Name: piezo high accuracy surgical osteal removal (phasor): a technique for improved cranial window surgery in mice
Uploaded: 2018-03-02T12:30:00.000+00:00
Duration: 6 min 9 s
Description: Watch this Scientific Journal Video about Piezo High Accuracy Surgical Osteal Removal PHASOR: A Technique for Improved Cranial Window Surgery in Mice at JoVE.com

塔玛拉 Zeric 帮助获得了该文中的光子图像。支持的大脑 & 行为, 帕金森和 JPB 基金会, R01 MH108186 和 R01 DA07418。F31 奖学金1F31MH109293-01A1 到 S C。

所有涉及动物的程序都是按照哥伦比亚大学医疗中心机构动物护理和使用委员会 (IACUC) 制定的标准进行的。在麻醉下, 用氯胺酮100毫克/千克和甲苯噻嗪10毫克/千克注射腹腔的颈椎脱位进行安乐死。所有外科手术都是以不育的方式进行的 (外科医生戴着头帽、口罩、消毒手套和干净的一次性实验室大衣), 每次使用时都要用高压的手术工具。
1. 术前步骤
<ol><li>高压釜所有手术器械, 确保不育。</li>
	<li>麻醉 C57bl6 小鼠与异氟醚 (4% 的诱导和 1.5-2% 的手术)。每10分钟捏一下后脚趾, 以确保适当的麻醉深度。增加麻醉, 如果发声或后肢退缩被看到。</li>
	<li>将兽医眼润滑剂应用于眼部, 防止干燥。</li>
	<li>去除头皮上的头发, 用3分钟的应用脱毛凝胶或剃须用电动剃须刀。</li>
	<li>把鼠标放在一个加热垫或校准的水循环毯设置到38°C 的立体定向框架。</li>
	<li>管理丁丙诺啡 (0.1 毫克/千克) 皮下提供术前镇痛。</li>
	<li>用棉尖涂抹器三交替应用洗必泰或 betadine 和70% 乙醇消毒头皮。</li>
	<li>注射100毫升的布比卡因稀释50% 在生理盐水下的头皮, 以提供局部麻醉。</li>
</ol>2. 压电颅窗手术
<ol><li>使用蒸压无菌手术器械, 通过切割与手术剪刀在一个圆形的模式, 删除一个1厘米的头皮圆的头骨。从头骨上抬起头皮, 露出骨头上的骨膜组织。</li>
	<li>用镊子的尖端刮掉任何残留的骨膜组织的外露骨。这是很重要的, 以确保牙科水泥不会脱落后。</li>
	<li>设置压电手术单元在最低设置振动。请注意, 较高的设置可能会破坏血管由于剧烈的振动。</li>
	<li>把不育的圆形尖端固定在手机上。 注: 4 毫米圆形尖端非常适合手术。大的尖端尺寸有助于手术的速度, 因为整个窗口可以同时变薄。</li>
	<li>用冰冷的人工脑脊液 (ACSF) 填充10毫升注射器, 并将其握在不抱压电手机的手里。</li>
	<li>用1毫升/分钟的速度将 ACSF 从注射器上滴下, 将头骨灌溉. 或者, 用蠕动泵定位在头骨上以释放另一只手的使用。 注: 冷 ACSF 的灌溉对于防止由于高频振动引起的摩擦造成的过热非常重要。</li>
	<li>轻轻地将振动手术尖端应用到头骨上, 用一个轻的圆形运动 (每1秒3个圆圈), 将骨骼薄到所需的深度, 以执行薄头骨准备 (5 到10分钟)。 注: 在这里, 4 毫米的窗户被制成稀释到20µm。然而, 窗口的大小会因手术提示的大小而异。骨骼的厚度可以根据所需的深度来调整, 这取决于应用尖端到头骨的时间长短。</li>
	<li>轻轻调整手机的角度, 改变刀尖的角度, 同时对颅骨施加压力, 使头骨均匀地薄。</li>
	<li>要执行完整的骨清除, 而不留下任何薄骨后, 薄骨, 直到裂缝可见周围的窗口区域。用镊子除去残留的薄骨薄片, 而不损害底层硬脑膜。</li>
	<li>在冷 ACSF 中浸泡1毫米的止血胶原泡沫。用镊子把湿泡沫拿出来放在薄头骨上。允许它坐在瘦头骨约三十年代, 以阻止任何微出血的发展。</li>
	<li>使用镊子放置一个4毫米玻璃盖玻片侧在窗口, 已被稀释到骨骼。</li>
	<li>使用一个小塑料或木棉尖头涂抹器的背面应用大约100µL 的牙科丙烯酸的头骨, 以保持窗口到位。</li>
</ol>3. 手术后护理
<ol><li>将鼠标从手术器械上取出, 放在加热的笼子里进行恢复。持续监视它, 直到它恢复了完全意识 (由正常的移动性、步态和行为评估)。</li>
	<li>每天两次对动物进行三天的评估, 以观察手术后疼痛的表现, 包括活动减少、仪容整理、食物和用水量、守卫行为 (如跛行或驼背) 或增加的侵略性。在完全恢复之前, 不要将动物归还其他动物的公司。</li>
	<li>手术3天后, 将丁丙诺啡 (0.05–0.1 毫克/千克) 皮下作为镇痛 8–12 h。在手术后密切监视窗户的区域, 以预防裂开、炎症和感染迹象。如果动物不返回基线喂养和梳理行为, 咨询兽医关于可能的干预或安乐死。</li>
</ol>

