Funding for this work was provided by: AG007218 and NIH F32 AG031606.
Images were generated in the Core Optical Imaging Facility, which is supported by UTHSCSA, NIH-NCI P30 CA54174 (CTRC at UTHSCSA) and NIH-NIA P01AG19316.

注:所有动物的程序批准了得克萨斯健康科学中心的圣安东尼奥大学的机构动物护理和使用委员会均与ARRIVE指南一致。 1.麻醉皮质准备<ol><li>鼠标放置在一个感应室用2-3％异氟烷与氧混合以诱导麻醉。观察呼吸速率减小，鼠标感应。捏鼠标的爪子来确定鼠标是否准备好移动到鼻锥。注意:麻醉水平是在任何在体内制备中的关键步骤和应注意不要诱导的水平，这将导致全球缺血。 </li><li>一旦老鼠被充分麻醉，动物转移到手术/影像平台，将鼠标的鼻子的鼻锥和应用1-1.2％异氟烷维持麻醉状态。确保鼠标躺在温度合作ntrolled加热垫在整个剩余的程序保持体温（37℃+/- 0.5°C）。将审核药膏过来的目光，以防止干燥，而在麻醉下。 </li><li>监视使用使用提供与该系统的尾部或脚夹一个脉冲血氧定量系统鼠标的生理学。检查呼吸率维持50-65次/分钟之间。检查心脏速率保持在300到450 BPM和氧饱和度被保持在97％-98％，以确保动物的长期存活。 </li><li>当鼠标充分麻醉，采用电动推剪剃光头发过头盖骨，去除残留的头发，干净的聚维酮碘，接着乙醇棉签。重复此过程直至三次，以确保一无菌手术环境。 </li></ol> 2.手术过程<ol><li>与充分清洗和剃头皮，制成5毫米的切口，在小鼠的头皮以显示颅fissu水库和定位前囟门。 </li><li>使用无菌棉涂敷器以除去任何剩余的筋膜覆盖在颅骨。 </li><li>胶水定制不锈钢环（ 图1）与组织粘合剂到骨上覆使用前囟的立体坐标顶叶:-1至-3毫米和横向:2-4毫米。注:通常胶水的位置环到骨头后设置在2分钟内。 </li><li>加盖环到立体定位架（ 图2），以稳定的小鼠，并防止在成像期间移动。 </li><li>在手术分级解剖显微镜，慢慢钻使用速度控制DREMEL状工具（Meisinger3.9毫米钻头），确保保持该地区的水平，因为它是在钻颅一个1-2毫米的部分。使用Z字形图案实现这一点。为了避免热量积聚，集钻速低，经常休息。 </li><li>当颅头骨变得光亮美观，继续细化使用利用相同的Z字形图案的解剖刀刀片保持减薄表面水平颅骨，以便顺利去除薄层颅头骨的。使用手术刀刀片的一角做小的线性招用轻压一次删除薄层骨。继续此，直到血管显然是通过解剖显微镜可见。 </li><li>如果实验者穿刺通过或中断期间薄型加工颅骨，安乐死的动物由于底层皮质可能损坏。 注意:小鼠头骨是大约300微米厚，并且包括两个薄层密质骨（一个外部，一个内部层）和一层松质骨的密质骨的两层之间。密质骨的外部层和大部分的松质骨的正导致紧凑骨其余的大致为50μm层5mm的钻孔区域内除去（ 见图2B）。 Visualiz脉管系统的通货膨胀将保证最终原封减薄颅骨是大约50微米厚。因此头骨是仍然存在的时候薄到这一厚度。 </li></ol> 3.显微镜建立<ol><li>使用倒置显微镜系统（常规的，共焦或双光子系统），其具有一个客观逆变器。注意:也可以利用一个标准直立显微镜。的限制因素将是该阶段与目标之间的空间。修改到阶段可能是必要的，以实现这种设置。 </li><li>固定手术/成像平台，位于一旁的显微镜底座一个定制的阶段。