这项工作是由加拿大自然科学和工程研究理事会（NSERC）发现格兰特＃40352支持，校园艾伯塔省创新程序委员会主席，阿尔伯塔省老年痴呆症研究计划，以MHM，并NSERC CREATE在BIF博士生奖学金和AIHS研究生奖学金到MK。我们感谢浦明·沃对这个协议的发展和外科培训，并贝·米尔扎·阿加和狄鞘的养殖。

<html>下面的协议如下莱斯布里奇动物护理委员会（ACC）的指导方针的大学，并根据加拿大动物保护协会（CCAC）的标准进行的。 1.准备<ol><li>对于长期研究阶段，高压灭菌所有打开的手术用品，并确保不育是保持整个手术。如果需要多次手术，手术之间的反应釜。 </li><li>确保有充足的缓冲脑的手头上（至少50 mL）中。该溶液是由134 mM氯化钠，5.4 mM的钾，氯化镁1mM的六水合物，1.8mM的氯化钙二水合物，和5mM HEPES钠，pH 7.4平衡的用氯化-5 M氢。 </li><li>鼠标放置在感应室，并与3麻醉 - 4％异氟烷。 1.0请 - 2.0％异氟烷在手术过程中的维护。进一步降低到低至0.4 - 0.8％成像期间S = "外部参照"&gt; 5，13，14，15，提供了适当的麻醉维持。这些低麻醉水平要求的鼠标（约每5分钟）的警惕和频繁的监测，以确保它仍然areflexic对疼痛刺激。 注:脱水可由于高的表面成为问题鼠标的体积比和长时间使用异氟醚的加剧。 <ol><li>使用盐水的皮下注射，每10g体重0.1mL的，每1 - 2小时。当充分地水合，鼠标会小便一次，每1 - 2小时。 </li></ol></li><li>密切监视鼠标，以确保整个手术和成像一致麻醉。不要离开鼠标无人值守和照顾它永远不会恢复意识。 </li><li>传送麻醉小鼠在头保持器设置和地点上设置为37℃的热调节的加热垫。安全上最佳匹配的在齿架小时。 </li><li>旋转鼠标的向左头约30°，以暴露所述头部的右横向侧并与耳棒（ 图1A）的钝端固定小鼠的头部。 </li><li>应用眼药膏，以防止角膜干燥。 </li><li>为了减少脑水肿，（4毫克/千克）肌内注射地塞米松。 </li><li>抹在4％氯己定（3次）和70％乙醇（3次）浸渍棉签手术区域的皮肤上。每次使用棉签只有一次。 </li><li>唐外科手套并覆盖有粘合剂保鲜膜的动物。由利多卡因注射提供局部麻醉剂（8 - 10毫克/千克; 2％肾上腺素）皮下在开颅手术部位。等待3 - 5分钟，使药物被吸收到组织中。 </li></ol> 2.取出皮肤和头骨缩回肌肉<ol><li>而下一个观察颅骨执行几乎所有的这些程序解剖显微镜（ 例如 0.7-4.5倍的功率，视情况而定）。 </li><li>用镊子提起中线左侧1毫米（仅在耳后），并用手术剪刀做一个小的水平切口。 </li><li>对右耳进行5 - 6 mm的横向切割，然后朝头部的头端切开。 </li><li>在初始切口处，插入剪刀，并沿切线10毫米。 </li><li>切割右耳和右眼附近的皮肤，以暴露头骨和颞肌的右侧。确保暴露区域的最宽部分至少为7 mm。如果手术区域需要延长，请进一步修剪皮肤。 </li><li>通过在颅骨和皮肤之间放入几滴氰基丙烯酸丁酯胶来固定切口周围的皮肤。确保组织已经预先用棉花涂抹器或滚动的组织干燥以增加粘合剂的功效。 </li><li>用棉签擦拭表面以圆周运动的头骨移除从颅骨骨膜。确保不仍然是完全干燥的头骨。 </li><li>使用弹簧剪刀和镊子，分离从头骨的颞肌;切割和横向缩回肌肉直至到达鳞骨（ 图1C）。要格外小心，不要损坏沿靠近眼睛的鳞骨的水平运行颞浅静脉，否则赔了可能发生。 </li><li>控制用凝胶泡沫预浸泡在脑出血缓冲器。对于严重的出血使用热cauterizer。降丁基氰基丙烯酸酯胶到出血部位以下与热cauterizer治疗。确保的区域是通过使用棉尖涂药器或轧制组织以增加粘合剂的效力预先干燥。 </li></ol> 3.开颅手术注:切除颅骨的过程中，外科医生必须保持勤奋和硬脑膜，以避免不必要的复杂性。包括故障排除步骤应该出现并发症。 <ol><li>标记前囟的任用细尖标记或切一小三角形片胶带并在前囟点的角部（ 图2A）来定向底层脑区的位置。 </li><li>之前固定所述头板确保颅骨完全干燥。颅骨将快速风干脑缓冲器浸泡凝胶泡沫的应用之后，如果需要更多的干燥，可使用棉尖拭子。这个步骤是用于头板固定（ 图1B）是至关重要的。 注意:如果头骨骨膜左，或者如果头骨是不是粘在头上盘前干的，它可能会脱落。如果发生这种情况，温和地去除头板，然后重新开始。出血在此过程中，可能会发生;允许几分钟它凝结，然后轻轻取出。不推荐使用此过程要重复两次以上。 </li> &lt;LI&gt;应用乙基氰基丙烯酸酯胶围绕头部板16的底部边缘，和胶水头板在开颅手术区域（ 图1D和图2A）。 </li><li>与牙科水泥只剩下开颅手术区域暴露填补了头骨和头板之间的开口空间。等待牙科用粘固剂干燥和硬化，通常为5 - 10分钟（ 图2B）。 <ol><li>一旦水泥设置，简单地用脑缓冲填充井，并允许浸泡3 - 5分钟。使用卷纸钻孔（ 图2C）之前去除脑缓冲器。 </li></ol></li><li>通过轻轻颅骨的表面与牙钻得分勾勒出手术区域。使用风钻（设置为最大20 PSI的），具有FG¼毛刺，并用一个可变速度脚踏板控制的。 </li><li>轻轻地追查沿原得分钻深化它，E确保钻头不会穿过颅骨进入大脑（ 图2D ）。每隔几分钟轮流，钻孔和用蘸有滚烫的组织颅骨表面。这将减少颅骨的加热和干燥从机械摩擦和长时间的暴露。 注意:使用湿凝胶泡沫后，颅骨将迅速空气干燥。