这项工作是由巴黎第六大学和皮埃尔·法布尔创新基金的支持。

所有的程序均按照照顾和使用实验动物的欧盟标准进行，与当地的伦理委员会进行动物实验（巴黎大区 - 巴黎委员会，授权4270）的批准。 1.麻醉<ol><li> （最多至1mg / 10g体重）腹膜内（27号针和1毫升注射器）到成年小鼠手术前注入戊巴比妥的致死剂量。 </li></ol> 2.血管灌注注:没有必要血管灌注过程中兽医药膏敷眼睛。这个过程是快速（5-10分钟），并在动物死亡结束。确认缺乏应对与脚趾捏的。 <ol><li>使用虹膜剪，使一个切口，约4厘米长，入腹壁和腹膜，只是肋条笼之下。 </li><li>做一个小切口（几毫米长）的膜片，然后continuË沿肋骨的整个长度隔膜的切口以暴露胸膜腔。 </li><li>提起胸骨路程，钳止血胸骨尖;把脖子上的止血钳。精心修剪的脂肪组织连接胸骨心脏。 </li><li>穿过左心室15号针头灌注到心脏的顶点。 </li><li>最后，用剪刀剪下肝叶之一来创建一个出口。 注:另一种插座可以通过使用虹膜剪创建到右心房切口创建。 </li><li>灌注动物用一个泵以2.5毫升/分钟的速率操作25至50ml的磷酸盐缓冲盐水（PBS）的。肝脏应作为灼血被替换为PBS。 </li><li>约五分钟后，一旦从肝脏中的流体是完全清楚，停止灌注。 </li><li>如果免疫染色或常规染色计划，灌注所述动物用50ml的多聚甲醛（PFA;在PBS中的4％），15分钟。 注意:小心，PFA烟雾是有毒的。用PFA动物灌注应在通风橱中进行。 </li></ol> 3.脑的分离和Willis环<ol><li> 脑的隔离 <ol><li>用一双手术剪取下头上。 </li><li>请与虹膜剪正中切口，沿颈部到鼻子皮肤。 </li><li>切去皮肤暴露的头骨和清除残留的肌肉和脂肪组织与虹膜剪。 </li><li>虹膜剪的锋利端放入枕大孔一侧，并沿头骨到外耳道（也称为耳道）的内表面上小心地将它们。 </li><li>重现对侧在3.1.4中描述的切口，使沿顶叶间骨的内表面切成矢状缝的开始的中线。 </li><li>工厂虹膜剪刀额骨，右两眼之间，在矢状缝，然后打开它们一分为二头骨。 </li><li>提起出大脑，抢夺嗅球，并使用虹膜剪刀剪下其腹面的神经连接。 </li><li>取出大脑，并放置在含有冰冷的PBS为牛隔离60毫米的培养皿。完全沉浸在PBS的大脑。如果大脑固定用4％PFA（用于随后的切片和免疫染色或定期染色），保持它在4％PFA的浴中于4℃24小时。 </li></ol></li><li> Willis环的分离 注:解剖显微镜需要奶牛隔离。大脑应该在整个过程保持在4℃。 <ol><li>把脑倒挂（ 即在其背水面），以可视化的奶牛。 </li><li>用小镊子在嗅叶的基部抢前脑动脉（ACA）（ <str翁&gt;一个，图1）和施加压力，以从容器中连续分离它们。使用相同的步骤，以切割在b中的大脑中动脉（MCA）（ 图1）。 </li><li>使用镊子锋利的两端提起并取下主要干道形成了从皮质牛。 </li><li>抬起后交通动脉（PCA）的开始从大脑断开它们，通过抓握大脑中动脉（MCA）与钳子。拿起前动脉（ACA和MCA），轻轻地拉他们在多前 - 背方向视交叉。为了防止牛中断，中断处理与其他血管的过程。 </li><li>重复步骤3.2.2和3.2.3上级和后小脑动脉（SCA）/（PCA）（ 三，图1）和用于基底动脉（BA）（D，图1），在一个dorsal-拉动它们前方向。停在过程中的描述我的结束ñ3.2.4。 </li><li>通过使用镊子轻轻拉动取下整个牛。将牛在60毫米培养皿中充满了冰冷的PBS并用两个镊子取出任何剩余的连接脑组织，拿着牛的地方小针。 </li><li>保持收获的母牛在-80℃下用于RNA纯化后续处​​理（RNA提取产量大量的RNA - 约500纳克）或蛋白质提取。 注意:牛可维持离体 24小时，通过调整对分离的小鼠嗅动脉开发的器官浴系统7。 </li></ol></li></ol><img alt="图1" src="/files/ftp_upload/54352/54352fig1.jpg" /> 图1:对小鼠脑突出的牛 ，牛是从两个内部颈动脉（MCA），这是从两个大脑前动脉（ACA）派生形成的腹面示意图。基底动脉（BA）分支成后（PCA）和优异的（SCA）的脑动脉和两个椎动脉（VA）。