注:该平台是用实验室插孔允许在客观手术/成像平台的垂直运动进行。实验室千斤顶被安装到固定到四个圆柱形磁极的板。 （参见图2）。 </li><li>定位包含20X客观逆变器在客观颅窗口。使用外部光源通过寻找通过显微镜的目镜找到颅窗口和在成像区域定位目标。注意:成像区域将通过脉管系统的存在来表示。 </li><li>用于水性目标，用人工脑脊髓液（ACSF）（130毫摩尔NaCl; 30毫氯化钾; 12毫KH 2 PO 4; 200mM的碳酸氢钠 ; 30毫米的HEPES;和100mM葡萄糖）作为介质，由于电势漏入颅腔成像期间（ 图3）。 </li></ol> 4.玫瑰红染料的制备，管理和中风的感应<ol><li>制备新鲜的20毫克/毫升溶液玫瑰红的人工脑脊髓液（ACSF）;筛选和给药前消毒。不要重复使用或存放玫瑰孟加拉一旦有好有坏。使每个实验新鲜溶液。 </li><li>给0.1ml的尾静脉注射玫瑰红，而的S用561纳米激光罐头颅窗口以确保足够的注射溶液。注意:玫瑰红将5秒内注射在大脑的血管后可视化。整个容器应充满玫瑰红。 </li><li>继足够的注射玫瑰红染料选择基于血管直径（40-80微米）血栓形成一个合适的容器，以确保特定的病变体积的可重复性。通过观察血液的流动方向的动脉和静脉之间区分:动脉会从较大直径移动到较小直径血管，静脉从较小移动到较大直径的血管。这是很容易实现可视化，一旦玫瑰红被注入。 </li><li>如下更改设定显微镜: <ol><li>增加的停留时间。注意:这将取决于显微镜系统上正在使用的不同而不同。 </li><li>增加激光功率为100％。 </li><li>在1帧/秒的使用时间采集图像序列最大扫描速度。 </li></ol></li><li>扫描鼠标，直到稳定的凝块是在容器内而形成。注意:这通常是在5分钟内连续扫描而实现（参见图4）。 </li><li>以下的使用玫瑰红凝块形成的诱导，从摄像区域返回到解剖显微镜删除鼠标。小心地从颅头骨取下不锈钢环。检查颅窗口任何出血。如果发生出血，终止实验。 </li><li>使用6.0单丝缝合关闭切口在颅骨。放置抗生素软膏沿缝合线，以防止感染。注入BUPRENEX（0.05毫克/千克）经皮下，每12小时对疼痛管理三天。 </li><li>返回鼠标恢复室下去除麻醉，直到完全清醒和自由​​移动。 </li><li>返回鼠标干净笼子进一步调查在以后的时间。 </li></ol>五。在随后几天的纵向成像<ol><li>采用以下方法来在随后几天后photothrombosis进行纵向成像。 <ol><li>麻醉小鼠中的方法第1节中所述。 </li><li>经充分的麻醉通过去除残留的缝线重新皮肤上覆之前钻出影像领域重新打开头皮。 </li><li>使用无菌棉涂敷器以除去任何剩余的筋膜覆盖在颅骨。 </li><li>胶水定制不锈钢环（ 图1）与组织粘合剂到骨头上覆先前拍摄视场。 </li><li>加盖环到立体定位架（ 图2），以稳定的小鼠，并防止在成像期间移动。 </li><li>找到血管先前颅骨变薄底层。使用尾静脉注射FITC-葡聚糖的验证先前诱导凝块的存在。 </li><li>使用6.0单丝缝合关闭的在Cision公司的头骨。放置抗生素软膏沿缝合线，以防止感染。注入BUPRENEX（为0.05mg / kg）的皮下每12小时进行疼痛管理的三天。 </li><li>返回鼠标恢复室下去除麻醉，直到完全清醒和自由​​移动。 </li></ol></li></ol> 6.确认行程感应（验尸） <ol><li>在研究结束时，验证使用2,3,5-氯化三苯基四唑（TTC）染色， 如图5搏出量，完整的方法可以在12找到。 </li></ol>

