Name: a polished and reinforced thinned-skull window for long-term imaging of the mouse brain
Uploaded: 2012-03-07T12:00:00
Description: Watch this Scientific Journal Video about A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain at JoVE.com

这项工作是由美国心脏协会（博士后奖学金AYS）和国立卫生研究院（MH085499，EB003832，并OD006831 DK）的支持。我们感谢贝思·弗里德曼和巴勃罗·布林德手稿上的评论。

1。准备手术， 我 <ol><li>清洁及手术Maxizyme超声波清洗机的牛奶混合物中超声分散的手术工具。高压灭菌之前，每个实验的外科手术工具。 </li><li>确保所有必要的试剂和耗材可用。表2中提供的试剂和耗材清单。试剂和耗材，在接触到暴露组织应该是无菌的，在可能的情况下。 </li><li>诱导麻醉。表1描述了适合生存研究的典型麻醉药。为缺乏脚趾掐反射的检查，以确保手术的麻醉平面。鼠...

<ol>
	<li>Cetin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereotaxic+gene+delivery+in+the+rodent+brain.">Stereotaxic gene delivery in the rodent brain.</a> Nature Protocols. 1, 3166-3173 (2006).</li><li>Kleinfeld, D., Delaney, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distributed+representation+of+vibrissa+movement+in+the+upper+layers+of+somatosensory+cortex+revealed+with+voltage+sensitive+dyes.">Distributed representation of vibrissa movement in the upper layers of somatosensory cortex revealed with voltage sensitive dyes.</a> Journal of Comparative Neurology. 375, 89-108 (1996).</li><li>Driscoll, J. D., Yuste, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+two-photon+imaging+of+blood+flow+in+cortex.">Quantitative two-photon imaging of blood flow in cortex.</a> Imaging in Neuroscience and Development. , 927-937 (2011).</li><li>Drew, P. J., Shih, A. Y., Kleinfeld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluctuating+and+sensory-induced+vasodynamics+in+rodent+cortex+extends+arteriole+capacity.">Fluctuating and sensory-induced vasodynamics in rodent cortex extends arteriole capacity.</a> Proceedings of the National Academy of Sciences U.S.A. 108, 8473-8473 (2011).</li><li>Mostany, R., Portera-Cailliau, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Method+for+2-Photon+Imaging+of+Blood+Flow+in+the+Neocortex+through+a+Cranial+Window.">A Method for 2-Photon Imaging of Blood Flow in the Neocortex through a Cranial Window.</a> J. Vis. Exp. (12), e678-e678 (2008).</li><li>Zhang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+reversible+changes+in+dendritic+spine+structure+in+vivo+gated+by+the+degree+of+ischemia.">Rapid reversible changes in dendritic spine structure in vivo gated by the degree of ischemia.</a> Journal of Neuroscience. 25, 5333-5338 (2005).</li><li>Takano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte-mediated+control+of+cerebral+blood+flow.">Astrocyte-mediated control of cerebral blood flow.</a> Nature Neuroscience. 9, 260-267 (2006).</li><li>Drew, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+optical+access+through+a+polished+and+reinforced+thinned+skull.">Chronic optical access through a polished and reinforced thinned skull.</a> Nature Methods. 7, 981-984 (2010).</li><li>Marker, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+thin-skull+window+technique+for+chronic+two-photon+in+vivo+imaging+of+murine+microglia+in+models+of+neuroinflammation.">A thin-skull window technique for chronic two-photon in vivo imaging of murine microglia in models of neuroinflammation.</a> Journal of Visualized Experiments. (43), e2059-e2059 (2010).</li><li>Feng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neuronal+subsets+in+transgenic+mice+expressing+multiple+spectral+variants+of+GFP.">Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP.</a> Neuron. 28, 41-51 (2000).</li><li>Martin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+neural-hemodynamic+coupling+and+the+hemodynamic+response+function+in+the+awake+rat.">Investigating neural-hemodynamic coupling and the hemodynamic response function in the awake rat.</a> Neuroimage. 32, 33-48 (2006).</li><li>Shih, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+microscopy+as+a+tool+to+study+blood+flow+and+neurovascular+coupling+in+the+rodent+brain.">Two-photon microscopy as a tool to study blood flow and neurovascular coupling in the rodent brain.