<ol>
	<li>Beckmann, N., 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=Age-dependent+cerebrovascular+abnormalities+and+blood+flow+disturbances+in+APP23+mice+modeling+Alzheimer's+disease.">Age-dependent cerebrovascular abnormalities and blood flow disturbances in APP23 mice modeling Alzheimer's disease.</a> J Neurosci. 23 (24), 8453-8459 (2003).</li><li>Sadasivan, C., Fiorella, D. J., Woo, H. H., Lieber, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+factors+effecting+cerebral+aneurysm+pathophysiology.">Physical factors effecting cerebral aneurysm pathophysiology.</a> Ann Biomed Eng. 41 (7), 1347-1365 (2013).</li><li>Ritz, K., Denswil, N., Stam, O., van Lieshout, J., Daemen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cause+and+mechanisms+of+intracranial+atherosclerosis.">Cause and mechanisms of intracranial atherosclerosis.</a> Circulation. 130 (16), 1407-1414 (2014).</li><li>Tulamo, R., Fr&#246;sen, J., Hernesniemi, J., Niemel&#228;, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+changes+in+the+aneurysm+wall:+a+review.">Inflammatory changes in the aneurysm wall: a review.</a> J Neurointerv Surg. 2 (2), 120-130 (2009).</li><li>Yamada, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+amyloid+angiopathy:+emerging+concepts.">Cerebral amyloid angiopathy: emerging concepts.</a> J Stroke. 17 (1), 17-30 (2015).</li><li>Oy, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracranial+atherosclerotic+stroke:+specific+focus+on+the+metabolic+syndrome+and+inflammation.">Intracranial atherosclerotic stroke: specific focus on the metabolic syndrome and inflammation.</a> Curr Atheroscler Rep. 8 (4), 330-336 (2006).</li><li>Lee, H. J., Dietrich, H. H., Han, B. H., Zipfel, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+ex+vivo+model+for+the+study+of+cerebrovascular+function+utilizing+isolated+mouse+olfactory+artery.">Development of an ex vivo model for the study of cerebrovascular function utilizing isolated mouse olfactory artery.</a> J Korean Neurosurg Soc. 57 (1), 1-5 (2015).</li><li>Hosaka, K., Downes, D. P., Nowicki, K. W., Hoh, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+murine+intracranial+aneurysm+model:+aneurysm+formation+and+rupture+by+elastase+and+hypertension.">Modified murine intracranial aneurysm model: aneurysm formation and rupture by elastase and hypertension.</a> J Neurointerv Surg. 6 (6), 474-479 (2013).</li><li>Gauthier, S. A., Sahoo, S., Jung, S. S., Levy, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+cerebrovascular+cells+as+a+cell+culture+model+for+cerebral+amyloid+angiopathy:+isolation+of+smooth+muscle+and+endothelial+cells+from+mouse+brain.">Murine cerebrovascular cells as a cell culture model for cerebral amyloid angiopathy: isolation of smooth muscle and endothelial cells from mouse brain.</a> Methods Mol Biol. 849, 261-274 (2012).</li><li>Choi, S., Kim, J., Kim, K., Suh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+in+vitro+culture+of+vascular+endothelial+cells+from+mice.">Isolation and in vitro culture of vascular endothelial cells from mice.</a> Korean J Physiol Pharmacol. 19 (1), 35-42 (2015).</li><li>Peters, D. G., Kassam, A. B., Yonas, H., O'Hare, E. H., Ferrell, R. E., Brufsky, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+transcript+analysis+in+small+quantitiesof+mRNA+by+SAGE-Lite.">Comprehensive transcript analysis in small quantitiesof mRNA by SAGE-Lite.</a> Nucleic Acids Res. 27 (24), (1999).</li><li>Badhwar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stanimirovic,+Hamel,+&amp;+Haqqani+The+proteome+of+mouse+cerebral+arteries.">Stanimirovic, Hamel, &amp; Haqqani The proteome of mouse cerebral arteries.</a> J Cereb Blood Flow Metab. 34 (6), 1033-1046 (2014).</li><li>Castro, L., Brito, 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=Striatal+neurones+have+a+specific+ability+to+respond+to+phasic+dopamine+release.">Striatal neurones have a specific ability to respond to phasic dopamine release.</a> J Physiol. 591 (13), 3197-3214 (2013).</li><li>H&#252;bscher, D., Nikolaev, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+transgenic+mice+expressing+FRET+biosensors.">Generation of transgenic mice expressing FRET biosensors.</a> Methods Mol Biol. 1294, 117-129 (2015).</li></ol>

