我们要感谢团连接和Gillson Longenbaugh基金会为这个项目提供资金。我们还要感谢她的关键意见凯瑟琳刘汉铨博士和她DXA的设备使用。最后，我们想在DXA的股骨骨分析，结合生物学和药理学部，织小野博士，感谢她的建议。

1。小鼠脊髓横断模型<ol><li>成人，男，C57BL6小鼠（约20-25克）麻醉用氯胺酮（200毫克/千克）和甲苯噻嗪的组合（10毫克/公斤）。无菌条件下，所有的外科手术是在体制，IACUC批准的手术套房。 </li><li>一旦深深麻醉，背部的皮毛修剪使用电动快船。剃光回是第一次与70％的乙醇碘溶液擦洗。 </li><li>初始切口之前，首先是阴刻回领域渗透的浓度（0.25％，&lt;1ml/kg），以尽量减少手术后的疼痛与局部麻醉（Marcaine）。 </li><li>使用小剪刀，一个小的开放是在皮肤上，在各地的二级地区。这是扩大开放与同一组的剪刀，纵向延伸的T2区。除了通过使用猛犬夹具的皮肤边缘，然后举行。 </li><li>然后用微型剪刀明确从8胸椎水平（T8）脊髓背椎板肌肉组织。 T8可以识别，丹山和Wrathall（1998年）10描述。简言之，T13可以识别背棘突。使用一对＃5杜蒙钳，可以触摸/最后一根肋骨，识别和计算到T8。一旦清除，骨Rongeurs用于执行T8背椎板切除。 </li><li>全脊髓横断病变：一旦脊髓是暴露在T8，另外一组是用来消毒微型剪刀切断手术显微镜下，在垂直的平面的脊髓长轴的线。我们使用＃5杜蒙钳轻轻地解除对脊髓的一极，以确定病变的完整性。 </li><li>脊髓横断，无菌明胶海绵小块，浸泡在无菌生理盐水（0.9％）是经过精心放置到病灶腔，以促进止血。 </li><li>额外的明胶海绵一块，然后放置在暴露自旋人线。皮肤，然后用无菌，不锈钢手术订书钉封闭。科目，然后返回自己的家乡笼，放在纸巾上，以防止床上用品材料的愿望，并用了一段时间约12小时的加热垫回暖。主题也提供了水凝胶包（ClearH2O）和笼子里的地板上的食物颗粒在早期恢复。受伤科目能够获得食物/在自己的笼子里的水一旦从麻醉中恢复。 </li><li>所有受伤的受试者接受每日两次人工膀胱后送（约12小时的间隔），使用期限（40天）的研究Crede手动方法修改。 </li><li>损伤小鼠还提供了3天（0.5毫升），每天两次腹腔注射0.9％生理盐水，以帮助维持水化，每日两次注射阿片类药物丁丙诺啡（0.05毫克/千克）控制手术后的疼痛，会期5天。如果动物表现出的迹象Øf痛后的最初5天期间，他们将收到额外丁丙诺啡（0.05毫克/千克）每天直到疼痛的迹象（行动不便，驼背的身材，没有新郎，发声时处理）已经解决了。 </li></ol> 2。纵向评估骨质密度使用IVIS Lumina的XR spinally横断小鼠相同队列<ol><li>开始后第10天 - SCI，持续10天至40天，我们评估了生活的权利和左股骨和胫骨，麻醉学科（横断科学和未受伤的年龄匹配的对照）。 </li><li>在一天的扫描，受试者从该机构的Vivarium面积的安置房，其中卡尺的IVIS Lumina的XR是在自己的笼子转移。使用相同的氯胺酮/甲苯​​噻嗪鸡尾酒以前在SCI的交付使用，所有受试者麻醉。这种鸡尾酒为1到1.5小时内保证麻醉状态;足够的scann持续时间ING程序。 </li><li>调查初始化的卢米纳XR设备，并允许内部的摄像头达到工作温度（-90℃）（〜10分钟）。 </li><li>主体（切断或控制）麻醉后，轻轻地放在内卢米纳XR动物平台。高倍率镜头插入设备，允许在股骨和胫骨地区（实地查看2.4X2.4cm高倍率镜头）的重点。如果这个问题不正确定位的视野之外，门被打开的主题，直到左或右后肢为中心的重新定位（参见图2A为妥善安置后肢）。 </li><li>一旦适当的位置，X射线的功能可以被执行。选择的能源下拉列表中选择合适的动物 （生活的主题，35千伏为100uA，过滤X -射线） </li><li>一旦动物是在合适的位置，使检查标记X射线在X射线的特征控制面板。 