Name: production and isolation of axons from sensory neurons for biochemical analysis using porous filters
Uploaded: 2014-07-08T23:45:00.000+00:00
Duration: 12 min
Description: Watch this Scientific Journal Video about Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters at JoVE.com

This project was supported by grant MOP62827 from the Canadian Institutes of Health Research.

注:以下步骤是按照批准的动物护理的加拿大议会的指引。所有的努力都是为了尽量减少在手术过程中的痛苦和动物的痛苦。 1，涂料的过滤器注意:当培养过滤器滤芯被放置在多孔板的孔中，两个隔室被定义:顶部隔室插入件上面的区域和底部隔室是插入图1A.ab低于1。外植体接种在顶部室，​​其中它们附着到过滤器的顶侧;许多轴突生长穿过过滤器并延伸于过滤器的底侧，底室图1A.c内。标准工作体积（ 即用于将涂布液，洗涤液，培养基，等等）是2毫升的底部隔室和1ml的顶部隔室和将要在协议中被称为";标准卷"。加通过将移液管的尖端在插入和阱的侧面之间的间隙开始的底部隔室的解决方案。另外，倾斜插入到一侧，而分配的解决方案，以防止气泡被截留在过滤器的底部。 <ol><li>开始电镀的前一天涂覆的过滤器。在无菌组织培养罩，放置24毫米过滤器滤芯在6孔接收板。孵育刀片与聚-D-赖氨酸的水溶液（1毫克/毫升）1小时，在室温下使用标准卷:2毫升在底室和1ml的顶部。 </li><li>吸聚-D-赖氨酸和用清水冲洗一次。吸出的水，盖上盖子，离开板在引擎盖的O / N。干在液体吸出，一定要去除的液体残余的成为直属过滤器捕获。 </li><li>第二天早上，层粘连蛋白稀释在水中（10微克/毫升），混合和温暖在37°C。孵育刀片，层粘连蛋白1个小时的CEL升培养箱中37℃下，使用标准卷。 </li><li>吸液层粘连蛋白和胶原蛋白加水（0.1毫克/毫升）并保温1小时，在室温。在这种情况下使用2毫升的底部隔室和2ml的顶室中。 </li><li>吸胶原蛋白并用无菌水冲洗一次。该过滤器现在已经准备好接受文化媒体和背根神经节外植体，如步骤3中所述。 </li></ol> 2，解剖胚胎背根神经节（DRG）植<ol><li>由IP注射圣阿韦坦（250毫克/千克）的麻醉为期13天的怀孕雌性小鼠。等待10-15分钟，并检查是否缺少角膜和踏板撤出反射。一旦反射消失，进行颈椎脱位。 </li><li>消毒所有手术器械和女性腹部用70％乙醇。允许工具晾干。保持腹部的皮肤和切断通过皮肤和肌肉层，以暴露腹腔。 </li><li>从宫颈和卵巢分离切除子宫。在免缝勒罩，从用剪刀子宫取出胚胎和收集它们在培养皿中充满了冰冷L15介质。置于冰上。 注:请参阅有关进一步详情，有关如何获得13天的妊娠期17,18的胚胎先前的技 ​​术报告。 </li><li>在显微镜下，躺在一边的胚胎，并使用小弹簧剪刀去掉头部。使沿侧面切口丢弃胸部，腹部，四肢和尾巴。保持背部含脊椎，躺在腹侧，并删除所有内脏器官。 </li><li>用小弹簧剪刀，从沿中线其腹侧切开脊柱暴露了整个脊髓。握住胴体下来与一对镊子（＃3），并使用第二对通过其最喙方面保持脊髓。 </li><li>慢慢地，轻轻地拉扯电源线远离脊椎腔和夹断采用超薄钳（＃5）从脊髓病种付费。集团按病种付费，以收集有关在盘子的底部。 </li><li>通过用200微升的枪头，已被切割，以扩大其开放吸气收集分组病种付费。放置的DRG中的管和直到DRG收集完成在冰上离开。 注:白色/透明秘诀是首选，因为病种付费不遵守这个技巧的墙壁（相对于标准黄芽）。有媒体法拉盛提示使用前进一步防止病种付费粘到墙壁。 </li></ol> 3，播种和背根神经节外植体的维护上过滤器<ol><li>在37℃下加完整的DRG媒体使用标准量的涂层刀片。与NGF的15纳克/毫升补充底部隔间介质，同时保持顶部隔室NGF-免费。 注:虽然NGF有效扩散穿过过滤器和小时内其浓度达到平衡，我们已经观察到应用NGF在车厢底部直接播种前显著增加在B轴突产量过滤器的ottom表面。 </li><li>从管使用1,000微升移液器与修整，以扩大其开放小费转移病种付费的过滤器。 </li><li>采集病种付费之前绘制介质插入针尖2到3倍。这防止它们粘到尖端的内侧。相同的尖端可以被重新用于所有的孔中。使用未着色的技巧（与标准蓝色提示）降低DRG粘在针尖表面。 </li><li>转移病种付费，设置吸管在800微升。自吸过滤器的顶部约200微升的媒体，然后去含DRG管和吸管小音量上下 - 重悬已经沉入底部病种付费。吸出整个体积和吸管病种付费淹没尖端插入中间。 </li><li>一旦所有插件被加载，关闭板盖，摇动板10倍，每个涡流之间的引擎盖板凳上轻轻拍打它。这些运动帮助提高产量轴索DRG外植体下沉和连接到过滤器。这也有助于分散均匀植让不同外植体的轴突不重叠。 注:在前面的步骤中描述的外植体接种程序外植体附着的整体比率显著增加至基板（高达75％）。最显著，移液过程容易地防止从外植体浮在介质表面而不是沉到所述插入件的底部。 </li><li>对于生化分析，种子按病种付费的过滤器每一个胚胎（约30）。对于形态分析，可以使用一半或三分之一的病种付费，从一个胚胎（10-15）。