Name: diffusion imaging in the rat cervical spinal cord
Uploaded: 2015-04-07T14:00:00.000+00:00
Duration: 10 min 46 s
Description: Watch this Scientific Journal Video about Diffusion Imaging in the Rat Cervical Spinal Cord at JoVE.com

我们感谢凯尔斯特赫利克，娜塔莎·威尔金斯和马特Runquist实验协助。通过研究和教育计划基金，推进更健康养老威斯康星州威斯康星医学院的一个组成部分，与克雷格·H·尼尔森基金会资助。

注:伦理学声明:威斯康星医学院的机构护理和使用委员会（IACUC）和克莱门特J. Zablocki VA医学中心批准的所有程序。 1.动物准备和监控<ol><li>麻醉大鼠在感应腔，用5％异氟烷在医用空气。当翻正反射是不存在，并且挤压后爪不产生撤回反射，减少麻醉至2％，在头部的第一俯卧动物转移到扫描仪床。保持2％异氟醚通过在整个过程的鼻锥设备，并保持医用空气以约1升/分钟的流速。取少量润滑软膏，以老鼠的眼睛，以避免损伤角膜，而在麻醉下。 </li><li>放置一个呼吸监测皮带围绕牢固老鼠的躯干。皮带连接到一个呼吸门控系统。前推进老鼠放入扫描仪孔，CHEC<html> k是呼吸监测计算机，以确保呼吸周期是清楚和一致。必要时调整皮带，因为这一步是必要的图像质量。 </li><li>监视和通过直肠探头和温暖的空气加热系统保持动物的体温在37℃。通过调整麻醉介于1.2和2％的水平维持每分钟30-45次呼吸的呼吸速率。 </li><li>定位在所述头部保持器的大鼠的咬入杆和螺钉在耳棒（ 图1），和头滑入一正交线圈体积直至颈椎被定位在线圈的中心。 注:老鼠的肩膀可以防止进一步恶化到线圈。 </li><li>推进大鼠和配套支架插入扫描仪孔。如果适用的话，调整调谐和线圈到适当频率的匹配电容器，并根据由所述线圈供应商提供的说明进行操作阻抗。 </li></ol>e_title"&gt; 2。MRI扫描参数注:这里所描述的方法使用了9.4 t水平孔小动物的制度，但适用于小动物磁共振成像系统等领域的优势。 <ol><li>使用MRI系统的自动化程序来检测共振频率的，迭代地改善磁场的接收器增益的均匀性（匀场）中，射频功率的校准和调节。 </li><li>使用该系统的软件界面，获得一个默认的三平面定位扫描，以确保正确定位。 <ol><li>点击"新建扫描"，选择tripilot，并点击"交通灯"，以获取图像。 </li><li>确保颈椎的中心与所述磁体的两个中心和MRI线圈的中心对准。居中脊柱磁铁，在推或拉的摇篮，重新获取验证搜索扫描。 </li><li>要调整positioÑ​​颈椎相对于所述MRI线圈的，从磁铁重新定位取出支架。如果有必要，重复这个过程，直到立场是一贯的。如果动物被重新定位，重复步骤2.1。 </li></ol></li><li>添加新的回波平面扩散加权自旋回波序列（DtiEpi）到当前摄像协议。 <ol><li>配置和使用，除了以下的默认设置与DWI序列获得弥散加权成像: </li><li>打开切片位置的图形界面，以规定12片的厚度为0.75毫米。定向片垂直于颈髓的主轴线。确保不同动物之间或跨越使用小脑作为内部参考的基部不同的成像会话一致切片定位。 </li><li>设置饱和度乐队"开"。第4位的饱和带与脊髓外部的厚度为10毫米，以尽量减少从这些组织的信号，并减少其潜在的诱导构件（ 图3）。设置呼吸门控&#39;的&#39;（&#39;触发模块"）来。 注:自定义呼吸门控要求的脉冲序列编程的知识和经验。如果这是不可用，一个解决办法是，减小条带的数量3-5和对TR至1秒，以确保所有的条带被在中间的动物的呼吸获得。重复该全序列与片的另一子集，以获得颈髓的全面覆盖。 </li><li>单击工具箱图标，然后单击"编辑方法。"EPI的段数设置为4.更改相位编码方向左右。其他默认设置应该是:回波间隔= 0.3234毫秒，每EPI段总回声列车长度= 32。 