Name: retrograde neuroanatomical tracing of phrenic motor neurons in mice
Uploaded: 2018-02-22T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice at JoVE.com

我们感谢罗伯特 Graffin 和宝琳 Duhant 的技术支持。

实验性议定书是按照欧洲共同体理事会动物实验指令 (2010/63/欧盟、86/609/欧洲经济共同体和 87-848/欧洲经济共同体) 进行的, 并得到了纳尔大学动物伦理委员会 (伦理项目 n°17-284) 的批准。).图 1描述了两种方法: 膈或经胸注射。在研究中使用雄性 C57bl/6J 小鼠 (n=18), 年龄从3个月到4月。
1. CTB 溶液的制备
<ol><li>膈注射:<ol>
<li>将 CTB 在无菌水中的能量溶解到浓度为 0.2% (w/v)。</li>
		<li>负载7.5 µL 的 CTB 溶液 (0.2% 瓦特/v) 在无菌 10-µL 微量泵与附加33口径针 (钝或短斜角) 为每个鼠标。</li>
	</ol>
</li>
	<li>经胸注射:<ol>
<li>将 CTB 在无菌水中的能量溶解到浓度为 0.1% (w/v)。</li>
		<li>负载20µL CTB 溶液 (0.1% 瓦特/v) 在一个无菌 500-µL 胰岛素注射器与斜面27口径针为每只老鼠。 注: 请务必使用蒸馏水和无菌水, 并适当溶解 CTB 粉。该解决方案可以保持一个月摄氏4摄氏度 (不要冻结)。沉淀可能在几天后形成。使用前用吸管将溶液带入室温, 并混合使用。</li>
	</ol>
</li>
</ol>2. 胸腔注射前的准备工作
<ol><li>使用高压釜或玻璃珠灭菌器, 在手术前消毒手术工具。准备一件干净的长凳做手术, 并处理手术工具。 注意: 在手术过程中使用的任何材料都应该是无菌的。不能灭菌的仪器, 如微量泵, 应用消毒剂 (必泰) 擦拭, 或者只应使用一次性。外科医生应该在手术开始前用消毒剂 (洗必泰擦洗) 洗手。外科医生应该戴上无菌手套、面罩和干净的长袍。</li>
	<li>称量动物, 并管理适当的麻醉剂量: 腹腔注射麻醉鸡尾酒 (如氯胺酮100毫克/千克和甲苯噻嗪5毫克/千克)。</li>
	<li>捏脚趾和/或检查眼睑反射的损失, 以确定是否正确麻醉鼠标。应用兽医软膏保护角膜。</li>
	<li>仔细剃须腹部皮肤 (为膈程序) 或右胸皮肤 (经胸手术), 使用电剪。剃得好, 足够宽, 以防止头发进入手术领域。</li>
	<li>通过局部应用10% 碘溶液在剃毛地区确保无菌条件。用碘溶液擦洗手术部位, 小心地从中心部位擦洗到周围。</li>
	<li>在整个手术中使用恒温垫保持动物的体温。</li>
</ol>3. 用膈方法进行胸腔注射
<ol><li>将麻醉鼠标放在加热垫上仰卧位。将卷纱垫放在动物的脖子下。对于成年鼠标, 请使用0.5 英寸厚度的滚动纱布垫。把这个垫子贴到手术板上, 以免发生运动。</li>
	<li>使用手术刀刀片, 切割腹侧皮肤沿中线: 做一个切口从剑突过程到脐区域, 而伸展皮肤侧向与另一只手, 使皮肤绷紧。不要对刀片施加太多的压力, 以免损害底层器官。</li>
	<li>使用小剪刀, 小心地把皮肤从腹部肌肉的切口周围剥离。这个程序将有助于缝合的肌肉和皮肤分开的手术结束。</li>