获得的X射线图像。确保整个股骨和胫骨都可见（见图2b）。原始图像数据自动保存到硬盘驱动器。代表。TIFF文件也被保存。鼠标，然后返回到其家乡笼和允许下恢复研究者观察。然后重复这个过程与未来的鼠标。 注：生活图像软件默认显示转化的X射线图像。为了显示原始的X射线图像，删除复选标记旁边的X射线吸收更正/过滤工具。当X射线数据已被吸收纠正，你可以通过比较信号强度测量的ROI评估相对的骨质密度。投资回报率强度的增加与组织密度增加。 </li></ol> 3。 IVIS X射线扫描图像分析<ol><li>对生活的图像图标双击打开该软件。 </li><li>通过点击加载X射线图像<st荣&gt;“浏览”按钮。在出现的对话框中，选择感兴趣的的文件夹，然后单击确定。生活的图像浏览器显示选定的数据与用户的ID，标签信息，和摄像头的配置信息。打开数据，下列操作之一：双击数据行，右键单击数据名称并选择快捷菜单上的负荷 ，选择数据行，并单击“ 加载 ”，或双击缩略图。显示图像和工具调色板。以绿色突出显示在浏览器中打开数据。 </li><li>点击工具面板上的投资回报率工具 。在投资回报率的工具，从类型下拉列表中选择测量投资回报率。负载3的投资回报率的在本次实验中使用，请按一下广场的图标并加载3平方。 </li><li>用一把尺子，测量股骨的长度。调整长度的平方两个全股骨长度的1 / 8次。调整这两个广场的宽度要1/24th全股骨长度。你的统治者，测量1 / 8从股骨近端的距离，并位于广场，所以它是股骨内为本。位于第二方阵，以便它是从远端股骨（图3）的总股骨长度1/4th。这些投资回报率可以用来衡量近端和股骨远端地区。 </li><li>使用标尺，测量胫骨的长度。调整的第三个正方形的长度，胫骨总长度的 1 / 8次。调整宽度，胫骨总长度的1 / 30日。位于广场中心和胫骨的距离胫骨近端（见图3）的总长度1 / 8 </li><li>点击“ 测量 ”图标（铅笔和直尺）。出现的投资回报率强度测量的X射线图像中出现的投资回报率的测量表。 导出此表to你所需的位置。csv文件。这将允许您使用Excel打开表。 </li><li>重复这与所有你保存的图像。 </li><li>整合到一个Excel工作表中的所有数据。确定通过使用Microsoft Excel或SigmaPlot 11.0软件（SYSTAT软件）学生的t检验统计学意义。 </li></ol> 4。验尸骨密度分析： <ol><li>继收购最终纵X射线扫描内IVIS Lumina的XR，小鼠，随后深深Beuthanasia（戊巴比妥75毫克/公斤）麻醉。一旦已经达到深麻醉，小鼠transcardially灌注冰冷的磷酸盐缓冲生理盐水heparain（40毫克/升），以抽血。 </li><li>一旦exsanguinated，消费都股骨。要特别小心删除尽可能多的软组织，这已被证明影响密度测量11。包装水浸湿纱布和存储的股骨- 20C，直到您准备对它们进行分析。 </li></ol> 5。使用一个Hologic公司QDR 4000骨密度DXA测量分析<ol><li>解冻纱布浸泡股骨骨密度套件和转让。 </li><li>校准设备制造商的协议，确保在可接受的限度内，BMC和BMD值下降。 </li><li>放置在机器的黄铜准直。这允许用户限制X射线束的大小和角度，把重点放在一个特定的目标。 </li><li>淹没到充满水（左髁，与股骨平行的床），位置只是激光的权利培养皿中解冻股骨。 </li><li>输入你要扫描的股骨的简历（一个描述性标题，其中包括动物识别信息，治疗等） 。 </li><li>输入扫描选择菜单，然后选择regionA高清晰度 。说明你的扫描参数：扫描地区2 × 0.7489英寸，0.01英寸的行距和0.00499英寸的点分辨率。 注：监控扫描，同时它是正在进行中。由于X射线束光栅扫描采样，监测，以确保有足够的水，在会议厅内，完全覆盖骨样品。 </li><li>分析选择菜单下输入分析 。