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作者宣称，他们有没有竞争的财务权益。

翻译实验中风的病理生理学从动物到人类应用的能力一直饱受失败。然而，使用动物模型，如photothrombosis模式，允许中风病理生理学认识的提高和新的治疗方法提供神经保护下面中风的探索。皮质小招的光化学模型制作microinfarctions是临床相关的亚临床或"沉默"中风13-15，因而具有较高的患病率和影响约4％的美国人口（约11万人），每年16。无声中风不具有经典卒中症状存在于较大的行程，如瘫痪，感觉丧失和说话困难，例如，在大脑中动脉（MCA）阻塞或短暂性脑缺血发作（TIA）17看到。此外，无声中风是腔隙性卒中，其中不同由单个穿透动脉阻塞在更深的大脑结构或脑干内引起的，临床上也有运动，感觉或混合赤字18体现。亚临床或"沉默"一般行程不显示任何外在的症状，往往不知道他们甚至中风。无声中风导致亚临床减少认知功能由赤字存储器，决策，和行为变化表现。随着时间的推移，多个无声中风导致称为血管或多发梗塞性痴呆的记忆丧失的临床显著迹象。然而，无声中风的脑损伤可以利用神经成像进行检测，并在未来19放置一个病人的风险TIA和主要行程。 该photothrombosis模型允许在血栓形成的体内模型再现的COMBI中使用光敏染料玫瑰红（RB）生产的一个完整的，麻醉鼠标全国共聚焦显微镜。有在体内 photothrombosis模型的许多优点。这种方法的一个优点是预定义使用立体定位坐标行程的位置的能力;允许一个跨越动物研究特定的细胞群。此外，病变的大小和体积的再现性良好的控制，通过改变激光的强度，并控制用于 ​​血管大小被照射20利用此方法。这种方法还允许更改在梗死周围神经传递的详细研究，并对应对侧皮层4。虽然单个无声中风引起最小缺陷时，该模型的再现性允许以诱导多种沉默笔划在特定区域的能力，这可以被用来模仿各种脑机能障碍如血管性痴呆。多重静音中风和临床上明显缺陷之间的阈值可以开发特定中确定通过使用这种方法的脑的FIC领域为好。最后，该模型允许在同一动物的纵向研究允许被观察急性和慢性影响。 然而，有，在使用photothrombosis模型一些缺点。一个缺点包括生产被标注为相比焦点行程4的其他车型有一个小的缺血半暗带一个病灶。其次，血管性和细胞毒性水肿的形成是可能的，因为感应photothrombosis的，这更类似于创伤性脑损伤比焦行程4中可能发生的损害。 当使用photothrombosis模型有一个一些必须在整个实验过程进行监测的因素。该动物的生理状态在所有成像过程进行监控是至关重要的。它公知的是麻醉水平可影响生理圣动物，具有过麻醉引起的ATU心脏速率和氧气输送减少到动物体内。这是一个重要的考虑因素，因为这将减少氧气的可用性造成缺血的脑。因此，利用一个系统来监测动物的生理状况将允许的同时的非侵入性的记录:动脉血氧饱和度（SPO 2）;心脏率;呼吸频率;脉冲腹胀（局部血液流量和信号质量指示符）;呼吸腹胀（代用品intraplueral压力）;和核心温度在小鼠和大鼠。与任何动物模型中工作时，减少在翻译的实验结果在临床斯托克好处意外的困惑这正在成为控制麻醉越来越重要混淆。的选择，持续时间和麻醉深度可以在实验性卒中的动物模型产生极大的影响。研究表明，麻醉剂可以减少梗塞州市泽甚至可能提供脑缺血21-23,24一些保护。此外，在生产的活性氧的变化也被证明在比较使用氟烷，异丙酚25的研究。这变乱是重要的，因为在与中风相关的神经元死亡的主要假设之一是产生活性氧种。 利用体内显微镜来研究大脑笔划的响应的一个复杂因素是所述成像深度达到的限制。在使用共焦显微镜我们的实验室中，可以实现在成像深度在100-200微米的范围内，同时使用双光子显微镜可以增加这个深度之间400-500微米。这些的困惑正在缓解由目标与发展规模减小增加工作距离和。例如，梯度折射率（GRIN）的微透镜​​是microendoscopes机智和35-1,000微米h之间直径最小的可用日期。这种类型的探针不能插入到组织中，而不会引起侵入损伤并具有低的数值孔径。相比传统的光学显微镜目标26由于低NA的分辨率较差。 综上所述，玫瑰红photothrombosis模型诱导的小尺寸的梗塞和是研究在两个明确定义的细胞群体中的急性和慢性阶段的梗塞的细胞应答是有用的。该模型表明看到的局灶性缺血脑缺血如下细胞至关重要的特性，因此在评估神经保护/神经再生疗法有用。

