Name: a versatile murine model of subcortical white matter stroke for the study of axonal degeneration and white matter neurobiology
Uploaded: 2016-03-17T13:00:00.000+00:00
Duration: 8 min 36 s
Description: Watch this Scientific Journal Video about A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology at JoVE.com

SN和MDD从NIH K08 NS083740和神经病学的加州大学洛杉矶分校部门的支持。 AJG承认由仪博士和谢尔登·阿德尔森G.医学研究基金会和拉里·希尔布鲁姆L.基金会的支持。九龙十分感谢美国心脏协会14BFSC17760005 ASA-Bugher卒中中心的支持。 ILL，EGS和STC是由美国国立卫生研究院R01 NS071481支持。 JDH确认来自美国国立卫生研究院K08 NS083740支持。

在这个协议中使用的动物是按照由加州大学洛杉矶分校的动物护理和使用委员会的批准大学的程序进行。 注:确定目标人群小鼠开始。在以前的研究中，只有雄性野生型C57 / BL6小鼠已被使用，但是各种转基因或基因敲除小鼠也可以使用。注意，立体定向坐标基于C57 / BL6解剖。建议每个用户最初验证中风白质的定位。 1.白质行程感应 - 内侧斜角方法<ol><li>通过使用0.5毫米毛细管使得远端直径为15-25微米11之间制备拉玻璃吸管开始。 </li><li>在无菌的0.9％生理盐水27.4毫克/毫升（130μM） - 制备L-NiO的无菌10微升等分试样（（1）-iminoethyl -L-鸟氨酸盐酸N（5））。 </li><li>预填拉玻璃吸管与由固定用玻璃吸管的小体积的L-仁王（2-5微升）至管连接到真空管线。躺在工作台上吸管平并插入拔出端插入L-仁王解决方案。 <ol><li>直到吸管0.5毫米部的至少2mm填充应用真空。关闭真空和撤回移液器。将它放在一旁，直到步骤1.12。 </li></ol></li><li>鼠标放置在感应腔和诱导使用标准的32％异氟烷鼠标麻醉通过蒸发器流入（5升/分钟吸入用5升/分钟的氧气和0.5升/分钟N 2），持续1分钟或直至深麻醉。鼠标转移到配备有立体显微镜立体定位装置。提供一种使用32％异氟烷维持麻醉通过蒸发器流入（2L /分吸入用5升/分钟的氧气和0.5升/分钟N 2）和一鼻锥体。检查麻醉深度与脚趾捏。 </li><li>注射臂调整到36°。 </li><li>阿菲XA拉玻璃吸管持有人一个低量的压力喷射系统的末端，并将其连接到立体设置的注入手臂。 </li><li>涂抹麻醉动物用凡士林和地点人工泪液软膏超过两只眼睛胡须。通过将无菌悬垂在动物的头用5-10厘米开幕的头准备一个无菌手术领域。剃皮毛覆颅骨准备aspetic手术的表面。清洁交替优碘和70％酒精棉签头皮。 </li><li>请用无菌剪刀罚款1.5厘米的头皮正中切口，显露颅骨表面。干后用消毒棉签头骨和1-3X的放大倍率用显微镜立体，用无菌微点工具可以删除任何覆盖骨膜组织。 </li><li>标记为前囟门用细点标记的参考点。 </li><li>使用无菌细STIP手术钻头钻2毫米ellipitical开颅手术0;在后方前囟门开始，向前延伸刚出中线。去除骨碎片和上覆的软组织，以便大脑皮层可以可视化。 </li><li>保持手术视野和皮质表面湿润通过间歇应用无菌生理盐水滴。 </li><li>贴上拉玻璃吸管立体定向仪的注射器手臂。对准前囟吸管的远端和零的立体定向坐标。 </li><li>前进移液器到第一前/后（A / P）和内侧/外侧（M / L） 表1坐标提供。 </li><li>推进吸管皮质表面和零背/腹（D / V）的测量。 </li><li>慢慢通过吸管进入脑，直到达到表1的第一D / V坐标。 </li><li>使用低容量压力喷射系统20毫秒脉冲设置为20磅，注入100 NL L-仁王的入脑，并等待5分钟，以防止反流了吸管轨道。 <ol><li>在立体定向显微镜的目镜和3X的倍率使用校准掩模版。 </li><li>因此，置换共有0.100毫米3（0.5毫米的长度在0.5毫米直径的移液管中，对应于100 NL）从拉玻璃吸管为每组坐标。通过使用划板，测量和标准化，因为每个设置取决于所使用的放大率和体重秤。 </li><li>对于每一个在注射过程中准确体积测量，接近与显微镜倾斜移液器从侧面使空气流体弯月面具有矢状图。弯液面应该出现在这两个吸移管的内壁和外壁的同一焦平面。 </li></ol></li><li>慢慢抽出吸管和重复步骤1.13-1.16.3在第二和第三组在表1中提供的坐标。 </li><li>最后一次注射后，取出吸管，并放置足够的骨蜡填补了开颅手术部位。一个pproximate头皮伤口的边缘，并与皮肤粘合剂结合。 </li><li>注入0.1毫升0.5％麻卡因成使用无菌​​地下30针，以防止以防止与头皮切口相关联的局部疼痛的伤口边缘。 </li><li>返回动物壳体和供应术后抗生素（0.48毫克/毫升复方新诺明，或0.5毫克/毫升左氧氟沙星）的饮用水5天。 </li></ol> 2.白质行程感应 - 后路斜角方法<ol><li>执行步骤1.1-1.12为在内侧倾斜的方式协议中，除了调整立体设置面向从前到后45度的注入手臂。 </li><li>前进移液器向第一A / P和M / L的坐标在表2中提供。 </li><li>完成剩余的步骤1.14-1.20作为在横向成角度的方法的协议。 </li></ol> 3.逆行神经标签<ol><li>制备无菌的10在0.9％的生理盐水54.8毫克/毫升的L-仁王的微升等分试样。 </li><li>制备20％Fluororuby（或20％的生物素化葡聚糖胺或2％荧光金）的0.9％生理盐水的无菌等分试样。 </li><li>稀释一起1:1 27.4毫克/毫升的L- NiO和10％Fluororuby的最终浓度。 </li><li>步骤1.3-1.23执行行程协议如上。 </li><li>如前所述8灌注固定，cryosectioning和显微可视化组织切片的原生荧光示踪剂。 </li></ol> 4.组织处理的免疫<ol><li>在适当的中风后间隔时间从3小时到中风后14天内，通过安乐死过量的异氟醚或当地IACUC小鼠批准的程序。 </li><li>使用倾斜的剪刀打开胸腔，并插入一个23政蝴蝶针进入左心室。 </li><li>放置在用细剪刀，以允许灌注流体流出轨道右心房的小切口。 </li><li>在室温为10毫升/分钟的速率用30-40毫升冷的磷酸盐缓冲盐水后30-40毫升冷的4％多聚甲醛的Transcardially灌注。 </li><li>斩首鼠标和使用无菌剪刀向后打开颅骨取出大脑，然后轻轻地用刮勺除去覆颅骨，将脑入冷的4％PFA中24小时，然后转移到30％蔗糖的PBS中48小时。 </li><li>制备40使用低温恒温器微米浮动切片，并如前所述6-8执行抗体的处理。在这项研究中，使用下列抗体:兔抗神经丝200（1:500稀释度）;兔抗波形蛋白（1:500）;山羊抗GFAP（1:500）;兔抗伊巴-1（1:1000）。 </li></ol> 5.组织处理的蛋白质或RNA分析<ol><li>在适当的中风后间隔时间从3小时到中风后14天，异氟醚通过安乐死过量或当地IACUC批准的程序。 </li><li>斩首用无菌剪刀向后打开颅骨鼠标和取出大脑，然后轻轻地用锅铲除去覆头骨。 </li><li>在大脑的前面插入一个无菌4毫米刮刀切断嗅球和视神经。轻轻抬起脑出颅骨并放入冰冷的夹层缓冲液（1×Hank氏平衡盐溶液，25mM的HEPES-KOH，pH 7.4的35毫摩尔葡萄糖，4mM的碳酸氢钠，和0.01毫克/毫升环己酰胺）。 </li><li>用脑块，并育新刀片，制成含有中风并放入冷夹层缓冲器2-3毫米的板坯。 </li><li>在解剖显微镜下，确定在所注入的半球白质底层运动皮层。在较长的脑卒中后的时间间隔，该区域可在视觉上由局灶性坏死和髓鞘苍白鉴定。 注:在早期中风后的时间间隔，L-仁王的注射1微升10％混合固绿可以让中风的视觉识别（ <strong&gt;图4A）。 </li><li>在解剖显微镜下的指导，并用新鲜的手术刀，小心解剖的白质含有行程，由任一坚牢绿标签或组织损失确定的区域。根据需要取出覆皮层和纹状体底层。 </li></ol>

