Name: contrast enhanced ultrasound imaging for assessment of spinal cord blood flow in experimental spinal cord injury
Uploaded: 2015-05-07T14:00:00.000+00:00
Duration: 9 min 19 s
Description: Watch this Scientific Journal Video about Contrast Enhanced Ultrasound Imaging for Assessment of Spinal Cord Blood Flow in Experimental Spinal Cord Injury at JoVE.com

We acknowledge Stephanie Gorgeard, Thierry Scheerlink (Toshiba France), and Christophe Lazare (Bracco France).

注:在本手稿中描述的方法被批准由医学，巴黎，法国（CEEALV / 2011-08-01）的拉里布瓦西埃学院的生物伦理委员会。 1.仪器准备<ol><li>准备和清理的导管插入以下工具:微型镊子，微型剪刀，微细血管钳，剪刀大，手术线（黑色编织丝绸4-0）和地下14导尿管。 Heparinize用肝素溶液（5,000单位/毫升）的导管。 </li><li>准备和清理的椎板以下工具:剪刀大，手术刀和骨刀。用一个定制的骨刀，旨在减少椎板（ 图1）期间损害脊髓的风险执行椎板切除术。 </li><li>设置用于定位的动物的和稳定的三维帧。定做的框架是建立与外部固定器霍夫曼3在相关的镊子的元素，WHICH与被弯曲，以适应动物的腰椎。 </li><li>准备用于脊髓损伤生物力学的重量降装置（冲击）。 注:定制嵌塞设备的设计采用三维软件和打印3D。 </li><li>打开超声机上。 </li><li>制备的试剂盒，所述造影剂的重建。 注:该套件包括1瓶含有25mg冻干粉末，含有5ml氯化钠和一个微型尖峰转移系统（ 图2）1预填充注射器。详述的步骤的造影剂的重构下面（在第5）。 </li></ol> 2.颈内静脉穿刺置管（图3） <ol><li> 麻醉 ，用4％的异氟醚的动物。发生在仰卧位动物。确认正确麻醉，确保动物没有响应时，爪子都捏了钳子。适用兽医药膏眼睛，防止干燥而UND呃麻醉。 </li><li>剃颈部和清洁皮肤。使在颈部的中线切口。缩回sternocleidomastoidian肌肉，以便找到颈内静脉。拧紧一个连字在静脉的喙部。 </li><li>上静脉，1厘米以下结扎申请微血管钳。通过另一个线程周围的静脉，刚刚与结随时可以收紧时，夹子松开夹子下面。 </li><li>打开静脉（venotomy）钳和吻端结扎之间的墙。在静脉腔内引入地下14导管将其推向心脏。 </li><li>当它碰到夹紧，松开后者与导管推进一步。固定导管的静脉，紧紧拧紧结上与导管内静脉。 </li><li>通过抽出，在导管少量的静脉血，并随后再与肝素化盐水冲洗它评估导管的通畅。这可以防止阻塞的catheter一个潜在的血块。 </li><li>连接软管的导管进一步注入造影剂（微泡）的。保持它关闭（密封），直到准备使用。 </li></ol> 3.访问的脊柱，椎板和鼠定位（在3D帧） <ol><li>将动物在一个平面俯卧水平位置。剃并清洁动物背部（胸部区域）。 </li><li>通过触诊（ 图4）确定的最后一个肋（在大鼠的第十三次）。这允许人们估计第十三次胸椎（ThXIII）的位置。 </li><li>使4cm的皮肤切口上的中线，集中在ThXIII。打开皮肤切口，以及底层的囊中。观察背部肌肉腱膜以及椎脊柱过程的提示。 </li><li>通过仔细触诊肋骨第十三次定位ThXIII脊柱过程。 注:第十三次肋骨连接到ThXIII，因此代表易失水TE解剖标志为ThXIII鉴定。这一步使ThXII的本地化THIX棘突，以及L1和L2（第一和第二腰椎椎）。 </li><li>切开肌肉腱膜和分离在任一侧上的肌肉在棘突的椎板和小关节从THIX暴露到L2。通过从横突分离肌肉暴露L1和L2的横向方面。 </li><li>钩的三维帧以固定位置（ 图5）对动物的门牙。夹紧L1和L2椎体与改性钳子。连接改性镊子三维帧，以稳定的动物。 </li><li>轻轻拉动尾部保持腰椎的镊子，以加强整个脊椎和提升从板凳上胸部。 注意:使用上述结构的动物应该能够呼吸。此外，尽管肋骨，脊柱和脊髓的呼吸运动线也应该保持不动。 </li><li>取出棘突processess从THIX到ThXII。轻轻插入骨刀的下刃ThXII的左椎板下方，然后关闭该骨刀，以切割薄片（ 图6）。 </li><li>重复同样的动作为右椎板和删除先后后弓。重复前面的步骤为椎体ThXI到THIX以实现四级椎板。每个椎骨取出两个小关节。 注意:在整个过程中，清洁手术视野从局部出血。对于这一点，用棉签和灌溉用温水盐水。