这项工作是由来自医疗系统工程研究所（IMSE）从GIST GIST加州理工学院联合基金（K04592）的资助，并通过韩国NRF的基础科学研究计划由科学，信息和通信技术及未来规划部提供资金支持（NRF-2013R1A2A2A01067890）。

所有程序均按照科学与技术（GIST）的光州研究所的机构准则进行的，所有的程序是由机构动物护理和使用委员会批准GIST。 1.预损毁步骤<ol><li> 利用AAV-GFP内囊前肢区的识别 <ol><li>按照机构和国家指导方针 - （13周〜400克，11）楼和处理SD大鼠。 </li><li>消毒用适当的消毒器（蒸汽或等离子体灭菌器）的所有外科手术工具和电极。在121℃，使用蒸汽灭菌器作为消毒30分钟和干燥30分钟设置。 </li><li>通过肌内注射麻醉用氯胺酮盐酸盐（100毫克/千克）和赛拉嗪（7毫克/千克）的混合物中的动物。通过捏爪子检查麻醉深度。通过在一个加热垫保持体温在37.5±0.5°C体内动物。 </li><li>放置动物用耳棒和嘴座立体定位框架。 </li><li>清洁和消毒手术部位用70％酒精和聚维酮碘溶液。渗入在预期颅骨切开区域的头皮下2％利多卡因盐酸盐，以减少手术的痛苦。 </li><li>适用兽医眼药膏，以防止眼睛干燥。放置无菌披盖在动物的手术部位。保持在无菌条件下的所有过程。 </li><li>执行使用手术刀2cm的中线颅骨切开并用导线拉钩双边缩回皮肤。干后用棉签和30％过氧化氢的头骨。 </li><li>请使用手电钻片在运动皮层的前肢区域的孔（:从囟+2.5，ML:AP±2.5从中线），并清除与微currette病毒注射的道。 </li><li>在冰上和负载1微升的解冻的AAV-GFP（2×10 12病毒分子/ ml）的病毒在10微升的注射器。放置立体定位框架上的注射器。 </li><li>移动针的预先制作孔和降低针1mm深入硬脑膜。 </li><li>注入使用高精度微泵病毒缓慢（0.1微升/分钟）和留在原地针另外10分钟以使病毒扩散出来。 </li><li>清洗用生理盐水冲洗手术部位后，固定用3-0尼龙缝合伤口;从立体定位框架释放鼠，并将其转移到一个回收室。通过肌内注射，术后疼痛控制管理酮洛芬（2毫克/千克）。 </li><li>保持体温（37℃）加热垫和管理第二代头孢类抗生素（0.1％，1毫升）通过经由作为必要皮下注射肌内注射和2％的盐酸利多卡因。不要离开无人看管的动物，直到它已经恢复了足够的意识来维持胸骨斜卧。单家动物，直到完全康复。 </li><li>继2 - 第三周回升，深深地通过在引擎盖肌内注射麻醉用盐酸氯胺酮（300毫克/千克）的过量的老鼠。缺乏脚趾掐响应，脉搏和呼吸的确认动物死亡。放置在引擎盖大鼠仰卧。 </li><li>通过&#39;Y&#39;形切口，打开腹腔打开胸腔。紧紧地钳止血钳降主动脉破裂和大鼠心脏的血液引流的右心房。发起灌注到心脏的用冷的1％多聚甲醛左心室5分钟（10毫升/分钟），随后加入4％多聚甲醛30分钟（10毫升/分钟）。 </li><li>移除胎体使用一对剪刀的大鼠头部。使从颈部到鼻子正中切口，用剪刀或咬骨钳使颅骨暴露除去颈部肌肉。轻轻地从大脑解剖的头骨和杜拉斯的。 </li><li>提取大脑，并放置在大鼠脑在填充有4％多聚甲醛过夜50ml锥形管中。第二天，洗涤脑用1×PBS的3倍，并放置在30％的蔗糖溶液。 </li><li>后大脑完全沉到30％的蔗糖溶液的底部，放置在与OCT化合物中的cryomold大脑在cryotome -20℃。切片在冠状平面中的大脑的厚度为40μm和200μm的间隔。 </li><li>执行使用滑动方法14 GFP免疫组织化学染色。申请初级抗体（1:抗绿色荧光蛋白，兔IgG级分的200），以脑切片在4℃下过夜。关于第二日，洗净，用1％磷酸盐缓冲盐水与吐温20（PBST）洗涤3次，并应用第二抗体（1:500山羊抗兔IgG抗体（H + L））1小时。冲洗用1％PBST 3次的幻灯片。放置在脑切片的护罩玻璃。 </li><li>使用荧光显微镜（激发波长470纳米，发射波长525纳米，放大倍率5倍），观察AAV-GFP转导的轴突内囊。比较大鼠脑图谱15转导的轴突的位置，以确定转导的轴突的立体坐标</li></ol></li><li> 预损毁的光强度调节适合囊梗死模型 <ol><li> 光学神经系统的接口建设 <ol><li>切27号脊椎针头的适当长度（4厘米）内使用切割钻头管心针。 注:切割可以压缩和粉碎脊椎针头;移除管心针和抛光脊椎针头尖端以除去脊椎针头的破碎部和保持脊椎针头的内口径。 </li><li>剥离光纤的护套的适当长度（10厘米）（125微米用62.5微米芯）一侧接插线。 </li><li>将unjacked光网络误码率到金属管（外径:3.8毫米，内径3.3毫米和长度:17mm）的，然后在纤维周围夹紧。金属管是有用的，以填补在光纤和脊椎针头的轮毂之间的空间。夹紧带压紧金属管的下1/2的两倍。 </li><li>应用在光纤可热固化的环氧树脂和光纤插入脊椎针头。施加附加的环氧树脂在轮毂的空的空间。固化环氧树脂，在100℃下20分钟进行稳定的固定。 </li><li>切割光纤即伸出脊椎针头中，并在使用金刚石研磨（抛光）片脊椎针头的尖端抛光该光纤。 </li><li>接插线的FC / PC连接器部分连接到绿色激光系统的耦合器，测量从使用数字光功率和能量计的光纤的前端的光强度。 </li></ol></li></ol></li></ol> 2. Photothrombotic梗死损毁内囊<ol><li>消毒用适当的消毒器（蒸汽或等离子体灭菌器）的所有外科手术工具和电极。在121℃，使用蒸汽灭菌器作为消毒30分钟和干燥30分钟设置。 </li><li>麻醉动物（〜400克，11 - 13周）通过肌肉注射氯胺酮盐酸盐（100毫克/千克）和赛拉嗪（7毫克/千克）的混合物中。通过捏爪子检查麻醉深度。通过动物的身下加热垫保持体温在37.5±0.5℃。 </li><li>放置动物用耳棒和嘴座立体定位框架。 </li><li>清洁和消毒手术部位用70％酒精和聚维酮碘溶液。渗入在预期颅骨切开区域的头皮下2％利多卡因盐酸盐，以减少手术的痛苦。适用兽医眼药膏，以防止眼睛干燥。 </li><li>应用无菌悬垂在一个进制和暴露手术部位。保持在无菌条件下的所有过程。 </li><li>执行2厘米的中线颅骨切开并用导线拉钩双边缩回皮肤。干后用棉掉期和过氧化氢的头骨。 </li><li>调节鼻夹的高度，直到前囟和lambda都在同一水平对齐。关键步骤:该对准是关键的正确看待更深的结构，如在主试验中的内囊执行梗塞病灶。 </li><li>做一个孔（直径2毫米; AP:-2.04从囟; ML:±3.0从中线）用钻头诱导photothrombosis。 </li><li>抛光和清洁光学接口的光学纤维尖端。修复ONI的立体框架无弯曲。检查ONI的前端与前和光学接口的插入之后明确擦拭它。 </li><li>事先测量从光纤的末端的激光强度的光学的插入l接口到大鼠脑的目标部位。调整激光强度到3.5毫瓦，通过手术前步骤所确认，在光纤的前端。 </li><li>插入ONI进入内囊的目标区域（-7.8毫米从手术前工序确认）通过钻孔。 </li><li>保持photothrombosis期间体温在37.5±0.5℃。较低的体温可能不会产生梗死的预期程度。通过尾静脉注射玫瑰红（2毫升/千克）。 </li><li>打开孟加拉玫瑰红注射后的532纳米绿光激光器90秒1分钟。照射后，轻轻地从大脑中删除ONI。清洗手术部位后，固定伤口保护与3-0尼龙缝合伤口;从立体定位框架释放鼠，并将其转移到一个回收室。 </li><li>对于假手术组（SOG），执行相同的病变制作过程中，除了盐水注射（0.2ml毫升/ 100克）代替玫瑰孟加拉。 </li><li>保持体温（37℃）下加热垫手术后和通过肌内注射施用抗生素（第二代头孢菌素，0.1％，1毫升）。不要离开无人看管的动物，直到它已经恢复了足够的意识，以保持胸骨斜卧。不要手术后动物回归由其他动物占据了笼子，直到完全康复。 注:在相同的步骤中进行的初步实验，以找到从1毫瓦的最佳的光强到5毫瓦和可能需要的程序以获取病变的令人满意的程度在不同​​的条件下。 </li></ol> 3.荚膜梗死损毁评估<ol><li> 行为测试和实验动物分组 <ol><li>执行单粒料达到如Whishaw 等人描述的任务。14冲程造型后每天评估前肢的运动缺陷为1周。执行单个颗粒达成任务使用透明有机玻璃（30×15×35厘米高）用1厘米宽的狭缝和在前壁的中部的前面一个食品成品（SPRT）在食品限制动物（90％的控制体重）。 </li><li>放置食品货架倾斜对侧优选前肢上的颗粒。辖每节20丸3周。 注:SPRTs的成功数定义为在动物掌握食物颗粒，并把它放进嘴里没有放弃它范围。 </li><li>计算分数为成功达到的百分比，其由下式定义: <img alt="公式1" src="/files/ftp_upload/53281/53281equation1.jpg" /> 注:我们把动物分为3组:假手术组（SOG），中度恢复组（MRG），差恢复组（PRG）。如果一个中风后SPRT得分&gt; 50％，我们归类大鼠作为MRG，这表明相当病变的存在，但た的不完全破坏rget。如果笔划后SPRT得分&lt;50％的行程预SPRT得分相比，我们分类的组作为PRG，这表明在目标完全损毁。 </li></ol></li><li> 梗塞损毁的Neurohistological确认 <ol><li>如前所述，用4％多聚甲醛进行心脏灌注。后的脑在30％蔗糖溶液完全下沉，在一厚度为10微米，并用切片机或cryotome 4的200微米间距进行冠状切片。 </li><li>染色用H＆E尼氏，Luxol坚牢蓝-PAS，神经丝蛋白-L或胶质纤维酸性蛋白染色，并观察组织学的研究结果，以确定可以覆盖在所述目标区域中的内囊，观察染色的整个广度的最佳的光强4,17。 </li><li>使用ImageJ软件，测量大脑幻灯片内囊的光化学梗塞面积的体积。<ol><li>为了测量梗死区的体积，启动"ImageJ的"软件。要打开的文件堆放，选择"图像到堆栈"（"图像"→"栈"→"图像叠图"）。编辑文件名，选择"设置规模"（"分析"→"设置规模"）来编辑的规模。 </li><li>在"插件"中，选择"测量栈"来计算图像的体积或面积。将2个图像的间隔距离为"切片间隔"。让所有图像的ROI的图纸（感兴趣区域），然后单击"度量"。 注:该软件的ImageJ的"自动计算面积和每个图像的数量和它们的总体积。 </li></ol></li></ol></li></ol>

