The authors acknowledge the following funding sources, the Natural Sciences and Engineering Research Council of Canada (NSERC), the Dorothy Pitts Research Fund (NG), and the Nicky and Thor Eaton Research Fund. The authors acknowledge Kevin DeSimone, and Aman Goyal and for their knowledge in MRI acquisition and analysis expertise.

1.参与者和死后的大脑建立注:使用3个T磁共振成像扫描仪具有32通道头线圈和所有的MRI扫描被收购所有图像在RT进行，约20℃。所有与会者都用右手，并签署知情同意书。每个参加者是在良好的健康与神经系统疾病史。实验方案获得批准，遵循约克大学人类受试者审查委员会的指导方针。 <ol><li>要求每个参与者填写并签署，详细MRI安全准则和神经影像学协议病人的知情同意书。 </li><li>对于每个参与者，将耳塞的每只耳朵和保护他们的头垫，以尽量减少头部运动。 </li><li>对于验尸脑成像，确保之前的神经影像学大脑是固定的，包含在该MRI头部线圈的夹缝之中袋或容器。将死后的大脑中的头部线圈，其z轴（优于INFerior）对准用扫描器的孔。脑干（后）应朝向扫描仪床脚。 </li><li>将真空垫手周围的验尸的大脑获得更多支持。 </li></ol> 2，本地化和处方皮层下注:丘脑是位于位于中脑和大脑皮层之间的大脑中央的双裂结构。位于背侧丘脑中，人的LGN是一个小的皮质下结构，扩展最大至10毫米。 <ol><li>要注册一个新的参与者，打开核磁共振成像软件并点击左上角的患者标签。然后点击注册。 </li><li>在合适的患者填写信息，然后单击考试选项卡。 </li><li>为了获得一个定位扫描，点击考试资源管理器选项卡上创建一个新的协议。观察设置窗口在屏幕上，单击常规选项卡，并输入以下参数:收购时间28秒，采集矩阵160×160，1片，厚1.6毫米的各向同性体素尺寸，FOV = 260毫米，相的FoV = 100％，切片分辨率= 69％，相位和切片部分相傅里叶= 6/8，TR = 3.15毫秒，TE = 1.37毫秒，翻转角= 8°。 </li><li>叠加用于获取对PD图象在定位覆盖丘脑内皮层下细胞核以及周围结构（图4）的切片选择框。 </li></ol> 3.高分辨率结构参数<ol><li>创建获取高分辨率的PD加权扫描一个新的协议。在屏幕上的设置窗口中，单击常规选项卡，并输入在冠状方向以下参数:采集时间179秒，采集矩阵512×512，0.3×0.3×1 立方毫米像素大小，TR = 3.25秒，TE = 32毫秒，翻转角= 120°，交错片采集，读取的FoV = 160毫米，相的FoV = 100％，平行成像（GRAPPA）与为2的加速因子。 <ol><li>使用快速自旋回波序列，与5。第一回波在32毫秒的回声列车长度为有效呼应此序列。减少带宽（BW），以最小的可能，40赫兹/像素，以最大化信噪比。为了减少扫描时间，选择18片，每次1毫米厚，与FOV = 160毫米。该板提供了足够的覆盖范围感兴趣皮质下区域。 注:为了保证可靠的识别皮层下结构，获得5次操作，上述参数。总的扫描持续时间仅为〜15分钟（ 图5）。脂肪的饱和度没有采用。 </li></ol></li><li>在验尸脑成像，可靠识别皮层下结构可以只在一个扫描中观察到的唯一〜3分钟以下的相同的扫描协议为在3.1（图6）的总持续时间。 </li></ol> 4.图像分析注:要分析MRI数据，使用免费提供的FMRIB的软件库（FSL）包可供下载<a href="https://www.fmrib.ox.ac.uk/fsl/">（https://www.fmrib.ox.ac.uk/fsl/）。</a> <ol><li>打开一个终端窗口，并从扫描仪对每个PD卷到NIfTI格式与DICOM到NIfTI转换器转换成原始DICOM文件。都可以免费下载其中一些（例如<a href="https://www.nitrc.org/projects/mricron">，https://www.nitrc.org/projects/mricron）。</a>在命令行中键入dc​​m2nii其次是每个PD加权像奔跑的目录。 </li><li>在终端窗口得到原始的PD扫描的参数。在命令行随后在NIfTI格式PD扫描类型fslinfo。 </li><li>创建高分辨率空白图像目标卷具有该分辨率的两倍和由参数从fslinfo从原来的PD扫描给定的一半的体素尺寸。是数据输入该命令的命令如下: fslcreatehd &lt;xsize&gt; &lt;ysize&gt; &lt;ž大小&gt; &lt;TSIZE&gt; &lt;xvoxsize&gt; &lt;yvoxsize&gt; &lt;zvoxsize&gt; &lt;TR&gt; &lt;xorigin&gt; &lt;yorigin&gt; &lt;zorigin&gt; &lt;数据类型&gt; &lt;headername&gt; 注:例如，如果与在3.1中所述的下列参数原来的PD扫描收集（ 即 512×512矩阵，18片，0.3×0.3×1 立方毫米像素大小，TR = 3.25 S），键入以下在命令窗口中: fslcreatehd 1024×1024 36 1 0.15 0.15 0.5 3.25 0 0 0 4 blankhr.nii.gz </li><li>定义使用单位矩阵的转变。键入任何文本编辑器程序的文本文件保存为"identity.mat"，看起来像这样: 0 0 0 1 0 0 0 1 0 0 0 1 </li><li>使用调情命令应用转换，上采样每个原始PD加权运行从512的总分辨率增加一倍，达到1024矩阵，减半体素大小在每个维度造成0.15×0.15的分辨率×0.5 立方毫米。在终端窗口中的每个PD卷，键入以下命令调情改变每个运行原始和输出的名称: 调情-interp正弦-in originalPD.nii.gz -ref blankhr.nii.gz -applyxfm -init identity.mat退房手续highresPD.nii.gz 注:其中originalPD.nii.gz是源卷，blankhr.nii.gz是所需的输出分辨率，并且highresPD.nii.gz是输出卷的名称。 </li><li>将所有的高分辨率图像到一个新的文件夹，然后导航到在一个终端窗口。 </li><li>对于每一个参与者，串接所有采样的局部放电图像转换成使用fslmerge一个4D文件。在终端窗口中输入: fslmerge -t concat_highresPD * .nii.gz 注:此创建一个名为concat_highresPD.nii.gz四维文件。 </li><li>使用mcflirt 10动态校正串联文件。该工具允许一个强大的自动注册线性（仿射）间及多式联运的大脑图像。选择4级校正，其利用正弦内插（内部），为进一步优化过程获得更高的精度。在终端窗口中输入: mcflirt -in concat_highresPD退房手续mcf_concat_highresPD.nii.gz -stages 4 -plots 注:此创建一个名为mcf_concat_highresPD.nii.gz四维文件。 </li><li>最后，创建3D意味着使用图像fslmaths。在终端窗口中输入: fslmaths mcf_concat_highresPD.nii.gz -Tmean mean_highresPD.nii.gz 注:此创建一个名为mean_highresPD.nii.gz一个3D文件，是高品质的</li><li>可视化使用fslview命令的最终结果的3D高清晰度的图像。在其中，您的图片所在的目录，键入一个终端窗口如下: fslview mean_highresPD.nii.gz。" </li><li>有关检查ROI的强度分布。创建使用fslview的ROI（这可以是跨LGN的区域画出的实施例的垂直线）。在fslview加载高分辨率的PD图象。单击工具选项卡上，然后点击单个图像选项卡上的放大图像绘制的ROI。然后，单击文件选项卡随后创建模板标签。绘制在感兴趣的ROI的线。通过单击文件，然后另存为保存的投资回报率。投资回报率的强度比较，并在问题的其他投资回报之内重复线路口罩多个领域。 </li><li>使用AFNI的3dmaskdump命令来分析所得的图像的强度。投资回报率面具在那里的图像，使用以下命令在终端窗口提取图像亮度和位置的目录（给出result_mask.txt）: 3dmaskdump -o result_mask.txt -noijk -xyz -mask ROI_linemask.nii.gz PDaverage_image.nii.gz </li></ol>

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The authors have nothing to disclose.