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M., Zhu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+innovative+transparent+cranial+window+based+on+skull+optical+clearing.">An innovative transparent cranial window based on skull optical clearing.</a> Laser Physics Letters. 9, (2012).</li><li>Yang, X., 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=Skull+Optical+Clearing+Solution+for+Enhancing+Ultrasonic+and+Photoacoustic+Imaging.">Skull Optical Clearing Solution for Enhancing Ultrasonic and Photoacoustic Imaging.</a> IEEE Trans Med Imaging. 35, (2016).</li><li>Vercellotti, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Piezoelectric+surgery+in+implantology:+a+case+report--a+new+piezoelectric+ridge+expansion+technique.">Piezoelectric surgery in implantology: a case report--a new piezoelectric ridge expansion technique.</a> Int J Periodontics Restorative Dent. 4, 358-365 (2000).</li><li>Vercellotti, T., De Paoli, S., Nevins, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+piezoelectric+bony+window+osteotomy+and+sinus+membrane+elevation:+introduction+of+a+new+technique+for+simplification+of+the+sinus+augmentation+procedure.">The piezoelectric bony window osteotomy and sinus membrane elevation: introduction of a new technique for simplification of the sinus augmentation procedure.</a> Int J Periodontics Restorative Dent. 6, 561-567 (2001).</li><li>Vercellotti, T., Podesta, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthodontic+microsurgery:+a+new+surgically+guided+technique+for+dental+movement.">Orthodontic microsurgery: a new surgically guided technique for dental movement.</a> Int J Periodontics Restorative Dent. 4, 325-331 (2007).</li><li>Stubinger, S., Stricker, A., Berg, B. 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J., Rao, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+Study+of+Piezoelectric+and+Rotary+Osteotomy+Technique+for+Third+Molar+Impaction.">Comparative Study of Piezoelectric and Rotary Osteotomy Technique for Third Molar Impaction.</a> J Contemp Dent Pract. 18, 60-64 (2017).</li><li>Pavlikova, 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=Piezoelectric+surgery+prevents+brain+tissue+damage:+an+experimental+study+on+a+new+rat+model.">Piezoelectric surgery prevents brain tissue damage: an experimental study on a new rat model.</a> Int J Oral Maxillofac Surg. 40, 840-844 (2011).</li></ol>