体内细胞应答的描述的技术允许可视化之后立即感应玫瑰红的photothrombosis在一个完整的小鼠。玫瑰红（4,5,6,7-四氯-2&#39;，4&#39;，5&#39;，7&#39;-四碘）是用于诱导在动物模型缺血性中风（小鼠和大鼠）的光敏染料。通过减薄头骨与564纳米的激光以下推注的RB的尾静脉和随后的照明，血栓诱导引起生理行程1。该方法最初是由布鲁姆和El-萨班在1977年描述，并通过华生在20世纪80年代中期以后1,2改编。简言之，玫瑰红照射绿色激发光（在我们的情况下，561纳米的激光），其产生产生活性氧种，随后激活组织因子，凝血级联的引发剂。凝血级联的诱导产生缺血莱离子是病理学相关的临床搏3。 行程具有复杂的病理生理由于许多不同的细胞类型，包括神经元，神经胶质细胞，内皮细胞和免疫系统的相互作用。选择最好的技术来研究特定的细胞过程需要多重考虑。实验技术大致可分为三类: 在体外 ， 体内和在计算机芯片与每一个具有优点和缺点体外研究有除去它们的天然环境中的细胞的主要缺点，因此可能无法再现见于一个完整的影响，。活的动物， 在体内技术提供对疾病状态具有增加的平移意义增强实验复制。 在硅片 ，一般是指一种疾病或细胞过程的计算机建模，并且同时日益用来研究对考试潜在的药物相互作用PLE，收集的任何信息仍然必须在活细胞或组织测试。 中风在实验室环境下的理想模式应该表现出类似的病理特征为那些出现在人群中。虽然有中风的人群中常见的生理特点，也有因受伤经历了种类繁多的差异。中风人群中发生的小的或大血管阻塞，出血性病变，和动脉 - 动脉或心栓塞导致在不同的梗死体积，以及在有关每一病理学机制的差异。利用动物中风模型的优点在于，模拟人类中风的特征再现的梗塞的产生。最常见的动物卒中模型包括动脉闭塞使用:大脑中动脉闭塞（栓塞血管内或长丝的方法），该模型远端缺血和photothrombosis模型。其优点是每个模型的D-缺点已在别处综述（参见图4和5）。全球局部缺血模型（MCAO），而相对容易地进行比是局灶性卒中模型不太相关人类中风。此外，这些方法都是在诱导可重复的脑梗塞病灶高度可变。该photothrombosis模型是高度可再现的，只要实验者控制他们的实验以及，提供了明显的优势MCAO模型。然而，由于微脉管损伤的模型已经描述，以显示一个最小的缺血半暗带，其中细胞被认为是挽救6,7的区域。此外，血管性水肿和细胞毒性水肿形成也可诱导的成像区域的下面照射。尽管有这些限制的技术中提供了新的认识以下中风8，9，10，11的许多生理过程。

