Name: a microbiomechanical system for studying varicosity formation and recovery in central neuron axons
Uploaded: 2018-04-30T13:00:00.000+00:00
Duration: 9 min 58 s
Description: Watch this Scientific Journal Video about A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons at JoVE.com

所有动物实验都是按照国家卫生研究院动物使用指南进行的。这项工作部分是由国立卫生研究院 (R01NS093073 和 R21AA024873) 提供给 c。

下面描述的所有方法都已由俄亥俄州立大学的机构动物护理和使用委员会 (IACUC) 批准。
1. 盖玻片准备
<ol><li>将一个或多个12毫米或25毫米的盖玻片盒放入含有70% 硝酸的玻璃烧杯中, 并在室温下一夜之间孵化盖玻片。 注意: 不要在同一个烧杯中洗25毫米的盖玻片12毫米盖玻片。最好分开洗。</li>
	<li>将所有盖玻片转换为4升烧杯, 填充2.5 升ddH2 O, 并摇动包含盖玻片的烧杯, 在 100 rpm 处为1小时。在摇晃的时候用橡皮筋来控制烧杯。重复冲洗与新鲜的 2.5 L ddH2O 四次。</li>
	<li>将清洗过的盖玻片转到带有金属盖的干烧瓶中。烘烤的盖玻片在一个烤箱在225°c 6 小时, 让他们冷却到室温。 注:25 毫米盖玻片容易折断。将盖玻片分开, 晾干, 然后用烤箱烘烤。</li>
	<li>将干燥和清洁的盖玻片在室温下存放在灭菌塑料容器中, 直到使用。</li>
	<li>
外套盖玻片如下:
	<ol>
<li>在离心管中制备新的涂层溶液。</li>
		<li>将每个盖玻片放入一个24井 (或6井) 板的一个井中。将盖玻片涂层溶液(表 1) 的 30 (或 100) µL 到12毫米 (或25毫米) 盖玻片和涡流。在井中加入1毫升无菌磷酸盐缓冲盐水 (PBS, 组织培养级)。这些板可以立即使用或储存在4摄氏度在无菌 PBS 长达一周。</li>
		<li>在解剖的那天, 取出 PBS。空气干燥和安置板材在紫外光 (30W) 之下为 2-4 h。 注: 最后一步应在细胞培养层流罩上的紫外线照射下完成。引擎盖的门应该稍微抬起, 以允许气流干燥盖玻片。</li>
	</ol>
</li>
</ol>2. 孕鼠/大鼠海马神经元的解剖、分离和培养
<ol><li>
培养海马神经元的解剖与分离
	<ol>
<li>解冻蛋白酶酶溶液 (3 毫克/毫升蛋白酶23在 SLDS,表 1) 和预热前电镀介质 (表 2) 在37°c。</li>
		<li>根据上一出版物14中描述的方法, 解剖海马并用 SLDS 冲洗。 注: 四海马为一个24井或一个6井板提供足够的电池。</li>
		<li>去除 SLDS, 加入2毫升蛋白酶酶溶液, 并在37摄氏度, 5% CO2上孵化样品15分钟。</li>
		<li>用5-10 毫升电镀培养基将海马外植体清洗两次。添加5毫升电镀培养基。用1毫升塑料尖端的吸管产生一个小漩涡, 将海马外植体分离成单个细胞。吸管上下大约40次, 避免形成氧泡, 直到溶液变得混浊, 没有大块的外植体依然存在。 注: 切记在洗涤过程中留一些培养基, 在整个过程中, 海马外植体应保持在培养基中浸泡。</li>
		<li>离心细胞在 1125 x g 3 分钟. 用介质淹没的细胞去除上清液。并用重悬145毫升的电镀培养基中的细胞。添加1毫升/井的细胞悬浮到一个24井板 (3 毫升/井到6井板)。 注: 145 毫升电镀介质用于产生细胞悬浮, 细胞密度低。这一体积将允许电镀细胞成六24井板, 八6井板, 或各种组合的24或6井板, 取决于要进行的实验。24井板的每个井都有约 3 x 104单元9。</li>
		<li>将电镀介质中的细胞孵化37摄氏度, 5% CO2用于 2-4 h, 然后取出所有电镀介质, 并用新鲜的维护介质替换。</li>
		<li>2天 (2 天体外, 2DIV), 用2µM (arabinosylcytosine) 溶解于维持介质中, 将一半的培养基替换为抑制成纤维细胞、内皮和胶质干细胞生长的介质。将细胞暴露在两天后, 用新鲜的维护介质替换介质。</li>
	</ol>
</li>
	<li>
细胞形态学可视化的转染 注: 染细胞与荧光结构的绿色荧光蛋白 (GFP), 黄色荧光蛋白 (YFP), mCherry,等, 以照亮个别神经元的形态学。<ol>
<li>将构造从股票 (1 µg/µL) 稀释成最的介质 (50 µL 中的结构的0.8 µg) 和涡。准备1建筑稀释为每个井。 注: 一个24井板需要0.8 µg 的结构, 在50µL 的最适记忆介质。一个6井板需要2.4 µg 的结构在150µL 的记忆介质。</li>