止血系统发生在几分钟之内。 </li></ol> 4. CEU探头定位<ol><li>覆盖硬脑膜超声波凝胶。这允许有效传输探针和脊髓（ 图7）之间的超声波。 </li><li>稳定的超声探头机智公顷钳可随后连接到3D-帧由一个铰接臂。手动定位探头。确保探头导向，以获得纵向斜切片矢状位。在一个正确的位置，脊髓是在图像上严格水平和脊髓的中央管是沿着脊髓的全段可见。 注:定位应由超声波机的屏幕上显示的实时B模式图像被引导。超声波探头的焦距应与脊髓中央管对齐。此时，脊髓的后方面是可访问的，这将最终允许冲击器的定位。 </li><li>当最佳，锁定铰接臂以稳定的位置。 </li></ol> 5.准备造影剂 - 微泡重建<ol><li>使用商业重构试剂盒的内容，并通过紧固它氩弧焊连接的柱塞杆htly到注射器（顺时针）。打开传送系统水疱并取下注射器尖端帽。打开传送系统帽和连接注射器输送系统（系紧）。 </li><li>取下小瓶保护磁盘。小瓶滑入的透明套</li><li>传输系统并按牢牢锁定小瓶到位。 </li><li>清空注射器的内容物到小瓶通过推动柱塞杆上。剧烈振荡20秒以混合所有的内容在小瓶，得到白色乳白色均相液体。 </li><li>倒置制度和退出仔细造影剂进入注射器。拧下传输系统中的注射器。重建（指示）后，将1ml所得分散体包含8微升六氟化硫的微泡。绘制微泡的悬浮液加入100毫升注射器。插入100毫升注射器进入电动泵。盖上盖子。 </li><li>开始重新的恒定的搅拌构成的微泡。得到恒定的搅拌用注射器，它保持了微泡悬浮液的缓慢旋转。通过柔性管连接泵的颈静脉导管。超声机器设置为"谐波模式"。 注:后者对应于其中微气泡可以特异性检测和可视化的方式。该模式具有低机械指数，其不破坏微泡，而不是B模式。 </li><li>通过注入造影剂的第一剂（400微升）吹扫导管。在这第一次输液，检查微气泡真的出现在超声屏幕上。这证实了整个电路（从注射器到老鼠的血液）是完整和开放的。 </li><li>超声机设定为"B模式"形象化脊髓实质和残留在血流中少数微泡的破坏。的"B型"的高频移植史密茨高能量的微泡，这让他们崩溃。 </li><li>让动物静静地躺着大约30分钟。此期间允许血液动力参数的稳定化。 </li></ol>在完整的脊髓6.评估的SCBF <ol><li>超声机器设置为"谐模式"。同时开始（1）输注造影剂（400微升）和（2）时计的。 注:在输注，微泡在血液中的浓度应增加，使脊髓（ 图8）的对比度想象。由于微气泡被迅速破坏，微泡的血液浓度开始下降，一旦注射完成，其产生在脊髓的对比度可视逐渐降低。 </li><li> 1分钟后，选择（按）超声机上的"剪辑保存"按钮。这将使一个保存的r 1分钟AW超声数据及摄像录像（被超声屏幕上先前显示）。 </li><li>超声机设定为"B模式"。这将消除剩余的微泡。 </li></ol> 7.实验SCI <ol><li>使用连接到3D-帧的显微，定位重锤落下撞击装置，使冲击器的前端开始与硬脑膜（对脊髓中线）接触，在THX和ThXI之间的交界处（ 图9） 。 注意:此程度应以与超声装置观察到脊髓的段的中间。撞针和撞击的主体是直径8mm。撞击的尖端，这将产生伤害，为3毫米的直径。 </li><li>将冲击装置的前锋在10厘米高的位置。通过释放撞击器的前锋诱导实验SCI。这名前锋下降，释放日Ë冲击，伤及脊髓。所述定制嵌塞提供相当于10克重的冲击，从10cm的高度落下。 </li></ol> SCBF 5分钟后SCI的评估8. <ol><li>重复在第6（SCBF的评估）描述的步骤。微泡将无法穿过受损微血管和损伤震中将保持暗（ 图10）。 </li></ol> 9.牲<ol><li>安乐死的动物与腹腔注射死刑戊巴比妥（100毫克）。 </li></ol> 10.定量SCBF通过离线分析<ol><li>启动用于定量（超声波机）的超扩展软件。选择" 文件 "，然后选择之前保存的原始数据，并打开相关文件。按（选择）了" 志Q"按钮，激活" 量化模式 "。塞莱CT" 设置的投资回报率 "（按钮），选择圆形。 </li><li>选择"Draw投资回报率 "（按钮）和脊髓（ 图11）画的兴趣7相邻的圆形区域（ROI）。打开菜单" 装修 "，并选择功能" 价值曲线 "。观察显示多个曲线的软件，每一个对应于微泡浓度的ROI内的更改。 注:每条曲线有一个"灌注deperfusion"轮廓。曲线的第一阶段是平坦的且对应于微泡的到来之前的期间。在第二阶段中，微泡的浓度迅速增加作为输液的结果。在第三阶段，该阶段开始时，输注完成后，微泡的浓度逐渐降低，因为它们在血液中disintegratse。 </li><li>将第一条垂直线的C的第二阶段的开始urve并选择" 设置 "。该通知从哪里开始分析软件。 </li><li>将第二垂直线在记录结束，并再次选择" 设定 "。该通知软件在哪里停下分析。 </li><li>看看"CV"菜单并记录"AUC"值，其对应于" 曲线下面积 "进行分析。该值正比于相应的ROI内的SCBF。 </li></ol>