	<li>用小剪刀进行剖腹手术。通过在脐的水平上执行一个纽扣切口打开腹腔。切割腹部肌肉沿白色线 ("linea") 由剑突过程。</li>
	<li>为了形象化的腹部表面的隔膜, 收回腹部肌肉使用商业或自制拉钩。 注意: 这些拉钩可以由形状为 L 挂钩19的 midi 大小的回形针组成。</li>
	<li>伸展开腹的两侧, 并把拉钩到板凳大衣上。优化视野的光照, 使隔膜的腹面不再在半影中。最强大的照明设备为这个目的是一个 LED 灯与定向光束。</li>
	<li>一方面使用软镊子, 举起 xyphoid 阑尾, 拉下肝脏的侧面裂片 (图 2A)。另一方面使用小剪刀, 小心切断悬吊韧带, 不损害胆囊或隔膜 (图 2B)。</li>
	<li>一方面用软镊子, 举起 xyphoid 附录。另一方面抓住哈密尔顿注射器。目标三的注射地点在右 hemidiaphragm 分别覆盖胸骨, 内侧和脚区域 (图 3A, 插入图 3B)。</li>
	<li>由于膜片厚度约为0.37 毫米小鼠7, 插入针不超过1或2毫米超过隔膜片, 以防止肺部损害在呼吸期间。通过每个站点的右膜片 (图 4B) 提供2.5 µL 的 CTB 解决方案 (0.2% 瓦特/v)。不需要膜片的稳定。</li>
	<li>对三个同侧注射点 (图 1A) 重复此过程, 并为 PhMN 池的双向标记 contralaterally。</li>
	<li>闭合剖腹手术部位, 缝合腹部肌肉与可吸收4-0 缝合。使用被打断的针间距为3毫米, 主要皮肤闭合与9.0 毫米伤口剪辑。收紧主食, 以防止动物拔下主食。间隔约5毫米的空间钉。不要过度收紧伤口夹, 因为这会导致愈合受损。</li>
	<li>用蒸馏水清洁微型注射器, 防止堵塞。慢慢地起草和驱逐2-3 次。不要把空气抽进注射器。</li>
</ol>4. 胸腔内注射用经胸法
注意: 此方法灵感来自4中描述的过程, 并适合于鼠标。
<ol><li>在加热垫 (图 4A) 上, 将麻醉鼠标放在左侧压疮位置。</li>
	<li>用手动触诊 (图 4B) 识别肘部区域下的第六和第七根肋骨。</li>
	<li>当助手扩展右前-和后肢 (图 5A) 时, 插入注射器 cranially 定向和切下的第六或第七肋, 3 毫米深从皮肤, 与斜角向下 (图 1B和图 5B)。</li>
	<li>轻轻抬高针, 以确认, 通过解除肋骨, 它是很好地定位到胸腔 (图 5C, 插入图 1B)。</li>
	<li>用两个手指在胸壁上施加轻微的压力, 稳定胸部的呼吸运动。</li>
	<li>在拿着注射器的同时, 在单个注射中提供20µL 的 CTB 溶液 (0.1% 瓦特/v)。</li>
</ol>5. 术后护理
<ol><li>术后立即将鼠标放置在恒温垫上的右侧压疮位置, 以恢复。这确保了示踪剂传播在胸腔的右侧, 并允许监测动物的呼吸。</li>
	<li>管理1毫升无菌盐水溶液。提供后续注射, 如果动物出现脱水和/或无精打采。</li>
	<li>在手术后的头两天, 每天两次在术后0.1 毫克/公斤的丁丙诺啡, 以尽量减少任何潜在的疼痛。 注: 许多机构建议对侵入性手术进行多模镇痛, 如这里所描述的剖腹手术。这可能包括使用非甾体抗炎 (NSAID) 药物和/或局部麻醉剂在切口部位。这些药物通常是在第一个切口之前提供的, 以减少出现的疼痛。</li>
	<li>每天监视动物直至安乐死。</li>
</ol>