该研究描述了为了得到皮质下区域的高分辨率的PD加权图像采集和分析技术的优化协议。一些扫描参数进行了测试，并修改与最显著那些关于矩阵大小，体素尺寸和带宽，以增加信噪比和减少收购的数量，在能够确定的高分辨率皮质结构的关键步骤。在与人类的生活中找到最佳参数一起使用，这项研究测试的理想条件下的核磁共振成像扫描仪的绝对限制，没有运动伪影和患者的时间限制通过扫描?...

这项研究的目的是测试结构性MRI的分辨率限制在没有生理噪声。质子密度（PD）的加权图像在死后的大脑获得在较长的持续时间（2〜24小时，会话），以确定需要被登记的图像的最小数目并取平均值，以解决皮层下结构。为了比较，PD加权图像在活人在若干会话还获得。具体地，其目的是要确定它是否有可能在一个最好的情况下，以解决人类LGN的所有六个单独的层，这是大约1毫米厚的（图1）。 <img alt="图1" src="/files/ftp_upload/53309/53309fig1.jpg" /> 图1.人类外侧膝状体层，原理图LGN的层状结构。大细胞（M）的层由较大的神经元的细胞大小和更小的细胞密度是负责解决运动当然轮廓（层1-2，描绘为深灰色）。小细胞亚核层（P）是由较小的神经元细胞的尺寸和更大的细胞密度是负责解决精细形式和颜色（层4-6，描绘为浅灰色）。比例条1毫米。图的基础上染色的人LGN 12。 在MRI中的空间分辨率时，矩阵尺寸增大得到改善，而当字段的视图（FOV）和切片厚度被减少。然而，增加的分辨率降低了信号噪声比（SNR），这是成比例的体素的体积。信噪比也正比于测量次数的平方根。在人类的生活，尽管多个图像可以通过许多单独的成像会被收购，最终分辨率由生理噪声的限制，诸如呼吸，循环脉动和头部运动。 高- 分辨率（0.35毫米面体素），PD被收购加权扫描。 PD扫描提高灰色和白色的对比在丘脑1，并导致在最小化T1和T2 2的效果的图像。其图像是依赖于质子在水和大分子的形式，例如在成像体积的蛋白质和脂肪的密度。在一个组织中的结果在图像上亮信号质子由于磁化2的较高纵向分量的数量增加。 收集的PD加权扫描，因为它们提供皮层下结构与周围组织更高的对比度。其他的对比，如T1和T2加权像导致难以划定，如LGN皮层下结构，由于较小的对比噪声比，所确定的ƒ1,3。 同样，早期的研究发现福尔马林的PD加权像固定验尸的大脑resulte比T1和T2加权图像，也有类似的灰质和白质图像强度3,4 d在灰质和白质之间更高的对比度差异。底层生物物理因素可以解释这些差异。氢质子T1（纵向）和T2（横向）弛豫时间取决于水如何在组织内移动。固定剂，例如通过交联蛋白质福尔马林工作。被还原的不同组织类型之间的水流动性之间的差异，当固定剂的使用。减压T1组织对比度已固定后已经观察到，而在脑组织的增加而固定内质子的相对密度的差异，从而提供更好的对比度分化3,4。 使用1.5T的5,6,7以前的研究已经确定了LGN在PD加权扫描，并在3个T扫描仪8,9。要能精确地勾画的程度是至关重要的获得这些扫描在LGN。为了保持皮层下核的全覆盖，18 PD加权切片丘脑内获得。每个卷被重新采样到第1024矩阵两次，（0.15毫米的平面内的体素尺寸），级联，运动校正和平均化，以产生皮层下结构的高分辨率3D图像。所需以下切片处方的PD图像的最佳数量为5，从而减少扫描时间为小于15分钟，在活的人类。仅1 PD图象被要求明确地划分皮层下区域中的死后脑，减少扫描时间少于3分钟（ 图2 和 3）。 整整福尔马林固定死后的大脑标本采自于谁岁时死于心肺骤停82岁的女人扫描。审查病历显示，她有:慢性阻塞性肺疾病，心绞痛，三搭桥手术8年死前，子宫癌与子宫切除术7年之前死亡，高脂血症，青光眼，和白内障手术。死后脑样品浸没固定在10％中性缓冲福尔马林中至少3周，在4℃C.The死后的大脑进行扫描具有相同的成像协议，以及与其他参数超过许多小时的过程中进行的图像质量比较。仅优化参数将描述的协议。

