Name: photothrombosis-induced focal ischemia as a model of spinal cord injury in mice
Uploaded: 2015-07-16T15:00:00.000+00:00
Duration: 8 min 27 s
Description: Watch this Scientific Journal Video about Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice at JoVE.com

这项工作是由美国国立卫生研究院[格兰特没有支持。 R01NS069726]和美国心脏协会的资助以资助格兰特[格兰特没有。 13GRNT17020004]到SD。

注:小鼠（C57BL / 6J，男）岁 10 - 19周后在该研究中使用。所有的程序均按照美国国立卫生研究院指南实验动物的护理和使用执行和批准了密苏里州机构动物护理和使用委员会的大学（IACUC）。 1.手术前<ol><li>手术前的高压釜一天消毒所有的手术器械。裹在121℃下的仪器和高压灭菌15磅30分钟，接着干燥（121℃，15磅，30/30循环）的30分钟。将在清洁无菌的环境仪器，直到进一步使用。 </li><li>准备新鲜的玫瑰红（RB）溶液（20毫克/毫升无菌生理盐水）在手术前的每一次。以完全溶解的RB，涡流管再接着超声处理5分钟，在50/60赫兹为19 W.裹管在铝箔和保护其不受光输出功率，直至河畔期间进一步使用盖瑞。 </li><li>制备氯胺酮/甲苯​​噻嗪的混合物在无菌盐水中。添加125微升甲苯噻嗪（原液浓度:20毫克/毫升）和325微升ketmaine的（原液浓度:100毫克/毫升）和550微升无菌生理盐水，使1毫升麻醉剂混合物的最终体积。 </li><li>预暖恒温加热垫。 </li><li>预温的金属卤化物灯（光源FN1落射荧光显微镜）处理30分钟，以稳定的灯功率。 </li><li>调整照明区域的大小的直径的使用10X物镜和一个分划板通过调整视场光阑的直立FN1落射荧光显微镜1毫米。 </li></ol> 2.手术过程<ol><li>麻醉小鼠用氯胺酮（130毫克/千克B.重量）和甲苯噻嗪的剂量（10毫克/千克B.重量）。基于鸡尾酒在1.3，每克鼠标重量B. 4微升。是必要的。消毒注射部位用酒精棉球，并通过帧内施用麻醉剂-peritoneal（IP）的路由。小心不要将麻醉药注入到血管或肌肉，因为这会耽误动物的诱导和恢复。 </li><li>应用人工泪液软膏鼠标的双眼，以防止干燥，然后将动物在加热垫，以防止体温过低。 </li><li>动物的制备<ol><li>通过脚趾捏响应检查动物进行适当的手术麻醉的水平。 </li><li>一旦动物已经达到麻醉手术水平，使用的是电动毛发修剪器夹上围绕动物的中线的背侧表面上的毛发。擦洗手术部位用70％乙醇，随后通过优碘溶液三次。遮盖的部位用无菌手术悬垂至下一个步骤。 </li></ol></li><li>手术瘦骨暴露脊髓<ol><li>鼠标放置在通过外科手术平台（ 图1A）上的恒温加热垫俯卧姿势。正确保护鼠标使用吻钳姿势维持一个细长颈区（ 图1A，B）。 </li><li>做一个切口（约1厘米长）使用沿着从胸椎到T9 T12延长背中线手术剪。走到一边的皮肤暴露手术区。 </li><li>使用手术刀，仔细清除肌肉，露出背刺在T9 - T12椎体。停在每一步运用所用无菌棉签轻压止血。单独T10 - T12椎骨从周围的肌肉和使用椎夹具来稳定和防止任何移动它们固定（ 图1A，B）。 </li><li>使用高速钻骨抛光钻头，小心翼翼地轻轻薄T10 T11或脊椎的背部表面可视化的SC（ 图1C）的背水面后脊柱静脉和其他小型船只。 </li><li>为了防止热损伤是由于减薄过程期间产生的热，应用生理盐水的温柔络绎不绝随着不断的吸取出碎片。 </li><li>使用解剖刀小心平滑骨表面，直到主容器是清晰可见。注意不要在此过程中损伤脊髓。 </li><li>一旦血管被可视化，管理的RB在通过眶后窦路线使用胰岛素注射器的剂量为30毫克/公斤（体重）。 </li><li>使用激光多普勒流量计3分钟后以下的RB注射测量血流量，如果必要（ 图2A，B）。在保持整个过程的无菌。 </li></ol></li></ol> 3.感应PT的<ol><li>将动物在XY位置可调级过一个实验室千斤顶可调节高度。调整鼠标的位置，从而T11脊髓的暴露区域是直属FN1落射荧光显微镜（ 图3A）的10倍的目标。 </li><li>设置里的力量向右源在12％，并与在减薄脊髓的中间直径为0.75mm照射T11区域（注:此区域包括后脊柱静脉和其他毛细血管）与绿色光（波长540 - 580纳米，这是通过在显微镜过滤立方体），通过10倍的目标2分钟完成。拍摄图像的开头和照射端（ 图3B，C）和在此点记录实验的时间。 </li><li>再次测量血流量为10分钟，如果必要通过将激光多普勒探针的相同位置的脊髓上面在2.4.8（ 图2A，B）。 </li><li>照射检查是否有出血及后若没有发现进行缝合动物。缝合浅筋膜连同使用可吸收的缝合线或4-0大小丝线对脊髓的任一侧上的肌肉。小心不要破坏暴露SC。缝合用4-0丝线缝合皮肤。应用聚维酮碘或碘到缝合后皮肤的边缘。 </li></ol> 4.手术后护理<ol><li>缝合后，放置在加热垫进行恢复的动物。恢复后通过观察两个后肢肢体运动检查动物的神经功能缺损的迹象。不要让动物无人看管，直到它已经恢复了意识足以维持胸骨斜卧。 </li><li>将动物转移到饲养笼。不返回已经历手术给其他动物的公司，直到完全恢复的动物。 </li><li>检查动物定期。在情况严重的神经功能障碍，提供适当的照顾就像疏散膀胱，止痛药的管理（丁丙诺啡，0.05 - 0.1毫克/千克）。检查脱水和管理生理盐水皮下在严重的情况。一般，丁丙诺啡（0.1毫克/千克）将缝合，以减轻疼痛，在外科手术部位后给药。 </li><li>如果动物是不能立即手术后安乐死，我们会放在笼地板含水量高的饮食，让动物能轻易达到的食物。 </li></ol> 5.穿心灌注，尼氏染色和免疫染色<ol><li> Transcardially灌注动物如前面所述17-20。 <ol><li>麻醉动物在协议中前面描述和用磷酸盐缓冲盐水（PBS，pH 7.4）中，随后冰冷的4％多聚甲醛（PFA）的PBS transcardially灌注。 </li><li>灌注后，取出脊髓（SC）和后修复它在4％PFA的PBS在4°CO / N。传送固定​​的SC到PBS中，用30％蔗糖，并保持它为2 - 3天，直到它下沉到管的底部。 </li><li>使用低温恒温器切断脊髓成30微米厚的切片并顺序将它们放置在明胶包被的载玻片或在一个48孔板用0.01M PBS中。 </li></ol></li><li>尼氏染色:要检查所造成的PT伤害进行尼氏staini纳克上脊髓切片如前所述17-20。 <ol><li>简单地说，收集玻片五分之一的脊髓切片和染色用0.25％甲酚紫。取染色的切片（ 图4）的图像。 </li></ol></li><li>免疫组化:正如先前使用浮动部分的方法17,18,20描述。 <ol><li>兔抗的NeuN抗体（1:300），和兔抗:简单地说，通过将O / N在4℃下用兔抗神经胶质纤维酸性蛋白（GFAP）多克隆抗体（300 1）染色脊髓切片Iba1抗体（1:500），随后加入驴抗兔的Alexa 568缀合的抗体（1:400），用于在室温下4小时的二次抗体。采取用荧光显微镜（ 图5）的图像。 </li></ol></li></ol>