		<li>将脂质体介导的转染试剂稀释到µL 介质中 (50 µL 中的1.5 的转染试剂) 和涡流。准备1转染稀释为每个井。 注:24 井板需要1.5 µL 的转染试剂, 在50µL 的最适记忆介质。一个6井板需要4.5 µL 的转染试剂在150µL 的最适记忆介质。</li>
		<li>准备1:1 混合物 (100 µL) 的结构, 在更低的记忆介质和转染试剂的最适记忆介质和涡流。室温下将混合物孵化20分钟。 注: 如果使用 24-井板 (300 µL 6 井板), 每一个井, 应有约100µL 的解决方案, 包含结构, 转染试剂, 和最适合的记忆介质。</li>
		<li>卸下一半的介质 (500 µL 为24井板, 1.5 毫升6井板) 从每个井和转移这个媒介成一个干净的锥形管。把这个管子放在孵化器里。然后将100µL 混合物 (步骤 2.2.3) 添加到井中的剩余介质中。允许细胞在37摄氏度孵育20-30 分钟。</li>
		<li>在孵化过程中 (步骤 2.2.4), 在锥形管 (步骤 2.2.4) 的介质中添加一卷新鲜的维护介质。将混合物放入同一孵化器 (37 °c 和 5% CO2)。</li>
		<li>在孵化后, 从井中移除包含转染溶液的所有介质, 并将其替换为锥形管 (步骤 2.2.5) 中旧的和新鲜的维护介质的1:1 混合物。 注意: 根据构造的大小, 单元格可以在12小时内开始表达;对于较大的构造, 在达到构造的峰值表达式之前, 可能需要3-4 天。</li>
	</ol>
</li>
</ol>3. 设置膨化吸管装置
注: 此设置有五基本组件: 玻璃吸管、油管、注射器、机器人和缓冲器 (汉克)。任何具有生理相关盐浓度的缓冲液都可用于此装置。
<ol><li>使用微拉出器和表 3中的规范, 在45µm 直径的开口尖端上拉一个硼硅吸管。 注: 在实验条件下, 发现45µm 是适用于吸管开口直径的合适尺寸。开口尺寸稍有变化不应显著影响结果。重要的是要注意的是, 更大的偏离这个数字应该仍然工作的静脉曲张诱导通过调整注射器的高度与吸管通过油管, 但这将需要重新校准的压力值。</li>
	<li>将吸管的未拔出端插入薄橡胶管。 注: 油管壁应薄而刚性, 以尽量减少流体阻力, 确保注射器的高度与流体通过吸管的压力直接成正比, 阻力极小。油管长度不得超过0.6 米, 以进一步减少阻力。</li>
	<li>将油管的另一端连接到一个10毫升的塑料注射器上, 通过连接器与可转动的阀门相连。 注: 塑料注射器需要保持在一个恒定的, 可测量的高度, 以确保一个准确的措施, 假定压力流动从吸管。高度190毫米被使用了, 因为这提供极小的压力, 但可靠地诱导静脉曲张在轴突。如果压力导致单元格从盖玻片表面分离, 也可以使用其他高度值。建议在整个实验中使用相同的设置。</li>
	<li>将吸管插入机器人的螺钉顶部, 将吸管固定到位。拧紧连接在机器人臂上的金属, 平顶螺钉, 使其保持在所附橡胶管的上方的吸管的未拔出端。在45°角上用含有神经元的盖玻片的表面将吸管倾斜。 注: 应构造机器人, 使吸管可以在不收缩橡胶管的情况下举行。机器人必须允许在 x、y 和 z 方向的吸管运动, 以便准确地将其放置在细胞的范围内。下面提供了一种图形以及该设备的照片 (图 1)。</li>
	<li>打开荧光滤光片, 开启光圈, 确保荧光点可以通过目标来观察。使用机器人, 将吸管移入一个位置, 将针尖与荧光点对线, 并将吸管降低到刚好高于细胞培养皿高度 (35 毫米 x 10 毫米) 的位置。一旦定位到位, 打开透射光并关闭荧光。</li>
	<li>使用镊子, 添加一个盖玻片 (与细胞朝向向吸管) 从细胞培养板到细胞培养皿包含约2毫升的汉克的缓冲在室温下。</li>
	<li>将包含盖玻片的文化菜盘放到范围内。</li>
	<li>使用精细对焦旋钮和20X 目标, 聚焦在四个以上的细胞 (约0.4 毫米) 的平面上。使用机器人定位吸管尖端, 使其集中, 并在中心, 左侧的平面上看到通过眼睛片断。 注意: 树突是投射, 应该与它们起源的细胞体在同一帧中。为诱发轴突静脉曲张, 应遵循从细胞体到内侧和远端轴突的较长的投射。</li>
</ol>4. 使用伸缩膜系统校准吸管的压力
<ol><li>在含有 microfabricated 硅膜 (直径500µm 和50µm 厚) 的系统上设置与上述参数完全相同的膨化吸管装置, 并将显微镜聚焦在膜表面。 注: 本系统设计用于测量小压力值, 并在10之前发布了系统说明。这种测量只需要做一次的膨化实验, 如果使用完全相同的膨化的设置。</li>
	<li>将显微镜聚焦在微粒的阵列上 (直径为4µm, 在中心之间测量10µm)。转动截止阀, 允许液体从吸管流出。用共焦显微镜检查膜的挠度, 以响应流体压力。</li>