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已经描述了若干皮层下行程的前模型，包括内皮素-1的焦点注射进入内囊，皮层下白质和纹状体在大鼠12-14和鼠标6,15。更近的小焦点冲程模型已经利用在颈动脉16胆固醇微栓子注射和单个穿透动脉17的光化学闭塞。每个模型都有有利有弊5。目前描述的模型产生具有许多模仿人类腔隙性脑梗塞，包括轴突异常和损失，髓鞘退化，焦距坏死核心和临床赤字是最小的，并演示了较快的恢复依赖于7年龄特点的病变。鼠靶向脑白质允许多种，可以支持机制研究遗传操作的。 关键该协议的步骤包括利用立体定向精确坐标。由于鼠类白质体积小和应变变化应变，可以根据使用鼠标的年龄和应变需要的立体坐标修订。注射的体积的控制也很重要，因为这是直接相关的梗塞的大小。注射较大将产生上覆皮层和纹状体底层染指大招。 使用此处描述的方法的ET-1的焦点注射已经报道，但内皮素-1显示出具有对少突胶质细胞的分化和成熟10,18混杂中风后白质生物学研究直接旁分泌的影响。相反，L-仁王方法目标内皮细胞单独同时产生相同的病变，并消除了对参与损伤反应的细胞的任何混杂旁分泌的影响。左旋仁王不直接细胞毒性和选择素（数据未显示）泰德剂量由初步剂量递增实验确定。后倾斜的方法的开发是为了更精确地从初级运动皮质倒勾轴突产生的最大行为缺损，可以归因于白质损伤。内侧倾斜的做法也损害运动皮层的神经轴突，但更多的横向延伸，涉及到底层的轴突初级感觉皮层。 笔划病变在最初24小时的相当迅速膨胀。由七天梗塞的大小为最大，我们没有观察到显著病斑生长超出该时间。额外细胞事件和轴突变性会发生超出这个初始阶段，但如由坏死核心测定梗塞大小不会在没有任何介入的显著变化。 在行程的时间神经解剖学示踪剂的共注射标识神经元经历缺血性轴索损伤。无论使用配有IAL或后倾斜的做法，受损轴突投射神经元细胞体仍然安然无恙。这将创建一个有用的模型来研究中枢神经系统的神经细胞缺血性离断伤的效果。我们主要利用示踪剂逆行包括葡聚糖胺和荧光金，这既显示出优良的摄取中风损伤轴突。通过采用多种转基因和基因敲除小鼠品系中，本规程的用户可以调查涉及脑白质损伤中风响应和修复神经元基因的具体作用。

Stroke affecting the subcortical white matter is a common clinical entity, accounting for up to 25% of clinical strokes annually in the US 1. Ischemic damage to white matter also occurs silently at a significantly higher rate and contributes to the development of vascular dementia 2,3. Presently, patients with this form of cerebral ischemia have few, if any treatment choices. Despite the clinical importance of this disease, few clinically relevant animal models exist 4,5. 
The goal of this protocol is to produce a focal ischemic lesion within the murine white matter. This murine model of human disease allows the specific study of axonal injury response to stroke and how the cellular elements of white matter, namely oligodendrocytes and astrocytes along with axons, respond to and repair after stroke.
Previous reports have described a model of subcortical white matter stroke using endothelin-1 (ET-1) 6 that is similar to the one described here. Several key changes to the experimental protocol have been made thereby the potential uses of this model have expanded 7,8. This protocol provides a reliable and modifiable strategy to produce a focal stroke within mouse brain white matter. 
The major advantages of this model are the use of a chemical endothelial nitric oxide synthase (eNOS) inhibitor N(5)-(1)-iminoethyl-L-ornithine HCl (L-Nio) 9 with no known paracrine effects on cellular elements of white matter which had been a complication of models using endothelin-1 10. In addition, the stereotactic targeting of white matter in the mouse allows the use of any variety of transgenic or knockout strains, greatly expanding the available tools to determine the effect of stroke on brain white matter. Here, two variations on this technique are described and demonstrate some of the additional variations that can be utilized to enhance the understanding of axonal and white matter damage and repair after stroke.

