Name: unilateral pyramidotomy of the corticospinal tract in rats for assessment of neuroplasticity-inducing therapies
Uploaded: 2014-12-15T14:00:00.000+00:00
Duration: 8 min 41 s
Description: Watch this Scientific Journal Video about Unilateral Pyramidotomy of the Corticospinal Tract in Rats for Assessment of Neuroplasticity-inducing Therapies at JoVE.com

这项工作是由国际脊髓研究信托与信托Rosetrees支持。

伦理学声明:所有的程序均符合由英国内政部的指导方针和动物（科学程序）1986年法令。 1.手术和伤害注意:使用大鼠于220 g重的可以帮助术后恢复，如果医生发现的动物往往失去自己的术前体重的10％以上。 <ol><li>手术前，测试动物的前爪偏好与缸饲养试验15或蒙托亚staicase测试24。最好，切金字塔/科委对应的主导前爪。 注:对于前两天手术，将以下物品放入笼子，让老鼠熟悉，这将是从手术中恢复过程中有益的食物:水凝胶包，湿捣烂，和高热量的食物补充剂。可以从不同的供应商，这些食品补充剂无菌版本。 </li><li>麻醉女L存器罩住大鼠（诱导和维持5％1〜2％）中的氧，用异氟烷（在200和250g体重）（流速:1.5升/分钟）。注:麻醉剂的选择是很重要的。吸入麻醉剂允许更快调节麻醉深度大于氯胺酮，这也是常用的。异氟烷允许更好地控制呼吸频率和深度。麻醉剂的选择是异氟醚。注意:使用大鼠于220 g重的可以帮助术后恢复，如果医生发现的动物往往失去自己的术前体重的10％以上。 </li><li>辖卡洛芬皮下作为镇痛时将动物麻醉，以减轻炎症和提供镇痛。注:卡洛芬是在锥体束术根据我们的兽医的意见，给出我们的实验室。在手术过程中的阿片类药物可能会导致进一步的呼吸抑制。 </li><li>使用家庭手术过程中保持这些动物在37℃下吸热层系统和直肠温度探头。 </li><li>确认完全麻醉通过检查的爪子掐撤回反射和瞬目反射。 注:可选:为了对抗期间手术，气管插管和通风呼吸问题可能是可行的。然而，要在手术过程中被移位记住，气管的需要。 </li><li>一旦麻醉，将大鼠在仰卧位，剃腹侧颈部和用1％的氯己定擦拭和/或酒精擦拭消毒它。应用无菌手术毯在动物和保持区域无菌在任何时候。 </li><li>一旦麻醉，将大鼠在仰卧位，剃腹侧颈部和用1％的氯己定和酒精擦拭消毒它。应用无菌手术毯在动物和保持区域无菌在任何时候。 注意:任选的是，老鼠的头部可在手术过程中被固定在仰卧位置的立体框架来禁用头部， 即流动性枕骨。 </li><li>使从下巴一个2至3厘米长的中线切口，以使用无菌手术刀（＃10）胸骨几乎喙端。可选，适用于小斗牛犬夹收回皮肤。 </li><li>钝解剖组织的上层，如腺（ 如颌下腺和腮腺）和肌肉使用反向动作与钝剪刀和齿钳覆盖气管。始终保持对中线。该组织应该很容易分开。 </li><li>一旦气管露出，取代它向一侧。中线是两个白色的脂肪垫下方可见，标志着中线的喙结束。 </li><li>喙的白色脂肪垫，钝解剖组织，直到头骨（的basioccipital骨）的腹面为止。 </li><li>插入长齿拉钩（齿长度5.5毫米或16毫米）至保持移位到一侧（最多1厘米）的气管和头骨基部露出。气管位移OB结构呼吸。 注:对于一些动物，松开拉钩在多次手术中去除应变的气管， 例如 2分钟，每隔10分钟。钩拉钩可使用的长齿拉钩代替，以减少位移的应变。 </li><li>仔细烧灼小可见血管的喙去年底对喉部，避免出血。 注意:为了防止烧灼喉返神经血管精心拉离组织壁他们烧灼之前。 </li><li>调整显微镜的手术的其余部分。 </li><li>取下骨膜覆盖用细镊子的颅底。调整拉钩来实现对钻孔的最佳视图。 </li><li>感觉basioccipital骨细镊子的表面凹凸不平，并指出海拔在中线，它涵盖了基底动脉。吻侧，头骨变得提出一个轻微的凸起。 </li><li>根据哪一方病变需要，钻约为1mm横向到中侧运动的中线孔。 注:请记住，老鼠是在仰卧位，其左边可能是在调查右手边！ </li><li>一旦孔被制成，放大朝具有小垂直向上和向下-运动用钻头在中线直到基底动脉清晰可见和放大侧向至少2毫米。 </li><li>确保基底动脉清晰可见。金字塔可以通过其稍稍鼓出的形状（ 图2）和由接壤的血管来鉴定。 </li><li>用细镊子取出任何剩余的骨头碎片。 </li><li>申请一滴盐酸多沙普仑，呼吸兴奋剂，在舌头上。 </li><li>用26号针头纵向打开硬脑膜和细镊子横向到基底动脉。沉浸在脑脊液（CSF）和棉签的任何出血。 </li><li>避免仁州Ry的基底动脉，使切割大约1.5毫米宽，横跨金字塔为0.5mm垂直于基底动脉（ 图2）来中断CST纤维深与Vannas弹簧剪刀的宽度。 注:标记microscissors事先在0.5毫米的提示。一旦硬脑膜打开基底动脉可以稍微移动到一侧允许金字塔更好的访问。 </li><li>重复使用26号针头的尖端，以确保病变包括靠近所述基底动脉的纤维切断。 </li><li>停止与光压力棉签应用于出血。任选地，该切口可覆盖有明胶海绵。 </li><li>取出拉钩，更换组织，只缝合皮肤3-0薇乔缝线。 </li><li>手术后，保持动物在培养箱在32℃下，直到完全清醒和管理用5ml盐水皮下，如果需要的话。不要让动物无人值守。 </li><li>给Carporfen皮下作为抗炎和作为镇痛用于手术后疼痛1天术后。 </li><li>手术后至少一次之后，每周密切一周监控动物。 </li><li>至于做手术，地方水化凝胶包，泥婴儿食品，湿捣烂干州城笼地板上后需要。提供水瓶长嘴（缺少球轴承阀门）。 </li></ol>

