我们感谢安娜丽莎滑稽的，有见地的建议和意见，Maurizio Grassano的滨海Boido和埃尔米拉Pajaj拍摄。这项工作是由FP7-MC-214003-2（居里夫人首次培训网络AXREGEN）和COMPAGNIA迪圣保罗，gliarep项目。

1。手术前<ol><li>称量玫瑰红的1.5毫升管中，溶解在无菌生理盐水溶液中，直到达到最终浓度为15毫克/毫升。通过0.2微米的过滤器过滤灭菌，并将其存储在黑暗中，在室温下长达两个月。 </li><li>所有的手术器械消毒灭菌。应消毒手术区不到一小时才开始手术。 </li><li>记录小鼠体重调整剂量注入孟加拉玫瑰红。女CD1小鼠12周龄本协议注入10微升/克动物体重。孟加拉玫瑰红需要的量，以产生所需的皮质病变的大小，可以很容易地确定的一组独立的预备实验，通过测试不同剂量（通常为2微升/克，5微升/克或10微升/ g体重）。请注意，孟加拉玫瑰红的量被注入高度依赖于实验条件下，特别是不同的光源使用光线照射的持续时间。另外，在本研究中，10微升/克（150微克/克）的剂量发现是必要的诱导photothrombosis曝光时的光15分钟，而其余各组报告，50微克/克6,8和100微克/克10腹腔注射足以诱导光化学病变。 </li></ol> 2。麻醉过程<ol><li>透明感应室（诱导的3.5-4％，保持1.5-2％）在50％（体积/体积）oxygen/50％（体积/体积）的一氧化二氮的混合气体与异氟醚麻醉小鼠。气体麻醉允许快速唤醒的动物和麻醉气体的水平，能够容易地调整。另外，小鼠也可以用氯胺酮 - 甲苯噻嗪的混合物麻醉。 </li><li>达到深麻醉时，取出麻醉动物感应室，将其放置在立体框架，并用面罩维持麻醉。调整isoflura的NE剂量达到足够的麻醉水平。监控整个过程中的呼吸速率，并确保它是恒定的（每分钟呼吸40 - 60）。 </li><li>用脚趾捏，以确保动物是深深麻醉。 </li><li>应用眼药膏，以防止眼睛干燥。 </li><li>轻轻插入直肠温度探头监控整个手术过程。鼠标体温维持在37±0.5℃下，设置相应的反馈控制的加热垫</li></ol> 3。手术目标区域照明<ol><li>用电动剃须刀刮胡子鼠标头皮。 </li><li>牢固地固定在头部和耳棒插入到外部鼻道。要小心，不要伤害耳膜。 </li><li>与交替挥笔70％乙醇和优碘用棉签消毒皮肤表面上。 </li><li>用手术刀做一个切口，沿中线从眼睛LEVEL的脖子。应用皮肤拉钩保持颅骨外露。 </li><li>轻轻缩回periostium，颅骨的边缘，用手术刀让颅骨表面晾干，用无菌棉签。确定前囟门和lambda。前囟门上放置一个玻璃微作为参考点，然后将其移动到您感兴趣的区域的坐标。本文选择感兴趣的区域大致是严厉形，约2毫米横向居中前囟门，占地约30毫米，其中包括了很大一部分的感觉皮层的小鼠脑图谱富兰克林paxinos 11。 </li><li>标记利益与参考点的位置，并把一根光纤与颅骨表面紧密接触，避免光的散射，但注意不要施加压力。照射范围可以任意申请头骨上口罩光纤GUI的尖端小光圈，或者上限限制德。 </li></ol> 4。孟加拉玫瑰红注射液和激活<ol><li>负载的玫瑰红溶液1 ml注射器和计算的量要根据在10μl/克体重的剂量注入。 </li><li>继续一个缓慢腹腔注射。 </li><li>让染料的漫反射，并进入血流。 5分钟后，冷光源切换。避免任何其他光源照明的动物。我们使用光纤照明灯150瓦的强度。 </li><li>经过15分钟的照明，停止光照射，并缝合伤口。五分钟的照明已经产生梗死和10分钟可能是足够的，以获得最大的效果12。为了尽量减少可变性，我们选择照亮15分钟。 </li></ol> 5。缝合<ol><li>去除皮肤拉钩和应用无菌生理盐水，以避免脱水。 </li><li>关闭使用反向切割needl的伤口e和丝绸或尼龙缝合线。 </li><li>中断麻醉交付，小心地取出鼠标从立体定位仪，并把它放在一个预热加热垫，直到它完全清醒，然后再返回到它的笼子。人体温度应仔细监测整个程序限制的可变性，在梗塞扩展。的浓度和给药途径，孟加拉玫瑰红，仍然可以在血液中检测到几个小时后注射13。不想电热毯暖灯，以避免潜在的二次损害（孟加拉玫瑰在绿色光谱吸收波长为）。 </li></ol>