<table>
	<tbody>
		<tr>
			<td>Magnetom Trio 3T&#160; MRI</td>
			<td>Siemens (Erlangen, Germany).</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum cushion hand</td>
			<td>Siemens</td>
			<td>Mat No: 4765454</td>
			<td>Manufactured by: Johannes-Stark-Stk. 8 D-92224 Amberg</td>
		</tr>
	</tbody>
</table>

high-resolution structural magnetic resonance imaging of the human subcortex in vivo and postmortem

这项研究的重点是测试一个死后的大脑结构MRI相比，活体人脑的分辨率极限。结构的MRI的体内分辨率最终由生理噪声，包括脉动，呼吸和头部运动的限制。虽然成像硬件的不断提高，但仍难以解决结构上的毫米级。例如，在外侧膝状体初级视觉感觉通路突触（LGN），一个视觉继电器和控制核中，通常被组织成六个交错单眼层丘脑。神经影像学研究一直无法可靠地区别这些层由于它们的小尺寸是小于1毫米厚。 结构的MRI的分辨极限，在死后的大脑进行了测试用的多个图像平均长持续时间（〜24小时）。的目的是测试是否有可能解决各个升所述LGN在没有生理噪声的艾尔斯。质子密度（PD）1加权脉冲序列用于具有不同分辨率和其他参数来确定必要要登记的图像的最小数目并取平均值以可靠区分LGN和其他皮质下区域。结果进行了比较，以在活的人脑获取的图像。 体内受试者进行扫描，以确定对区分皮层下结构，在临床应用中有用的PD所需的最小扫描次数生理噪声的附加 ​​效果。

一旦皮层下被规定丘脑内，PD加权图像中的切片选择框（图4）收集。信噪比通过增加在两个死后和体内扫描平均次数提高。以确定的图像质量，从不同的扫描平均数信噪比是由在脑外的某些区域的平均脑区域的信号除以标准偏差进行比较。信噪比被计算为SNR = 0.655 *μ 组织 /σ 空气 11，其中，μ 组织表示脑区域内的ROI的平均像...

Watch this Scientific Journal Video about High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem at JoVE.com

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

Here we present a protocol to determine the minimum number images that needed to be registered and averaged to resolve subcortical structures and test whether the individual layers of the LGN could be resolved in the absence of physiological noise.

高分辨率结构磁共振人类皮质下的成像<I&gt;在体内</I&gt;与死亡

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of ...

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Neuroscience

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

15.3K 次观看.  York University. Here we present a protocol to determine the minimum number images that needed to be registered and averaged to resolve subcortical structures and test whether the individual layers of the LGN could be resolved in the absence of physiological noise. 

视频: High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

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 ...