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R., Soni, D., Irwin, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inosine+stimulates+extensive+axon+collateral+growth+in+the+rat+corticospinal+tract+after+injury.">Inosine stimulates extensive axon collateral growth in the rat corticospinal tract after injury.</a> Proc Natl Acad Sci U S A. 96, 13486-13490 (1999).</li><li>Zhou, L., Baumgartner, B. J., Hill-Felberg, S. J., McGowen, L. R., Shine, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophin-3+expressed+in+situ+induces+axonal+plasticity+in+the+adult+injured+spinal+cord.">Neurotrophin-3 expressed in situ induces axonal plasticity in the adult injured spinal cord.</a> J Neurosci. 23, 1424-1431 (2003).</li></ol>

作者什么都没有透露。

我们所描述的锥体束损伤模型。皮质脊髓束被切断在脑干的水平。从技术角度来看这个手术需要大量的谨慎性和准确性。脑干容纳呼吸中心和心血管中心中的多个核，如疑核和孤嵌入在金字塔下方的网状结构。如果切口是由过深，这可能导致突然的呼吸困难，这可能是致命的。我们给Dopram刚切，增加呼吸活动，以防止突然停止呼吸之前。如果呼吸停止，值得通风动物为以下五个分钟，因为它可能会恢复氧气。此外，应绝对避免在基底动脉的损害。在最初几天伤后的并发症有:脑干出血的另一个重要的影响作用，相当消瘦，严重的呼吸diffic由于大量的伤害，包括橄榄只是横向的金字塔，由于气管移位和动物的严重的方向和平衡问题ulties。这些并发症可以被看作端点和动物可能会受到人道杀害。在第一周期间密切监测手术后是必需的。补充食物可以以抗衡体重减轻给出。气道炎症（刺耳呼吸）在一到两天正常解析。一般情况下，年轻的动物更容易操作的是他们的脖子少深。此外，他们更弹性，较少有呼吸并发症。 它需要一些练习来达到CST完整的病变有锥体束。同样，在两项研究由管家等 16，其中规定了由Benowitz 等 28复制前的实验，最初报道的高死亡率，而他们的一些病变是不完整的。行为缺陷也将是可变的。我们建议一个新的外科医生进行很多实践锥体束手术进行任何全面的实验来测试候选人治疗前。如何防止不完全的病变追溯切用注射器针头，确保在本协议描述的皮质纤维接近基底动脉被切断。其他选项是如Zhou 等人，2003年29描述了一个倾斜的玻璃吸移液管吸移病灶部位。的细钨线，也可用于切割区域22。另一种为锥体束是削减颈椎C3背部CST单方面，然后切断与一个腹办法CST 18 ipsilesional C2腹侧部。但可以说，一个完整的病变是没有必要的，如果我们的目标是研究对侧CST发芽;如果大多数金字塔被损毁，并从未病变侧的发芽响应发生这可以是足够了。 的锥体束病变模型的一个目的是要烧蚀的皮质脊髓束的一个侧面，并使用神经可塑性诱导疗法刺激完整的非受伤纤维的可塑性和发芽。为了测试神经保护治疗这种模式是不适合和其他损伤模型，如挫伤受伤应该被使用。 pyramidotomies的成果进行评估最常使用的行为测试和解剖。水平阶梯测试，缸饲养试验和蒙托亚楼梯测试是最常用的行为评估。在文献中描述的其它试验是颗粒到达测试，爬绳试验，步态分析和胶带测试3,17-19。然而，大多数行为测试的结果往往只监测最多42天postlesion 3,4,11,17,18,20-22。从过去的经验，啮齿动物恢复大部分功能，在稍后的时间点未经任何处理的干预。我们已经表明塔吨的水平阶梯测试和蒙托亚楼梯颗粒达到试验检测残疾高达稍后的时间点。它们可以用于测量功能恢复因治疗。病变完整性被PKC伽玛染色（其标签在背柱CST纤维）脊髓延髓在宫颈级别或病变部位的横截面铬花青染色评判。生物素化的葡聚糖胺可注入的对侧皮层，以评估完整纤维穿过中线的发芽到去神经脊髓区域。 总之，锥体束模式是很好的评估，如果手术成功掌握了治疗药物中枢神经系统损伤的神经可塑性诱导能力。它评估的完整纤维的可塑性。下一个步骤将是使用一个更临床相关的模型如挫伤损伤评估，以克服更大injuri药物的能力ES影响多个大片。