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The authors declare that they have no competing financial conflict of interest. The ultrasound machine was graciously lent by the Toshiba France company. The Vueject pump was graciously lent by the Bracco France company.

尽管我们已经描述了如何使用CEU在大鼠脊髓挫伤模型，该协议可以被修改，以适应其他的实验的目标或SCI模型。我们选择（损伤，15分钟后的SCI之前）来衡量SCBF只在两个时间点，然而时间点的数目和SCBF测量之间的延迟可以适于满足其他研究的需要。例如，在我们以前的工作如图17所示 ，我们已经测量SCBF在五个连续的时间点在整个第一小时后的SCI。值得注意的是，假手术组（无SCI）中，我...

外伤性脊髓损伤（SCI）是一种破坏性的条件，导致电机显著障碍，感觉和自治职能。迄今为止，还没有治疗已显示出它在患者的效率。对于这样的原因，以确定新的技术，这将提高潜在的治疗方法的评估，并可以进一步阐明损伤pathiophysiology 1是非常重要的。 SCI被分为两个连续的阶段，称为初级和继发性损伤。原发性损伤对应于最初的机械损伤。而继发性损伤团的各种生物事件（如炎症，氧化应激和缺氧）的级联进一步向初始损伤，组织损伤，因此，神经功能缺损2,3的逐渐扩大。 在脊髓损伤的急性期，神经保护疗法旨在减少继发性损伤的病理和sh乌尔德从而提高神经系统的结果。在众多的继发性损伤的事件，缺血起着至关重要的作用4,5。在SCI震中的水平，受损的脑实质微血管阻碍了有效的脊髓血流量（SCBF）。此外，SCBF也显著在围绕损伤震中，具体被称为"局部缺血半影区"的区域中的区域减小。如果SCBF不能迅速这些区域内恢复，局部缺血可导致补充实质坏死和进一步的神经组织损伤。因为即使是轻微的组织保存可以有功能的重大影响，这是主要的兴趣来开发药物和治疗方法，可以减少缺血后SCI。为了突出这一现象，以前的工作表明，只能保存10％髓鞘的轴突就足以使走在猫后SCI 6。 虽然有几种方法已被描述，以评估SCBF，所述Ÿ都有显著的局限性。例如，使用放射性微球7,8-和C14-iodopyrine放射自显影9需要后续动 ​​物牺牲并不能重复在稍后的时间点。氢气清除法10取决于脊柱内电极，其可进一步损伤脊髓的插入。而激光多普勒成像，光电容积描记14,15 和体内光镜16具有测量11-13的一个非常有限的深度/面积。 我们的团队此前曾表明，超声造影（CEU）成像，可用于评估实时体内大鼠脊髓实质17 SCBF变化。要注意的是类似的技术是由Huang 等人施加是很重要的。在脊髓18的猪模型。 CEU应用于超声成像的具体方式，它允许灰度形态IM关联年龄的血流19的空间分布（通过常规的B模式获得）。该SCBF成像和定量依赖于血管内注射回声造影剂。造影剂是由六氟化硫微泡（平均直径约2.5微米，90％的具有直径小于6微米）由磷脂稳定。微泡反映从而增强血液回声和组织根据其血流量增大对比度探头发射的超声波束。因此，能够根据反射信号的强度，以评估在感兴趣的特定区域的血液流动。微泡也安全的，他们已经在人类中被临床应用。六氟化硫被快速清除（平均终末半衰期为12分钟）和80％以上的施用六氟化硫被内注射后2分钟回收在呼出的空气。该协议提供了使用IM CEU一个简单的方法老化，以评估在大鼠SCBF变化。