The authors have nothing to disclose.

我们在这里描述为Willis环的隔离可再现协议。涉及牛最常见的脑血管疾病是CAA相关血管病变，颅内动脉粥样硬化和颅内动脉瘤，所有这些都影响动脉血管的壁。的危险因素是众所周知的，但这些脑疾病的分子发病机制仍然知之甚少和用于预测它们发生特定的生物标记物所缺乏的。目前的方法相当大的兴趣了隔离转基因小鼠的牛的肉眼观察结果与分子变化联系起来。例如，通过在其特定的基因被敲?...

脑底动脉环（circulus动脉脑瘤），也被称为威利斯（COW），Willisor威利斯多边形的环圈）最早是由托马斯·威利斯在1664描述的是周围设视交叉和下丘脑，可以循环系统吻合被看作是一个中心毂供给血液到脑和周围结构。血经颈内动脉和椎动脉进入这个结构，它通过内部中，后脑动脉流出圈。每个这些动脉的具有左右分支圆上的任一侧。基底，后连通，并且前交通动脉完全的圆（ 图1和图2）。血流受损的任何流出动脉的风险由血液进入从颈和脑动脉的圆，从而保证有足够的血液供给到b中的合并减少雨。这种结构也可作为在颈内动脉的严重闭塞性疾病侧支血流的主要途径。 几种类型的脑血管疾病有他们的牛奶原产地。最常见的是脑淀粉样血管病（CAA）相关血管病变，颅内动脉粥样硬化和颅内动脉瘤。1，2，3，这些疾病可能导致灌注不足是由于血管扩张，和脑和/或蛛网膜下腔出血最终转化为缺血性或出血性中风或充其量短暂性缺血发作。在诊断过程，包括神经影像学，可能与血管造影术相结合的最新进展，已使人们有可能在临床诊断这些主要脑血管疾病，而不需要一个脑活检。然而，有效和特定的治疗（药理学或血管内），目前缺乏，因此，有必要定义新分子靶点。 的新颖的药物靶标用于预防这些疾病在人类中鉴定需要的动物模型并分离脑脉管系统包括牛的方法。这些模型应提供的证据和线索的具体变化，包括炎性改变，在颅内动脉瘤，CAA或颅内动脉粥样硬化的动物模型的大血管壁发生。4，5，6 我们已经建立了小鼠牛隔离的方法，以促进在阿尔茨海默氏病（AD）和相关疾病容器炎症研究，例如CAA。该隔离鼠标牛方法，为脑血管炎症基因表达的疾病进展过程中的评估开发。再加上检测软脑膜及脑膜动脉壁内的β淀粉样蛋白的沉积，这种方法可以更容易地阻止矿山在脑血管壁的炎症基因表达和Aβ肽的积累之间的可能关系。大脑，包括蛛网膜下腔的柔脑膜和软脑膜的血管网，是大动脉形成Willis环的扩展。此处所描述的方法可以用于任何小鼠谱系的牛隔离和可用于在大型船只的小鼠脑脉管系统的所有类型的筛选（ 例如 ，基因表达，蛋白质产生和翻译后蛋白修饰）的。