	<li>利用线性模型确定与膜挠度相关的压力。 注: 虽然最近的一项研究发现 w =-3.143 0.69 µm 的偏转是为190毫米注射器高度获得的, 这产生了压力 0.25 @ 0.06 nN/µm2从目前的吸管设置, 这是在生理和病理范围内, 虽然注射器高度不是唯一的决定因素。其他因素也会影响神经元的确切压力值, 包括吸管的开口、与细胞相对的吸管尖端的定位 (距离和角度), 甚至是导管的灵活性4,10。</li>
</ol>5. 膨化诱导静脉曲张
<ol><li>一旦吸管就位, 将显微镜聚焦在包含感兴趣区域的平面上。将单元格和进程放置在成像场的左半部分 (通过相机的实时捕获) 在膨化区域内, 而右半部分位于膨化区域之外。 注意: 由于神经元轴突和树突的广泛生长, 神经元的所有过程不太可能在同一膨化区域内。这实际上是这个系统的一个优点, 它允许检查膨化的局部效应。有两种膨化方式可用于诱导静脉曲张: 长时间膨化和脉冲膨化。</li>
	<li>
膨化
	<ol>
<li>长时间膨化<ol>
<li>将注射器放置在高度, 190 毫米以上的盖玻片包含培养的神经元4。开始时间推移成像的兴趣区域与优化曝光时间, 揭示清晰的神经元形态学。以2秒间隔 (三十年代总计) 捕获至少15帧作为基线。该间隔可以根据实验进行调整。在16帧, 打开注射器的可转截止阀, 允许流体流动75帧 (150s)。在75帧, 关闭阀门, 使流体流动停止。停止视频捕获。 注: 这应诱发神经元轴突静脉曲张在5-10 秒内 7-9 DIV, 而较旧的神经元需要较长的时间来形成静脉曲张。</li>
		</ol>
</li>
		<li>脉冲 (2 s 持续时间) 膨化<ol>
<li>将注射器放置在适当的高度 (190 毫米)。开始时间推移成像和捕获至少15帧 (三十年代) 在2秒的时间间隔。在16帧, 打开注射器的阀门, 允许流体流动, 然后在2秒后关闭阀门. 等待5-20 秒 (取决于要测试的频率), 然后再打开阀门。重复开启和关闭阀门以产生流体脉冲, 并持续150-300 秒的总周期. 持续时间推移成像20分钟, 以捕获恢复阶段。 注: 当间隔时间长于十年代时, 脉冲产生的静脉曲张大大减少。在二十年代的时间间隔内, 脉冲4不能诱发静脉曲张。为获得可靠、一致的静脉曲张形成, 建议注射器高度应在190毫米左右. 注射器高度和流体压力不应提高到细胞开始从盖玻片表面分离的地步。</li>
		</ol>
</li>
		<li>使用前一天, 清洁设置 (吸管, 油管, 和注射器) 通过流动约10毫升70% 乙醇通过设置。在乙醇清洗后, 通过设置以使系统在当天使用时, 流10毫升的汉克缓冲器 (或所需的缓冲器)。在完成当天的实验后, 清洁以前用乙醇描述的设置, 以防止污染物在一夜之间生长。</li>
	</ol>
</li>
</ol>6. 恭维方法
注: 膨化试验可以结合各种不同的技术, 在介绍部分讨论。在这里, 重点是与膨化试验最常用的核心技术, 包括高分辨率荧光 timelapse 成像, 钙成像和恢复化验。下文将讨论这些问题。
<ol><li>
按照下面的协议进行高分辨率荧光时间推移成像
	<ol>
<li>用100X 油透镜跟踪神经元内荧光标记蛋白的运动。神经元用适当的 cDNA 结构进行转染。捕获150帧 (总 300s), 间隔为2秒。 注意: 有时, 多色时移成像是同时对多个荧光标记的蛋白质的运动进行图像处理的。</li>
		<li>通过视频捕获软件或通过 ImageJ (NIH) 生成 kymograph。 注意: 使用 kymograph 可以确定旅行的速度和方向性。* 一定要注意到从捕获的区域中的躯体所在的方向。对躯体的旅行是逆行的, 而从躯体到轴突尖端的旅行顺。</li>
		<li>重复此过程后膨化, 或一个 kymograph 可以创建使用图像捕捉期间膨化试验。 注意: 膨化对线粒体和突触蛋白的轴突传输的影响在最近的一篇论文4中显示。</li>
	</ol>
</li>
	<li>
钙成像
	<ol>
<li>增加1µL (为24井板材) 或3µL (为6井板材) 5 毫米 Fluo-4 是对文化媒介在一个井和孵化在37°c 为30分钟。</li>
		<li>用1毫升的缓冲器洗两次细胞。 注: 加载的神经元通过 FITC 过滤器集发出绿色荧光。基因座表示细胞内有明显钙的区域。当与膨化结合时, 可以看到强度的增加 (内部钙含量的增加)。</li>
	</ol>
</li>
	<li>
静脉曲张恢复 注: 在膨化实验之后, 可以进行20分钟的恢复实验。<ol>
<li>捕获300帧 (1200 s) 的图像, 其间隔为4秒。 注意: 用可溶性 YFP/mCherry/GFP 转染的细胞应在成像会话结束时显示中等水平 (少于 50%) 的恢复。轴突恢复取决于多种因素, 如培养神经元的发育阶段4。这也可以与上面描述的高分辨率荧光成像和钙成像相结合。</li>
	</ol>
</li>
</ol>

