PDK是支持由CIHR经营授予（澳门币86523）

1。外科技术<ol><li>使用无菌技术操作，并按照您的特定机构的动物使用协议，应进行实验。活体组织接触到的工具和材料（解决方案，测试物质，示踪剂，针等）必须是无菌的，以防止感染动物福利和研究的潜在的负面影响和不利影响。 </li></ol> 2。麻醉<ol><li>老鼠将被麻醉的兽医异氟醚蒸发器系统。使用医疗级氧气在0.8 L / min的异氟醚气体蒸发的速度。放置在附加麻醉中的动物和异氟醚浓度在4％拨号，直到呼吸速度减慢和动物是稳重。 </li><li>接下来，气体流量开关防毒面具附件立体框架，放置在立体定位仪上的动物。关闭异氟醚的浓度下降到2％，并监测麻醉。大型动物（&gt; 300克），可能需要较高浓度的异氟醚。麻醉手术过程中应监测和异氟醚用量相应调整。应不断评估的深度和呼吸频率，并应脚趾捏评价了深深的痛苦的情况下（每5分钟）。 </li><li>手术完成后，关闭异氟醚和切除脑立体定位装置前的几分钟，让动物呼吸的氧气。涵盖动物用手术毯和/或使用在手术过程中的受规管的加热毯，应保持体温。 </li></ol> 3。手术方式<ol><li>湿用70％乙醇使皮草更容易削减的头顶部的皮毛。取出使用剪或锋利的剪刀的眼睛之间的毛皮。碘洗涤剂溶液交替应用（Proviodine，优碘，等），其次是70％的乙醇，清洁切口面积的三倍。保持角膜湿润，整个眼科眼膏（泪Naturale PM）角膜手术。遍布手动打开和关闭眼睑对角膜表面的药膏。 </li><li>使用11号刀片，沿头部中线在前面的眼睛后面的眼睛1厘米约0.5厘米的切口。在使用产钳的眼睛缩回皮瓣，轻轻梳理，手术刀背面底层的结缔组织。然后，收回皮瓣横向和向下，并在同一个手术牵开，可贴在立体定位仪的基础的地方。 </li><li>同时用锋利的钳拉覆筋膜，沿眼眶骨的优越RIM切口。这将退出筋膜覆盖在眼睛的轨道。眼眶骨的边缘，可以清楚地划分使用钳推覆轨道上的筋膜。使用烧灼设备或手术刀，继续切口向后走向的眼轨道后的限制。使用骨切口指南优越的轨道。下一步继续切口向前走向前极限轨道。最好的做法是用一个小的烧灼设备，以防止从通信的底层血管，静脉窦出血​​切口优越的轨道。 </li><li>如果出血发生时，可以采取以下几个步骤。首先，使用无菌手术拭子或棉签施加压力。如果继续出血，适用于冷，无菌磷酸盐缓冲液（PBS）的区域使用滴管，并保持压力。轻微出血停止使用此程序后的几秒钟。如果出血持续，适用于牵引与手术棉签或棉棒组织，以确定出血源，并迅速烧灼受损船只。载后出血，使用冷无菌PBS清洁血液​​的手术区。手术区应定期清洗，以这种方式，以便更好地可视化轨道的眼睛结构。 </li><li>一旦切口已清理，使用产钳和手术刀背面清洁的结缔组织在后面的眼睛，覆盖在眶内容。回的第11号手术刀以及提示是罚款。这将打开眶腔提供一个更大的手术窗口工作时，其曲率和罚款提示眼操纵在轨道结构的理想工作。使用杜蒙＃7B尖锐弯曲的锯齿钳的更深的部分。此外，锯齿帮助夹持结构。 </li><li>接下来，删除的结缔组织包围位于中线附近的三叉神经眼科部，并使用镊子取出神经。这一步是没有必要的，但是消除神经，将提供一个ACCES更大的窗口s到视神经稍后。 </li><li>去除神经后，用钳收回血管完全烧灼船只。