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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+chemokine+release+supports+a+bimodal+mechanism+of+spinal+cord+ischemia+and+reperfusion+injury.">The evolution of chemokine release supports a bimodal mechanism of spinal cord ischemia and reperfusion injury.</a> Circulation. 126 (11 Suppl 1), S110-S117 (2012).</li><li>Jia, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+in+spinal+cord+injury+and+antioxidant-based+intervention.">Oxidative stress in spinal cord injury and antioxidant-based intervention.</a> Spinal Cord. 50 (4), 264-274 (2012).</li></ol>

The authors have nothing to disclose.

在这项研究中，我们描述了SC缺血的光化学模型。由于进展遗传工程一直存在于市售的转基因小鼠的激增已经使得有可能研究涉及在SC缺血病理生理学特异性基因的影响。这项研究的目的是开发脊髓缺血可重现小鼠模型。在这里，我们适应皮质PT型号诱导脊髓损伤的小鼠。手术后脊柱静脉和毛细血管对小鼠的背侧处的T11胸椎水平被暴露。然后RB，市售的光敏染料，通过眶后窦路线被注入以达到所希望的血管分布。暴露的血管，然后由绿色光照射来诱导血栓的和以后的梗塞形成。我们从组织学和免疫组化方法结果显示，PT诱导脊髓和reacti梗死已经胶质增生在梗死周围区域。神经功能缺损像后肢麻痹也观察到。这些数据表明，PT是一个合适的模型，SCI后研究病理生理学和细胞死亡的机制。在协议中的关键步骤是使用一个高速钻至椎骨的薄的表面上的SC的背表面上的血管的可视化。这一步应该认真执行作为应用程序的过剩压力可能导致钻头进入脊髓腔和损坏SC。另一方面，不均匀变薄可能导致不当的照明，并且可以产生不规则的梗塞。为了解决这个问题，在显微镜下骨的钻孔的每个短步骤后的表面的频繁检查建议评估骨的厚度，并进一步评估使用钻头。使用无菌盐水的建议被洗出的碎片以及用于更好的可视化的暴露表面。维护利弊在整个手术过程坦无菌，适当的术后护理的动物可以改善动物的生存和增加实验的成功率。 我们当前的PT的模型不需要购买任何昂贵的仪器，作为配备有一个落射荧光显微镜用光源任何实验室（如汞灯，金属卤化物灯，或488纳米波长的激光）就可以执行此过程。此外，这种技术提供了在梗塞通过调整孔径大小的尺寸控制相对于其他的SC缺血模型，如主动脉的组合闭塞，左锁骨和乳内动脉25和改性主动脉阻断法26是复杂的，是非常侵入。在我们的模型高速钻椎体进行可视化的薄背表面被选为替代椎板切除，方法的选择，许多实验室诱导脊髓损伤。椎板涉及这可能导致过度出血由于椎体血管横切和这可能掩盖场成像椎骨的切割。即使一些协议建议使用棉签椎板切除可能导致压缩过程中，以清除出血过多可能造成额外的伤害SC。进一步脊髓的暴露表面能来与血液及其组分以及切割骨头，可以增加不必要的变异性，以实验的锋利的边缘直接接触。使用当前的PT模型，可以通过简单的操作光源，暴露的表面的曝光和区域的持续时间的强度来生成梗死具有不同的尺寸和深度。虽然目前的研究在SC产生缺血上T11的中央区域，这种方法也可以产生在沿延髓到尾不同位置的梗塞以及脊髓，其中米格的横向方向HT受益理解缺血对截瘫的区域专用效果。另一方面，虽然照明是脊髓的表面上，光可以穿透到一定深度的组织和损伤也可诱发在灰质。如玫瑰红是分布在整个循环系统中，如果动物种类是相同的，以及患者的年龄和体重相似，我们预期一致病变会产生如皮质缺血引起由PT。 PT-诱导缺血的另一主要优点是动物非常低死亡率。低死亡率意味着长期存活研究可以进行，这可能是在解开缺血性损伤的生存和运动功能恢复的时间效应非常有用。这种模式也可以帮助理解这通常发生晚于损伤14,19,27-29的慢性期的细胞修复机制。这种模式也产生相当的运动功能障碍，可bë用来评估对功能恢复神经保护剂的效果。另外，该模型也将允许的SCI后的病理变化，如轴突变性和再生，神经元和星形细胞的Ca 2+信号传导并且在使用双光子显微镜活体小鼠超载的研究。 像所有其他型号的SCI，PT是不是毫无缺点。这种技术的缺点是类似于见于皮质PT。少的缺点包括缺乏解剖学清晰缺血半暗带，这是许多神经保护药物的目标，以及缺乏灌注。它公知的是再灌注缺血的特征在于像增加产量的活性氧，炎性细胞浸润和增加的细胞因子的产生30-32的变化。缺乏再灌注PT意味着再灌注损伤的SC所带来的变化仍将难以用这个模型来研究。但是，使用PT诱发缺血的利大于弊这种技术提供了研究人员一个易于执行，并在小鼠产生SCI的高度重复性模式。