按照提示突出的投资回报率作为整个股骨。报表页面会拿出计算的BMC（克）和BMD（GMS / 厘米 2） 。 </li><li>重复这些步骤，分析余下的股骨。 </li><li>整合到一个Excel工作表中的所有数据，并进行统计分析（t检验）。 </li></ol> 6。代表性的成果： 相对鼠标脊髓损伤后的胫骨和股骨骨密度的损失时，天真的小鼠相比采用上述方法检测。有后可检测骨质密度显著下降短短10天（12％，P &lt;0.0005），高达15％的骨骼密度在40天的损失（P &lt;0.0005，图4）。观察伤后40天（下降7％，P &lt;0.05，图5）在股骨骨密度损失。这些结果提供了证据使用的非侵入性的X射线成像的脊髓损伤后的骨质密度变化的纵向观察。 为了比较什么是当前可用的方法的疗效，我们分析这些小鼠切除股骨使用DXA的成像损伤后40天。图6可以看出，在数据输出的代表性。我们发现，有一个在SCI小鼠骨矿物质含量的显著损失相比，天真的（减少12％，P &lt;0.05，图7）。骨密度没有显著变化，但也遵循了类似的趋势（图8）。这些结果与文献中发现的;皮卡德等观察减少了13.5％，在BMC（P &lt;0.001），但没有显著DECR缓解骨密度（2008皮卡德）。 <img alt="图1" src="/files/ftp_upload/3246/3246fig1.jpg" /> 图1实验时间轴。 <img alt="图2" src="/files/ftp_upload/3246/3246fig2.jpg" /> 图2左后肢代表方向：一）照片和B），X射线。 <img alt="图3" src="/files/ftp_upload/3246/3246fig3.jpg" /> 图3。近端股骨和胫骨的地区内的投资回报率的尺寸和方向。 <img alt="图4" src="/files/ftp_upload/3246/3246fig4.jpg" /> 图4。脊髓损伤后的骨质密度在胫骨近端10，20，30和伤后40天的损失（N = 5）相比，年龄匹配naives（N = 5 ）。误差棒代表教育统筹局局长; ** P &lt;0.005，*** P &lt;0.0005。 <img alt="图5" src="/files/ftp_upload/3246/3246fig5.jpg" /> <st伤后10，20，30和40天荣&gt;图5。脊髓损伤后骨的密度在股骨近端的损失（N = 5）相比，年龄相匹配的naives（N = 5 ）。误差棒代表教育统筹局局长; * P &lt;0.05。 <img alt="图6" src="/files/ftp_upload/3246/3246fig6.jpg" /> 图6。脊髓损伤后骨的密度在伤后10，20，30和40天的股骨远端损失（N = 5）相比，年龄匹配天真对照组（n = 5 ）。误差棒代表教育统筹局局长; * P &lt;0.01-0.05; ** P &lt;0.001-0.01; *** P &lt;0.0001-0.001; **** P &lt;0.0001。 <img alt="图7" src="/files/ftp_upload/3246/3246fig7.jpg" /> 图7。DXA数据显示BMC和BMD输出的形象代表。 <img alt="图8" src="/files/ftp_upload/3246/3246fig8.jpg" /> 图8。DXA测量分析在脊髓损伤小鼠股骨的骨矿含量（克）伤后40天（N = 5），与年龄相匹配的naives（N = 5） 。误差棒代表教育统筹局局长; * P &lt;0.05。 <img alt="图9" src="/files/ftp_upload/3246/3246fig9.jpg" /> 图9在脊髓损伤小鼠股骨的骨密度DXA测量分析（毫克/厘米2）伤后40天（N = 5），与年龄相匹配的naives（N = 5） 。误差棒代表教育统筹局局长;无显着性差异。