<table><tbody><tr><td>L-N5-(1-Iminoethyl)ornithine, Dihydrochloride</td><td>Calbiochem</td><td>400600-20MG</td><td></td></tr><tr><td>Isoflurane</td><td>Phoenix Pharmaceutical, Inc.</td><td>NDC 57319-559-06</td><td></td></tr><tr><td>Capillary tubes</td><td>World Precision Instruments</td><td>50-821-807</td><td></td></tr><tr><td>Picospritzer</td><td>Parker Instrumentation</td><td>Picospritzer II</td><td></td></tr><tr><td>Stereotactic setup</td><td>Kent Scientific</td><td>KSC51725</td><td></td></tr><tr><td>Pipette puller</td><td>KOPF</td><td>Model 720</td><td></td></tr><tr><td>Stereomicroscope SZ51</td><td>Olympus</td><td>88-124</td><td></td></tr><tr><td>Fine scissors</td><td>Fine Scientific Tools</td><td>14084-08</td><td></td></tr><tr><td>Forceps</td><td>Harvard Apparatus</td><td>PY2 72-8547</td><td></td></tr><tr><td>Curved Forceps</td><td>Harvard Apparatus</td><td>PY2 72-8598</td><td></td></tr><tr><td>Blunt dissection tool</td><td>Fine Scientific Tools</td><td>10066-15</td><td></td></tr><tr><td>Drill</td><td>Dremel</td><td>8220-1/28</td><td></td></tr><tr><td>Drill bits</td><td>Fine Scientific Tools</td><td>19007-05</td><td></td></tr><tr><td>Vetbond</td><td>3M</td><td>1469SB&amp;nbsp;</td><td></td></tr><tr><td>Marcaine</td><td>HOSPIRA</td><td>NDC 0409-1610-50</td><td></td></tr><tr><td>Trimethoprim-Sulfamethaxole</td><td>STI Pharmacy</td><td>NDC 54879-007-16</td><td></td></tr><tr><td>Fluororuby</td><td>Fluorochrome Inc</td><td>30 mg</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Fisher</td><td>O4042-500</td><td></td></tr><tr><td>Sucrose</td><td>Fisher</td><td>BP220-10</td><td></td></tr><tr><td>Cryostat</td><td>Leica CM3050 S</td><td>14047033518</td><td></td></tr><tr><td>Glass slides</td><td>Fisher</td><td>12-544-7</td><td></td></tr><tr><td>Fast Green&amp;nbsp;</td><td>Sigma</td><td>F7252-5G</td><td></td></tr><tr><td>Dissection microscope</td><td>Nikon</td><td>SMZ1500</td><td></td></tr><tr><td>23 G butterfly needle</td><td>Fisher</td><td>14-840-35</td><td></td></tr><tr><td>10x Hank&#039;s Balanced Salt Solution</td><td>Life Technologies</td><td>14065056</td><td></td></tr><tr><td>1 M HEPES-KOH, pH 7.4</td><td>Affymetrix</td><td>16924</td><td></td></tr><tr><td>D-Glucose</td><td>Sigma</td><td>G8270</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma</td><td>S5761</td><td></td></tr><tr><td>Cyclohexamide</td><td>Sigma</td><td>01810</td><td></td></tr></tbody></table>

a versatile murine model of subcortical white matter stroke for the study of axonal degeneration and white matter neurobiology

中风影响白质占的临床冲程演示高达25％，在费率可能5-10倍更大默默地发生，并且显著血管性痴呆的发展有助于。存在焦白质中风的几个型号，这缺乏适当的模式阻碍了参与这类中风后损伤反应和修复neurobiologic机制的理解。其他皮层下中风模型的主要限制是，它们不灶性限制梗塞的白质或已经主要在非鼠物种得到验证。这限制应用各种各样的鼠研究工具来研究白质中风的神经生物学的能力。在这里，我们提出了一个方法，为可靠的生产使用的是不可逆的eNOS抑制剂局部注射鼠白质的焦点中风。我们还提出对一般协议，包括两个独特的立体几个变化变型中，逆行神经追踪，以及新鲜组织标记和解剖，大大扩大该技术的潜在应用。这些变化允许多种方法来分析的行程这个共同的和充分研究形式neurobiologic影响。