外伤性脊髓损伤（SCI）是影响SC的感觉和植物神经功能毁灭性的临床情况。患者存活SCI往往留下衰弱截瘫的显著影响他们的日常活动和生活1的质量。实验SCI车型已经在科学考察中不可或缺的工具来了解脊髓损伤的病理生理学和相关的神经修复过程。这些模型也被用于测试的目的是在功能恢复各种实验神经保护干预的临床前功效。目前，大部分车型SCI在实践应用中使用的物理钝力机械破坏和伤害的SC。这些方法包括SC 2挫伤，压缩，错位和横断。有人建议，该一次侧机械损伤的缺血集形式的继发性损伤在受伤的SC后<html> 3,4。继发缺血的病因包括广泛组织变性，脑实质出血，有时被阻塞的血管通过组织水肿5-7。作为继发性损伤的SC的完整性被进一步影响的结果，神经元和胶质细胞严重受损功能和活力和凋亡导致在损伤的慢性阶段对梗死增长，类似于缺血半影中风后的生长8,9。几种机制一样兴奋性中毒，自由基的产生，和炎症已经报道负责缺血性细胞死亡以下脊髓损伤10,11。此外，SC缺血是胸腹主动脉瘤修复手术，往往导致截瘫的病人12,13的严重并发症。尽管有如此高的临床影响，目前可脊髓缺血具有高重复性非常少的机型。 核苷酸"&gt; Photothrombosis（PT）是一种常用的方法，在大脑中14-20局灶性缺血的诱导。该技术是相当的非侵入性的，高度可再现的，并产生一个精确的局灶性缺血病灶在脑17的露出面积-21。这是通过光敏染料像玫瑰红（RB）16-20,22或赤藓红的B全身给药达到23接着血管适当光源局部照射。染料的光敏化引起的自由基的生成而扰乱平滑血管内皮的完整性，并引起血小板聚集，随后形成血栓。血流的阻塞由血栓导致在由容器24供给的区域的梗塞。由于减轻控制对强度和辐照的持续时间这一步骤产生高度均匀和可再现的梗塞。此外，这种方法可用于诱导梗死T对于不同的解剖位置，使缺血的作用空间（ 例如 ，灰质与白质）的理解。 目前研究的目的是开发SC缺血小鼠容易和高度可再现的模式。我们描述SC缺血的小鼠模型的PT的过程。结果，从组织学和免疫证明，PT能有效诱导SC梗死，神经元缺失和胶质增生反应。

<table><tbody><tr><td>Rose Bengal</td><td>Sigma-Aldrich</td><td>330000</td><td>20 mg/ml in sterile saline</td></tr><tr><td>C57BL/6J</td><td>Jackson lab</td><td>664</td><td>22 - 25 g</td></tr><tr><td>Ketamine&amp;nbsp;</td><td>VEDCO</td><td>NDC-50989-996-06</td><td>100 mg/ml</td></tr><tr><td>Xylazine&amp;nbsp;</td><td>VEDCO</td><td>NDC-50989-234-11</td><td>100 mg/ml</td></tr><tr><td>Betadine solution</td><td>Purdue</td><td>NDC-67618-150-01</td><td>10% povidone iodine topical solution</td></tr><tr><td>Normal saline</td><td>Abott Laboratories</td><td>04930-04-10</td><td>For diluting RB, anaesthesia and for preventing tissue from drying</td></tr><tr><td>Artificial tears ointment&amp;nbsp;</td><td>Rugby</td><td>NDC-0536-6550-91</td><td>83% white petrolatum</td></tr><tr><td>Ethanol</td><td>Decon labs.Inc</td><td>2716</td><td>70% ethanol for disinfection</td></tr><tr><td>Metal halide lamp</td><td>EXFO, Canada</td><td>X-Cite 120 PC&amp;nbsp;</td><td>Set power at 12%</td></tr><tr><td>Spring scissors&amp;nbsp;</td><td>Fine Science Tool</td><td>15000-10</td><td>for minor dissection</td></tr><tr><td>Scissors (angled to side)</td><td>Fine Science Tool</td><td>14063-011</td><td>No. 3 handle</td></tr><tr><td>Standard scalpel</td><td>Fine Science Tool</td><td>10003-12</td><td>for removing muscle</td></tr><tr><td>Scalpel blade</td><td>Feather</td><td>2976</td><td>No. 10</td></tr><tr><td>Forceps (curved)</td><td>Fine Science Tool</td><td>11150-10</td><td>for holding tissue</td></tr><tr><td>Forceps (straight)</td><td>Fine Science Tool</td><td>11151-10</td><td>for holding tissue</td></tr><tr><td>Needle holder&amp;nbsp;</td><td>Fine Science Tool</td><td>12002-12</td><td>for suturing</td></tr><tr><td>Tissue adhesive glue</td><td>3M Vetbond</td><td>1469SB</td><td>to adhere to edges of the cut skin</td></tr><tr><td>Monofilament polypropylene&amp;nbsp;</td><td>USSC Sutures</td><td>VP-521</td><td>Size = 4-0 (for fascia)</td></tr><tr><td>Perma-hand silk</td><td>Ethicon</td><td>683G</td><td>Size = 4-0 (for skin)</td></tr><tr><td>Micro drill</td><td>Roboz Surgical Instrument Co. Inc.</td><td>RS-6300</td><td>with bone polishing drill bit</td></tr><tr><td>Laser doppler flowmeter</td><td>Moor Instruments</td><td>moorVMS-LDF1</td><td>for monitoring change in blood flow</td></tr><tr><td>Heating pad</td><td>Fine Science Tool</td><td>21052-00</td><td>to prevent hypothermia</td></tr><tr><td>Lab-Jack</td><td>Fisher scientific&amp;nbsp;</td><td>14-673-50</td><td>4 x 4 in plate to adjust the height of the animal</td></tr><tr><td>X-Y gliding stage&amp;nbsp;</td><td>Amscope</td><td>GT100</td><td>for positioning the animal under microscope&amp;nbsp;&amp;nbsp;</td></tr></tbody></table>

