Name: the analysis of neurovascular remodeling in entorhino-hippocampal organotypic slice cultures
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这项工作得到了巴塞尔大学生物医学系，以及瑞士国家科学基金会（31003A_141007）的支持。马库斯Saxer提供了技术支持。

动物实验是按照2010年9月22日的欧盟理事会指令（2010/63 /欧盟）和进行了审查，并通过瑞士当局允许的。 1，建立器官Entorhino，海马切片培养<ol><li>使用静态培养法4,11日龄4（P4），鼠幼仔准备entorhino，海马脑片培养（EHOSCs）。在这里，使用C57BL6和CB6F1小鼠。 <ol><li>以每只小鼠幼崽大约30分钟的片培养的准备， 即 3小时为6小鼠幼崽一窝。进行无菌条件下的所有步骤中的层流工作台中用无菌手术器械。 </li><li>为了避免从沿隔 - 时间轴的切片的位置的可能的干扰，只能使用海马室间隔上半年的切片。如果没有另外说明，进行所有的步骤在RT。 </li></ol></li><li>切断吨他头一台P4鼠标小狗手术剪刀。不需要麻醉的这一步。 <ol><li>删除周围的脑颅骨，并确定大脑半球和中脑的尾端之间的裂隙。小心取出脑延髓部分，从头骨的脑，并将其放置在由补充有L-谷氨酰胺的稳定形式冰冷的筹备中。 </li></ol></li><li>继续解剖立体显微镜下用脑沉浸在冰冷的筹备中。从半球皮质除去残留的脑膜。识别海马在半球的内侧尾部表面，并与来自大脑的其余部分相邻的内嗅皮质中分离它一起。 <ol><li>转移组织到组织切碎机切成400μm的无菌条件下的横向厚片到海马的隔 - 时间轴。 </li></ol></li><li>广场上CE切片LL培养插入（约6％插片），并培养他们在6孔板，每培养液（47毫升灰熊，将25ml Eagle培养基中井1毫升，25毫升马血清1毫升Glutamax的一，1毫升无菌的10％葡萄糖溶液，pH7.3）中，在湿润的气氛中，用5％CO 2，37℃。第二天，然后隔日达1星期换一次培养基。 </li></ol> 2， 体外缺氧及缺氧缺糖再灌注<ol><li>请参阅表1的程序的确切时间表。文化EHOSCs暴露在低氧或缺氧缺糖前5天（DIV5）。有2％B27补充剂，1％Glutamax的和额外的0.1％葡萄糖的Neurobasal介质:在该时间点从含有25％马血清，以无血清培养基具有以下组成的孵化介质切换平台。 </li><li>制备OGD介质与参考7 .The终浓度的组分详述将是以下:0.3毫的CaCl 2，70 mM氯化钠，5.25毫碳酸氢钠 ，70 mM的氯化钾，1.25毫的NaH 2 PO 4，2mM的MgSO 4干燥，和10mM蔗糖，在pH 6.8。加入1 ml OGD介质的两个6孔组织培养板的每个孔中。 <ol><li>灌注OGD中与N 2 1小时的缺氧室，然后密封，并保持O / N的孵化器在37℃。 </li><li>使用厌氧条为缺氧的指标。考虑介质是无氧时带的颜色从粉红变为白色。 </li><li>缺氧诱导，用无血清培养基中的葡萄糖，而不是缺氧缺糖培养基。在使用前，灌注在低氧室如上详述的OGD介质与N 2的平台。 </li></ol></li><li>前条带被暴露于OGD，再灌注OGD介质用N 2 30分钟。加入2微升的1毫克/毫升的碘化丙啶（PI），每孔溶液（2微克/毫升的最终浓度）的切片30分钟，并选择只有健康的切片实验由低数字PI阳性细胞的指示。 </li><li>使用OGD曝光和再灌注在图1中所示的时间表。剥夺或者氧气只（缺氧）或氧气和葡萄糖（OGD）的切片15分钟。 <ol><li>对于缺氧诱导或缺氧缺糖的，转成片缺糖中，并保持在低氧室15分钟。确保所有的留在刀片的两侧流体被吸走，当培养物是从常规培养基以OGD介质和背面切换。 </li><li>低氧或缺氧缺糖后，更换OGD介质含氧无血清培养基中，并培养被允许恢复为3,24或48小时在无血清培养基中常规培养器在37℃下用5％的CO 2。之前再添加固定的PI细胞死亡的决心。 </li></ol></li></ol>的Entorhino，海马脑切片培养3药物治疗<ol><li>文化性ËEHOSCs 5天（DIV5）在培养液暴露在药物治疗之前。开始无论在缺氧缺糖或立即当片被转移到无血清培养基后缺氧缺糖药物治疗。 </li><li>治疗防止缺氧缺糖。 <ol><li> OGD诱导后加入100μM6-氰基-7 - 硝基喹喔啉-2,3二酮（CNQX）或1μM的河豚毒素（TTX），以在OGD诱导培养基中（15分钟），或者立即进行30分钟。然后，在孵育常氧无血清培养基用于固定前48小时。 </li></ol></li><li>治疗诱导兴奋死亡。 <ol><li> 5天后，在培养介质中培养开关血清含有100μM的（RS）-α氨基-3 - 羟基-5 - 甲基-4 - 异唑丙酸（AMPA）处理30分钟的培养基。 </li><li>除去含有AMPA通过抽吸（小心移除插入物的任何残余培养基）培养基中，然后转移切片新鲜平板与正常无血清培养基中，并让他们48小时，直到固定。 </li></ol></li></ol> 4，固定和免疫染色Entorhino，海马脑切片培养<ol><li>碘化丙啶（PI）染色。 <ol><li>加入PI进行30分钟至活切片的培养基中，以2微克/毫升的浓度。查看现场的文化与倒置显微镜配备了荧光光学和选择进一步处理切片。对于与免疫组化相结合PI染色，之前培养的固定加PI溶液30分钟。 </li></ol></li><li>固定的切片。 <ol><li>从6孔板中除去培养基，并加入3毫升新制备的4％多聚甲醛（PFA）的各孔。然后将6孔板在冰箱和修复在o / n 4℃。 </li><li>除去PFA溶液翌日并用磷酸盐缓冲盐水（PBS）洗片3-4次。 </li></ol></li><li>从细胞培养物ı除去切片nsert和为自由浮动切片免疫组织粘连过程。 <ol><li>仔细分离和使用艺术画笔的大小为0或更小的文化刀片取出切片。传送每个单独的切片于96孔板中填充有封闭缓冲液（PBS，3％正常山羊血清（NGS）和2％的Triton-X100）的孔中。由轨道摇床保持在封闭缓冲液，将切片在恒定搅拌下2小时，在室温。 </li></ol></li><li>片文化的染色<ol><li>在PBS中含有1％NGS和0.5％的Triton-X100 200:用于标记的血管使用多克隆抗层粘连蛋白的稀释度为1。 </li><li>在PBS中含有1％NGS和0.5％的Triton-X100 500:用于标记的神经元用相关蛋白2（MAP2）的抗微管的稀释度为1。 </li><li>孵育48小时的初级抗体的切片在4℃下以恒定的搅拌。培养结束后，把它们洗干净的PBS中含有0.5％的Triton-X100的3-4倍。 </li&gt; <li>在PBS中含有1％NGS和0.5％的Triton-X100 500:稀释的二抗1。孵育切片2小时在RT与Alexa的荧光团共轭次级抗体的黑暗。使用适当的山羊抗兔或羊抗​​鼠第二抗体缀合的Alexa 350或488。 </li><li>去除二级抗体溶液并用Tris缓冲盐水（TBS）洗涤切片三次;它们安装在载玻片和盖玻片的Mowiol幻灯片。 </li></ol></li><li>染色切片的显微镜</li><li>查看切片染色无论是在一个标准的显微镜落射荧光装置或用共聚焦显微镜。取的图像与适当的相机。对于一些图像，反转，以产生深染的船只在明亮的背景荧光造影（ 图5和图6）。 </li></ol> 5，确定在文化区管密度<ol><li>量化vesse如在参考文献8中描述的基于容器过路升密度。 </li><li>使用染色层粘连蛋白对这些测量的片。叠加的记录图像的放大10倍以一个6×6的网格，其中每个网格等于每平方100×100微米，为0.25mm 2总计字段。 </li><li>叠加三个这样的网格中，CA3，DG和欧盟地区（ 图2）。算交叉网格的线的船只。 </li><li>获得的结果具有良好的统计相关性，从3小鼠幼仔用每只小鼠幼崽3片进行测量中的至少3个独立的实验中，每一个都包括数据。 </li><li>确定船舶口岸从电网各区域的平均值和方差分析和Bonferroni修正为事后用适当的统计软件测试（P &lt;0.05定义为显著）进行统计分析。 </li></ol>