没有披露。

手术可以通过改变手术技巧来修改。有许多不同大小的小费, 可以应用到手机。更改笔尖的大小或形状将导致不同大小的窗口。除了4毫米的尖端, 我们还尝试了3毫米的尖端, 并发现它也工作良好。即使与冰冷 ACSF 的灌溉, 我们无法得到良好的结果, 因为过于狭窄 (约0.25 毫米) 的提示, 由于集中振动造成太多的热量和燃烧的骨骼导致损害的基础硬脑膜。我们还没有尝试过许多其他可用的提示, 我们预计其他实验室可能会找到新的应用程序, 为这些不同的提示。虽然手术相对简单, 但我们发现有几个步骤可能需要进行故障排除。第一个是, 振动尖端产生了大量的湍流在 ACSF, 这减少能见度, 并产生困难, 确定骨深度在骨细化步骤的手术。我们建议, 如果很难看到骨头有多薄, 休息一下, 用不育的棉签涂抹在骨头的一侧, 使头骨和窗户干燥。不要将棉签直接涂抹在窗户上, 因为粗糙的表面会损害到薄骨。检查深度后, 重新应用 ACSF 并继续手术。我们还发现, 如果硬脑膜受损, 通常是由于外科医生给小费施加了太多的压力。更轻柔地握住手机, 施加更少的力就有可能解决这个问题。如果外科医生用力刮骨上的尖端, 它会导致在薄骨骨折, 并损害头骨。最后, 如果在瘦骨后, 底层的硬脑膜出现擦伤, 很可能是由于来自振动的过热。提高 ACSF 流的速率将解决此问题。
相量的主要限制是手机可以瘦骨, 但不能去除硬脑膜上的所有骨。金刚石涂层尖端有小颠簸。薄头骨所需的摩擦会擦伤硬脑膜, 如果用来去除所有的骨头, 会导致出血。因此, 需要使用镊子来去除剩余的薄层。虽然目前正在进行的辩论是否通过一个薄薄的头骨成像产生较少的炎症和小胶质细胞和反应性星形胶质的增殖较少, 在某些情况下, 一个没有骨头的窗口是首选, 例如, 以提供更深入的成像3.
我们为小鼠的光子成像优化了颅骨窗的制备。我们知道, 相量是第一次应用压电技术在啮齿动物的颅窗手术中进行光学成像。我们发现, 使用压电手术在小鼠共享的优势, 增加速度和减少不良事件也报告在人类7,8,9,10。压电装置的使用使手术速度更快 (图 2A), 而与高速旋转钻头相比, 出血的不良事件减少 (图 2B)。我们还发现, 在我们的实验室里, 新的外科医生比传统的颅骨窗手术方法更容易学到相量。在外科医生中, 速度和易用性的优势可能不同。薄头骨的准备工作通常需要30-45 分钟, 用钻头和手术刀2来薄骨, 而相量法通常需要少于10分钟。
我们发现, 在用相量制备的窗口中, 我们可以在运动皮层的4层锥体神经元中显示钙的瞬变, 这是用腺相关病毒编码钙指示器 JRGECO1a (AAV9。jRGECO1a. WPRE. SV40). 我们还发现, 与用旋转钻头和手术刀制作的薄壁颅骨窗相比, 成像区域更大, 而用旋转钻头和手术刀制作的薄头骨有一个20µm 直径的成像窗口2.用相量, 我们能够快速地薄3-4 毫米的面积。这与用端口4报告的图像窗口类似。这个更大的窗口保留了快速成像报告的好处为稀薄的头骨窗口准备用牙钻和手术刀。此外, 用相量编制的窗口可以立即成像, 而不是传统的骨移除窗口, 可能需要几周的时间来愈合, 然后窗口才能最佳地成像3。
该技术可用于研究硬脑膜下血管血流的变化。将相量的免疫反应与其他颅窗方法进行比较, 是今后研究的重要课题。这将是很重要的, 以确定相量是否产生较少的炎症相比, 现有的方法。我们希望, 在这里记录的压电手术技术将允许更多的实验室执行颅骨窗口和成功地使用多光子成像在体内。
确保手机设置为在最低设置上振动, 并且 ACSF 以恒定速率进行灌溉。冰冷 ACSF 必须不断地应用, 否则会加热并损坏硬脑膜。我们发现, ACSF 必须以至少1毫升/分钟的速度应用. 要么使用手里握着的注射器, 要么使用蠕动泵, 而不是手机内的内置灌溉。手机的灌溉系统 ACSF 太用力, 产生太多的湍流, 这将大大削弱能见度。

颅窗手术制备啮齿动物为光子成像在体内已成为神经科学的一项重要技术。骨的去除或细化是必要的, 以准备的小鼠光学成像与光子显微镜。通过完全移除骨骼区域以暴露基础硬脑膜1, 或者通过将骨骼区域细化而不完全从硬脑膜2中取出来执行此手术。薄头骨方法可能产生较少的炎症和小胶质细胞的激活3 , 但提供了一个较浅的深度的成像, 一个较小的成像窗口大小 (200 µm) 和有限的时间段期间, 该窗口可以成像由于骨骼再生2.添加一个抛光和增强玻璃窗口 (端口) 可以增加成像的大小和成像周期, 但很难执行4。
目前的两种手术都使用高速旋转钻头来瘦身或从头骨中取出骨头。薄头骨技术还使用手术刀后, 进一步瘦骨2。端口技术要求用砂砾4进行高速抛光的额外步骤。在高速旋转钻头中, 空气动力涡轮或电动机使钻头在高速旋转。作为旋转钻头的骨骼和软组织, 有破坏硬脑膜和下血管的危险。手术的成功取决于外科医生的技能。除了这些用机械手术方法准备的窗户外, 还开发了一种用不同溶液光学清除头骨的化学方法5,6。然而, 由于压电手术是一种机械的手术方法, 我们在这里的比较将限于其他机械方法。
压电手术装置利用超声振动分解矿化骨而不损害底层软组织, 从而为快速瘦大面积骨提供了一种方法。在压电外科手机中, 涡轮被一堆陶瓷圆盘所取代, 当电流被应用时, 圆盘在超声波频率下振动。振动通过手机转移到金刚石涂层尖端, 通过骨骼切开, 而不会损伤软组织, 这比不区分组织类型的旋转钻头的优势。压电手术最初是为人类使用的托马斯 Vercellotti, 并导致改善牙科和颅 maxilofacial 手术7,8,9,10,11.
压电手术已被用于制造大鼠截骨术, 并被磁共振成像 (MRI) 和组织学发现比传统的牙科钻具明显减少12。作者认为, 压电手术是安全的, 以消除骨近软脑组织。然而, 老鼠有一个更薄的硬脑膜, 更容易受损, 这项研究并没有为慢性光学成像准备窗户。慢性影像学要求血管不受损, 而血液凝块不在窗口下形成。对硬脑膜的损伤导致炎症, 导致窗口云, 激活小胶质细胞和活性星形胶质的增殖。在这里, 我们有优化的压电手术小鼠, 以创建瘦头骨和完整的骨清除颅窗适合慢性成像。我们将这项手术技术与高速旋转钻机制备的颅骨窗手术进行了比较。