使用提出的模型，白质底层前肢感觉运动皮层能够可靠地针对性。此化学诱导中风模型产生焦轴突和髓磷脂损失，星形胶质细胞增生，和小神经胶质细胞（ 图1），如在人类腔隙性脑梗死通常看到。通过使用三次注射，临床有用的模型建立与前肢运动任务7月初，障碍和脑组织经验，一个小而显著部分缺血的免疫，免疫和生化技术是在定量层面可行，可靠。在中风诱导后早期时间点（小时），在轴突分子组织的变化可以被检测（ 图2）。在7天，行程完成其成熟轴索损（ 图1a）的限定区。在我们的手中，这种方法产生的焦白质病变approximately 800微米水平直径和延伸沿胼胝体的前后轴约1毫米。由7天，平均总梗塞大小大约0.200毫米3和将具有椭圆形状。我们观察到约10％的变异笔触大小两种动物之间和部分，取决于其中有椭圆形的部分之间发生。在梗死面积进一步增长很少见到超过7天。 在显著神经元标记的葡聚糖胺结果的同时注射的在层5和层6具有轴突通过行程的区域突出（ 图3）的神经元细胞体的加法。不是由步骤（细针的通路）的假方面受损上方皮质神经元，进行远端轴索损害，并且可以通过包含与L仁王制备示踪物来鉴定。这种方法是使用<html> d可卒中后8展示轴突起始段的动态变化。 而受到中风的组织的区域的小尺寸限制了使用的其它常规技术，如2,3,5-氯化三苯基四唑（TTC）染色，脑白质行程区可以在新鲜组织来鉴定。增加了一个共同的染料如坚牢绿产生组织的可识别的区域，可以在解剖显微镜下解剖（ 图4）。一旦解剖这个组织可以用于与蛋白质印迹或免疫沉淀，或用于RNA分离和分析（ 图4C）蛋白分析。通过使用各种转基因小鼠线，各种创新的方法可以用来研究焦点白质行程包括细胞命运的映射研究和激光捕获显微切割（ 图4C）后，神经生物学。 帐篷"FO: - together.within页保留="1"&gt; <img alt="图1" src="/files/ftp_upload/53404/53404fig1.jpg" /> 图1:焦白质行程同时使用内侧和后路角度的途径为神经丝免疫荧光标记（A，红色）演示了行程七天后轴突损失的使用方法内侧的程度。使用后倾斜的做法，脑白质病变行程是仅高于侧脑室（B，C），针对性和显示强烈的小胶质细胞（B）和星形胶质细胞反应性（C）。两个星形胶质细胞中间丝标记，波形蛋白（红色）和胶质纤维酸性蛋白（GFAP，绿色），无一不透露行程（C）后变化的白质（纤维）星形胶质细胞的形态。比例尺= 500微米。 <a href="https://www.jove.com/files/ftp_upload/53404/53404fig1large.jpg" target="_blank">请点击此处查看大版本</a>这个数字的锡永。 <img alt="图2" src="/files/ftp_upload/53404/53404fig2.jpg" /> 图2:脑白质行程改变轴突微区组织内白质行程感应，轴突微区组织的节点上，通过测试-IV血影（红色），并在paranode标记，通过contactin相关蛋白标记的3小时（CASPR，绿），被破坏（箭头B）。对侧白质轴突显示常规节点，paranodal和juxtaparanodal组织（A和C），而同侧白质显示节点和paranodal伸长率是典型的与失去axoglial接触（B和D）轴突。比例尺=5μm以下。 <img alt="图3" src="/files/ftp_upload/53404/53404fig3.jpg" /> 图3:逆行神经Labelin摹与脑白质中风标识单个神经元与轴索损害。卒中诱导的实时荧光葡聚糖胺（红色）联合注射允许与中风受伤轴突的单个神经元的鉴定。大部分的标签发生在5层内的轴突和6个神经元在初级感觉皮质覆中风。形象代表中风后7天。比例尺= 500微米。 <a href="https://www.jove.com/files/ftp_upload/53404/53404fig3large.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/53404/53404fig4.jpg" /> 图4:在中风诱导的使用时间在快速绿色生化和转录化验联合注射白质病变卒中显微切割允许受伤的地区早期识别在24小时（A，上图）。 Immunoblotti纳克特定轴突蛋白可以被执行以表明在笔划单独（A，下图）的区域减少。在较长的时间点，白质中风（左图）的区域可被灶性解剖无特异性标记（B）。右上图显示的去除解剖区域后，白质区域，而右下角的面板显示的包含行程（B）白质解剖区域。 PCR利用RNA从解剖区域从白质（C）中分离特定的少突胶质细胞的基因。 IL =同侧; CL =对侧; C =控制; S =行程。 <table fo:keep-together.within-page="1"><tbody><tr height="21"><td height="21" style="height:21px;"> 注射 </td><td> 前/后 </td><td> 内侧/外侧 </td><td> 背/腹 </td></tr><tr height="21"><td align="right" heigHT ="21"的风格="高度:21px;"&gt; 1 </td><td align="right"> 0.22 </td><td align="right"> 0.22 </td><td align="right"> -2.10 </td></tr><tr height="21"><td align="right" height="21" style="height:21px;"> 2 </td><td align="right"> 0.70 </td><td align="right"> 0.15 </td><td align="right"> -2.16 </td></tr><tr height="21"><td align="right" height="21" style="height:21px;"> 3 </td><td align="right"> 1.21 </td><td align="right"> 0.15 </td><td align="right"> -2.18 </td></tr></tbody></table> 表1:横向成角度的方法（单位mm）立体定向坐标。 <table fo:keep-together.within-page="1"><tbody><tr height="21"><td height="21" style="height:21px;"> 注射 </td><td> 前/后 </td><td> 内侧/外侧 </td><td> 背/腹 </td></tr><tr height="21"><td align="right" heighT ="21"的风格="高度:21px;"&gt; 1 </td><td align="right"> -0.75 </td><td align="right"> -0.96 </td><td align="right"> -2.10 </td></tr><tr height="21"><td align="right" height="21" style="height:21px;"> 2 </td><td align="right"> -1.00 </td><td align="right"> -0.96 </td><td align="right"> -2.05 </td></tr><tr height="21"><td align="right" height="21" style="height:21px;"> 3 </td><td align="right"> -1.25 </td><td align="right"> -0.96 </td><td align="right"> -2.00 </td></tr></tbody></table> 表2:对于后验斜角方法（单位mm）立体定向坐标。