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动物实验是按照1986年11月24日（86/609 / EEC）欧洲共同体理事会指令，并通过瑞士当局审查和批准。作者什么都没有透露。

用这里介绍的方法，海马脑切片培养物可被用作一个通用的工具损伤后，研究塑料的改变的神经组织。虽然器官切片文化已被用于在过去的研究神经元的反应缺血后6，7的血管改变的同时进行研究的新局面显著增强了该方法的应用潜力。 OGD或单独缺氧诱导可以通过在器官切片培养物，而简单的装置来施加，并导致在神经组织​​可靠和可再现的损伤。此外，切片培养方法允许不仅研究缺?...

中枢神经系统是特别敏感的损失或减少的氧和葡萄糖的供应通过脉管。血液供应到大脑，即使是很短的中断能引起领导的典型中风病证的相关脑区的功能永久性丧失。除了在主影响区域的神经元丧失，存在通常是通过二次伤害附加迟发性神经元损失。遗憾的是到现在为止，没有任何神经保护治疗继发性神经元死亡的减少是可用1。学习继发性损害的机制研究工作依赖于使用脑缺血就像大脑中动脉闭塞和各种血栓闭塞技术的动物模型（对于最近的综述见2）。同时，也由于限制和使用动物模型，各种中枢神经系统组织器官切片文化的伦理问题已使用，允许STUDY神经元的反应不同类型的伤害3-5的。 对于学习，模仿缺血性脑损伤的条件下神经元的反应，已经开发了氧糖剥夺模型系统（OGD）。在这个模型中，切片培养物暂时暴露在缺乏葡萄糖，并已达到平衡，用氮气在不存在氧的平台。通过这样的处理，因此能够诱导神经元损伤和损失，这是相当一个类似于体内 6,7缺血性损伤后观察到的，在海马，这种处理诱导的神经元损失特别是在CA1区，而不是在CA3区和海马齿状回。与此相反，在切片培养血管反应的研究至今还没有得到广泛开展。一个显而易见的原因是在切片养殖模式，缺乏循环和血管的灌注。然而，我们已经有可能保持b前面所示lood血管中枢片培养了好几天8,9。 这种方法的总体目标是，不仅可以监测神经元缺氧缺糖后的命运，但延长的研究，这是损伤反应的重要组成部分血管的命运和重塑。直至现在这样的研究需要使用动物实验（德容等人，1999;卡瓦利亚等人，2001）。在该协议在这里提出，我们将详细介绍如何这样的研究可以做到entorhino，海马脑片培养的挑战无论是与缺氧或兴奋毒性损伤后两个神经元的存活和血管反应的分析。该协议是基于先前发表的研究报告对这个问题10，并且可以为有意在中枢神经系统的神经血管的相互作用任何实验室用。

