我们想为她出色的技术援助和马赫什·库马尔特立组织的视频拍摄准备感谢Kaltwasser布丽塔。

1。动物<ol><li>根据美国国立卫生研究院的保养和使用实验动物的指导方针进行了实验和地方当局的批准。对于所有的实验中，C57BL6 / J野生型小鼠，载脂蛋白- / - （ApoE基因KO）和SV129小鼠（12-16周龄，体重26-30克，每实验组5-6动物）。 </li></ol> 2。彩色乳胶脑血管染色<ol><li>准备为25μl炭黑油墨（茨博士，德国）的混合物用0.5毫升的胶乳化合物（贝碧欧，法国）以1:20的比例在EP管，并在水浴中，将混合物在37℃下预热。收藏报错在2 ml注射器的混合物与28-30G的针前麻醉动物。 </li><li>溶解50毫克到1毫升无菌生理盐水的papavarine盐酸盐粉末。收集的溶液中的胰岛素注射器。从另一个无菌胰岛素注射器的针折断。这种针放置在端部的的20米长的PE10管。立即附加此PE10管上的针的胰岛素注射器含有papavarine盐酸盐溶液被注入，以确保通过股静脉血管舒张和适当的填补船只。 </li><li>准备另外两个胰岛素注射器各含1毫升的生理盐水。胰岛素注射器的注射液可以顺利交付的准确位置。然而，由于高粘度的乳液，另一种可移动的更广泛的针1毫升注射器应该被使用。 </li><li>以所有的注射器和消毒解剖仪器靠近的操作阶段。传播的手术的布店表周围的操作阶段。的操作阶段，应充分地照射，如有必要，也可以使用在手术显微镜。 </li><li>立即测量C57BL6 / J小鼠的体重，然后用5％的汽化在70％N 2 O和O 2的 30％的异氟烷诱导麻醉。继续用1％的蒸发，异氟醚麻醉。经过适当的positionin克鼠标在它的背部和四肢固定的，评估麻醉深度。然后在左侧股静脉切开皮肤，找到静脉。插入针的尖锐的尖端放在PE10管，并在100微升（每分钟）（50毫克/公斤体重）的速率缓慢​​注入papavarine盐酸盐溶液。 </li><li>在注射之后，切断的腹腔和刺穿隔膜。切肋骨，暴露心脏，而不会损坏或瘀伤。夹胸降主动脉的动脉钳。车型右心房的大幅削减超过允许静脉血排出。进入左心室注入2毫升盐水，随后通过手动有轻微压力超过18-20秒注入着色胶乳。使用预充式注射器此起彼伏。避免注入任何气泡。 </li><li>注射后的乳胶，动物离开5分钟。杀头的动物，小心地取出整个大脑。照顾好，不要伤到血管构筑和科尔特X，而去除颅骨和脑膜。将大脑中的一个60毫米的细胞培养皿中含有6-8毫升的4％PFA拍摄照片。将测量标尺下透明的菜。以25倍的放大倍数在手术显微镜摄像头的背侧和腹侧脑组织表面的照片。应采取照片之间的陪替氏培养皿和显微镜的物镜，在90°角，以量化的原始距离。 </li><li>重复该过程在另一个4-5动物。 </li></ol> 3。脑血管染色的碳黑色墨水的混合物<ol><li>要比较的结果与我们的协议的的胶乳基于脑血管染色，用900μl的鹈鹕Scribtol施瓦茨墨水（CB2）在1.5ml EP管中制备的混合物中加入100μl，茨博士Stempelfarbe墨（CB1）。准备两个EP管中，使总体积为2毫升CB1和CB2的混合物按1:9的比例。在两个单独的胰岛素注射器（1收集混合物毫升）。盖上注射器，并将其放置在水浴中预热至37℃。收藏2毫升的生理盐水另外两个胰岛素注射器，并将其放置在水浴中。 </li><li>麻醉动物，并重复相同的过程，除了注射papavarine盐酸如上所述。继左心室注射2毫升salaine，注入2毫升的混合物的碳黑色墨水，而不是着色胶乳。执行注射每毫升超过18-20秒的手有轻微的压力。夹胸降主动脉去打针之前。 </li><li>牺牲的动物，消除大脑和拍照。 </li><li>对于长期保存的大脑，把大脑中的4％PFA过夜，然后逐渐增加浓度的蔗糖培养，直到浮动的大脑完全淹没（5％，15％和30％）。有色乳胶灌注的动物的大脑也可以以同样的方式被保留。 </li><li>执行4-5动物的过程中。比较差异脑血管解剖，由于不同遗传背景或品系，重复的协议，在同等数量的ApoE KO和SV129小鼠，分别。 </li><li>研究缺血条件下的血管解剖，引起局灶性脑缺血45分钟或90分钟，根据腔内阻塞的MCA深麻醉下（在手术的详细描述超出了本文的范围，是一个标准的协议;读者可以参考Doeppner等人，2011）。在再灌注期间的末尾计划（1天或5天，），执行血管染色CB1 + CB2油墨只与和染色全脑，用2％TTC溶液，在37℃下为5-10分钟，以确定梗死体积。由线粒体酶（特别是琥珀酸脱氢酶），TTC减少到红色的有色的甲。 TTC染色后，污渍深红色的代谢活跃的组织，而心肌梗死的组织仍然unstai的非执行董事，并出现白色的，由于功能失调，变性线粒体酶。在如上所述的相同的方式收集的图像。 </li></ol> 4。研究脑血管新界的<ol><li>要分析的大体解剖脑血管，图像的大脑背表面任何沾上了颜色的乳液或CB1和CB2油墨可以分析ImageJ软件（基于Java的公共领域，用于图像处理和分析软件）。脑灌注彩色乳胶可能会出现不同程度的血管染色位于背表面上，，而CB1 + CB2灌注染色腹侧和背侧表面上的所有船只。 </li><li> ImageJ软件中打开图像文件。确定所有的ACA和MCA之间的吻合点。的吻合血管直径的部位是最窄的或最近的分支点的ACA和MCA分支之间的一半距离，分别为2的点被定义为。 <s&gt;绘制的假想线（吻合线路），由标记的帮助下绘制分割线工具和插件“虚线”的吻合点。 </li><li>设置规模在毫米。测量的吻合线之间的距离，和在4毫米，尾鳍使用直线绘图工具和“测量”工具的大脑额极的中线。做同样的尾部从额极，6毫米。分析3-4每只动物的图像，并计算平均值。不同种类的动物之间的比较值，，识别脑血管解剖结构的变化。在C57BL6 / J的动物，ACA和MCA之间的吻合线可以追溯更接近中线在4毫米超过6毫米，尾端从额极。载脂蛋白E基因敲除小鼠在这方面不存在显着的差异。与此相反，在SV129小鼠的吻合线将在于进一步的和平行的中​​线在4毫米和6毫米尾端从正面极。 </li><li>要分析的血管解剖我Ň缺血的条件下，进行上述同样的计算。确定梗死边框的红色和白色代表代谢活跃的组织和未染色的组织坏死的组织保证金。测量的距离在4毫米和6毫米的尾鳍从额极的大脑从中线梗死边界。收集平均值动物图像，每3-4。不同缺血再灌注期间的结果进行比较。 </li></ol>