注:相位编码中设置的左右方向，而不是前 - 后会减少运动的污染其他结构。 </li><li>使用以下geometriCAL设置。矩阵大小= 128×128，并且在平面视场的= 25.6 X25.6毫米，导致平面内的空间分辨率= 0.200 X0.200毫米。确保层厚= 0.75毫米。切片为了=&#39;交错&#39;，层间距= 0毫米。 </li><li>使用下面的扩散加权设置:DW测量模式=&#39;DW对比度&#39;，扩散梯度的持续时间（δ）= 7毫秒，扩散梯度分离（Δ）= 12毫秒，b值数= 8，所需的b值= 0 ，250，500，750，1000，1500，2500 3500毫米/秒2，扩散方向= 2，扩散加权方向= [1 0 0]号和[0 0 1]（制成为在平面平行且正交于脊髓轴）。 注:使用这些设置，我们实现了b值高达3500秒/毫米2。硬件规格和其他系统性能特征可能限制b值，由于扩散梯度的持续时间（δ）和扩散梯度分离（Δ）是依赖于克radient的表现，这对我们的系统有:（最大梯度强度:440吨/米，最大压摆率:3440 T /米/秒）。用于测量峭度的，2 b值，具有至少2000秒/毫米2的较高b值，被推荐的。 </li><li>使用下面的时间设置。回波时间（TE）= 27毫秒（通过输入0设置为最小），重复时间（TR）= 1800毫秒。 </li></ol></li><li>收购准备序列。上面列出的参数，总采集时间是约25分钟。 </li><li>在所有的扫描，监测呼吸门控的软件，并调整"触发"之间的延迟时间（软件检测呼气的）和信号对MRI系统，使得仅出现在呼吸周期的静态（静止的）部分（ 图收购2A，灰线的稳定部分）。 100-400毫秒之间的触发延时需要根据动物的呼吸模式。这将有助于减少ARTI与呼吸运动（ 图3e）发生的事实。 </li><li>如果可以，用定制的"反向光点"设置为"ON"，这就要求采集时间的25分钟重复上述步骤。 注意:如果自定义的"反向标志"序9（第3步骤期间所需的易感性伪影校正）是不可用的，只有一个单一的EPI相位编码方向是可能的，而相反的光点序列修饰允许相位编码方向的选择（右-to-向左或从左向右）。 </li><li>当成像完成后，从支架上取下动物，并返回到它的笼子里。不要让动物无人看管，直到它恢复足够的意识，保持胸骨斜卧。 </li></ol> 3.图像处理<ol><li>从系统中DICOM格式导出数据直接从系统（首选）或使用自定义或锡尔将数据转换为NIFTI格式D-第三方软件。 </li><li>进行敏感性伪影校正。 <ol><li>提取B =从每个0卷扫描到一个文件中，使用带有FSL或其他MRI软件包工具。是必需的一个文件的每个相位编码方向。 注意:例如，如果每次扫描由8扫描不同b值与扩散加权在横向方向上，其次是8扫描的长度方向扩散加权的，则该图像文件中包含B = 0的扫描中的1 次和第第 9卷，并且可以提取并组装用下面的壳代码: fslroi $ {}起来_dwi_masked.nii.gz temp1中0 1 fslroi $ {}起来TEMP2 _dwi_masked.nii.gz 8 1 fslroi $ {}下降_dwi_masked.nii.gz TEMP3 0 1 fslroi $ {}下降_dwi_masked.nii.gz temp4 8 1 fslmerge -t blip_both temp1中TEMP2 TEMP3 temp4 （其中，在这种情况下，$向上和向下$是与正常的扫描和反向相位编码方向，分别地）。 </li&gt; <li>使用FSL 10,11的&#39;充值&#39;命令创建降低图像失真文物修正文件。该修正适用于将用于创建参数映射的原始的DWI图像。 注:使用说明该命令可以在这里找到<a href="http://fsl.fmrib.ox.ac.uk/fsl/fslwiki/TOPUP/TopupUsersGuide">http://fsl.fmrib.ox.ac.uk/fsl/fslwiki/TOPUP/TopupUsersGuide</a> 。