<table><tbody><tr><td>External Fixator Hoffman 3</td><td>Stryker, Kalamazoo, USA</td><td></td><td>Modular system used to build the custom made 3D frame and the jointed arm holding the ultrasound probe</td></tr><tr><td>Toshiba Applio</td><td>Toshiba, Tokyo, Japan</td><td></td><td>Ultrasound machine</td></tr><tr><td>Sonovue</td><td>Bracco, Milan, Italy</td><td></td><td>Contrast agent : microbubbles</td></tr><tr><td>Vueject pump</td><td>Bracco, Milan, Italy</td><td></td><td>Electric pump for infusion of microbubbles bolus</td></tr><tr><td>Aquasonic Ultrasound Gel</td><td>Parker Laboratories, Fairfield, NJ, USA</td><td></td><td>Ultrasound gel used to transmit the ultrasound waves</td></tr><tr><td>Isovet</td><td>Piramal Healthcare, Mumbai, India</td><td></td><td>Isoflurane used for anesthesia</td></tr><tr><td>Ultra Extend</td><td>Toshiba, Tokyo, Japan</td><td></td><td>Software used for quantification of spinal cord blood flow</td></tr><tr><td>Mastercraft Five-piece Mini-pliers Set, Product #58-4788-6</td><td>Canadian Tire, Toronto, Canada</td><td></td><td>Set of pliers for Do-it-yourself job</td></tr></tbody></table>

contrast enhanced ultrasound imaging for assessment of spinal cord blood flow in experimental spinal cord injury

降低脊髓血流量（SCBF）（ 即缺血）起着外伤性脊髓损伤（SCI）的病理生理关键作用，并进行相应的治疗神经保护的一个重要目标。虽然有几种方法已被描述，以评估SCBF，它们都具有显著局限性。克服后者，我们提出使用实时超声造影成像（CEU）。在这里，我们描述这种技术在脊髓损伤的大鼠模型挫伤的应用程序。甲颈静脉导管，首先注入在重复注射造影剂，六氟化硫包封的微泡氯化钠溶液。脊柱然后稳定与定做的三维帧和脊髓硬膜通过椎板切除术在THIX-ThXII露出。超声波探头然后定位在硬膜（涂有超声凝胶）的后方面。为了评估禁忌基线SCBF，单次静脉注射（400微升）圣剂施加穿过完好脊髓微血管记录其通过。的重降装置随后被用于生成SCI的可重复实验挫伤模型。造影剂重新注入15分钟以下的伤害，以评估后SCI SCBF变化。 CEU允许实时体内的变化SCBF以下脊髓损伤的评估。在未受伤的动物，超声造影显示沿完好的脊髓不均匀血流量。此外，15分钟后SCI，有缺血的关键在震中的水平，而在SCBF较偏远地区的完好保存依然。在相邻的震中（既延髓和尾部）的区域，SCBF被显著降低。这对应于先前描述的"局部缺血半影区"。这个工具是用于评估疗法旨在限制缺血和随后的SCI所产生的组织坏死的影响重大利益的。

与上面描述的协议，有可能映射沿纵向脊髓矢状段SCBF。 在完整的脊髓，似乎有实质（ 图12）内SCBF不规则性。这可以通过radiculo髓动脉变量分布（RMA）的从一个动物进行说明到另一个。 RMA指节段性即到达脊髓前动脉（ASA），因此提供血液供应脊髓实质内动脉。与此相反，根性动脉对应节段性动脉，这没有达到ASA和因此不提供脊髓血液供应。因此，在脊髓节段所在?...