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作者没有什么可透露的。

本文所描述的协议可以适用于任何成年小鼠的应变或任何实验范式, 其中的 PhMN 电路的完整性应评估。例如, 肌萎缩侧索硬化 (ALS) 和颈脊髓损伤 (cSCI) 是与 PhMN 丢失、膈轴突顺变性和随后的呼吸折衷有关的条件。动物模型的 ALS 或 cSCI 模拟病理学和功能性呼吸衰竭观察到人类疾病。在这些模型中, 组织学技术经常被用来评估 PhMN 生存后, 针对 PhMN 神经保护18,20</...

本研究的目的是提出一种可靠的方法来识别膈运动神经元 (PhMN) 在小鼠脊髓部分。在胸腔内注射荧光神经解剖学示踪剂作为传递方法, 以达到膈肌神经的投射到膜片上, 并使用逆行运输沿膈轴突标记膈细胞体。本文介绍两种胸腔内分娩技术: 膈与胸肺。
膈运动神经元是脊髓中继细胞, 其轴突汇聚成膈神经, 最终支配膈肌。这些是较低的马达神经元接受吸气驱动从球呼吸中心和中继到隔膜神经肌肉连接 (NMJ)。PhMN 结构成两个马达柱, 一个为每个 hemicord, 运行沿中颈椎脊柱。在包括人类在内的大多数哺乳动物物种中, 膈电机列从 C3 级别扩展到 C61,2,3。我们和其他人已经证实, PhMN 集中在大鼠和小鼠脊髓的 C3-C5 水平4,5,6,7,8。膈细胞的地形图分布不随机;在膈肌运动池 (C3) 的颅骨部分, 支配的运动神经元分布较密, 而支配脚部的运动神经元则更是尾鳍 (C5)9。此外, PhMN 在腹角灰质中有不同的聚集。在 C3 水平, 膈细胞的簇在侧向, 然后他们在一个延髓方向转移并且被发现 ventromedially 在最尾部水平10,11。
考虑到他们在灵感中的重要作用, 准确地识别 PhMN 在健康的脊髓中是至关重要的, 但在病理条件下, 如退行性疾病或脊髓外伤损伤, 也应遵循他们的命运。由于 PhMN 与其他颈椎运动神经元的形态学不同, PhMN 的识别依赖于在初级呼吸系统中心 (8) 的水平, 或在隔膜 NMJ7或在膈神经4。追踪器由神经纤维占去并且运载到膈肌细胞身体在颈椎, 在那里它可以被视觉化使用直接或间接检测系统。逆行或顺示踪剂在商业上有广泛的共轭。值得注意的是, 每个追踪器都被赋予无、低或高的跨突触追踪能力。
在目前的研究中, 我们选择了霍乱毒素的β亚基 (CTB) 功能性与 Alexa 555 (从今以后称为 CTB-荧光) 作为荧光标签, 允许直接可视化 PhMN 的冰冻脊髓部分。CTB 通常被描述为 monosynaptic 示踪器, 尽管实验数据倾向于显示 transneuronal 通道12。CTB 有能力将苷 GM1 在神经末端的等离子膜上。CTB 是通过网格蛋白依赖或独立的机制内化, 并通过反式高尔基网络进入内质网, 在逆行的时尚13, 14.内部化和逆行传输似乎依赖于肌动蛋白骨架15、16以及微管网络17。
为了证明 CTB 作为逆行神经解剖学示踪贴标膜片-PhMN 电路的用处, CTB 荧光被交付 intrapleurally。CTB 使用两种技术进行管理: 第一个包括剖腹手术和多膈注射;第二个, 较少侵入, 使用了独特的经胸注射。四天后, 荧光标记的 PhMNs 从健康和 spinally 受伤 (C4) 动物的颈椎脊髓中定量。