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没有利益冲突的声明。

这项研究提出了一种新的非侵入性方法，评估范围内个人骨骼密度变化（胫骨和股骨）的纵向，在小鼠的单个队列，用卡尺IVIS Lumina的XR成像系统。 SCI的生产年龄相匹配的，没有受伤，对照组小鼠相比，超过40天的实验期间内spinally横断小鼠的单个队列研究时，在股骨和胫骨的骨质密度减少。股骨我们的研究结果证实，验尸，通过D​​XA的使用，表明卡钳仪器IVIS Lumina的XR的应用程序可以作为一个敏感的测量工具，用来评估SCI的长期和渐进的的影响骨质流失。 使用这个系统的调查的一个潜在好处是纵向遵循以下脊髓损伤的动物后肢骨骼病理变化发展的能力。一个单一的队列长的时间周期，以评估的能力提供了ST荣的优势，以研究者的条款：1）与动物购买和长期护理相关费用。取而代之的是研究需要大量的动物，需要在特定时间点牺牲，以评估时间依赖性变化，可以遵循这些病理结果在相同的队列超过同一时期，2）变异：除了受伤科目的单一队列内产生的结果比较与对照组群，可以评估和整个研究的程度相同的动物;再次降低内动物的变异以及需要较大的时间点之间比较，同伙的实验对象。 虽然Caliper的IVIS Lumina的XR我们提供关于脊髓损伤引起的骨质疏松症的发展具有重要的纵向信息，也有限制，应考虑：1），而这个平台，使我们能够生成高分辨率X射线DAT在以下脊髓损伤的小鼠后肢骨骼，其效用可能是有限的小型啮齿动物，如小鼠。内卢米纳XR允许的最大主体高度为2.8厘米。该高度以上的任何事情都不能成功使用的卢米纳XR组件X射线成像。在我们手中，这已经排除了我们的能力，研究老鼠在200-250克的重量。虽然它可能会根据这个重量范围或其他啮齿类动物如仓鼠或沙鼠的形象大鼠，这将需要额外的测试由PI。 2）联合的方向。这是至关重要的，调查人员精心安排，使成像会议的成功之间的比较轻松重现方向四肢。肢体位置的标准化将使研究者生成和应用标准化的投资回报率，可以保存和跨学科和时间应用。 整体而言，我们相信，IVIS Lumina的XR提供了一个极好的平台与模型在小鼠SCI依赖骨质疏松症的发展。骨质流失的纵向学习能力，在相同的鼠标队列将允许我们：1）更好地了解骨质流失和除盐的时间性质，脊髓损伤后，2）确定这些变化是否稳定随着时间的推移，3）探索，可用性转基因和空的鼠标线，学习能力，基本这些病理变化，在还原的方式，和4）的分子机制，很可能是最重要的优势，开始测试设计新颖的干预措施，以防止骨质疏松症在早期阶段的发展的伤害，或扭转这种变化一旦骨质疏松症已经开发。最后，Lumina的XR的，除了提供优良的X射线成像的潜力，也可以用来研究在动物活体发光和荧光信号。人们可以很容易地想象，结合本系统内提供的其他方式，来看待一个MULtitude因素决定骨动力学机制SCI或任何其他疾病的过程中，骨质疏松症的结果。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td> Beuthanasia </td><td>巴特勒施恩​​动物 健康</td><td> 001848 </td><td></td></tr><tr><td> Ketathesia（氯胺酮 盐酸盐） </td><td>巴特勒施恩​​动物 健康</td><td> 023061 </td><td></td></tr><tr><td>甲苯噻嗪</td><td>巴特勒施恩​​动物 健康</td><td> 037849 </td><td></td></tr><tr><td>布比卡因</td><td>巴特勒施恩​​动物 健康</td><td> 021801 </td><td></td></tr><tr><td>明胶海绵; 7MM </td><td> Fisher Scientific则</td><td> NC0085178 </td><td>为了促进 止血时 手术</td></tr><tr><td> IVIS Lumina的XR </td><td>卡尺生命科学</td><td> 135400 </td><td></td></tr><tr><td> ZFOV，2.5厘米领域 查看镜头</td><td>卡尺生命科学</td><td> 127285 </td><td>绝对必要的 到产生高 放大x光 图像骨 结构</td></tr><tr><td> QDR骨 密度仪</td><td> Hologic公司</td><td></td><td>模型采用不再 在生产</td></tr></tbody></table>

longitudinal evaluation of mouse hind limb bone loss after spinal cord injury using novel, in vivo, methodology

