files/ftp_upload/1694/1694fig3.jpg" alt="图3" /> 图3。3D投影成像血管堆栈。灰色色调代表一个体积容器面具，创建的基础上的结构图像。 2 PO测量的值是颜色编码。比例尺为200微米。自然出版集团的许可重印 7 。

<ol>
	<li>Kleinfeld, D., Friedman, B., Lyden, P. D., Shih, A. Y., Chen, J., Xu, Z., Xu, X., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+occlusion+to+surface+and+deep+vessels+in+neocortex+via+linear+and+nonlinear+optical+absorption,+Animal+Models+of+Acute+Neurological+Injuries.">Targeted occlusion to surface and deep vessels in neocortex via linear and nonlinear optical absorption, Animal Models of Acute Neurological Injuries.</a> Contemporary Neuroscience Series. , (2007).</li><li>Mostany, R., Portera-Cailliau, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Craniotomy+Surgery+Procedure+for+Chronic+Brain+Imaging.">A Craniotomy Surgery Procedure for Chronic Brain Imaging.</a> J Vis Exp. , (2008).</li><li>Lebedev, A. Y., Cheprakov, A. V., Sakadzic, S., Boas, D. A., Wilson, D. F., Vinogradov, S. A., A, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+Phosphorescent+Probes+for+Oxygen+Imaging+in+Biological+Systems.">Dendritic Phosphorescent Probes for Oxygen Imaging in Biological Systems.</a> Applied Materials &#38; Interfaces. , (2009).</li><li>Finikova, O. S., Lebedev, A. Y., Aprelev, A., Troxler, T., Gao, F., Garnacho, C., Muro, S., Hochstrasser, R. M., Vinogradov, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+microscopy+by+two-photon-excited+phosphorescence.">Oxygen microscopy by two-photon-excited phosphorescence.</a> Chemphyschem. 9, 1673-1679 (2008).</li><li>Sakad&#382;i&#263;, S., Yuan, S., Dilekoz, E., Ruvinskaya, S., Vinogradov, S. A., Ayata, C., Boas, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+imaging+of+cerebral+partial+pressure+of+oxygen+and+blood+flow+during+functional+activation+and+cortical+spreading+depression.">Simultaneous imaging of cerebral partial pressure of oxygen and blood flow during functional activation and cortical spreading depression.</a> Appl Opt. 48, D169-D177 (2009).</li><li>Yaseen, M. A., Srinivasan, V. J., Sakadzic, S., Wu, W., Ruvinskaya, S., Vinogradov, S. A., Boas, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+monitoring+of+oxygen+tension+in+cortical+microvessels+with+confocal+microscopy.">Optical monitoring of oxygen tension in cortical microvessels with confocal microscopy.</a> Opt Express. 17, 22341-22350 (2009).</li><li>Sakadzic, S., Roussakis, E., Yaseen, M. A., Mandeville, E. T., Srinivasan, V. J., Arai, K., Ruvinskaya, S., Devor, A., Lo, E. H., Vinogradov, S. A., Boas, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+high-resolution+measurement+of+partial+pressure+of+oxygen+in+cerebral+vasculature+and+tissue.">Two-photon high-resolution measurement of partial pressure of oxygen in cerebral vasculature and tissue.</a> Nat Methods. 7, 755-759 (2010).</li></ol>

我们证明了两个PO2皮质微血管的基础上磷光氧依赖淬火的测量应用。虽然基于CCD成像的第一种方法提供广泛的领域观点氧分压监测，测量皮质微血管的氧气分压双光子显微镜的基础上提供了毛细血管的决议，并允许在深度成像。这两种方法提供高速度和高信号噪声的测量。此外，PO2磷光寿命测量主要是在实验过程中，这通常是其他光学成像技术，有一个对比机制的基础上强度的关注，在组织光学参数的变化不敏感。主办仪器允许动态交付的氧气和脑组织的新陈代谢，这将导致更好地了解在正常和病变脑的神经血管耦合的定量分析

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cerebral blood oxygenation measurement based on oxygen-dependent quenching of phosphorescence

监测脑血流和组织氧合的时空特征是为更好地理解代谢的神经血管的关系是至关重要的。新氧分压PO2同步监测的角度较大的领域，具有较高的空间和/或时间分辨率的测量方式的发展将使到正常的大脑的运作有更深的了解，也将产生重大影响，如神经血管疾病的诊断和治疗中风，阿尔茨海默氏症，和头部受伤。 光学成像方式，都表现出很大的潜力，提供高时空分辨率和PO2定量成像，在可见光和近红外光谱范围内的血红蛋白吸收的基础上。然而，多光谱测量脑血氧依赖通过高散射脑组织中光子迁移。组织光学参数，在实验过程中可能发生的动态变化的估计和建模，通常需要为准确估计血氧。另一方面，估计的氧分压（PO2）基于磷光氧依赖淬火应不显着影响，在组织光学参数的变化，并提供了一​​个绝对的PO2措施。利用氧敏感染料的实验系统已被证明在体内灌注组织的研究以及监测在组织培养中的氧气含量，表明磷光淬火是一个强有力的技术，能够准确的氧气成像PO2在生理范围。 在这里，我们展示了两种不同的成像方式如何执行上的磷光寿命成像的皮层血管PO2测量。在第一个示范，我们目前在老鼠的皮质表面的角度宽视场成像PO2。这种成像方式比较简单的实验装置，基于CCD相机和一个脉冲的绿色激光。监测的皮层扩散性抑制，基于Oxyphor R3的染料的磷光寿命的一个例子。在第二个演示中，我们提出了一个高分辨率的双光子PO2在皮质鼠标的微型血管成像。实验装置包括一个定制的双光子显微镜，飞秒激光，电光调制​​器，光子计数光电倍增管。我们目前的成像例如皮质微血管包括毛细血管氧分压异质性，使用新型具有增强PTP - C343染料的双光子激发截面。 点击这里查看相关文章<a href="https://www-jove-com.remotexs.ntu.edu.sg/Details.php?ID=1731">的合成与氧在生物系统成像的磷光纳米探针的</a>校准。

Watch this Scientific Journal Video about Cerebral Blood Oxygenation Measurement Based on Oxygen-dependent Quenching of Phosphorescence at JoVE.com

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

我们提出了一个基于磷光氧依赖淬火脑血管的氧分压（PO2）测量的实验过程。动物准备和成像过程概述了两个大的领域的观点基于CCD成像PO2在大鼠和双光子激发的小鼠PO2成像。

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 
...

cerebral-blood-oxygenation-measurement-based-on-oxygen-dependent

Nanyang Technological University

Research

JoVE Journal

Neuroscience

14.6K 次观看.  Massachusetts General Hospital and Harvard Medical School. 我们提出了一个基于磷光氧依赖淬火脑血管的氧分压（PO2）测量的实验过程。动物准备和成像过程概述了两个大的领域的观点基于CCD成像PO2在大鼠和双光子激发的小鼠PO2成像。

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

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

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

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 ...

科研

神经科学

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
	One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
	Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
	We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

同时脑电图，实时测量的啮齿动物的大脑皮质中的神经元活动的乳酸浓度与Optogenetic的操作

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

的实时成像<em&gt;果蝇</em&gt;幼虫神经母细胞

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

Isolation and Cannulation of Cerebral Parenchymal Arterioles

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

This manuscript describes a simple and reproducible protocol for isolation of intracerebral arterioles (a group of blood vessels encompassing parenchymal arterioles, penetrating arterioles and pre-capillary arterioles) from mice, to be used in pressure myography, immunofluorescence, biochemistry, and molecular studies.

分离及脑实质小动脉插管

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance ...

A Rat Model of Mild Intrauterine Hypoperfusion with Microcoil Stenosis

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

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

轻度宫内灌注圈狭窄的大鼠模型

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

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

新鲜人脑组织中毛细血管的分离

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

Quantitative Autoradiographic Method for Determination of Regional Rates of Cerebral Protein Synthesis In Vivo

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it is thought that dysregulation of protein synthesis in the nervous system may be a core phenotype in some developmental disorders. Accurate measurement of rates of cerebral protein synthesis in animal models is important for understanding these disorders. The method that we have developed was designed to be applied to the study of awake, behaving animals. It is a quantitative autoradiographic method, so it can yield rates in all regions of the brain simultaneously. The method is based on the use of a tracer amino acid, L-[1-14C]-leucine, and a kinetic model of the behavior of L-leucine in the brain. We chose L-[1-14C]-leucine as the tracer because it does not lead to extraneous labeled metabolic products. It is either incorporated into protein or rapidly metabolized to yield 14CO2 which is diluted in a large pool of unlabeled CO2 in the brain. The method and the model also allow for the contribution of unlabeled leucine derived from tissue proteolysis to the tissue precursor pool for protein synthesis. The method has the spatial resolution to determine protein synthesis rates in cell and neuropil layers, as well as hypothalamic and cranial nerve nuclei. To obtain reliable and reproducible quantitative data, it is important to adhere to procedural details. Here we present the detailed procedures of the quantitative autoradiographic L-[1-14C]-leucine method for the determination of regional rates of protein synthesis in vivo.

Protein synthesis is&#160;a critical biological process for cells. In brain, it is required for adaptive changes. Measurement of rates of protein synthesis in the intact brain requires careful methodological considerations. Here we present the L-[1-14C]-leucine quantitative autoradiographic method for determination of regional rates of cerebral protein synthesis in vivo.

定量自动放射法测定维沃脑蛋白合成区域速率

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

爆炸性神经损伤对压生啮鼠中脑动脉的影响

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...

Cheek Injection Model for Simultaneous Measurement of Pain and Itch-related Behaviors

Itch was defined as &#34;an unpleasant cutaneous sensation that provokes a desire to scratch&#34;&#160;by Rothman in 1941. In mouse models, scratch bouts are typically counted to evaluate itch induced by pruritogens. However, previous reports have shown that algesic substances also induce scratching behaviors in a mouse neck injection model, which is the most common test used for scratching behaviors. This finding makes it difficult to study itch in mice. &#160;In contrast, capsaicin, a common algogen, reduced scratching behaviors in some neck injection experiments. Therefore, the effect of pain on scratching behaviors remains unclear. It is thus necessary to develop a method to concurrently investigate itch and pain sensation using behavioral tests. Here, a cheek injection model is introduced which can be used to simultaneously measure pain- and itch-related behaviors. In this model, pruritogens induce scratching behaviors while algesic substances induce wiping behaviors. Using this model, lysophosphatidic acid (LPA), an itch mediator found in cholestatic patients with itch, is shown to exclusively induce itch but not pain. However, in mouse models, LPA has been reported to be both a pruritogen and an algogen. Investigation into the effects of LPA in a mouse cheek injection model showed that LPA only induced scratching, but not wiping behaviors. This indicates that LPA acts as a pruritogen similarly in mice and humans, and demonstrates the utility of a cheek injection model for itch research.

Typically, the mouse neck injection model is used for evaluate pruritogen-induced scratch behaviors. However, the model provides information only on itch, not pain. Here, a cheek injection model is introduced in mice which can be used to simultaneously measure pain and itch-related behaviors.

用于同时测量疼痛和瘙痒相关行为的切面注射模型

Itch was defined as &#34;an unpleasant cutaneous sensation that provokes a desire to scratch&#34;&#160;by Rothman in 1941. In mouse models, scratch bouts ...