Watch this Scientific Journal Video about A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology at JoVE.com

A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology

Here we present methodology for the production of a focal stroke in murine white matter by local injection of an irreversible endothelial nitric oxide synthase (eNOS) inhibitor (L-Nio). Presented are two stereotactic variations, retrograde neuronal tracing, and fresh tissue labeling and dissection that expand the potential applications of this technique.

皮层下的多功能小鼠模型白质行程轴索变性和白质神经生物学的研究

Stroke affecting white matter accounts for up to 25% of clinical stroke presentations, occurs silently at rates that may be 5-10 fold greater, and ...

a-versatile-murine-model-subcortical-white-matter-stroke-for-study

Universidad San Sebastian

Research

JoVE Journal

Medicine

8.0K 次观看.  University of California, Los Angeles. Here we present methodology for the production of a focal stroke in murine white matter by local injection of an irreversible endothelial nitric oxide synthase (eNOS) inhibitor (L-Nio). Presented are two stereotactic variations, retrograde neuronal tracing, and fresh tissue labeling and dissection that expand the potential applications of this technique.

视频: A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology

Mouse Models of Periventricular Leukomalacia

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature infants and the most common antecedent of cerebral palsy. PVL is characterized by periventricular white matter damage with prominent oligodendroglial injury. Hypoxia/ischemia with or without systemic infection/inflammation are the primary causes of PVL. We use P6 mice to create models of neonatal brain injury by the induction of hypoxia/ischemia with or without systemic infection/inflammation with unilateral carotid ligation followed by exposure to hypoxia with or without injection of the endotoxin lipopolysaccharide (LPS). Immunohistochemistry of myelin basic protein (MBP) or O1 and electron microscopic examination show prominent myelin loss in cerebral white matter with additional damage to the hippocampus and thalamus. Establishment of mouse models of PVL will greatly facilitate the study of disease pathogenesis using available transgenic mouse strains, conduction of drug trials in a relatively high throughput manner to identify candidate therapeutic agents, and testing of stem cell transplantation using immunodeficiency mouse strains.

We established mouse models of periventricular leukomalacia (PVL), the predominant brain injury in premature infants characterized by periventricular white matter lesions. Hypoxia/ischemia with/without systemic infection are the primary causes of PVL. Unilateral carotid ligation and hypoxia exposure with/without lipopolysaccharide injection creates PVL-like lesions in P6 mice.