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The authors have nothing to disclose.

这里提出的囊梗死模型表明，在前肢的功能显着和持久的运动障碍有针对性的病变。皮质下囊中风之前的机型都表现出了足够的程度运动功能障碍和快速恢复过程6,8,9的。在这个意义上，该模型类似于表现出长期的功能障碍的临床荚膜梗塞例。 在一个外接荚膜梗塞模型的发展中的最重要的步骤是:1），以正确地识别旨在禁止内部胶囊内的功能的主体部的躯体表示; 2）识别绿色激光，它可与邻近的灰质结构最小侵入破坏内囊的整个广度的最优强度;和3），以精确地放置光纤在目标结构。虽然呈现技术可诱发外接具有高的复制率（&gt; 70％）D囊梗死模型，破坏完整性的定位和程度小的差异覆盖内囊的整个宽度可能占不同的运动障碍。 尽管躯体组织15的争议皮质脊髓束坐落在人类内囊的后肢的前半部分。与此相反，一直没有等效分类或在啮齿动物内囊的躯体组织的详细解释。关于躯体组织的知识的缺乏往往导致梗塞损毁的不正确的目标与囊梗死模型中不同的电机结果的内囊中。然而，我们确定在内部胶囊的尾部的GFP转导的轴突，这可能代表前肢运动纤维的路径。此外，损毁这一领域正在示威的编前肢技能达到的显着和持续的赤字。因此，我们建议对损毁立体定向内囊尾侧部分，以提高囊梗死模型的有效性。 光强度的预调整是强制性的，以产生在中风模型梗塞病灶的均匀程度，因为动物品系，体重，轻ONI的源和类型可以生成梗死的不同的尺寸。因此，在实验动物一脉相承的，体重采用不同的光照强度初步实验应该进行，直到满意梗塞病灶与最小的光的强度来实现的。 可以摧毁囊纤维的整个广度强的光强度（前 - 后和背腹程度），该对应于与向邻近结构损害最小前肢区域被认为是最佳的光强度。该forelim内囊的B区由优丘脑和视束inferiorly界。因此，ONI插入深度应准确摧毁IC的整个范围中的背腹方向，与上和下邻近结构的同时保存。 ONI的不准确的放置导致在IC，这导致电动机赤字作为内囊的其余锥体纤维的突触可塑性的结果的快速恢复的不完全破坏。在串行的组织学检查，在持久性运动障碍的诱导最混杂因素是ONI，从而导致了不破坏PLIC 4,16的整个广度的不正确的定位。因此，仔细应注意以达到正确的目标。最近，布拉西等人报告说，持久的纯运动缺陷可以通过在后内囊作出梗塞病灶产生使用内皮素-1（ET-1）17。然而，ET-1可以通过ET-1的扩散破坏邻近的灰质结构。 行为测试是在实验室中的立即可用的基准，以评估在内部胶囊梗塞病灶的形成。然而，一个星期后，梗死损毁电机性能评估建议将动物分为低分化恢复组。温和恢复被定义为增加由&gt; 50％的损毁前的分数相比，业绩得分，而恢复差被定义为&lt;50％的回收率。间的前肢马达行为测试中，单个的颗粒达到的任务是对中风引起的电机性能14的定量和定性测量的最灵敏的测试之一。任务定量测量达到成功，同时提供前肢使用的分析，如抓和检索一个食品沉淀。趋近运动的定性分析也有助于通过区分真正的功能恢复或补偿20来区分中风恢复的质量。在这里，我们简要介绍了SPRT的定量测量;然而，建议进一步的详细分析使用基于一帧接一帧分析拍摄和得分定性分析。 这里介绍的技术不必局限于外接囊梗死模型的诱导。该技术可应用于在脑白质其他领域，例如胼胝体，前连合和神经结构之间连接光纤的梗塞病灶的诱导。基于白质的光学特性的微小ONI和光化学技术的组合是可能破坏目标结构使用对邻近结构的损害最小。例如，腔隙性梗死可以很容易地产生通过靶向涉及电机，认知和记忆功能的皮层下结构ð。当目标结构是大的，可能需要ONI和不同的定位和成角度的轨迹多次插入产生病变的所希望的程度。 有在该技术一些局限性。该技术是，足以证明在PLIC和随后的恢复梗塞损毁的结果。然而，这种模式并不能反映人的WMS的全谱因为白质破坏光化学略有人类WMS不同。因此，神经生物学或MRI影像表现可以以光化学损毁的早期阶段表现出不同的特征。因此，该模型应当适当使用权衡模型的优点和缺点。从技术上讲，不是所有的手术能够产生标记和永久电机赤字在这个模型，因为它需要非常精确的程序。小号pecifically，需要训练有素，经验丰富的双手生产高重复性在这个模型的产生。 总之，组合使用光化学技术中，光强度的优化，和正确定位的是生产一种外接荚膜梗塞模型一种有用的技术。这种模式将有助于不仅要研究的行为，电路WMS，和细胞水平，而且评估新的治疗和康复干预措施的有效性。