<table><tbody><tr><td>Glass-bead sterilizer Steri 250</td><td>Keller</td><td>31-101</td><td></td></tr><tr><td>Small scissors</td><td>F.S.T.</td><td>14058-00</td><td></td></tr><tr><td>Soft tweezers</td><td>F.S.T.</td><td>11042-08</td><td></td></tr><tr><td>Scalpel blades</td><td>Swann Morton</td><td>No.11 or 15</td><td></td></tr><tr><td>Cholera toxin subunit beta conjugated to Alexa Fluor 555</td><td>Life Technologies</td><td>C22843</td><td>Bring at room temperature before use&amp;nbsp;</td></tr><tr><td>10ul Hamilton syringue, removable needle</td><td>Sigma-Aldrich</td><td>701RN</td><td></td></tr><tr><td>33-gauge needle for Hamilton syringue, 20mm length, point style 4</td><td>Filter Service</td><td>7803-05</td><td></td></tr><tr><td>500ul insulin syringue MyJector, 27-gauge</td><td>Terumo</td><td>BS05M2713</td><td></td></tr><tr><td>Orientable LED lamp</td><td>V.W.R.</td><td>631-0995</td><td></td></tr><tr><td>Resorbable 4/0 sutures</td><td>S.M.I. AG</td><td>15151519</td><td></td></tr><tr><td>Needle holder</td><td>F.S.T.</td><td>12002-14</td><td></td></tr><tr><td>9mm autoclips</td><td>Bioseb</td><td>205016</td><td></td></tr><tr><td>Autoclip 9mm applier</td><td>Bioseb</td><td>MikRon 9mm</td><td></td></tr></tbody></table>

retrograde neuroanatomical tracing of phrenic motor neurons in mice

膈运动神经元是由 C3 到 C6 水平的颈椎运动神经元。轴突投射到膈神经支配呼吸隔膜。在脊髓切片中, 膈运动神经元不能从其他运动神经元中识别出形态学或生物化学标准。我们提供描述的程序, 以可视化膈运动神经元细胞体的小鼠, 后胸腔注射霍乱毒素亚基β (CTB) 共轭荧光。该荧光神经解剖学示踪剂有能力被抓住在隔膜神经肌肉交界, 被携带 retrogradely 沿膈轴突和到达膈细胞体。比较了胸腔 CTB 分娩的两种方法: 膈与经胸注射。这两种方法都是成功的, 并导致了类似数量的 CTB 标记的膈运动神经元。总之, 这些技术可用于可视化或量化的膈运动神经元的各种实验研究, 如那些关注膈膈电路。

研究中包括雄性 C57bl/6J 小鼠 (n=18), 年龄从3个月到4月。在实验的0天, 8 只小鼠接受了单边 C4 挫伤, 右侧, 根据发布的协议7,18。作为假手术, 10 只小鼠在 C4 顶部进行了椎板切除术, 无挫伤。3天, 根据上述两种不同的程序, 对 CTB 荧光胸腔注射的小鼠进行了准备。7天, 所有小鼠在麻醉后被安乐死 (氯胺酮100毫克/千克和甲苯噻嗪5毫克/千?...

Watch this Scientific Journal Video about Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice at JoVE.com

Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice

本文描述了荧光共轭霍乱毒素亚单位β胸腔内分娩后小鼠膈运动神经元识别的一种协议。比较两种方法注射胸腔: 膈与经胸的方法。

小鼠膈运动神经元逆行神经解剖学追踪的研究

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic ...

retrograde-neuroanatomical-tracing-of-phrenic-motor-neurons-in-mice

Research

JoVE Journal

Neuroscience

12.6K 次观看.  Université de Namur. 本文描述了荧光共轭霍乱毒素亚单位β胸腔内分娩后小鼠膈运动神经元识别的一种协议。比较两种方法注射胸腔: 膈与经胸的方法。

视频: Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

重复性小鼠坐骨神经挤压和再生的后续评估整装肌肉分析

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

科研

神经科学

Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes

We describe a technique for retrograde labeling of motor neurons in Drosophila. We use an oil-dissolved lipophilic dye and deliver a small droplet to an embryonic fillet preparation by a microinjector. Each motor neuron whose membrane is contacted by the droplet can then be rapidly labeled. Individual motor neurons are continuously labeled, enabling fine structural details to be clearly visualized. Given that lipophilic dyes come in various colors, the technique also provides a means to get adjacent neurons labeled in multicolor. This tracing technique is therefore useful for studying neuronal morphogenesis and synaptic connectivity in the motor neuron system of Drosophila.

Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes

We describe a method for retrograde tracing of the Drosophila embryonic motor neurons using lipophilic fluorescent dyes.

使用亲脂荧光染料的果蝇胚胎运动神经元的逆行追踪

We describe a technique for retrograde labeling of motor neurons in Drosophila. We use an oil-dissolved lipophilic dye and deliver a small droplet to an ...

发育生物学

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and morphological levels. Compound muscle action potential (CMAP) recording following phrenic nerve stimulation can be used to quantitatively assess functional diaphragm innervation by PhMNs of the cervical spinal cord in vivo in anesthetized rats and mice. Because CMAPs represent simultaneous recording of all myofibers of the whole hemi-diaphragm, it is useful to also examine the phenotypes of individual motor axons and myofibers at the diaphragm NMJ in order to track disease- and therapy-relevant morphological changes such as partial and complete denervation, regenerative sprouting and reinnervation. This can be accomplished via whole-mount immunohistochemistry (IHC) of the diaphragm, followed by detailed morphological assessment of individual NMJs throughout the muscle. Combining CMAPs and NMJ analysis provides a powerful approach for quantitatively studying diaphragmatic innervation in rodent models of CNS and PNS disease.

Compound muscle action potential recording quantitatively assesses functional diaphragm innervation by phrenic motor neurons. Whole-mount diaphragm immunohistochemistry assesses morphological innervation at individual neuromuscular junctions. The goal of this protocol is to demonstrate how these two powerful methodologies can be used in various rodent models of spinal cord disease.

隔膜的神经支配膈运动神经元的功能和形态评估

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and ...

Intramuscular Injections Along the Motor End Plates: A Minimally Invasive Approach to Shuttle Tracers Directly into Motor Neurons

Diseases affecting the integrity of spinal cord motor neurons are amongst the most debilitating neurological conditions. Over the last decades, the development of several animal models of these neuromuscular disorders has provided the scientific community with different therapeutic scenarios aimed at delaying or reversing the progression of these conditions. By taking advantage of the retrograde machinery of neurons, one of these approaches has been to target skeletal muscles in order to shuttle therapeutic genes into corresponding spinal cord motor neurons. Although once promising, the success of such gene delivery approach has been hampered by the sub-optimal number of transduced motor neurons it has so far shown to yield. Motor end plates (MEPs) are highly specialized regions on the skeletal musculature that are in direct synaptic contact to the spinal cord &#945; motor neurons. In this regard, it is important to note that, so far, the efforts to retrogradely transfer genes into motor neurons were made without reference to the location of the MEP region in the targeted muscles. Here, we describe a simple protocol 1) to reveal the exact location of the MEPs on the surface of skeletal muscles and 2) to use this information to guide the intramuscular delivery and subsequent optimal retrograde transport of retrograde tracers into motor neurons. We hope to utilize the results from these tracing experiments in further studies into investigating retrograde transport of therapeutic genes to spinal cord motor neurons through the targeting of MEPs.

The efficacy of intramuscular uptake and retrograde transport of molecules to corresponding motor neurons depends on the location of the injection sites with respect to the motor end plates (MEPs). Here, we describe how to locate MEPs on skeletal muscles to optimise retrograde transport of tracers into motor neurons.

肌肉注射沿运动终板:微创方法来接送示踪剂直接为运动神经元

Diseases affecting the integrity of spinal cord motor neurons are amongst the most debilitating neurological conditions. Over the last decades, the ...

Laser-guided Neuronal Tracing In Brain Explants

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake through electroporation in brain explants with targeted laser illumination and matching filter goggles during the procedure. The described technique of in vitro electroporation by itself yields relatively good visual control for targetting certain areas of the brain. By combining it with laser excitation of fluorescent genetic markers and their read-out through band-passing filter goggles, which can pick up the emissions of the genetically labeled cells/nuclei and the fluorescent tracing dye, a researcher can substantially increase the accuracy of injections by finding the area of interest and controlling for the dye-spread/uptake in the injection area much more efficiently. In addition, the laser illumination technique allows to study the functionality of a given neurocircuit by providing information about the type of neurons projecting to a certain area in cases where the GFP expression is linked to the type of transmitter expressed by a subpopulation of neurons.

We describe a technique to label neurons and their processes via anterograde or retrograde tracer injections into brain nuclei using an in vitro preparation. We modified an existing method of in vitro tracer electroporation by taking advantage of fluorescently labeled mouse mutants and basic optical equipment in order to increase labeling accuracy.