<table><tbody><tr><td>Minimum Essential Medium MEM</td><td>Gibco</td><td>11012-044</td><td></td></tr><tr><td>Glutamax</td><td>Gibco</td><td>35050-061</td><td>stabilized form of L-glutamine</td></tr><tr><td>Millicell cell culture inserts</td><td>Millipore</td><td>PICM03050</td><td></td></tr><tr><td>Basal medium Eagle&amp;nbsp;</td><td>Gibco</td><td>41010-026</td><td></td></tr><tr><td>Horse serum</td><td>Gibco</td><td>26050-088</td><td></td></tr><tr><td>Neurobasal medium</td><td>Gibco</td><td>21103-049</td><td></td></tr><tr><td>B27 supplement</td><td>Gibco</td><td>17504-044</td><td></td></tr><tr><td>Anaerobic strips</td><td>Sigma-Aldrich</td><td>59886</td><td></td></tr><tr><td>Propidium iodide solution</td><td>Sigma-Aldrich</td><td>P4864</td><td></td></tr><tr><td>AMPA</td><td>R&amp;amp;D systems</td><td>0169-10</td><td></td></tr><tr><td>CNQX</td><td>R&amp;amp;D systems</td><td>0190/10</td><td></td></tr><tr><td>TTX</td><td>R&amp;amp;D systems</td><td>1078/1</td><td></td></tr><tr><td>polyclonal anti-laminin</td><td>Sigma-Aldrich</td><td>L9393&amp;nbsp;</td><td></td></tr><tr><td>anti-MAP2</td><td>Abcam</td><td>ab11267</td><td></td></tr><tr><td>Alexa anti-mouse 350</td><td>Molecular Probes</td><td>A11045</td><td></td></tr><tr><td>Alexa anti-mouse 488</td><td>Molecular Probes</td><td>A11001</td><td></td></tr><tr><td>Alexa anti-rabbit 350</td><td>Molecular Probes</td><td>A11046</td><td></td></tr><tr><td>Alexa anti-rabbit 488</td><td>Molecular Probes</td><td>A11008</td><td></td></tr><tr><td>Statistics software</td><td>GraphPad Software</td><td>GraphPad Prism</td><td></td></tr><tr><td>McIlwain tissue chopper</td><td>Ted Pella</td><td>10180</td><td></td></tr><tr><td>Hypoxia chamber</td><td>Billups-Rothenberg</td><td>MIC-101</td><td></td></tr></tbody></table>

the analysis of neurovascular remodeling in entorhino-hippocampal organotypic slice cultures

缺血性脑损伤中最常见的和毁灭性的条件影响正常的脑功能，常常导致持续的功能缺陷，在受影响的病人。尽管密集的研究努力，但仍可以减少神经元损伤，并保护在从延迟次级死亡的缺血区的神经元用没有有效的治疗选择。这方面的研究通常涉及使用复杂的和有问题的动物模型。挑战与氧和葡萄糖剥夺（OGD）Entorhino，海马脑片培养是建立在模仿脑缺血体外模型。本研究的新颖的方面是，对脑血管的变化进行了研究，除了神经元的变化和两者的神经隔腔和血管隔室中的反应可以进行比较和相关性。在这个协议中提出的方法，大大拓宽了潜在的应用程序或ganotypic切片培养方法。 OGD或单独缺氧诱导可以通过在器官切片培养物，而简单的装置来施加，并导致在神经组织​​可靠和可再现的损伤。与此形成鲜明对比的是复杂的和有问题的动物实验中诱发中风和缺血体内 。通过扩大分析，包括血管可以提供关于如何保护和恢复大脑功能的新途径反应的研究。这里介绍的切片培养方法可能发展成一个有吸引力的和重要的工具，缺血性脑损伤的研究，并可能是用于测试目的的神经保护潜力的治疗措施是有用的。

缺氧缺糖和缺氧诱导神经元死亡和血管减少特别是在海马CA1区 。 OGD或氧气剥夺单独15分钟诱导很强的诱导细胞死亡的作为如前面7描述在海马（ 图3）类似的CA1区通过碘化丙啶染色可见特异性。用标记物来可视化血管的网络，人们发现，血管密度和组织似乎类似于在除CA1区的文化的大部分地区OGD挑战后的控制。有血管密度降低和血管网络被部分破坏（ 图3，E<...