<ol>
	<li>Meng, H., Peng, 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=Nuclear+contrast+angiography:+A+simple+method+for+morphological+characterization+of+cerebral+arteries.">Nuclear contrast angiography: A simple method for morphological characterization of cerebral arteries.</a> Brain Research. 1261, 75-81 (2009).</li><li>Maeda, K., Hata, R., 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=Differences+in+the+cerebrovascular+anatomy+of+C57black/6+and+SV129+mice.">Differences in the cerebrovascular anatomy of C57black/6 and SV129 mice.</a> Neuroreport. 9 (7), 1317-1319 (1998).</li><li>Coyle, P., Jokelainen, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsal+cerebral+arterial+collaterals+of+the+rat.">Dorsal cerebral arterial collaterals of the rat.</a> The Anatomical Record. 203, 397-404 (1982).</li><li>Coyle, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsal+cerebral+collaterals+of+stroke-prone+spontaneously+hypertensive+rats+(SHRSP)+and+Wistar+Kyoto+rats+(WKY).">Dorsal cerebral collaterals of stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar Kyoto rats (WKY).</a> The Anatomical Record. 218, 40-44 (1987).</li><li>Hasan, M. 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R., Nagel, F., 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=TAT-Hsp70-mediated+neuroprotection+and+increased+survival+of+neuronal+precursor+cells+after+focal+cerebral+ischemia+in+mice.">TAT-Hsp70-mediated neuroprotection and increased survival of neuronal precursor cells after focal cerebral ischemia in mice.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 29, 1187-1196 (2009).</li><li>Todo, K., Kitagawa, K., 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=Granulocyte-macrophage+colony-stimulating+factor+enhances+leptomeningeal+collateral+growth+induced+by+common+carotid+artery+occlusion.">Granulocyte-macrophage colony-stimulating factor enhances leptomeningeal collateral growth induced by common carotid artery occlusion.</a> Stroke. 39 (6), 1875-1882 (2008).</li><li>Sugiyama, Y., Yagita, 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=Granulocyte+colony-stimulating+factor+enhances+arteriogenesis+and+ameliorates+cerebral+damage+in+a+mouse+model+of+ischemic+stroke.">Granulocyte colony-stimulating factor enhances arteriogenesis and ameliorates cerebral damage in a mouse model of ischemic stroke.</a> Stroke. 42 (3), 770-775 (2011).</li><li>Busch, H. J., Buschmann, I. R., 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=Arteriogenesis+in+hypoperfused+rat+brain.">Arteriogenesis in hypoperfused rat brain.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 23 (5), 621-628 (2003).</li><li>Maeda, K., Hata, R., 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=Regional+metabolic+disturbances+and+cerebrovascular+anatomy+after+permanent+middle+cerebral+artery+occlusion.">Regional metabolic disturbances and cerebrovascular anatomy after permanent middle cerebral artery occlusion.</a> in C57black/6 and SV129 mice. 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没有利益冲突的声明。