<table><tbody><tr><td colspan="4">Reagents</td></tr><tr><td>Rose Bengal</td><td>Sigma</td><td>330000</td><td></td></tr><tr><td>Isoflurane Anesthetic</td><td>MWI Veterinary Supply</td><td>088-076</td><td></td></tr><tr><td>Vetbond</td><td>1469SB</td><td>1469SB</td><td></td></tr><tr><td>aCSF&amp;nbsp;</td><td>126 mM NaCl, 2.5 mM KCl, 1.25 mM NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 2 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 2 mM CaCl&lt;sub&gt;2&lt;/sub&gt;, 10 mM glucose and 26 mM NaHCO&lt;sub&gt;3&lt;/sub&gt; (pH 7.4).</td><td></td><td></td></tr><tr><td colspan="4">[header]</td></tr><tr><td colspan="4">Equipment</td></tr><tr><td>Dissecting Scissors</td><td>Bioindustrial Products</td><td>500-410</td><td></td></tr><tr><td>Operating scissors 14 cm</td><td>Bioindustrial Products</td><td>12-055</td><td></td></tr><tr><td>Forceps Dumont High Tech #5 style, straight</td><td>Bioindustrial Products</td><td>TWZ-301.22</td><td></td></tr><tr><td>LabJack 132X80</td><td>Optosigma Co</td><td>123-6670</td><td></td></tr><tr><td>Platform for Labjack 8X 8</td><td>Optosigma Co</td><td>145-1110</td><td></td></tr><tr><td>Ear bar holder from stereotaxic setup</td><td>Stoelting/Cyborg</td><td>51654</td><td></td></tr><tr><td>Dispomed Labvent Rodent anesthesia machine</td><td>DRE, Inc.</td><td>15001</td><td></td></tr><tr><td>Tech IV Isoflurane vaporizer</td><td>DRE, Inc.</td><td>34001</td><td></td></tr><tr><td>F Air Canister</td><td>DRE, Inc</td><td>80120</td><td></td></tr><tr><td>Bain circuit breathing tube</td><td>DRE, Inc</td><td>86111B</td><td></td></tr><tr><td>Rodent adapter for bain tube</td><td>DRE, Inc</td><td>891000</td><td></td></tr><tr><td>O&lt;sub&gt;2&lt;/sub&gt; regulator for oxygen tanks</td><td>DRE, Inc</td><td>CE001E</td><td></td></tr><tr><td>Rodent induction chamber</td><td>DRE, Inc</td><td>15004C</td><td></td></tr><tr><td>Ethicon Silk 6-0; 18 in with P-3 needle</td><td>Suture Express</td><td>1639G</td><td></td></tr><tr><td>Objective inverter Optical Adapter</td><td>LSM technologies</td><td></td><td></td></tr><tr><td>Foredom drill Dual voltage 110/120</td><td>Foredom</td><td>134.53</td><td></td></tr><tr><td>Meisinger 3.9 mm drill bit</td><td>Meisinger</td><td></td><td>(Ref#310 104 001 001 009)</td></tr></tbody></table>

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.