<ol>
	<li>Lopez, A. D., Mathers, C. D., Ezzati, M., Jamison, D. T., Murray, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+and+regional+burden+of+disease+and+risk+factors.">Global and regional burden of disease and risk factors.</a> Lancet. 367, 1747-1757 (2001).</li><li>Mathers, C. D., Boerma, T., Ma Fat, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+and+regional+causes+of+death.">Global and regional causes of death.</a> Br. Med. Bull. 92, 7-32 (2009).</li><li>Rosenblum, W. I., El-Sabban, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+aggregation+in+the+cerebral+microcirculation:+effect+of+aspirin+and+other+agents.">Platelet aggregation in the cerebral microcirculation: effect of aspirin and other agents.</a> Circ. Res. 40, 320-328 (1977).</li><li>Watson, B. D., Dietrich, W. D., Busto, R., Wachtel, M. S., Ginsberg, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+reproducible+brain+infarction+by+photochemically+initiated+thrombosis.">Induction of reproducible brain infarction by photochemically initiated thrombosis.</a> Ann. Neurol. 17, 497-504 (1985).</li><li>Bergeron, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducing+photochemical+cortical+lesions+in+rat+brain.">Inducing photochemical cortical lesions in rat brain.</a> Curr. Protoc. Neurosci. Chapter 9, Unit 9 16 (2003).</li><li>Lee, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photochemically+induced+cerebral+ischemia+in+a+mouse+model.">Photochemically induced cerebral ischemia in a mouse model.</a> Surg. Neurol. 67, 620-625 (2007).</li><li>Dietrich, W. D., Watson, B. D., Busto, R., Ginsberg, M. D., Bethea, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photochemically+induced+cerebral+infarction.+I.+Early+microvascular+alterations.">Photochemically induced cerebral infarction. I. Early microvascular alterations.</a> Acta Neuropathol. 72, 315-325 (1987).</li><li>Schroeter, M., Jander, S., Stoll, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+induction+of+focal+cerebral+ischemia+in+mice+by+photothrombosis+of+cortical+microvessels:+characterization+of+inflammatory+responses.">Non-invasive induction of focal cerebral ischemia in mice by photothrombosis of cortical microvessels: characterization of inflammatory responses.</a> J. Neurosci. Methods. 117, 43-49 (2002).</li><li>Kitagawa, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+ischemia+after+bilateral+carotid+artery+occlusion+and+intraluminal+suture+occlusion+in+mice:+evaluation+of+the+patency+of+the+posterior+communicating+artery.">Cerebral ischemia after bilateral carotid artery occlusion and intraluminal suture occlusion in mice: evaluation of the patency of the posterior communicating artery.</a> J. Cereb. Blood Flow Metab. 18, 570-579 (1998).</li><li>Sigler, A., Goroshkov, A., Murphy, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hardware+and+methodology+for+targeting+single+brain+arterioles+for+photothrombotic+stroke+on+an+upright+microscope.">Hardware and methodology for targeting single brain arterioles for photothrombotic stroke on an upright microscope.</a> J. Neurosci. Methods. 170, 35-44 (2008).</li><li>Franklin, K. B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates. , (1997).</li><li>Piao, M. S., Lee, J. K., Jang, J. W., Kim, S. H., Kim, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+of+photochemically+induced+spinal+cord+injury.">A mouse model of photochemically induced spinal cord injury.</a> J. Korean Neurosurg. Soc. 46, 479-483 (2009).</li><li>Silva, V. M., Corson, N., Elder, A., Oberdorster, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rat+ear+vein+model+for+investigating+in+vivo+thrombogenicity+of+ultrafine+particles+(UFP).">The rat ear vein model for investigating in vivo thrombogenicity of ultrafine particles (UFP).