示例代码以在这种情况下使用的命令如下: 充值--imain = blip_both_nlmf_b0images_masked.nii --datain = .. / topup_data.txt --config =。/ b02b0_ratspine.cnf --out = topup_splines_nlmf --iout = $出去 - 详细--logout = topuplog.log dwiup =`LS $ {}上升* dwi_nlmFilt.nii` dwidown =`LS $ {}下来* dwi_nlmFilt.nii` applytopup --imain = $ {} dwiup，$ {} dwidown = --datain .. / topup_data.txt --method =江淮--inindex = 1，$ IND --topup = topup_splines_nlmf --out = DWI _ $ {出} -v 复制和编辑$ {} FSLDIR的/etc/flirtsch/b02b0.cnf默认文件的大鼠旋人帘线由通过10倍减少的每个在--warpres和--fwhm线的数值。 </li></ol></li><li>如果使用扩散加权图像沿至少6个非正交方向收购（使用DTI计划，Paravision或类似的定制设计），使用的软件包，如佛山照明的扩散工具箱12或13的Camino计算标准DTI参数映射。如果没有，则使用自定义过程用于产生有用的指标，其采用扩散加权仅沿两个方向，例如，如在步骤3.4向前表示。 </li><li>通过加载充值输出校正DWI文件到fslview，选择"文件 - &gt;创建规则"从菜单中。使用铅笔工具绘制的感兴趣区域内的一个组织类型（例如通用，背WM，或腹外侧WM）。保存此文件，并重复任何其它所需的投资回报，以备后用。 注:从脊髓等程序段的投资回报已被记录在案14,15 </s达&gt;，可能是最好的高级用户。 </li><li>使用ROI文件以掩蔽DWI文件，然后将ROI使用以下命令中的每个图像体积内计算平均信号: fslstats -t DWI_corrected.nii.gz -k GM_mask.nii.gz -M </li><li>复制的第一个8结果转换成数值计算程序如MATLAB，作为横向的信号矢量（例如称之为sig_T），和第二8的结果作为矢量为纵向信号（sig_L），其中，图8是B-数使用的值。 <ol><li>在一个数值计算程序复制的b值作为8 b值的向量。的b值在横向和纵向方向是相同的。如果可能的话，将有效b值，而不是标称b值，应该从扫描仪，它是从步骤2.3.5为"有效果B值"中列出的参数窗口获得。 </li><li>使用数值计算程序的曲线拟合工具箱，以适应信号与b值数据为thË通过在命令提示符下键入cftools所需的模型。要做到这一点，请单击"数据..."，然后选择以Y型数据信号矢量和b值x数据。点击"配件..."和"拟合类型"下选择"自定义公式"，然后单击"新建"和"一般方程"，进入一个方程拟合。 </li></ol></li><li>以适合于标准扩散模型，输入公式: S0 * EXP（-x。* D）"（1） </li><li>以适合一个模型，其中包括扩散和第二阶项（峰度K）来测量从高斯扩散16的偏差，输入公式: S0 * EXP（-x。* D +（1/6）*（X * D）^ 2 * K）"（2） </li><li>点击"确定"和"应用"。观察的估计值为扩散系数（D）和峰度（K）的输出窗口上。在"数据集:"选择中，选择与方程使用sig_T（或sig_L）数据（1）（2），然后点击"应用"。 </li><li>计算使用横向和纵向扩散系数的各向异性指数（AI）: AI =（ 平负-D T）/（平负+ D T）（3） 这是类似的分数各向异性（FA）从DTI模型计算。峰度的各向异性指数也可以使用横向和纵向峭度代替扩散率计算出来的。 注意此方法给出的模型参数如K T，D T，等等。另外，也可以使用曲线拟合工具箱的命令行操作的每个体素的脊柱内，以从模型创建的每个参数的值映射。替代拟合方法可以用于和其他地方详细描述。17 </li></ol>