<table border="0" cellpadding="0" cellspacing="0" width="692">
	<tbody>
		<tr height="46">
			<td height="46" style="height:47px;width:173px;">Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:173px;">D8537</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:173px;">Dumont #55 Forceps</td>
			<td style="width:173px;">Fine Science Tools</td>
			<td style="width:173px;">11295-51</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:173px;">Hardened Fine Iris Scissors&#160;</td>
			<td style="width:173px;">Fine Science Tools</td>
			<td style="width:173px;">14090-11</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:173px;">Scissors - Straight / Sharp / Sharp&#160;&#160; 16.5 cm</td>
			<td style="width:173px;">Fine Science Tools</td>
			<td style="width:173px;">14002-16</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:173px;">Dumont #7b Forceps&#160;</td>
			<td style="width:173px;">Fine Science Tools</td>
			<td style="width:173px;">11270-20</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:173px;">Stereoscopic Zoom Microscope</td>
			<td style="width:173px;">Nikon</td>
			<td style="width:173px;">SMZ745T</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:173px;">CellBIND Surface 60mm Culture Dish</td>
			<td style="width:173px;">Corning</td>
			<td style="width:173px;">#3295</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:173px;">Peristaltic Pump - MINIPULS 3</td>
			<td style="width:173px;">Gilson</td>
			<td style="width:173px;">M312</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:173px;">Pentobarbital Sodique</td>
			<td style="width:173px;">Ceva Sant&#233; Animale</td>
			<td style="width:173px;">FR/V/2770465 3/1992</td>
			<td style="width:173px;"></td>
		</tr>
	</tbody>
</table>

protocol for isolating the mouse circle of willis

脑底动脉环（circulus动脉脑瘤）或威利斯（COW）的圆是围绕着视交叉和下丘脑的血液供应到大脑和周围结构的循环吻合。它已牵涉几种脑血管疾病，包括脑淀粉样血管病（CAA）相关血管病变，颅内动脉粥样硬化和颅内动脉瘤。这些疾病的基本为新颖的药物靶标对他们的预防识别的分子机制的研究需要的动物模型。其中一些模型可以是转基因，而其他将涉及脑血管的隔离，包括这里所描述的CoW.The方法适合于在任何小鼠谱系牛隔离，并具有相当大的潜力用于筛选（基因，蛋白质生产的表达，翻译后蛋白修饰，分泌组分析等 ）上的大血管鼠标cerebro-研究血管。它也可以用于体外研究，通过调整对分离的小鼠嗅动脉开发的器官浴系统。

<html>在PBS-灌注小鼠被杀害，并且在协议中的第3.2节所述的奶牛被隔离。当正确地进行清扫，奶牛应该出来在一块和应稍透明由于在脉管没有残留血液。 <img alt="图2" src="/files/ftp_upload/54352/54352fig2.jpg" /> 图2: 隔离后的母牛鼠标。在一个10厘米的培养皿中的牛（一）概述（二）...

Watch this Scientific Journal Video about Protocol for Isolating the Mouse Circle of Willis at JoVE.com

Protocol for Isolating the Mouse Circle of Willis

We describe here a reproducible protocol for isolating the mouse circle of Willis.

协议隔离威利斯的鼠标圈

The cerebral arterial circle (circulus arteriosus cerebri) or circle of Willis (CoW) is a circulatory anastomosis surrounding the optic chiasma and ...

protocol-for-isolating-the-mouse-circle-of-willis

Research

JoVE Journal

Neuroscience

13.0K 次观看.  Sorbonne Universités. We describe here a reproducible protocol for isolating the mouse circle of Willis.

视频: Protocol for Isolating the Mouse Circle of Willis

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

NeuroStar TMS的设备进行美国FDA批准用于治疗抑郁症的议定书"

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

科研

神经科学

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

多鼠神经解剖学磁共振成像

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. 
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

隔离啮齿类动物或人类的鼻嗅干细胞

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae.
The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential1,2. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems3-7. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown8.
To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice9. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described2,10-12. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

Flow cytometry is a powerful tool allowing for the isolation and study of specific cell populations. This protocol describes steps for isolating LacZ-expressing cells from cochlear tissues from neonatal transgenic mice. Dissociated cochlear cells were labeled using fluorescent-conjugated substrates of &#946;-galactosidase prior to separation via flow cytometry.

隔离LacZ基因表达的小鼠内耳组织用流式细胞仪检测细胞

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues ...