</a> Journal of Cerebral Blood Flow and Metabolism. , (2011).</li><li>Kobat, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+tissue+multiphoton+microscopy+using+longer+wavelength+excitation.">Deep tissue multiphoton microscopy using longer wavelength excitation.</a> Optics Express. 17, 13354-13364 (2009).</li><li>Holtmaat, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=high-resolution+imaging+in+the+mouse+neocortex+through+a+chronic+cranial+window.">high-resolution imaging in the mouse neocortex through a chronic cranial window.</a> Nature Protocols. 4, 1128-1144 (2009).</li><li>Xu, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choice+of+cranial+window+type+for+in+vivo+imaging+affects+dendritic+spine+turnover+in+the+cortex.">Choice of cranial window type for in vivo imaging affects dendritic spine turnover in the cortex.</a> Nature Neuroscience. 10, 549-551 (2007).</li><li>Nimmerjahn, A., Kirchhoff, F., Helmchen, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resting+microglial+cells+are+highly+dynamic+surveillants+of+brain+parenchyma+in+vivo.">Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo.</a> Science. 308, 1314-1318 (2005).</li><li>Davalos, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATP+mediates+rapid+microglial+response+to+local+brain+injury+in+vivo.">ATP mediates rapid microglial response to local brain injury in vivo.</a> Nature Neuroscience. 8, 752-758 (2005).</li><li>Ascenzi, A., Fabry, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technique+for+dissection+and+measurement+of+refractive+index+of+osteons.">Technique for dissection and measurement of refractive index of osteons.</a> The Journal of Biophysical and Biochemical Cytology. 6, 139-142 (1959).</li><li>Stosiek, C. <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+two-photon+calcium+imaging+of+neuronal+networks.">In vivo two-photon calcium imaging of neuronal networks.</a> Proceedings of the National Academy of Sciences U.S.A. 100, 7319-7324 (2003).</li><li>Grinvald, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+architecture+of+cortex+revealed+by+optical+imaging+of+intrinsic+signals.">Functional architecture of cortex revealed by optical imaging of intrinsic signals.</a> Nature. 324, 361-364 (1986).</li><li>Dunn, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+imaging+of+cerebral+blood+flow+using+laser+speckle.">Dynamic imaging of cerebral blood flow using laser speckle.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 21, 195-201 (2001).</li><li>Villringer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+perfusion+of+the+rat+brain+cortex:+An+in+vivo+confocal+microscopy+study.">Capillary perfusion of the rat brain cortex: An in vivo confocal microscopy study.</a> Circulation Research. 75, 55-62 (1994).</li><li>Denk, W., Strickler, J. H., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+laser+scanning+fluorescence+microscopy.">Two-photon laser scanning fluorescence microscopy.</a> Science. 248, 73-76 (1990).</li><li>Srinivasan, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+coherence+tomography+for+the+quantitative+study+of+cerebrovascular+physiology.">Optical coherence tomography for the quantitative study of cerebrovascular physiology.</a> Journal of Cerebral Blood Flow &#38; Metabolism. 31, 1339-1345 (2011).</li><li>Hu, S., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoacoustic+imaging+and+characterization+of+the+microvasculature.">Photoacoustic imaging and characterization of the microvasculature.</a> Journal of Biomedical Optics. 15, 011101-011101 (2010).</li><li>Flecknell, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laboratory animal anesthesia. , (1987).</li></ol>

通过一个端口“窗口中的双光子成像需要通过传输薄骨和硬脑膜，衰减激光和光学像差增加了更大的深度8。然而，尽管这个缺点，成像深度可达250微米以下脑膜表面可以达到900 nm激发。大成像深度可能在原则上有可能与较长的激发波长13。这种方法的主要优点是皮质炎症的情况下，可能存在短暂的涉及全开颅14，15的方法。一个精心准备的端口窗口应显示血管生成或炎症没...