<table><tbody><tr><td>Piezosurgery Touch</td><td>Mectron</td><td>5120062</td><td>Piezosurgery GP model has the same settings</td></tr><tr><td>Circular 4mm flat piezosurgery tip (# OT11)</td><td>Mectron</td><td>3370019</td><td>This tip was ideal for our windows but there are many other tips of different sizes availible.</td></tr><tr><td>Stereotax frame</td><td>Kopf</td><td>963</td><td></td></tr><tr><td>Mouse adaptor</td><td>Stoelting</td><td>51625</td><td></td></tr><tr><td>Peristaltic pump for irrigation.</td><td>Cole-Parmer</td><td>WU-77120-42</td><td>Makes it easier to irrigate and frees up the other hand to provide stability. Irrigation can be performed by hand with a syringe if necessary.</td></tr><tr><td>Avitene Ultrafoam</td><td>Bard-Davol</td><td>1050020</td><td>Important to stop any minor bleeding instantly.</td></tr><tr><td>C&amp;amp;B Metabond</td><td>Parkell</td><td>S380</td><td>Much stronger than regular dental acrylic.</td></tr><tr><td>Artificial cerebro spinal fluid (ACSF)</td><td>Tocris</td><td>3525</td><td></td></tr><tr><td>Puralube opthalmic ointment</td><td>Dechra</td><td>17033-211-38</td><td></td></tr><tr><td>Mice</td><td>JAX</td><td>664</td><td></td></tr><tr><td>Prairie Ultima multiphoton microscope</td><td>Bruker</td><td></td><td></td></tr><tr><td>JRGECO1a (AAV9.Syn.NES-jRGECO1a.WPRE.SV40)</td><td>UPENN Vector Core</td><td></td><td></td></tr></tbody></table>

piezo high accuracy surgical osteal removal (phasor): a technique for improved cranial window surgery in mice

光子显微镜已广泛应用于成像神经元体内。反复成像需要植入颅骨窗口或反复细化颅骨。颅窗手术通常是用高速旋转钻头进行的, 许多调查人员发现它具有挑战性, 以防止钻头损坏细腻的硬脑膜和血管。需要进行广泛的训练和练习, 以去除骨骼而不损伤底层组织, 从而使颅骨窗手术困难, 耗时, 并产生组织损伤。压电外科, 广泛用于颌面和牙科手术, 利用超声振动去除骨不损伤软组织。我们开发了一种应用压电手术改善小鼠颅骨窗手术的方法, 用于制备光子成像。在我们的实验室比较发现, 该方法需要较少的手术时间, 并有较低的平均率的硬脑膜出血比颅窗手术与旋转钻机。