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.

本研究的目的是为了产生在使用PT模型小鼠脊髓缺血。骨脊髓（T10 - T12）以上的所希望的区域之后被减薄，玫瑰红通过眶后窦途径注射，和局部缺血诱导的PT 图1A，B示出的鼠标放置在一个特制的手术。在手术过程中的平台。将小鼠保持在适当位置由口鼻部夹钳和两个可调脊椎动物夹具来稳定脊髓图1C示出了减薄窗口T10的脊髓以上- T12。主要血管及其分支可以清楚地显现。以确认缺血的诱导，血流变化，使用激光多普勒流量计之前和PT后，测定（ 图2A，B）。为了分析，在％血流量的减少使用photothrombosis之前的基线血流，计算。血流后光照立即下降到约20％，低于W¯¯第i的基础水平之前照明。 图3B中，C表示脊髓血管在开始和PT的端荧光图像。照明2分钟诱发血块在血管（ 图3C），表明诱导缺血，用激光多普勒流量计的测量一致。检查所造成的PT的损伤，处死小鼠3天PT和尼氏染色，后。以下Nissl染色拍摄的图像显示梗塞区域，可以从周围的区域被明确划分，这表明脊髓组织损伤和细胞死亡的PT之后（ 图4）。下进行的NeuN，GFAP和Iba1免疫染色。的NeuN阳性神经元丢失在灰质缺血核心（ 图5A），而GFAP的表达在缺血核心的边界增加（ 图5B，也看到了加框区域）。该Iba1 +小胶质细胞表现出铁道部弧面phology（ 即 ，扩大的细胞体具有较短和较少的工序，见盒装区域）随着增加Iba1表达（ 图5C）。虽然有一个组织损失在缺血中心区域由于浮动部分染色，在整个梗死周围区域的增加GFAP和Iba1表达可以清楚地观察。这些结果表明，神经元死亡和反应性胶质增生的SC缺血半暗带。另一方面，大量的功能缺陷，观察该损伤小鼠中， 即 ，禁用后肢运动1天PT之后，指示后肢肢体麻痹（见动画 ）。 <img alt="图1" src="/files/ftp_upload/53161/53161fig1.jpg" /> 图1. PT诱导缺血模型中脊髓。（A）中的手术平台的脊髓的PT的照片。插图:扩大椎夹。（二）鼠标举行一个吻夹具和两个特制的夹子椎在舞台上。注意，骨变薄在T10 - T12区和两个金属椎体夹具被用来稳定脊髓（℃）的变焦式表示在T10-11为与薄化骨脊髓上方的区域的图像感应PT的。请注意，主血管及其分支。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53161/53161fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53161/53161fig2.jpg" /> 图2.脊髓血流量测量。 （A）使用激光多普勒流量计和立体定位装置来定位探针脊髓表面血流测定的设置。（B）的PT前，后脊髓血流量测定。在该实验中，PT诱导与光源2分钟以12％的输出功率照射。照射表面的直径为0.75毫米，在脊髓的中间。血流记录长达5分钟的PT之前，以获得稳定的信号和最多10分钟之后的PT。从每只小鼠的数据被标准化为值之前的光照明。该图显示的数据来自3只小鼠的平均值。箭头表示PT的开始。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53161/53161fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53161/53161fig3.jpg" />一图3. PT-引起脊髓缺血。（A）照片鼠标放置在脊髓的显微镜诱导PT。鼠标的位置可以在三维使用在XY滑动台和一个实验室千斤顶进行调整。从客观10X光集中脊髓的表面上。（BC）的血管脊髓前（B）后（C）照明注入以下荧光图像玫瑰红。注意照射（C）2分钟（见箭头）后血块。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53161/53161fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53161/53161fig4.jpg" /> 图4. Nissl染色脊髓。一系列喙至尾脊髓横向切片Nissl染色图像，包括正常（节1和6）和PT诱导震中（第2 - 5）。处死小鼠的PT后3天。每个脊髓节30微米厚。两个部分之间的间隔为750微米。该虚线第三图像勾勒出梗死区域。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53161/53161fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/53161/53161fig5.jpg" /> 图5.免疫染色的NeuN，GFAP和Iba1的 。的NeuN（A）的荧光图像，GFAP（B）和Iba1（℃）染色从正常（上图）和PT-受伤（下面板）脊髓切片。被牺牲受伤的鼠标PT后3天。虚线从正常组织中分离的梗死区域。盒装地区显示GFAP和Iba1表达高分辨率图像的50比例尺微米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53161/53161fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="电影1" src="/files/ftp_upload/53161/53161figMovie.jpg" /> <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53161/Movie_JOVE.MP4">影片</a> 。 PT中脊髓诱导的行为缺陷。影片显示正常和PT-受伤小鼠关在笼子里的运动。请注意，两个后肢四肢鼠标习以为常脊髓的拖动，表明后肢肢体（截瘫）瘫痪。这部电影拍摄24小时后，PT中受伤的鼠标。