该方法的目的是诱导在动物模型以下推注的RB的通过尾静脉变薄颅骨和随后的照明用561纳米激光的缺血性中风（小鼠和大鼠）。 图4中的图像表明凝块形成的进展在单个容器内之后的区域的0，1，1.5和2分钟的照射。此前凝块形成全船是白色的，由于自由流动玫瑰红。以下的容器的照射感应有在容器的部分明显变黑并且指示的凝块形成（帧1和1.5分钟）的诱导。以下完全闭塞有玫瑰红染料（白色区域）的容器内的凝块（黑色区域）前述的显着积累。 2分钟时帧表明动脉完全闭塞。 为了验证缺血性中风TTC染色的存在可加以利用。 TTC是共mmonly用于染色脑梗死由红甲TTC产品中的健康组织的形成的检测。甲生产（白色组织）的缺乏表明梗死区。在图5中由方框表示的区域表明以下的容器直径大约为80毫米内产生的凝块从两个单独的动物中获得1至5天的典型病灶大小。在平板扫描仪和使用ImageJ的软件进行图像分析。感兴趣的区域可以在ImageJ的被吸引到测量每搏量的每个大脑的区域。 <img alt="图1" src="/files/ftp_upload/52794/52794fig1.jpg" /> 图1:不锈钢戒指三个视图（俯视图，侧视图和底视图）是示出施加到鼠标将其固定到立体定位支架的头骨的不锈钢环保持器。 图2:显微镜成像平台设置为RB photothrombosis的外科/成像平台包含用于麻醉管与鼻锥和被固定到动物颅骨期间减少动物的运动为不锈钢环的立体定位支架的支架成像。该平台置于顶部并固定到实验室千斤顶，允许在显微镜物镜定位鼠标垂直移动。实验室千斤顶然后连接到显微镜的阶段，其允许水平移动。显微镜载物台被置于上方并固定到四个圆柱形磁极。 <img alt="图3" src="/files/ftp_upload/52794/52794fig3.jpg" /> 图3:在目标INV图片的成像/手术平台的设计和方向erter。（A）左边的面板展示了麻醉小鼠（麻醉鼻锥被删除简要地拍照）的定位的形象代表。注意:使用自定义钢环来连接鼠标头骨降低整个成像过程的呼吸文物的贡献。右边的图像展示在解剖显微镜下皮质窗口的图像。从冠状视图展示了头骨的各层相对于硬膜和减薄区域的相对于全颅骨厚度的厚度薄头骨制备（B）的示意图。 <img alt="图4" src="/files/ftp_upload/52794/52794fig4.jpg" /> 图4:图片的玫瑰红血块形成含有玫瑰红染料的单船那是通过尾巴注入VEI代表图像。n中的鼠标。图像表明凝块形成的进展在容器内之后的区域的0，1，1.5和2分钟的照射。注意玫瑰红染料（白色）在2分钟帧证明动脉的完全闭塞的凝块（黑色）前述的积累。 <img alt="图5" src="/files/ftp_upload/52794/52794fig5.jpg" /> 图5:RB所致损伤的2,3,5-氯化三苯基四唑（TTC）图像代表性的图像示出在第1天及5后photothrombosis感应。处死小鼠和大脑迅速按照标准方法除去，切成1毫米冠状切片并用TTC染色。 TTC是一种常用的染色为脑梗塞的由红甲TTC产品中的健康组织的形成的检测。甲生产（白色组织）的缺乏表明梗死区。该由方框表示的区域表明以下的容器内产生直径约为80μm的凝块获得的典型病灶大小。 <img alt="图6" src="/files/ftp_upload/52794/52794fig6.jpg" /> 图6:玫瑰红光化学过程的示意图。

Watch this Scientific Journal Video about Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke at JoVE.com

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 ...

rose-bengal-photothrombosis-confocal-optical-imaging-vivo-model

Nanyang Technological University

Research

JoVE Journal

Medicine

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

15.0K 次观看.  The University of Texas Health Science Center San Antonio. 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. 

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

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

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 ...

Quantification of Cerebral Perfusion using Laser Speckle Imaging and Infarct Volume using MRI in a Pre-clinical Model of Posterior Circulation Stroke

Background: Basilar artery occlusion (BAO) is a subset of posterior circulation stroke that carries a mortality as high as 90%.&#160; The current clinical standard to diagnose ischemic stroke include computerized tomography (CT), CT angiography and perfusion and magnetic resonance imaging (MRI). Large animal pre-clinical models to accurately reflect the clinical disease as well as methods to assess stroke burden and evaluate treatments are lacking.
Methods: We describe a canine model of large vessel occlusion (LVO) stroke in the posterior circulation, and developed a laser speckle imaging (LSI) protocol to monitor perfusion changes in real time.&#160; We then utilized high b-value DWI (b=1800s/mm2) MRI to increase detection sensitivity. We also evaluated the ability of magnetic resonance angiography (MRA) to assess arterial occlusion and correlate with DSA. Finally, we verified infarct size from apparent diffusion coefficient (ADC) mapping with histology.&#160;
Results:&#160; Administration of thromboembolism occluded the basilar artery as tracked by DSA (n=7).&#160; &#160;LSI correlated with DSA, demonstrating a reduction in perfusion after stroke onset that persisted throughout the experiment, allowing us to monitor perfusion in real time.&#160; DWI with an optimized b-value for dogs illustrated the stroke volume and allowed us to derive ADC and magnetic resonance angiography (MRA) images. The MRA performed at the end of the experiment correlated with DSA performed after occlusion. Finally, stroke burden on MRI correlated with histology.
Conclusions: Our studies demonstrate real time perfusion imaging using LSI of a canine thromboembolic LVO model of posterior circulation stroke, which utilizes multimodal imaging important in the diagnosis and treatment of ischemic stroke.

