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 border="0" cellpadding="0" cellspacing="0" width="1154">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:397px;">Name of Reagent/ Equipment</td>
			<td style="width:229px;">Company</td>
			<td style="width:229px;"></td>
			<td style="width:299px;">Comments/Description</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:397px;">External Fixator Hoffman 3</td>
			<td style="width:229px;">Stryker, Kalamazoo, USA</td>
			<td style="width:229px;"></td>
			<td>Modular system used to build the custom made 3D frame and the jointed arm holding the ultrasound probe</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:397px;">Toshiba Applio</td>
			<td style="width:229px;">Toshiba, Tokyo, Japan</td>
			<td style="width:229px;"></td>
			<td>Ultrasound machine</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:397px;">Sonovue</td>
			<td style="width:229px;">Bracco, Milan, Italy</td>
			<td style="width:229px;"></td>
			<td>Contrast agent : microbubbles</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:397px;">Vueject pump</td>
			<td style="width:229px;">Bracco, Milan, Italy</td>
			<td style="width:229px;"></td>
			<td>Electric pump for infusion of microbubbles bolus</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:397px;">Aquasonic Ultrasound Gel</td>
			<td style="width:229px;">Parker Laboratories, Fairfield, NJ, USA</td>
			<td style="width:229px;"></td>
			<td>Ultrasound gel used to transmit the ultrasound waves</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:397px;">Isovet</td>
			<td style="width:229px;">Piramal Healthcare, Mumbai, India</td>
			<td style="width:229px;"></td>
			<td>Isoflurane used for anesthesia</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:397px;">Ultra Extend</td>
			<td style="width:229px;">Toshiba, Tokyo, Japan</td>
			<td style="width:229px;"></td>
			<td>Software used for quantification of spinal cord blood flow</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:397px;">Mastercraft Five-piece Mini-pliers Set, Product #58-4788-6</td>
			<td style="width:229px;">Canadian Tire, Toronto, Canada</td>
			<td style="width:229px;"></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

13.9K 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

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 ...

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 ...

Implanting Glass Spinal Cord Windows in Adult Mice with Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as multiple sclerosis. Here we present a low-cost and reproducible glass window implantation protocol that is suitable for intravital microscopy and studying the dynamics of spinal cord cytoarchitecture with subcellular resolution in live adult mice with EAE. Briefly, we surgically expose the vertebrae T12-L2 and construct a chamber around the exposed vertebrae using a combination of cyanoacrylate and dental cement. A laminectomy is performed from T13 to L1, and a thin layer of transparent silicone elastomer is applied to the dorsal surface of the exposed spinal cord. A modified glass cover slip is implanted over the exposed spinal cord taking care that the glass does not directly contact the spinal cord. To reduce the infiltration of inflammatory cells between the window and spinal cord, anti-inflammatory treatment is administered every 2 days (as recommended by ethics committee) for the first 10 days after implantation. EAE is induced only 2-3 weeks after the cessation of anti-inflammatory treatment.
Using this approach we successfully induced EAE in 87% of animals with implanted windows and, using Thy1-CFP-23 mice (blue axons in dorsal spinal cord), quantified axonal loss throughout EAE progression. Taken together, this protocol may be useful for studying the recruitment of various cell populations as well as their interaction dynamics, with subcellular resolution and for extended periods of time. This intravital imaging modality represents a valuable tool for developing therapeutic strategies to treat autoimmune demyelinating diseases such as EAE.

We describe a method for implanting and maintaining glass windows over the exposed spinal cords of adult mice for studying experimental autoimmune encephalomyelitis. These windows provide chronic optical access to the spinal cord for monitoring cell dynamics at the subcellular level in live animals using two-photon microscopy.

玻璃植入脊髓的Windows在成年小鼠实验性自身免疫性脑脊髓炎

Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as ...

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 ...

Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical Spinal Cord Injury

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. Clinically, more than 50% of SCI affect the cervical spine1. As a consequence of the primary injury, a cascade of secondary mechanisms including inflammation, apoptosis, and demyelination occur finally leading to tissue scarring and development of intramedullary cavities2,3. Both represent physical and chemical barriers to cell transplantation, integration, and regeneration. Therefore, shaping the inhibitory environment and bridging cavities to create a supportive milieu for cell transplantation and regeneration is a promising therapeutic target4. Here, a contusion/compression model of cervical SCI using an aneurysm clip is described. This model is more clinically relevant than other experimental models, since complete transection or ruptures of the cord are rare. Also in comparison to the weight drop model, which in particular damage the dorsum columns, circumferential compression of the spinal cord appears advantageous. Clip closing force and duration can be adjusted to achieve different injury severity. A ring spring facilitates precise calibration and constancy of clip force. Under physiological conditions, synthetic self-assembling peptides (SAP) self-assemble into nanofibers and thus, are appealing for application in SCI5. They can be injected directly into the lesion minimizing damage to the cord. SAPs are biocompatible structures erecting scaffolds to bridge intramedullary cavities and thus, equip the damaged cord for regenerative treatments. K2(QL)6K2 (QL6) is a novel SAP introduced by Dong et al.6 In comparison to other peptides, QL6 self-assembles into &#946;-sheets at neutral pH6.14 days after SCI, after the acute stage, SAPs are injected into the center of the lesion and neural precursor cells (NPC) are injected into adjacent dorsal columns. In order to support cell survival, transplantation is combined with continuous subdural administration of growth factors by osmotic micro pumps for 7 days.

Treating cervical spinal cord injury with both self-assembling peptides (SAP) and neural precursor cells (NPC), together with growth factors, is a promising approach to promote regeneration and recovery. A contusion/compression aneurysm clip rat model of cervical SCI and combined treatment involving SAP injection and NPC transplantation is established.

在实验颈脊髓损伤协同使用神经前体细胞，并自组装肽的

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

在脊髓损伤的实验挫伤型号神经干细胞移植

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

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 ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

新生儿小鼠脊髓压迫损伤模型

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...