这一步也是没有必要的，然而烧灼允许的船只，它向前移动，从而创造一个更大的窗口，一旦达到视神经。 </li><li>使用的镊子一双锐利，或有他们的秘诀弯曲向内，仔细挑选并删除在眼外肌，泪腺的一层薄的结缔组织钳。额外的眼部肌肉收回由前到后的轨道。一双镊子夹住近端的肌肉部分，并使用一个弧形的锯齿眼敷料钳的第二对适用于肌肉向外牵引。确保眼敷料钳导向，在同一个方向的肌肉时，拉，以防止撕裂。取出的最前部的肌肉，以这种方式的轨道（上斜肌）。肌肉会从深中解放出来的轨道内，以旋转的眼睛向外，其余的长度可回缩。 </li><li>与未来肌（内直肌）位于眼睛的背水面的中线附近的泪腺和哈氏腺叶之间重复步骤3.8。牵带下来，以保持肌肉的牵引。 </li><li>在泪腺表面上轻轻地取下任何剩余的结缔组织和腺体向上抬起，使用产钳。不要压缩或挤压腺体。为了收回的腺体，只有在单船后极需要被烧灼。电梯向上的腺体后结束，然后烧灼的船只。 </li><li>接下来，皮瓣泪腺开放轨道后，允许不受阻碍地进入肌肉覆盖在视神经。保持不断潮湿的地区使用无菌PBS，并与手术拭子或棉签干燥。 </li><li>再次使用尖锐镊子删除薄的结缔组织后轨道（提肌Palpebrae Superioris和上直肌）周围的肌肉和肌肉分开的基本捆绑。独立或异口同声地缩回肌肉，利用弯曲的锯齿状眼敷料钳，再次，在与肌纤维的拉动。将剩余的肌肉长度牵一起缩回步骤3.8和3.9上下牵带，适用于牵引肌肉。现在共有4肌肉牵。这将向前和向外转动眼球，以揭示视神经周围的脂肪含有鞘。 </li></ol> 4。访问视神经<ol><li>使用锋利的钳（精尖杜蒙）向上拉结缔组织视神经周围的脂肪鞘。作一纵形切开，用小Vannas弹簧剪刀。展开削减必要的。鞘中的脂肪将开始凸出，一旦削减。接下来删除小心从边缘向上提拉，切断新月形皮瓣组织结缔组织的襟翼。 </li><li>取出使用产钳拉脂肪Vannas弹簧剪刀切割，而脂肪覆视神经。保持该地区的清洁使用无菌PBS和手术棉签清洁切除组织中出现少量的血液。 </li><li>视神经是现在可见。为了访问的神经，周围神经脑膜鞘必须拆除而损坏的眼动脉，饲料视网膜内层。检查，脑膜使用镊子轻轻旋转鞘鞘血管格局。寻找一个没有血管的区域，并允许一个纵向切开脑膜鞘。 </li><li>使用精尖杜蒙钳，捏硬脑膜，向上拉。近创建硬脑膜形的三角楔的基础上，使用Vannas弹簧剪刀做一个小切口鞘。插入切口较低的刀片和剪刀切断鞘视神经，注意不要损伤血管横向切割方向平​​行。使用镊子和剪刀，悬垂性硬脑膜视神经两侧。 </li><li>唯一的剩余的神经覆盖蛛网膜。这是非常薄的和透明的。为了确定如果膜仍然存在，用锋利的钳捏面神经。蛛网膜是目前的，捏膜和上拉，以创建一个组织的三角楔。做一个小切口用类似剪刀步4.4的尖端。然后，插入下刀片，剪刀和一个纵行剪开蛛网膜。接下来，使用您的剪刀和镊子，悬垂性蛛网膜视神经两侧。 </li><li>使用微河畔gical挂钩，提高视神经的脑膜鞘。外缘周围的神经传递的钩提示，确保钩停留在接触与神经，使你没有赶上与此挂钩的脑膜膜和意外样。轻轻抬起视神经脑膜鞘和完全样钩支持点背后的神经。切断视神经残端现在有自由端，允许拆除的钩。 </li><li>如果RGC的是逆标，以量化的生存，地方的明胶海绵小块，浸泡在3％荧光金（或其他逆行示踪）在切断视神经残端（ <a href="http://www.jove.com/details.php?id=2261">见朱庇特的协议</a> ，2261 ）。 </li></ol> 5。关闭和恢复<ol><li>减轻额外的眼部肌肉牵引，并返回眼到一个中立的立场。虽然这样做，确保推入轨道的眼睛明胶海绵，以确保，映入眼帘的是旋转回原位，明胶海绵周围视神经残端仍然的。泪腺和额外的眼部肌肉返回到它们的自然位置。 </li><li>返回中线皮瓣，缝合伤口。眼科眼膏双眼。然后，关闭异氟醚源，并允许几分钟的动物呼吸的氧气。放置在一个加热灯恢复了激烈的笼或下方一个笼子里的动物。不要放置任何在回收笼的床上用品，以消除在恢复过程中的吸床上用品的机会。 </li><li>手术后的动物应设独立。手术后止痛药应根据动物保健当局的指引，管理，和动物手术后应仔细监测。 </li></ol> 6。代表性的成果： 横断postaxotomy 9-11受伤的视网膜神经节细胞的90％，14天之内的损失，视神经，结果。研资局死亡的主要机制是细胞凋亡的9，12 ​​。正常的视网膜神经节细胞的密度大约是2500个细胞/ mm 2 。 Epifluorescence或共焦成像可用于可视化逆行标记视网膜神经节细胞后，干切断。研资局凋亡延迟约4天，干切断后，留下了实验操作的时间窗口。在干切断，并用荧光金逆行标记后1天，研资局在视网膜和视网膜神经纤维层的轴突分册的神经节细胞层细胞体都清晰可见时成像flatmounted准备（图1A）。后干切断了14天，大部分的视网膜神经节细胞已经死亡，和几个剩余的视网膜神经节细胞是视网膜小胶质细胞（图1b）之间穿插。当视网膜神经节细胞发生凋亡，小胶质细胞吞噬死亡细胞和结果成为transcellularly荧光示踪标记，用来标记的视网膜神经节细胞13，14。示踪剂在小胶质细胞的吞噬体的外观，是尚存的视网膜神经节细胞的不同。小胶质细胞含有示踪剂，在高度集中和极其明亮的吞噬体比较大，散落在整个细胞质（图1C）。视网膜神经节细胞的染色更加分散的模式已逆行运其轴突备案细胞质中的小点状囊泡（图1C）。这些囊泡要小得多，有那么强烈的荧光，允许一个区别于小胶质细胞尚存的视网膜神经节细胞。此外，小胶质细胞胞体小得多，而且往往有星状或变形虫而不是有比较大的圆形胞体的视网膜神经节细胞的形态。视网膜神经节细胞的树突状树木还可以帮助他们从明亮的短流程的小神经胶质细胞分化时，量化细胞的存活。在视网膜的不同区域细胞的存活，可量化的和相应的显微面积密度（细胞/ 毫米 2）可推知，因为视网膜神经节细胞内神经节细胞层单层。 <img src="/files/ftp_upload/2241/2241fig1.jpg" alt="图1" /> 图1。Epifluorescence显微荧光金标记后视网膜神经节细胞和视神经残端示踪干切断应用。 （一）标记与示踪干切断视网膜神经节细胞和其轴突分册后1天，在细点状的方式。 （二）干切断后14天，90％的视网膜神经节细胞死亡，明亮的标记的小胶质细胞，吞噬死亡细胞示踪标记。 （三）说明了更高的放大倍率之间的视网膜神经节细胞和小胶质细胞（红色箭头）在14天postaxotomy的差别。 A和B的比例尺为50μm。规模在C栏是25微米。