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+tensor+imaging+of+the+mouse+brainstem+and+cervical+spinal+cord.">Diffusion tensor imaging of the mouse brainstem and cervical spinal cord.</a> Nat Protoc. 8 (2), 409-417 (2013).</li></ol>

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这里概述的方法可以在体内提供的大鼠脊髓高质量扩散加权图像。图像质量取决于许多因素，但脊髓具有很重要的几个独特的问题。 运动是一个重要的问题，即如果不纠正，将导致不能使用的图像。因此，它需要MRI会议期间仔细监测。如果图像伪影的初始扫描是与运动相一致观察，停止收购，并采取措施消除文物，因为这些是很难在后期处理来去除。确保呼吸道计算机从呼吸监测设备接收到一个强大的，定期的信号。呼吸带，可能需要进行调整以获得正确的张力，提供了一个一致的信号，但不限制动物的呼吸。维持麻醉在任何时候的适当水平; 1.5-2.0％，异氟醚已在我们的experien被使用CE。同样地，减少在动物和脊柱的整体运动是提供无伪影的图像的另一个重要方面。不像人类脊髓，其经历引起脑脊液脉动相关的心脏周期显著运动，在啮齿动物脑脊液脉动与呼吸周期18主要关联。虽然很难完全消除在帘线所有运动中，特别重要的是减少了运动的程度成为可能，这通常是通过试验和错误来实现。此外，大鼠各种神经损伤或病症可具有异常呼吸速率或其他生理并发症，可能需要的本文所概述的程序适应。 该修改用于呼吸门控脉冲序列，连同定制用于该目的的图像重建程序，使失真最小而引起不均匀磁场以致无法祛瘀的影响通过在MRI系统上执行的调整编同样地，图像质量取决于成像时间的持续时间。在我们的例子中，限制扩散的加权的仅沿两个方向上的数目使在总成像时间的减少。这种方法的一个限制是，它不再是全张量分析（DTI），这是标准的许多其他研究兼容。或者，使用较少的平均值和多扩散方向或b值可以允许更好的表征，同时保持相同的采集时间。以前的研究已经表明，在2方向的方法提供了与6-方向（DTI）的方法19一致的信息，但必须小心，以确保切片（和扩散方向）的取向准确地沿着并垂直于该线。然而，获取多个b值允许更好表征和峭度的数学拟合，推荐在使用单一的B-VALU的即此外，全序列，重复用颠倒的相位编码方向这减少磁场敏感伪影的效果，并提高了通过平均化图像的整体质量。最后，在我们的协议中使用的图像的分辨率提供的白质和灰质明确分离。具有更高分辨率的图像是可能的，虽然这往往出现以牺牲更长的扫描时间或更多神器的潜力。 在射频线圈，脉冲序列，和后处理方法的改进都将有改善脊髓成像在该方法的将来的修改的效果。例如，表面线圈可能有利于类似于在小鼠中观察到的改善的图像质量。20，这些措施有被作为生物标志物用于脊髓损伤的临床诊断和管理有用的高可能性。

磁共振成像（MRI）是一种非侵入性工具，它提供了一个窗口的脑和脊髓在健康和疾病。 MRI已经彻底改变了临床诊断，但它也适用于实验室研究的重要工具。神经损伤或疾病的动物模型提供了一个平台，了解病理生理学和推动发现疗法。在这份报告中，我们展示了MRI的应用脊髓损伤的大鼠模型来研究微观损伤1采用弥散张量成像（DTI）的潜在生物标志物。成像生物标志物的潜力发现将有助于患者脊髓损伤的诊断和管理。这些标记物都可能在他们的翻译中发现的疗法在临床前模型中发挥作用，并能观察或预后的临床环境。 DTI MRI是一种特殊形式的测量微观运动水分子（ 即扩散）。 DTI已经在神经系统是特别有利的，由于轴突的存在，其中扩散是不成比例更快沿轴突比垂直于它们，这提供了关于他们的取向和微结构的组合物的信息。从DTI衍生标量指数，其中包括在组织内的总体扩散的量度，平均弥散（MD），和定向扩散的依赖性的量度，分数各向异性（FA）2,3，已经看到了广泛的应用中表征的微观结构神经系统在健康和疾病4。这些指标揭示微观组织的功能，是无形的，通过其他大多数MRI方法。以前的努力证明，DTI检测颈髓大鼠1以下胸SCI中远程微观结构的变化。英国贸工部的变化远离病变可能反映了怎样的整个脊髓水库池塘损伤，并具有潜在的继发性损伤的一个标志物。 活体成像的大鼠脊髓提出了一些独特的挑战。最值得注意的是，脊髓受呼吸运动的影响，并且需要小心注意，以尽量减少使用多种方法的运动。在过去的研究中，固定设备扫描5中去除脊柱的运动。为颈髓成像，我们利用在一个头支架和耳棒，其中衰减的形式物理约束，但不消除由呼吸运动。此外，我们利用一个自定义的呼吸门控方案来以有效的方式进行同步的图像采集与呼吸周期。这些修饰使去除否则所造成的呼吸6大型整体运动引起的伪影。 DWI是微观运动，包括脑脊液流动和血液脉动，和运动这些小源高度敏感contamination的缓解也由呼吸门控方案。此外，脊髓具有小的横截面面积，并且表示视场的一小部分。用于颈椎成像，其中脊髓位于动物的身体内深处，圆柱形射频线圈有足够的信号穿透是需要图像颈髓具有高分辨率。在视场的降低是由外体积抑制（OVS），它也用于取消或弃土，从组织中的信号的脊髓外实现。这种方法被称为扰流梯度或外卷抑制，也有助于减少这些组织中残留的动物的运动，脑脊液流动，或血液脉动任何污染。 脊髓的排列也可以被利用来简化成像协议。在白质（WM）脊髓轴突几乎都取向为平行于脊髓的主轴线。日我们，而大脑的DWI需要至少沿6个方向，以确保该结果不依赖于磁铁（这个过程被称为扩散张量成像）内的位置的测量，在脊髓测量能够获得仅沿2个方向平行，并垂直于所述线7,8，以下简称为纵向和横向上。因此，扩散度及其他参数沿2个方向分别测定推论，并允许进入在健康和疾病或损伤的组织的微结构。