A canine model of LVO stroke was utilized to develop laser speckle imaging to monitor cerebral perfusion in real-time.&#160; Diffusion-weighted MRI was optimized to image infarct volume utilizing a high b-value, enabling ADC and MRA, correlated with DSA at the time of stroke.&#160; Finally, ADC reconstructions correlated with histological findings.

Background: Basilar artery occlusion (BAO) is a subset of posterior circulation stroke that carries a mortality as high as 90%.&#160; The current clinical ...

神经科学

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 ...

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, ...

Optimization of the Retinal Vein Occlusion Mouse Model to Limit Variability

Mouse models of retinal vein occlusion (RVO) are often used in ophthalmology to study hypoxic-ischemic injury in the neural retina. In this report, a detailed method pointing out critical steps is provided with recommendations for optimization to achieve consistently successful occlusion rates across different genetically modified mouse strains. The RVO mouse model consists primarily of the intravenous administration of a photosensitizer dye followed by laser photocoagulation using a retinal imaging microscope attached to an ophthalmic guided laser. Three variables were identified as determinants of occlusion consistency. By adjusting the wait time after rose bengal administration and balancing the baseline and experimental laser output, the variability across experiments can be limited and a higher success rate of occlusions achieved. This method can be used to study retinal diseases that are characterized by retinal edema and hypoxic-ischemic injury. Additionally, as this model induces vascular injury, it can also be applied to study the neurovasculature, neuronal death, and inflammation.

Here, we describe an optimized protocol for retinal vein occlusion using rose bengal and a laser-guided retinal imaging microscope system with recommendations to maximize its reproducibility in genetically modified strains.

优化视网膜静脉遮挡小鼠模型以限制变异性

Mouse models of retinal vein occlusion (RVO) are often used in ophthalmology to study hypoxic-ischemic injury in the neural retina. In this report, a ...

缺血性卒中的无创综合 PAUSAT 全脑成像

Integrated Photoacoustic, Ultrasound, and Angiographic Tomography (PAUSAT) for NonInvasive Whole-Brain Imaging of Ischemic Stroke

Presented here is an experimental ischemic stroke study using our newly developed noninvasive imaging system that integrates three acoustic-based imaging technologies: photoacoustic, ultrasound, and angiographic tomography (PAUSAT). Combining these three modalities helps acquire multi-spectral photoacoustic tomography (PAT) of the brain blood oxygenation, high-frequency ultrasound imaging of the brain tissue, and acoustic angiography of the cerebral blood perfusion. The multi-modal imaging platform allows the study of cerebral perfusion and oxygenation changes in the whole mouse brain after stroke. Two commonly used ischemic stroke models were evaluated: the permanent middle cerebral artery occlusion (pMCAO) model and the photothrombotic (PT) model. PAUSAT was used to image the same mouse brains before and after a stroke and quantitatively analyze both stroke models. This imaging system was able to clearly show the brain vascular changes after ischemic stroke, including significantly reduced blood perfusion and oxygenation in the stroke infarct region (ipsilateral) compared to the uninjured tissue (contralateral). The results were confirmed by both laser speckle contrast imaging and triphenyltetrazolium chloride (TTC) staining. Furthermore, stroke infarct volume in both stroke models was measured and validated by TTC staining as the ground truth. Through this study, we have demonstrated that PAUSAT can be a powerful tool in noninvasive and longitudinal preclinical studies of ischemic stroke.

作者聚焦：用于缺血性卒中的无创全脑成像的集成光声、超声和血管造影断层扫描 （PAUSAT）  

This work demonstrates the use of a multimodal ultrasound-based imaging platform for noninvasive imaging of ischemic stroke. This system allows for the quantification of blood oxygenation through photoacoustic imaging and impaired perfusion in the brain through acoustic angiography.

集成光声、超声和血管造影断层扫描 （PAUSAT） 用于缺血性卒中的无创全脑成像

Presented here is an experimental ischemic stroke study using our newly developed noninvasive imaging system that integrates three acoustic-based imaging ...