</a> Toxicol. Sci. 85, 983-989 (2005).</li><li>Watson, B. D., Prado, R., Dietrich, W. D., Ginsberg, M. D., Green, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photochemically+induced+spinal+cord+injury+in+the+rat.">Photochemically induced spinal cord injury in the rat.</a> Brain Res. 367, 296-300 (1986).</li><li>Van Reempts, J., Van Deuren, B., Van de Ven, M., Cornelissen, F., Borgers, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flunarizine+reduces+cerebral+infarct+size+after+photochemically+induced+thrombosis+in+spontaneously+hypertensive+rats.">Flunarizine reduces cerebral infarct size after photochemically induced thrombosis in spontaneously hypertensive rats.</a> Stroke. 18, 1113-1119 (1987).</li><li>Carmichael, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+of+focal+stroke:+size,+mechanism,+and+purpose.">Rodent models of focal stroke: size, mechanism, and purpose.</a> NeuroRx. 2, 396-409 (2005).</li><li>Kleinschnitz, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blocking+of+platelets+or+intrinsic+coagulation+pathway-driven+thrombosis+does+not+prevent+cerebral+infarctions+induced+by+photothrombosis.">Blocking of platelets or intrinsic coagulation pathway-driven thrombosis does not prevent cerebral infarctions induced by photothrombosis.</a> Stroke. 39, 1262-1268 (2008).</li><li>Porritt, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photothrombosis-induced+infarction+of+the+mouse+cerebral+cortex+is+not+affected+by+the+Nrf2-activator+sulforaphane.">Photothrombosis-induced infarction of the mouse cerebral cortex is not affected by the Nrf2-activator sulforaphane.</a> PLoS One. 7, e41090 (2012).</li><li>Baskin, Y. K., Dietrich, W. D., Green, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+effective+behavioral+tasks+for+evaluating+sensorimotor+dysfunction+following+traumatic+brain+injury+in+mice.">Two effective behavioral tasks for evaluating sensorimotor dysfunction following traumatic brain injury in mice.</a> J. Neurosci Methods. 129, 87-93 (2003).</li><li>Markgraf, C. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+histopathologic+consequences+of+photothrombotic+occlusion+of+the+distal+middle+cerebral+artery+in+Sprague-Dawley+and+Wistar+rats.">Comparative histopathologic consequences of photothrombotic occlusion of the distal middle cerebral artery in Sprague-Dawley and Wistar rats.</a> Stroke. 24, 286-292 (1993).</li><li>Wester, P., Watson, B. D., Prado, R., Dietrich, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+photothrombotic+'ring'+model+of+rat+stroke-in-evolution+displaying+putative+penumbral+inversion.">A photothrombotic 'ring' model of rat stroke-in-evolution displaying putative penumbral inversion.</a> Stroke. 26, 444-450 (1995).</li><li>Hu, X., Wester, P., Brannstrom, T., Watson, B. D., Gu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+and+reproducible+focal+cortical+ischemia+with+or+without+late+spontaneous+reperfusion+generated+by+a+ring-shaped,+laser-driven+photothrombotic+lesion+in+rats.">Progressive and reproducible focal cortical ischemia with or without late spontaneous reperfusion generated by a ring-shaped, laser-driven photothrombotic lesion in rats.</a> Brain Res. Brain Res. Protoc. 7, 76-85 (2001).</li><li>Maxwell, K. A., Dyck, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+reproducible+focal+ischemic+lesions+in+neonatal+mice+by+photothrombosis.">Induction of reproducible focal ischemic lesions in neonatal mice by photothrombosis.</a> Dev. Neurosci. 27, 121-126 (2005).</li><li>Kuroiwa, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+rat+model+of+photothrombotic+ischemia+and+infarction+within+the+caudoputamen.">Development of a rat model of photothrombotic ischemia and infarction within the caudoputamen.</a> Stroke. 40, 248-253 (2009).</li></ol>