脊髓损伤（SCI）是由骨质疏松症常伴有骨盆和下肢sublesional地区，导致更高的频率的骨折1。由于这些骨折常发生在已经失去了正常的感觉功能的地区，患者的骨折依赖病症的风险更大，包括死亡。 SCI依赖的损失都骨密度（BMD，克/ 厘米 2）和骨矿含量（BMC，克）已被归因于废用机械 2，异常神经信号 3，荷尔蒙的变化 4 。脊髓损伤引起的骨质疏松症的啮齿类动物模型的使用可以提供宝贵以下脊髓损伤有关骨质疏松症的发展的内在机制，以及为新疗法的 5-7（8审查）代的测试环境。 SCI小鼠模型的极大兴趣，因为他们允许还原机制BA通过使用空和转基因小鼠的SED评估。虽然这种模式提供了重要数据，仍然是需要微创，可靠，重现性好，可量化的方法，在确定以下脊髓损伤的骨质流失的程度，特别是随着时间的推移，在相同的实验动物队列，提高诊断，治疗方法，和/或脊髓损伤引起的骨质疏松症的预防的。 在啮齿类动物的骨骼密度测量的理想方法，将允许多个连续（​​随着时间的推移）的风险，以低层次的X射线辐射。这项研究描述了一个新的整体动物IVIS Lumina的XR（卡尺仪器），可用于提供低能量（1-3 milligray（MGY））高清晰度，高倍率的X射线图像扫描仪，使用随着时间的推移以下脊髓损伤的小鼠后肢骨骼。看到明显的骨质密度的损失后脊髓横断10天没有受伤，年龄匹配的对照相比，在小鼠的胫骨（天真）小鼠（下降了13％，P &lt;0.0005）。在股骨远端骨质密度的损失 - SCI后第10天检测，而直到伤后40天（下降7％，P &lt;0.05）检测的密度在股骨近端的损失。 SCI依赖小鼠股骨密度的损失验尸报告证实，通过使用双能X射线骨密度仪（DXA），目前的“金标准”的骨质密度测量。在40天使用IVIS Lumina的XR - SCI公司后我们发现在小鼠股骨的BMC 12％的损失。这绝不逊色于以前报告的BMC的损失为13.5％，皮卡尔和他的同事用鼠标股骨验尸报告后30天- SCI 9 DXA测量分析。我们的研究结果表明，IVIS Lumina的XR提供了一种新颖，high-resolution/high-magnification鼠标在执行长期的纵向测量，后肢骨密度以下脊髓损伤的方法。

Watch this Scientific Journal Video about Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology at JoVE.com

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

在成年小鼠股骨和胫骨的纵向检查的骨质流失，脊髓损伤后使用连续低剂量X射线扫描。整个研究过程中胫骨损耗检测，直到检测到伤后40天，而在股骨的骨损失。

鼠标使用新型脊髓损伤的后肢骨质流失的纵向评价，在体内，方法论

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher ...

longitudinal-evaluation-mouse-hind-limb-bone-loss-after-spinal-cord

Research

JoVE Journal

Medicine

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

15.0K 次观看.  University of Texas Health Science Center at Houston. 在成年小鼠股骨和胫骨的纵向检查的骨质流失，脊髓损伤后使用连续低剂量X射线扫描。整个研究过程中胫骨损耗检测，直到检测到伤后40天，而在股骨的骨损失。

