作者感谢与手稿准备援助伏尔泰Gungab。这项工作是支持的，部分补助：国家卫生研究院（T32 GM008440 EJ沃克，R01 NS27713轮候册年轻，P01 NS44155 WL杨和H.苏），R21 NS070153 H ·苏和美国心脏协会（AHA 10GRNT3130004到H苏）。

1。清除血液从血管<ol><li> 100毫克/千克10毫克/千克0.25 ml生理盐水稀释甲苯噻嗪对氯胺酮腹腔注射麻醉小鼠。 </li><li>一旦证实一爪子掐无反应，麻醉已通过膜片切割剪刀，略低于xyphoid过程，使整个胸部切口，剪刀切背，腹段的肋骨暴露胸腔之间。折叠起来胸骨和邻近胸壁和安全与止血暴露心脏。用镊子打开心包囊，暴露心脏。 </li><li>打开连接到温暖PBS加肝素（5单位/毫升）热水澡的解决方案，在37 ° C和22钝的针头流出管。灌注泵（100mmHg压力） </li><li>针插入左心室，靠近心脏的顶点。立即切开右心房，使全身的血液流出，并继续冲洗，直到血我从流通中删除的小号。 </li></ol> 2。铸造代理灌注到血管系统<ol><li>准备Microfil（流技术，公司卡佛，马）铸造每制造商的协议解决方案：混合与4毫升过滤的MV化合物的MV稀释液5毫升。加入450μl的催化剂（5％）（MV固化剂）。解决方案的工作时间是20分钟，但混合使用前，以确保适当的粘度。 </li><li>撤柜铸造成10ml注射器剂溶液，将一种生硬的20号针头注射器和填充的解决方案针。 </li><li>通过使用相同的孔冲洗血液进入左心室插入针。安全进针，止血钳对心脏的地方。 </li><li>慢慢进入左心室注入铸造剂溶液约3毫升/分钟。压力过大，可能会导致不正确的流量铸造代理人或潜在的血管破裂。 </li><li>寻找成功灌注的迹象，包括：可视化铸造在小肠和肝脏血管，远端肢体变色，鼻子和舌头变色剂。调整针，如果要达到适当的灌注。 </li></ol> 3。收集和组织处理<ol><li>大脑和4％多聚甲醛，放置过夜取出4 ° C。 </li><li>从多聚甲醛直接放置到越来越多地集中在室温乙醇脱水大脑：25％乙醇，1天，50％乙醇，1天，75％的乙醇，1天，95％乙醇，2天;和100％乙醇，2天。 </li><li>澄清沉浸在水杨酸甲酯在室温下2天灌注脑血管周围脑组织。 </li><li>整个unsectioned澄清脑血管可与水杨酸甲酯沉浸在在解剖显微镜（图1）（徕卡MZFL第三显微镜，徕卡显微系统，班诺克本，白细胞介素）或明场显微镜（图2）（徕卡马克/下的脑成像LS，莱卡微系统）。 </li><li> Reclarification可以做，如果组织没有完全清除。从水杨酸甲酯，脑转移，95％的乙醇2天，2天100％的乙醇，水杨酸甲酯2天。 </li></ol> 4。显微CT成像或组织学的应用，组织准备<ol><li>大脑可以使用后直接澄清或再水化后（图4）显微CT成像。 </li><li>为了准备脑组织作病理分析，补充水分通过串行稀释乙醇的大脑：100％乙醇，1天，95％乙醇，1天，75％乙醇，1天，50％乙醇，2天; 25％乙醇，2天;并存储在PBS。大脑可以被嵌入在使用一个标准程序石蜡。 </li></ol> 5。代表性的成果血管铸型后，可以看到cerebrovasculature，从宏观上脑表面（图1a）。实质内的血管树可以可视化的以下澄清（图1B）。详细的血管结构可成像和分析下一个明场显微镜（图2），解剖的范围（图3），并与微CT扫描（图4）。使用明场显微镜，血管可以被视为不同的焦平面，没有在更高的放大倍率（图2B）切片的大脑（图2A）以及个人微血管。解剖的范围下，人们可以看到一个焦平面整个血管结构，从而实现了更好的三个cerebrovasculature的三维结构（图3）可视化。在解剖显微镜下拍摄的图像可以显示大血管的形态和全脑血管，但缺乏与明场显微镜（图2B）拍摄的照片显示的细节。这项技术使我们能够检测不规则的船只，在我们的实验BAVM成年小鼠模型 2 。图5显示了一个微型CT扫描（图5A）和明场显微镜（图5B）检测相同的不规则血管的例子。总之，这数据显示，我们可以分析吨他同一个样品后血管铸型多种成像工具。 <img alt="图1" src="/files/ftp_upload/2958/2958fig1.jpg" /> 图1。整个鼠脑血管铸型。前（a）和（二）澄清水杨酸甲酯溶剂后。船只可以清楚地显示脑表面（A）和实质以下澄清程序（二）。 <img alt="图2" src="/files/ftp_upload/2958/2958fig2.jpg" /> 图2。明视显微镜图像澄清血管铸造大脑。大脑可以在各种飞机和在不同的放大倍率级别成像。比例尺为200μm（A）和50微米（二）。 <img alt="图3" src="/files/ftp_upload/2958/2958fig3.jpg" /> 图3。剖析1倍显微镜图像显示整个成年小鼠大脑cerebrovasculature从背视图。 &lt;IMG ALT =“图4”SRC =“/ files/ftp_upload/2958/2958fig4.jpg”/&gt; 图4。显微CT扫描相同的整体成年小鼠大脑从背视图图3所示的图像。 <img alt="图5" src="/files/ftp_upload/2958/2958fig5.jpg" /> 图5。血管铸型方法检测脑动静脉畸形中的应用。一）微铸造大脑血管的CT扫描显示区域的扩大，动静脉畸形不规则状大脑右半球的船只，从腹面观。显示在右边的区域放大后的图像（白盒）。 b）同不规则的血管可视化明场显微镜下，在50倍放大倍率。比例尺50微米。