在进行压电手术前, 从颅骨上取出残余的骨膜。一旦头骨不透明和平滑 (图 1a), 外科医生可能开始压电手术。当用振动压电尖去除骨骼时, 用冰冷 ACSF 对颅骨进行冲洗是至关重要的。当尖端的底部1毫米淹没在 ACSF (图 1b) 中时, 就能进行适当的灌溉。如果没有适当的灌溉, 骨骼会过热并损伤大脑。当颅骨被外科医生选择的深度稀释后, 骨头上的血管可能会留下一些残余的出血。为了迅速停止所有出血, 应用一个小1毫米的胶原泡沫浸泡在 ACSF 的窗口区域。让胶原蛋白坐在头骨上大约三十年代 (图 1c)。胶原质泡沫被除去后, 窗户会呈半透明状, 使硬脑膜中的血管清晰可见。硬脑膜将完好无损, 没有明显的瘀伤。(图 1d)。如果硬脑膜出现红色和发炎, 这很可能是由于血管破裂, 在手术中应用到尖端的压力太大。为了保护新的窗口并准备用于慢性成像, 必须在该区域放置一个玻璃盖玻片。适当应用的玻璃盖玻片将轻轻地坐在窗口的顶部, 不会造成任何损害的区域。玻璃盖玻片必须覆盖整个窗户。如果玻璃盖玻片正确地应用到头骨, 窗口将保持半透明的玻璃下。硬脑膜中的所有血管仍将可见 (图 1e)。牙科丙烯酸必须在玻璃盖玻片的周围使用, 永久地坚持它的头骨表面。玻璃盖玻片的边缘也必须覆盖在牙科丙烯酸(图 1f)。
我们发现, 压电手术通常比传统的颅骨窗快得多, 每次手术大约要10-12 分钟 (图 2A)。我们还发现, 有较少的并发症, 由于硬脑膜瘀伤和出血的眼睛观察 (图 2B)。一个适当准备好的窗口将允许在大脑皮层神经元的荧光指示器的光子成像在体内。我们选择了在4层皮层神经元 (图 3A-3B) 的细胞体中 JRGECO1a 红色钙指示器的图像。我们可以通过用相量制备的窗口观察这些细胞体内的钙瞬态。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56172/56172fig1.jpg"> 图 1: 压电手术.(a) 头骨经过适当的相量准备后。所有的骨膜组织已被删除, 留下一个平滑和干净的表面。(b) 正在进行的相量的代表性图像。1毫米的振动外科尖端被淹没在冰冷的 ACSF。该液体以1毫升/分钟的速度应用于颅骨。在颅骨上采用光循环运动, 通过超声振动使骨骼变薄。(c) 在头骨变薄后, 在冷 ACSF 中浸泡1毫米圆形胶原泡沫, 可以在窗户上休息, 以阻止骨骼中的微量出血。(d) 一个成功的窗口, 半透明硬脑膜显示所有完整的血管。大脑表面没有明显的损伤或瘀伤。(e) 将玻璃盖玻片放在窗户上, 以保护大脑的表面。(f) 将牙科丙烯酸应用于头骨, 将玻璃盖玻片永久固定在窗户上。在所有图像中缩放条形图1毫米。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56172/56172fig2.jpg"> 图2。相量或牙钻外科训练成功率的比较。() 每个手术的平均时间比牙科钻探手术 (p < 0.05, 两个尾 t 测试) n = 每组30次手术和每名外科医生平均10个手术.(B) 成功手术的百分比 (定义为: 如果外科医生的眼睛通过目标设置为350X 变焦) 对硬脑膜没有出血或明显损伤, 则相量比牙科钻孔手术 (每组30次手术) 高 (一尾 z 测试)。误差线显示 SD。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56172/56172fig3.jpg"> 图3。在活体中的光子成像在体内通过颅骨窗口稀释与相量.(A) 用红色钙指示器 JRGECO1a 观察到的钙瞬变在小鼠运动皮层体内的4层锥体神经元中被成像。缩放条 = 100 µm (B) 同一窗口, 但4层运动皮层的不同区域。用相量制备的 3 mm 窗 Headfixed 鼠标。刻度条 = 30 µm. 用光子显微镜成像, 40X 目标, 1040 nm 激发波长。

Watch this Scientific Journal Video about Piezo High Accuracy Surgical Osteal Removal PHASOR: A Technique for Improved Cranial Window Surgery in Mice at JoVE.com

Piezo High Accuracy Surgical Osteal Removal PHASOR: A Technique for Improved Cranial Window Surgery in Mice

压电手术已导致改善人颌面部和牙科手术。我们已经制定了一个优化压电手术的小鼠颅窗手术的协议。

压电高精度手术骨切除 (相量): 改进小鼠颅骨窗手术的技术

Multiphoton microscopy has been widely adapted for imaging neurons in vivo. Repeated imaging requires implantation of a cranial window or repeated ...

piezo-high-accuracy-surgical-osteal-removal-phasor-technique-for

Research

JoVE Journal

Neuroscience

Piezo High Accuracy Surgical Osteal Removal (PHASOR): A Technique for Improved Cranial Window Surgery in Mice

10.6K 次观看.  Columbia University Medical Campus. 压电手术已导致改善人颌面部和牙科手术。我们已经制定了一个优化压电手术的小鼠颅窗手术的协议。

视频: Piezo High Accuracy Surgical Osteal Removal PHASOR: A Technique for Improved Cranial Window Surgery in Mice

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

Optical coherence tomography (OCT) is a biomedical imaging technique with high spatial-temporal resolution. With its minimally invasive approach OCT has been used extensively in ophthalmology, dermatology, and gastroenterology1-3. Using a thinned-skull cortical window (TSCW), we employ spectral-domain OCT (SD-OCT) modality as a tool to image the cortex in vivo. Commonly, an opened-skull has been used for neuro-imaging as it provides more versatility, however, a TSCW approach is less invasive and is an effective mean for long term imaging in neuropathology studies. Here, we present a method of creating a TSCW in a mouse model for in vivo OCT imaging of the cerebral cortex.