<ol>
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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclosporin-A+treatment+attenuates+delayed+cytoskeletal+alterations+and+secondary+axotomy+following+mild+axonal+stretch+injury.">Cyclosporin-A treatment attenuates delayed cytoskeletal alterations and secondary axotomy following mild axonal stretch injury.</a> Dev. Neurobiol. 67 (14), 1831-1842 (2007).</li><li>Tang-Schomer, M. D., Johnson, V. E., Baas, P. W., Stewart, W., Smith, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+interruption+of+axonal+transport+due+to+microtubule+breakage+accounts+for+the+formation+of+periodic+varicosities+after+traumatic+axonal+injury.">Partial interruption of axonal transport due to microtubule breakage accounts for the formation of periodic varicosities after traumatic axonal injury.</a> Exp. Neurol. 233 (1), 364-372 (2012).</li><li>Donkin, J. J., Vink, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+cerebral+edema+in+traumatic+brain+injury:+therapeutic+developments.">Mechanisms of cerebral edema in traumatic brain injury: therapeutic developments.</a> Curr Opin Neurol. 23 (3), 293-299 (2010).</li><li>Gardner, A., Jukkola, P., Gu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelination+of+rodent+hippocampal+neurons+in+culture.">Myelination of rodent hippocampal neurons in culture.</a> Nat Protoc. 7 (10), 1774-1782 (2012).</li><li>Wang, Q., Zhang, X., Zhao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromechanical+stimulator+for+localized+cell+loading:+fabrication+and+strain+analysis.">Micromechanical stimulator for localized cell loading: fabrication and strain analysis.</a> J. Micromech. Microeng. 23, 015002 (2013).</li><li>Lu, Y. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viscoelastic+properties+of+individual+glial+cells+and+neurons+in+the+CNS.">Viscoelastic properties of individual glial cells and neurons in the CNS.</a> Proc. Natl. Acad. Sci. USA. 103 (47), 17759-17764 (2006).</li><li>Elkin, B. S., Azeloglu, E. U., Costa, K. D., Morrison, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+heterogeneity+of+the+rat+hippocampus+measured+by+atomic+force+microscope+indentation.">Mechanical heterogeneity of the rat hippocampus measured by atomic force microscope indentation.</a> J. Neurotrauma. 24 (5), 812-822 (2007).</li><li>Franze, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurite+branch+retraction+is+caused+by+a+threshold-dependent+mechanical+impact.">Neurite branch retraction is caused by a threshold-dependent mechanical impact.</a> Biophys. J. 97 (7), 1883-1890 (2009).</li><li>Camp&#224;s, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+cell-generated+mechanical+forces+within+living+embryonic+tissues.">Quantifying cell-generated mechanical forces within living embryonic tissues.</a> Nat. Methods. 11 (2), 183-189 (2014).</li></ol>

作者没有什么可透露的。

这 microbiomechanical 化验的程序是直接的。它将产生可靠的结果, 如果所有的步骤都仔细执行。有几个关键步骤, 如果执行不当, 将阻碍成功的数据收集。关键步骤从实际应用到膨化刺激的上游开始。仔细解剖, 培养和照顾的主要神经元培养是至关重要的。如果培养的神经元不健康, 它们就不会持续反应, 因为它们可能已经被灌输给压力。下游的文化, 初始设置和校准的膨化设备提供一致的压力, 这将诱?...

静脉曲张形成, 或肿胀/珠, 沿轴突, 是一个突出的特点变性观察到许多疾病或损伤的中枢神经系统, 包括多发性硬化症, 阿尔茨海默病, 帕金森病, 和创伤脑损伤1,2。尽管轴突静脉曲张对动作电位传播和突触传输有显著的生理影响3, 但如何生成静脉曲张仍然不得而知。最近, 利用新建立的 microbiomechanical 对啮齿动物培养的海马神经元进行了检测, 发现机械刺激能诱发这些神经元中的静脉曲张, 具有极具吸引力的特征。第一, 静脉曲张诱导迅速 (< 十年代), 这个过程是出乎意料的可逆的。其次, 静脉曲张的萌生取决于膨化压力的强度: 压力越大, 启动速度越快。第三, 静脉曲张的萌生取决于神经元的年龄。与较旧的神经元相比, 年轻神经元的轴突对机械应力的反应更大。第四, 静脉曲张沿海马神经元的轴突形成, 而这些神经元的树突和初始轴突段在同一膨化条件下无变化。因此, 我们的研究揭示了一种新的神经极性特征。这些发现与体外系统在生理上是相关的。使用体内模型进行轻度创伤性脑损伤 (mTBI), 我们表明, 轴突静脉曲张在小鼠体感皮层的多焦点模式下, 在闭合颅骨撞击后立即形成, 与我们的体内一致。结果4。值得注意的是, 我们的 mTBI 小鼠的染色和成像只提供了神经元形态学变化的快照, 因为执行体内的神经元形态学在机械撞击过程中的时间推移成像仍然是不可行的。
这种液体膨化系统不仅使我们能够捕捉到与机械应力引起的静脉曲张形成相关的独特特征, 而且还可以确定其内在机制。通过对不同细胞外溶液、mechanosensitive 离子通道不同候选者的阻滞剂和开启器的测试, 以及细胞电生理学, 我们确定了瞬态受体电位阳离子通道亚科 V 成员 4 (TRPV4)可渗透到 Ca2 +和 Na+并由膨化激活的通道主要负责检测轴突静脉曲张形成的初始机械应力4。这进一步证实了以 siRNA 淘汰赛方法。结合这一新的实验系统, 我们开发了海马神经元培养, 对于研究中枢神经元的微机械特性, 特别是与其他技术相结合, 具有重要的价值。
我们建立的这一微机械系统是独一无二的, 不同于以前存在的系统在几个主要方面。首先, 在这个系统中, 神经元在压缩和剪切的形式中经历了平面外的机械应力。在机械撞击过程中, 神经元的过程仍然附着在盖玻片表面, 不移动。这与其他主要涉及弯曲和平面拉伸 (或张力) 的实验系统不同, 例如, 捆绑轴突的偏转, 如移动字符串5,6或在 micropatterned 上生长的拉伸轴突。通道和伸缩膜7,8。此外, 虽然轴突静脉曲张也可以在这些化验中诱发像我们的液体膨化系统, 这些设置的过程需要更多的时间 (从10分钟到几个小时6,7,8), 并出现不可逆转。最后, 我们的系统使用局部液体膨化允许检查静脉曲张形成的空间特征 (e. g., 树突, 树枝状刺, 躯体, 轴突的初始段, 轴突终端), 除了它的世俗特征。利用该系统, 我们发现了轴突静脉曲张形成的几种意想不到的独特特征, 特别是快速发作、慢可逆性和轴突-枝晶极性。
本文所讨论的系统与多种分子和细胞生物学技术相适应。例如, 研究机械应激对神经元形态和功能的影响, 可与髓鞘共培养、荧光共振能量转移 (焦虑) 的时移成像和全内反射荧光 (TIRF) 一起使用,钙成像和膜片钳记录。本文重点讨论了系统的核心组件。海马神经元培养, 液体膨化设置, 高分辨率的轴突传输的时间推移成像, 和钙成像, 说明了逐步下一步。

<table><tbody><tr><td>12 mm coverslips&amp;nbsp;</td><td>Warner Instruments&amp;nbsp;</td><td>64-0702</td><td>for 24-well plate&amp;nbsp;</td></tr><tr><td>25 mm coverslips&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>12-545-102&amp;nbsp;</td><td>for 6-well plate&amp;nbsp;</td></tr><tr><td>Acetic acid</td><td>Fisher Scientific&amp;nbsp;</td><td>A38-212</td><td></td></tr><tr><td>Poly-D-lysine</td><td>Sigma&amp;nbsp;</td><td>P6407</td><td></td></tr><tr><td>Rat tail collagen&amp;nbsp;</td><td>Roche&amp;nbsp;</td><td>11 179 179 001&amp;nbsp;</td><td></td></tr><tr><td>10X PBS&amp;nbsp;</td><td>National Diagnostics&amp;nbsp;</td><td>CL-253</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific&amp;nbsp;</td><td>S373-500</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific&amp;nbsp;</td><td>P304-500</td><td></td></tr><tr><td>HEPES&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>BP410-500</td><td></td></tr><tr><td>D-glucose&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>D16-500</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher Scientific&amp;nbsp;</td><td>BP214-500</td><td></td></tr><tr><td>NaOH</td><td>Fisher Scientific&amp;nbsp;</td><td>SS255-1</td><td></td></tr><tr><td>Protease enzyme&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>P4032</td><td></td></tr><tr><td>FBS</td><td>Gibco&amp;nbsp;</td><td>26140</td><td></td></tr><tr><td>Sodium pyruvate&amp;nbsp;</td><td>Gibco&amp;nbsp;</td><td>11360-070</td><td></td></tr><tr><td>L-glutamine&amp;nbsp;</td><td>Gibco&amp;nbsp;</td><td>25030081</td><td></td></tr><tr><td>Penicillin/Streptomycin 100x (P/S)</td><td>Gibco&amp;nbsp;</td><td>15140122</td><td></td></tr><tr><td>MEM Earle&#039;s Salts&amp;nbsp;</td><td>Gibco&amp;nbsp;</td><td>11090</td><td></td></tr><tr><td>B27 supplement&amp;nbsp;</td><td>Gibco&amp;nbsp;</td><td>17504-044</td><td></td></tr><tr><td>Neurobasal&amp;nbsp;</td><td>Gibco&amp;nbsp;</td><td>21103-049</td><td></td></tr><tr><td>Arabinosylcytosine (Ara-C)</td><td>Sigma&amp;nbsp;</td><td>147-94-4</td><td></td></tr><tr><td>Opti-MEM media&amp;nbsp;</td><td>Gibco&amp;nbsp;</td><td>31985-070</td><td></td></tr><tr><td>Lipofectamine 2000</td><td>Invitrogen&amp;nbsp;</td><td>1854313</td><td>transfection reagent&amp;nbsp;</td></tr><tr><td>Borosilicate rods&amp;nbsp;</td><td>World Precision Instruments Inc.&amp;nbsp;</td><td>PG52151-4</td><td>for puffing pipette&amp;nbsp;</td></tr><tr><td>rubber tubing&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>14-169-1A</td><td></td></tr><tr><td>10cc plastic syringe and plunger</td><td>Becton Dickinson&amp;nbsp;</td><td></td><td></td></tr><tr><td>micromanipulator&amp;nbsp;</td><td>Sutter Instruments&amp;nbsp;</td><td></td><td></td></tr><tr><td>NaCl&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>S640-3</td><td></td></tr><tr><td>KCl&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>BP366-500</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher Scientific&amp;nbsp;</td><td>C70-500</td><td></td></tr><tr><td>Cell culture dish (35 mm x 10 mm)</td><td>Corning&amp;nbsp;</td><td>3294</td><td></td></tr><tr><td>Fluo-4 AM</td><td>Molecular Probes</td><td>F14201</td><td>for calcium imaging&amp;nbsp;</td></tr><tr><td>Mito-YFP construct&amp;nbsp;</td><td>Takara Bio Inc.&amp;nbsp;</td><td></td><td>for cell transfection&amp;nbsp;</td></tr><tr><td>YFP-N1 construct&amp;nbsp;</td><td>Takara Bio Inc.&amp;nbsp;</td><td></td><td>for cell transfection&amp;nbsp;</td></tr><tr><td>Model P-1000 Flaming/Brown Micropipette puller&amp;nbsp;</td><td>Sutter Instruments&amp;nbsp;</td><td></td><td></td></tr><tr><td>Eclipse TE2000-U Mcroscope&amp;nbsp;</td><td>Nikon</td><td></td><td></td></tr><tr><td>Plan Fluor ELWD 20x lens&amp;nbsp;&amp;nbsp;</td><td>Nikon</td><td>062933</td><td>objective&amp;nbsp;</td></tr><tr><td>Apo TIRF 100x/1.49 oil lens&amp;nbsp;</td><td>Nikon</td><td>MRD01991</td><td>objective&amp;nbsp;</td></tr></tbody></table>

a microbiomechanical system for studying varicosity formation and recovery in central neuron axons

轴突静脉曲张是在高异质性的轴轴上扩大结构。它们不仅存在于神经退行性疾病或损伤的大脑中, 还存在于正常大脑中。在这里, 我们描述了一个新建立的微机械系统快速, 可靠, 可逆诱发轴突静脉曲张, 使我们能够理解的机制, 控制静脉曲张形成和异构蛋白组成。该系统代表了一种新的方法来评估压缩和剪切应力对不同的神经元亚细胞间隔的影响, 不同于其他体外系统, 主要集中于伸展作用。重要的是, 由于系统的独特性, 我们最近提出了一种新的发现, 即加压液的应用可以通过瞬态受体电位通道的活化, 快速、可逆地诱发轴突静脉曲张。我们的生物力学系统可以方便地与药物灌注、活细胞成像、钙成像和膜片钳记录结合使用。因此, 该方法可用于研究 mechanosensitive 离子通道、轴突转运调节、轴突细胞骨架动力学、钙信号传递以及与颅脑损伤相关的形态学变化。

在膨化前, 轴突通常表现为小静脉曲张形成。随着我们的标准压力 (190 嗯2O 高度) 的膨胀, 轴突开始发展许多像珠样的静脉曲张。静脉曲张的形成部分是可逆的, 如轴突区域在10分钟恢复期后返回到其预膨胀状态 (图 2a B) 所示。经过较长时间的恢复 (> 20 分钟), 一些轴突完全恢复。膨化吸管的次优定位以及190毫米以上的注射器准确定?...

Watch this Scientific Journal Video about A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons at JoVE.com

A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons

本协议描述了一种生理上相关的加压液方法, 用于快速可逆地诱导神经元中的静脉曲张。

中枢神经元轴突静脉曲张形成和恢复的 Microbiomechanical 系统研究

Axonal varicosities are enlarged structures along the shafts of axons with a high degree of heterogeneity. They are present not only in brains with ...

a-microbiomechanical-system-for-studying-varicosity-formation

Research

JoVE Journal

Neuroscience

6.2K 次观看.  The Ohio State University. 本协议描述了一种生理上相关的加压液方法, 用于快速可逆地诱导神经元中的静脉曲张。

视频: A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons

Mechanical Manipulation of Neurons to Control Axonal Development

Cell manipulations and extension of neuronal axons can be accomplished with calibrated glass micro-fibers capable of measuring and applying forces in the 10-1000 &mu;dyne range1,2. Force measurements are obtained through observation of the Hookean bending of the glass needles, which are calibrated by a direct and empirical method3. Equipment requirements and procedures for fabricating, calibrating, treating, and using the needles on cells are fully described. The force regimes previously used and different cell types to which these techniques have been applied demonstrate the flexibility of the methodology and are given as examples for future investigation4-6. The technical advantages are the continuous 'visualization' of the forces produced by the manipulations and the ability to directly intervene in a variety of cellular events. These include direct stimulation and regulation of axonal growth and retraction7; as well as detachment and mechanical measurements on any type of cultured cell8.

Application and direct measurements of forces on neurons in the 2-1000 microdyne range are achieved with high precision using calibrated glass needles. This methodology can be used to control and measure several aspects of axonal development, including axonal initiation, axonal tension, velocity of axonal elongation, and force vectors.

机械操纵控制神经元轴突的发展

Cell manipulations and extension of neuronal axons can be accomplished with calibrated glass micro-fibers capable of measuring and applying forces in the ...

科研

神经科学

Laser Nanosurgery of Cerebellar Axons In Vivo

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.

Laser Nanosurgery of Cerebellar Axons In Vivo

Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo.

小脑轴突的激光Nanosurgery<em&gt;在体内</em

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

一个<em&gt;离体</em&gt;激光诱导脊髓损伤模型，以评估轴索变性的机制在实时

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Intravital Imaging of Axonal Interactions with Microglia and Macrophages in a Mouse Dorsal Column Crush Injury

Traumatic spinal cord injury causes an inflammatory reaction involving blood-derived macrophages and central nervous system (CNS)-resident microglia. Intra-vital two-photon microscopy enables the study of macrophages and microglia in the spinal cord lesion in the living animal. This can be performed in adult animals with a traumatic injury to the dorsal column. Here, we describe methods for distinguishing macrophages from microglia in the CNS using an irradiation bone marrow chimera to obtain animals in which only macrophages or microglia are labeled with a genetically encoded green fluorescent protein. We also describe a injury model that crushes the dorsal column of the spinal cord, thereby producing a simple, easily accessible, rectangular lesion that is easily visualized in an animal through a laminectomy. Furthermore, we will outline procedures to sequentially image the animals at the anatomical site of injury for the study of cellular interactions during the first few days to weeks after injury.

Two-photon intravital imaging can be used to investigate interactions among different cell types in the spinal cord in their native tissue environment in a bone marrow chimeric animal with a dorsal column traumatic spinal cord crush injury.

轴突的相互作用与小胶质细胞和巨噬细胞在小鼠背柱破碎损伤活体成像

Traumatic spinal cord injury causes an inflammatory reaction involving blood-derived macrophages and central nervous system (CNS)-resident microglia. ...

Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and accurately reconnect them with an appropriate target. Here a procedure is described to rapidly initiate, elongate, and precisely connect new functional neuronal circuits over long distances. The extension rates achieved reach over 1.2 mm/h, 30-60 times faster than the in vivo rates of the fastest growing axons from the peripheral nervous system (0.02 to 0.04 mm/h)28 and 10 times faster than previously reported for the same neuronal type at an earlier stage of development4. First, isolated populations of rat hippocampal neurons are grown for 2-3 weeks in microfluidic devices to precisely position the cells, enabling easy micromanipulation and experimental reproducibility. Next, beads coated with poly-D-lysine (PDL) are placed on neurites to form adhesive contacts and pipette micromanipulation is used to move the resulting bead-neurite complex. As the bead is moved, it pulls out a new neurite that can be extended over hundreds of micrometers and functionally connected to a target cell in less than 1 h. This process enables experimental reproducibility and ease of manipulation while bypassing slower chemical strategies to induce neurite growth. Preliminary measurements presented here demonstrate a neuronal growth rate far exceeding physiological ones. Combining these innovations allows for the precise establishment of neuronal networks in culture with an unprecedented degree of control. It is a novel method that opens the door to a plethora of information and insights into signal transmission and communication within the neuronal network as well as being a playground in which to explore the limits of neuronal growth. The potential applications and experiments are widespread with direct implications for therapies that aim to reconnect neuronal circuits after trauma or in neurodegenerative diseases.

This procedure describes how to rapidly initiate, extend and connect neurites organized in microfluidic chambers using poly-D-lysine-coated beads fixed to micropipettes that guide neurite elongation.

重建神经元电路:快速神经延伸和功能性神经元连接的新方法

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and ...

A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration

The degeneration of neurons occurs during normal development and in response to injury, stress, and disease. The cellular hallmarks of neuronal degeneration are remarkably similar in humans and invertebrates as are the molecular mechanisms that drive these processes. The fruit fly, Drosophila melanogaster, provides a powerful yet simple genetic model organism to study the cellular complexities of neurodegenerative diseases. In fact, approximately 70% of disease-associated human genes have a Drosophila homolog and a plethora of tools and assays have been described using flies to study human neurodegenerative diseases. More specifically the neuromuscular junction (NMJ) in Drosophila has proven to be an effective system to study neuromuscular diseases because of the ability to analyze the structural connections between the neuron and the muscle. Here, we report on an in vivo motor neuron injury assay in Drosophila, which reproducibly induces neurodegeneration at the NMJ by 24 h. Using this methodology, we have described a temporal sequence of cellular events resulting in motor neuron degeneration. The injury method has diverse applications and has also been utilized to identify specific genes required for neurodegeneration and to dissect transcriptional responses to neuronal injury.