<ol>
	<li>Bolland, B. J., Kanczler, J. M., Dunlop, D. G., Oreffo, R. O. <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+in+vivo+muCT+evaluation+of+neovascularisation+in+tissue+engineered+bone+constructs.">Development of in vivo muCT evaluation of neovascularisation in tissue engineered bone constructs.</a> Bone. 43, 195-202 (2008).</li><li>Walker, 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=Arteriovenous+malformation+in+the+adult+mouse+brain+resembling+the+human+disease.">Arteriovenous malformation in the adult mouse brain resembling the human disease.</a> Ann. Neurol. , (2011).</li><li>Iqbal, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+analysis+of+novel+mono-+and+multivalent+VHH-fragments+and+their+application+for+molecular+imaging+of+brain+tumours.">Kinetic analysis of novel mono- and multivalent VHH-fragments and their application for molecular imaging of brain tumours.</a> Br. J. Pharmacol. 160, 1016-1028 (2010).</li><li>Fukunaga, A., Kawase, T., Uchida, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+recovery+after+simultaneous+transplantation+with+neuro-epithelial+stem+cells+and+adjacent+mesenchymal+tissues+into+infarcted+rat.">Functional recovery after simultaneous transplantation with neuro-epithelial stem cells and adjacent mesenchymal tissues into infarcted rat.</a> 145, 473-480 (2003).</li><li>Chugh, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+cerebral+blood+volume+in+mouse+brain+regions+using+micro-computed+tomography.">Measurement of cerebral blood volume in mouse brain regions using micro-computed tomography.</a> Neuroimage. 47, 1312-1318 (2009).</li><li>Marxen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroCT+scanner+performance+and+considerations+for+vascular+specimen+imaging.">MicroCT scanner performance and considerations for vascular specimen imaging.</a> Med. Phys. 31, 305-313 (2004).</li><li>Barger, A. C., Beeuwkes, R., 3rd, L. a. i. n. e. y., L, L., Silverman, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothesis:+vasa+vasorum+and+neovascularization+of+human+coronary+arteries.+A+possible+role+in+the+pathophysiology+of+atherosclerosis.">Hypothesis: vasa vasorum and neovascularization of human coronary arteries. A possible role in the pathophysiology of atherosclerosis.</a> N. Engl. J. Med. 310, 175-177 (1984).</li><li>Fossett, D. T., Caputy, A. 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> Operative Neurosurgical Anatomy. , (2002).</li><li>Reynolds, D. G., Brim, J., Sheehy, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vascular+architecture+of+the+small+intestinal+mucosa+of+the+monkey+(Macaca+mulatta).">The vascular architecture of the small intestinal mucosa of the monkey (Macaca mulatta).</a> Anat. Rec. 159, 211-218 (1967).</li><li>Bernard, S., Luchtel, D. L., Polissar, N., Hlastala, M. P., Lakshminarayan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+size+of+bronchopulmonary+anastomoses+in+sheep+lung.">Structure and size of bronchopulmonary anastomoses in sheep lung.</a> Anat. Rec. A. Discov. Mol. Cell. Evol. Biol. 286, 804-813 (2005).</li><li>Brady, J. M., Cutright, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+technique+of+measuring+blood+vessel+volume+in+bone+applied+to+the+mandible+and+humerus+of+the+rat.">A new technique of measuring blood vessel volume in bone applied to the mandible and humerus of the rat.</a> Anat. Rec. 170, 143-146 (1971).</li><li>Evan, A. P., Dail, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efferent+arterioles+in+the+cortex+of+the+rat+kidney.">Efferent arterioles in the cortex of the rat kidney.</a> Anat. Rec. 187, 135-145 (1977).</li><li>Murphy, 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=Endothelial+Notch4+signaling+induces+hallmarks+of+brain+arteriovenous+malformations+in+mice.">Endothelial Notch4 signaling induces hallmarks of brain arteriovenous malformations in mice.</a> Proc. Natl. Acad. Sci. U. S. A. 105, 10901-10906 (2008).</li></ol>