直到最近，"灰质行程（GMS）模型"已经被专门用来了解中风的病理生理机制，并指导新治疗方法的发展。然而，出现了行程的患病率增加，影响在老年个体，这构成15皮层下白质-所有笔划1,2的25％。大量的研究已经进行使用GMS模型对中风，而有一些已经使用白质行程（WMS）模型的研究很少。在啮齿类动物中白质比在人类或灵长类白质基本上更少。因此，更难以在脑白质3选择性地访问和破坏靶区域。此外，没有有效的工具已经被开发迄今为止为选择性地破坏的靶向白质的计划的程度。因此，一直存在缺乏脑白质笔划研究适当的模型。 动物ST洛克模型经常用于监测马达恢复的新康复和治疗方法的发展的进展。它是理想的，利用表现出一个长期的神经功能缺损一致与人类中风4,5展示了解剖改变的动物模型。在这方面，马达赤字和脑以下梗塞损毁的广泛参与快速恢复可能不追求中风研究的现实的。以前荚膜梗塞模型已被制成由内皮素-1（ET-1）的内部颈动脉或脉络膜前动脉和扩散的闭塞进入内囊6-9。尽管如此，动脉闭塞，需要动脉小心剥离，但它产生的梗塞病灶的面积广，包括内囊，而不持久行为缺陷。此外，ET-1是不是扩散彻底摧毁内囊后肢，因此不太明显或持续BEHavioral赤字。 一个光化学梗死模型已被广泛地用于产生各种类型的皮质梗塞病灶和皮层下结构10的。该技术包括静脉内给药后焦距照明，这导致在小血管的血小板凝集和生成梗塞病灶10。光化学技术已被广泛使用，以创建GMS病变，而它很少被用来产生WMS病变5,11。对于这种技术，玫瑰红染料和光照射的组合已被证明是在靶结构的破坏是有用的，从而导致相应的功能缺损。的光化学技术的关键因素是光照射，因为它决定了梗塞病灶的大小。光照射结果在灰质和白质不同的效果，由于光线的散射是白龙马高出4倍tter灰质比12;因此，如果光强度具有足够低辐照度（&lt;1,140毫瓦/毫米2），一个能限制扩展到光化学病变影响的程度的白质（ 即 ，内囊）。例如，高能量的光可以诱导两个灰质和白质梗死，但是较低的能量光只能在白质诱导photothrombosis。另外，光能量的穿透是非常有限的。光能量大约99％失去了从外光13源1毫米。因此，预期能够准确定位，低能量的光只在与邻近的灰质的最小侵入白质诱导photothrombosis。 在这里，我们描述了一种新方法，在啮齿类动物中的内囊的前肢区域来创建梗塞病灶。我们描述的前肢区域的识别方法在内部CApsule，光照射，包括调整与光的传送，和梗塞病灶的生成的技术。我们还描述了用于评估囊建模的完整性行为测试。