收集的DRG成管之前必须作出这样的决定，从而使管的所有的DRG接种到一个单一的孔（不管是30，15或10的DRG）。 </li><li>保持文化在细胞培养箱中37℃，变化的媒体每2-3天。更换介质，第一抽吸车厢顶部，随后是底部隔室。加入新鲜的培养基中标准体积天开始与车厢底部。 注:为了防止细胞/神经轴突从干燥，不要在超过3刀片同时更换介质。在NGF的存在生长在过滤器按病种付费将延长长轴突，在培养2天达到了2毫米。 </li></ol> 4，营养因素放弃治疗神经生长因子撤出诱导轴突变性与轴突碎片首先出现大约12小时后停药（如果相衬和β-III-微管蛋白测定）。完整的碎片是由36小时实现的。多长时间的退化是留给继续进行，必须由最终用户根据实验设计选择。 <ol><li>为期2天的轴突延伸阶段后，从顶部和底部隔层缺乏NGF和含有抗神经生长因子抗体（1微克/毫升），以隔绝任何剩余（或内源性）神经生长因子改变媒体介质。 </li><li>返回板，细胞培养箱（37℃），以允许NGF戒断诱发的变性进行对时间的期望的量。 </li><li>继续蛋白提取（步骤6）审议通过免疫印迹或中频（第7步）直接检查过滤器。 </li></ol> 5，轴突横断来诱导沃勒变性此程序离开轴突上从它们的细胞体分离的过滤器的底侧，但在其他完好。此后，这些轴突迅速启动Wallerian变性。轴突碎片横断后首次出现了3个小时（如果相衬或β-III-微管蛋白的证明）; 9小时，轴突是完全分散，从过滤器被分离。多长时间的退化是留给继续进行，必须由最终用户根据实验设计选择。 <ol><li>刮除过滤器的顶侧，含有生长的DRG外植体，用细胞刮刀破碎。在X轴，Y轴和圆形运动着的刮板确保完全去除顶部材料方向。 </li><li>从含有已被刮离过滤器并更换为1ml含有NGF（10纳克/毫升）预热，完整的DRG媒体的浮动外植体的顶部隔室丢弃媒体。 </li><li>返回板对细胞培养箱中（37℃），以允许Wallerian变性进行的时间由最终用户选择的量。 </li><li>继续蛋白提取（步骤6）审议通过免疫印迹或中频（第7步）直接检查过滤器。 </li></ol> 6，轴突蛋白样品的提取<ol><li>填收集管（1.5毫升）与75微升Laemmli样品缓冲液中。保持管在冰上，而从整个一套过滤器收获轴突。 </li><li>用在PBS中的DRG外植体洗涤过滤器和刮除器的顶侧。从板中取出刀片，清洁过滤器的顶侧用棉尖涂药和从顶侧和底侧吸任何额外的PBS。 </li><li>将插入上攻下来的长凳上，用锋利的手术刀切滤出的插入。握住过滤器镊子，自下而上。做一个切口从周围到过滤器的使用剪刀的中心。 </li><li>在收集管上放置过滤器，并用血管钳，按中央过滤器的下管。虽然这样做，一个漏斗应形成。推漏斗形过滤器，以在管的底部，以便它浸没在Laemmli样品缓冲液中。 </li><li>完整蛋白质提取煮沸管4分钟。使用镊子将其置于倒置​​在管的顶部和执行简短的自旋（2000×g离心15秒）除去的过滤器。丢弃干燥过滤器。 </li><li>解决10微升的轴突蛋白制备的SDS-PAGE电泳后免疫印迹检测感兴趣的蛋白质。 注意:作为一个参考，从生长2天，并溶解在100μlRIPA 20外植体的轴突制备裂解缓冲液将4-6的蛋白质浓度微克/微升。 </li></ol>轴突7。对过滤器免疫荧光检查以下步骤概括的一般操作来执行中频染色的过滤器滤芯，β-III-微管蛋白的检测例证。固色剂，孵育时间，浓度及其他详情，应为其他抗原 - 抗体对进行优化。 <ol><li>利用以前使用的6孔板中，彻底清洗，对刀片的下面的洗涤和孵育。 </li><li>在PBS中简要地洗刀片。解决他们在PBS新鲜配制的4％多聚甲醛在室温在摇床上10分钟。 </li><li>固定后，刮去和清洁刀片的顶侧，以收集只生长在过滤器的底表面上的轴突。继续孵育标准中频。 </li><li>在封闭溶液（TBS，0.05％吐温20,5％脱脂奶粉），使用标准的卷，1小时，在室温下轻轻摇动孵育插入。 </li><li>转让插入到稀释于封闭缓冲液（抗β-III-微管蛋白，1:5000），使用标准的卷初级抗体。孵育的O / N（约18小时），在4℃下轻轻摇动。 </li><li>在PBS中（2×10在RT（分钟））洗两次第一抗体。 </li><li>转让插入到稀释于封闭缓冲液（山羊抗小鼠，1:2,000），使用标准的卷荧光标记的二抗。孵育涵盖了从光2小时在室温轻轻摇动。 </li><li>在PBS（3×10在RT（分钟））洗二次抗体3倍。 </li><li>如果完成后，需要插入出去，倒掉PBS从最后一次洗涤和清洁过滤器的上侧用棉尖涂药。从顶侧和底侧抽吸的液体。 </li><li>地方插入板凳上颠倒，切滤出的插入用锋利的手术刀。 </li><li>握住过滤器镊子，下行起来，并将其放置在载玻片。覆盖盖玻片和荧光兼容谋nting媒体。让干扁在寒冷的房间，涵盖了从光。检查并用荧光显微镜拍摄的图像。 注意:当储存在4℃下并避光下，这些制剂可以保持周与荧光的损失最小。 </li><li>可选:另外，如果能在已经从插入取出过滤器进行。要做到这一点，凑和固定后直接清洗过滤器的顶部。 <ol><li>采取过滤器出来的刀片与一个6孔板的底部锋利的手术刀和地方过滤器的轴突式。 </li><li>此后，在7.4-7.8和7.11所示为中频进行孵化。这种替代的优点是，它可以节省试剂，因为，例如，孵化卷可以下降到500微升（相比于使用时，过滤器被直接染色的插入，如图7.1-7.11 3毫升）中。 </li></ol></li></ol>