脑室周围白质软化的小鼠模型

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature ...

科研

神经科学

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

造型中风小鼠:在远端大脑中动脉永久性混凝

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

医学

Circumscribed Capsular Infarct Modeling Using a Photothrombotic Technique

Recent increase in the prevalence rate of white matter stroke demands specific research in the field. However, the lack of a pertinent animal model for white matter stroke has hampered research investigations. Here, we describe a novel method for creating a circumscribed capsular infarct that minimizes damage to neighboring gray matter structures. We used pre-surgery neural tracing with adeno-associated virus-green fluorescent protein (AAV-GFP) to identify somatotopic organization of the forelimb area within the internal capsule. The adjustment of light intensity based on different optical properties of gray and white matter contributes to selective destruction of white matter with relative preservation of gray matter. Accurate positioning of optical-neural interface enables destruction of entire forelimb area in the internal capsule, which leads to a marked and persistent motor deficit. Thus, this technique produces highly replicable capsular infarct lesions with a persistent motor deficit. The model will be helpful not only to study white matter stroke (WMS) at the behavioral, circuit, and cellular levels, but also to assess its usefulness for development of new therapeutic and rehabilitative interventions.

This manuscript describes a modeling technique of capsular infarct. Here we utilized a modified photothrombotic technique with low intensity of light after pre-surgery target mapping. Using this technique, we created a circumscribed capsular infarct model with persistent motor impairment.

外切囊梗死模型使用光化学技术

Recent increase in the prevalence rate of white matter stroke demands specific research in the field. However, the lack of a pertinent animal model for ...

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of motor and cognitive disabilities. Its high prevalence poses a serious health burden, as stroke is among the principal causes of long-term disability and death worldwide1. Recovery of neuronal function is, in most cases, only partial. So far, treatment options are very limited, in particular due to the narrow time window for thrombolysis2,3. Determining methods to accelerate recovery from stroke remains a prime medical goal; however, this has been hampered by insufficient mechanistic insights into the recovery process. Experimental stroke researchers frequently employ rodent models of focal cerebral ischemia. Beyond the acute phase, stroke research is increasingly focused on the sub-acute and chronic phase following cerebral ischemia. Most stroke researchers apply permanent or transient occlusion of the MCA in mice or rats. In patients, occlusions of the MCA are among the most frequent causes of ischemic stroke4. Besides proximal occlusion of the MCA using the filament model, surgical occlusion of the distal MCA is probably the most frequently used model in experimental stroke research5. Occlusion of a distal (to the branching of the lenticulo-striate arteries) MCA branch typically spares the striatum and primarily affects the neocortex. Vessel occlusion can be permanent or transient. High reproducibility of lesion volume and very low mortality rates with respect to the long-term outcome are the main advantages of this model. Here, we demonstrate how to perform a chronic cranial window (CW) preparation lateral to the sagittal sinus, and afterwards how to surgically induce a distal stroke underneath the window using a craniotomy approach. This approach can be applied for sequential imaging of acute and chronic changes following ischemia via epi-illuminating, confocal laser scanning, and two-photon intravital microscopy.

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Surgical occlusion of a distal middle cerebral artery branch (MCAo) is a frequently used model in experimental stroke research. This manuscript describes the basic technique of permanent MCAo, combined with the insertion of a lateral cranial window, which offers the opportunity for longitudinal intravital microscopy in mice.

横向慢性颅窗口准备启用<em&gt;在体内</em&gt;观察继末节小鼠大脑中动脉闭塞

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of ...

Rat Model of Widespread Cerebral Cortical Demyelination Induced by an Intracerebral Injection of Pro-Inflammatory Cytokines

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and death, caused by white matter lesions in the spinal cord and cerebellum, as well as by demyelination in grey matter. Whilst conventional models of experimental allergic encephalomyelitis are suitable for the investigation of the cell-mediated inflammation in the spinal and cerebellar white matter, they fail to address grey matter pathologies. Here, we present the experimental protocol for a novel rat model of cortical demyelination allowing the investigation of the pathological and molecular mechanisms leading to cortical lesions. The demyelination is induced by an immunization with low-dose myelin oligodendrocyte glycoprotein (MOG) in an incomplete Freund&#39;s adjuvant followed by a catheter-mediated intracerebral delivery of pro-inflammatory cytokines. The catheter, moreover, enables multiple rounds of demyelination without causing injection-induced trauma, as well as the intracerebral delivery of potential therapeutic drugs undergoing a preclinical investigation. The method is also ethically favorable as animal pain and distress or disability are controlled and relatively minimal. The expected timeframe for the implementation of the entire protocol is around 8 - 10 weeks.