如果需要更多干燥，请使用棉签拭子。 注意:头骨厚度不均匀。例如，顶叶嵴是最厚的区域，而中线附近的头骨区域和鳞状的地标相对较薄。 </li><li>在钻孔期间，通过用镊子或不动钻头轻轻地按压颅骨，定期检查颅骨的屈曲。当骨头开始弯曲时，停止钻孔并将整个窗口浸入脑缓冲液中。 注意:如果血液从一个区域冲出，可能表明硬脑膜已被损坏。如果是这样的话，放一个semI-湿区域上方凝胶泡沫，并尝试以吸收血液，同时轻轻用棉拭子末端将压力施加到凝胶泡沫。 </li><li>等待至少5分钟，头骨移除之前软化骨并减少硬脑膜粘到骨的机会，使得头骨移除过程更容易。 </li><li>而颅骨脑缓冲淹没执行头骨移除过程。 注意:如果该头骨的一部分保持附着顽固，一个＃11手术刀刀片可用于轻轻得分颅骨。要格外小心不要通过头骨到大脑穿刺刀。 </li><li>从前缘开始，轻轻撬动从使用镊子硬脑膜松散头骨。 注:如果在头骨移除过程中发生出血的量小，用移液管或注射器去除缓冲液，然后用新的缓冲区替换。 </li><li>一旦骨松动和硬脑膜"浮动"，紧紧握住用钳子骨和抬离骨硬脑膜。确保骨从来没有渗透到大脑。 </li><li>为了控制出血，滚组织的角落成一点，并从颅很好地去除大部分的缓冲区。快速应用凝胶泡沫，预先浸泡在缓冲液中，于出血部位，同时用棉签擦拭添加很轻的压力。 注:出血通常来自于骨或硬脑膜的表面的边缘;这两种情况都是正常的，如果没有大的血管受损出血会迅速停止。如果继续出血，血液可以填充整个窗口，形成覆盖所述成像区域中的凝块片。 <ol><li>以除去凝块片，小心地同时使血块周围的血管破裂的源完好从摄像区域拾取的凝结血块。注意不要从泄放源取出血块，因为这可能会导致更多的血液流失。灌溉大脑表面脑缓冲洗去任何血液。 </li><li>小心避免接触细腻的脑组织或添加异物到大脑;重复，直到出血停止，约2 - 5分钟（ 图2E）。 注:在这一点上，开颅手术准备准备颅窗口（见第5步）。如果需要，植入颅窗口（参见步骤4）之前，除去硬脑膜。 </li></ol></li></ol> 4.去除杜拉注:杜拉过程需要格外小心，可能需要超过15分钟。 <ol><li>从开颅客场战平多余的缓冲液。同时保持湿润表面，抓住一小块硬脑膜用镊子轻轻撕硬脑膜。 </li><li>使用镊子和剪刀春天轻轻撕开，并切去硬脑膜。 </li><li>降脑表面上更多的大脑缓冲浮动硬脑膜，并帮助它从大脑中分离出来。直到所有硬脑膜从颅窗网站上删除。当正确执行大脑就会显得非常干净的具有鲜明BLOOD血管和没有瑕疵（比较2F 图2E）。 注意:硬脑膜的某些区域连接到小动脉脑（ 例如 ，中线附近靠近颅顶联合区）的表面，去除这种可破裂动脉。在这种情况下，它可能是最好保留一小块硬脑膜完好在动脉之上的。该VSD可能无法穿透小面积，但是这是优选的有大出血。 </li><li>尽快从脉动最小化运动，并进一步防止肿胀固定在琼脂糖脑表面（参见步骤5）。 </li></ol> 5.准备颅窗<ol><li>喷雾盖玻璃用70％乙醇，并使用空气罐轻轻地干燥。确保玻璃与没有斑点或灰尘存在完全清洁。 </li><li>通过加热200毫克琼脂糖粉末溶解在15毫升脑缓冲器的制备1.3％的琼脂糖。设置microwAVE到高和热为10 - 15秒的时间，之间温和搅拌中，直到所有的琼脂溶解。 注意:确保没有气泡或颗粒存在，因为这些将干扰成像。 </li><li>放置在热琼脂糖温度计和冷却琼脂糖至刚好高于固化温度（〜40℃）。 注:在琼脂糖容器的外部运行冷水可加速冷却过程。轻轻搅拌连续，以确保没有气泡或颗粒存在。 </li><li>立即将琼脂之前，从颅很好地去除脑缓冲区。画了一个移液管的琼脂糖，并直接对大脑下降琼脂糖。快速放置在表面上的盖玻片固定琼脂糖盖玻片滴在了角落。 </li></ol> 6.安乐死注:根据我们的经验，这个过程需要经验的外科医生至少3 - 4实践手术达到大于90％成功率。缺乏经验的外科医生可能需要更多的实践。在开颅手术或硬脊膜切开，大脑可能蒙受损害，例如，如果通过骨到脑中钻拳。在开颅手术的边缘的一些轻微损坏可能是允许的。然而，如果大脑看起来并不"干净"与鲜红完好的血管和白色皮质，实验可能需要被终止。可怜的制剂的实例包括那些死者的血管，或在皮质上标有撕裂或破损的血管。如果这些迹象都存在，实验将不可能产生高质量的数据。无论手术/实验成功与否，人道在实验完成安乐死鼠标。 <ol><li>至少有3.5％的异氟烷​​麻醉深刻。然后，给予戊巴比妥钠的腹膜内注射300毫克/公斤。理想情况下，如果针插入到肝脏，死亡会非常快速（&lt;1分钟）。 </li><li>如果需要灌注，确认该动物是诉讼程序（见步骤1.3）之前，深度麻醉。 </li><li>可替代地，如果不需要灌注，等待至少5分钟，然后验证该小鼠是死的。确认没有呼吸和心跳，以及缺乏疼痛撤离及角膜反射消失。此外，观察四肢的淡蓝色/白色着色和变暗血管在皮层。 </li></ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesoscale+transcranial+spontaneous+activity+mapping+in+GCaMP3+transgenic+mice+reveals+extensive+reciprocal+connections+between+areas+of+somatomotor+cortex.">Mesoscale transcranial spontaneous activity mapping in GCaMP3 transgenic mice reveals extensive reciprocal connections between areas of somatomotor cortex.