<table border="1"><tbody><tr><td> 代理人 </td><td> 交货路线 </td><td> 剂量为鼠标 </td><td> 时间 </td><td> 笔记 </td><td> 源 </td><td> 参考价 </td></tr><tr><td> 巴比妥（戊巴比妥） </td><td>知识产权</td><td> 90微克/克</td><td> 15-60分钟</td><td>狭窄的安全保证金。工作到适当的麻醉剂量缓慢。所需补编诱导剂量的10％。 </td><td> 036093;巴特勒施恩</td><td> 7 </td></tr><tr><td> 氯胺酮（Ketaset）混二甲苯胺噻嗪（Anased） </td><td>知识产权</td><td> 60微克/克（金） 10微克/ G（X）（混合在同一注射器） </td><td> 20-30分钟</td><td>甲苯噻嗪联合注入作为肌肉松弛剂和止痛药。氯胺酮诱导剂量的50％只补充需要。 </td><td> （金）010177（X）033198;巴特勒施恩</td><td> 7 </td></tr><tr><td> 异氟醚（Isothesia） </td><td>吸入</td><td> 4％，平均诱导肺泡浓度（MAC）; 1-2％的维修MAC </td><td> 4-6小时。 </td><td>提供70％氧气和30％的一氧化二氮的混合物。长时间麻醉导致的缓慢复苏。 </td><td> 029403;巴特勒施恩</td><td> 26 </td></tr></tbody></table> 表1。推荐为生存研究麻醉剂。 <table border="1"><tbody><tr><td> 项目 </td><td> 公司 </td><td><stroNG&gt;目录＃/型号</td></tr><tr><td>优碘</td><td>巴特勒施恩</td><td> 6906950 </td></tr><tr><td>丁丙诺啡（Buprenex） </td><td>巴特勒施恩</td><td> 031919 </td></tr><tr><td> 2 MDA分子量右旋糖酐，异硫氰酸荧光素</td><td>西格玛</td><td> FD2000S </td></tr><tr><td>异丙醇</td><td>费舍尔</td><td> AC42383-0010 </td></tr><tr><td>乳酸林格氏液的解决方案</td><td>巴特勒施恩</td><td> 009846; </td></tr><tr><td>利多卡因溶液，2％（V / V） </td><td>巴特勒施恩</td><td> 002468 </td></tr><tr><td>盐水</td><td>巴特勒施恩</td><td> 009861 </td></tr><tr><td>手术牛奶</td><td>巴特勒施恩</td><td&gt; 014325 </td></tr><tr><td>德州红，70 kDa的分子量右旋糖酐</td><td> Invitrogen公司</td><td> D1864 </td></tr><tr><td> maxizyme </td><td>巴特勒施恩</td><td> 035646 </td></tr><tr><td> 一次性 </td></tr><tr><td>硬质合金旋转锉，1/2毫米针尖大小</td><td>罚款科学工具</td><td> 19007-05 </td></tr><tr><td>棉田尖端喷头</td><td> Fisher Scientific则</td><td> 23-400-100 </td></tr><tr><td>覆盖玻璃，没有。 0厚度</td><td>托马斯科学</td><td> 6661B40 </td></tr><tr><td>氰基丙烯酸酯胶</td><td> ND工业</td><td> 31428 H04308 </td></tr><tr><td>煤气除尘器</td><td>新蛋</td><td> N82E16848043429 </td></tr><tr><td>握水泥粉</td><td>登士柏</td><td> 675571 </td></tr><tr><td>握水泥溶剂</td><td>登士柏</td><td> 675572 </td></tr><tr><td>胰岛素注射器，0.3 mL体积29.5针头</td><td>巴特勒施恩</td><td> 018384 </td></tr><tr><td>螺母和螺栓，以确保头</td><td> digikey </td><td>螺母，H723-ND;螺栓，R2-56X1/4-ND </td></tr><tr><td>眼科软膏</td><td>巴特勒施恩</td><td> 039886 </td></tr><tr><td>手术刀片</td><td> Fisher Scientific则</td><td> 12-460-448 </td></tr><tr><td>螺钉，自攻＃000 </td><td>姬·莫里斯公司</td><td> FF000CE125 </td></tr><tr><td>硅水族馆密封胶</td><td> perfecto制造</td><td> 31001 </td></tr><tr><td>锡氧化物粉末</td><td>妈妈的矿物</td><td>殷拓，TINOX </td></tr><tr><td> 设备 </td></tr><tr><td>玻璃划线</td><td> Fisher Scientific则</td><td> 08-675 </td></tr><tr><td>解剖显微镜</td><td>卡尔·蔡司</td><td> OPMI-1的FC </td></tr><tr><td>电动钻</td><td> foredom或Osada </td><td> K.1020（Foredom）或EXL-M40的（Osada） </td></tr><tr><td>电剃刀</td><td>华尔</td><td>系列8900 </td></tr><tr><td>钳，杜蒙没有。 55 </td><td>罚款科学工具</td><td> 11255-20 </td></tr><tr><td>反馈调节热垫</td><td>金控</td><td> 40-90-8（直肠热敏电阻40-90-5D-02，H吃垫，40-90-2-07） </td></tr><tr><td>异氟醚蒸发器</td><td>美达</td><td> IsoTec4 </td></tr><tr><td>脉搏血氧仪</td><td>斯塔尔生命科学</td><td> MouseOx </td></tr><tr><td>螺丝刀，微型</td><td>阁楼韦德</td><td> 26B09.01 </td></tr><tr><td>立体框架</td><td> kopf仪器</td><td>型号900（鼠标麻醉面罩和非破裂耳酒吧） </td></tr><tr><td>手术剪，钝端</td><td>罚款科学工具</td><td> 14078-10 </td></tr><tr><td>超声波清洗机</td><td> Fisher Scientific则</td><td> 15-335-30 </td></tr></tbody></table> 表2。特定的试剂，耗材和设备的名单。