<ol>
	<li>Conde, C., Caceres, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+assembly,+organization+and+dynamics+in+axons+and+dendrites.">Microtubule assembly, organization and dynamics in axons and dendrites.</a> Nat. Rev. Neurosci. 10, 319-332 (2009).</li><li>Rasband, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+axon+initial+segment+and+the+maintenance+of+neuronal+polarity.">The axon initial segment and the maintenance of neuronal polarity.</a> Nat. Rev. Neurosci. 11, 552-562 (2010).</li><li>Lingor, P., Koch, J. C., Tonges, L., Bahr, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+degeneration+as+a+therapeutic+target+in+the+CNS.">Axonal degeneration as a therapeutic target in the CNS.</a> Cell Tissue Res. 349, 289-311 (2012).</li><li>Torre, E. R., Steward, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demonstration+of+local+protein+synthesis+within+dendrites+using+a+new+cell+culture+system+that+permits+the+isolation+of+living+axons+and+dendrites+from+their+cell+bodies.">Demonstration of local protein synthesis within dendrites using a new cell culture system that permits the isolation of living axons and dendrites from their cell bodies.</a> J. Neurosci. 12, 762-772 (1992).</li><li>Zheng, J. Q., 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=A+functional+role+for+intra-axonal+protein+synthesis+during+axonal+regeneration+from+adult+sensory+neurons.">A functional role for intra-axonal protein synthesis during axonal regeneration from adult sensory neurons.</a> J. Neurosci. 21, 9291-9303 (2001).</li><li>Willis, D., 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=Differential+transport+and+local+translation+of+cytoskeletal,+injury-response,+and+neurodegeneration+protein+mRNAs+in+axons.">Differential transport and local translation of cytoskeletal, injury-response, and neurodegeneration protein mRNAs in axons.</a> J. Neurosci. 25, 778-791 (2005).</li><li>Poon, M. M., Choi, S. H., Jamieson, C. A., Geschwind, D. H., Martin, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+process-localized+mRNAs+from+cultured+rodent+hippocampal+neurons.">Identification of process-localized mRNAs from cultured rodent hippocampal neurons.</a> J. Neurosci. 26, 13390-13399 (2006).</li><li>Schoenmann, Z., 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=Axonal+degeneration+is+regulated+by+the+apoptotic+machinery+or+a+NAD+-sensitive+pathway+in+insects+and+mammals.">Axonal degeneration is regulated by the apoptotic machinery or a NAD+-sensitive pathway in insects and mammals.</a> J. Neurosci. 30, 6375-6386 (2010).</li><li>Unsain, N., Higgins, J. M., Parker, K. N., Johnstone, A. D., Barker, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XIAP+regulates+caspase+activity+in+degenerating+axons.">XIAP regulates caspase activity in degenerating axons.</a> Cell Rep. 4, 751-763 (2013).</li><li>Ure, D. R., Campenot, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrograde+transport+and+steady-state+distribution+of+125I-nerve+growth+factor+in+rat+sympathetic+neurons+in+compartmented+cultures.">Retrograde transport and steady-state distribution of 125I-nerve growth factor in rat sympathetic neurons in compartmented cultures.</a> J. Neurosci. 17, 1282-1290 (1997).</li><li>Taylor, A. M., 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=Axonal+mRNA+in+uninjured+and+regenerating+cortical+mammalian+axons.">Axonal mRNA in uninjured and regenerating cortical mammalian axons.</a> J. Neurosci. 29, 4697-4707 (2009).</li><li>Minis, A., 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=Subcellular+transcriptomics-Dissection+of+the+mRNA+composition+in+the+axonal+compartment+of+sensory+neurons.">Subcellular transcriptomics-Dissection of the mRNA composition in the axonal compartment of sensory neurons.</a> Dev. Neurobiol. , (2013).</li><li>Niekerk, E. A., 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=Sumoylation+in+axons+triggers+retrograde+transport+of+the+RNA-binding+protein+La.">Sumoylation in axons triggers retrograde transport of the RNA-binding protein La.</a> Proc. Natl. Acad. Sci. U. S. A. 104, 12913-12918 (2007).</li><li>Huang, E. J., Reichardt, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophins:+roles+in+neuronal+development+and+function.">Neurotrophins: roles in neuronal development and function.</a> Annu. Rev. Neurosci. 24, 677-736 (2001).</li><li>Vargas, M. E., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+is+Wallerian+degeneration+in+the+CNS+so+slow.">Why is Wallerian degeneration in the CNS so slow.</a> Annu. Rev. Neurosci. 30, 153-179 (2007).</li><li>Waller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experiments+on+the+Section+of+the+Glossopharyngeal+and+Hypoglossal+Nerves+of+the+Frog,+and+Observations+of+the+Alterations+Produced+Thereby+in+the+Structure+of+Their+Primitive+Fibres.">Experiments on the Section of the Glossopharyngeal and Hypoglossal Nerves of the Frog, and Observations of the Alterations Produced Thereby in the Structure of Their Primitive Fibres.</a> Philosophical Transactions of the Royal Society of London. 140, 423-429 .</li><li>Albuquerque, C., Joseph, D. J., Choudhury, P., MacDermott, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection,+Plating,+and+Maintenance+of+Dorsal+Root+Ganglion+Neurons+for+Monoculture+and+for+Coculture+with+Dorsal+Horn+Neurons.">Dissection, Plating, and Maintenance of Dorsal Root Ganglion Neurons for Monoculture and for Coculture with Dorsal Horn Neurons.</a> Cold Spring Harbor Protocols. , (2009).</li><li>Leach, M. 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=The+culture+of+primary+motor+and+sensory+neurons+in+defined+media+on+electrospun+poly-L-lactide+nanofiber+scaffolds.">The culture of primary motor and sensory neurons in defined media on electrospun poly-L-lactide nanofiber scaffolds.</a> J Vis Exp. (48), (2011).</li><li>Sasaki, Y., Araki, T., Milbrandt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulation+of+nicotinamide+adenine+dinucleotide+biosynthetic+pathways+delays+axonal+degeneration+after+axotomy.">Stimulation of nicotinamide adenine dinucleotide biosynthetic pathways delays axonal degeneration after axotomy.</a> J. Neurosci. 26, 8484-8491 (2006).</li></ol>