</a> J Neurosci. 34 (48), 15931-15946 (2014).</li><li>Xie, Y., 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=Resolution+of+High-Frequency+Mesoscale+Intracortical+Maps+Using+the+Genetically+Encoded+Glutamate+Sensor+iGluSnFR.">Resolution of High-Frequency Mesoscale Intracortical Maps Using the Genetically Encoded Glutamate Sensor iGluSnFR.</a> J Neurosci. 36 (4), 1261-1272 (2016).</li><li>Chan, A. W., Mohajerani, M. H., LeDue, J. M., Wang, Y. T., 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=Mesoscale+infraslow+spontaneous+membrane+potential+fluctuations+recapitulate+high-frequency+activity+cortical+motifs.">Mesoscale infraslow spontaneous membrane potential fluctuations recapitulate high-frequency activity cortical motifs.</a> Nat Commun. 6, 7738 (2015).</li><li>Lim, D. H., 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=In+vivo+Large-Scale+Cortical+Mapping+Using+Channelrhodopsin-2+Stimulation+in+Transgenic+Mice+Reveals+Asymmetric+and+Reciprocal+Relationships+between+Cortical+Areas.">In vivo Large-Scale Cortical Mapping Using Channelrhodopsin-2 Stimulation in Transgenic Mice Reveals Asymmetric and Reciprocal Relationships between Cortical Areas.</a> Front Neural Circuits. 6, (2012).</li><li>Ferezou, I., 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=Spatiotemporal+dynamics+of+cortical+sensorimotor+integration+in+behaving+mice.">Spatiotemporal dynamics of cortical sensorimotor integration in behaving mice.</a> Neuron. 56 (5), 907-923 (2007).</li><li>Mohajerani, M. H., Aminoltejari, K., 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=Targeted+mini-strokes+produce+changes+in+interhemispheric+sensory+signal+processing+that+are+indicative+of+disinhibition+within+minutes.">Targeted mini-strokes produce changes in interhemispheric sensory signal processing that are indicative of disinhibition within minutes.</a> Proc Natl Acad Sci U S A. 108 (22), E183-E191 (2011).</li><li>Madisen, L., 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=Transgenic+mice+for+intersectional+targeting+of+neural+sensors+and+effectors+with+high+specificity+and+performance.">Transgenic mice for intersectional targeting of neural sensors and effectors with high specificity and performance.</a> Neuron. 85 (5), 942-958 (2015).</li></ol>