机构的动物护理和美国加州大学旧金山分校（UCSF）使用委员会批准使用实验动物的实验过程。

我们在这里报告成年小鼠cerebrovasculature可能与各种方式成像的铸造方法。这种方法应用到疾病模型BAVM，可不规则血管的三维分析。潜在的未来研究包括微型CT图像量化，组织病理分析，诊断血管疾病的进展。 遇到的常见问题及建议完整的脑血管灌注并不总是能够达到。有足够的压力来达到远端的血管没有破裂的左心房或主动脉是一个挑战。为了克服这个问题，采用一致的压力约为100mmHg。此外，调整针的位置，以确保进入主动脉铸造剂的适当流出。重要的是，颈动脉并不仅限于铸造剂，因此可以达到脑血管。改善小血管每相关的关键步骤融合是：（1）适当的血液从血管结算;（2）过滤铸造剂;（3）在铸造剂注射针的位置。应谨慎，以避免注射铸造成左心房和肺的代理。其他研究者已经成功灌注3小鼠和大鼠4 cerebrovasculature无需使用额外的程序。 Microfil灌注血管的手动分析counterstained hemotoxylin显示，93％的血管腔灌注 5 。务必丢弃或彻底清洁接触铸造剂，因为它会迅速聚合，工具不能用于多个科目使用任何工具。小鼠脑组织周围的澄清是具有挑战性的。沉浸在水杨酸甲酯，仍可能出现不透明的组织，使血管成像困难。确保适当的脱水和澄清，要解决这个问题。在酒精和小号甲基脑组织上放置一个摇杆alicylate治疗，可提高组织的澄清。 该技术的缺点这项技术的局限性包括：（1）有必要立即注入铸造剂溶液混合后，添加催化剂后，因为它会变粗迅速;（2）铸造剂填充大电导船容易，但它并不可靠填补较小的船只，如动脉（阻力血管）和毛细血管（营养血管），（3）它是不是最好不透X线显微CT成像相比之下，虽然一些群体更喜欢微型CT成像1,6 Microfil。然而，以下提出上述建议，包括适当的针的位置，船只的位置和灌注压，可实现完整的铸造较小的船只。各种分析工具的适应性使得这个代理一个有价值的工具，分析脑的结构，包括动脉和静脉循环，各种观点。 该技术的应用这种方法的优点是其广阔的应用潜力。投填充所有船只，包括动脉，静脉和毛细血管身体内的所有组织，因此可以分析不同种类的多个器官的血管结构。此前，其他团体，用于大 7 小 8船只在人类 9，猴， 羊 10， 大鼠 4,11,12， 小鼠 3,13灌注。我们现在表明使用这一技术在小鼠大脑微血管的结构分析，在健康和疾病状态，2的证据。此外，还有与灌注的不同大小和不同颜色的的船舶，以提高各器官的血管可视化不同粘度Microfil的解决方案。例如，红色，可以很容易地可视化在苍白的脑实质，和黄的对比以及与暗红色的心肌。此外，视物不透X线，血管，可成像显微CT扫描获得三维旋转图像。该组织也可用于石蜡切片和病理分析后成像。 结论我们在这里展示一个协议，使一个成年小鼠的大脑，它可以适应使用各种分析技术的研究形态结构的cerebrovasculature血管的血管铸。我们还提供了BAVM疾病状态模型，证明这种方法的应用为代表的扩大，不规则形状的船只，分流的血液从动脉到静脉环流。有多种血管铸型方法可用。简单的协议，这里我们提供了一个血管演员可能被用来作为一种工具来检查使用各种成像方式的成年小鼠大脑血管。