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

We present a method of creating a thinned-skull cortical window (TSCW) in a mouse model for in vivo OCT imaging of the cerebral cortex.

稀疏的头骨皮层的窗口技术<em在体内</em光学相干断层成像

Optical coherence tomography (OCT) is a biomedical imaging technique  with high spatial-temporal resolution. With its minimally invasive approach OCT  has ...

科研

神经科学

Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of therapies to enhance fracture healing.&#160; Periosteal cells rapidly migrate to an injury to supply new chondrocytes and osteoblasts for fracture healing. Traditionally, the efficacy of a cytokine to induce cell migration has only been conducted in vitro by performing a transwell or scratch assay. With advancements in intravital microscopy using multiphoton excitation, it was recently discovered that 1) P-SSCs express the migratory gene CCR5 and 2) treatment with the CCR5 ligand known as CCL5 improves fracture healing and the migration of P-SSCs in response to CCL5. These results have been captured in real-time. Described here is a protocol to visualize P-SSC migration from the calvarial suture skeletal stem cell (SSC) niche towards an injury after treatment with CCL5. The protocol details the construction of a mouse restraint and imaging mount, surgical preparation of the mouse calvaria, induction of a calvaria defect, and acquisition of time-lapse imaging.

This protocol describes the detection of CCL5-mediated periosteal skeletal stem cell migration in real-time using live animal intravital microscopy.

CCL5诱导小鼠骨膜骨骼干细胞迁移的实时成像

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of ...

发育生物学

The Mouse Round-window Approach for Ototoxic Agent Delivery: A Rapid and Reliable Technique for Inducing Cochlear Cell Degeneration

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research involving the cochlea and its associated neural pathways entails exposure of model cochleae to a variety of ototoxic agents. This allows investigators to study the effects of targeted damage to cochlear structures, and in some cases, the self-repair or regeneration of those structures. Various techniques exist for delivery of ototoxic agents to the cochlea. When selecting a particular technique, investigators must consider a number of factors, including the induction of inadvertent systemic toxicity, the amount of cochlear damage produced by the surgical procedure itself, the type of lesion desired, animal survivability, and reproducibility/reliability of results. Currently established techniques include parenteral injection, intra-peritoneal injection, trans-tympanic injection, endolymphatic sac injection, and cochleostomy with perilymphatic perfusion. Each of these methods has been successfully utilized and is well described in the literature; yet, each has various shortcomings. Here, we present a technique for topical application of ototoxic agents directly to the round window niche. This technique is non-invasive to inner ear structures, produces rapid onset of reliably targeted lesions, avoids systemic toxicity, and allows for an intra-animal control (the contra-lateral ear). Results stemming from this approach have helped deeper understanding of auditory pathophysiology, cochlear cell degeneration, and regenerative capacity in response to an acute injury. Future investigations may use this method to conduct interventional studies involving gene therapy and stem cell transplantation to combat hearing loss.

Various methods exist for introducing ototoxic agents to the cochleae of animal models. Presented is a surgical protocol for delivery of ototoxic agents to the round window niche. The procedure is reliable, creates targeted intra-cochlear lesions, and avoids mechanical damage to the microarchitecture. Examination of cochlear self-repair/regeneration is possible.

老鼠圆窗途径耳毒性剂递送:一个快速和可靠的技术诱导耳蜗细胞变性

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research ...

医学

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 ...

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their activity in vivo in behaving mice. This paper describes a method for the implantation of a chronic cranial window to allow for the longitudinal imaging of brain cells in awake, head-restrained mice. In combination with genetic strategies and viral injections, it is possible to label specific cells and regions of interest with structural or physiological markers. This protocol demonstrates how to combine viral injections to label neurons in the vicinity of GCaMP6-expressing astrocytes in the cortex for simultaneous imaging of both cells through a cranial window. Multiphoton imaging of the same cells can be performed for days, weeks, or months in awake, behaving animals. This approach provides researchers with a method for viewing cellular dynamics in real time and can be applied to answer a number of questions in neuroscience.

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

Presented here is a protocol for the implantation of a chronic cranial window for the longitudinal imaging of brain cells in awake, head-restrained mice.

植入颅窗以在清醒小鼠 中重复体内 成像

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their ...