视频: Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

Acute and Chronic Tactile Sensory Testing after Spinal Cord Injury in Rats

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory testing in rat SCI models is needed. However, until recently, no single testing approach had been validated for SCI so that standardized methods have not been implemented across labs. Additionally, available testing methods could not be implemented acutely or when severe motor impairments existed, preventing studies of the development of SCI-induced allodynia3. Here we present two validated sensory testing methods using von Frey Hair (VFH) monofilaments which quantify changes in tactile sensory thresholds after SCI4-5. One test is the well-established Up-Down test which demonstrates high sensitivity and specificity across different SCI severities when tested chronically5. The other test is a newly-developed dorsal VFH test that can be applied acutely after SCI when allodynia develops, prior to motor recovery4-5. Each VFH monofilament applies a calibrated force when touched to the skin of the hind paw until it bends. In the up-down method, alternating VFHs of higher or lower forces are used on the plantar L5 dermatome to delineate flexor withdrawal thresholds. Successively higher forces are applied until withdrawal occurs then lower force VFHs are used until withdrawal ceases. The tactile threshold reflects the force required to elicit withdrawal in 50% of the stimuli. For the new test, each VFH is applied to the dorsal L5 dermatome of the paw while the rat is supported by the examiner. The VFH stimulation occurs in ascending order of force until at least 2 of 3 applications at a given force produces paw withdrawal. Tactile sensory threshold is the lowest force to elicit withdrawal 66% of the time. Acclimation, testing and scoring procedures are described. Aberrant trials that require a retest and typical trials are defined. Animal use was approved by Ohio State University Animal Care and Use Committee.

We describe two tactile sensory testing methods for acute or chronic periods of spinal cord injury in rats. These validated procedures can detect the development and maintenance of allodynia-like sensations.

在大鼠脊髓损伤后的急性和慢性触觉测试

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory ...

科研

医学

Controlled Cervical Laceration Injury in Mice

Use of genetically modified mice enhances our understanding of molecular mechanisms underlying several neurological disorders such as a spinal cord injury (SCI). Freehand manual control used to produce a laceration model of SCI creates inconsistent injuries often associated with a crush or contusion component and, therefore, a novel technique was developed. Our model of cervical laceration SCI has resolved inherent difficulties with the freehand method by incorporating 1) cervical vertebral stabilization by vertebral facet fixation, 2) enhanced spinal cord exposure, and 3) creation of a reproducible laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm in depth without associated contusion. Compared to the standard methods of creating a SCI laceration such as freehand use of a scalpel or scissors, our method has produced a consistent lesion. This method is useful for studies on axonal regeneration of corticospinal, rubrospinal, and dorsal ascending tracts.

A novel technique to create a reproducible in vivo model of cervical spinal cord laceration injury in the mouse is described. This technique is based on spine stabilization by fixation of the cervical facets and laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm.

控制宫颈裂伤损伤小鼠

Use of genetically modified mice enhances our  understanding of molecular mechanisms underlying several neurological disorders  such as a spinal cord ...

A Contusion Model of Severe Spinal Cord Injury in Rats

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is described to obtain a consistent model of severe SCI. Use of a stereotactic frame and computer controlled impactor allows for creation of reproducible injury. Hypothermia and urinary tract infection pose significant challenges in the post-operative period. Careful monitoring of animals with daily weight recording and bladder expression allows for early detection of post-operative complications. The functional results of this contusion model are equivalent to transection models. The contusion model can be utilized to evaluate the efficacy of both neuroprotective and neuroregenerative approaches.

A contusion model of severe spinal cord injury is described. Detailed pre-operative, operative and post-operative steps are described to obtain a consistent model.

挫伤模型脊髓严重损伤的保护作用

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is ...