<table><tbody><tr><td>DC Temperature controller</td><td>WORLD PRECISION INSTRUMENTS, INC.</td><td>ATC1000</td><td></td></tr><tr><td>Digital Stereotaxic Instruments</td><td>STOELTING CO.</td><td>51900</td><td></td></tr><tr><td>Electrical Stimulator</td><td>CyberMedic Corp.</td><td>EMGFES 2000</td><td></td></tr><tr><td>Epoxy&amp;nbsp;</td><td>Precision Fiber Products, INC.</td><td>PFP-353ND1</td><td>Mix Ratio:&lt;br/&gt; 10(A):1(B-hardener) by weight&amp;nbsp;&lt;br/&gt; Curing Schedule:&lt;br/&gt; 1 min @150 &amp;deg;C&lt;br/&gt; 2 ~ 5 min @120 &amp;deg;C&lt;br/&gt; 5 ~ 10 min @100 &amp;deg;C&lt;br/&gt; 15 ~ 30 min @80 &amp;deg;C</td></tr><tr><td>Fiber Optic Scribe&amp;nbsp;</td><td>THORLABS, INC</td><td>S90R</td><td></td></tr><tr><td>Fiber patch cable</td><td>KOREA OPTRON Corp.</td><td></td><td>Outer diameter: 3 mm&lt;br/&gt; &amp;Oslash;200 &amp;micro;m&lt;br/&gt; 0.39 NA&lt;br/&gt; FC/PC-FC/PC&lt;br/&gt; 1 m</td></tr><tr><td>Laser Power Supply</td><td>CHANGCHUN NEW INDUSTRIES OPTOELECTRONICS TECH. CO., LTD.</td><td>MGL-FN-532nm-200mW-14010196</td><td></td></tr><tr><td>Crimp ring&amp;nbsp;</td><td>DAWOOTECH CO.,LTD.</td><td></td><td>Length: 19 mm&lt;br/&gt; Inner diameter: 3 mm&lt;br/&gt; Outer diameter: 3.8 mm&lt;br/&gt; Material: SUS</td></tr><tr><td>Micro4-micro syringe pump controller</td><td>WORLD PRECISION INSTRUMENTS, INC</td><td>95100</td><td></td></tr><tr><td>Optical Power Meter</td><td>THOLABS, INC</td><td>PM100D</td><td></td></tr><tr><td>Diamond lapping (polishing) sheet</td><td>THORLABS, INC</td><td>LF3D</td><td>Grit : 3 &amp;micro;m</td></tr><tr><td>Diamond lapping (polishing) sheet</td><td>THORLABS, INC</td><td>LF6D</td><td>Grit : 6 &amp;micro;m</td></tr><tr><td>Rose Bengal</td><td>SIGMA-ALDRICH CO. LLC.</td><td>330000</td><td></td></tr><tr><td>Needle for spinal anesthesia with pencil point tip (Spinal needle)&amp;nbsp;</td><td>B.BRAUN MELSUNGEN AG&amp;nbsp;</td><td>4502027</td><td>Size: 27 G&lt;br/&gt; Length: 88 mm&lt;br/&gt; Needle: 0.40 mm</td></tr><tr><td>Waterproof sandpaper&amp;nbsp;</td><td>DEERFOS CO.,LTD</td><td>CC261</td><td>Grit : 1,000 &amp;micro;m</td></tr><tr><td>Nanofil 10&amp;nbsp;&amp;micro;l syringe</td><td>WORLD PRECISION INSTRUMENTS, INC</td><td>NANOFIL</td><td></td></tr><tr><td>Nanofil 33&amp;nbsp;G BVLD needle</td><td>WORLD PRECISION INSTRUMENTS, INC</td><td>NF33BV-2</td><td></td></tr><tr><td>AAV-GFP virus</td><td>UNC Vector Core</td><td>AAV2-CamKIIa-eYFP</td><td>2 x 10&lt;sup&gt;12&lt;/sup&gt; virus molecules/ml</td></tr><tr><td>Anti-Green Fluorescent Protein, Rabbit IgG fraction</td><td>Life Technologies, INC</td><td>A11122</td><td>primary antibody (1:200)</td></tr><tr><td>Goat Anti-Rabbit IgG (H + L)</td><td>Life Technologies, INC</td><td>A11034</td><td>secondary antibody (1:500)</td></tr><tr><td>Ceftezole</td><td>GUJU Pharma CO.,LTD.</td><td>A27802741</td><td>0.1%, 1 ml</td></tr><tr><td>Lidocain hydrochloride injection</td><td>JEIL PHARMACEUTICAL CO.,LTD.</td><td>A04900271</td><td>2%, 1 ml</td></tr><tr><td>Hand Piece Drill</td><td>Seshin</td><td></td><td></td></tr><tr><td>Digital optical power and energy meter</td><td>THORLABS, INC</td><td>PM100D</td><td></td></tr><tr><td>Ketoprofen</td><td>UNIBIOTech</td><td></td><td></td></tr></tbody></table>

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.