In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus

Microglia, the only immune cells resident in the brain, actively participate in neural circuit maintenance by modifying synapses and neuronal excitability. Recent studies have revealed the differential gene expression and functional heterogeneity of microglia in different brain regions. The unique functions of the hippocampal neural network in learning and memory may be associated with the active roles of microglia in synapse remodeling. However, inflammatory responses induced by surgical procedures have been problematic in the two-photon microscopic analysis of hippocampal microglia. Here, a method is presented that enables the chronic observation of microglia in all layers of the hippocampal CA1 through an imaging window. This method allows the analysis of morphological changes in microglial processes for more than 1 month. Long-term and high-resolution imaging of the resting microglia requires minimally invasive surgical procedures, appropriate objective lens selection, and optimized imaging techniques. The transient inflammatory response of hippocampal microglia may prevent imaging immediately after surgery, but the microglia restore their quiescent morphology within a few weeks. Furthermore, imaging neurons simultaneously with microglia allows us to analyze the interactions of multiple cell types in the hippocampus. This technique may provide essential information about microglial function in the hippocampus.

In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus

This paper describes a method for chronic in vivo observation of the resting microglia in the mouse hippocampal CA1 using precisely controlled surgery and two-photon microscopy.

体内 小鼠海马体中小胶质细胞的慢性双光子成像

Microglia, the only immune cells resident in the brain, actively participate in neural circuit maintenance by modifying synapses and neuronal ...

Assessment of Thermal Damage from Robot-Drilled Craniotomy for Cranial Window Surgery in Mice

Cranial window surgery allows for the imaging of brain tissue in live mice with the use of multiphoton or other intravital imaging techniques. However, when performing any craniotomy by hand, there is often thermal damage to brain tissue, which is inherently variable surgery-to-surgery and may be dependent on individual surgeon technique. Implementing a surgical robot can standardize surgery and lead to a decrease in thermal damage associated with surgery. In this study, three methods of robotic drilling were tested to evaluate thermal damage: horizontal, point-by-point, and pulsed point-by-point. Horizontal drilling utilizes a continuous drilling schematic, while point-by-point drills several holes encompassing the cranial window. Pulsed point-by-point adds a &#34;2 s on, 2 s off&#34; drilling scheme to allow for cooling in between drilling. Fluorescent imaging of Evans Blue (EB) dye injected intravenously measures damage to brain tissue, while a thermocouple placed under the drilling site measures thermal damage. Thermocouple results indicate a significant decrease in temperature change in the pulsed point-by-point (6.90 &#176;C &#177; 1.35 &#176;C) group compared to the horizontal (16.66 &#176;C &#177; 2.08 &#176;C) and point-by-point (18.69 &#176;C &#177; 1.75 &#176;C) groups. Similarly, the pulsed point-by-point group also showed significantly less EB presence after cranial window drilling compared to the horizontal method, indicating less damage to blood vessels in the brain. Thus, a pulsed point-by-point drilling method appears to be the optimal scheme for reducing thermal damage. A robotic drill is a useful tool to help minimize training, variability, and reduce thermal damage. With the expanding use of multiphoton imaging across research labs, it is important to improve the rigor and reproducibility of results. The methods addressed here will help inform others of how to better use these surgical robots to further advance the field.

Cranial windows have become a ubiquitously implemented surgical technique to allow for intravital imaging in transgenic mice. This protocol describes the use of a surgical robot that performs semi-automated bone drilling of cranial windows and can help reduce surgeon-to-surgeon variability and partially mitigate thermal blood-brain barrier damage.

机器人钻孔开颅术对小鼠颅窗手术热损伤的评估

Cranial window surgery allows for the imaging of brain tissue in live mice with the use of multiphoton or other intravital imaging techniques. However, ...

Spatiotemporal Analysis of Microglial Ca2+ Activity at Single-Cell Resolution

Microglia are the sole resident immune cells in the central nervous system. Their morphology is highly plastic, changing depending on their activity. Under homeostatic conditions, microglia possess a highly ramified morphology. This facilitates their monitoring of the surrounding environment through the continuous extending and retracting of their processes. During brain injury and inflammation, however, microglia become activated and undergo dramatic morphological changes, retracting their ramified processes and swelling their cell body. This facilitates activities such as migration and phagocytosis, which microglia undertake to navigate the brain environment to a less pathological state.
This close relationship between microglial morphology and changes in their activity have enabled considerable insights into various microglial functions. However, such morphological and activity changes are themselves phenomena that can result from any number of intracellular signaling pathways. Moreover, the time-lag between stimulus and response, as well as the highly compartmentalized morphology of microglia, make it difficult to isolate the causative mechanisms that underpin function. To solve this problem, we developed a genetically modified mouse line in which a highly sensitive fluorescent Ca2+-indicator protein is specifically expressed in microglia.
After describing methods for in vivo microglial Ca2+ imaging, this paper presents a structured analysis approach that classifies this Ca2+ activity to rationally defined subcellular regions, thus ensuring that the spatial and temporal dimensions of the encoded information are meaningfully extracted. We believe that this approach will provide a detailed understanding of the intracellular signaling rules that govern the diverse array of microglial activities associated with both higher brain functions and pathological conditions.