作者什么都没有透露。

这对于大颅窗口创新协议使得能够在大脑皮质的时间和顶区同时成像。与光学成像结合，它可以帮助自发的和刺激诱发的活动中来揭示皮层区域内的神经动力学。这种膨胀的开颅手术还公开了一个大的扩展皮质脉管系统的网络，包括大脑中动脉（MCA）的近端，从而使在血流和血管外侧缺血性模型的直接操纵的体内成像。这种技术将是伟大的使用表达电压和钙指标蛋白23</...

开颅是使用神经科学家以显示脑的一部分的标准程序。由于电的曙光，开颅手术已经允许在神经科学领域前所未有的突破。与电极大脑皮层的密集映射导致基于这些地图的实验测试假说和理论。我们最近进入其中开颅正用于皮质血流1，2，3和神经血管结构4的体内成像，使曝光区5，6，7内皮层活动的实时可视化的新时代。虽然许多研究使用与开颅体内光学成像技术相结合，研究结构和皮层神经元，神经胶质的功能，和COR蒂卡尔脉管系统8， 图9，进一步调查由暴露皮质的小区域限定（但参阅10）。 该协议的目的是提供一种用于产生大的横向开颅，从中线暴露大脑皮层到鳞骨，并且延伸超出前囟和lambda提供的方法。这种大的开颅使得关联皮层的同时观看（脾后，扣，和顶叶），原发性和继发性马达，体感，视觉和听觉皮层。这种方法已经被预先加上电压敏感染料成像（VSDI）调查皮层区域如何多个自发的和刺激诱发的皮质活动5，11，12中彼此交互。此过程中最具挑战性的方面包括定位头动物的，固定所述头板，和避免出血，同时从顶骨分离颞肌。护理也必须在钻井和头骨移除过程作为头骨曲线以倾斜的角度拍摄。

<table><tbody><tr><td>Heating Pad</td><td>FHC</td><td>40-90-2</td><td></td></tr><tr><td>Fine Scissors</td><td>Fine Science Tools</td><td>14058-09</td><td></td></tr><tr><td>Forceps&amp;nbsp;</td><td>Fine Science Tools</td><td>11251-35</td><td>2 or more pairs are recommended</td></tr><tr><td>Spring scissors</td><td>Fine Science Tools</td><td>15000-00, 15000-10</td><td>1 pair should be designated for dura removal&amp;nbsp;</td></tr><tr><td>Jet tooth shade powder</td><td>LANG Dental</td><td>Jet Tooth Shade Powder</td><td>to be mixed with the Jet Liquid</td></tr><tr><td>Jet tooth shade liquid</td><td>LANG Dental</td><td>Jet Tooth Shade Liquid</td><td>to be mixed wihth the Jet Powder&amp;nbsp;</td></tr><tr><td>Drill Heads - Carbide Burs FG 1/4 389</td><td>Midwest Dental</td><td>385201</td><td></td></tr><tr><td>Agarose Powder</td><td>Sigma-Aldrich</td><td>A9793</td><td></td></tr><tr><td>Gelfoam</td><td>Sinclair Dental Canada</td><td>Pfizer Gelfoam</td><td></td></tr><tr><td>Isoflurane</td><td>Western Drug Distribution Centre Ltd</td><td>124125</td><td></td></tr><tr><td>Lidocaine 2% Epinephrine</td><td>Western Drug Distribution Centre Ltd</td><td>125299</td><td></td></tr><tr><td>Dexamethazone 5&amp;nbsp;mg/mL</td><td>Western Drug Distribution Centre Ltd</td><td>125231</td><td></td></tr><tr><td>Butyl cyanoacrylate glue (VetBond)</td><td>Western Drug Distribution Centre Ltd</td><td>12612</td><td></td></tr></tbody></table>

a large lateral craniotomy procedure for mesoscale wide-field optical imaging of brain activity

开颅是一种常用执行程序以暴露脑用于体内实验。在小鼠研究，大多数实验室利用小开颅，一般为3毫米×3毫米。此协议引入用于产生基本上更大7毫米×6mm的颅窗露出最一个大脑半球的在小鼠颞和顶叶皮层的方法（ 例如，囟2.5 - 4.5毫米，侧0 - 6毫米）。为了执行该手术，头必须倾斜大约30°，并且许多颞肌的必须缩回。由于大量的骨质去除，这种方法仅用于急性实验在整个手术和麻醉实验动物。 这种创新的大的横向颅窗口的主要优点是提供了皮质的两个内侧和外侧的区域同时访问。这个大单边颅窗口可用于研究细胞之间的神经动力学，以及通过组合多电极电生理记录，神经元活动（ 例如，内部或外部成像），和光遗传学刺激的成像作为不同的皮层区之间。此外，这种大开颅手术也暴露出皮层血管大面积的，允许横向皮质血管的直接操作。

<html>为了研究在单个半球内皮层区之间的相互作用，我们使用了跨越矢状窦和5延伸出的大的开颅 - 6毫米横向。此颅窗口包括初级（马达，体感，视觉，听觉），仲（电机，视觉）和关联（脾后，扣，顶叶协会）右脑半球（ 图3A）的皮层。对于这项工作，我们使用电压敏感染料（VSD）成像，这反映在膜电位3变化。该协议也将是其它?...

Watch this Scientific Journal Video about A Large Lateral Craniotomy Procedure for Mesoscale Wide-field Optical Imaging of Brain Activity at JoVE.com

A Large Lateral Craniotomy Procedure for Mesoscale Wide-field Optical Imaging of Brain Activity

该协议提出了在小鼠大脑皮质的时间和顶叶区域产生大单方面开颅的方法。这对于实时成像在皮质半球的一个广阔的区域是特别有用的。

大脑侧向颅骨切开术大脑活动的中尺度广角光学成像

The craniotomy is a commonly performed procedure to expose the brain for in vivo experiments. In mouse research, most labs utilize a small craniotomy, ...

a-large-lateral-craniotomy-procedure-for-mesoscale-wide-field-optical

Research

JoVE Journal

Neuroscience

12.2K 次观看.  University of Lethbridge. 该协议提出了在小鼠大脑皮质的时间和顶叶区域产生大单方面开颅的方法。这对于实时成像在皮质半球的一个广阔的区域是特别有用的。 

视频: A Large Lateral Craniotomy Procedure for Mesoscale Wide-field Optical Imaging of Brain Activity

A Craniotomy Surgery Procedure for Chronic Brain Imaging

Imaging techniques are becoming increasingly important in the study brain function. Among them, two-photon laser scanning microscopy has emerged as an extremely useful method, because it allows the study of the live intact brain. With appropriate preparations, this technique allows the observation of the same cortical area chronically, from minutes to months. In this video, we show a preparation for chronic in vivo imaging of the brain using two-photon microscopy. This technique was initially pioneered by Dr. Karel Svoboda, who is now a Howard Hughes Medical Institute Investigator at Janelia Farm. Preparations like the one shown here can be used for imaging of neocortical structure (e.g., dendritic and axonal dynamics),  to record neuronal activity using calcium-sensitive dyes, to image cortical blood flow dynamics, or for intrinsic optical imaging studies. Deep imaging of the neocortex is possible with optimal cranial window surgeries. Operating under the most sterile conditions possible to avoid infections, together with using extreme care to do not damage the dura mater during the surgery, will result in successful and long-lasting glass-covered cranial windows.


This video and protocol demonstrate how to implant a glass-covered cranial window in rodents. These preparations can be used for chronic in vivo two-photon imaging of the neocortex over time scales of months. It may also be used for other types of imaging, including optical intrinsic signal imaging.

一种慢性脑功能成像开颅手术过程

Imaging techniques are becoming increasingly important in the study brain function. Among them, two-photon laser scanning microscopy has emerged as an ...

科研

生物学

Longitudinal Intravital Imaging of Brain Tumor Cell Behavior in Response to an Invasive Surgical Biopsy

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized treatment determination. However, perturbation of the tumor architecture by biopsy and other invasive procedures has been associated with undesired effects on tumor progression, which need to be studied in depth to further improve the clinical benefit of these procedures. Conventional static approaches, which only provide a snapshot of the tumor, are limited in their ability to reveal the impact of biopsy on tumor cell behavior such as migration, a process closely related to tumor malignancy. In particular, tumor cell migration is the key in highly aggressive brain tumors, where local tumor dissemination makes total tumor resection virtually impossible. The development of multiphoton imaging and chronic imaging windows allows scientists to study this dynamic process in living animals over time. Here, we describe a method for the high-resolution longitudinal imaging of brain tumor cells before and after a biopsy in the same living animal. This approach makes it possible to study the impact of this procedure on tumor cell behavior (migration, invasion, and proliferation). Furthermore, we discuss the advantages and limitations of this technique, as well as the ability of this methodology to study changes in the cancer cell behavior for other surgical interventions, including tumor resection or the implantation of chemotherapy wafers.

Here we describe a method for high-resolution time-lapse multiphoton imaging of brain tumor cells before and after invasive surgical intervention (e.g., biopsy) within the same living animal. This method allows studying the impact of these invasive surgical procedures on tumor cells&#39; migratory, invasive, and proliferative behavior at a single cell level.

脑肿瘤细胞行为的纵向生命成像,以响应侵入性手术活检

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized ...

癌症研究

Targeted Labeling of Neurons in a Specific Functional Micro-domain of the Neocortex by Combining Intrinsic Signal and Two-photon Imaging

In the primary visual cortex of non-rodent mammals, neurons are clustered according to their preference for stimulus features such as orientation1-4, direction5-7, ocular dominance8,9 and binocular disparity9. Orientation selectivity is the most widely studied feature and a continuous map with a quasi-periodic layout for preferred orientation is present across the entire primary visual cortex10,11. Integrating the synaptic, cellular and network contributions that lead to stimulus selective responses in these functional maps requires the hybridization of imaging techniques that span sub-micron to millimeter spatial scales. With conventional intrinsic signal optical imaging, the overall layout of functional maps across the entire surface of the visual cortex can be determined12. The development of in vivo two-photon microscopy using calcium sensitive dyes enables one to determine the synaptic input arriving at individual dendritic spines13 or record activity simultaneously from hundreds of individual neuronal cell bodies6,14. Consequently, combining intrinsic signal imaging with the sub-micron spatial resolution of two-photon microscopy offers the possibility of determining exactly which dendritic segments and cells contribute to the micro-domain of any functional map in the neocortex. Here we demonstrate a high-yield method for rapidly obtaining a cortical orientation map and targeting a specific micro-domain in this functional map for labeling neurons with fluorescent dyes in a non-rodent mammal. With the same microscope used for two-photon imaging, we first generate an orientation map using intrinsic signal optical imaging. Then we show how to target a micro-domain of interest using a micropipette loaded with dye to either label a population of neuronal cell bodies or label a single neuron such that dendrites, spines and axons are visible in vivo. Our refinements over previous methods facilitate an examination of neuronal structure-function relationships with sub-cellular resolution in the framework of neocortical functional architectures.