CB1 + CB2通过人工注射灌注成功地进行了不密集的训练，因为它不涉及任何特定的设备意味着一定的压力2,3。灌注在我们的协议结果的异质性也可以忽略不计。动物的16个非缺血性的动物只有1和3 20缺血性动物显示不完整的灌注。在这种情况下，将泡在盐水灌注导致血管闭塞是最有可能的原因不成功的结果。 虽然灌注胶乳原本进行通过升主动脉2,3，替代路线的?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司名称 </td><td> 产品编号 </td></tr><tr><td> Scribtol施瓦茨（CB2） </td><td>鹈鹕，德国</td><td> 221 135 </td></tr><tr><td> Stempelfarbe（CB1） </td><td>德国赫利茨PBS AG， </td><td> 10417202 </td></tr><tr><td> Gedeo乳胶</td><td>法国贝碧欧， </td><td> 13042B </td></tr></tbody></table>

intravascular perfusion of carbon black ink allows reliable visualization of cerebral vessels

脑血管的解剖结构的一个重要因素就是对大脑血流动力学以及对缺血损伤的严重程度。脑血管系统动态响应各种病理生理状态与株之间的遗传操作的情况下，它表现出相当大的差异。从本质上讲，一个可靠的颅内血管染色技术研究缺血性中风的发病机制是必不可少的。直到最近，一组不同的技术已被用于可视化的脑血管包括注射低粘度树脂，ARALDITE F，明胶与各种染料1（ 即胭脂红，印度墨水）或胶乳与2或无3炭黑混合。白乳胶化合物通过升主动脉灌注已报道由科伊尔和Jokelainen 3。 Maeda 等人 2已修改的协议通过添加碳氮化Ň黑色墨盐水灌注后的大脑血管的对比度提高的可视化的胶乳化合物。然而，低效的灌注和船只填充不足经常经历由于高粘度胶乳化合物4。因此，我们已经描述了一个简单的和成本有效的技术，使用的混合物的两种市售碳黑色墨水（CB1和CB2）可视化的脑血管在可重复的方式5。我们已经表明，灌注与CB1 + CB2在小鼠的结果，在胶乳灌注5相比在更高的密度的显着较小的脑血管染色。在这里，我们描述的协议来识别之间的吻合点前（ACA）和大脑中动脉（MCA），研究不同遗传背景的小鼠的血管变化。最后，我们证明了我们的技术可行性相结合，在局灶性脑缺血小鼠模型中CB1 +CB2-介导的血管染色与TTC染色不同程度的缺血性损伤。

这里所描述的协议，克服了技术上的限制，传统的乳胶的可视化啮齿类动物的脑血管。 图1A表明，以下的彩色乳胶灌注，大血管的腹侧表面有污渍，使整个背表面不染。的结果也充满变数。只有一个动物，满分6示出在ACA和MCA大脑（数据未示出）的背表面上可见的部分染色。相反，CB1 +的CB2灌注结果以平等的方式（ 图1B）的小型和大型船舶在充分填充。染色是稳定的，直到...