The authors have nothing to disclose.

重要参数适应这种技术时要考虑到包括神经元群，将待研究的衬底，所述过滤器的孔径大小和表面积。所有这些参数会影响所获得的神经突制剂的特异性，质量和数量，并且必须由最终用户仔细考虑。在此协议中提出的例子，使用DRG神经元具有延伸仅轴突，从而给出纯的（特定的）轴突制剂的优点。 胚胎病种付费的轴突生长是非常快的比较，从中枢神经系统的神经元，使轴突样品后，只有2天的文化有所准备。为1微米的孔尺寸已经证明足够小以防止细胞体从迁移进入底部表面，同时允许轴突生长的有效通道。相对于较小的尺寸可供选择（ 即 0.4微米）它并不会给选择性薄轴突。对背根神经节外植体的研究中，24毫米滤芯（用于在6孔板上使用）产生足够数量的轴突蛋白样品的大多数技术，包括免疫沉淀，这往往需要大量的蛋白质输入的。但是，较小的尺寸（ 即过滤器滤芯12孔板）是适合于某些应用，特别是如果使用分离的细胞和/或免疫细胞化学实验。 虽然滤芯有效隔离从胞体突起的检查，一定要注意这两个隔间共享相同的培养基，限制了实验操作，以全细胞治疗是很重要的。其他的隔离方法，包括条块Campenot室或微流体室，以便区别对待不同的隔间。实验者必须确定这些电池舱隔离技术是对自己具体的实验设计最合适。 <p c姑娘="jove_content"&gt;替代方法来获得纯轴突样品包括条块分割的文化系统，如Campenot室或微流体装置或手动删除的外植体中心（含神经元胞体）。在条块分割的方法只提供了少量的轴突材料。它们的使用需要显著培训以及专门的设备，他们是费时。另一方面，除去外植体中心以获得富集的轴突样品是不现实的，需要专门知识和被限制在可以生长为外植体的神经元群体。与此相反，这里介绍的方法产生丰富而纯净的轴突制剂，速度快，并不需要广泛的培训。最重要的，它也可以与分离的神经元进行。 当然，过滤器插入件的隔离能力可能是有利的超越轴突变性。例如，再生的生长锥的生物化学可以很容易地研究了刮过滤器的底部与成年DRG外植体。在几个小时内，视神经切断后DRG神经元会重新生长锥中，如在本协议中所述，然后可以分离再生生长锥蛋白的纯化过滤器的底部。此外，通过该方法得到的轴索的样品可以通过生化方法比蛋白质印迹等进行分析。例如，轴突可以在RIPA或CHAPS裂解缓冲液进行溶解，得到的蛋白样品，可以进一步进行免疫沉淀或下拉实验9。或者，轴突的样品可以用于RNA提取处理，如先前所描述5。 提出的培养系统的优点不限于感觉神经元。例如，皮质神经元将延长树突和轴突，将形成突触上的过滤器7的底侧。因此，纯粹的轴突样品（富含突触）可以ISOLAT教育署从细胞体，以了解发生只在突触进程的详细生化分析。使用这种技术的简单，没有突出的关键步骤。然而，轴突长度和形态，是过滤器的涂层的质量非常敏感。因此，必须谨慎使用新鲜和均匀的涂层解决方案的时间指示值，以获得稳定一致的轴突样品。广泛使用这种技术将有助于推动我们的轴突的生理知识，在正常情况下和疾病。