Trypsin Digest Protocol to Analyze the Retinal Vasculature of a Mouse Model

Trypsin digest is the gold standard method to analyze the retinal vasculature 1-5. It allows visualization of the entire network of complex three-dimensional retinal blood vessels and capillaries by creating a two-dimensional flat-mount of the interconnected vascular channels after digestion of the non-vascular components of the retina. This allows one to study various pathologic vascular changes, such as microaneurysms, capillary degeneration, and abnormal endothelial to pericyte ratios. However, the method is technically challenging, especially in mice, which have become the most widely available animal model to study the retina because of the ease of genetic manipulations 6,7. In the mouse eye, it is particularly difficult to completely remove the non-vascular components while maintaining the overall architecture of the retinal blood vessels. To date, there is a dearth of literature that describes the trypsin digest technique in detail in the mouse. This manuscript provides a detailed step-by-step methodology of the trypsin digest in mouse retina, while also providing tips on troubleshooting difficult steps.

Trypsin digest is one of the most commonly used methods to analyze retinal vasculature. This manuscript describes the method in detail, including key alterations to optimize the technique and remove the non-vascular tissue while preserving the overall architecture of the vessels.

胰蛋白酶精华协议分析视网膜血管小鼠模型

Trypsin digest is the gold standard method to analyze the retinal vasculature 1-5. It allows visualization of the entire network of complex ...

A Comprehensive Protocol for Manual Segmentation of the Medial Temporal Lobe Structures

The present paper describes a comprehensive protocol for manual tracing of the set of brain regions comprising the medial temporal lobe (MTL): amygdala, hippocampus, and the associated parahippocampal regions (perirhinal, entorhinal,&#160;and parahippocampal proper). Unlike most other tracing protocols available, typically focusing on certain MTL areas (e.g., amygdala and/or hippocampus), the integrative perspective adopted by the present tracing guidelines allows for clear localization of all MTL subregions. By integrating information from a variety of sources, including extant tracing protocols separately targeting various MTL structures, histological reports, and brain atlases, and with the complement of illustrative visual materials, the present protocol provides an accurate, intuitive, and convenient guide for understanding the MTL anatomy. The need for such tracing guidelines is also emphasized by illustrating possible differences between automatic and manual segmentation protocols. This knowledge can be applied toward research involving not only structural MRI investigations but also structural-functional colocalization and fMRI signal extraction from anatomically defined ROIs, in healthy and clinical groups alike.

The present work provides a comprehensive set of guidelines for manually tracing the medial temporal lobe (MTL) structures. This protocol can be applied to research involving structural and/or combined structural-functional magnetic resonance imaging (MRI) investigations of the MTL, in both healthy and clinical groups.

一个全面协议的内侧颞叶结构的手动分割

The present paper describes a comprehensive protocol for manual tracing of the set of brain regions comprising the medial temporal lobe (MTL): amygdala, ...

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol (TRAP) Assay

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and lifespan. In addition, telomerase protects the mitochondria from oxidative stress and confers resistance to apoptosis, suggesting its possible importance for the surviving of non-mitotic, highly active cells such as neurons. We previously demonstrated the ability of novel telomerase activators to increase telomerase activity and expression in the various mouse brain regions and to protect motor neurons cells from oxidative stress. These results strengthen the notion that telomerase is involved in the protection of neurons from various lesions. To underline the role of telomerase in the brain, we here compare the activity of telomerase in male and female mouse brain and its dependence on age. TRAP assay is a standard method for detecting telomerase activity in various tissues or cell lines. Here we demonstrate the analysis of telomerase activity in three regions of the mouse brain by non-denaturing protein extraction using CHAPS lysis buffer followed by modification of the standard TRAP assay.
In this 2-step assay, endogenous telomerase elongates a specific telomerase substrate (TS primer) by adding TTAGGG 6 bp repeats (telomerase reaction). The telomerase reaction products are amplified by PCR reaction creating a DNA ladder of 6 bp increments. The analysis of the DNA ladder is made by 4.5% high resolution agarose gel electrophoresis followed by staining with highly sensitive nucleic acid stain.
Compared to the traditional TRAP assay that utilize 32P labeled radioactive dCTP&#39;s for DNA detection and polyacrylamide gel electrophoresis for resolving the DNA ladder, this protocol offers a non-toxic time saving TRAP assay for evaluating telomerase activity in the mouse brain, demonstrating the ability to detect differences in telomerase activity in the various female and male mouse brain region.