科研

神经科学

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are characteristic to neurons in both normal and diseased brain. Towards this, a great deal of effort has been placed on building high-resolution neuroanatomical maps1-3. With the expansion of molecular genetics and advances in light microscopy has come the ability to query not only neuronal morphologies, but also the molecular and cellular makeup of individual neurons and their associated networks4. Major advances in the ability to mark and manipulate neurons through transgenic and gene targeting technologies in the rodent now allow investigators to 'program' neuronal subsets at will5-6. Arguably, one of the most influential contributions to contemporary neuroscience has been the discovery and cloning of genes encoding fluorescent proteins (FPs) in marine invertebrates7-8, alongside their subsequent engineering to yield an ever-expanding toolbox of vital reporters9. Exploiting cell type-specific promoter activity to drive targeted FP expression in discrete neuronal populations now affords neuroanatomical investigation with genetic precision.
Engineering FP expression in neurons has vastly improved our understanding of brain structure and function. However, imaging individual neurons and their associated networks in deep brain tissues, or in three dimensions, has remained a challenge. Due to high lipid content, nervous tissue is rather opaque and exhibits auto fluorescence. These inherent biophysical properties make it difficult to visualize and image fluorescently labelled neurons at high resolution using standard epifluorescent or confocal microscopy beyond depths of tens of microns. To circumvent this challenge investigators often employ serial thin-section imaging and reconstruction methods10, or 2-photon laser scanning microscopy11. Current drawbacks to these approaches are the associated labor-intensive tissue preparation, or cost-prohibitive instrumentation respectively.
Here, we present a relatively rapid and simple method to visualize fluorescently labelled cells in fixed semi-thick mouse brain slices by optical clearing and imaging. In the attached protocol we describe the methods of: 1) fixing brain tissue in situ via intracardial perfusion, 2) dissection and removal of whole brain, 3) stationary brain embedding in agarose, 4) precision semi-thick slice preparation using new vibratome instrumentation, 5) clearing brain tissue through a glycerol gradient, and 6) mounting on glass slides for light microscopy and z-stack reconstruction (Figure 1).
For preparing brain slices we implemented a relatively new piece of instrumentation called the 'Compresstome' VF-200 (http://www.precisionary.com/products_vf200.html). This instrument is a semi-automated microtome equipped with a motorized advance and blade vibration system with features similar in function to other vibratomes. Unlike other vibratomes, the tissue to be sliced is mounted in an agarose plug within a stainless steel cylinder. The tissue is extruded at desired thicknesses from the cylinder, and cut by the forward advancing vibrating blade. The agarose plug/cylinder system allows for reproducible tissue mounting, alignment, and precision cutting. In our hands, the 'Compresstome' yields high quality tissue slices for electrophysiology, immunohistochemistry, and direct fixed-tissue mounting and imaging. Combined with optical clearing, here we demonstrate the preparation of semi-thick fixed brain slices for high-resolution fluorescent imaging.

Here we describe a rapid and simple method to image fluorescently labeled cells in semi-thick brain slices. By fixing, slicing, and optically clearing brain tissue we describe how standard epifluorescent or confocal imaging can be used to visualize individual cells and neuronal networks within intact nervous tissue.

一个快速高分辨率的荧光成像的方法在半厚的脑片

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

高分辨率人类中脑功能磁共振成像方法

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

高分辨率实时成像发展中的神经上皮细胞的行为

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

The Use of Magnetic Resonance Spectroscopy as a Tool for the Measurement of Bi-hemispheric Transcranial Electric Stimulation Effects on Primary Motor Cortex Metabolism

Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of neurological and psychiatric disorders such as stroke and depression. Yet, the mechanisms underlying its ability to modulate brain excitability to improve clinical symptoms remains poorly understood 33. To help improve this understanding, proton magnetic resonance spectroscopy (1H-MRS) can be used as it allows the in vivo quantification of brain metabolites such as &#947;-aminobutyric acid (GABA) and glutamate in a region-specific manner 41. In fact, a recent study demonstrated that 1H-MRS is indeed a powerful means to better understand the effects of tDCS on neurotransmitter concentration 34. This article aims to describe the complete protocol for combining tDCS (NeuroConn MR compatible stimulator) with 1H-MRS at 3 T using a MEGA-PRESS sequence. We will describe the impact of a protocol that has shown great promise for the treatment of motor dysfunctions after stroke, which consists of bilateral stimulation of primary motor cortices 27,30,31. Methodological factors to consider and possible modifications to the protocol are also discussed.

This article aims to describe a basic protocol for combining transcranial direct current stimulation (tDCS) with proton magnetic resonance spectroscopy (1H-MRS) measurements to investigate the effects of bilateral stimulation on primary motor cortex metabolism.

磁共振波谱的用途，用于对初级运动皮层新陈代谢双半球经颅电刺激效果的测量工具一

Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of ...