A Novel Vertebral Stabilization Method for Producing  Contusive Spinal Cord Injury

Clinically-relevant animal cervical spinal cord injury (SCI) models are essential for developing and testing potential therapies; however, producing reliable cervical SCI is difficult due to lack of satisfactory methods of vertebral stabilization. The conventional method to stabilize the spine is to suspend the rostral and caudal cervical spine via clamps attached to cervical spinous processes. &#160;However, this method of stabilization fails to prevent tissue yielding during the contusion as the cervical spinal processes are too short to be effectively secured by the clamps (Figure 1). &#160;Here we introduce a new method to completely stabilize the cervical vertebra at the same level of the impact injury. &#160;This method effectively minimizes movement of the spinal column at the site of impact, which greatly improves the production of consistent SCIs. &#160;We provide visual description of the equipment (Figure 2-4), methods, and a step-by-step protocol for the stabilization of the cervical 5 vertebra (C5) of adult rats, to perform laminectomy (Figure 5) and produce a contusive SCI thereafter. &#160;Although we only demonstrate a cervical hemi-contusion using the NYU/MASCIS impactor device, this vertebral stabilization technique can be applied to other regions of the spinal cord, or be adapted to other SCI devices. &#160;Improving spinal cord exposure and fixation through vertebral stabilization may be valuable for producing consistent and reliable injuries to the spinal cord. &#160;This vertebral stabilization method can also be used for stereotactic injections of cells and tracers, and for imaging using two-photon microscopy in various neurobiological studies.

Vertebral stabilization is necessary for minimizing variability, and for producing consistent experimental spinal cord injuries.&#160; Using a customized stabilizing apparatus in conjunction with the NYU/MASCIS impactor device, we have demonstrated here the proper equipment and procedure for generating reproducible hemi-contusive cervical (C5) spinal cord injuries in adult rats.

一种新的椎稳定制备方法挫伤脊髓损伤

Clinically-relevant animal cervical spinal cord injury (SCI) models are essential for developing and testing potential therapies; however, producing ...

Evaluation of Respiratory Muscle Activation Using Respiratory Motor Control Assessment (RMCA) in Individuals with Chronic Spinal Cord Injury

During breathing, activation of respiratory muscles is coordinated by integrated input from the brain, brainstem, and spinal cord. When this coordination is disrupted by spinal cord injury (SCI), control of respiratory muscles innervated below the injury level is compromised1,2 leading to respiratory muscle dysfunction and pulmonary complications. These conditions are among the leading causes of death in patients with SCI3. Standard pulmonary function tests that assess respiratory motor function include spirometrical and maximum airway pressure outcomes: Forced Vital Capacity (FVC), Forced Expiratory Volume in one second (FEV1), Maximal Inspiratory Pressure (PImax) and Maximal Expiratory Pressure (PEmax)4,5. These values provide indirect measurements of respiratory muscle performance6. In clinical practice and research, a surface electromyography (sEMG) recorded from respiratory muscles can be used to assess respiratory motor function and help to diagnose neuromuscular pathology. However, variability in the sEMG amplitude inhibits efforts to develop objective and direct measures of respiratory motor function6. Based on a multi-muscle sEMG approach to characterize motor control of limb muscles7, known as the voluntary response index (VRI)8, we developed an analytical tool to characterize respiratory motor control directly from sEMG data recorded from multiple respiratory muscles during the voluntary respiratory tasks. We have termed this the Respiratory Motor Control Assessment (RMCA)9. This vector analysis method quantifies the amount and distribution of activity across muscles and presents it in the form of an index that relates the degree to which sEMG output within a test-subject resembles that from a group of healthy (non-injured) controls. The resulting index value has been shown to have high face validity, sensitivity and specificity9-11. We showed previously9 that the RMCA outcomes significantly correlate with levels of SCI and pulmonary function measures. We are presenting here the method to quantitatively compare post-spinal cord injury respiratory multi-muscle activation patterns to those of healthy individuals.

Evaluation of Respiratory Muscle Activation Using Respiratory Motor Control Assessment RMCA in Individuals with Chronic Spinal Cord Injury

The purpose of this publication is to present our original work on a multi-muscle surface electromyographic approach to quantitatively characterize respiratory muscle activation patterns in individuals with chronic spinal cord injury using vector-based analysis.

评价呼吸肌慢性脊髓损伤个人使用呼吸电机控制评估（RMCA）

During breathing, activation of respiratory muscles is coordinated by integrated input from the brain, brainstem, and spinal cord. When this coordination ...

