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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这篇文章出版费用由布鲁克公司被部分赞助。

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。

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			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
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			<td height="21" style="height:21px;width:216px;">Small animal imaging RF coil</td>
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			<td height="21" style="height:21px;width:216px;">Respiratory gating system</td>
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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

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

在体内脊髓的鼠标线使用双光子显微镜成像

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

科研

神经科学

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

在斑马鱼幼虫脊髓横断

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

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 ...

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 ...

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

影像学检查HT能纤维在小鼠脊髓使用CLARITY / CUBIC技术

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

胸腺1-gmcmp6 转基因小鼠在原代体血源皮质中的成像神经活性

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

拉米内切手术和脊髓窗口植入小鼠

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

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 ...

Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. Injection directly into spinal cord tissue bypasses these barriers, providing access to cell bodies or synapses where molecules can be incorporated. Combining viral vector technology with this method allows for introduction of target genes into nervous tissue for the purpose of gene therapy or tract tracing. Here a virus engineered for highly efficient retrograde transport (HiRet) is introduced at the synapses of propriospinal interneurons (PNs) to encourage specific transport to neurons in the spinal cord and brainstem nuclei. Targeting PNs takes advantage of the numerous connections they receive from motor pathways such as the rubrospinal and reticulospinal tracts, as well as their interconnection with each other throughout spinal cord segments. Representative tracing using the HiRet vector with constitutively active green fluorescent protein (GFP) shows high fidelity details of cell bodies, axons and dendritic arbors in thoracic PNs and in reticulospinal neurons in the pontine reticular formation. HiRet incorporates well into brainstem pathways and PNs but shows age dependent integration into corticospinal tract neurons. In summary, spinal cord injection using viral vectors is a suitable method for introduction of proteins of interest into neurons of targeted tracts.

This protocol demonstrates injection of a retrogradely transportable viral vector into rat spinal cord tissue. The vector is taken up at the synapse and transported to the cell body of target neurons. This model is suitable for retrograde tracing of important spinal pathways or targeting cells for gene therapy applications.

直接注射慢病毒载体突出大鼠脊髓中的多种运动途径

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. ...

Thoracic Spinal Cord Hemisection Surgery and Open-Field Locomotor Assessment in the Rat

Spinal cord injury (SCI) causes disturbances in motor, sensory, and autonomic function below the level of the lesion. Experimental animal models are valuable tools to understand the neural mechanisms involved in locomotor recovery after SCI and to design therapies for clinical populations. There are several experimental SCI models including contusion, compression, and transection injuries that are used in a wide variety of species. A hemisection involves the unilateral transection of the spinal cord and disrupts all ascending and descending tracts on one side only. Spinal hemisection produces a highly selective and reproducible injury in comparison to contusion or compression techniques that is useful for investigating neural plasticity in spared and damaged pathways associated with functional recovery. We present a detailed step-by-step protocol for performing a thoracic hemisection at the T8 vertebral level in the rat that results in an initial paralysis of the hindlimb on the side of the lesion with graded spontaneous recovery of locomotor function over several weeks. We also provide a locomotor scoring protocol to assess functional recovery in the open-field. The locomotor assessment provides a linear recovery profile and can be performed both early and repeatedly after injury in order to accurately screen animals for appropriate time points in which to conduct more specialized behavioral testing. The hemisection technique presented can be readily adapted to other transection models and species, and the locomotor assessment can be used in a variety of SCI and other injury models to score locomotor function.

The rat thoracic spinal hemisection is a valuable and reproducible model of unilateral spinal cord injury to investigate the neural mechanisms of locomotor recovery and treatment efficacy. This article includes a detailed step-by-step guide to perform the hemisection procedure and to assess locomotor performance in an open-field arena.

胸脊髓截断手术和大鼠开放场运动评估

Spinal cord injury (SCI) causes disturbances in motor, sensory, and autonomic function below the level of the lesion. Experimental animal models are ...