激光制导神经跟踪在脑外植体

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake ...

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of individual cells. Since an increase of intracellular calcium (Ca2+) in the cytosol often occurs because of the electrical activity, or in response to a myriad of other stimuli, this process can be monitored by the imaging of cells loaded with fluorescent calcium-sensitive dyes.&#160; However, it is difficult to image this response in an individual cell type within whole tissue because these dyes are taken up by all cell types within the tissue. In contrast, genetically encoded calcium indicators (GECIs) can be expressed by an individual cell type and fluoresce in response to an increase of intracellular Ca2+, thus permitting the imaging of Ca2+ signaling in entire populations of individual cell types. Here, we apply the use of the GECIs GCaMP3/6 to the mouse neuromuscular junction, a tripartite synapse between motor neurons, skeletal muscle, and terminal/perisynaptic Schwann cells. We demonstrate the utility of this technique in classic ex vivo tissue preparations. Using an optical splitter, we perform dual-wavelength imaging of dynamic Ca2+ signals and a static label of the neuromuscular junction (NMJ) in an approach that could be easily adapted to monitor two cell-specific GECI or genetically encoded voltage indicators (GEVI) simultaneously. Finally, we discuss the routines used to capture spatial maps of fluorescence intensity. Together, these optical, transgenic, and analytic techniques can be employed to study the biological activity of distinct cell subpopulations at the NMJ in a wide variety of contexts.

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Here we present a protocol to image calcium signaling in populations of individual cell types at the murine neuromuscular junction.

前体小鼠膈肌三方突触细胞特异性钙信号的影像学研究

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of ...

A Mouse Model of Direct Anastomosis via the Prespinal Route for Crossing Nerve Transfer Surgery

Crossing nerve transfer surgery has been a powerful approach for repairing injured upper extremities in patients with brachial plexus avulsion injuries. Recently, this surgery was creatively applied in the clinical treatment of brain injury and achieved substantial rehabilitation of the paralyzed arm. This functional recovery after the surgery suggests that peripheral sensorimotor intervention induces profound neuroplasticity to compensate for the loss of function after brain damage; however, the underlying neural mechanism is poorly understood. Therefore, an emergent clinical animal model is required. Here, we simulated clinical surgery to establish a protocol of direct anastomosis of bilateral brachial plexus nerves via the prespinal route in mice. Neuroanatomical, electrophysiological, and behavioral experiments helped identify that the transferred nerves of these mice successfully reinnervated the impaired forelimb and contributed to accelerating motor recovery after brain injury. Therefore, the mouse model revealed the neural mechanisms underlying rehabilitation upon crossing nerve transfer after central and peripheral nervous system injuries.

We simulated clinical surgery to establish a protocol of direct anastomosis of bilateral brachial plexus nerves via the prespinal route in mice, contributing to the study of the neural mechanisms underlying rehabilitation upon crossing nerve transfer after central and peripheral nervous system injuries.

通过椎前途径直接吻合的小鼠模型用于跨神经转移手术

Crossing nerve transfer surgery has been a powerful approach for repairing injured upper extremities in patients with brachial plexus avulsion injuries. ...

Stereotaxic Surgical Approach to Microinject the Caudal Brainstem and Upper Cervical Spinal Cord via the Cisterna Magna in Mice

Stereotaxic surgery to target brain sites in mice is commonly guided by skull landmarks. Access is then obtained via burr holes drilled through the skull. This standard approach can be challenging for targets in the caudal brainstem and upper cervical cord due to specific anatomical challenges as these sites are remote from skull landmarks, leading to imprecision. Here we outline an alternative stereotaxic approach via the cisterna magna that has been used to target discrete regions of interest in the caudal brainstem and upper cervical cord. The cisterna magna extends from the occipital bone to the atlas (i.e., the second vertebral bone), is filled with cerebrospinal fluid, and is covered by dura mater. This approach provides a reproducible route of access to select central nervous system (CNS) structures that are otherwise hard to reach due to anatomical barriers. Furthermore, it allows for direct visualization of brainstem landmarks in close proximity to the target sites, increasing accuracy when delivering small injection volumes to restricted regions of interest in the caudal brainstem and upper cervical cord. Finally, this approach provides an opportunity to avoid the cerebellum, which can be important for motor and sensorimotor studies.