<ol>
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这个手术过程中有许多变化，在本议定书中的几个步骤是没有必要的。它是只需要收回，覆盖在视神经以获得神经肌肉。然而，这个结果在一个非常有限的工作空间周围神经的横断关键的最后阶段更加困难。在某些情况下是可取的转染细胞和视神经残端回缩所有眼外肌，泪腺是有利于在这种情况下所提供的增加的存取空间。 在协议中最重要的步骤是步骤4.3-4.6。重要的是，不?...

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<td>Stereotaxic Frame</td>
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<td>Stoelting, Kopf, WPI</td>
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<td>Rat Gas Mask</td>
<td>&nbsp;</td>
<td>Stoelting, Kopf, WPI</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Anesthesia System</td>
<td>&nbsp;</td>
<td>VetEquip</td>
<td>901806</td>
<td>&nbsp;</td>
<tr>
<td>Isoflurane (PrAErrane)</td>
<td>&nbsp;</td>
<td>Baxter Corp</td>
<td>DIN 02225875</td>
<td>&nbsp;</td>
<tr>
<td>Surgical Microscope</td>
<td>&nbsp;</td>
<td>WPI, Zeiss, Leica</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fluorogold -(Hydroxystilbamidine bis(methanesulfonate)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>39286</td>
<td>&nbsp;</td>
<tr>
<td>Gelfoam</td>
<td>&nbsp;</td>
<td>Pharmacia &amp; Upjohn</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Tears Naturale P.M.</td>
<td>&nbsp;</td>
<td>Alcon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Proviodine</td>
<td>&nbsp;</td>
<td>Medline</td>
<td>MDS093945H</td>
<td>&nbsp;</td>
<tr>
<td>Vannas spring scissors</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>15000-00</td>
<td>&nbsp;</td>
<tr>
<td>Fine tip Dumont forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11252-00</td>
<td>&nbsp;</td>
<tr>
<td>Micro surgical hook</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>10062-12</td>
<td>&nbsp;</td>
<tr>
<td>Eye dressing serrated forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11152-10</td>
<td>&nbsp;</td>
<tr>
<td>Dumont #7b sharp curved serrated forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11270-20</td>
<td>&nbsp;</td>
<tr>
<td>Cauterizer</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>18010-00</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

optic nerve transection: a model of adult neuron apoptosis in the central nervous system

视网膜神经节细胞（RGC的）是中枢神经系统的神经元，输出的视觉信息从视网膜到大脑，通过视神经。视神经眼轨道内可以访问，并完全切断（axotomized），切割研资局在整个人口的轴突。视神经横断重现模型在1-4成人中枢神经系统的细胞凋亡的神经细胞死亡。这种模式是特别有吸引力，因为眼睛的玻璃体腔行为作为药物输送到视网膜的胶囊，允许通过人工注射的实验操作。通过玻璃体液体化学品的扩散，确保他们的行为后，整个研资局人口。此外，视网膜神经节细胞可以选择性地转用短干扰RNA（siRNAs）的质粒或病毒载体的视神经5-7或注入自己的目标，上丘8向量的切端。这使研究人员能够学习所需的神经元群的细胞凋亡的机制，没有其他旁观者神经元或神经胶质细胞周围的混杂影响。另外一个好处是与细胞的存活，可在伤后量化的易用性和准确性。视网膜是一个平面，分层组织和RGC的是在最里层，神经节细胞层的本地化。随着时间的推移，可跟踪的视网膜神经节细胞的生存，申请时间干切断视神经切开结束了荧光示踪（3％荧光金），或上丘（研资局的目标）前一周注入示踪剂干切断。示踪逆行运输，标签研资局在整个人口。由于神经节细胞层是单层（一个细胞厚），研资局的密度可量化的扁平式的组织中，无体视需要。视神经横断导致的90％受伤的视网膜神经节细胞的凋亡，14天以内 postaxotomy 9-11。研资局凋亡有一个特点的时间进程，使细胞死亡是推迟3-4天postaxotomy后，这些细胞迅速退化。这提供了一个针对在细胞凋亡有关的途径的实验操作的时间窗口。