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay

Telomerase is expressed in the neonatal brain and also in distinct regions of the adult brain. We present a non-toxic time saving TRAP assay for the analysis of telomerase activity in various regions of the mouse brain and detection of differences in telomerase activity between male and female mouse brains.

在小鼠大脑的各个区域端粒酶活性:非放射性端粒酶重复序列扩增法（TRAP）分析

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and ...

Receptor Autoradiography Protocol for the Localized Visualization of Angiotensin II Receptors

This protocol describes receptor binding patterns for Angiotensin II (Ang II) in the rat brain using a radioligand specific for Ang II receptors to perform receptor autoradiographic mapping.
Tissue specimens are harvested and stored at -80 &#176;C. A cryostat is used to coronally section the tissue (brain) and thaw-mount the sections onto charged slides. The slide-mounted tissue sections are incubated in 125I-SI-Ang II to radiolabel Ang II receptors. Adjacent slides are separated into two sets: &#39;non-specific binding&#39; (NSP) in the presence of a receptor saturating concentration of non-radiolabeled Ang II, or an AT1 Ang II receptor subtype (AT1R) selective Ang II receptor antagonist, and &#39;total binding&#39; with no AT1R antagonist. A saturating concentration of AT2 Ang II receptor subtype (AT2R) antagonist (PD123319, 10 &#181;M) is also present in the incubation buffer to limit 125I-SI-Ang II binding to the AT1R subtype. During a 30 min pre-incubation at ~22 &#176;C, NSP slides are exposed to 10 &#181;M PD123319 and losartan, while &#39;total binding&#39; slides are exposed to 10 &#181;M PD123319. Slides are then incubated with 125I-SI-Ang II in the presence of PD123319 for &#39;total binding&#39;, and PD123319 and losartan for NSP in assay buffer, followed by several &#39;washes&#39; in buffer, and water to remove salt and non-specifically bound radioligand. The slides are dried using blow-dryers, then exposed to autoradiography film using a specialized film and cassette. The film is developed and the images are scanned into a computer for visual and quantitative densitometry using a proprietary imaging system and a spreadsheet. An additional set of slides are thionin-stained for histological comparisons.
The advantage of using receptor autoradiography is the ability to visualize Ang II receptors in situ, within a section of a tissue specimen, and anatomically identify the region of the tissue by comparing it to an adjacent histological reference section.

Here we present a protocol to describe the localization of angiotensin II Type 1 receptors in the rat brain by quantitative, densitometric, in vitro receptor autoradiography using an iodine-125 labeled analog of angiotensin II.

受体放射自显影协议血管紧张素Ⅱ受体的本地化的可视化

This protocol describes receptor binding patterns for Angiotensin II (Ang II) in the rat brain using a radioligand specific for Ang II receptors to ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

一个简单的一步解剖协议成人全安装准备<em&gt;果蝇</em&gt;脑

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

A Protocol for the Administration of Real-Time fMRI Neurofeedback Training

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control neuroplastic processes and correct abnormal function, or even shift functions from damaged tissue to physiologically healthy brain regions, hold the potential to dramatically improve overall health. Of the current neuroplastic interventions in development, neurofeedback training (NFT) from functional Magnetic Resonance Imaging (fMRI) has the advantages of being completely non-invasive, non-pharmacologic, and spatially localized to target brain regions, as well as having no known side effects. Furthermore, NFT techniques, initially developed using fMRI, can often be translated to exercises that can be performed outside of the scanner without the aid of medical professionals or sophisticated medical equipment. In fMRI NFT, the fMRI signal is measured from specific regions of the brain, processed, and presented to the participant in real-time. Through training, self-directed mental processing techniques, that regulate this signal and its underlying neurophysiologic correlates, are developed. FMRI NFT has been used to train volitional control over a wide range of brain regions with implications for several different cognitive, behavioral, and motor systems. Additionally, fMRI NFT has shown promise in a broad range of applications such as the treatment of neurologic disorders and the augmentation of baseline human performance. In this article, we present an fMRI NFT protocol developed at our institution for modulation of both healthy and abnormal brain function, as well as examples of using the method to target both cognitive and auditory regions of the brain.

The ability to induce and/or control neural plasticity may be critical in future treatments for neurologic disorders and the recovery from brain injury. In this paper, we present a protocol on the use of neurofeedback training with functional magnetic resonance imaging to modulate human brain function.

实时 fMRI 神经训练管理协议

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control ...