没有利益冲突的声明。

修改和替换由于其在562 nm处的吸收峰从过滤的氙弧灯，绿光激光器最初被选择照射在感光玫瑰红。虽然仍然使用激光介导激发recently5，它可以取代冷光源灯泡，也能确保染料激发10,15。冷光源光纤更易于操作，更便宜比激光源。然而，应该注意的是激光器被普遍用来针对单个表面小动脉开颅手术后在体内的特定血管凝血10。 交付敏感?...

在21世纪的开始，缺血性中风是一种破坏性疾病，长期残疾和全球死亡原因的第二位，其中中风占约5,7万人死亡，在2004年2代表第二个原因。尽管被放在许多努力，但仍然没有有效的治疗方法，可改善卒中后功能恢复。卒中动物模型被广泛用于中风研究的领域中，因为它们允许缺血性损伤的病理生理学机制的建模和测试不同的神经保护策略在体内的疗效。这些模型大多通过中断（暂时或永久）大脑中动脉内的血流量，而其他车型的开发，研究在特定区域，通常是电动机和体感皮层的病变体积小，旨在引起广泛梗死。然而，有几个因素可能会导致产生交流在实验中风的研究，其中包括所使用的小鼠品系ERTAIN变异程度，在研究中的动物的年龄和性别，并最重要的是，该技术通过诱导局部缺血损伤。对于后一点，手术的持续时间和侵袭性的（ 即开颅手术的需要），以及需要操作者能够可靠地诱导缺血性病变的外科手术技术人员的关键决定因素，一个成功的体内中风研究的相关。 最初提出的概念photothrombosis布鲁姆和El-萨班在1977年3和其应用在大鼠脑沃森等人成为著名的。于1985年4技术大幅度提高，并设置当前模型3的基础- 6。光化学方法的目的是通过光激活的光敏感染料先前交付进入血液系统，WH诱发皮质梗死ICH导致局部血管血栓形成暴露在光天化日之下的地区。当循环染料在适当的波长的照明冷光源，它释放出能量的氧分子，从而能够产生大量的反应性高的单重态氧产品。这些氧中间体诱导血管内皮细胞的膜脂质过氧化，导致血小板粘附和聚集，并最终确定局部脑血流量中断7的血栓的形成。 photothrombosis是一个非规范性脑缺血模型，不吸留或打破只有一个动脉因为它通常发生在人类卒中，但诱导病变的比较浅的容器，这会导致在暴露在光线下的区域中选择性的血流中断。由于这个原因，这种方法可能适合皮层可塑性的细胞和分子的研究。这种技术的主要优点驻留在其简单的执行。此外，photothrombosis可以容易地进行在每只动物约40分钟，包括21分钟的等待时间（3分钟麻醉; 1分钟到剃的头皮;动物放置在立体定位仪上3至5分钟，2分钟，擦洗头皮消毒液，做一个切口，清洁的头骨; 2到4分钟，将冷光源光纤，1分钟，注入孟加拉玫瑰红溶液，5分钟等待腹腔扩散，15分钟的照明; 5分钟，清洗伤口，缝合动物）。此外，没有手术专业知识是必要执行此技术通过简单的照明完整的头骨，引起病变。不同于古典动脉闭塞，这个方法决定选择性地照射区域内的软脑膜和脑实质微血管闭塞，没有侧支血管留在目标区域的氧气供应，降低病变之间的变异。 其特殊的性质，尽管光化学损伤股脑中风发生的重要机制。类似于人类卒中的动脉闭塞，在照射区域7的血小板聚集和凝块的形成决定的血流中断。同样，这种模式也分享必不可少的炎症反应，如在大脑中动脉闭塞8。然而，因为以及delimitated的边界，半影区，它对应于一个区域的部分保存下来的代谢，是非常减少或不存在的后光化学病变。这种边界清晰，可以方便的在缺血性或完整的皮质区细胞反应研究。 photothrombosis小鼠模型是特别适合于在各种转基因动物的中风研究。事实上，经典机型不适合所有菌株和长时间的研究在C57BL / 6小鼠品系死亡率高的比例，可能会导致偏见9。

<table border="1">
<tbody>
 <tr>
 <td>MATERIAL NAME</td>
 <td>COMPANY </td>
 <td>CATALOGUE NUMBER</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Solutions and chemicals</td>
 </tr>
 <tr>
 <td>Rose Bengal </td>
 <td>Sigma, Italy </td>
 <td>330000 </td>
 <td></td>
 </tr>
 <tr>
 <td>Isoflurane Vet </td>
 <td>Merial </td>
 <td> 103120022 </td>
 <td></td>
 </tr>
 <tr>
 <td>Betadine </td>
 <td>Asta Medica </td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Paraformaldehyde </td>
 <td>Sigma-Aldrich </td>
 <td> 158127 </td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Surgical material and equipment</td>
 </tr>
 <tr>
 <td>Fluosorber Filter </td>
 <td>Havard apparatus </td>
 <td> 340415 </td>
 <td></td>
 </tr>
 <tr>
 <td>150W fiber optic illuminator</td>
 <td>Photonic</td>
 <td>PL3000</td>
 <td></td>
 </tr>
 <tr>
 <td>Temperature Controller for Plate TCAT-2DF </td>
 <td>Havard apparatus</td>
 <td>727561 </td>
 <td></td>
 </tr>
 <tr>
 <td>Stereotaxic Instrument </td>
 <td>Stoelting</td>
 <td> 51950 </td>
 <td></td>
 </tr>
 <tr>
 <td>Operating microscope </td>
 <td>Takagi </td>
 <td> OM8 </td>
 <td></td>
 </tr>
 <tr>
 <td>Heating pad </td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Oxygen and nitrogen gas</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Surgery Tools</td>
 <td>World precision instrument </td>
 <td></td>
 <td>Optic fiber taps and mask are custom-made </td>
 </tr>
 </tbody>
</table>