Watch this Scientific Journal Video about The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures at JoVE.com

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

一种协议，用于entorhino，海马脑片培养，允许再生缺血性脑损伤的多个方面，提出了。通过研究，除了在神经血管的变化，以改变神经元，该协议是一个多功能的工具损伤后，研究塑料改变神经组织。

神经血管重塑的分析Entorhino，海马脑切片培养

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

the-analysis-neurovascular-remodeling-entorhino-hippocampal

Research

JoVE Journal

Neuroscience

9.7K 次观看.  University of Basel. 一种协议，用于entorhino，海马脑片培养，允许再生缺血性脑损伤的多个方面，提出了。通过研究，除了在神经血管的变化，以改变神经元，该协议是一个多功能的工具损伤后，研究塑料改变神经组织。 

视频: The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

器官切片文化为产后神经再生的研究

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

科研

发育生物学

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved 1. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.

海马脑片的器官型检查神经元损伤的文化模式

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

神经科学

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

器官型海马切片培养

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

对于高分辨率延时成像神经细胞迁移的一个器官型产后脑切片检测

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

器官切片文化研究少突胶质细胞动力学和髓鞘

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

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. ...

Short-Term Free-Floating Slice Cultures from the Adult Human Brain

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of slice cultures in neuroscience, studies using adult nervous tissue to prepare such cultures are still scarce, particularly those from human subjects. The use of adult human tissue to prepare slice cultures is particularly attractive to enhance the understanding of human neuropathologies, as they hold unique properties typical of the mature human brain lacking in slices produced from rodent (usually neonatal) nervous tissue. This protocol describes how to use brain tissue collected from living human donors submitted to resective brain surgery to prepare short-term, free-floating slice cultures. Procedures to maintain and perform biochemical and cell biology assays using these cultures are also presented. Representative results demonstrate that the typical human cortical lamination is preserved in slices after 4 days in vitro (DIV4), with expected presence of the main neural cell types. Moreover, slices at DIV4 undergo robust cell death when challenged with a toxic stimulus (H2O2), indicating the potential of this model to serve as a platform in cell death assays. This method, a simpler and cost-effective alternative to the widely used protocol using membrane inserts, is mainly recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases. Finally, although the protocol is devoted to using cortical tissue collected from patients submitted to surgical treatment of pharmacoresistant temporal lobe epilepsy, it is argued that tissue collected from other brain regions/conditions should also be considered as sources to produce similar free-floating slice cultures.

A protocol to prepare free-floating slice cultures from adult human brain is presented. The protocol is a variation of the widely used slice culture method using membrane inserts. It is simple, cost-effective, and recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases.

来自成人大脑的短期自由浮动切片培养

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of ...

A Model of Epileptogenesis in Rhinal Cortex-Hippocampus Organotypic Slice Cultures

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s neuroprotective and therapeutic potential. Organotypic slices are prepared from explanted tissue and represent a complex multicellular ex vivo environment. They preserve the three-dimensional cytoarchitecture and local environment of brain cells, maintain the neuronal connectivity and the neuron-glia reciprocal interaction. Hippocampal organotypic slices are considered suitable to explore the basic mechanisms of epileptogenesis, but clinical research and animal models of epilepsy have suggested that the rhinal cortex, composed of perirhinal and entorhinal cortices, play a relevant role in seizure generation.
Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Recordings of spontaneous activity from the CA3 area under perfusion with complete growth medium, at physiological temperature and in the absence of pharmacological manipulations, showed that these slices depict evolving epileptic-like events throughout time in culture. Increased cell death, through propidium iodide uptake assay, and gliosis, assessed with fluorescence-coupled immunohistochemistry, was also observed. The experimental approach presented highlights the value of rhinal cortex-hippocampus organotypic slice cultures as a platform to study the dynamics and progression of epileptogenesis and to screen potential therapeutic targets for this brain pathology.

Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Under a gradual and controlled deprivation of serum, these slices depict evolving epileptic-like events and can be considered an ex vivo model of epileptogenesis. This system represents an excellent tool for monitoring the dynamics of spontaneous activity, as well as for assessing the progression of neuroinflammatory features throughout the course of epileptogenesis.

莱纳尔皮质-希波坎普斯有机切片文化中的癫痫发生模型

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s ...

Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell resolution using electrophysiology, microscopy, and pharmacology. However, a major consideration in the design of in vitro experiments is the extent to which different slice preparations recapitulate naturalistic patterns of neural activity as observed in vivo. In the intact brain, the hippocampal network generates highly synchronized population activity reflective of the behavioral state of the animal, as exemplified by the sharp-wave ripple complexes (SWRs) that occur during waking consummatory states or non-REM sleep. SWRs and other forms of network activity can emerge spontaneously in isolated hippocampal slices under appropriate conditions. In order to apply the powerful brain slice toolkit to the investigation of hippocampal network activity, it is necessary to utilize an approach that optimizes tissue health and the preservation of functional connectivity within the hippocampal network. Mice are transcardially perfused with cold sucrose-based artificial cerebrospinal fluid. Horizontal slices containing the hippocampus are cut at a thickness of 450 &#956;m to preserve synaptic connectivity. Slices recover in an interface-style chamber and are transferred to a submerged chamber for recordings. The recording chamber is designed for dual surface superfusion of artificial cerebrospinal fluid at a high flow rate to improve oxygenation of the slice. This protocol yields healthy tissue suitable for the investigation of complex and spontaneous network activity in vitro.

This protocol describes the preparation of horizontal hippocampal-entorhinal cortex (HEC) slices from mice exhibiting spontaneous sharp-wave ripple activity. Slices are incubated in a simplified interface holding chamber and recordings are performed under submerged conditions with fast-flowing artificial cerebrospinal fluid to promote tissue oxygenation and the spontaneous emergence of network-level activity.

急性小鼠脑切片研究自发海马网络活动

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell ...

Horizontal Hippocampal Slices of the Mouse Brain

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as well as the manifestation of severe brain disorders, including Alzheimer&#8217;s disease and epilepsy. The hippocampus receives a high degree of intra- and inter-connectivity, securing a proper communication with internal and external brain structures. This connectivity is accomplished via different informational flows in the form of fiber pathways. Brain slices are a frequently used methodology when exploring neurophysiological functions of the hippocampus. Hippocampal brain slices can be used for several different applications, including electrophysiological recordings, light microscopic measurements as well as several molecular biological and histochemical techniques. Therefore, brain slices represent an ideal model system to assess protein functions, to investigate pathophysiological processes involved in neurological disorders as well as for drug discovery purposes.
There exist several different ways of slice preparations. Brain slice preparations with a vibratome allow a better preservation of the tissue structure and guarantee a sufficient oxygen supply during slicing, which present advantages over the traditional use of a tissue chopper. Moreover, different cutting planes can be applied for vibratome brain slice preparations. Here, a detailed protocol for a successful preparation of vibratome-cut horizontal hippocampal slices of mouse brains is provided. In contrast to other slice preparations, horizontal slicing allows to keep the fibers of the hippocampal input path (perforant path) in a fully intact state within a slice, which facilitates the investigation of entorhinal-hippocampal interactions. Here, we provide a thorough protocol for the dissection, extraction, and acute horizontal slicing of the murine brain, and discuss challenges and potential pitfalls of this technique. Finally, we will show some examples for the use of brain slices in further applications.

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.

鼠脑水平海马片

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as ...