In vivo Macrophage Imaging Using MR Targeted Contrast Agent for Longitudinal Evaluation of Septic Arthritis

Macrophages are key-cells in the initiation, the development and the regulation of the inflammatory response to bacterial infection. Macrophages are intensively and increasingly recruited in septic joints from the early phases of infection and the infiltration is supposed to regress once efficient removal of the pathogens is obtained. The ability to identify in vivo macrophage activity in an infected joint can therefore provide two main applications: early detection of acute synovitis and monitoring of therapy.
In vivo noninvasive detection of macrophages can be performed with magnetic resonance imaging using iron nanoparticles such as ultrasmall superparamagnetic iron oxide (USPIO). After intravascular or intraarticular administration, USPIO are specifically phagocytized by activated macrophages, and, due to their magnetic properties, induce signal changes in tissues presenting macrophage infiltration. A quantitative evaluation of the infiltrate is feasible, as the area with signal loss (number of dark pixels) observed on gradient echo MR images after particles injection is correlated with the amount of iron within the tissue and therefore reflects the number of USPIO-loaded cells.
We present here a protocol to perform macrophage imaging using USPIO-enhanced MR imaging in an animal model of septic arthritis, allowing an initial and longitudinal in vivo noninvasive evaluation of macrophages infiltration and an assessment of therapy action.

In vivo Macrophage Imaging Using MR Targeted Contrast Agent for Longitudinal Evaluation of Septic Arthritis

We demonstrate how to perform macrophage MR imaging using ultrasmall superparamagnetic contrast agent (USPIO) in septic arthritis, allowing an initial and longitudinal in vivo non-invasive evaluation of macrophages infiltration and an assessment of therapy efficacy.

在体内的巨噬细胞成像采用MR靶向造影剂化脓性关节炎的纵向评价

Macrophages are key-cells in the initiation, the development and the regulation of the inflammatory response to bacterial infection. Macrophages are ...

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

控制皮层影响模型创伤性脑损伤

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter connected to a radio transmitter into the femoral artery of rats that underwent a T4 spinal cord transection with or without grafting of embryonic brainstem-derived neural stem cells expressing green fluorescent protein. Compared to other methods such as cannula insertion or tail-cuff, telemetry is advantageous to continuously monitor blood pressure and heart rate in freely moving animals. It is also capable of long term multiple data acquisitions. In spinal cord injured rats, basal cardiovascular data under unrestrained condition and autonomic dysreflexia in response to colorectal distension were successfully recorded. In addition, cardiovascular parameters before and after SCI can be compared in the same rat if a transmitter is implanted before a spinal cord transection. One limitation of the described telemetry procedure is that implantation in the femoral artery may influence the blood supply to the ipsilateral hindlimb.

We present a protocol for using a radio-telemetric system to record cardiovascular parameters in T4 spinal cord transected rats eight weeks after embryonic brainstem neural stem cell grafting into the lesion site. Telemetry is an advanced technique to accurately evaluate cardiovascular function in conscious freely moving spinal cord injured rats.

无线电遥测系统，以监测大鼠脊髓横断和胚胎神经干细胞移植在心血管功能

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter ...

Calibrated Forceps Model of Spinal Cord Compression Injury

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The calibrated forceps model of compression injury is a convenient, low cost, and very reproducible animal model for SCI. We used a pair of modified forceps in accordance with the method published by Plemel et al. (2008) to laterally compress the spinal cord to a distance of 0.35 mm. In this video, we will demonstrate a dorsal laminectomy to expose the spinal cord, followed by compression of the spinal cord with the modified forceps. In the video, we will also address issues related to the care of paraplegic laboratory animals. This injury model produces mice that exhibit impairment in sensation, as well as impaired hindlimb locomotor function. Furthermore, this method of injury produces consistent aberrations in the pathology of the SCI, as determined by immunohistochemical methods. After watching this video, viewers should be able to determine the necessary supplies and methods for producing SCI of various severities in the mouse for studies on SCI and/or treatments designed to mitigate impairment after injury.

Spinal cord injury models should be highly reproducible. We demonstrate that the calibrated forceps compression model of spinal cord injury is an easy to use surgical method for generating reproducible injuries to the murine spinal cord.

脊髓压迫损伤的校准钳型

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The ...

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 ...