A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration

Here we describe a simple and widely accessible method to injure segmental nerves in Drosophila larvae to visualize and quantify neurodegeneration of motor neurons at the neuromuscular junction (NMJ) of third instar larvae.

一个简单的神经元机械损伤方法学研究<em&gt;果蝇</em&gt;运动神经元变性

The degeneration of neurons occurs during normal development and in response to injury, stress, and disease. The cellular hallmarks of neuronal ...

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various intrinsic and extrinsic inhibitory factors involved in the restriction of axon regeneration have been identified. However, simply removing these inhibitory cues is insufficient for successful regeneration, indicating the existence of additional regulatory machinery. Drosophila melanogaster, the fruit fly, shares evolutionarily conserved genes and signaling pathways with vertebrates, including humans. Combining the powerful genetic toolbox of flies with two-photon laser axotomy/dendriotomy, we describe here the Drosophila sensory neuron &#8211; dendritic arborization (da) neuron injury model as a platform for systematically screening for novel regeneration regulators. Briefly, this paradigm includes a) the preparation of larvae, b) lesion induction to dendrite(s) or axon(s) using a two-photon laser, c) live confocal imaging post-injury and d) data analysis. Our model enables highly reproducible injury of single labeled neurons, axons, and dendrites of well-defined neuronal subtypes, in both the peripheral and central nervous system.

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

Here, we present a protocol using the Drosophila sensory neuron - dendritic arborization (da) neuron injury model, which combines in vivo live imaging, two-photon laser axotomy/dendriotomy, and the powerful fly genetic toolbox, as a platform for screening potential promoters and inhibitors of neuroregeneration.

一种研究外周和中枢神经系统 Neuroregeneration 的果蝇体内损伤模型

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various ...

医学

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

In neurons, microtubule orientation has been a key assessor to identify axons that have plus-end out microtubules and dendrites that generally have mixed orientation. Here we describe methods to label, image, and analyze the microtubule dynamics and growth during the development and regeneration of touch neurons in C. elegans. Using genetically encoded fluorescent reporters of microtubule tips, we imaged the axonal microtubules. The local changes in microtubule behavior that initiates axon regeneration following axotomy can be quantified using this protocol. This assay is adaptable to other neurons and genetic backgrounds to investigate the regulation of microtubule dynamics in various cellular processes.

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

A protocol for imaging the dynamic microtubules in vivo using fluorescently labeled End binding protein has been presented. We described the methods to label, image, and analyze the dynamic microtubules in the Posterior lateral microtubule (PLM) neuron of C. elegans.

在体内卡诺哈布德炎电子神经元微管动力学和方向评估

In neurons, microtubule orientation has been a key assessor to identify axons that have plus-end out microtubules and dendrites that generally have mixed ...

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal transport have devastating consequences for individual neurons and their networks, and contribute to a plethora of neurological disorders. As many of these conditions involve both cell autonomous and non-autonomous mechanisms, and often display a spectrum of pathology across neuronal subtypes, methods to accurately identify and analyze neuronal subsets are imperative.
This paper details protocols to assess in vivo axonal transport of signaling endosomes and mitochondria in sciatic nerves of anesthetized mice. Stepwise instructions are provided to 1) distinguish motor from sensory neurons in vivo, in situ, and ex vivo by using mice that selectively express fluorescent proteins within cholinergic motor neurons; and 2) separately or concurrently assess in vivo axonal transport of signaling endosomes and mitochondria. These complementary intravital approaches facilitate the simultaneous imaging of different cargoes in distinct peripheral nerve axons to quantitatively monitor axonal transport in health and disease.

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Using transgenic fluorescent mice, detailed protocols are described to assess in vivo axonal transport of signaling endosomes and mitochondria within motor and sensory axons of the intact sciatic nerve in live animals.

扩展轴突转运体内成像工具包

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal ...

Inducing Axonal Varicosities with a Micromechanical Stimulus on Neurons

Source: Servello, D., et al. <a href="https://app.jove.com/v/57202">A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons.</a> J. Vis. Exp. (2018).
This video demonstrates the induction of axonal varicosities by applying micromechanical stimulus on neurons. The system comprises a micropipette attached to a syringe containing buffer via tubing. Using a micromanipulator, the micropipette is positioned above the neurons. Fluid pressure is then applied to induce varicosities in axons.

中枢神经元轴突静脉曲张形成和恢复的 Microbiomechanical 系统研究

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机械操纵控制神经元轴突的发展

小脑轴突的激光Nanosurgery

一个

轴突的相互作用与小胶质细胞和巨噬细胞在小鼠背柱破碎损伤活体成像

重建神经元电路:快速神经延伸和功能性神经元连接的新方法

一个简单的神经元机械损伤方法学研究

一种研究外周和中枢神经系统 Neuroregeneration 的果蝇体内损伤模型

在体内卡诺哈布德炎电子神经元微管动力学和方向评估

扩展轴突转运体内成像工具包

通过对神经元的微机械刺激诱导轴突静脉曲张