photothrombotic ischemia: a minimally invasive and reproducible photochemical cortical lesion model for mouse stroke studies

光化学中风模型的目的是通过在一个给定的皮层区的先前注入的光敏感的染料的光激活诱导的局部缺血损伤。照明，染料被激活，并产生单重态氧，损害元件的内皮细胞的膜，随后的血小板聚集和血栓形成，最终确定了局部血流量的中断。布鲁姆和El-萨班于1977年，最初提出这种方法，后来在1985年提高沃森在大鼠脑并设置当前模型的基础。此外，供应量增加的转基因小鼠线进一步提供的提高利息上photothrombosis模型。简单地说，光敏染料（玫瑰红）腹膜内注射，并进入血流。当冷光源照明，染料被激活，诱导血管内皮损伤，血小板活化和血栓形成，造成局部血流中断。光源可应用于完整的头骨，无需开颅手术，可以针对任何皮质区的利益在一个可重复的和非侵入性的方式。鼠标，然后缝合，并允许醒来。局部缺血损伤的评价可以迅速通过三苯基四唑鎓氯化物或甲酚紫染色。该技术生产出体积小，以及分隔的边界，这是非常有利于精确的电池特性或功能的研究梗死。此外，它是特别适合用于研究细胞和分子在转基因小鼠中脑的可塑性响应相关。

此协议将产生皮质病变，皮质解剖后，已是可见的，肉眼（ 图1A-1C）。光化学病变发展浅表和深部皮质层组织是半透明到足以允许孟加拉玫瑰红的光激活的。脑梗塞的程度的测量，可快速地执行通过组织染色与三苯基氯化四氮唑（TTC）在4％多聚甲醛（PFA）固定后对新鲜组织或甲酚紫。 TTC孵育之前，新鲜解剖的脑定位在脑切片机，划分成1毫米厚的片（ 图1C）。 TTC标记为?...

Watch this Scientific Journal Video about Photothrombotic Ischemia: A Minimally Invasive and Reproducible Photochemical Cortical Lesion Model for Mouse Stroke Studies at JoVE.com

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

的Photothrombosis是一种快速，微创技术领域的兴趣，高度可重复的方式诱导小以及分隔梗死。它特别适用于为研究细胞和分子在转基因小鼠中脑的可塑性响应相关。

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 ...

photothrombotic-ischemia-minimally-invasive-reproducible

Research

JoVE Journal

Medicine

34.7K 次观看.  University of Turin. 的Photothrombosis是一种快速，微创技术领域的兴趣，高度可重复的方式诱导小以及分隔梗死。它特别适用于为研究细胞和分子在转基因小鼠中脑的可塑性响应相关。

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

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

蛛网膜下腔出血的小鼠模型

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

科研

医学

Lesion Explorer: A Video-guided, Standardized Protocol for Accurate and Reliable MRI-derived Volumetrics in Alzheimer's Disease and Normal Elderly