Watch this Scientific Journal Video about Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice at JoVE.com

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

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) ...

photothrombosis-induced-focal-ischemia-as-model-spinal-cord-injury

Universidad San Sebastian

Research

JoVE Journal

Medicine

13.4K 次观看.  University of Missouri. 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. 

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

Photothrombotic Ischemia: A Minimally Invasive and Reproducible Photochemical Cortical Lesion Model for Mouse Stroke Studies

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected light-sensitive dye. Following illumination, the dye is activated and produces singlet oxygen that damages components of endothelial cell membranes, with subsequent platelet aggregation and thrombi formation, which eventually determines the interruption of local blood flow. This approach, initially proposed by Rosenblum and El-Sabban in 1977, was later improved by Watson in 1985 in rat brain and set the basis of the current model. Also, the increased availability of transgenic mouse lines further contributed to raise the interest on the photothrombosis model. Briefly, a photosensitive dye (Rose Bengal) is injected intraperitoneally and enters the blood stream. When illuminated by a cold light source, the dye becomes activated and induces endothelial damage with platelet activation and thrombosis, resulting in local blood flow interruption. The light source can be applied on the intact skull with no need of craniotomy, which allows targeting of any cortical area of interest in a reproducible and non-invasive way. The mouse is then sutured and allowed to wake up. The evaluation of ischemic damage can be quickly accomplished by triphenyl-tetrazolium chloride or cresyl violet staining. This technique produces infarction of small size and well-delimited boundaries, which is highly advantageous for precise cell characterization or functional studies. Furthermore, it is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

Photothrombosis is a quick, minimally-invasive technique for inducing small and well-delimited infarction in areas of interest in highly reproducible manner. It is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

光化学缺血：一种微创和可重复性的光化学皮质病变模型小鼠中风研究

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected ...

科研

医学

Reproducable Paraplegia by Thoracic Aortic Occlusion in a Murine Model of Spinal Cord Ischemia-reperfusion

Background 
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered advancements to decrease the occurrence this injury.&#160;The current model demonstrates reproducible lower&#160;extremity paralysis following thoracic aortic occlusion.
Methods 
Adult male C57BL6 mice were anesthetized with isoflurane. Through a cervicosternal incision the aorta was exposed. The descending thoracic aorta and left subclavian arteries were identified without entrance into pleural space. Skeletonization of these arteries was followed by immediate closure (Sham) or occlusion for 4 min (moderate ischemia) or 8 min (prolonged ischemia). The sternotomy and skin were closed and the mouse was transferred to warming bed for recovery.&#160; Following recovery, functional analysis was obtained at 12 hr intervals until 48 hr.
Results 
Mice that underwent sham surgery showed no observable hind&#160;limb deficit. Mice subjected to moderate ischemia for 4 min had minimal functional deficit at 12 hr followed by progression to complete paralysis at 48 hr. Mice subjected to prolonged ischemia had an immediate paralysis with no observable hind-limb movement at any point in the postoperative period. There was no observed intraoperative or post&#160;operative mortality.
Conclusion 
Reproducible lower extremity paralysis whether immediate or delayed can be achieved in a murine model. Additionally, by using a median sternotomy and careful dissection, high survival rates, and reproducibility can be achieved.