Watch this Scientific Journal Video about Intravascular Perfusion of Carbon Black Ink Allows Reliable Visualization of Cerebral Vessels at JoVE.com

Intravascular Perfusion of Carbon Black Ink Allows Reliable Visualization of Cerebral Vessels

分析啮齿类动物的脑解剖实验中风研究中起着重要的作用。在这方面，一直被认为是血管内灌注有色乳胶作为标准工具数年。然而，这种技术意味着不同的技术限制，破坏了它的可重复性。在这里，我们将介绍一个简单的方法来可视化脑血管在一个可重复的方式。注射液的混合物的两种市售碳黑色油墨通过在足够的填充具有高对比度的可视化的脑血管的左心肌心室结果。我们已经成功地应用这种技术来识别不同的遗传背景的小鼠脑血管领土之间的吻合点。最后，我们提供的证据表明，这种新的和简单的方法，容器染色可与氯化三苯基四氮唑（TTC）染色 - 一种广泛使用的工具，来观察和分析小鼠梗死体积。

血管内灌注的碳黑色墨水可实现可靠的可视化脑血管

The anatomical structure of cerebral vessels is a key determinant for brain hemodynamics as well as the severity of injury following ischemic insults. The ...

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Neuroscience

15.3K 次观看.  University of Duisburg-Essen Medical School. 分析啮齿类动物的脑解剖实验中风研究中起着重要的作用。在这方面，一直被认为是血管内灌注有色乳胶作为标准工具数年。然而，这种技术意味着不同的技术限制，破坏了它的可重复性。在这里，我们将介绍一个简单的方法来可视化脑血管在一个可重复的方式。注射液的混合物的两种市售碳黑色油墨通过在足够的填充具有高对比度的可视化的脑血管的左心?...

视频: Intravascular Perfusion of Carbon Black Ink Allows Reliable Visualization of Cerebral Vessels

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 ...

科研

神经科学

Automated Interactive Video Playback for Studies of Animal Communication

Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, parameter-based computer animation offers the opportunity to independently manipulate any number of behavioral, morphological, or spectral characteristics in the context of realistic, moving images of animals on screen. A major limitation of conventional playback, however, is that the visual stimulus lacks the ability to interact with the live animal. Borrowing from video-game technology, we have created an automated, interactive system for video playback that controls animations in response to real-time signals from a video tracking system. We demonstrated this method by conducting mate-choice trials on female swordtail fish, Xiphophorus birchmanni. Females were given a simultaneous choice between a courting male conspecific and a courting male heterospecific (X. malinche) on opposite sides of an aquarium. The virtual male stimulus was programmed to track the horizontal position of the female, as courting males do in the wild. Mate-choice trials on wild-caught X. birchmanni females were used to validate the prototype's ability to effectively generate a realistic visual stimulus.

Video playback is a widely used technique in animal behavior. We created and evaluated a program that applies rules-based, interactive playback of 3-D computer animations in response to real-time, automated data on subject behavior.

自动化研究动物交流的交互式视频播放

Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

逆行荧光染料标记允许有针对性的外单细胞记录技术鉴定神经元<em&gt;在体内</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

可视化和遗传操作在小鼠大脑皮层和使用海马树突和棘<em&gt;在子宫内</em&gt;电穿孔

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

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 ...

Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to stimuli remain to be fully understood. To overcome the difficulty of accessing the brainstem in the whole animal, isolation of the brainstem and part of the spinal cord is performed. This preparation is maintained in artificial cerebro-spinal fluid where gases, concentrations, and temperature are controlled and monitored. The output signal from the respiratory network is recorded by a suction electrode placed on the fourth ventral root. In this manner, stimuli can be directly applied onto the brainstem, and the effect can be recorded directly. The signal recorded is linked to the inspiratory signal sent to the diaphragm via the phrenic nerve, and can be described as bursts (around 8 bursts per minute). Analysis of these bursts (frequency, amplitude, length, and area under the curve) allows precise characterization of the stimulus effect on the respiratory network. The main limitation of this method is the viability of the preparation beyond the early post-natal stages. Thus, this method greatly focuses on the study of the whole network without the peripheral inputs in the newborn rat.

The central respiratory drive is located in the brainstem. Spontaneous respiratory motor output from an isolated brainstem-spinal cord is recorded by placing an electrode on the fourth ventral root. This experimental approach is valuable for pharmacological investigations or the assessment of respiratory challenges and genetic manipulations on rhythmic motor behavior.

电从新生儿鼠害隔离脑干，脊髓准备允许呼吸中枢网络输出记录

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to ...

Purification of Mouse Brain Vessels

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and immune cells between the brain and the blood. Moreover, the huge neuronal metabolic demand requires a moment-to-moment regulation of blood flow. Notably, abnormalities of these regulations are etiological hallmarks of most brain pathologies; including glioblastoma, stroke, edema, epilepsy, degenerative diseases (ex: Parkinson&#8217;s disease, Alzheimer&#8217;s disease), brain tumors, as well as inflammatory conditions such as multiple sclerosis, meningitis and sepsis-induced brain dysfunctions. Thus, understanding the signaling events modulating the cerebrovascular physiology is a major challenge. Much insight into the cellular and molecular properties of the various cell types that compose the cerebrovascular system can be gained from primary culture or cell sorting from freshly dissociated brain tissue. However, properties such as cell polarity, morphology and intercellular relationships are not maintained in such preparations. The protocol that we describe here is designed to purify brain vessel fragments, whilst maintaining structural integrity. We show that isolated vessels consist of endothelial cells sealed by tight junctions that are surrounded by a continuous basal lamina. Pericytes, smooth muscle cells as well as the perivascular astrocyte endfeet membranes remain attached to the endothelial layer. Finally, we describe how to perform immunostaining experiments on purified brain vessels.

We describe a protocol allowing the purification of the mouse brain's vascular compartment. Isolated brain vessels include endothelial cells linked by tight junctions and surrounded by a continuous basal lamina, pericytes, vascular smooth muscle cells, as well as perivascular astroglial membranes.

对小鼠脑血管净化

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and ...

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 ...

Intracerebroventricular and Intravascular Injection of Viral Particles and Fluorescent Microbeads into the Neonatal Brain

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the hematogenous route, which involves infection of the endothelial cells and pericytes of the brain. The second is the intracerebroventricular (ICV) route. Once within the central nervous system (CNS), viruses may spread to the subarachnoid space, meninges, and choroid plexus via the cerebrospinal fluid. In experimental models, the earliest stages of CNS viral distribution are not well characterized, and it is unclear whether only certain cells are initially infected. Here, we have analyzed the distribution of cytomegalovirus (CMV) particles during the acute phase of infection, termed primary viremia, following ICV or intravascular (IV) injection into the neonatal mouse brain. In the ICV injection model, 5 &#181;l of murine CMV (MCMV) or fluorescent microbeads were injected into the lateral ventricle at the midpoint between the ear and eye using a 10-&#181;l syringe with a 27 G needle. In the IV injection model, a 1-ml syringe with a 35 G needle was used. A transilluminator was used to visualize the superficial temporal (facial) vein of the neonatal mouse. We infused 50 &#181;l of MCMV or fluorescent microbeads into the superficial temporal vein. Brains were harvested at different time points post-injection. MCMV genomes were detected using the in situ hybridization method. Fluorescent microbeads or green fluorescent protein expressing recombinant MCMV particles were observed by fluorescent microscopy. These techniques can be applied to many other pathogens to investigate the pathogenesis of encephalitis.

Here, we describe a simple method of intracerebroventricular and intravascular injection of viral particles or fluorescent microbeads into the neonatal mouse brain. The localization pattern of the virus and nanoparticles could be detected by microscopic evaluation or by in situ hybridization.

病毒颗粒和荧光微球侧脑室和血管内注入新生儿脑

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the ...

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 ...