<table border="1"><tbody><tr><td> 名称 </td><td> 公司 </td><td> 目录编号 </td><td> 注释 </td></tr><tr><td> Microfil </td><td> FlowTech，公司</td><td> MV130 </td><td> 4个月的保质期</td></tr><tr><td>水杨酸甲酯</td><td> Fisher Scientific则</td><td> O3695 - 500 </td><td>澄清组织</td></tr></tbody></table>

cerebrovascular casting of the adult mouse for 3d imaging and morphological analysis

血管造影是脑血管疾病，如脑动静脉畸形（BAVMs），在临床诊断和管理的关键。动物模型研究etiopathology和潜在的治疗脑血管疾病的的必要。成像的大型动物的血管是比较容易的。然而，发展船舶小鼠脑疾病模型成像方法是可取的，由于转基因小鼠线路的成本和可用性。成像小鼠脑血管树是一个挑战。在人类和较大的动物，评估在大血管angioarchitecture的黄金标准（电导）级X射线导管造影对比，不适合小老鼠的方法。 在这篇文章中，我们提出了一个脑铸造的方法，产生了整个血管床的耐用的骨架，包括动脉，静脉和毛细血管，可分析使用许多Different方式。鼠标cerebrovasculature微血管全面铸造可困难;然而，在这一步一步协议解决这些挑战。通过intracardial灌注血管铸型材料，身体的所有船只都铸造。大脑可以被删除，使用有机溶剂的水杨酸甲酯澄清。三脑血管三维成像，可以可视化，简单，成本低廉与任何传统的明场显微镜或解剖显微镜。铸造cerebrovasculature也可以使用微型计算机断层扫描（显微CT）1成像和量化。此外，铸造脑成像后，可以嵌入在石蜡病理分析。 与其他技术相比，这种血管铸型方法的好处是其广泛的适应各种分析工具，包括明的微观分析，CT扫描由于透X线的字符的材料，以及组织学和免疫组化分析teristic。这种组织的有效利用，可以节省动物的使用和降低成本。最近，我们已经证明这种方法的可视化在微观层面 2在成年BAVM的小鼠模型的不规则血管，微CT扫描成像畸形血管提供额外的图像应用程序。虽然这种方法也有缺点，可能并不适合于所有类型的分析，它是一种简单，实用的技术，可以很容易地教训和广泛应用于全身各处血管的血管铸型。

Watch this Scientific Journal Video about Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis at JoVE.com

Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

在这篇文章中，我们提出一个简单的实用技术，为脑血管铸造，很容易执行，并可以利用图像的成年鼠脑血管树。

成年鼠脑铸造三维成像和形态分析

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). ...

cerebrovascular-casting-adult-mouse-for-3d-imaging-morphological

Research

JoVE Journal

Neuroscience

21.4K 次观看.  University of California, San Francisco. 在这篇文章中，我们提出一个简单的实用技术，为脑血管铸造，很容易执行，并可以利用图像的成年鼠脑血管树。

视频: Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

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 ...