Obtaining in vivo human brain tissue volumetrics from MRI is often complicated by various technical and biological issues. These challenges are exacerbated when significant brain atrophy and age-related white matter changes (e.g. Leukoaraiosis) are present. Lesion Explorer (LE) is an accurate and reliable neuroimaging pipeline specifically developed to address such issues commonly observed on MRI of Alzheimer's disease and normal elderly. The pipeline is a complex set of semi-automatic procedures which has been previously validated in a series of internal and external reliability tests1,2. However, LE's accuracy and reliability is highly dependent on properly trained manual operators to execute commands, identify distinct anatomical landmarks, and manually edit/verify various computer-generated segmentation outputs.
LE can be divided into 3 main components, each requiring a set of commands and manual operations: 1) Brain-Sizer, 2) SABRE, and 3) Lesion-Seg. Brain-Sizer's manual operations involve editing of the automatic skull-stripped total intracranial vault (TIV) extraction mask, designation of ventricular cerebrospinal fluid (vCSF), and removal of subtentorial structures. The SABRE component requires checking of image alignment along the anterior and posterior commissure (ACPC) plane, and identification of several anatomical landmarks required for regional parcellation. Finally, the Lesion-Seg component involves manual checking of the automatic lesion segmentation of subcortical hyperintensities (SH) for false positive errors.
While on-site training of the LE pipeline is preferable, readily available visual teaching tools with interactive training images are a viable alternative. Developed to ensure a high degree of accuracy and reliability, the following is a step-by-step, video-guided, standardized protocol for LE's manual procedures.

Lesion Explorer (LE) is a semi-automatic, image-processing pipeline developed to obtain regional brain tissue and subcortical hyperintensity lesion volumetrics from structural MRI of Alzheimer's disease and normal elderly. To ensure a high level of accuracy and reliability, the following is a video-guided, standardized protocol for LE's manual procedures.

病变资源管理器:一个视频引导，标准化为老年痴呆症准确，可靠的MRI衍生Volumetrics和正常老年人协议

Obtaining in vivo human brain tissue volumetrics from MRI is often complicated by various technical and biological issues. These challenges are ...

Minimally Invasive Thumb-sized Pterional Craniotomy for Surgical Clip Ligation of Unruptured Anterior Circulation Aneurysms

Less invasive surgical approaches for intracranial aneurysm clipping may reduce length of hospital stay, surgical morbidity, treatment cost, and improve patient outcomes. We present our experience with a minimally invasive pterional approach for anterior circulation aneurysms performed in a major tertiary cerebrovascular center and compare the results with an aged matched dataset from the Nationwide Inpatient Sample (NIS). From August 2008 to December 2012, 22 elective aneurysm clippings on patients &#8804;55 years of age were performed by the same dual fellowship-trained cerebrovascular/endovascular neurosurgeon. One patient (4.5%) experienced transient post-operative complications. 18 of 22 patients returned for follow-up imaging and there were no recurrences through an average duration of 22 months. A search in the NIS database from 2008 to 2010, also for patients aged &#8804;55 years of age, yielded 1,341 hospitalizations for surgical clip ligation of unruptured cerebral aneurysms. Inpatient length of stay and hospital charges at our institution using the minimally invasive thumb-sized pterional technique were nearly half that of NIS (length of stay: 3.2 vs 5.7 days; hospital charges: $52,779 vs. $101,882). The minimally invasive thumb-sized pterional craniotomy allows good exposure of unruptured small and medium-sized supraclinoid anterior circulation aneurysms. Cerebrospinal fluid drainage from key subarachnoid cisterns and constant bimanual microsurgical techniques avoid the need for retractors which can cause contusions, localized venous infarctions, and post-operative cerebral edema at the retractor sites. Utilizing this set of techniques has afforded our patients with a shorter hospital stay at a lower cost compared to the national average.

Minimally invasive thumb-sized pterional craniotomy for aneurysm clipping has afforded our patients with a shorter hospital stay at a lower cost compared to the national average.

微创拇指大小翼点入开颅未破裂前循环动脉瘤的手术夹子结扎

Less invasive surgical approaches for intracranial aneurysm clipping may reduce length of hospital stay, surgical morbidity, treatment cost, and improve ...

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular weight metabolites in tissues of the treated host to achieve long-term survival. The semipermeable membrane allows engrafted encapsulated cells to avoid rejection by the immune system. The encapsulation procedure was designed to enable a controlled release of bioactive compounds, such as insulin, other hormones, and cytokines. Here we describe a method for encapsulation of catabolic cells, which consume lipids for heat production and energy dissipation (thermogenesis) in the intra-abdominal adipose tissue of obese mice. Encapsulation of thermogenic catabolic cells may be potentially applicable to the prevention and treatment of obesity and type 2 diabetes. Another potential application of catabolic cells may include detoxification from alcohols or other toxic metabolites and environmental pollutants.

Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

封装产热前脂肪细胞移植到脂肪组织仓库

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular ...