Watch this Scientific Journal Video about Contrast Enhanced Ultrasound Imaging for Assessment of Spinal Cord Blood Flow in Experimental Spinal Cord Injury at JoVE.com

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

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 ...

contrast-enhanced-ultrasound-imaging-for-assessment-spinal-cord-blood

Research

JoVE Journal

Medicine

14.0K 次观看.  Faculté de Médecine Paris Diderot Paris VII, U942. 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.

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

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

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 ...

行为学

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

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.

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 ...

Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) below the site of injury. The secondary ischemia that develops following the initial mechanical insult is a serious complication of the SCI and severely impairs the function and viability of surviving neuronal and non-neuronal cells in the SC. In addition, ischemia is also responsible for the growth of lesion during chronic phase of injury and interferes with the cellular repair and healing processes. Thus there is a need to develop a spinal cord ischemia model for studying the mechanisms of ischemia-induced pathology. Focal ischemia induced by photothrombosis (PT) is a minimally invasive and very well established procedure used to investigate the pathology of ischemia-induced cell death in the brain. Here, we describe the use of PT to induce an ischemic lesion in the spinal cord of mice. Following retro-orbital sinus injection of Rose Bengal, the posterior spinal vein and other capillaries on the dorsal surface of SC were irradiated with a green light resulting in the formation of a thrombus and thus ischemia in the affected region. Results from histology and immunochemistry studies show that PT-induced ischemia caused spinal cord infarction, loss of neurons and reactive gliosis. Using this technique a highly reproducible and relatively easy model of SCI in mice can be achieved that would serve the purpose of scientific investigations into the mechanisms of ischemia induced cell death as well as the efficacy of neuroprotective drugs. This model will also allow exploration of the pathological changes that occur following SCI in live mice like axonal degeneration and regeneration, neuronal and astrocytic Ca2+ signaling using two-photon microscopy.

Photothrombosis is a minimally invasive and highly reproducible procedure to induce focal ischemia in the spinal cord and serves as a model of spinal cord injury in mice.

Photothrombosis引起的局部脑缺血脊髓损伤的小鼠模型

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) ...

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 ...

Disruption of the Blood-Spinal Cord Barrier Using Low-Intensity Focused Ultrasound in a Rat Model

Low-intensity focused ultrasound (LIFU) uses ultrasonic pulsations at lower intensities than ultrasound and is being tested as a reversible and precise neuromodulatory technology. Although LIFU-mediated blood-brain barrier (BBB) opening has been explored in detail, no standardized technique for blood-spinal cord barrier (BSCB) opening has been established to date. Therefore, this protocol presents a method for successful BSCB disruption using LIFU sonication in a rat model, including descriptions of animal preparation, microbubble administration, target selection&#160;and localization, as well as BSCB disruption visualization and confirmation. The approach reported here is particularly useful for researchers who need a fast and cost-effective method to test and confirm target localization and precise BSCB disruption in a small animal model with a focused ultrasound transducer, evaluate the BSCB efficacy of sonication parameters, or explore applications for LIFU at the spinal cord, such as drug delivery, immunomodulation, and neuromodulation. Optimizing this protocol for individual use is recommended, especially for advancing future preclinical, clinical, and translational work.

Disruption of the blood-spinal cord barrier (BSCB) can be successfully achieved with the intravenous administration of microbubbles and the application of low-intensity focused ultrasound (LIFU). This protocol details the opening of the BSCB using LIFU in a rodent model, including equipment setup, microbubble injection, target localization, and BSCB disruption visualization.

在大鼠模型中使用低强度聚焦超声破坏血脊髓屏障

Low-intensity focused ultrasound (LIFU) uses ultrasonic pulsations at lower intensities than ultrasound and is being tested as a reversible and precise ...