Metabolomic Analysis of Rat Brain by High Resolution Nuclear Magnetic Resonance Spectroscopy of Tissue Extracts

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics and proteomics have been complemented by comprehensive quantitative analysis of the metabolite pool present in biological systems. This strategy, termed metabolomics, strives to provide a global characterization of the small-molecule complement involved in metabolism. While the genome and the proteome define the tasks cells can perform, the metabolome is part of the actual phenotype. Among the methods currently used in metabolomics, spectroscopic techniques are of special interest because they allow one to simultaneously analyze a large number of metabolites without prior selection for specific biochemical pathways, thus enabling a broad unbiased approach. Here, an optimized experimental protocol for metabolomic analysis by high-resolution NMR spectroscopy is presented, which is the method of choice for efficient quantification of tissue metabolites. Important strengths of this method are (i) the use of crude extracts, without the need to purify the sample and/or separate metabolites; (ii) the intrinsically quantitative nature of NMR, permitting quantitation of all metabolites represented by an NMR spectrum with one reference compound only; and (iii) the nondestructive nature of NMR enabling repeated use of the same sample for multiple measurements. The dynamic range of metabolite concentrations that can be covered is considerable due to the linear response of NMR signals, although metabolites occurring at extremely low concentrations may be difficult to detect. For the least abundant compounds, the highly sensitive mass spectrometry method may be advantageous although this technique requires more intricate sample preparation and quantification procedures than NMR spectroscopy. We present here an NMR protocol adjusted to rat brain analysis; however, the same protocol can be applied to other tissues with minor modifications.

The neurochemistry of mammalian brain is changed in many neurological and systemic diseases. Characteristic profiles of cerebral metabolites can be efficiently obtained based on crude extracts of brain tissue. To this end, high-resolution NMR spectroscopy is employed, enabling detailed quantitative analysis of metabolite concentrations (metabolomics).

大鼠脑组织被提取物的高分辨核磁共振代谢组学分析

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics ...

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has been heavily studied. While many imaging studies treat the hippocampus as a single unitary neuroanatomical structure, it is, in fact, composed of several subfields that have a complex three-dimensional geometry. As such, it is known that these subfields perform specialized functions and are differentially affected through the course of different disease states. Magnetic resonance (MR) imaging can be used as a powerful tool to interrogate the morphology of the hippocampus and its subfields. Many groups use advanced imaging software and hardware (&#62;3T) to image the subfields; however this type of technology may not be readily available in most research and clinical imaging centers. To address this need, this manuscript provides a detailed step-by-step protocol for segmenting the full anterior-posterior length of the hippocampus and its subfields: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus (DG), strata radiatum/lacunosum/moleculare (SR/SL/SM), and subiculum. This protocol has been applied to five subjects (3F, 2M; age 29-57, avg. 37). Protocol reliability is assessed by resegmenting either the right or left hippocampus of each subject and computing the overlap using the Dice&#39;s kappa metric. Mean Dice&#39;s kappa (range) across the five subjects are: whole hippocampus, 0.91 (0.90-0.92); CA1, 0.78 (0.77-0.79); CA2/CA3, 0.64 (0.56-0.73); CA4/dentate gyrus, 0.83 (0.81-0.85); strata radiatum/lacunosum/moleculare, 0.71 (0.68-0.73); and subiculum 0.75 (0.72-0.78). The segmentation protocol presented here provides other laboratories with a reliable method to study the hippocampus and hippocampal subfields in vivo using commonly available MR tools.

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The goal of this manuscript is to study the hippocampus and hippocampal subfields using MRI. The manuscript describes a protocol for segmenting the hippocampus and five hippocampal substructures: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus, strata radiatum/lacunosum/moleculare, and subiculum.

高分辨率<em&gt;在体内</em&gt;使用3T磁共振成像手动分割协议为人类海马子字段

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has ...