科研

神经科学

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

急性片的成年小鼠前脑的神经细胞迁移的延时成像

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

可视化和遗传操作在小鼠大脑皮层和使用海马树突和棘<em&gt;在子宫内</em&gt;电穿孔

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Methods for the Modulation and Analysis of NF-&#954;B-dependent Adult Neurogenesis

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult hippocampus has been viewed as a very static anatomical region of the mammalian brain. However, recent findings have demonstrated that the dentate gyrus of the hippocampus is an area of tremendous plasticity in adults, involving not only modifications of existing neuronal circuits, but also adult neurogenesis. This plasticity is regulated by complex transcriptional networks, in which the transcription factor NF-&#954;B plays a prominent role. To study and manipulate adult neurogenesis, a transgenic mouse model for forebrain-specific neuronal inhibition of NF-&#954;B activity can be used.
In this study, methods are described for the analysis of NF-&#954;B-dependent neurogenesis, including its structural aspects, neuronal apoptosis and progenitor proliferation, and cognitive significance, which was specifically assessed via a dentate gyrus (DG)-dependent behavioral test, the spatial pattern separation-Barnes maze (SPS-BM). The SPS-BM protocol could be simply adapted for use with other transgenic animal models designed to assess the influence of particular genes on adult hippocampal neurogenesis. Furthermore, SPS-BM could be used in other experimental settings aimed at investigating and manipulating DG-dependent learning, for example, using pharmacological agents.

Methods for the Modulation and Analysis of NF-&amp;#954;B-dependent Adult Neurogenesis

Methods for the manipulation and analysis of NF-&#954;B-dependent adult hippocampal neurogenesis are described. A detailed protocol is presented for a dentate gyrus-dependent behavioral test (termed the spatial pattern separation-Barnes maze) for the investigation of cognitive outcome in mice. This technique should also help enable investigations in other experimental settings.

方法对NF-κB依赖的成年神经发生的调制与分析

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult ...

Stimulus-specific Cortical Visual Evoked Potential Morphological Patterns

This paper presents a methodology for the recording and analysis of cortical visual evoked potentials (CVEPs) in response to various visual stimuli using 128-channel high-density electroencephalography (EEG). The specific aim of the described stimuli and analyses is to examine whether it is feasible to replicate previously reported CVEP morphological patterns elicited by an apparent motion stimulus, designed to simultaneously stimulate both ventral and dorsal central visual networks, using object and motion stimuli designed to separately stimulate ventral and dorsal visual cortical networks.&#160; Four visual paradigms are presented: 1. Randomized visual objects with consistent temporal presentation. 2. Randomized visual objects with inconsistent temporal presentation (or jitter).&#160; 3. Visual motion via a radial field of coherent central dot motion without jitter.&#160; 4. Visual motion via a radial field of coherent central dot motion with jitter.&#160; These four paradigms are presented in a pseudo-randomized order for each participant.&#160; Jitter is introduced in order to view how possible anticipatory-related effects may affect the morphology of the object-onset and motion-onset CVEP response.&#160; EEG data analyses are described in detail, including steps of data exportation from and importation to signal processing platforms, bad channel identification&#160;and removal, artifact rejection, averaging, and categorization of average CVEP morphological pattern type based upon latency ranges of component peaks. Representative data show that the methodological approach is indeed sensitive in eliciting differential object-onset and motion-onset CVEP morphological patterns and may, therefore, be useful in addressing the larger research aim. Given the high temporal resolution of EEG and the possible application of high-density EEG in source localization analyses, this protocol is ideal for the investigation of distinct CVEP morphological patterns and the underlying neural mechanisms which generate these differential responses.&#160; &#160; &#160;&#160;