神经元的轴突舱显示了很大程度的躯体树突状隔间功能独立的。在投射神经元，轴突包含了大部分神经蛋白1,2。轴突的生理和病理生理的研究已经获得了在神经科学界的势头，因为最近的研究表明，早期轴突功能障碍似乎是神经退行性疾病和神经精神障碍3的 ​​一个共同特征。这很可能是一个更好地了解正常和病理条件下轴突独家机制将揭示早期事件驱动功能障碍灯。 本协议描述了如何使用培养插入轴承的多孔膜（过滤器），以获得大量的纯粹性轴索材料，适用于生化和免疫组化分析。使用过滤器的插入，研究轴突生物学最早是由管家和实施同事4，并进一步向它的当前配置开发的特维斯和他的同事5。该技术现在处于当前使用通过研究轴突和树突生物学的不同方面，在每种情况下轻微的修改，以适应研究神经细胞的群体，所执行的处理和生物化学分析的类型，使用6-9。本协议不打算以适应这样的各种应用程序的所有备选方案，而是提供了一个简单的方法，是适用于大多数应用。特别是，这里所描述的协议使用三重涂层，最大限度地提高产量轴索用于生化分析和是研究文献中描述的轴突变性，其它涂层最合适的生产小于缩回和退化速度从营养因子剥夺9时轴突。 在此过程中，神经元细胞体保持隔离之上的过滤器的wh异亮氨酸的轴突穿过的孔，并沿其底表面上生长。纯轴突（免费神经胶质细胞和神经元细胞体污染物）在过滤器的底部表面生长，可用于采集生化分析或可固定在原位 ，并通过免疫细胞化学技术检测9。该过程依赖于使用胚胎感觉神经元从背根神经节（DRG）。胚胎背根节被广泛地用于研究轴突生物学，因为来自这些细胞的神经突起进行体外快速和强劲增长时，在神经生长因子（NGF）保持，并且当失去这个因素，因为它们迅速地进行变性。此外，DRG神经元缺乏树突，以便所有通过这种方法收集的突起是纯粹的轴突。 过滤器滤芯代表相比，用于研究轴突其他方法有很大的优势。研究依赖于使用显微镜来区分发生在从那些在胞体的过程xons提供有限的生化信息。作为另一个例子，条块培养系统（ 例如 Campenot室10或微流体装置11）是用于成像的方法和差分处理细胞区室的有用的，但仅提供少量的轴突材料制成的，排除了使用这些技术对于需要生化分析相对大量的样品。此外，它们的使用需要显著培训，以及专业的设备，他们是费时。常用于植隔离轴突另一种方法是轴突样品采集之前手动删除植中心（含神经元胞体）。虽然这可以产生大量的轴突富集制剂，在该制剂的轴突可以笼罩在神经胶质细胞和来自6个孔中（如一个最小的实验的一个例子）快速除去20外植体的体连续是费时和可行性的限制。 与此相反，这里提出的方法产生，然后可以通过几乎任何生化技术，通常被用于分析全细胞溶胞产物，如蛋白质印迹，免疫沉淀6，northern印迹5质谱6，RNA纯化进行分析丰富和纯轴索制剂7,12,13，等等。此外，该协议可以用于免疫荧光（IF）的技术，因为它可以固定轴突在过滤器的底侧上生长由中频进一步加以研究。这种方法大大方便了轴突的具体过程的分析，因为没有细胞体中的最后准备。这是一个显著的优势，因为从细胞体高免疫荧光信号往往破坏检查和较弱的信号由薄的结构，如神经轴突起源的分析。 两个应用程序的培养方法，研究轴突变性ARE中所述;发育修剪和损伤引起的退行性变的模型的模型（通常被称为Wallerian变性）。发育修剪的造型是基于一个事实，即在体 ​​内的感觉神经元竞争限制目标源性神经生长因子和金额那些未能得到足够的神经营养支持简并14。这种现象可以通过从培养基中，其中，因为发生在体内 ，启动轴突变性，然后进行细胞体破坏吸NGF来模拟在胚胎背根神经节培养物。建模Wallerian变性，过滤器与细胞体的顶侧被刮掉。这已经增长到过滤器成为从细胞体物理上分离并随后经受谁最先报道的现象在1850年16称为Wallerian变性15的快速和刻板退化过程，A.沃勒命名后的底侧的轴突。

<table><tbody><tr><td>CD-1 mouse, E13 timed pregnancy</td><td>Charles River</td><td></td><td>In house timed pregnancies can be done by mating mice and checking for vaginal plugs (Day E0)</td></tr><tr><td>Falcon cell culture inserts</td><td>Corning</td><td>353102</td><td>1 &amp;micro;m pore size, fits 6-well receiving plate</td></tr><tr><td>Falcon cell culture companion plates</td><td>Corning</td><td>353502</td><td>Receiving plates for inserts</td></tr><tr><td>Poly-D-lysine</td><td>Sigma-Aldrich</td><td>P6407</td><td>Used at 1 mg/ml in water. Can re-use once.</td></tr><tr><td>Laminin</td><td>Sigma-Aldrich</td><td>L2020</td><td>Used at 10 &amp;micro;g/ml in water. Take 100x stock from -80 &amp;deg;C and thaw O/N at 4 &amp;deg;C&amp;nbsp;</td></tr><tr><td>PureCol bovine collagen solution</td><td>Advanced Biomatrix</td><td>5005-B</td><td></td></tr><tr><td>Leibovitz&#039;s L-15 Medium</td><td>Invitrogen</td><td>11415-064</td><td>Can be replaced by DMEM</td></tr><tr><td>Neurobasal medium</td><td>Invitrogen</td><td>21103-049</td><td></td></tr><tr><td>B-27 serum-free supplement</td><td>Invitrogen</td><td>17504-044</td><td></td></tr><tr><td>5-Fluoro-2&amp;prime;-deoxyuridine (FDU)</td><td>Sigma-Aldrich</td><td>F0503</td><td></td></tr><tr><td>NGF (2.5S, beta subunit)</td><td>Cedarlane</td><td>CLMCNET-001</td><td></td></tr><tr><td>Anti-NGF antibody</td><td>Prepared in-house</td><td></td><td>As described in: Acheson, A. &lt;em&gt;et al.&lt;/em&gt; Detection of brain-derived neurotrophic factor-like activity in fibroblasts and Schwann cells: inhibition by antibodies to NGF. Neuron. &lt;strong&gt;7&lt;/strong&gt;, 265-75, (1991).</td></tr><tr><td>Alomone Labs</td><td>AN-240</td><td>Commercial option</td><td></td></tr><tr><td>Anti-tubulin antibody</td><td>Millipore</td><td>MAB5564</td><td></td></tr><tr><td>Alexa Fluor 488 goat anti-mouse antibody</td><td>Invitrogen</td><td>A-11029</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;DRG culture medium&lt;/strong&gt;</td></tr><tr><td colspan="4">Neurobasal Medium&lt;br/&gt; 2% B27 Neurobasal Supplement&lt;br/&gt; 2 mM l-Glutamine&lt;br/&gt; 1% Penicillin-streptomysin&lt;br/&gt; 10 &amp;micro;M FDU&lt;br/&gt; 15 ng/ml NGF (bottom compartment only)</td></tr><tr><td colspan="4">&lt;strong&gt;1x Laemmli sample buffer&lt;/strong&gt;</td></tr><tr><td colspan="4">2% SDS&lt;br/&gt; 50 mM DTT&lt;br/&gt; 60 mM Tris (pH 6.8)&lt;br/&gt; 5% Glycerol&lt;br/&gt; 0.01% (w/v) Bromophenol blue</td></tr></tbody></table>

production and isolation of axons from sensory neurons for biochemical analysis using porous filters

