Name: the analysis of neurovascular remodeling in entorhino-hippocampal organotypic slice cultures
Uploaded: 2014-10-23T14:00:00
Description: Watch this Scientific Journal Video about The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures at JoVE.com

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?...

<ol>
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Exp Neurol. 169, 135-147 (2001).</li><li>Bonnici, B., Kapfhammer, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+regeneration+of+intrinsic+spinal+cord+axons+in+a+novel+spinal+cord+slice+culture+model.">Spontaneous regeneration of intrinsic spinal cord axons in a novel spinal cord slice culture model.</a> Eur J Neurosci. 27, 2483-2492 (2008).</li><li>Laake, J. H., Haug, F. M., Wieloch, T., Ottersen, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+in+vitro+model+of+ischemia+based+on+hippocampal+slice+cultures+and+propidium+iodide+fluorescence.">A simple in vitro model of ischemia based on hippocampal slice cultures and propidium iodide fluorescence.</a> Brain Res Brain Res Protoc. 4, 173-184 (1999).</li><li>Rytter, A., Cronberg, T., Aszt&#233;ly, F., Nemali, S., Wieloch, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+hippocampal+organotypic+tissue+cultures+exposed+to+in+vitro+&#8216;ischemia&#8217;+show+selective+and+delayed+CA1+damage+that+is+aggravated+by+glucose.">Mouse hippocampal organotypic tissue cultures exposed to in vitro &#8216;ischemia&#8217; show selective and delayed CA1 damage that is aggravated by glucose.</a> J Cereb Blood Flow Metab. 23, 23-33 (2003).</li><li>Bendfeldt, K., Radojevic, V., Kapfhammer, J., Nitsch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+fibroblast+growth+factor+modulates+density+of+blood+vessels+and+preserves+tight+junctions+in+organotypic+cortical+cultures+of+mice:+a+new+in+vitro+model+of+the+blood-brain+barrier.">Basic fibroblast growth factor modulates density of blood vessels and preserves tight junctions in organotypic cortical cultures of mice: a new in vitro model of the blood-brain barrier.</a> J Neurosci. 27, 3260-3267 (2007).</li><li>Camenzind, R. S., 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=Preservation+of+transendothelial+glucose+transporter+1+and+P-glycoprotein+transporters+in+a+cortical+slice+culture+model+of+the+blood-brain+barrier.">Preservation of transendothelial glucose transporter 1 and P-glycoprotein transporters in a cortical slice culture model of the blood-brain barrier.</a> Neuroscience. 170, 361-371 (2010).</li><li>De Jong, G. I., 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=Cerebral+hypoperfusion+yields+capillary+damage+in+the+hippocampal+CA1+area+that+correlates+with+spatial+memory+impairment.">Cerebral hypoperfusion yields capillary damage in the hippocampal CA1 area that correlates with spatial memory impairment.</a> Neuroscience. 91, 203-210 (1999).</li><li>Cavaglia, M., 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+variation+in+brain+capillary+density+and+vascular+response+to+ischemia.">Regional variation in brain capillary density and vascular response to ischemia.</a> Brain Res. 910, 81-93 (2001).</li><li>Chip, S., Nitsch, C., Wellmann, S., Kapfhammer, J. 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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。

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			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
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			<td height="42" style="height:42px;width:216px;">Minimum Essential Medium MEM</td>
			<td style="width:71px;">Gibco</td>
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			<td style="width:71px;">Gibco</td>
			<td>35050-061</td>
			<td>stabilized form of L-glutamine</td>
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			<td height="21" style="height:21px;width:216px;">Millicell cell culture inserts</td>
			<td style="width:71px;">Millipore</td>
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			<td height="21" style="height:21px;width:216px;">Neurobasal medium</td>
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			<td height="42" style="height:42px;width:216px;">Propidium iodide solution</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td>P4864</td>
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			<td height="42" style="height:42px;width:216px;">polyclonal anti-laminin</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td>L9393&#160;</td>
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			<td style="width:119px;">ab11267</td>
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			<td>A11045</td>
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			<td height="42" style="height:42px;width:216px;">Alexa anti mouse 488</td>
			<td style="width:71px;">Molecular Probes</td>
			<td>A11001</td>
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			<td height="42" style="height:42px;width:216px;">Alexa anti rabbit 350</td>
			<td style="width:71px;">Molecular Probes</td>
			<td>A11046</td>
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			<td height="42" style="height:42px;width:216px;">Alexa anti rabbit 488</td>
			<td style="width:71px;">Molecular Probes</td>
			<td>A11008</td>
			<td></td>
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			<td height="42" style="height:42px;width:216px;">Statistics software</td>
			<td style="width:71px;">GraphPad Software</td>
			<td style="width:119px;">GraphPad Prism</td>
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			<td height="21" style="height:21px;width:216px;">McIlwain tissue chopper</td>
			<td style="width:71px;">Ted Pella</td>
			<td align="right" style="width:119px;">10180</td>
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			<td height="21" style="height:21px;width:216px;">Hypoxia chamber</td>
			<td>Billups-Rothenberg</td>
			<td style="width:119px;">MIC-101</td>
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</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 ...

Research

JoVE Journal

Neuroscience

The Organotypic Hippocampal Slice Culture Model for Examining 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 ...

科研

神经科学

Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus

切片的制备，组织器官型培养，并记录在视交叉上核时钟基因活性的荧光素酶

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the ...

Organotypic Hippocampal Slice Cultures

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 ...

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

器官型小脑文化：细胞凋亡的挑战和检测

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. ...

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

从大脑皮层的器官型切片制备全细胞记录

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex.  Because of the ...

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

器官型培养的神经元雪崩多电极阵列记录

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

胚胎腹侧中脑器官型片文化：一个系统来研究多巴胺能神经元发展在体外</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

浦肯野细胞树突形态的器官切片培养分析

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

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

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. ...