The lack of mechanistic understanding of spinal cord ischemia-reperfusion injury has hindered further adjuncts to prevent paraplegia following high risk aortic operations. Thus, the development of animal models is imperative. This manuscript demonstrates reproducible lower extremity paralysis following thoracic aortic occlusion in a murine model.

可重放截瘫胸主动脉阻断脊髓缺血再灌注的小鼠模型

Background
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered ...

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 ...

A Tissue Displacement-based Contusive Spinal Cord Injury Model in Mice

Producing a consistent and reproducible contusive spinal cord injury (SCI) is critical to minimizing behavioral and histological variabilities between experimental animals. Several contusive SCI models have been developed to produce injuries using different mechanisms. The severity of the SCI is based on the height that a given weight is dropped, the injury force, or the spinal cord displacement. In the current study, we introduce a novel mouse contusive SCI device, the Louisville Injury System Apparatus (LISA) impactor, which can create a displacement-based SCI with high injury velocity and accuracy. This system utilizes laser distance sensors combined with advanced software to produce graded and highly-reproducible injuries. We performed a contusive SCI at the 10th thoracic vertebral (T10) level in mice to demonstrate the step-by-step procedure. The model can also be applied to the cervical and lumbar spinal levels.

We introduce a tissue displacement-based contusive spinal cord injury model that can produce a consistent contusive spinal cord injury in adult mice.

基于组织位移的小鼠脊髓损伤模型

Producing a consistent and reproducible contusive spinal cord injury (SCI) is critical to minimizing behavioral and histological variabilities between ...

Induction of Complete Transection-Type Spinal Cord Injury in Mice

Spinal cord injury (SCI) largely leads to irreversible and permanent loss of function, most commonly as a result of trauma. Several treatment options, such as cell transplantation methods, are being researched to overcome the debilitating disabilities arising from SCI. Most pre-clinical animal trials are conducted in rodent models of SCI. While rat models of SCI have been widely used, mouse models have received less attention, even though mouse models can have significant advantages over rat models. The small size of mice equates to lower animal maintenance costs than for rats, and the availability of numerous transgenic mouse models is advantageous for many types of studies. Inducing repeatable and precise injury in the animals is the primary challenge for SCI research, which in small rodents requires high-precision surgery. The transection-type injury model has been a commonly used injury model over the last decade for transplantation-based therapeutic research, however a standardized method for inducing a complete transection-type injury in mice does not exist. We have developed a surgical protocol for inducing a complete transection type injury in C57BL/6 mice at thoracic vertebral level 10 (T10). The procedure uses a small tip drill instead of rongeurs to precisely remove the lamina, after which a thin blade with rounded cutting edge is used to induce the spinal cord transection. This method leads to reproducible transection-type injury in small rodents with minimal collateral muscle and bone damage and therefore minimizes confounding factors, specifically where behavioral functional outcomes are analyzed.

This protocol describes how to create a precise laminectomy for induction of stable transection-type spinal cord injury in the mouse model, with minimal collateral damage for spinal cord injury research.

小鼠完全转型脊髓损伤的诱导

Spinal cord injury (SCI) largely leads to irreversible and permanent loss of function, most commonly as a result of trauma. Several treatment options, ...

神经科学

Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis

Stroke is a leading cause of death and acquired adult disability in developed countries. Despite extensive investigation for novel therapeutic strategies, there remain limited therapeutic options for stroke patients. Therefore, more research is needed for pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Given the inability of in vitro models to reproduce the complexity of the brain, experimental stroke models are essential for the analysis and subsequent evaluation of novel drug targets for these mechanisms. In addition, detailed standardized models for all procedures are urgently needed to overcome the so-called replication crisis. As an effort within the ImmunoStroke research consortium, a standardized photothrombotic mouse model using an intraperitoneal injection of Rose Bengal and the illumination of the intact skull with a 561 nm laser is described. This model allows the performance of stroke in mice with allocation to any cortical region of the brain without invasive surgery; thus, enabling the study of stroke in various areas of the brain. In this video, the surgical methods of stroke induction in the photothrombotic model along with&#160;histological analysis are demonstrated.

Described here is the photothrombotic stroke model, where a stroke is produced through the intact skull by inducing permanent microvascular occlusion using laser illumination after administration of a photosensitive dye.