In this paper, we present a protocol to investigate differential cortical visual evoked potential morphological patterns through stimulation of ventral and dorsal networks using high-density EEG. Visual object and motion stimulus paradigms, with and without temporal jitter, are described. Visual evoked potential morphological analyses are also outlined.&#160;&#160;

刺激特定的皮质视觉引发潜在形态模式

This paper presents a methodology for the recording and analysis of cortical visual evoked potentials (CVEPs) in response to various visual stimuli using ...

Morphological and Functional Evaluation of Ribbon Synapses at Specific Frequency Regions of the Mouse Cochlea

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have indicated that hair cell synapses may be the initial targets in sensorineural hearing loss (SNHL). Such studies have proposed the concept of cochlear &#34;synaptopathy&#34;, which refers to alterations in ribbon synapse number, structure, or function that result in abnormal synaptic transmission between IHCs and SGNs. While cochlear synaptopathy is irreversible, it does not affect the hearing threshold. In noise-induced experimental models, restricted damage to IHC synapses in select frequency regions is employed to identify the environmental factors that specifically cause synaptopathy, as well as the physiological consequences of disturbing this inner ear circuit. Here, we present a protocol for analyzing cochlear synaptic morphology and function at a specific frequency region in adult mice. In this protocol, cochlear localization of specific frequency regions is performed using place-frequency maps in conjunction with cochleogram data, following which the morphological characteristics of ribbon synapses are evaluated via synaptic immunostaining. The functional status of ribbon synapses is then determined based on the amplitudes of auditory brainstem response (ABR) wave I. The present report demonstrates that this approach can be used to deepen our understanding of the pathogenesis and mechanisms of synaptic dysfunction in the cochlea, which may aid in the development of novel therapeutic interventions.

This manuscript describes an experimental protocol for evaluating the morphological characteristics and functional status of ribbon synapses in normal mice. The present model is also suitable for noise-induced and age-related cochlear synaptopathy-restricted models. The correlative results of previous mouse studies are also discussed.

小鼠耳蜗特定频率区域带状带突起的形态和功能评估

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have ...

Fluorescence Angiography for Evaluation of Aneurysm Perfusion and Parent Artery Patency in Rat and Rabbit Aneurysm Models

Brain aneurysm treatment focuses on achieving complete occlusion, as well as preserving blood flow in the parent artery. Fluorescein sodium and indocyanine green are used to enable the observation of blood flow and vessel perfusion status, respectively. The aim of this study is to apply FVA to verify real-time blood flow, vessel perfusion status and occlusion of aneurysms after induction of sidewall aneurysms in rabbits and rats, as well as to validate the procedure in these species.
Twenty sidewall aneurysms were created in 10 rabbits by suturing a decellularized arterial vessel pouch on the carotid artery of a donor rabbit. In addition, 48 microsurgical sidewall aneurysms were created in 48 rats. During follow-up at one month after creation, the parent artery/aneurysm complex was dissected and FVA was performed using an intravenous fluorescein (10%, 1 mL) injection via an ear vein catheterization in rabbits and a femoral vein catherization in rats. Aneurysms were then harvested, and patency was evaluated macroscopically.
Macroscopically, 14 out of 16 aneurysms in rabbits indicated no residual parent artery perfusion with totally occluded luminae, however 11 (79%) were detected by FVA. Four aneurysms were excluded due to technical problems. In rats, residual aneurysm perfusion was macroscopically observed in 25 out of 48 cases. Of the 23 without macroscopic evidence of perfusion, FVA confirmed the incidence of 22 aneurysms (96%). There were no adverse events associated with FVA. Fluorescein is easily applicable and no special equipment is needed. It is a safe and extremely effective method for evaluating parent artery integrity and aneurysm patency/residual perfusion in an experimental setting with rabbits and rats. FVA using fluorescein as a contrast agent appears to be effective in controlling patency of aneurysms and the underlying vessel and can even be adapted to bypass surgery.