这里介绍的方法的目的是创建一个持久的运动障碍外切囊梗塞。因此，重要的是正确地判断在手术前的工序中的内部胶囊内的目标。内囊锥体纤维的躯体映射尚未解决日期。正确地识别内部胶囊内的目标，前肢区域必须划定。 AAV-GFP的注射入运动皮层的前肢区域可以跟踪锥体纤维的轴突内囊（图1）。其它神经示踪剂，如生物素化的葡聚糖胺（BDA），可以用于相同的目的。内部胶囊内的目标的立体坐标可以通过跟踪轴突突起从运动皮层到内囊的前肢区域起源阐明。 图1.源自运动皮层的前肢区域的AAV-GFP。GFP转导的轴突纤维的注射后2周的内囊的前肢区的鉴定在丘脑（箭头）的腹外侧核示出并内囊（箭头）的尾部分。虚线表示的内囊的轮廓，和数字是指从囟的距离。河马，海马; CPU，尾壳核; VL，腹外侧核; IC，内囊。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53281/53281fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 根据动物的品系和体重，种类和直径的最佳的光强可以是不同的<html>光纤。因此，最佳的光强度应该确定分别先于主梗塞损毁实验。使用光化学过程中，光强度可逐渐增加，直到病变的程度覆盖内囊的整个宽度，而不通过比较病理程度破坏邻近的灰质结构（图2）。最佳的光强可以被验证的梗塞病灶和位置。 <img alt="图2" src="/files/ftp_upload/53281/53281fig2.jpg" /> 图2. 程度的梗塞病灶的横跨激光光的不同强度从2毫瓦到光化学损毁后5毫瓦2星期，最佳光强度被认为是为3mW和4毫瓦之间在这个实验设置。箭头表示梗塞病灶​​。/53281/53281fig2large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 我们优选使用在其中光纤被包含在薄的金属管（脊椎针头）的军情局。光纤可以从光纤，这是可能产生沿着光学纤维束的其他神经损伤的侧面产生最小的光散射。光纤的包套也是有利，以防止该光纤的弯曲在更深的目标，以及向ONI附加到立体定位框架（图3）。 <img alt="图3" src="/files/ftp_upload/53281/53281fig3.jpg" /> 图3. 光学神经接口（ONI）的建设。（一）脊椎穿刺针的切割。 （二）剥离光纤。 （C＆D）的锚定金属管被插在剥离光纤和局促到光纤固定于脊椎针头的中心。 （E）的环氧加光纤被插入脊椎针头。 （f）在环氧树脂在100℃硬化20分钟。 （G）的光纤是在脊椎针头的末端裂解。 （h）本光纤被抛光。 （I）的光强度从光纤的尖端测量。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53281/53281fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 该光化学过程会产生重复性的病变和位置与在〜运动障碍70％的成功。典型荚膜梗塞病灶包括荚膜纤维（图4A）的ventrodorsal尺寸。另外，梗塞病灶沿内囊因囊纤维内部增加光散射的前后轴线延伸（图4B）位于内囊下的光道由白质纤维;因此，它通常是由一个增加的光强度的辐射损伤。连续切片和染色都必须确认整个体积和程度梗死。梗死体积为0.63±0.37 立方毫米。为了评估囊纤维，神经纤维的破坏和Luxol快蓝PAS染色是有益的。 <img alt="图4" src="/files/ftp_upload/53281/53281fig4.jpg" /> 图4. Photothrombosis后囊梗死3周的微观形貌。囊梗死的微观形貌photothrombosis 3周后。 A）在大鼠脑冠状切面的脑片。箭头指示包含光纤在丘脑和高达内囊针的管道。 B）冠状脑片showi串行尼氏染色<html>纳克梗塞病灶的整个范围内囊。箭头表示梗死病灶。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53281/53281fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 建模的成功可以通过行为测试使用单一粒料到达任务进行评估。 1周以下梗死损毁后的行为表现是一个很好的指南，以确认准确的损毁，伴随SPRT的持续和显着的障碍，尽管每天的单颗粒达到培训（图5）。一旦电机赤字显示在PRG，期间3疗效观察个月的神经功能缺损持续存在。假手术组没有表现出SPRT表现术后显著下降。 上传/ 53281 / 53281fig5.jpg"/&gt; 图5. 变化球团单囊Infart 4,20 后到达成绩 。实验组（PRG和MRG）显著展出与假手术组（SOG）相比梗死损毁后立即降低分数。该MRG表现出SPRT表演逐步复苏，而PRG随着时间的推移呈现出持续性运动障碍。欧普，光化学梗塞损毁; PRG，可怜的恢复组; MRG，适度恢复组。采用方差重复测量分析确定统计学意义。 + SOG与MRG; * SOG与PRG。数据是平均值±SEM。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53281/53281fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Circumscribed Capsular Infarct Modeling Using a Photothrombotic Technique at JoVE.com

Circumscribed Capsular Infarct Modeling Using a Photothrombotic Technique

这份手稿描述囊梗塞的建模技术。在这里，我们利用与手术后前目标映射光的低强度改良光化学技术。使用这种技术，我们创造了持续性运动障碍外接囊梗死模型。

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 ...