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is required to shape functional neuronal circuits. The protocol described here explains how to use cell culture inserts bearing a porous membrane (filter) to obtain large amounts of pure axonal preparations suitable for examination by conventional biochemical or immunocytochemical techniques. The functionality of these filter inserts will be demonstrated with models of developmental pruning and Wallerian degeneration, using explants of embryonic dorsal root ganglion. Axonal integrity and function is compromised in a wide variety of neurodegenerative pathologies. Indeed, it is now clear that axonal dysfunction appears much earlier in the course of the disease than neuronal soma loss in several neurodegenerative diseases, indicating that axonal-specific processes are primarily targeted in these disorders. By obtaining pure axonal samples for analysis by molecular and biochemical techniques, this technique has the potential to shed new light into mechanisms regulating the physiology and pathophysiology of axons. This in turn will have an impact in our understanding of the processes that drive degenerative diseases of the nervous system.

从E13.5小鼠背根神经节外植体长出了大量关于过滤器依次涂有聚-D-赖氨酸，层粘连蛋白和胶原蛋白（ 图1B，左），达到了2毫米内培养2天。该基板组合产生特别旺盛的增长;其他组合产生的轴突是相当短（ 图1B，右）。此外，在保持病种付费的过滤器比延长生长在塑料图1C病种付费更多和更长的轴突。 过滤器具有1微米的直径，在该协议中使用的，保留在过滤器上的上侧的细胞体，同时允许轴突生长通过毛孔和延伸在底表面上的孔隙。这显然是由过滤器染色的细胞核（DAPI）和β-III-微管蛋白图1D荧光显微图所示。在完整的过滤器，可以观察到细胞核，无论是从神经元和非神经元细胞，集中在从它的外植体和中心部分照射。然而，当过滤器的顶侧被刮除，细胞核消失，仅可以检测到轴突，在过滤器底侧上。 图1E示出了一个过滤器底部最初持续约17外植体生长的一个例子。在这个例子中，上侧的固定之前进行清洗，因此，免疫荧光信号表示在过滤器的底侧纯轴突。从轴突制剂（过滤器的下侧）的蛋白质提取是不含核蛋白图1F的，这表明该制剂是无细胞体。或者，用RNA提取操作时，能够确认轴索制备的，通过比较差分定位的mRNA的存在的全细胞裂解物和轴突制剂之间（ 即 γ-肌动蛋白）的纯度（如由他人5 </s达&gt;）。 感觉神经元的发育修剪可以模拟在过滤器中进行营养因子剥夺。后在NGF的存在轴突延长2-3天，介质被改变为1缺乏NGF和含有抗-NGF抗体，将螯合任何残留的NGF 图2A中 。在期望的时间点，过滤器处理用于生物化学或免疫细胞化学实验。 图2B显示过滤器前后剥夺染色β-III-微管蛋白底边的典型例子。经过12小时剥夺很少有轴突碎片，虽然有些轴突开始出现卷边。然而，广泛的轴索变性是剥夺36后实现的。 Wallerian变性引起的感觉神经元的轴突横断容易建模的刮过滤器的顶侧上生长的外植体的过滤器，使所述远端婆背后该增加在过滤器上图3A的底部侧的轴突rtion。 图3B示出的过滤器染色的β-III-微管蛋白的底侧的典型实例之前和在过滤器切断后。在切断后3小时，几乎没有碎片，虽然一些轴突开始出现卷边。 7.5小时，最轴突零散或完全消失。外植体与烟酰胺腺嘌呤二核苷酸（NAD +，5mM的，30分钟预处理）的预处理可防止Wallerian变性引起的过滤器，因为它在其它的体外和体内模型19。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51795/51795fig1highres.jpg" width="500" /> 图1。滤芯生产大量的纯轴突的准备。插入（一）A）架构在一个良好的插入，（b）及与背根神经节外植体（c）中B）的过滤器与聚-D-赖氨酸，层粘连蛋白和胶原蛋白的产生生长更多和更长的轴突相比，这些生长与单独的聚-D-赖氨酸或聚-D外植体的连续涂层的最终配置-赖氨酸和胶原蛋白。对图像进行平铺使用图像处理软件。C）在相同的基板涂覆条件重叠，低倍率的图片制作，维护过滤器上的DRG外植体显示出比生长在塑料。ð植相当多的旺盛轴突生长）通过刮制得的轴突准备该过滤器的顶侧是缺乏细胞核，起源自约17植和生长在24毫米滤波器的底侧轴突E）的实施例。多达30外植体可以很容易地生长在这些准备工作，产生足够的轴突材料共同的生化途径。裂解液F）的免疫印迹科尔<html> ected从过滤器顶部与底部表明，组蛋白H3，核标记，仅限于过滤器顶部。在不同的样品中总蛋白含量归一化。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51795/51795fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51795/51795fig2highres.jpg" width="500" /> 图2。轴索变性的研究引起营养因子撤出过滤器滤芯。 A）架 ​​构概述一个典型的NGF剥夺实验。背根神经节外植体长出大量的轴突中神经生长因子的存在下，与变质后NGF剥夺B）从保持在NGF剥夺或神经生长因子为12或36小时外植体的轴突制剂在不同放大倍率的代表性图像（5X和20X的目标）。"https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51795/51795fig2highres.jpg"目标="_blank"&gt;请点击这里查看该图的放大版本。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51795/51795fig3highres.jpg" width="500" /> 图3。华勒轴索变性的过滤器滤芯由轴突切断术诱导的研究。 A）架 ​​构概述一个典型的干切断实验。轴突切断是通过刮过滤器的顶侧，而使切断轴突（不含细胞体）的底侧B）从外植体完好性轴索制剂的代表性图像或之后切断后3或7.5小时在存在或不存在来实现5 mM的NAD +的。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51795/51795fig3highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters at JoVE.com

Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

This article describes a neuronal culture system that can be used to obtain large quantities of pure axonal samples for biochemical and immunocytochemical analyses. This technique will allow an improved understanding of normal axonal physiology and signaling pathways leading to neurodegeneration.

