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?...

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。

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			<td>Reagents</td>
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		<tr>
			<td>Rose Bengal</td>
			<td>Sigma</td>
			<td>330000</td>
			<td></td>
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			<td>Isoflurane Anesthetic</td>
			<td>MWI Veterinary Supply</td>
			<td>088-076</td>
			<td></td>
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			<td>Vetbond</td>
			<td>1469SB</td>
			<td>1469SB</td>
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			<td>aCSF&#160;</td>
			<td>126 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 2 mM MgCl2, 2 mM CaCl2, 10 mM glucose and 26 mM NaHCO3 (pH 7.4).</td>
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			<td>[header]</td>
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			<td>Equipment</td>
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			<td>Dissecting Scissors</td>
			<td>Bioindustrial Products</td>
			<td>500-410</td>
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			<td>Bioindustrial Products</td>
			<td>12-055</td>
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			<td>Optosigma Co</td>
			<td>123-6670</td>
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			<td>Platform for Labjack 8X 8</td>
			<td>Optosigma Co</td>
			<td>145-1110</td>
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			<td>Ear bar holder from stereotaxic setup</td>
			<td>Stoelting/Cyborg</td>
			<td>51654</td>
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			<td>Dispomed Labvent Rodent anesthesia machine</td>
			<td>DRE, Inc.</td>
			<td>15001</td>
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			<td>Tech IV Isoflurane vaporizer</td>
			<td>DRE, Inc.</td>
			<td>34001</td>
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			<td>F Air Canister</td>
			<td>DRE, Inc</td>
			<td>80120</td>
			<td></td>
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			<td>Bain circuit breathing tube</td>
			<td>DRE, Inc</td>
			<td>86111B</td>
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			<td>Rodent adapter for bain tube</td>
			<td>DRE, Inc</td>
			<td>891000</td>
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			<td>O2 regulator for oxygen tanks</td>
			<td>DRE, Inc</td>
			<td>CE001E</td>
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			<td>Rodent induction chamber</td>
			<td>DRE, Inc</td>
			<td>15004C</td>
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			<td>1639G</td>
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			<td>Foredom drill Dual voltage 110/120</td>
			<td>Foredom</td>
			<td>134.53</td>
			<td></td>
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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...

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 ...

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Research

JoVE Journal

Medicine

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

14.8K 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. 