circumscribed-capsular-infarct-modeling-using-photothrombotic

Nanyang Technological University

Research

JoVE Journal

Medicine

7.8K 次观看.  Gwangju Institute of Science and Technology. 这份手稿描述囊梗塞的建模技术。在这里，我们利用与手术后前目标映射光的低强度改良光化学技术。使用这种技术，我们创造了持续性运动障碍外接囊梗死模型。 

视频: Circumscribed Capsular Infarct Modeling Using a Photothrombotic Technique

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

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow (ischemia) 1. Middle cerebral artery (MCA) delivers a great majority of blood to the lateral surface of the cortex 2, is the most common site of human stroke 3, and ischemia within its territory can result in extensive dysfunction or death 1,4,5. Survivors of ischemic stroke often suffer loss or disruption of motor capabilities, sensory deficits, and infarct. In an effort to capture these key characteristics of stroke, and thereby develop effective treatment, a great deal of emphasis is placed upon animal models of ischemia in MCA.
Here we present a method of permanently occluding a cortical surface blood vessel. We will present this method using an example of a relevant vessel occlusion that models the most common type, location, and outcome of human stroke, permanent middle cerebral artery occlusion (pMCAO). In this model, we surgically expose MCA in the adult rat and subsequently occlude via double ligature and transection of the vessel. This pMCAO blocks the proximal cortical branch of MCA, causing ischemia in all of MCA cortical territory, a large portion of the cortex. This method of occlusion can also be used to occlude more distal portions of cortical vessels in order to achieve more focal ischemia targeting a smaller region of cortex. The primary disadvantages of pMCAO are that the surgical procedure is somewhat invasive as a small craniotomy is required to access MCA, though this results in minimal tissue damage. The primary advantages of this model, however, are: the site of occlusion is well defined, the degree of blood flow reduction is consistent, functional and neurological impairment occurs rapidly, infarct size is consistent, and the high rate of survival allows for long-term chronic assessment.

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

We describe a highly reproducible method for the permanent occlusion of a rodent major cerebral blood vessel. This technique can be accomplished with very little peripheral damage, minimal blood loss, a high rate of long-term survival, and consistent infarct volume commensurate with the human clinical population.

永久性脑血管闭塞<em&gt;通过</em&gt;双人结扎横断

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow ...

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

玫瑰红Photothrombosis共聚焦光学成像<em&gt;在体内</em&gt;:单支脑卒中模型

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...

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

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

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

Photothrombosis引起的局部脑缺血脊髓损伤的小鼠模型

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

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of stroke than relying on indirect measurements, and will be a potent and robust vascular research tool. We use an optical imaging approach that labels thrombi with a molecular imaging thrombus marker&#160;&#8212; a Cy5.5 near-infrared fluorescent (NIRF) probe that is covalently linked to the fibrin strands of the thrombus by the fibrin-crosslinking enzymatic action of activated coagulation factor XIIIa during the process of clot maturation. A micro-computed tomography (microCT)-based approach uses thrombus-seeking gold nanoparticles (AuNPs) functionalized to target the major component of the clot: fibrin. This paper describes a detailed protocol for the combined in vivo microCT and ex vivo NIRF imaging of thromboemboli in a mouse model of embolic stroke. We show that in vivo microCT and fibrin-targeted glycol-chitosan AuNPs (fib-GC-AuNPs) can be used for visualizing both in situ thrombi and cerebral embolic thrombi. We also describe the use of in vivo microCT-based direct thrombus imaging to serially monitor the therapeutic effects of tissue plasminogen activator-mediated thrombolysis. After the last imaging session, we demonstrate by ex vivo NIRF imaging the extent and the distribution of residual thromboemboli in the brain. Finally, we describe quantitative image analyses of microCT and NIRF imaging data. The combined technique of direct thrombus imaging allows two independent methods of thrombus visualization to be compared: the area of thrombus-related fluorescent signal on ex vivo NIRF imaging vs. the volume of hyperdense microCT thrombi in vivo.

This protocol describes the application of combined near-infrared fluorescent (NIRF) imaging and micro-computed tomography (microCT) for visualizing cerebral thromboemboli. This technique allows the quantification of thrombus burden and evolution. The NIRF imaging technique visualizes fluorescently labeled thrombus in excised brain, while the microCT technique visualizes thrombus inside living animals using gold-nanoparticles.

结合近红外荧光成像和微计算机断层扫描直接可视化脑血栓

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of ...