生物工程

Analysis of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage with High Frequency Transcranial Duplex Ultrasound

Cerebral vasospasm that occurs in the weeks after subarachnoid hemorrhage, a type of hemorrhagic stroke, contributes to delayed cerebral ischemia. A problem encountered in experimental studies using murine models of SAH is that methods for in vivo monitoring of cerebral vasospasm in mice are lacking. Here, we demonstrate the application of high frequency ultrasound to perform transcranial Duplex sonography examinations on mice. Using the method, the internal carotid arteries (ICA) could be identified. The blood flow velocities in the intracranial ICAs were accelerated significantly after induction of SAH, while blood flow velocities in the extracranial ICAs remained low, indicating cerebral vasospasm. In conclusion, the method demonstrated here allows functional, noninvasive in vivo monitoring of cerebral vasospasm in a murine SAH model.

The aim of this manuscript is to present a sonography-based method that allows in vivo imaging of blood flow in cerebral arteries in mice. We demonstrate its application to determine changes in blood flow velocities associated with vasospasm in murine models of subarachnoid hemorrhage (SAH).

高频颅内双体超声波的苏巴拉赫诺德出血的穆林模型脑血管痉挛分析

Cerebral vasospasm that occurs in the weeks after subarachnoid hemorrhage, a type of hemorrhagic stroke, contributes to delayed cerebral ischemia. A ...

Real-Time Assessment of Spinal Cord Microperfusion in a Porcine Model of Ischemia/Reperfusion

Spinal cord injury is a devastating complication of aortic repair. Despite developments for the prevention and treatment of spinal cord injury, its incidence is still considerably high and therefore, influences patient outcome. Microcirculation plays a key role in tissue perfusion and oxygen supply and is often dissociated from macrohemodynamics. Thus, direct evaluation of spinal cord microcirculation is essential for the development of microcirculation-targeted therapies and the evaluation of existing approaches in regard to spinal cord microcirculation. However, most of the methods do not provide real-time assessment of spinal cord microcirculation. The aim of this study is to describe a standardized protocol for real-time spinal cord microcirculatory evaluation using laser-Doppler needle probes directly inserted in the spinal cord. We used a porcine model of ischemia/reperfusion to induce deterioration of the spinal cord microcirculation. In addition, a fluorescent microsphere injection technique was used. Initially, animals were anesthetized and mechanically ventilated. Thereafter, laser-Doppler needle probe insertion was performed, followed by the placement of cerebrospinal fluid drainage. A median sternotomy was performed for exposure of the descending aorta to perform aortic cross-clamping. Ischemia/reperfusion was induced by supra-celiac aortic cross-clamping for a total of 48 min, followed by reperfusion and hemodynamic stabilization. Laser-Doppler Flux was performed in parallel with macrohemodynamic evaluation. In addition, automated cerebrospinal fluid drainage was used to maintain a stable cerebrospinal pressure. After completion of the protocol, animals were sacrificed, and the spinal cord was harvested for histopathological and microsphere analysis. The protocol reveals the feasibility of spinal cord microperfusion measurements using laser-Doppler probes and shows a marked decrease during ischemia as well as recovery after reperfusion. Results showed comparable behavior to fluorescent microsphere evaluation. In conclusion, this new protocol might provide a useful large animal model for future studies using real-time spinal cord microperfusion assessment in ischemia/reperfusion conditions.

Spinal cord microcirculation plays a pivotal role in spinal cord injury. Most methods do not allow real-time assessment of spinal cord microcirculation, which is essential for the development of microcirculation-targeted therapies. Here, we propose a protocol using Laser-Doppler-Flow Needle probes in a large animal model of ischemia/reperfusion.

猪缺血/再灌注模型中脊髓微灌注的实时评估

Spinal cord injury is a devastating complication of aortic repair. Despite developments for the prevention and treatment of spinal cord injury, its ...

Laser Doppler Needle Probe Insertion: A Technique for Real-time Quantification of Blood Flow within the Spinal Cord in a Porcine Model

Source: Behem, C. R. et al., <a href="https://www.jove.com/v/62047">Real-Time Assessment of Spinal Cord Microperfusion in a Porcine Model of Ischemia/Reperfusion.</a> J. Vis. Exp. (2020).
This video demonstrates the placement of a laser Doppler needle probe in the spinal cord of a porcine model. This helps in the continuous quantification of blood supply to the spinal cord, an important assessment in spinal cord injury.

激光多普勒针探针插入:一种在猪模型中实时量化脊髓内血流的技术

Source: Behem, C. R. et al., Real-Time Assessment of Spinal Cord Microperfusion in a Porcine Model of Ischemia/Reperfusion. J. Vis. Exp. (2020).
This ...