<table><tbody><tr><td>Small animal imaging RF coil</td><td>Doty</td><td>SAIP400-H-38-S</td><td></td></tr><tr><td>Respiratory gating system</td><td>SA Instruments</td><td>1030</td><td></td></tr><tr><td>MR scanner</td><td>Bruker</td><td>Biospec 94/30 USR</td><td></td></tr></tbody></table>

diffusion imaging in the rat cervical spinal cord

Magnetic resonance imaging (MRI) is the state of the art approach for assessing the status of the spinal cord noninvasively, and can be used as a diagnostic and prognostic tool in cases of disease or injury. Diffusion weighted imaging (DWI), is sensitive to the thermal motion of water molecules and allows for inferences of tissue microstructure. This report describes a protocol to acquire and analyze DWI of the rat cervical spinal cord on a small-bore animal system. It demonstrates an imaging setup for the live anesthetized animal and recommends a DWI acquisition protocol for high-quality imaging, which includes stabilization of the cord and control of respiratory motion. Measurements with diffusion weighting along different directions and magnitudes (b-values) are used. Finally, several mathematical models of the resulting signal are used to derive maps of the diffusion processes within the spinal cord tissue that provide insight into the normal cord and can be used to monitor injury or disease processes noninvasively.

正确的步骤，以尽量减少运动伪影导致大鼠脊髓型颈椎病的高品质的弥散加权图像。使用自定义呼吸门控（ 图2），饱和从组织不希望的信号的脊柱外侧（ 图3B＆amp; C），以及磁场磁化率失真校正产生扩散加权图像，如在图4和5。不当或未选通的图像会导致重影（ 图3E）的形式文物，而正确的门是免费的文物。 横跨12片的扩散加权图像的视觉检测揭示了涉及其微观脊髓的功能。具体而言，在组织导致在扩散加权图像更大的信号损耗，这是加剧具有更大扩散加权（b值）快扩散。随着扩散权重进行perpendicular脊髓轴，白质沿该线的周边显示明亮，因为扩散是缓慢的，限制在垂直于轴突。与此相反，该线的中心区域内的灰质出现暗的，因为它是由轴突和细胞体未全部沿单一方向排列的。相比较而言，扩散加权在并联方向导致白质与较暗的外观，因为扩散是快速沿轴突，而灰质是相对较亮。值得注意的是，单独的扩散加权图像被示出为不同的b值，由于平行和垂直的方向上具有不同b值白质和灰质之间的最佳对比度是重要的。 综合所有使用的数学形式主义扩散加权图像就可以显示扩散参数的地图。从白质和灰质的平均信号被暗算的DIF融合权重系数（b值）为平行和垂直的方向。这个量化数据强化了图4所示的扩散加权图像，具体而言，白质具有一个在扩散加权（纵向或横向）的方向上有很强的依赖性，而灰质是较少依赖于方向。同样地，使用等式扩散峭度的产量的扩散参数（ 图6B），它突出这一相同的依赖定量映射拟合信号中的每个体素。白质具有高程度的各向异性的两个扩散（AID）和峰度测量（AIK）。因此，横向的扩散和峭度表明，从组织学研究已知脊髓底层的显微结构。这些扩散参数，这将在现场获得的，但麻醉动物，反映微观组织性质如轴突密度和直径。在改变所造成的伤害和疾病本身的措施将是无创性评估损害的后果，并承诺治疗的效果非常有用。因此大鼠脊髓型颈椎病的弥散加权成像可能成为脊髓损伤和脊髓疾病的临床前研究的一个工具。 <img alt="图1" src="/files/ftp_upload/52390/52390fig1highres.jpg" /> 图1:线圈和持有人的脊髓型颈椎病的MRI设计的自定义正交卷线圈（多蒂科学公司）用于图像颈椎具有高灵敏度和均匀性。麻醉和医用空气是由所指示的气体口递送到鼻锥，它适合舒适周围的大鼠的鼻子。呼出和过量的气体被轻微真空下排气管线捕获。鼠的头被固定在咬合杆周围放置门齿和耳杆放置德利奇ately内耳道。其他生理监测组件，包括呼吸监测器和温度传感器未示出。 <img alt="图2" src="/files/ftp_upload/52390/52390fig2highres.jpg" /> 图2:呼吸门控方案 。一个典型的呼吸迹线（灰色）和触发器（红色）从选通单元被示意性示出（A）中 。在典型的实现选通（B）的，单触发器用于获取所有切片（垂直线;在这里12示出）在时间均匀间隔的重复时间（TR）内。如果TR超过典型期间，可能会发生呼吸期间几片，并容易受到运动（红色）。在修改后的方案（C）中 ，片的一个子集被触发（此处6中示出），接着在延迟之后迅速获得与随后的触发后获得的其他条带。有效中，TR为通过重新排列序列内的延迟相同的两种方案之间。 <img alt="图3" src="/files/ftp_upload/52390/52390fig3highres.jpg" /> 图3:MRI片定位，饱和度带，和运动控制轴十二片被安排的侦察图像（A）最前片位于距脑干和小脑的交点一致的距离上。饱和带（B）的加入来消除感兴趣的区域之外的干扰信号。不扩散权重（C）和一个带有扩散加权（D）所采用的定制选通方案的图像清晰地显示了线的解剖，是免费的文物。与非优化的门控方案或不当呼吸门控，扩散加权图像显示伪影（E），为丢失<html>电源线内的信号，或多个"鬼"的线，将损坏的后续分析，外<a href="https://www.jove.com/files/ftp_upload/52390/52390fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52390/52390fig4highres.jpg" /> 图4:代表弥散加权像使用文本描述的优化，高品质的弥散加权图像与扩散加权横向应用（A）和纵向（B）脊髓主轴获得。不同b值示为每个方向提供用于说明性目的的白色和灰色物质之间的最佳对比度。对于每一个方向或b值，所有12个片约90秒被收购。<html>加载/ 52390 / 52390fig4large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图5" src="/files/ftp_upload/52390/52390fig5highres.jpg" /> 图5:反相编码校正过程中左栏显示了单片成像与DWI序列在本协议（"昙花一现达"的形象）概述。中间一列给出了序列获得第二次用"反向的blip"设置为&#39;上&#39;。请注意如何将显得捉襟见肘，在第一图像特征出现压缩在中间一栏。右列显示了使用充值纠正扩散加权图像。顶行是所述非扩散加权图像，中间行是与在横向方向上施加扩散加权的例子，且最底行是与在纵向方向上施加扩散加权的例子。//www.jove.com/files/ftp_upload/52390/52390fig5large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图6" src="/files/ftp_upload/52390/52390fig6highres.jpg" /> 图6:计算扩散和峭度的地图的归一化信号（图像强度）绘制（A），因为扩散加权（b值），用于在横向（T）和纵向（L）的扩散编码方向的函数。高质量的地图扩散（D）的（B），峰度（K）和各向异性（AI）是从每个像素的信号，计算并显示脊髓组织的独特功能。具体而言，就是在白质和灰质之间的参数，以及白质区域的区域差异明显的区别。 <a href="https://www.jove.com/files/ftp_upload/52390/52390fig6large.jpg" target="_blank">请点击此处为vi</a>EW这个数字的放大版本。