a polished and reinforced thinned-skull window for long-term imaging of the mouse brain

在体内的皮质功能成像需要光纤接入无脑颅内环境的破坏。我们提出了一个方法，在小鼠颅骨，跨越直径几毫米个月的稳定形成抛光和钢筋颅骨变薄（端口）窗口。手举行演习，以实现光学清晰度头骨10至15微米的厚度变薄，然后覆盖氰基丙烯酸酯胶和玻璃盖：1）提供刚性，2）抑制骨的再生和3）减少光散射从骨表面上的违规行为。由于颅骨没有违反，任何可能会影响正在研究过程中的炎症，会大大减少。可以实现利用双光子激光扫描显微镜成像深度可达250微米以下的皮质表面。这个窗口非常适合研究脑血流和细胞的功能，在麻醉和清醒的准备。它进一步提供运portunity操纵细胞的活性，利用光遗传学或破坏由照射循环光敏剂的目标船只的血流量。

Watch this Scientific Journal Video about A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain at JoVE.com

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

我们提出了一个方法，形成一个横跨毫米，是没有大脑的炎症个月稳定在小鼠颅骨的影像窗口。这种方法非常适合于使用双光子显微镜的血液流通，细胞动力学，细胞/血管结构的纵向研究。

抛光和钢筋，颅骨变薄窗口，为长期的小鼠脑成像

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

Research

JoVE Journal

Neuroscience

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

慢性成像小鼠的视觉皮层使用减薄，颅骨的制备

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

科研

神经科学

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

活体鼠脑微循环显微镜使用一个封闭的颅窗口

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, ...

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

多鼠神经解剖学磁共振成像

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Extinction Training During the Reconsolidation Window Prevents Recovery of Fear

灭绝培训在彭兰霞窗口无法还原的恐惧

Fear is maladaptive when it persists long after circumstances have become safe. It is therefore crucial to develop an approach that persistently prevents ...

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

长期的嗅觉适应性的分子读数<em&gt; C.线虫</em

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

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

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

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 ...

Improved Preparation and Preservation of Hippocampal Mouse Slices for a Very Stable and Reproducible Recording of Long-term Potentiation

长时程增强一个非常稳定的和可重复的记录和保存海马鼠标片制备工艺的改进

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

双光子<em&gt;在体内</em&gt;树突棘中的鼠标皮质用稀疏的头颅成像准备

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Long-term Time Lapse Imaging of Mouse Cochlear Explants

老鼠耳蜗外植体的长期时间推移成像

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

琼脂糖微室的长期钙成像<em&gt;秀丽隐杆线虫</em

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...