皮质脊髓束（CST）是一个主要的机动车道内，在人类的脊髓。损伤后脊髓损伤造成相当大的损失灵巧此道。 CST是细电机运动在人类，如数字移动和远端肌肉的控制尤为重要。还发现在啮齿类虽然脊髓内其解剖学位置是不同的，人类的和CST损伤较少禁用1-4。 一种在体内 CST损伤模型涉及单侧锥体束，其中在腹侧脑干1棱锥被切断延髓到十字交叉（ 图1）。金字塔是在延髓的腹侧清晰的解剖特征，通过它的CST运行它decussates之前。在大鼠中，大多数纤维（约95％）交互对生在髓质的尾端和脊髓的背内侧部分此后找到。然而，approximat伊利10％decussated纤维在背外侧列中运行。纤维的剩余〜5％留不交叉同侧和旅行的腹内侧部分5,6。锥体束后脊髓失去其从对侧CST直接输入（ 图1）。 该锥体束模式是针对未病变CST的完好纤维的反应测试，以治疗特别好。它允许可塑性横跨中线的完整纤维在脊髓，其支配神经支配区和发芽的评估。皮质脊髓束已经观察到在脊髓损伤模型相当发芽并介导尤其前肢恢复7-10。一个可能的治疗目的是加强这方面的前肢恢复。一疗法的功效可与行为评估，所述未病变道的解剖追踪的技术和终端电生理实验-3,11,12-进行测试。&lt;/ P&gt; 相比，脊椎损伤的大多数其他临床前模型的优点在于，它特别影响一个道相对于其他脊椎损伤往往影响多个束。在一个明确的，肤浅的解剖位置只有一个道被损伤。另一个优点是，锥体束是一个可再现的损伤模型。它是一个合适的在体内的资源为研究人员测试潜在神经可塑性诱导疗法，在受伤或影响电机系统13,14病症之后恢复运动功能，其目的的效果。

<table><tbody><tr><td>Carprofen</td><td>Norbrook</td><td>Vm No; 02000/4229</td><td>give 5 mg/kg twice daily</td></tr><tr><td>Homeothermic Blanket System</td><td>Harvard Instruments</td><td>507222F</td><td></td></tr><tr><td>Isoflurane</td><td>Abbott</td><td>B506</td><td></td></tr><tr><td>Fine Scissors- Tough Cut</td><td>Fine Science Tools</td><td>14058-09</td><td></td></tr><tr><td>Forceps</td><td>Fine Science Tools</td><td>11019-12</td><td></td></tr><tr><td>Long-toothed Alm retractors</td><td>Fine Science Tools</td><td>17009-07</td><td></td></tr><tr><td>cautery system kit</td><td>Harvard Instruments</td><td>726067</td><td></td></tr><tr><td>fine Dumont forceps</td><td>Fine Science Tools</td><td>11251-10</td><td></td></tr><tr><td>Carbon Steel burrs</td><td>Fine Science Tools</td><td>19007-07</td><td></td></tr><tr><td>Dopram V-drops</td><td>Pfizer</td><td></td><td>apply 1 drop to tongue</td></tr><tr><td>Vannas Spring Scissors</td><td>Fine Science Tools</td><td>15000-03</td><td></td></tr><tr><td>gelfoam</td><td>Equimedical</td><td>EQU705001</td><td></td></tr><tr><td>3-0 Vicryl sutures</td><td>Ethicon</td><td></td><td></td></tr><tr><td>operating microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Compact Anaesthesia System Isoflurane K/F Single Gas</td><td>VetTech Solutions</td><td></td><td></td></tr><tr><td>Scales</td><td>Ohaus</td><td>NVT3201</td><td></td></tr><tr><td>Carbon Steel Scalpel blades No. 10</td><td>Swann-Morton</td><td>201</td><td></td></tr><tr><td>25g needles</td><td>Terumo</td><td>NN-2525R</td><td></td></tr><tr><td>syringes (1 ml and 5 ml)</td><td>Terumo</td><td>SS+01T1 / SS*05SE1</td><td></td></tr><tr><td>Saline (Sodium Chloride 0.9%)</td><td>Fresenius Kabi</td><td>Pl 08828/0178</td><td></td></tr><tr><td>cotton buds</td><td>Johnson and Johnson</td><td>5000207582502</td><td>sterilize before use</td></tr></tbody></table>

unilateral pyramidotomy of the corticospinal tract in rats for assessment of neuroplasticity-inducing therapies

皮质脊髓束（CST），可以完全在啮齿动物脑干的延髓金字塔单方面切断。 CST就是一个马达道具有用于在人中远端肌肉控制重视，并在较小程度上，在啮齿类动物。一个金字塔导致脊髓主要是脊髓导致短暂的电机残疾前肢的对侧的CST神经支配的损失单方面削减和持续灵巧的损失。皮质脊髓束的同侧预测是次要的。我们修订我们的手术方法，以增加损伤完整性的机会。我们描述术后护理。在蒙托亚楼梯颗粒达到测试与这里显示的水平梯试验赤字检测多达伤后8周。只检测瞬时在气缸饲养试验赤字。因此，气缸测试可能只适合于检测短期恢复。我们展示如何，电生理和解剖，可以评估病变和塑料的变化。我们还描述了如何分析跨中线萌到贫困地区从无恙CST纤维。它是具有挑战性的，以获得完整的&gt; 90％的病变始终由于靠近基底动脉在延髓和生存率可低。另一种手术方法和行为测试在本协议中的描述。的锥体束模型是评估神经可塑性诱导治疗，其中损伤后增加完整纤维的发芽的良好工具。