Watch this Scientific Journal Video about Diffusion Imaging in the Rat Cervical Spinal Cord at JoVE.com

Diffusion Imaging in the Rat Cervical Spinal Cord

The goal of this protocol is to obtain high-quality diffusion weighted magnetic resonance imaging (DWI) of the rat spinal cord for noninvasive characterization of tissue microstructure. This protocol describes optimizations of the MRI sequence, radiofrequency coil, and analysis methods to enable DWI images free from artifacts.

在大鼠颈脊髓弥散成像

Magnetic resonance imaging (MRI) is the state of the art approach for assessing the status of the spinal cord noninvasively, and can be used as a ...

diffusion-imaging-in-the-rat-cervical-spinal-cord

Research

JoVE Journal

Neuroscience

11.6K 次观看.  Medical College of Wisconsin. The goal of this protocol is to obtain high-quality diffusion weighted magnetic resonance imaging (DWI) of the rat spinal cord for noninvasive characterization of tissue microstructure. This protocol describes optimizations of the MRI sequence, radiofrequency coil, and analysis methods to enable DWI images free from artifacts.

视频: Diffusion Imaging in the Rat Cervical Spinal Cord

Using Laser Doppler Imaging and Monitoring to Analyze Spinal Cord Microcirculation in Rat

Laser Doppler flowmetry (LDF) is a noninvasive method for blood flow (BF) measurement, which makes it preferable for measuring microcirculatory alterations of the spinal cord. In this article, our goal was to use both Laser Doppler imaging and monitoring to analyze the change of BF after spinal cord injury. Both the laser Doppler image scanner and the probe/monitor were being employed to obtain each readout. The data of LDPI provided a local distribution of BF, which gave an overview of perfusion around the injury site and made it accessible for comparative analysis of BF among different locations. By intensely measuring the probing area over a period of time, a combined probe was used to simultaneously measure the BF and oxygen saturation of the spinal cord, showing overall spinal cord perfusion and oxygen supply. LDF itself has a few limitations, such as relative flux, sensitivity to movement, and biological zero signal. However, the technology has been applied in clinical and experimental study due to its simple setup and rapid measurement of BF.