生产和轴突隔离从感觉神经元的生化分析使用多孔过滤器

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is ...

production-isolation-axons-from-sensory-neurons-for-biochemical

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

14.2K 次观看.  Montreal Neurological Institute, McGill University. This article describes a neuronal culture system that can be used to obtain large quantities of pure axonal samples for biochemical and immunocytochemical analyses. This technique will allow an improved understanding of normal axonal physiology and signaling pathways leading to neurodegeneration. 

视频: Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

Axoplasm Isolation from Rat Sciatic Nerve

Isolation of pure axonal cytoplasm (axoplasm) from peripheral nerve is crucial for biochemical studies of many biological processes. In this article, we demonstrate and describe a protocol for axoplasm isolation from adult rat sciatic nerve based on the following steps: (1) dissection of nerve fascicles and separation of connective tissue; (2) incubation of short segments of nerve fascicles in hypotonic medium to release myelin and lyse non-axonal structures; and (3) extraction of the remaining axon-enriched material. Proteomic and biochemical characterization of this preparation has confirmed a high degree of enrichment for axonal components.

We demonstrate a protocol for axoplasm isolation from adult rat sciatic nerve based on dissection of nerve fascicles and incubation in hypotonic medium to release myelin and lyse non-axonal structures, followed by extraction of the remaining axon-enriched material.

从大鼠坐骨神经轴浆隔离

Isolation of pure axonal cytoplasm (axoplasm) from peripheral nerve is crucial for biochemical studies of many biological processes. In this article, we ...

科研

神经科学

Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that break away from axon terminals when the cortical tissue is homogenized. The SNs retain pre- and postsynaptic characteristics, which makes them useful in the study of synaptic transmission. They retain the molecular machinery used in neuronal signaling and are capable of uptake, storage, and release of neurotransmitters.
The production and isolation of active SNs can be problematic using medias like Ficoll, which can be cytotoxic and require extended centrifugation due to high density, and filtration and centrifugation methods, which can result in low activity due to mechanical damage of the SNs. However, the use of discontinuous Percoll-sucrose density gradients to isolate SNs provides a rapid method to produce good yields of translationally active SNs. The Percoll-sucrose gradient method is quick and gentle as it employs isotonic conditions, has fewer and shorter centrifugation spins and avoids centrifugation steps that pellet SNs and cause mechanical damage.

A method to prepare translationally active, intact synaptoneurosomes (SNs) from mouse brain cortex is described. The method uses a discontinuous Percoll-sucrose density gradient allowing for the quick preparation of active SNs.

准备使用一个不连续Percoll蔗糖密度梯度的从小鼠皮质Synaptoneurosomes

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that ...

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

培养成年的背根神经节神经元轴突再生的遗传研究

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

隔离的感觉神经元中<em&gt;海兔丫</em谷氨酸能电流膜片钳记录

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

Identification of Specific Sensory Neuron Populations for Study of Expressed Ion Channels

Sensory neurons transmit signals from various parts of the body to the central nervous system. The soma for these neurons are located in the dorsal root ganglia that line the spinal column. Understanding the receptors and channels expressed by these sensory afferent neurons could lead to novel therapies for disease. The initial step is to identify the specific subset of sensory neurons of interest. Here we describe a method to identify afferent neurons innervating the muscles by retrograde labeling using a fluorescent dye DiI (1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindocarbocyanine perchlorate). Understanding the contribution of ion channels to excitation of muscle afferents could help to better control excessive excitability induced by certain disease states such as peripheral vascular disease or heart failure. We used two approaches to identify the voltage dependent ion channels expressed by these neurons, patch clamp electrophysiology and immunocytochemistry. While electrophysiology plus pharmacological blockers can identify functional ion channel types, we used immunocytochemistry to identify channels for which specific blockers were unavailable and to better understand the ion channel distribution pattern in the cell population. These techniques can be applied to other areas of the nervous system to study specific neuronal groups.

Afferent sensory neurons signal sensory information from the periphery to the central nervous system. Identifying specific afferent neurons will help in understanding their physiology. We describe a method of retrograde labeling to identify afferent neurons, and study the voltage-gated ion channels in these neurons using patch clamp electrophysiology and immunocytochemistry.

具体的感觉神经元的人群离子通道的表达研究鉴定

Sensory neurons transmit signals from various parts of the body to the central nervous system. The soma for these neurons are located in the dorsal root ...

A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. Without sensory innervation, daily oral activities would cause irreparable damage. Despite its importance, the roles of innervation in tooth development and maintenance have been largely overlooked. Several studies have demonstrated that DP cells secrete extracellular matrix proteins and paracrine signals to attract and guide TG axons into and throughout the tooth. However, few studies have provided detailed insight into the crosstalk between the DP mesenchyme and neuronal afferents. To address this gap in knowledge, researchers have begun to utilize co-cultures and a variety of techniques to investigate these interactions. Here, we demonstrate the multiple steps involved in co-culturing primary DP cells with TG neurons dispersed on an overlying transwell filter with large diameter pores to allow axonal growth through the pores. Primary DP cells with the gene of interest flanked by loxP sites were utilized to facilitate gene deletion using an Adenovirus-Cre-GFP recombinase system. Using TG neurons from the Thy1-YFP mouse allowed for precise afferent imaging, with expression well above background levels by confocal microscopy. The DP responses can be investigated via protein or RNA collection and analysis, or alternatively, through immunofluorescent staining of DP cells plated on removable glass coverslips. Media can be analyzed using techniques such as proteomic analyses, although this will require albumin depletion due to the presence of fetal bovine serum in the media. This protocol provides a simple method that can be manipulated to study the morphological, genetic, and cytoskeletal responses of TG neurons and DP cells in response to the controlled environment of a co-culture assay.