Stereotaxic Surgical Approach to Microinject the Caudal Brainstem and Upper Cervical Spinal Cord via the Cisterna Magna in Mice

Stereotaxic surgery to target brain sites in mice commonly involves access through the skull bones and is guided by skull landmarks. Here we outline an alternative stereotaxic approach to target the caudal brainstem and upper cervical spinal cord via the cisterna magna that relies on direct visualization of brainstem landmarks.

立体定位手术方法 ，通过 大水池在小鼠中显微注射尾部脑干和上颈椎脊髓

Stereotaxic surgery to target brain sites in mice is commonly guided by skull landmarks. Access is then obtained via burr holes drilled through the skull. ...

靶向小鼠交感神经元：一种腰交感神经切除术方法

Surgical Lumbar Sympathectomy in Mice

Peripheral nerve injuries are common, and full functional recovery after injury is achieved in only 10% of patients. The sympathetic nervous system plays many critical roles in maintaining bodily homeostasis, but it has rarely been studied in the context of peripheral nerve injury. The extent of postganglionic sympathetic neuronal functions in distal targets in the periphery is currently unclear. To better explore the role of sympathetic innervation of peripheral targets, a surgical &#34;knock-out&#34; model provides an alternative approach. Although this can be achieved chemically, chemical destruction of postganglionic sympathetic neurons can be nonspecific and dose-dependent. The use of a surgical lumbar sympathectomy in mice, once thought to be &#34;virtually not practicable&#34; in small animals, allows for specific targeting of postganglionic sympathetic neurons that innervate the hind limbs. This manuscript describes how to surgically remove the L2-L5 lumbar sympathetic ganglia from a mouse as a survival surgery, which reliably decreases the hind paw sweat response and the number of sympathetic axons in the sciatic nerve.

作者聚焦：探索交感神经元的可塑性

This manuscript presents a protocol for surgically removing the postganglionic lumbar sympathetic neurons from a mouse. This procedure will facilitate a multitude of studies aimed at investigating the role of sympathetic innervation in distal tissue targets.

小鼠手术腰交感神经切除术

Peripheral nerve injuries are common, and full functional recovery after injury is achieved in only 10% of patients. The sympathetic nervous system plays ...

小鼠膈运动神经元逆行神经解剖学追踪的研究

摘要

探索更多视频

此视频中的章节

重复性小鼠坐骨神经挤压和再生的后续评估整装肌肉分析

使用亲脂荧光染料的果蝇胚胎运动神经元的逆行追踪

隔膜的神经支配膈运动神经元的功能和形态评估

肌肉注射沿运动终板:微创方法来接送示踪剂直接为运动神经元

激光制导神经跟踪在脑外植体

前体小鼠膈肌三方突触细胞特异性钙信号的影像学研究

通过椎前途径直接吻合的小鼠模型用于跨神经转移手术

立体定位手术方法，通过大水池在小鼠中显微注射尾部脑干和上颈椎脊髓

小鼠手术腰交感神经切除术

小鼠手术腰交感神经切除术

小鼠膈运动神经元逆行神经解剖学追踪的研究

摘要

探索更多视频

此视频中的章节

重复性小鼠坐骨神经挤压和再生的后续评估整装肌肉分析

使用亲脂荧光染料的果蝇胚胎运动神经元的逆行追踪

隔膜的神经支配膈运动神经元的功能和形态评估

肌肉注射沿运动终板:微创方法来接送示踪剂直接为运动神经元

激光制导神经跟踪在脑外植体

前体小鼠膈肌三方突触细胞特异性钙信号的影像学研究

通过椎前途径直接吻合的小鼠模型用于跨神经转移手术

立体定位手术方法 ，通过 大水池在小鼠中显微注射尾部脑干和上颈椎脊髓

小鼠手术腰交感神经切除术

小鼠手术腰交感神经切除术

立体定位手术方法，通过大水池在小鼠中显微注射尾部脑干和上颈椎脊髓