A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA thrombolysis) rarely produces benefits in mechanical occlusion-based experimental models, which dominate preclinical stroke research. This split between the bench and bedside suggests the need to employ tPA-responsive models in preclinical stroke research. To this end, a simple and tPA-reactive thrombotic stroke model is invented and described here. This model consists of transient occlusion of the unilateral common carotid artery and delivery of 7.5% oxygen through a face mask in adult mice for 30 min, while maintaining the animal rectal temperature at 37.5 &#177; 0.5 &#176;C. Although reversible ligation of the unilateral carotid artery or hypoxia each suppressed cerebral blood flow only transiently, the combination of both insults caused lasting reperfusion deficits, fibrin and platelet deposition, and large infarct in the middle cerebral artery-supplied territory. Importantly, tail-vein injection of recombinant tPA at 0.5, 1, or 4 hr post-tHI (10 mg/kg) provided time-dependent reduction of the mortality rate and infarct size. This new stroke model is simple and can be standardized across laboratories to compare experimental results. Further, it induces thrombosis without craniectomy or introducing pre-formed emboli. Given these unique merits, the tHI model is a useful addition to the repertoire of preclinical stroke research.

Thromboembolic stroke models are vital tools for optimizing the recanalization therapy. Here we report a murine thrombotic stroke model based on transient cerebral hypoxic-ischemic (tHI) insult, which triggers thrombosis and infarction, and responds favorably to tissue plasminogen activator (tPA)-mediated fibrinolysis in a therapeutic window similar to those in stroke patients.

基于短暂性脑缺氧缺血性血栓性中风模型

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA ...

Minimally Invasive Transverse Aortic Constriction in Mice

Minimally invasive transverse aortic constriction (MTAC) is a more desirable method for the constriction of the transverse aorta in mice than standard open-chest transverse aortic constriction (TAC). Although transverse aortic constriction is a highly functional method for the induction of high pressure in the left ventricle, it is a more difficult and lengthy procedure due to its use of artificial ventilation with tracheal intubation. TAC is oftentimes also less survivable, as the newer method, MTAC, neither requires the cutting of the ribs and intercostal muscles nor tracheal intubation with a ventilation setup. In MTAC, as opposed to a thoracotomy to access to the chest cavity, the aortic arch is reached through a midline incision in the anterior neck. The thyroid is pulled back to reveal the sternal notch. The sternum is subsequently cut down to the second rib level, and the aortic arch is reached simply by separating the connective tissues and thymus. From there, a suture can be wrapped around the arch and tied with a spacer, and then the sternal cut and skin can be closed. MTAC is a much faster and less invasive way to induce left ventricular hypertension and enables the possibility for high-throughput studies. The success of the constriction can be verified using high-frequency trans-thoracic echocardiography, particularly color Doppler and pulsed-wave Doppler, to determine the flow velocities of the aortic arch and left and right carotid arteries, the dimension of the blood vessels, and the left ventricular function and morphology. A successful constriction will also trigger significant histopathological changes, such as cardiac muscle cell hypertrophy with interstitial and perivascular fibrosis. Here, the procedure of MTAC is described, demonstrating how the resulting flow changes in the carotid arteries can be examined with echocardiography, gross morphology, and histopathological changes in the heart.

Minimally invasive transverse aortic constriction (MTAC) conserves the essentials of regular transverse aortic constriction (TAC) while eliminating the use of a ventilator with tracheal intubation. It proves to be a highly desirable method for high-throughput studies on left ventricular overload, particularly in translational studies.

微创横向主动脉缩窄小鼠

Minimally invasive transverse aortic constriction (MTAC) is a more desirable method for the constriction of the transverse aorta in mice than standard ...

Reproducible Motor Deficit Following Aortic Occlusion in a Rat Model Of Spinal Cord Ischemia

Spinal cord ischemia is a fatal complication following thoracoabdominal aortic aneurysm surgery. Researchers can investigate the strategies for preventing and treating this complication using experimental models of spinal cord ischemia. The model described here demonstrates varying degrees of paraplegia that relate to the length of occlusion following thoracic aortic occlusion in a rat spinal cord ischemia model.
A 2-Fr. balloon-tipped catheter was advanced through the femoral artery into the descending thoracic aorta until the catheter tip was placed at the left subclavian artery in anesthetized male Sprague-Dawley rats. Spinal cord ischemia was induced by inflating the catheter balloon. After a set period of occlusion (9, 10, or 11 min), the balloon was deflated. Neurologic assessment was performed using the motor deficit index at 24 h after surgery, and the spinal cord was harvested for histopathological examination.
Rats that underwent 9 min of aortic occlusion showed mild and reversible motor impairment in the hind limb. Rats subjected to 10 min of aortic occlusion presented with moderate but reversible motor impairment. Rats subjected to 11 min of aortic occlusion displayed complete and persistent paralysis. The motor neurons in the spinal cord sections were more preserved in rats subjected to shorter duration of aortic occlusion.
Researchers can achieve a reproducible hind limb motor deficit following thoracic aortic occlusion using this spinal cord ischemia model.