We describe the isolation, dispersion and plating of dental pulp (DP) primary cells with trigeminal (TG) neurons cultured atop overlying transwell filters. Cellular responses of DP cells can be analyzed with immunofluorescence or RNA/protein analysis. Immunofluorescence of neuronal markers with confocal microscopy permits the analysis of neurite outgrowth responses.

研究对牙髓寄生虫信号反应的神经质生长的共同培养方法

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. ...

发育生物学

Designing Porous Silicon Films as Carriers of Nerve Growth Factor

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the protein does not cross the blood-brain barrier, owing to its chemical properties, and thus requires long-term delivery to the brain to have a biological effect. This work describes fabrication of nanostructured PSi films as degradable carriers of NGF for sustained delivery of this sensitive protein. The PSi carriers are specifically tailored to obtain high loading efficacy and continuous release of NGF for a period of four weeks, while preserving its biological activity. The behavior of the NGF-PSi carriers as a NGF delivery system is investigated in vitro by examining their capability to induce neuronal differentiation and outgrowth of PC12 cells and dissociated DRG neurons. Cell viability in the presence of neat and NGF-loaded PSi carriers is evaluated. The bioactivity of NGF released from the PSi carriers is compared to the conventional treatment of repetitive free NGF administrations. PC12 cell differentiation is analyzed and characterized by the measurement of three different morphological parameters of differentiated cells; (i) the number of neurites extracting from the soma (ii) the total neurites' length and (iii) the number of branching points. PC12 cells treated with the NGF-PSi carriers demonstrate a profound differentiation throughout the release period. Furthermore, DRG neuronal cells cultured with the NGF-PSi carriers show an extensive neurite initiation, similar to neurons treated with repetitive free NGF administrations. The studied tunable carriers demonstrate the long-term implants for NGF release with a therapeutic potential for neurodegenerative diseases.

Here, we present a protocol to design and fabricate nanostructured porous silicon (PSi) films as degradable carriers for the nerve growth factor (NGF). Neuronal differentiation and outgrowth of PC12 cells and mice dorsal root ganglion (DRG) neurons are characterized upon treatment with the NGF-loaded PSi carriers.

多孔硅薄膜作为神经生长因子载体的设计

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the ...

生物工程

Purification of Fibroblasts and Schwann Cells from Sensory and Motor Nerves in Vitro

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and motor phenotypes involved in different patterns of neurotrophic factor gene expression and other biological processes, affecting nerve regeneration. The present study has established a protocol to obtain highly purified rat sensory and motor SCs and fibroblasts more rapidly. The ventral root (motor nerve) and the dorsal root (sensory nerve) of neonatal rats (7-days-old) were dissociated and the cells were cultured for 4-5 days, followed by isolation of sensory and motor fibroblasts and SCs by combining differential digestion and differential adherence methods sequentially. The results of immunocytochemistry and flow cytometry analyses showed that the purity of the sensory and motor SCs and fibroblasts were &#62;90%. This protocol can be used to obtain a large number of sensory and motor fibroblasts/SCs more rapidly, contributing to the exploration of sensory and motor nerve regeneration.

Here, we present a method to purify fibroblasts and Schwann cells from sensory and motor nerves in vitro.

从体外感觉和运动神经中纯化成纤维细胞和施万细胞

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and ...

Microfluidics-Assisted Selective Depolarization of Axonal Mitochondria

Mitochondria are the primary suppliers of ATP (adenosine triphosphate) in neurons. Mitochondrial dysfunction is a common phenotype in many neurodegenerative diseases. Given some axons' elaborate architecture and extreme length, it is not surprising that mitochondria in axons can experience different environments compared to their cell body counterparts. Interestingly, dysfunction of axonal mitochondria often precedes effects on the cell body. To model axonal mitochondrial dysfunction in vitro, microfluidic devices allow treatment of axonal mitochondria without affecting the somal mitochondria. The fluidic pressure gradient in these chambers prevents diffusion of molecules against the gradient, thus allowing for analysis of mitochondrial properties in response to local pharmacological challenges within axons. The current protocol describes the seeding of dissociated hippocampal neurons in microfluidic devices, staining with a membrane-potential sensitive dye, treatment with a mitochondrial toxin, and the subsequent microscopic analysis. This versatile method to study axonal biology can be applied to many pharmacological perturbations and imaging readouts, and is suitable for several neuronal subtypes.

The present protocol describes the seeding and staining of neuronal mitochondria in microfluidic chambers. The fluidic pressure gradient in these chambers allows for the selective treatment of mitochondria in axons to analyze their properties in response to pharmacological challenges without affecting the cell body compartment.

微流体辅助轴突线粒体选择性去极化

Mitochondria are the primary suppliers of ATP (adenosine triphosphate) in neurons. Mitochondrial dysfunction is a common phenotype in many ...

Isolation of Cells from Sensory Ganglia

Source: Kaelberer, M. M., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/53917">A Method to Target and Isolate Airway-innervating Sensory Neurons in Mice.</a> J. Vis. Exp. (2016).
This video demonstrates the isolation of cells from the sensory ganglia of mice. Ganglia are subjected to enzymatic digestion to dissolve the extracellular matrix. Single cells are then collected via filtering through a cell strainer followed by density gradient centrifugation for debris removal. Finally, washing and centrifugation are done to collect the isolated cells for future analysis.

从感觉神经节中分离细胞

All procedures involving sample collection have been performed in accordance with the institute&#8217;s IRB guidelines.
1. Sensory Ganglia Dissociation

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