Here we present a combination of laser Doppler perfusion imaging (LDPI) and laser Doppler perfusion monitoring (LDPM) to measure spinal cord local blood flows and oxygen saturation (SO2), as well as a standardized procedure for introducing spinal cord trauma on rat.

激光多普勒成像与监测在大鼠脊髓微循环分析中的应用

Laser Doppler flowmetry (LDF) is a noninvasive method for blood flow (BF) measurement, which makes it preferable for measuring microcirculatory ...

科研

行为学

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to return to normal in a large majority of patients2, which is mainly due to current lack of clinical treatment for acute stroke. This necessitates understanding the physiological effects of cerebral ischemia on brain tissue over time and is a major area of active research. Towards this end, experimental progress has been made using rats as a preclinical model for stroke, particularly, using non-invasive methods such as 18F-fluorodeoxyglucose (FDG) coupled with Positron Emission Tomography (PET) imaging3,10,17. Here we present a strategy for inducing cerebral ischemia in rats by middle cerebral artery occlusion (MCAO) that mimics focal cerebral ischemia in humans, and imaging its effects over 24 hr using FDG-PET coupled with X-ray computed tomography (CT) with an Albira PET-CT instrument. A VOI template atlas was subsequently fused to the cerebral rat data to enable a unbiased analysis of the brain and its sub-regions4. In addition, a method for 3D visualization of the FDG-PET-CT time course is presented. In summary, we present a detailed protocol for initiating, quantifying, and visualizing an induced ischemic stroke event in a living Sprague-Dawley rat in three dimensions using FDG-PET.

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Brain damage resulting from cerebral ischemia may be non-invasively imaged and studied in rats using pre-clinical positron emission tomography coupled with the injectable radioactive probe, 18F-fluorodeoxyglucose. Further, the use of modern software tools that include volume of interest (VOI) brain templates dramatically increase the quantitative information gleaned from these studies.

无创成像和脑缺血分析生活中的大鼠使用正电子发射断层扫描<sup&gt; 18</sup&gt; F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to ...

医学

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

一个<em&gt;离体</em&gt;激光诱导脊髓损伤模型，以评估轴索变性的机制在实时

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

神经科学

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

一个程序，植入脊柱商会纵<em&gt;在体内</em&gt;鼠标脊髓成像

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

Contrast Enhanced Ultrasound Imaging for Assessment of Spinal Cord Blood Flow in Experimental Spinal Cord Injury

Reduced spinal cord blood flow (SCBF) (i.e., ischemia) plays a key role in traumatic spinal cord injury (SCI) pathophysiology and is accordingly an important target for neuroprotective therapies. Although several techniques have been described to assess SCBF, they all have significant limitations. To overcome the latter, we propose the use of real-time contrast enhanced ultrasound imaging (CEU). Here we describe the application of this technique in a rat contusion model of SCI. A jugular catheter is first implanted for the repeated injection of contrast agent, a sodium chloride solution of sulphur hexafluoride encapsulated microbubbles. The spine is then stabilized with a custom-made 3D-frame and the spinal cord dura mater is exposed by a laminectomy at ThIX-ThXII. The ultrasound probe is then positioned at the posterior aspect of the dura mater (coated with ultrasound gel). To assess baseline SCBF, a single intravenous injection (400 &#181;l) of contrast agent is applied to record its passage through the intact spinal cord microvasculature. A weight-drop device is subsequently used to generate a reproducible experimental contusion model of SCI. Contrast agent is re-injected 15 min following the injury to assess post-SCI SCBF changes. CEU allows for real time and in-vivo assessment of SCBF changes following SCI. In the uninjured animal, ultrasound imaging showed uneven blood flow along the intact spinal cord. Furthermore, 15 min post-SCI, there was critical ischemia at the level of the epicenter while SCBF remained preserved in the more remote intact areas. In the regions adjacent to the epicenter (both rostral and caudal), SCBF was significantly reduced. This corresponds to the previously described &#8220;ischemic penumbra zone&#8221;. This tool is of major interest for assessing the effects of therapies aimed at limiting ischemia and the resulting tissue necrosis subsequent to SCI.