Watch this Scientific Journal Video about Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System at JoVE.com

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

视神经横断是一个成人中枢神经系统损伤的广泛使用的模型。视网膜神经节细胞的90％（视网膜神经节细胞），其轴突完全横断（干切断）干切断后14天之内死亡。这种模式很容易经得起实验操作和高度重现。

视神经横断：一个成人在中枢神经系统的神经元凋亡模型

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be ...

optic-nerve-transection-model-adult-neuron-apoptosis-central-nervous

Research

JoVE Journal

Neuroscience

20.5K 次观看.  University of Toronto. 视神经横断是一个成人中枢神经系统损伤的广泛使用的模型。视网膜神经节细胞的90％（视网膜神经节细胞），其轴突完全横断（干切断）干切断后14天之内死亡。这种模式很容易经得起实验操作和高度重现。

视频: Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals through the vitreous fluid ensures that they act upon the entire RGC population. Viral vectors, plasmids or short interfering RNAs (siRNAs) can also be delivered to the vitreous chamber in order to infect or transfect retinal cells 5-12. The high tropism of Adeno-Associated Virus (AAV) vectors is beneficial to target RGCs, with an infection rate approaching 90% of cells near the injection site 6, 7, 13-15. Moreover, RGCs can be selectively transfected by applying siRNAs, plasmids, or viral vectors to the cut end of the optic nerve 16-19 or injecting vectors into their target the superior colliculus 10. This allows researchers to study apoptotic mechanisms in the injured neuronal population without confounding effects on other bystander neurons or surrounding glia. RGC apoptosis has a characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a window for experimental manipulations directed against pathways involved in apoptosis. Manipulations that directly target RGCs from the transected optic nerve stump are performed at the time of axotomy, immediately after cutting the nerve. In contrast, when substances are delivered via an intraocular route, they can be injected prior to surgery or within the first 3 days after surgery, preceding the initiation of apoptosis in axotomized RGCs. In the present article, we demonstrate several methods for experimental manipulations after optic nerve transection.

Optic Nerve transection is a widely used model of adult CNS injury. This model is ideal for performing a number of experimental manipulations that target the retina globally or directly target the injured neuronal population of retinal ganglion cells.

在哺乳动物的中枢神经系统视神经横断后的实验操作方法

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be ...

科研

神经科学

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

在中央小鼠中枢神经系统的干细胞移植的多模态成像

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10.
Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (&Psi;) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (<a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a>). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (<a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/</a>). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production.
In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

从中枢神经系统转导细胞的慢病毒载体生产

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

标记的单细胞在中枢神经系统<em&gt;果蝇</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

啮齿动物的中枢神经系统药物直接脑室交货

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are tightly regulated, affecting the final dynamics of protein abundance. Various regulatory mechanisms exist at the translation step, rendering mRNA levels alone an unreliable indicator of gene expression. In addition, local regulation of mRNA translation has been particularly implicated in neuronal functions, shifting &#39;translatomics&#39; to the focus of attention in neurobiology. The presented method can be used to bridge transcriptomics and proteomics.
Here we describe essential modifications to the technique of polyribosome fractionation, which interrogates the translatome based on the association of actively translated mRNAs to multiple ribosomes and their differential sedimentation in sucrose gradients. Traditionally, working with in vivo samples, particularly of the central nervous system (CNS), has proven challenging due to the restricted amounts of material and the presence of fatty tissue components. In order to address this, the described protocol is specifically optimized for use with minimal amount of CNS material, as demonstrated by the use of single mouse spinal cord and brain. Briefly, CNS tissues are extracted and translating ribosomes are immobilized on mRNAs with cycloheximide. Myelin flotation is then performed to remove lipid rich components. Fractionation is performed on a sucrose gradient where mRNAs are separated according to their ribosomal loading. Isolated fractions are suitable for a range of downstream assays, including new genome wide assay technologies.