The protocol presented here allows the reproduction of a widespread grey matter demyelination of both cortical hemispheres in adult male Dark Agouti rats. The method comprises of intracerebral implantation of a catheter, subclinical immunization against myelin oligodendrocyte glycoprotein, and intracerebral injection of a pro-inflammatory cytokine mixture through the implanted catheter.

由亲炎细胞因子的内脑注射诱导的广度脑皮质脱叶的鼠模型

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and ...

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, ...

Modeling Stroke in Mice: Transient Middle Cerebral Artery Occlusion via the External Carotid Artery

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options are limited to a small proportion of stroke patients within the first hours after stroke. Novel therapeutic strategies are being extensively investigated, especially to prolong the therapeutic time window. These current investigations include the study of important pathophysiological pathways after stroke, such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Over the last decade, there has been increasing concern about the poor reproducibility of experimental results and scientific findings among independent research groups. To overcome the so-called &#8220;replication crisis&#8221;, detailed standardized models for all procedures are urgently needed. As an effort within the &#8220;ImmunoStroke&#8221; research consortium (https://immunostroke.de/), a standardized mouse model of transient middle cerebral artery occlusion (MCAo) is proposed. This model allows the complete restoration of the blood flow upon removal of the filament, simulating the therapeutic or spontaneous clot lysis that occurs in a large proportion of human strokes. The surgical procedure of this &#8220;filament&#8221; stroke model and tools for its functional analysis are demonstrated in the accompanying video.

Different models of middle cerebral artery occlusion (MCAo) are used in experimental stroke research. Here, an experimental stroke model of transient MCAo via the external carotid artery (ECA) is described, which aims to mimic human stroke, in which the cerebrovascular thrombus is removed due to spontaneous clot lysis or therapy.

模拟小鼠中风:通过颈外动脉的短暂性脑中动脉闭塞

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options ...

建立经颅大脑中动脉闭塞小鼠模型

A Modified Transcranial Middle Cerebral Artery Occlusion Model to Study Stroke Outcomes in Aged Mice

In experimental stroke research, middle cerebral artery occlusion (MCAO) with an intraluminal filament is widely used to model ischemic stroke in mice. The filament MCAO model typically exhibits a massive cerebral infarction in C57Bl/6 mice that sometimes includes brain tissue in the territory supplied by the posterior cerebral artery, which is largely due to a high incidence of posterior communicating artery atresia. This phenomenon is considered a major contributor to the high mortality rate observed in C57Bl/6 mice during long-term stroke recovery after filament MCAO. Thus, many chronic stroke studies exploit distal MCAO models. However, these models usually produce infarction only in the cortex area, and consequently, the assessment of post-stroke neurologic deficits could be a challenge. This study has established a modified transcranial MCAO model in which the MCA at the trunk is partially occluded either permanently or transiently via a small cranial window. Since the occlusion location is relatively proximal to the origin of the MCA, this model generates brain damage in both the cortex and striatum. Extensive characterization of this model has demonstrated an excellent long-term survival rate, even in aged mice, as well as readily detectable neurologic deficits. Therefore, the MCAO mouse model described here represents a valuable tool for experimental stroke research.

作者聚焦:中风研究的创新技术和未来方向 

This protocol demonstrates a unique mouse stroke model with a medium-sized infarct and an excellent survival rate. This model allows preclinical stroke researchers to extend the ischemia duration, use aged mice, and assess long-term functional outcomes.

改进的经颅大脑中动脉闭塞模型研究老年小鼠脑卒中结局

In experimental stroke research, middle cerebral artery occlusion (MCAO) with an intraluminal filament is widely used to model ischemic stroke in mice. ...

皮层下的多功能小鼠模型白质行程轴索变性和白质神经生物学的研究

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改进的经颅大脑中动脉闭塞模型研究老年小鼠脑卒中结局

改进的经颅大脑中动脉闭塞模型研究老年小鼠脑卒中结局