A Magnetic Resonance Imaging Protocol for Stroke Onset Time Estimation in Permanent Cerebral Ischemia

MRI provides a sensitive and specific imaging tool to detect acute ischemic stroke by means of a reduced diffusion coefficient of brain water. In a rat model of ischemic stroke, differences in quantitative T1 and T2 MRI relaxation times (qT1 and qT2) between the ischemic lesion (delineated by low diffusion) and the contralateral non-ischemic hemisphere increase with time from stroke onset. The time dependency of MRI relaxation time differences is heuristically described by a linear function and thus provides a simple estimate of stroke onset time. Additionally, the volumes of abnormal qT1 and qT2 within the ischemic lesion increase linearly with time providing a complementary method for stroke timing. A (semi)automated computer routine based on the quantified diffusion coefficient is presented to delineate acute ischemic stroke tissue in rat ischemia. This routine also determines hemispheric differences in qT1 and qT2 relaxation times and the location and volume of abnormal qT1 and qT2 voxels within the lesion. Uncertainties associated with onset time estimates of qT1 and qT2 MRI data vary from &plusmn; 25 min to &plusmn; 47 min for the first 5 hours of stroke. The most accurate onset time estimates can be obtained by quantifying the volume of overlapping abnormal qT1 and qT2 lesion volumes, termed 'Voverlap' (&plusmn; 25 min) or by quantifying hemispheric differences in qT2 relaxation times only (&plusmn; 28 min). Overall, qT2 derived parameters outperform those from qT1. The current MRI protocol is tested in the hyperacute phase of a permanent focal ischemia model, which may not be applicable to transient focal brain ischemia.

A protocol for stroke onset time estimation in a rat model of stroke exploiting quantitative magnetic resonance imaging (qMRI) parameters is described. The procedure exploits diffusion MRI for delineation of the acute stroke lesion and quantitative T1 and T2 (qT1 and qT2) relaxation times for timing of stroke.

脑缺血中风发作时间估计的磁共振成像协议

MRI provides a sensitive and specific imaging tool to detect acute ischemic stroke by means of a reduced diffusion coefficient of brain water. In a rat ...

High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. Transcytosis across the BBB regulates the delivery of hormones, metabolites, and therapeutic antibodies to the brain parenchyma. Here, we present a protocol that combines immunofluorescence of free-floating sections with laser scanning confocal microscopy and image analysis to visualize subcellular organelles within endothelial cells at the BBB. Combining this data-set with 3D image analysis software allows for the semi-automated segmentation and quantification of capillary volume and surface area, as well as the number and intensity of intracellular organelles at the BBB. The detection of mouse endogenous immunoglobulin (IgG) within intracellular vesicles and their quantification at the BBB is used to illustrate the method. This protocol can potentially be applied to the investigation of the mechanisms controlling BBB transcytosis of different molecules in vivo.

Here we present a microscopy-based protocol for high-resolution imaging and a three-dimensional reconstruction of the mouse neurovascular unit and blood-brain barrier using brain free-floating sections. This method allows for the visualization, analysis, and quantification of intracellular organelles at the BBB.

血脑屏障的高分辨率共聚焦成像: 成像、3D 重建和 Transcytosis 的量化

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. ...

Diffusion Tensor Magnetic Resonance Imaging in Chronic Spinal Cord Compression

Chronic spinal cord compression is the most common cause of spinal cord impairment in patients with nontraumatic spinal cord damage. Conventional magnetic resonance imaging (MRI) plays an important role in both confirming the diagnosis and evaluating the degree of compression. However, the anatomical detail provided by conventional MRI is not sufficient to accurately estimate neuronal damage and/or assess the possibility of neuronal recovery in chronic spinal cord compression patients. In contrast, diffusion tensor imaging (DTI) can provide quantitative results according to the detection of water molecule diffusion in tissues. In the present study, we develop a methodological framework to illustrate the application of DTI in chronic spinal cord compression disease. DTI fractional anisotropy (FA), apparent diffusion coefficients (ADCs), and eigenvector values are useful for visualizing microstructural pathological changes in the spinal cord. Decreased FA and increases in ADCs and eigenvector values were observed in chronic spinal cord compression patients compared to healthy controls. DTI could help surgeons understand spinal cord injury severity and provide important information regarding prognosis and neural functional recovery. In conclusion, this protocol provides a sensitive, detailed, and noninvasive tool to evaluate spinal cord compression.

Here, we present a protocol for the application of diffusion tensor imaging parameters to evaluate spinal cord compression.

扩散张量磁共振成像在慢性脊髓压迫中的应用

Chronic spinal cord compression is the most common cause of spinal cord impairment in patients with nontraumatic spinal cord damage. Conventional magnetic ...