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

This protocol illustrates essential modifications of polyribosome fractionation in order to study the translatome of in vivo CNS samples. It allows global assessment of translation and transcription regulation through the isolation and comparison of total RNA to ribosome bound RNA fractions.

在中枢神经系统Translatome由多核糖体分馏体内拷问

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

在啮齿动物中枢神经系统的体内光遗传学刺激

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to monitor the disease course in the follow-up period. Changes in the VEP parameters closely correlate with pathological damage in the optic nerve. This protocol provides a detailed description about the rodent model of optic nerve microinjection, in which a partial demyelination lesion is produced in the optic nerve. VEP recording techniques are also discussed. Using skull implanted electrodes, we are able to acquire reproducible intra-session and between-session VEP traces. VEPs can be recorded on individual animals over a period of time to assess the functional changes in the optic nerve longitudinally. The optic nerve demyelination model, in conjunction with the VEP recording protocol, provides a tool to investigate the disease processes associated with demyelination and remyelination, and can potentially be employed to evaluate the effects of new remyelinating drugs or neuroprotective therapies.

Focal demyelination is induced in the optic nerve using lysolecithin microinjection. Visual evoked potentials are recorded via skull electrodes implanted over the visual cortex to examine the signal conduction along the visual pathway in vivo. This protocol details the surgical procedures underlying electrode implantation and optic nerve microinjection.

在实验视神经脱髓鞘模型大鼠视觉诱发电位记录

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to ...

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique non-linear deformations, dramatically hampering one's ability to evaluate cellular morphology, distribution and connectivity in the central nervous system (CNS). However, optical clearing techniques are changing the way tissues are visualized. These approaches permit one to probe deeply into intact organ preparations, providing tremendous insight into the structural organization of tissues in health and disease. Techniques such as Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) achieve this goal by providing a matrix that binds important biomolecules while permitting light-scattering lipids to freely diffuse out. Lipid removal, followed by refractive index matching, renders the tissue transparent and readily imaged in 3 dimensions (3D). Nevertheless, the electrophoretic tissue clearing (ETC) used in the original CLARITY protocol can be challenging to implement successfully and the use of a proprietary refraction index matching solution makes it expensive to use the technique routinely. This report demonstrates the implementation of a simple and inexpensive optical clearing protocol that combines passive CLARITY for improved tissue integrity and 2,2&#8242;-thiodiethanol (TDE), a previously described refractive index matching solution.

Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.

鼠标中枢神经系统的光透明使用被动CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique ...

Partial Optic Nerve Transection in Rats: A Model Established with a New Operative Approach to Assess Secondary Degeneration of Retinal Ganglion Cells

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection is considered a useful and reproducible model. Compared with other optic nerve injury models used commonly for assessing secondary degeneration, e.g. complete optic nerve transection and optic nerve crush models, the partial optic nerve transection model is superior as it distinguishes primary from secondary degeneration in situ. Therefore, it serves as an excellent tool for evaluating secondary degeneration. This study describes a novel operative approach of partial optic nerve transection by directly accessing the area of the retrobulbar optic nerve through the orbital lateral wall of the eyeball. Moreover, we present a newly designed, low cost surgical instrument to assist with transection. As demonstrated by the representative results in distinguishing the boundary of primary and secondary injury areas, the new approach and instrument ensures high efficiency and stability of the model by providing adequate space for surgical operation. This in turn makes it easy to separate the meningeal sheath and ophthalmic vessels from the optic nerve before transection. An additional benefit is that this space-saving operative approach improves the investigators&#39; ability to administer drugs, carriers, or selective RGC tracers to the stump of the partially transected optic nerve, allowing the exploration of mechanisms behind secondary injury in RGCs, in a new way.

Secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. This study describes an innovative operative approach for partial optic nerve transection. The use of this space-saving operative approach extends the model's application range, and allows exploration of secondary injury mechanisms in RGCs in a new way.

大鼠部分视神经切断术: 建立一种新的手术方法评价视网膜神经节细胞二次变性的模型

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection ...