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。眼内注射的注射器制备<ol><li>首先，执行人工注射的注射器系统组装。 10μL护士气密1701汉密尔顿注射器用于注射。删除任何注射器针头或RN螺母目前。 </li><li>插入的PEEK杯煤灰套圈，套圈，压缩接头。然后将其插入注射器年底完成装修和松散顶部的RN螺母，螺钉。 </li><li>的1 / 16英寸的PEEK管的一端插入到压缩接头，通过在RN螺母开放。确保PEEK管完全就位。护士螺母拧紧，压缩套圈，密封PEEK管。 </li><li>执行步骤3.2双RN玻璃耦合两端。通过紧缩的护士螺母，将PEEK管自由端的双RN玻璃耦合的一端。 </li><li>一把拉住玻璃微吸管将形成针注射系统和桶。使用1.5 mm外径的厚壁玻璃毛细管管制作的玻璃吸液管，玻璃吸管插入双RN玻璃耦合的自由端。护士螺母拧紧，修复到位的吸管。 </li><li>拉玻璃微量的精尖通常是太小了，执行人工注射。为了创造一个适当的直径的尖端，打破玻璃吸管的尖端视觉引导下，用手术显微镜。磨砂的玻璃标本幻灯片结束非常适合打破吸管。移液器保持在一个角度和擦沿玻璃结霜的提示创建一个突破。理想的情况下，最终尖应略有斜面。它可能需要几分钟的尝试产生一个很好的提示，所以拉多个移液器前使用。 </li><li>喷射系统是液压的。因此，注射器，PEEK管连接器，双RN玻璃耦合器和玻璃微量矿物油在使用前必须填写。使用底漆从汉密尔顿注射器有限公司（PRMKIT）试剂盒，填补与矿物油吸注射器。使用大直径的针，让稠油容易通过。更换适合每桶10μL汉密尔顿注射器内与汉密尔顿90030针针。下一步，放置一个橡胶隔在汉密尔顿90030针，以提供印章，同时注入矿物油。 </li><li>汉密尔顿90030针吸注射器插入回到每桶10μL汉密尔顿注射器喷射系统。按注射器结束对隔紧紧地创建一个密封。 </li><li>慢慢压低柱塞。你会看到通过喷射系统的玻璃元素的矿物油通过，终于填补了玻璃吸管。通过喷射系统推油，以去除气泡沿道的任何。如果灌装注射器是矿物油，慢慢退出针，同时不断配药油，以防止气泡的形成。笔芯的吸注射器和注射一次。 </li><li>当整个系统是用矿物油和泡沫填充，插入原来的10μL汉密尔顿注射器的柱塞。为了从系统中删除额外的矿物油压低，直到旅行结束柱塞。擦拭玻璃微吸管干净，清洁注射器用70％乙醇年底。 </li><li> Withdraw为2μL桶大关注射器的柱塞。玻璃微吸管年底将填补与空气之间的矿物油和所需的液体被注入提供一个缓冲地带。吸管桶，现在可以充满，放置在理想​​的解决方案的微年底，撤回的汉密尔顿注射器的柱塞。不要超过4-5μL的溶液注入成年大鼠眼。 </li></ol> 4。眼内注射程序：全球定位视网膜<ol><li>与动物麻醉和固定在立体定位框架（第2部分所述），使用Alcaine眼药水局部麻醉角膜。打开用手指和地方上的角膜表面麻醉的解决方案之一滴眼睑。 </li><li>注射需要两个人之一插入玻璃体腔玻璃吸管和保持吸液管的位置，和另一个压低提供了理想的解决方案注射器的柱塞。 </li><li>在手术显微镜，握的玻璃微管和双护士用手指玻璃耦合器。传播与另一只手的手指在眼皮，形成一个“V”对面的注射部位的形状。应用牵引眼睑，眼睛会升高轨道，露出了后面的角膜缘的后表面。通过创建与对面的注射部位的手指一个“V”字形，形成一个摇篮眼睛提供注射时的稳定性。 </li><li>通过眼结膜的玻璃微吸管尖端轻轻插入，在一个地区缺乏血管，并通过巩膜，在一个向下的角度。你应该感到一个小的“砰砰”，当吸管穿透巩膜。在稍微向下的角度插入吸管，减少击球时的镜头插入针的机会。 </li><li>拿着注射器的人现在应该注入郁闷柱塞2μL注射器上标记的溶液4μL。注射应采取合共约一到两秒。在一个非常缓慢的的速度注入&gt; 3秒，减少了初步的解决方案通过玻璃体腔蔓延。您将可以看到注入的解决方案，通过在显微镜下玻璃体腔冲洗，从而验证程序的成功。 </li><li>保持约5秒钟的微管稳定，然后退出在同一方向，针插入。结膜瓣回小开放，帮助密封巩膜穿刺。 </li><li>在恢复过程中，以防止角膜干燥，并返回的动物恢复笼盖眼科眼膏（泪Naturale PM）的角膜表面。 </li><li>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> 5。有选择地针对视网膜神经节细胞视神经残端<ol><li>应用示踪剂，药品，质粒，siRNAs的病毒载体视神经断端，干切断后，直接针对视网膜神经节细胞，通过逆行运输。这是通过先执行视神经横断根据“视神经横断议定书”22。 </li><li>有两个主要的方法来完成此过程：用明胶海绵，或直接到视神经注射。 </li><li>明胶海绵的方法是类似逆行追踪的，然而，当它是可取的标签前的视网膜神经节细胞逆行示踪剂注入上丘1个星期之前，以干切断视神经提供试验性治疗。这是非常重要的细胞追踪，因为在某些情况下可干扰神经示踪逆行运输的实验物质，反之亦然。 </li><li>视神经后立即被切断，切断视神经残端放置在实验溶液中浸泡的明胶海绵小块。眶内容，然后返回到先前的位置。当眼睛恢复到中立的立场，有必要使用倒推入轨道的明胶海绵片钳，以确保它仍然在视神经年底。 </li><li>注射技术是更有效地交付的物质，必须获得轴突的细胞质，以便逆行轴浆流运输。这包括siRNA和质粒，并在某些情况下，病毒载体。 5-10μL汉密尔顿注射器注入切断视神经所需的解决方案。 </li><li>视神经与精尖杜蒙钳手柄的边缘。插入视神经针年底，平行的神经，直到锥不再可见。插入针头将包裹各方的神经。一个用短斜面的针头是可取的。 </li><li>一旦在Needle是插入到神经，用细尖镊子轻轻挤压神经的两侧，并同时转动四分之一圈顺时针或逆时针旋转的注射器注入一季度的解决方案。 </li><li>继续重复步骤5.7，直到完成一个完整的旋转针和注射器注射的全部内容。为了获得最佳结果，共注入10μL使用气密性的10μL汉密尔顿注射器解决方案。 </li><li>从神经取出注射器的尖端。保留的过剩注射液，从轨道内的神经回流，因为这将形成一个由轴突末端吸收池。 </li><li>眶内容返回到原来的位置，关闭伤口，让动物恢复。 </li><li>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： 眼内注射细胞在视网膜上的所有目标从全球来看，和受伤的视网膜神经节细胞的传递物质以及神经节细胞位于最内层的视网膜神经元层，相邻的玻璃体腔。视网膜神经节细胞，也可以直接切断视神经残端提供物质针对性。使用图1所示的神经断端的肽，药物，载体，质粒，或siRNA荧光金逆行标记视网膜神经节细胞，可以有针对性。图1A - C的演示一个Cy3标记的标记肽，前到上丘注射荧光金标记的视网膜神经节细胞somata本地化。 Cy3标记标记肽分别注入后立即干切断视神经，视网膜成像活着，以防止从浸出组织在固定的肽。图1D - F演示Cy3标记标记的siRNA和axotomized RGC的荧光金逆行示踪本地化。 Cy3标记标记的siRNA被注射到视神经残端后立即干切断和视网膜成像活着。 <img src="/files/ftp_upload/2261/2261fig1.jpg" alt="图1" /> 图1。flatmounted视网膜视网膜神经节细胞Epifluorescence的照片，在1天或者标记肽或到视神经残端的siRNAs后干切断和注射。 （一）荧光金逆行标记axotomized RGC的1天postaxotomy（二）在下面的标记肽的逆行运输，干切断和肽注射到视神经后1天的视网膜神经节细胞的荧光Cy3标记。 （三）覆盖（A）和（二）展示Cy3标记标记肽的荧光金标记的视网膜神经节细胞的选择性本地化。 （DF）的细胞的荧光金标记的视网膜神经节细胞（四）机构也充满了标记Cy3标记的siRNA注射到视神经残端24小时前（五），覆盖（六）所示。在交流的比例尺为50μm。在DF规模的酒吧是20微米。

<ol>
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视神经横断是一个成人中枢神经系统神经细胞凋亡的高度重复性的模型。在这个手稿中表现出的实验操作许可证伤后RGC的凋亡机制的研究。 全球定位视网膜，眼内注射是有用的。这个过程需要一定的实践，关键是不要伤害了的玻璃吸管尖端的镜头。晶状体的损害已经造成生长因子的释放，改变细胞的存活和再生20，21。同样重要的是要小心地插入和退出提示的方向平?...

A correction to the article Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS was made.

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

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methods for experimental manipulations after optic nerve transection in the mammalian cns

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。病毒载体，质粒或短干扰RNAs（siRNAs），也可以传递到玻璃体腔感染或转染视网膜细胞 5-12 。腺相关病毒（AAV）载体的高向性是有利于目标的视网膜神经节细胞，感染率接近90％的细胞注射部位6，7，13-15附近。此外，视网膜神经节细胞，可以选择性地转，运用到自己的目标上丘 10的siRNA，质粒或病毒载体的削减 16-19视神经或注射载体结束。这使研究人员能够在受伤的神经元群细胞凋亡的机制研究没有其他旁观者神经元或神经胶质细胞周围的混杂影响。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窗口。操作直接针对视网膜神经节细胞从横断视神经残端后，立即切割神经干切断时间。相比之下，通过人工途径传递物质，它们可以被注射，手术前或在手术后第3天前开始axotomized RGC的凋亡。在本文中，我们展示了视神经横断后的实验操作的几种方法。

Watch this Scientific Journal Video about Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS at JoVE.com

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

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。这种模式是执行的全球目标的视网膜或直接目标受伤的视网膜神经节细胞神经元群的实验操作的理想选择。

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 ...

methods-for-experimental-manipulations-after-optic-nerve-transection

Research

JoVE Journal

Neuroscience

13.3K 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。这种模式是执行的全球目标的视网膜或直接目标受伤的视网膜神经节细胞神经元群的实验操作的理想选择。

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

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

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. Moreover, RGCs can be selectively transfected by applying short interfering RNAs 
(siRNAs), plasmids, or viral vectors to the cut end of the optic nerve 5-7 or injecting vectors into their target, the superior colliculus 8. 
This allows researchers to study apoptotic mechanisms in the desired neuronal population without confounding effects on other bystander neurons or surrounding glia. 
An additional benefit is the ease and accuracy with which cell survival can be quantified after injury. The retina is a flat, layered tissue and RGCs are localized in 
the innermost layer, the ganglion cell layer. The survival of RGCs can be tracked over time by applying a fluorescent tracer (3% Fluorogold) to the cut end of the 
optic nerve at the time of axotomy, or by injecting the tracer into the superior colliculus (RGC target) one week prior to axotomy. The tracer is retrogradely transported, labeling 
the entire RGC population. Because the ganglion cell layer is a monolayer (one cell thick), RGC densities can be quantified in flat-mounted tissue, without the need 
for stereology. Optic nerve transection leads to the apoptotic death of 90% of injured RGCs within 14 days postaxotomy 9-11. 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 time window for 
experimental manipulations directed against pathways involved in apoptosis.

Optic Nerve transection is a widely used model of adult CNS injury. Ninety percent of retinal ganglion cells (RGCs) whose axons are completely transected (axotomy) die within 14 days after axotomy. This model is easily amenable to experimental manipulations and highly reproducible.

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 ...

科研

神经科学

An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible vision loss. Examples of such diseases in human include traumatic optic neuropathy and optic nerve degeneration in glaucoma. It is characterized by typical changes in the optic nerve head, progressive optic nerve degeneration, and loss of retinal ganglion cells, if uncontrolled, leading to vision loss and blindness.
The optic nerve crush (ONC) injury mouse model is an important experimental disease model for traumatic optic neuropathy, glaucoma, etc. In this model, the crush injury to the optic nerve leads to gradual retinal ganglion cells apoptosis. This disease model can be used to study the general processes and mechanisms of neuronal death and survival, which is essential for the development of therapeutic measures. In addition, pharmacological and molecular approaches can be used in this model to identify and test potential therapeutic reagents to treat different types of optic neuropathy.
Here, we provide a step by step demonstration of (I) Baseline retrograde labeling of retinal ganglion cells (RGCs) at day 1, (II) Optic nerve crush injury at day 4, (III) Harvest the retinae and analyze RGC survival at day 11, and (IV) Representative result.

This protocol shows how to retrogradely label retinal ganglion cells, and how to subsequently make an optic nerve crush injury in order to analyze retinal ganglion cell survival and apoptosis. It is an experimental disease model for different types of optic neuropathy, including glaucoma.

一个视神经挤压伤的小鼠模型，研究视网膜神经节细胞生存

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible ...

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory system is characterized by its ability to give rise to new neurons even in adult animals. This particular ability is partly due to the presence of OECs which create a favorable microenvironment for neurogenesis. This property of OECs has been used for cellular transplantation such as in spinal cord injury models. Although the peripheral nervous system has a greater capacity to regenerate after nerve injury than the central nervous system, complete sections induce misrouting during axonal regrowth in particular after facial of laryngeal nerve transection. Specifically, full sectioning of the recurrent laryngeal nerve (RLN) induces aberrant axonal regrowth resulting in synkinesis of the vocal cords. In this specific model, we showed that OECs transplantation efficiently increases axonal regrowth.
OECs are constituted of several subpopulations present in both the olfactory mucosa (OM-OECs) and the olfactory bulbs (OB-OECs). We present here a model of cellular transplantation based on the use of these different subpopulations of OECs in a RLN injury model. Using this paradigm, primary cultures of OB-OECs and OM-OECs were transplanted in Matrigel after section and anastomosis of the RLN. Two months after surgery, we evaluated transplanted animals by complementary analyses based on videolaryngoscopy, electromyography (EMG), and histological studies. First, videolaryngoscopy allowed us to evaluate laryngeal functions, in particular muscular cocontractions phenomena. Then, EMG analyses demonstrated richness and synchronization of muscular activities. Finally, histological studies based on toluidine blue staining allowed the quantification of the number and profile of myelinated fibers.
All together, we describe here how to isolate, culture, identify and transplant OECs from OM and OB after RLN section-anastomosis and how to evaluate and analyze the efficiency of these transplanted cells on axonal regrowth and laryngeal functions.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

嗅鞘细胞移植周围神经损伤后，以评估功能恢复

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

Motor Nerve Transection and Time-lapse Imaging of Glial Cell Behaviors in Live Zebrafish

The nervous system is often described as a hard-wired component of the body even though it is a considerably fluid organ system that reacts to external stimuli in a consistent, stereotyped manner, while maintaining incredible flexibility and plasticity. Unlike the central nervous system (CNS), the peripheral nervous system (PNS) is capable of significant repair, but we have only just begun to understand the cellular and molecular mechanisms that govern this phenomenon. Using zebrafish as a model system, we have the unprecedented opportunity to couple regenerative studies with in vivo imaging and genetic manipulation. Peripheral nerves are composed of axons surrounded by layers of glia and connective tissue. Axons are ensheathed by myelinating or non-myelinating Schwann cells, which are in turn wrapped into a fascicle by a cellular sheath called the perineurium. Following an injury, adult peripheral nerves have the remarkable capacity to remove damaged axonal debris and re-innervate targets. To investigate the roles of all peripheral glia in PNS regeneration, we describe here an axon transection assay that uses a commercially available nitrogen-pumped dye laser to axotomize motor nerves in live transgenic zebrafish. We further describe the methods to couple these experiments to time-lapse imaging of injured and control nerves. This experimental paradigm can be used to not only assess the role that glia play in nerve regeneration, but can also be the platform for elucidating the molecular mechanisms that govern nervous system repair.

Although the peripheral nervous system (PNS) is capable of significant repair after injury, little is known about the cellular and molecular mechanisms that govern this phenomenon. Using live, transgenic zebrafish and a reproducible nerve transection assay, we can study dynamic glial cell behaviors during nerve degeneration and regeneration.

电机神经切断和时间推移成像胶质细胞行为在现场斑马鱼

The  nervous system is often described as a hard-wired component of the body even  though it is a considerably fluid organ system that reacts to external ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

谷氨酸和GABA快速微离子导入：一个有用​​的工具，调查突触整合

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and postsynaptic projections to the visual cortex. Based on its distinct anatomical structure and convenient accessibility, it has become the favored structure for studies on neuronal survival, axonal regeneration, and synaptic plasticity. Recent advancements in MR imaging have enabled the in vivo visualization of the retino-tectal part of this projection using manganese mediated contrast enhancement (MEMRI). Here, we present a MEMRI protocol for illustration of the visual projection in mice, by which resolutions of (200 &#181;m)3 can be achieved using common 3 Tesla scanners. We demonstrate how intravitreal injection of a single dosage of 15 nmol MnCl2 leads to a saturated enhancement of the intact projection within 24 hr. With exception of the retina, changes in signal intensity are independent of coincided visual stimulation or physiological aging. We further apply this technique to longitudinally monitor axonal degeneration in response to acute optic nerve injury, a paradigm by which Mn2+ transport completely arrests at the lesion site. Conversely, active Mn2+ transport is quantitatively proportionate to the viability, number, and electrical activity of axon fibers. For such an analysis, we exemplify Mn2+ transport kinetics along the visual path in a transgenic mouse model (NF-&#954;B p50KO) displaying spontaneous atrophy of sensory, including visual, projections. In these mice, MEMRI indicates reduced but not delayed Mn2+ transport as compared to wild type mice, thus revealing signs of structural and/or functional impairments by NF-&#954;B mutations.
In summary, MEMRI conveniently bridges in vivo assays and post mortem histology for the characterization of nerve fiber integrity and activity. It is highly useful for longitudinal studies on axonal degeneration and regeneration, and investigations of mutant mice for genuine or inducible phenotypes.

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

This video illustrates a method, using a clinical 3 T scanner, for contrast-enhanced MR imaging of the na&#239;ve mouse visual projection and for repetitive and longitudinal in vivo studies of optic nerve degeneration associated with acute optic nerve crush injury and chronic optic nerve degeneration in knock-out mice (p50KO).

在视神经纤维通过诚信小鼠对比增强MRI 活体成像

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

在斑马鱼幼虫脊髓横断

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

Minimally-invasive Technique for Injection into Rat Optic Nerve

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central nervous system in a minimally-invasive surgery without bone removal. This technique describes an approach to visualization and direct injection of the optic nerve following minor fascial dissection from the orbital ridge, using a conjunctival traction suture to gently pull the eye down and out. Representative examples of an injected optic nerve show successful injection of dyed beads.

Direct injection into the rat optic nerve is useful for regenerative research. We demonstrate a minimally-invasive technique for direct injection into a rat optic nerve that does not involve opening the skull. Using this method, surgical complications are minimized and recovery is more rapid.

微创技术注入到老鼠视神经

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central ...

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 ...

Exposure of the Pig CNS for Histological Analysis: A Manual for Decapitation, Skull Opening, and Brain Removal

Pigs have become increasingly popular in large-animal translational neuroscience research as an economically and ethically feasible substitute to non-human primates. The large brain size of the pig allows the use of conventional clinical brain imagers and the direct use and testing of neurosurgical procedures and equipment from the human clinic. Further macroscopic and histological analysis, however, requires postmortem exposure of the pig central nervous system (CNS) and subsequent brain removal. This is not an easy task, as the pig CNS is encapsulated by a thick, bony skull and spinal column. The goal of this paper and instructional video is to describe how to expose and remove the postmortem pig brain and the pituitary gland in an intact state, suitable for subsequent macroscopic and histological analysis.

The goal of this paper and instructional video is to describe how to expose and remove the postmortem pig brain and pituitary gland in an intact state, suitable for subsequent macroscopic and histological analysis.