存活率。 在美国进行的一项代表性的研究，我们有16个出20名女性李斯特连帽大鼠（200〜250克）生存率。最常见的并发症是呼吸，因为气管位移或损坏在脑干的呼吸中枢的困难只是背侧到金字塔（核孤，暧昧和parabrachilis）。类似的结果已经在文献中报道16。 行为的结果。 的行为测试一系列已被用来评估锥体束在啮齿动物后感觉运动结果:在水平梯试验中，单粒料到达测试，缸饲养试验和蒙托亚楼梯测试是最常用的行为评估。在文献中描述的其它试验是颗粒到达测试，爬绳试验，步态分析和胶带测试3,17-19。大多数这些显示损伤后的功能性运动障碍1周。目前，在文献中描述的多数研究只能执行行为测试42天4,11,13,20,21，或者用于更短的时间3,18,22。我们已经进行行为测试用于在延长的时间周期和所看到的基本上完全恢复的马达功能后8周锥体束对某些行为测试的，除了蒙托亚楼梯测试和水平阶梯测试。这可能是有几个原因。首先，受伤CST喙病灶上可能会形成在脑干神经元充当间接中继到运动神经元在脊髓突触。其次，未受伤（健侧）CST可以发芽，形成突触在脑干或脊髓神经元作为继电器运动神经元。第三，其他备用电机大片，如网状脊髓，前庭或红核脊髓束可能会接管一些功能。因此，持久吨reatment效果难以检测与许多行为测试在锥体束模式。一些行为测试在这个模型然而可能的伤害，这是关系到一个处理后的早期指示加速恢复。 缸饲养试验 3,4,23 基于缸饲养试验对应的主导前爪的CST受伤。将大鼠置于一个有机玻璃圆筒和饲养行为是观察期为3分钟。伤前有轻微偏爱使用一种前爪，占主导地位的前爪，期间垂直探测（ 图3A）。伤病影响的主导方和使用对侧前爪下降到28％1周伤后（重复测量ANOVA P &lt;0.05;事后分析发现动物有在第1周，2和3，P &lt;0.05显著赤字， Fisher的LSD）。 4周伤后的恢复动物的ð使用受影响的前肢用于后轮的45％，和6周后，这是57％（n = 16的平均值±示出标准误差）。使用气缸试验锥体束后42天同样，斯达克等人（4）评估了小鼠和报告，在使用受影响的前爪在整个测试期间一个显著下降。 蒙托亚楼梯测试 24 只需要训练前本次测试之前伤害适应环境。要在测试环节动物增加的动力是食品限制在每鼠15克食物的前一天晚上。食物剥夺大鼠被放置在蒙托亚楼梯15分钟。在每一侧，有7个步骤与井，各含有3粒料楼梯。每侧检索粒料的总数被记​​录。检索颗粒与对侧/前爪受影响的人数从61％伤前显著下降至3％3天伤后（ 图3B）。检索粒料的能力仍然在第4周伤后显著障与检索粒料的20％。老鼠表现出显著赤字高达8星期只有29％吃在那个时间点的颗粒（重复测量ANOVA P &lt;0.05， 事后分析发现动物对对侧方显著赤字可言周，P &lt;0.05，Fisher的LSD）。所述ipsilesional前爪受到影响长达1周伤后;然而，这种复苏在第二周（重复测量ANOVA P &lt;0.05， 事后分析发现动物有3节和第1周显著赤字的ipsilesional前爪，P &lt;0.05，Fisher的LSD）。所述ipsilesional前爪的初始缺陷可能由皮质脊髓束通过切口金字塔运行，随后发芽和恢复的同侧部的损毁进行说明。 横梯形图测试25 只需要训练前进行此测试。只走三次在1米长的梯子，用不规则的横档间距。影片将在后面的慢镜头和脚滑倒，并为每个爪子失误总数分析量化。在图3C的曲线图表示未命中和受影响的和受影响较小侧（前爪+后爪），以百分比来采取步骤的总数的卡瓦的总数。老鼠有误差分别1周伤后用10％或患侧11％，受到的影响较小侧显著增加。这个赤字是持久的对侧/患侧多达8个星期10％的错误做，但是，2周解析ipsilesional /受影响较小的一侧（重复测量方差分析， 事后分析发现动物有显著赤字可言周在对侧方和显著赤字在1周和3对ipsilesional边p &lt;0.05，Fisher的LSD）。 电生理检测 在一个终端电的建立将动物麻醉腹膜内用尿烷。在整个过程中，它保持在37℃±1℃。其腹侧颈部区域，它的前肢和胸部区域被剃光和消毒用碘酒擦洗。如先前所述和桡神经暴露在上臂由腹侧方法切开皮肤和胸大肌金字塔暴露。桡神经的末端被切断，神经被放置在两个银线钩电极在矿物油浴中。任一金字塔，刺激与同心双极电极在不同深度具有5个脉冲以300Hz和增加刺激幅度。录音总是从禁用臂（ 图4）的桡神经进行。 没有受伤阿尼姆ALS有在延迟12至20毫秒之间在径向神经强烈反应时对侧金字塔进行刺激（ 图4B）。同侧的刺激只会导致偶尔的激活（ 图4C）。动物，其中有以前收到pyramidotomies，在对侧的手臂记录显示，在桡神经无反应时，损伤呼吸道的病变（ 图4B&#39;）以上的刺激。然而，CST纤维在金字塔的刺激同侧电极导致了桡神经更强活化相比未受伤的动物在第12周伤后（ 图4C&#39;）的记录。 解剖的结果。 动物根据动物附表1（科学程序）法1986年之前两周安乐死10周术后，未受伤的CST被曝与生物素葡聚糖胺（BDA）注 ​​射到运动皮层26同侧残疾人臂（ 图1）。这使得未受伤的纤维，它越过中线和供应对侧脊髓（ 图5C，D）的定量。未受伤的动物只有几个纤维横过中线，而这增加了响应于伤害。横颈脊髓切片用蛋白激酶C（PKC）伽马评估失神经支配的损伤CST（ 图5E）16的比例。可替代地，对病变的完整性可通过注入BDA示​​踪剂进入电动机皮质上的锥体束损伤ipsilesional侧进行评估。在对侧背内侧CST缺乏标签和同侧腹内侧CST提供单侧完全性横断证据。 <img alt="图1" fo:conte<html> NT-WIDTH ="4英寸"SRC ="/文件/ ftp_upload / 51843 / 51843fig1highres.jpg"WIDTH ="400"/&gt; 图1:示意图显示，在大鼠和科技委在金字塔单边干切断的皮质脊髓束顶部图像:皮质神经细胞起源于皮质层5锥体细胞。生物素化的葡聚糖胺（以绿色示出）之前两个星期的实验结束注入对侧皮层。中间的图像:该束上形成的髓质腹面并在spinomedullary结大多数轴突的交互对生的金字塔。单侧锥体束在尾髓质（显示为红色）进行。较低的图像:大多数脊髓的背内侧和背外侧部分向下运行的皮质脊髓束。少数保持同侧和运行ventromedially。 1843fig2highres.jpg"WIDTH ="500"/&gt; 图2:大鼠脑腹面观 。金字塔是与众不同的，因为他们提出，平行凸（黄框）。金字塔的边界是由基底动脉（BAS）通过中线和横向的凸中途基底动脉和动脉paraolivary（POL）之间的碱，（C）中可见内侧限定。开颅手术进行了基底动脉和横向延伸到paraolivary动脉（在C红色框）。一个金字塔垂直切到基底动脉（红线在C中，切口上示出在A和B试样）。因为CST开始交互对生，并因椎动脉被掀起中线放置太靠近尾端的椎动脉（垂直）病灶将是不完整的。 （C）图片由乔治Paxinos 27改编自"金鼠神经系统"。注:此图显示了图像的左手侧的病变:这是动物的钻机HT锥体束，因为它是仰卧。但是请注意，在视频显示病变的动物的左锥体束。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51843/51843fig3highres.jpg" width="500" /> 图3:在缸饲养试验代表性的成果，蒙托亚楼梯颗粒达到测试和水平梯形测试没有通过损伤后所有测试检测到电机赤字。然而，没有赤字可以与在第4周伤后气缸饲养试验来检测，而持续的赤字可以与蒙托亚楼梯测试和水平梯试验8周伤后（每组n =，平均值和标准误差16来检测示出）。星号表示P &lt;0.05相对于受伤前的基线（Fisher的LSD测试）。 <a href="https://www.jove.com/files/ftp_upload/51843/51843fig3highres.jpg" target="_blank">请点击</a>这里查看这个数字的放大版本。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51843/51843fig4highres.jpg" width="500" /> 图4:电评估皮质脊髓束和前锥体束后（A）示出的建立用于终端的实验。我们刺激金字塔与5脉冲喙损毁和十字交叉的部位不同深度的一列火车。红色系的损伤道。我们从上侧对侧桡神经记录。（B）和（C）表示实施例的记录与ipsilesional刺激或对侧刺激前或12周后锥体束。 Ipsilesional刺激诱 ​​发多种复合动作电位（在跟踪尖峰）的桡神经损伤（B）之前。这些是由损伤废除和活动不returñ在12周（B&#39;）。对侧刺激唤起了罕见的单一化合物动作电位伤前（C）。 12周伤活动后明显增加（C&#39;），这可能是由于完整的轴突横贯性脊髓内的中线和塑料变化发芽。 <a href="https://www.jove.com/files/ftp_upload/51843/51843fig4highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/51843/51843fig5highres.jpg" width="500" /> 图5:锥体束后解剖学评价（A，B，C，D）的生物素化葡聚糖胺被用于通过其注入相应于未病变CST运动皮层跟踪皮质脊髓束（见绿于图1）。背内侧，对CS的腹内侧部件T分别清晰可见（A，背内侧部分放大中的B）。（C）和（D）是纤维横跨中线发芽到失神经侧灰质响应于治疗的从（A）的放大倍数。为了评估宫颈级别4（12周伤后的组织）的病变横截面的完整性被切断，并用抗PKC伽玛（E）。科技委上的图像的左手侧是完好无损，可以看出在整个中线鼓出的受损背CST的结构崩解，大概是因为进入去神经侧。

Watch this Scientific Journal Video about Unilateral Pyramidotomy of the Corticospinal Tract in Rats for Assessment of Neuroplasticity-inducing Therapies at JoVE.com

Unilateral Pyramidotomy of the Corticospinal Tract in Rats for Assessment of Neuroplasticity-inducing Therapies

皮质脊髓束的主要感觉运动束之一，可以单方面地损伤在啮齿类脑干为了测试神经可塑性诱导疗法的中枢神经系统。此手术（"锥体束"）和术后评估本协议描述。

皮质脊髓束的大鼠单侧锥体束的神经可塑性诱导疗法的评估

The corticospinal tract (CST) can be completely severed unilaterally in the medullary pyramids of the rodent brainstem. The CST is a motor tract that has ...

unilateral-pyramidotomy-corticospinal-tract-rats-for-assessment

Research

JoVE Journal

Neuroscience

15.8K 次观看.  King's College London. 皮质脊髓束的主要感觉运动束之一，可以单方面地损伤在啮齿类脑干为了测试神经可塑性诱导疗法的中枢神经系统。此手术（"锥体束"）和术后评估本协议描述。 

视频: Unilateral Pyramidotomy of the Corticospinal Tract in Rats for Assessment of Neuroplasticity-inducing Therapies

A Novel Approach to Assess Motor Outcome of Deep Brain Stimulation Effects in the Hemiparkinsonian Rat: Staircase and Cylinder Test

Deep brain stimulation of the subthalamic nucleus is an effective treatment option for Parkinson&#39;s disease. In our lab we established a protocol to screen different neurostimulation patterns in hemiparkinsonian (unilateral lesioned) rats. It consists of creating a unilateral Parkinson&#39;s lesion by injecting 6-hydroxydopamine (6-OHDA) into the right medial forebrain bundle, implanting chronic stimulation electrodes into the subthalamic nucleus and evaluating motor outcomes at the end of 24 hr periods of cable-bound external neurostimulation. The stimulation was conducted with constant current stimulation. The amplitude was set 20% below the individual threshold for side effects. The motor outcome evaluation was done by the assessment of spontaneous paw use in the cylinder test according to Shallert and by the assessment of skilled reaching in the staircase test according to Montoya. This protocol describes in detail the training in the staircase box, the cylinder test, as well as the use of both in hemiparkinsonian rats. The use of both tests is necessary, because the staircase test seems to be more sensitive for fine motor skill impairment and exhibits greater sensitivity to change during neurostimulation. The combination of the unilateral Parkinson model and the two behavioral tests allows the assessment of different stimulation parameters in a standardized way.

Deep brain stimulation (DBS) is an effective treatment option for Parkinson's disease. We established a study design to screen novel stimulation paradigms in rats. The protocol describes the use of the staircase test and cylinder test for motor outcome assessment in DBS treated hemiparkinsonian rats.

一种新的方法，以评估在帕金森病大鼠脑深部电刺激效应的运动结果:楼梯和缸测试

Deep brain stimulation of the subthalamic nucleus is an effective treatment option for Parkinson&#39;s disease. In our lab we established a protocol to ...

科研

行为学

Preparing Undercut Model of Posttraumatic Epileptogenesis in Rodents

Partially isolated cortex ("undercut") is an animal model of posttraumatic epileptogenesis. The surgical procedure involves cutting through the sensorimotor cortex and the underneath white matter (undercut) so that a specific region of the cerebral cortex is largely isolated from the neighboring cortex and subcortical regions1-3. After a latency of two or more weeks following the surgery, epileptiform discharges can be recorded in brain slices from rodents1; and electrical or behavior seizures can be observed in vivo from other species such as cat and monkey4-6. This well established animal model is efficient to generate and mimics several important characteristics of traumatic brain injury. However, it is technically challenging attempting to make precise cortical lesions in the small rodent brain with a free hand. Based on the procedure initially established in Dr. David Prince's lab at the Stanford University1, here we present an improved technique to perform a surgery for the preparation of this model in mice and rats. We demonstrate how to make a simple surgical device and use it to gain a better control of cutting depth and angle to generate more precise and consistent results. The device is easy to make, and the procedure is quick to learn. The generation of this animal model provides an efficient system for study on the mechanisms of posttraumatic epileptogenesis.

Partially isolated cortex (&#x201C;undercut&#x201D;) is an efficient animal model of posttraumatic epileptogenesis. Here we demonstrate how to make a novel surgical device and use it to make more precise and consistent lesions to generate this model.

准备在鼠害外伤性癫痫的底切模型

Partially isolated cortex ("undercut") is an animal model of posttraumatic epileptogenesis. The surgical procedure involves cutting through the ...

神经科学

Repetitive Transcranial Magnetic Stimulation to the Unilateral Hemisphere of Rat Brain

Previous rodent models of repetitive transcranial magnetic stimulation (rTMS) adopted whole-brain stimulation instead of unilateral hemispheric rTMS, which is unlike the protocols used for human subjects. We report a successful application of rTMS to the unilateral hemisphere of rat brain. The rTMS was delivered with a low-frequency (1 Hz), high-frequency (20 Hz), or sham stimulation protocol to one side of the brain by using a small 25-mm figure-8 coil. We placed the center of the coil 1 cm lateral to the vertex on the biauricular line and angulated the coil 45&#176; to the ground to minimize a potential direct effect of rTMS on the contralateral cortex. We also used an in-house water cooling system to enable repetitive magnetic stimulation for more than 20 min, even at a 20-Hz stimulation frequency. Increases in the transcriptions of immediate early genes (Arc, Junb, and Egr2) were greater after rTMS than after sham stimulation. After 5 consecutive days of 20-min 1-Hz rTMS, bdnf mRNA expression was significantly higher in stimulated cortex than in contralateral side. The model presented herein will elucidate the molecular mechanisms of rTMS by allowing analysis of the inter-hemispheric difference in its effect.

We applied repetitive transcranial magnetic stimulation (rTMS) to the unilateral hemisphere of rat brain, by placing a 25-mm figure-8 coil 1 cm lateral to the vertex on the biauricular line and angulating the coil by 45&#176;. An in-house water cooling system was used for rTMS for more than 20 min.

重复经颅磁刺激对大鼠大脑的单边半球

Previous rodent models of repetitive transcranial magnetic stimulation (rTMS) adopted whole-brain stimulation instead of unilateral hemispheric rTMS, ...

The Knob Supination Task: A Semi-automated Method for Assessing Forelimb Function in Rats

Tasks that accurately measure dexterity in animal models are critical to understand hand function. Current rat behavioral tasks that measure dexterity largely use video analysis of reaching or food manipulation. While these tasks are easy to implement and are robust across disease models, they are subjective and laborious for the experimenter. Automating traditional tasks or creating new automated tasks can make the tasks more efficient, objective, and quantitative. Since rats are less dexterous than primates, central nervous system (CNS) injury produces more subtle deficits in dexterity, however, supination is highly affected in rodents and crucial to hand function in primates. Therefore, we designed a semi-automated task that measures forelimb supination in rats. Rats are trained to reach and grasp a knob-shaped manipulandum and turn the manipulandum in supination to receive a reward. Rats can acquire the skill within 20 &plusmn; 5 days. While the early part of training is highly supervised, much of the training is done without direct supervision. The task reliably and reproducibly captures subtle deficits after injury and shows functional recovery that accurately reflects clinical recovery curves. Analysis of data is performed by specialized software through a graphical user interface that is designed to be intuitive. We also give solutions to common problems encountered during training, and show that minor corrections to behavior early in training produce reliable acquisition of supination. Thus, the knob supination task provides efficient and quantitative evaluation of a critical movement for dexterity in rats.

This manuscript describes a semi-automated task that quantifies supination in rats. Rats reach, grasp, and supinate a spherical manipulandum. The rat is rewarded with a pellet if the turn angle exceeds a criterion set by the user. This task increases throughput, sensitivity to injury, and objectivity compared to traditional tasks.

旋钮旋任务: 一种半自动评估大鼠前肢功能的方法

Tasks that accurately measure dexterity in animal models are critical to understand hand function. Current rat behavioral tasks that measure dexterity ...

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical insult to secondary injury processes, including apoptosis and inflammation. Animal modeling has been valuable in the search to unravel injury mechanisms and evaluate potential neuroprotective therapies. This protocol describes the controlled cortical impact (CCI) model of focal, open-head TBI. Specifically, parameters for producing a mild unilateral cortical injury are described. Behavioral consequences of CCI are analyzed using the adhesive tape removal test of bilateral sensorimotor integration. Regarding experimental therapy for TBI pathology, this protocol also illustrates a process for transplanting cultured cells into the brain. Neural cell cultures derived from human induced pluripotent stem cells (hiPSCs) were chosen for their potential to show superior functional restoration in human TBI patients. Chronic survival of hiPSCs in the host mouse brain tissue is detected using a modified DAB immunohistochemical process.

This protocol demonstrates methodologies for a mouse model of open-skull traumatic brain injury and transplantation of cultured human induced pluripotent stem cell-derived cells into the injury site. Behavioral and histologic tests of outcomes from these procedures are also described in brief.

人类诱导多能干细胞衍生神经细胞治疗移植小鼠脑损伤控制皮质影响模型

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical ...

发育生物学

Performing Permanent Distal Middle Cerebral with Common Carotid Artery Occlusion in Aged Rats to Study Cortical Ischemia with Sustained Disability

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, obesity and diabetes. Accordingly, when evaluating therapies for stroke in animals, it is important to select a model with excellent face validity. Ischemic stroke accounts for 80% of all strokes, and the majority of these occur in the territory of the middle cerebral artery (MCA), often inducing infarcts that affect the sensorimotor cortex, causing persistent plegia or paresis on the contralateral side of the body. We demonstrate in this video a method for producing ischemic stroke in elderly rats, which causes sustained sensorimotor disability and substantial cortical infarcts. Specifically, we induce permanent distal middle cerebral artery occlusion (MCAO) in elderly female rats by using diathermy forceps to occlude a short segment of this artery. The carotid artery on the ipsilateral side to the lesion was then permanently occluded and the contralateral carotid artery was transiently occluded for 60 min. We measure the infarct size using structural T2-weighted magnetic resonance imaging (MRI) at 24 hr and 8 weeks after stroke. In this study, the mean infarct volume was 4.5% &#177; 2.0% (standard deviation) of the ipsilateral hemisphere at 24 hr (corrected for brain swelling using Gerriet&#8217;s equation, n = 5). This model is feasible and clinically relevant as it permits the induction of sustained sensorimotor deficits, which is important for the elucidation of pathophysiological mechanisms and novel treatments.