Real-Time Monitoring and Modulation of Blood Pressure in a Rabbit Model of Ischemic Stroke

Control of blood pressure, in terms of both absolute values and its variability, affects outcomes in ischemic stroke patients. However, it remains challenging to identify the mechanisms that lead to poor outcomes or evaluate measures by which these effects can be mitigated because of the prohibitive limitations inherent to human data. In such cases, animal models can be utilized to conduct rigorous and reproducible evaluations of diseases. Here we report refinement of a previously described model of ischemic stroke in rabbits that is augmented with continuous blood pressure recording to assess the impacts of modulation on blood pressure. Under general anesthesia, femoral arteries are exposed through surgical cutdowns to place arterial sheaths bilaterally. Under fluoroscopic visualization and roadmap guidance, a microcatheter is advanced into an artery of the posterior circulation of the brain. An angiogram is performed by injecting the contralateral vertebral artery to confirm occlusion of the target artery. With the occlusive catheter remaining in position for a fixed duration, blood pressure is continuously recorded to allow for tight titration of blood pressure manipulations, whether through mechanical or pharmacological means. At the completion of the occlusion interval, the microcatheter is removed, and the animal is maintained under general anesthesia for a prescribed length of reperfusion. For acute studies, the animal is then euthanized and decapitated. The brain is harvested and processed to measure the infarct volume under light microscopy and further assessed with various histopathological stains or spatial transcriptomic analysis. This protocol provides a reproducible model that can be utilized for more thorough preclinical studies on the effects of blood pressure parameters during ischemic stroke. It also facilitates effective preclinical evaluation of novel neuroprotective interventions that might improve care for ischemic stroke patients.

Continuous arterial blood pressure recording allows the investigation of impacts of various hemodynamic parameters. This report demonstrates the application of continuous arterial blood pressure monitoring in a large animal model of ischemic stroke for determination of stroke pathophysiology, impact of different hemodynamic factors, and the assessment of novel treatment approaches.

玫瑰红Photothrombosis共聚焦光学成像

摘要

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此视频中的章节

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

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

Quantification of Cerebral Perfusion using Laser Speckle Imaging and Infarct Volume using MRI in a Pre-clinical Model of Posterior Circulation Stroke

纤维蛋白富集和 tPA 敏感的光血栓形成卒中模型

小鼠模型中风:光血栓形成的焦皮质病变

优化视网膜静脉遮挡小鼠模型以限制变异性

集成光声、超声和血管造影断层扫描（PAUSAT）用于缺血性卒中的无创全脑成像

集成光声、超声和血管造影断层扫描（PAUSAT）用于缺血性卒中的无创全脑成像

集成光声、超声和血管造影断层扫描（PAUSAT）用于缺血性卒中的无创全脑成像

缺血性脑卒中兔模型中血压的实时监测和调节

玫瑰红Photothrombosis共聚焦光学成像

摘要

探索更多视频

此视频中的章节

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

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

Quantification of Cerebral Perfusion using Laser Speckle Imaging and Infarct Volume using MRI in a Pre-clinical Model of Posterior Circulation Stroke

纤维蛋白富集和 tPA 敏感的光血栓形成卒中模型

小鼠模型中风:光血栓形成的焦皮质病变

优化视网膜静脉遮挡小鼠模型以限制变异性

集成光声、超声和血管造影断层扫描 （PAUSAT） 用于缺血性卒中的无创全脑成像

集成光声、超声和血管造影断层扫描 （PAUSAT） 用于缺血性卒中的无创全脑成像

集成光声、超声和血管造影断层扫描 （PAUSAT） 用于缺血性卒中的无创全脑成像

缺血性脑卒中兔模型中血压的实时监测和调节

集成光声、超声和血管造影断层扫描（PAUSAT）用于缺血性卒中的无创全脑成像

集成光声、超声和血管造影断层扫描（PAUSAT）用于缺血性卒中的无创全脑成像

集成光声、超声和血管造影断层扫描（PAUSAT）用于缺血性卒中的无创全脑成像