A method is described for labeling neurons with fluorescent dyes in predetermined functional micro-domains of the neocortex. First, intrinsic signal optical imaging is used to obtain a functional map. Then two-photon microscopy is used to label and image neurons within a micro-domain of the map.

有针对性的特定功能的微域的新皮质相结合的内源信号和双光子成像标记的神经元

In the primary visual cortex of non-rodent mammals, neurons are clustered according to their preference for stimulus features such as orientation1-4, ...

神经科学

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology rely on the injection of a suspension of glioma cells into the brain parenchyma following incision of the dura-mater. Whereas the cells have to be injected superficially to be accessible to intravital two-photon microscopy, superficial injections fail to recapitulate the physiopathological conditions. Indeed, escaping through the injection tract most tumor cells reach the extra-dural space where they expand abnormally fast in absence of mechanical constraints from the parenchyma.
Our improvements consist not only in focally implanting a glioma spheroid rather than injecting a suspension of glioma cells in the superficial layers of the cerebral cortex but also in clogging the injection site by a cross-linked dextran gel hemi-bead that is glued to the surrounding parenchyma and sealed to dura-mater with cyanoacrylate. Altogether these measures enforce the physiological expansion and infiltration of the tumor cells inside the brain parenchyma. Craniotomy was finally closed with a glass window cemented to the skull to allow chronic imaging over weeks in absence of scar tissue development.
Taking advantage of fluorescent transgenic animals grafted with fluorescent tumor cells we have shown that the dynamics of interactions occurring between glioma cells, neurons (e.g. Thy1-CFP mice) and vasculature (highlighted by an intravenous injection of a fluorescent dye) can be visualized by intravital two-photon microscopy during the progression of the disease.
The possibility to image a tumor at microscopic resolution in a minimally compromised cerebral environment represents an improvement of current GBM animal models which should benefit the field of neuro-oncology and drug testing.

We have established a cortical orthotopic glioblastoma model in mice for intravital two-photon microscopy that recapitulates the biophysical constraints normally at play during the growth of the tumor. A chronic glass window replacing the skull above the tumor enables the follow-up of the tumor progression over time by two-photon microscopy.

原位胶质母细胞瘤小鼠模型的维护脑实质的物理约束，并适用于活体双光子显微镜

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology ...

医学

Laser Nanosurgery of Cerebellar Axons In Vivo

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.

Laser Nanosurgery of Cerebellar Axons In Vivo

Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo.

小脑轴突的激光Nanosurgery<em&gt;在体内</em

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

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

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

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

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

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

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

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 &#956;m of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer&#39;s disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer&#39;s disease.

纵<em&gt;在体内</em&gt;的Cerebrovasculature影像:相关性疾病CNS

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

Translational Brain Mapping at the University of Rochester Medical Center: Preserving the Mind Through Personalized Brain Mapping

The Translational Brain Mapping Program at the University of Rochester is an interdisciplinary effort that integrates cognitive science, neurophysiology, neuroanesthesia, and neurosurgery. Patients who have tumors or epileptogenic tissue in eloquent brain areas are studied preoperatively with functional and structural MRI, and intraoperatively with direct electrical stimulation mapping. Post-operative neural and cognitive outcome measures fuel basic science studies about the factors that mediate good versus poor outcome after surgery, and how brain mapping can be further optimized to ensure the best outcome for future patients. In this article, we describe the interdisciplinary workflow that allows our team to meet the synergistic goals of optimizing patient outcome and advancing scientific understanding of the human brain.

This article provides an overview of a multi-modal brain mapping program designed to identify regions of the brain that support critical cognitive functions in individual neurosurgery patients.

罗切斯特大学医学中心的转化脑图:通过个性化大脑映射保护大脑

The Translational Brain Mapping Program at the University of Rochester is an interdisciplinary effort that integrates cognitive science, neurophysiology, ...