小鼠模型中风:光血栓形成的焦皮质病变

Stroke is a leading cause of death and acquired adult disability in developed countries. Despite extensive investigation for novel therapeutic strategies, ...

Establishing a Mouse Contusion Spinal Cord Injury Model Based on a Minimally Invasive Technique

Using minimally invasive methods to model spinal cord injury (SCI) can minimize behavioral and histological differences between experimental animals, thereby improving the reproducibility of the experiments.
These methods need two requirements to be fulfilled: clarity of the surgical anatomical pathway and simplicity and convenience of the laboratory device. Crucially for the operator, a clear anatomical pathway provides minimally invasive exposure, which avoids additional damage to the experimental animal during the surgical procedures and allows the animal to maintain a consistent and stable anatomical morphology during the experiment.
In this study, the use of a novel integrated platform called the SCI coaxial platform for spinal cord injury in small animals to expose the T9 level spinal cord in a minimally invasive way and stabilize and immobilize the vertebra of mice using a vertebral stabilizer is researched, and, finally, a coaxial gravity impactor is used to contuse the spinal cord of mice to approach different degrees of T9 spinal cord injury. Finally, histological results are provided as a reference for the readers.

Minimally invasive techniques and a simple laboratory device improve the reproducibility of the spinal cord injury model by reducing operative damage to the experimental animals and allowing anatomical morphology maintenance. The method is worthwhile because the reliable results and reproducible procedure facilitate investigations of the mechanisms of disease reparation.

基于微创技术的小鼠挫伤脊髓损伤模型的建立

Using minimally invasive methods to model spinal cord injury (SCI) can minimize behavioral and histological differences between experimental animals, ...

Establishment of Central Cord Syndrome Model in C57BL/6J Mouse

Animal models of central cord syndrome (CCS) could substantially benefit preclinical research. Identifiable anatomical pathways can give minimally invasive exposure approaches and reduce extra injury to experimental animals during operation, enabling the maintenance of consistent and stable anatomical morphology during experiments to minimize behavioral and histological differences between individuals to improve the reproducibility of experiments. In this study, the C6 level spinal cord was exposed using a spinal cord injury coaxial platform (SCICP) and combination with a minimally invasive technique. With the assistance of a vertebral stabilizator, we fixed the vertebrae and compressed the spinal cords of C57BL/6J mice with 5 g/mm2 and 10 g/mm2 weights with SCICP to induce different degrees of C6 spinal cord injury. In line with the previous description of CCS, the results reveal that the lesion in this model is concentrated in the gray matter around the central cord, enabling further research into CCS. Finally, histological results are provided as a reference for the readers.

The present protocol simulating central cord syndrome (CCS) in mice has improved repeatability and minimized operation damage to the experimental animals, avoiding disrupting the anatomical structure excessively. The strategy in this study is advantageous because it allows for research into injury mechanisms by producing consistent results.

C57BL/6J小鼠中索综合征模型的建立

Animal models of central cord syndrome (CCS) could substantially benefit preclinical research. Identifiable anatomical pathways can give minimally ...

Modeling Photothrombotic Stroke in Mouse Model: A Laser Illumination Technique Through Mouse Skull Following Photosensitive Dye Administration to Induce Photothrombosis

Source: Llovera, G. et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/62536">Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis.</a> J. Vis. Exp. (2021)
This video demonstrates the procedure to generate photothrombotic stroke in a mouse model by laser illumination of an intact skull following administration of photosensitive dye.

小鼠模型中的光血栓形成中风建模:光敏染料给药后通过小鼠颅骨的激光照明技术诱导光血栓形成

Source: Llovera, G. et al. Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis. J. Vis. Exp. (2021)
This video demonstrates the procedure ...

JoVE Encyclopedia of Experiments

生物学

Rodent Models

Adult

Inducing a Spinal Cord Contusion in a Mouse Model Using a Spinal Cord Injury Coaxial Platform

<a href='https://app-jove-com.bdigitaluss.remotexs.co/v/64538/establishing-mouse-contusion-spinal-cord-injury-model-based-on'>Source: Elzat, E. Y. Y., et al.,&#160;Establishing a Mouse Contusion Spinal Cord Injury Model Based on a Minimally Invasive Technique. J. Vis. Exp.&#160;(2022).</a>This video demonstrates the method for inducing a contusion injury at the T9 level spinal cord in a mouse model, using a spinal cord injury coaxial platform. A weight is dropped on the T9 level spinal cord that creates a contusion that immediately causes tissue discoloration and swelling.