Ferric Chloride-induced Murine Thrombosis Models

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, diabetes, obesity, cancer and chronic autoimmune rheumatologic disorders. Thrombi are the cause of most heart attacks, strokes and extremity loss, making thrombosis an extremely important public health problem. Since these thrombi stem from inappropriate platelet activation and subsequent coagulation, targeting these systems therapeutically has important clinical significance for developing safer treatments. Due to the complexities of the hemostatic system, in vitro experiments cannot replicate the blood-to-vessel wall interactions; therefore, in vivo studies are critical to understand pathological mechanisms of thrombus formation. To this end, various thrombosis models have been developed in mice. Among them, ferric chloride (FeCl3) induced vascular injury is a widely used model of occlusive thrombosis that reports platelet activation and aggregation in the context of an aseptic closed vascular system. This model is based on redox-induced endothelial cell injury, which is simple and sensitive to both anticoagulant and anti-platelets drugs. The time required for the development of a thrombus that occludes blood flow gives a quantitative measure of vascular injury, platelet activation and aggregation that is relevant to thrombotic diseases. We have significantly refined this FeCl3-induced vascular thrombosis model, which makes the data highly reproducible with minimal variation. Here we describe the model and present representative data from several experimental set-ups that demonstrate the utility of this model in thrombosis research.

We report a refined procedure of the ferric chloride (FeCl3)-induced thrombosis models on carotid and mesenteric artery as well as vein, characterized efficiently using intravital microscopy to monitor time to occlusive thrombi formation.

三氯化铁诱导小鼠模型的血栓形成

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, ...

A Fibrin-Enriched and tPA-Sensitive Photothrombotic Stroke Model

An ideal thromboembolic stroke model requires certain properties, including relatively simple surgical procedures with low mortality, a consistent infarction size and location, precipitation of platelet:fibrin intermixed blood clots similar to those in patients, and an adequate sensitivity to fibrinolytic treatment. The rose bengal (RB) dye-based photothrombotic stroke model meets the first two requirements but is highly refractory to tPA-mediated lytic treatment, presumably due to its platelet-rich, but fibrin-poor clot composition. We reason that combination of RB dye (50 mg/kg) and a sub-thrombotic dose of thrombin (80 U/kg) for photoactivation aimed at the proximal branch of middle cerebral artery (MCA) may produce fibrin-enriched and tPA-sensitive clots. Indeed, the thrombin and RB (T+RB)-combined photothrombosis model triggered mixed platelet:fibrin blood clots, as shown by immunostaining and immunoblots, and maintained consistent infarct sizes and locations plus low mortality. Moreover, intravenous injection of tPA (Alteplase, 10 mg/kg) within 2 h post-photoactivation significantly decreased the infarct size in T+RB photothrombosis. Thus, the thrombin-enhanced photothrombotic stroke model may be a useful experimental model to test novel thrombolytic therapies.

Traditional photothrombotic stroke (PTS) models mainly induce dense platelet aggregates of a high resistance to tissue plasminogen activator (tPA)-lytic treatment. Here a modified murine PTS model is introduced by co-injecting thrombin and photosensitive dye for photoactivation. The thrombin-enhanced PTS model produces mixed platelet:fibrin clots and is highly sensitive to tPA-thrombolysis.

纤维蛋白富集和 tPA 敏感的光血栓形成卒中模型

An ideal thromboembolic stroke model requires certain properties, including relatively simple surgical procedures with low mortality, a consistent ...

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, ...

Injection of Hydrogel Biomaterial Scaffolds to The Brain After Stroke

Stroke is the leading cause of disability and the fifth-leading cause of death in the United States. Approximately 87% of all strokes are ischemic strokes and are defined as the sudden blockage of a vessel supplying blood to the brain. Within minutes of the blockage, cells begin to die and result in irreparable tissue damage. Current therapeutic treatments focus on clot removal or lysis to allow for the reperfusion and prevent more severe brain damage. Although transient brain plasticity may salvage some of the damaged tissue over time, significant fractions of patients are left with neurological deficits that will never resolve. There is a lack of therapeutic options to treat neurological deficits caused by stroke, emphasizing the need to develop new strategies to treat this growing patient population. Injectable biomaterials are currently being designed to enhance brain plasticity and improve endogenous repair through the delivery of active agents or stem cells. One method to test these approaches is to utilize a rodent stroke model, inject the biomaterial into the stroke core, and assess repair. Knowing the precise location of the stroke core is imperative for the accurate treatment after stroke, therefore, a stroke model that results in a predictable stroke location is preferable to avoid the need for imaging prior to injection. The following protocol will cover how to induce a photothrombotic stroke, how to inject a hydrogel in a controlled and precise manner, and how to extract and cryosection the brain while keeping the biomaterial intact. In addition, we will highlight how these same hydrogel materials can be used for the co-delivery of stem cells. This protocol can be generalized to the use of other injectable biomaterials into the stroke core.

Stroke is a global issue with minimal treatment options and no current clinical therapy for regenerating the lost brain tissue. Here we describe methods for creating precise photothrombotic stroke in the motor cortex of rodents and subsequent injection of hydrogel biomaterials to study their effects on tissue regeneration after stroke.

中风后向大脑注射水凝胶生物材料支架

Stroke is the leading cause of disability and the fifth-leading cause of death in the United States. Approximately 87% of all strokes are ischemic strokes ...

生物学

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.remotexs.ntu.edu.sg/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 ...

外切囊梗死模型使用光化学技术

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