Here we present a protocol to produce permanent distal middle cerebral artery occlusion in elderly female rats with simultaneous occlusion of the carotid arteries to generate large cortical infarcts and sustained deficits. We show confirmation of the lesion size using structural MRI at 24 hr and 8 weeks after stroke.

执行永久远端大脑中与老年大鼠颈总动脉闭塞研究皮层缺血具有持续残疾

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, ...

医学

Rodent Behavioral Testing to Assess Functional Deficits Caused by Microelectrode Implantation in the Rat Motor Cortex

Medical devices implanted in the brain hold tremendous potential. As part of a Brain Machine Interface (BMI) system, intracortical microelectrodes demonstrate the ability to record action potentials from individual or small groups of neurons. Such recorded signals have successfully been used to allow patients to interface with or control&#160;computers, robotic limbs, and their own limbs. However, previous animal studies have shown that a microelectrode implantation in the brain not only damages the surrounding tissue but can also result in functional deficits. Here, we discuss a series of behavioral tests to quantify potential motor impairments following the implantation of intracortical microelectrodes into the motor cortex of a rat. The methods for open field grid, ladder crossing, and grip strength testing provide valuable information regarding the potential complications resulting from a microelectrode implantation. The results of the behavioral testing are correlated with endpoint histology, providing additional information on the pathological outcomes and impacts of this procedure on the adjacent tissue.

We have shown that a microelectrode implantation in the motor cortex of rats causes immediate and lasting motor deficits. The methods proposed herein outline a microelectrode implantation surgery and three rodent behavioral tasks to elucidate potential changes in the fine or gross motor function due to implantation-caused damage to the motor cortex.

大鼠运动皮质微电极植入所致功能缺损的啮齿动物行为检测

Medical devices implanted in the brain hold tremendous potential. As part of a Brain Machine Interface (BMI) system, intracortical microelectrodes ...

生物工程

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

大鼠慢性经颅电刺激及 Intracortical 记录

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

Spinal Cord Lateral Hemisection and Asymmetric Behavioral Assessments in Adult Rats

Incomplete spinal cord injury (SCI) often leads to impairments of sensorimotor functions and is clinically the most frequent type of SCI. Human Brown-S&#233;quard syndrome&#160;is a common type of incomplete SCI caused by a lesion to one half of the spinal cord which results in paralysis and loss of&#160;proprioception&#160;on the same (or ipsilesional) side as the injury, and loss of pain and temperature sensation on the opposite (or contralesional) side. Adequate methodologies for producing a spinal cord lateral hemisection (HX) and assessing neurological impairments are essential to establish a reliable animal model of Brown-S&#233;quard syndrome. Although lateral hemisection model plays a pivotal role in basic and translational research, standardized protocols for creating such a hemisection and assessing unilateralized function are lacking. The goal of this study is to describe step-by-step procedures to produce a rat spinal lateral HX at the 9th thoracic (T9) vertebral level. We, then, describe a combined behavior scale for HX (CBS-HX) that provides a simple and sensitive assessment of asymmetric neurological performance for unilateral SCI. The CBS-HX, ranging from 0 to 18, is composed of 4 individual assessments which include unilateral hindlimb stepping (UHS), coupling, contact placing, and grid walking. For CBS-HX, the ipsilateral and contralateral hindlimbs are assessed separately. We found that, after a T9 HX, the ipsilateral hindlimb showed impaired behavior function whereas the contralateral hindlimb showed substantial recovery. The CBS-HX effectively discriminated behavioral functions between ipsilateral and contralateral hindlimbs and detected temporal progression of recovery of the ipsilateral hindlimb. The CBS-HX components can be analyzed separately or in combination with other measures when needed. Although we only provided visual descriptions of the surgical procedures and behavioral assessments of a thoracic HX, the principle may be applied to other incomplete SCIs and at other levels of the injury.

Here we describe surgical procedures to produce a reliable spinal cord lateral hemisection (HX) at the 9th thoracic level in adult rats and neurobehavioral assessments designed for detecting asymmetric deficits after such a unilateral injury.

成年大鼠脊髓侧性横断与不对称行为评估

Incomplete spinal cord injury (SCI) often leads to impairments of sensorimotor functions and is clinically the most frequent type of SCI. Human ...

Multifactorial Assessment of Motor Behavior in Rats after Unilateral Sciatic Nerve Crush Injury

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In addition, in the research field of movement disorders, sciatic crush injury has been employed to trigger a dystonia-like phenotype in genetically predisposed DYT-TOR1A rodent models of dystonia. To achieve consistent, reproducible and comparable results after a sciatic nerve crush injury, a standardized method for inducing the nerve crush is essential, in addition to a standardized phenotypical characterization. Attention must be paid not only to the specific assortment of behavioral tests, but also to the technical requirements, the correct execution and consecutive data analysis. This protocol describes in detail how to perform a sciatic nerve crush injury and provides a behavioral test battery for the assessment of motor deficits in rats that includes the open field test, the CatWalk XT gait analysis, the beam walking task, and the ladder rung walking task.

We provide a protocol for the assessment of motor behavior via a behavioral test battery in rats after sciatic nerve crush injury.

单边坐骨神经粉碎损伤后大鼠运动行为的多因素评估

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In ...