We present a protocol to efficiently evaluate aneurysm perfusion and vessel patency of sidewall aneurysm in rats and rabbits, using fluorescein-based fluorescence video angiography (FVA). With a positive predictive value of 92.6%, it is a simple but very effective and economical method with no special equipment required.

荧光血管造影用于评估大鼠和兔动脉瘤模型中的动脉灌注和父动脉鉴赏

Brain aneurysm treatment focuses on achieving complete occlusion, as well as preserving blood flow in the parent artery. Fluorescein sodium and ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

坐骨神经粉碎损伤大鼠模型中功能评估的3D运动学分析

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Cochlear Surface Preparation in the Adult Mouse

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous etiologies such as exposure to excessive noise, the use of ototoxic medications, bacterial or viral ear infections, head injuries, and the aging process. Loss of sensory hair cells is a common pathological feature of the varieties of acquired hearing loss. Additionally, the inner hair cell synapse can be damaged by mild insults. Therefore, surface preparations of cochlear epithelia, in combination with immunolabeling techniques and confocal imagery, are a very useful tool for the investigation of cochlear pathologies, including losses of ribbon synapses and sensory hair cells, changes in protein levels in hair cells and supporting cells, hair cell regeneration, and determination of report gene expression (i.e., GFP) for verification of successful transduction and identification of transduced cell types. The cochlea, a bony spiral-shaped structure in the inner ear, holds the auditory sensory end organ, the organ of Corti (OC). Sensory hair cells and surrounding supporting cells in the OC are contained in the cochlear duct and rest on the basilar membrane, organized in a tonotopic fashion with high-frequency detection occurring in the base and low-frequency in the apex. With the availability of molecular and genetic information and the ability to manipulate genes by knockout and knock-in techniques, mice have been widely used in biological research, including in hearing science. However, the adult mouse cochlea is miniscule, and the cochlear epithelium is encapsulated in a bony labyrinth, making microdissection difficult. Although dissection techniques have been developed and used in many laboratories, this modified microdissection method using cell and tissue adhesive is easier and more convenient. It can be used in all types of adult mouse cochleae following decalcification.

This article presents a modified cochlear surface preparation method that requires decalcification and use of a cell and tissue adhesive to adhere the pieces of cochlear epithelia to 10 mm round cover slips for immunohistochemistry in adult mouse cochleae.

成年小鼠中的科利耳表面制备

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous ...

Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study of this movement in situ was only possible using radioisotopic pulse-labeling, which permits analysis of axonal transport in whole nerves with a temporal resolution of days and a spatial resolution of millimeters. To study neurofilament transport in situ with higher temporal and spatial resolution, we developed a hThy1-paGFP-NFM transgenic mouse that expresses neurofilament protein M tagged with photoactivatable GFP in neurons. Here we describe fluorescence photoactivation pulse-escape and pulse-spread methods to analyze neurofilament transport in single myelinated axons of tibial nerves from these mice ex vivo. Isolated nerve segments are maintained on the microscope stage by perfusion with oxygenated saline and imaged by spinning disk confocal fluorescence microscopy. Violet light is used to activate the fluorescence in a short axonal window. The fluorescence in the activated and flanking regions is analyzed over time, permitting the study of neurofilament transport with temporal and spatial resolution on the order of minutes&#160;and microns, respectively. Mathematical modeling can be used to extract kinetic parameters of neurofilament transport including the velocity, directional bias and pausing behavior from the resulting data. The pulse-escape and pulse-spread methods can also be adapted to visualize neurofilament transport in other nerves. With the development of additional transgenic mice, these methods could also be used to image and analyze the axonal transport of other cytoskeletal and cytosolic proteins in axons.

We describe fluorescence photoactivation methods to analyze the axonal transport of neurofilaments in single myelinated axons of peripheral nerves from transgenic mice that express a photoactivatable neurofilament protein.

切除小鼠神经神经纤维神经丝的成像与分析

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study ...