Contrast Enhanced Ultrasound imaging is a reliable in-vivo tool for quantifying spinal cord blood flow in an experimental rat spinal cord injury model. This paper contains a comprehensive protocol for application of this technique in association with a contusion model of thoracic spinal cord injury.

超声造影成像脊髓血流量的评估实验脊髓损伤

Reduced spinal cord blood flow (SCBF) (i.e., ischemia) plays a key role in traumatic spinal cord injury (SCI) pathophysiology and is accordingly an ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

细胞动力学在小鼠脊髓双光子成像

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues throughout the acute injury phase. In fact, endothelial cells appear to be the first to die after a contusive SCI. The early vascular events, including increased permeability of the blood-spinal cord barrier (BSCB), induce vasogenic edema and contribute to detrimental secondary injury events caused by complex injury mechanisms. Targeting the vascular disruption, therefore, could be a key strategy to reduce secondary injury cascades that contribute to histological and functional impairments after SCI. Previous studies were mostly performed on postmortem samples and were unable to capture the dynamic changes of the vascular network. In this study, we have developed an in vivo duo-color two-photon imaging method to monitor acute vascular dynamic changes following contusive SCI. This approach allows detecting blood flow, vessel diameter, and other vascular pathologies at various sites of the same rat pre- and post-injury. Overall, this method provides an excellent venue for investigating vascular dynamics.

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

We introduce an in vivo imaging method using two different fluorescent dyes to track dynamic spinal vascular changes following a contusive spinal cord injury in adult Sprague-Dawley rats.

在体内挫伤脊髓损伤后影像血管动力学的双色法

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues ...

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 ...

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter implantation. The technical limitation of this surgical technique lies in the finesse of the hands. The injection is rapid, especially for a trained experimenter, and since tissue disruption with this technique is minimal, repeated injections are possible; moreover immune reaction to foreign tools (e.g. catheter) does not occur, thereby giving a better and more specific read out of spinal cord modulation. Since the application of the substance is largely limited to the target region of the spinal cord, drugs do not need to be applied in large dosages, and more importantly unwanted effects on other tissue, as observed with a systemic delivery, could be circumvented1,2. Moreover, we combine this technique with in vivo transfection of nucleic acid with the help of polyethylenimine (PEI) reagent3, which provides tremendous versatility for studying spinal functions via delivery of pharmacological agents as well as gene, RNA, and protein modulators.

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a simple and rapid technique of intrathecal needle puncture for a localized transfection of siRNA in the lumbar spinal cord in mouse under short lasting light anesthesia.

体内转染和基因敲除在脊髓通过快速无创腰椎鞘内注射小鼠

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter ...

生物学

幼年大鼠腰椎穿刺 AAV9 递送用于 CNS 基因治疗

Intrathecal Vector Delivery in Juvenile Rats via Lumbar Cistern Injection

Gene therapy is a powerful technology to deliver new genes to a patient for the treatment of disease, be it to introduce a functional gene, inactivate a toxic gene, or provide a gene whose product can modulate the biology of the disease. The delivery method for the therapeutic vector can take many forms, ranging from intravenous infusion for systemic delivery to direct injection into the target tissue. For neurodegenerative disorders, it is often desirable to skew transduction towards the brain and/or spinal cord. The least invasive approach to target the entire central nervous system involves injection into the cerebrospinal fluid (CSF), allowing the therapeutic to reach a large fraction of the central nervous system. The safest approach to deliver a vector into the CSF is the lumbar intrathecal injection, where a needle is introduced into the lumbar cistern of the spinal cord. This technique, also known as a lumbar puncture, has been widely used in neonatal and adult rodents and in large animal models. While the technique is similar across species and developmental stages, subtle differences in size, structure, and elasticity of tissues surrounding the intrathecal space require accommodations in the approach. This article describes a method for performing lumbar puncture in juvenile rats to deliver an adeno-associated serotype 9 vector. Here, 25-35 &#181;L of vector were injected into the lumbar cistern, and a green fluorescent protein (GFP) reporter was used to evaluate the transduction profile resulting from each injection. The benefits and challenges of this approach are discussed.

作者聚焦：评估幼年大鼠的鞘内基因治疗疗效

A surgical procedure is described to perform injections into the lumbar cistern of the juvenile rat. This approach has been used for the intrathecal delivery of gene therapy vectors, but it is anticipated that this approach can be used for a variety of therapeutics, including cells and drugs.

通过腰椎池注射在幼年大鼠中鞘内载体给药

Gene therapy is a powerful technology to deliver new genes to a patient for the treatment of disease, be it to introduce a functional gene, inactivate a ...

在大鼠颈脊髓弥散成像

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