Craniotomy Procedure for Visualizing Neuronal Activities in Hippocampus of Behaving Mice

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit functions in systems neuroscience. However, high absorbance and scattering of light in mammalian tissue limit intravital imaging mostly to superficial brain regions, leaving deep-brain areas, such as the hippocampus, out of reach for optical microscopy. In this video, we show the preparation and implantation of the custom-made imaging window to enable chronic in vivo imaging of the dorsal hippocampal CA1 region in head-fixed behaving mice. The custom-made window is supplemented with an infusion cannula that allows targeted delivery of viral vectors and drugs to the imaging area. By combining this preparation with wide-field imaging, we performed a long-term recording of neuronal activity using a fluorescent calcium indicator from large subsets of neurons in behaving mice over several weeks. We also demonstrated the applicability of this preparation for voltage imaging with single-spike resolution. High-performance genetically encoded indicators of neuronal activity and scientific CMOS cameras allowed the recurrent visualization of subcellular morphological details of single neurons at high temporal resolution. We also discuss the advantages and potential limitations of the described method and its compatibility with other imaging techniques.

This article demonstrates the preparation of a custom-made imaging window supplemented with infusion cannula and its implantation onto the CA1 region of the hippocampus in mice.

行为小鼠希波坎帕的神经元活动可视化的颅骨切除术

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit ...

In Vivo Fiber-Coupled Pre-Clinical Confocal Laser-scanning Endomicroscopy (pCLE) of Hippocampal Capillaries in Awake Mice

The goal of this protocol is to describe fiber-optic-bundle-coupled pre-clinical confocal laser-scanning endomicroscopy (pCLE) in its specific application to elucidate capillary blood flow effects during seizures, driven by mural cells. In vitro and in vivo cortical imaging have shown that capillary constrictions driven by pericytes can result from functional local neural activity, as well as from drug application, in healthy animals. Here, a protocol is presented on how to use pCLE to determine the role of microvascular dynamics in neural degeneration in epilepsy, at any tissue depth (specifically in the hippocampus). We describe a head restraint technique that has been adapted to record pCLE in awake animals, to address potential side-effects of anesthetics on neural activity. Using these methods, electrophysiological and imaging recordings can be conducted over several hours in deep neural structures of the brain.

In Vivo Fiber-Coupled Pre-Clinical Confocal Laser-scanning Endomicroscopy pCLE of Hippocampal Capillaries in Awake Mice

Whereas multiphoton imaging is only effective at limited depths from the tissue&#8217;s surface, it is possible to achieve 3 &#181;m resolution imaging at any depth via pCLE. Here, we present a protocol to conduct pCLE imaging to measure microvascular dynamics in the hippocampus of ictal and wild-type mice.

清醒小鼠海马毛细血管的体内纤维耦合临床前共聚焦激光扫描内窥镜 （pCLE）

The goal of this protocol is to describe fiber-optic-bundle-coupled pre-clinical confocal laser-scanning endomicroscopy (pCLE) in its specific application ...

大脑侧向颅骨切开术大脑活动的中尺度广角光学成像

摘要

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此视频中的章节

一种慢性脑功能成像开颅手术过程

脑肿瘤细胞行为的纵向生命成像,以响应侵入性手术活检

有针对性的特定功能的微域的新皮质相结合的内源信号和双光子成像标记的神经元

原位胶质母细胞瘤小鼠模型的维护脑实质的物理约束，并适用于活体双光子显微镜

小脑轴突的激光Nanosurgery

横向慢性颅窗口准备启用

纵

罗切斯特大学医学中心的转化脑图:通过个性化大脑映射保护大脑

行为小鼠希波坎帕的神经元活动可视化的颅骨切除术

清醒小鼠海马毛细血管的体内纤维耦合临床前共聚焦激光扫描内窥镜（pCLE）

大脑侧向颅骨切开术大脑活动的中尺度广角光学成像

摘要

探索更多视频

此视频中的章节

一种慢性脑功能成像开颅手术过程

脑肿瘤细胞行为的纵向生命成像,以响应侵入性手术活检

有针对性的特定功能的微域的新皮质相结合的内源信号和双光子成像标记的神经元

原位胶质母细胞瘤小鼠模型的维护脑实质的物理约束，并适用于活体双光子显微镜

小脑轴突的激光Nanosurgery

横向慢性颅窗口准备启用

纵

罗切斯特大学医学中心的转化脑图:通过个性化大脑映射保护大脑

行为小鼠希波坎帕的神经元活动可视化的颅骨切除术

清醒小鼠海马毛细血管的体内纤维耦合临床前共聚焦激光扫描内窥镜 （pCLE）

清醒小鼠海马毛细血管的体内纤维耦合临床前共聚焦激光扫描内窥镜（pCLE）