Spatiotemporal Analysis of Microglial Ca2+ Activity at Single-Cell Resolution

In this paper, we describe a protocol for in vivo imaging of microglial Ca2+ activity and subsequent analysis of its spatiotemporal dynamics. This method enables thorough characterization of how microglia respond to changes in the brain environment, appropriately capturing the fine spatiotemporal scales at which such events occur.

Microglia are the sole resident immune cells in the central nervous system. Their morphology is highly plastic, changing depending on their activity. ...

自由移动小鼠的 FUN 和纤维光度测定的综合方案

Simultaneous Focused Ultrasound Neuromodulation and Fiber Photometry Recording in Free-Moving Mouse

Focused ultrasound neuromodulation (FUN) represents a promising approach for non-invasive perturbation of neuronal circuits at deep brain regions. It is compatible with most of the existing modalities for monitoring brain functions in vivo. Integration with brain function recording modalities not only enables us to address orders and disorders of specific brain functions with closed-loop feedback but also provides us with mechanistic insights about FUN itself. Here, we provide a modified, simple, dependable, and robust protocol for the simultaneous application of FUN and fiber photometry GCaMP6s fluorescence recording in free-moving mice. This involves the fabrication of a well-sized single transducer and its temporary placement on the mice, along with the secure fixation of a fiber optical implant to facilitate the smooth passage of the transducer. The combination of FUN and fiber photometry provides for the optical recording of neural circuitry responses upon FUN in real-time in deep brain regions. To demonstrate the efficiency of this protocol, Thy1-GCaMP6s mice were used as an example to record the neuroactivity in the anterior thalamic nucleus during FUN while the mice are freely moving. We believe that this protocol can promote the widespread use of FUN in both the neuroscience field and the biomedical ultrasound field.

作者聚焦:纤维光度法和聚焦超声神经调控的整合用于研究自由移动小鼠的神经调节

The protocol includes transducer manufacturing, parameters reporting, surgical procedure, and signal recording for the entire operational workflow of concurrent focused ultrasound neuromodulation and fiber photometry recording in free-moving mice.

自由移动小鼠的同步聚焦超声神经调控和纤维光度记录

Focused ultrasound neuromodulation (FUN) represents a promising approach for non-invasive perturbation of neuronal circuits at deep brain regions. It is ...

压电高精度手术骨切除 (相量): 改进小鼠颅骨窗手术的技术

摘要

探索更多视频

此视频中的章节

稀疏的头骨皮层的窗口技术

CCL5诱导小鼠骨膜骨骼干细胞迁移的实时成像

老鼠圆窗途径耳毒性剂递送:一个快速和可靠的技术诱导耳蜗细胞变性

行为小鼠希波坎帕的神经元活动可视化的颅骨切除术

植入颅窗以在清醒小鼠中重复体内成像

体内小鼠海马体中小胶质细胞的慢性双光子成像

机器人钻孔开颅术对小鼠颅窗手术热损伤的评估

Spatiotemporal Analysis of Microglial Ca²⁺ Activity at Single-Cell Resolution

自由移动小鼠的同步聚焦超声神经调控和纤维光度记录

自由移动小鼠的同步聚焦超声神经调控和纤维光度记录

压电高精度手术骨切除 (相量): 改进小鼠颅骨窗手术的技术

摘要

探索更多视频

此视频中的章节

稀疏的头骨皮层的窗口技术

CCL5诱导小鼠骨膜骨骼干细胞迁移的实时成像

老鼠圆窗途径耳毒性剂递送:一个快速和可靠的技术诱导耳蜗细胞变性

行为小鼠希波坎帕的神经元活动可视化的颅骨切除术

植入颅窗以在清醒小鼠 中重复体内 成像

体内 小鼠海马体中小胶质细胞的慢性双光子成像

机器人钻孔开颅术对小鼠颅窗手术热损伤的评估

Spatiotemporal Analysis of Microglial Ca2+ Activity at Single-Cell Resolution

自由移动小鼠的同步聚焦超声神经调控和纤维光度记录

自由移动小鼠的同步聚焦超声神经调控和纤维光度记录

植入颅窗以在清醒小鼠中重复体内成像

体内小鼠海马体中小胶质细胞的慢性双光子成像

Spatiotemporal Analysis of Microglial Ca²⁺ Activity at Single-Cell Resolution