This study demonstrates the technique to make a minimally invasive and easily reproducible model of spinal cord ischemia in rats. Various degrees of hind limb motor deficit can be produced by controlling the aortic occlusion time.

主动脉阻塞后脊髓缺血大鼠模型中的可再生电机缺陷

Spinal cord ischemia is a fatal complication following thoracoabdominal aortic aneurysm surgery. Researchers can investigate the strategies for preventing ...

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an increased use of mouse models in orthopedic research was noted, most probably because mouse models offer a large number of genetically-modified strains and special antibodies for the analysis of molecular mechanisms of fracture healing. To control the biomechanical conditions, well-characterized osteosynthesis techniques are mandatory, also in mice. Here, we report on the design and use of a closed bone healing model to stabilize femur fractures in mice. The intramedullary screw, made of medical-grade stainless steel, provides through fracture compression an axial and rotational stability compared to the mostly used simple intramedullary pins, which show a complete lack of axial and rotational stability. The stability achieved by the intramedullary screw allows the analysis of endochondral healing. A large amount of callus tissue, received after stabilization with the screw, offers ideal conditions to harvest tissue for biochemical and molecular analyses. A further advantage of the use of the screw is the fact that the screw can be inserted into the femur with a minimally invasive technique without inducing damage to the soft tissue. In conclusion, the screw is a unique implant that can ideally be used in closed fracture healing models offering standardized biomechanical conditions.

This protocol describes a minimally invasive osteosynthesis technique using an intramedullary screw for standardized stabilization of femur fractures, which can be used to analyze endochondral bone healing in mice.

标准化生物力学条件下小鼠软骨骨折愈合的微创模型分析

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an ...

Electromagnetic Navigation Transthoracic Nodule Localization for Minimally Invasive Thoracic Surgery

The increased use of chest computed tomography (CT) has led to an increased detection of pulmonary nodules requiring diagnostic evaluation and/or excision. Many of these nodules are identified and excised via minimally invasive thoracic surgery; however, subcentimeter and subsolid nodules are frequently difficult to identify intra-operatively. This can be mitigated by the use of electromagnetic transthoracic needle localization. This protocol delineates the step-by-step process of electromagnetic localization from the pre-operative period to the postoperative period and is an adaptation of the electromagnetically guided percutaneous biopsy previously described by Arias et al. Pre-operative steps include obtaining a same day CT followed by the generation of a three-dimensional virtual map of the lung. From this map, the target lesion(s) and an entry site are chosen. In the operating room, the virtual reconstruction of the lung is then calibrated with the patient and the electromagnetic navigation platform. The patient is then sedated, intubated, and placed in the lateral decubitus position. Using a sterile technique and visualization from multiple views, the needle is inserted into the chest wall at the prechosen skin entry site and driven down to the target lesion. Dye is then injected into the lesion and, then, continuously during needle withdrawal, creating a tract for visualization intra-operatively. This method has many potential benefits when compared to the CT-guided localization, including a decreased radiation exposure and decreased time between the dye injection and the surgery. Dye diffusion from the pathway occurs over time, thereby limiting intra-operative nodule identification. By decreasing the time to surgery, there is a decrease in wait time for the patient, and less time for dye diffusion to occur, resulting in an improvement in nodule localization. When compared to electromagnetic bronchoscopy, airway architecture is no longer a limitation as the target nodule is accessed via a transparenchymal approach. Details of this procedure are described in a step-by-step fashion.

Presented here is a protocol for lung nodule localization using dye marking via electromagnetically navigated transthoracic needle access. The technique described here can be accomplished in the peri-operative period to optimize nodule localization and to successful resection when performing minimally invasive thoracic surgery.

电磁导航经胸结节定位用于微创胸外科手术

The increased use of chest computed tomography (CT) has led to an increased detection of pulmonary nodules requiring diagnostic evaluation and/or ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

重建显微外科缺血再灌注损伤研究的临床前大鼠模型

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...