财政支持由美国国立卫生研究院U01-NS094340（Cullen），T32-NS043126（Harris）和F31-NS090746（Katiyar）提供），Michael J.Fox基金会（治疗管道计划＃9998（Cullen））， Penn医学神经科学中心试点奖（Cullen），国家科学基金会（研究生研究奖学金DGE-1321851（Struzyna和Adewole）），退伍军人事务部（RR＆D优异评估＃B1097-I（Cullen）），美国协会的神经外科医生和神经外科医生大会（2015-2016 - 神经损伤与重症监护（Petrov）Codman研究金）和美国陆军医学研究和物资司令部（＃W81XWH-13-207004（Cullen）和W81XWH-15-1- 0466（Cullen））。

作者没有什么可以披露的。

CNS损伤和疾病通常导致构成脑连接的长途轴突途径的丧失或功能障碍，伴有或不伴有神经元变性。由于中枢神经系统有限的能力来促进神经发生和再生，所以复合化。尽管追求修复策略，如生长因子，细胞和生物材料传递作为个体或组合方法，这些技术不能同时解释神经元细胞的退化和轴突连接的损失14,22。技术上的这些差距限制了以受控和持续的方式修复，改变和探测神经网络的能力。因此，?...

中枢神经系统疾病和疾病（CNS）的常见特征，如创伤性脑损伤（TBI），脊髓损伤（SCI），中风，阿尔茨海默病和帕金森病，是轴突途径和神经元细胞的断开损失1，2，3，4，5，6 。例如，当缺血性中风未经治疗时，估计轴突以每分钟轴突7英里的速度丢失5 。在TBI的情况下，每年约有170万人在美国经历，轴突变性可能在创伤后几年可能继续发生，因为初始损伤导致长期的神经变性状态4 。加剧这些有害影响，中枢神经系统受到严重限制城市再生1，7，8，9。受伤后，出现抑制环境，其特征在于缺乏远距离靶点的指导性指导，阻止神经突生长的髓磷脂相关抑制剂的存在，以及由反应性星形胶质细胞形成胶质瘢痕8,10,11,12。胶质瘢痕可作为再生的生化和物理障碍，硫酸软骨素蛋白多糖等分子阻碍轴突长出8,11。此外，即使在成人CNS中发现了神经干细胞，新神经元的产生也是有限的，因为在嗅球中仅发现了神经发生的一致证据，海马脊髓区域，脑室周围区域和脊髓中央管道13,14 。这些障碍阻碍了损伤或疾病后遗失的神经元和白质结构的功能恢复，导致这些病症经常改变生命和延长的效果。 尽管成人中枢神经系统缺乏再生能力，但是已经证明，如果向宿主神经元15,16,17,18提供足够的环境线索，则轴突再生是可能的。研究人员尝试提供和操纵生长因子（ 如神经生长因子，表皮生长因子，神经胶质依赖性生长因子和神经营养因子-3）和其他指导分子，以刺激可塑性和轴突再生14 ，/ sup&gt; 18，19 。即使这些研究已经证实成年轴突能够响应生长因子，这些策略受到血脑屏障的低渗透性和促进再生所需的特定空间和时间梯度的限制14,18,19。其他方法依赖于CNS神经元中再生相关转录因子的过度活化。例如，Stat3转录因子的过度表达刺激视神经中的轴突再生20 。然而，生物分子递送和转录因子的过表达都不能代替丢失的神经元群体。基于细胞的策略主要集中在将神经干细胞（NSCs）移植到CNS中，利用其替代CNS神经元的能力，释放营养因子，并支持在损伤后发生的神经发生的尝试17 。尽管如此，仍然存在阻碍这种方法的紧迫挑战，包括移植的神经细胞存活，与主机整合的阻碍能力，并且在空间上保持受限制的区域6,14,17,21。此外，单独的细胞传递不能恢复受损或丢失的轴突途径的细胞结构。解决细胞和药物/化学品输送策略面临的问题的另一种方法是将这些方法与生物材料14,22,23的使用相结合。生物材料如水凝胶能够模拟细胞外基质（ECM）的生物化学和物理性质，有助于细胞递送d保留在受伤区域内，并传递生长因子和其他生物活性分子与受控释放22 。这些基于生物材料的策略的有吸引力的特征已经导致在支架移植到损伤区域24,25,26,27,28,29,30之后体内轴突再生的证据。然而，无细胞生物材料策略不能代替丢失的神经元群体;当用作神经元，胶质细胞或神经元前体细胞的递送载体时，生物材料不能重建长距离轴突网络。开发解决轴突途径退化和与CNS损伤和疾病相关的神经元损失的方法的挑战仍然是&lt;sup class ="xref"&gt; 31。 我们的研究小组以前报告了可植入的微组织工程神经网络（micro-TENNs）的发展，这是一种由活性支架组成的神经细胞体，其限于琼脂糖水凝胶-EGC微柱的一端或两端，其中对齐的轴突束延伸穿过该三维（3D）包装1,10,31,32的整个内部。这种技术和以前的方法之间的主要区别之一是微TENN 的细胞结构在体外完全产生，并在之后33,34,35,36,37,38 ，sup class ="xref"&gt; 39，40，41。 体外制备提供了广泛的空间和时间控制细胞表型和取向，机械/物理性质，生物化学提示和外源性因素，这有利于这些支架与植入后的主体整合41,42 。 Micro-TENN是解剖学上的启发，因为它们模拟脑神经解剖学，显示类似于桥接不同功能区域的轴突区域（ 图1A ） 1 。因此，这种策略可能能够在植入损伤区域后物理上替代丢失的白质束和神经元。这种技术也受到发育机制的启发，其中由径向胶质细胞和开创性轴突形成的"天然生物支架"作为细胞的寻路指南分别从室下区和轴突向外移动43 。这些机制在微TENN的对齐轴突区域中重现，这可以通过轴突介导的轴突生长呈现神经细胞迁移和轴突再生的生物通路（ 图1C ） 43 。此外，该策略利用微TENN神经元和原生电路之间的突触整合，形成可能有助于功能恢复的新继电器（ 图1B ）。突触形成的能力也可以允许这种方法根据网络反馈调节CNS并响应宿主组织的能力。例如，可以通过突触相互作用刺激生物支架中的光遗传活性神经元来调节宿主神经元（ 图1D ）。 此外，基于生物材料的管状结构微TENN的作用为细胞粘附，生长，神经突延伸和信号传导提供了足够的环境，而构建体的微型尺寸可能允许微创植入，并提供部分隔离的微环境，用于逐渐融入脑中。事实上，最近的出版物已经证明了微型TENN在植入大鼠脑后模拟神经通路的潜力。在立体定位显微注射之后，我们以前报道了微TENN神经元存活的证据，轴突结构的维持和轴突延伸到主体皮质至少1个月的体内 10,31。此外，用突触蛋白标记提供了与天然组织突触整合的组织学证据10,31。总的来说，微型TENN可能独特地适用于重建和调制损坏CNS通过替换丢失的神经元，与主机电路进行突触整合，恢复损失的轴突细胞结构，并且在某些情况下，为再生轴突提供适当的寻路提示。 <img alt="图1" src="/files/ftp_upload/55609/55609fig1.jpg" /> 图1:微组织工程神经网络（微型TENN）发展背后的原理和启示。 （ A ）Micro-TENN模拟大脑连接体（紫色）的细胞结构，其中功能上不同的区域以单向（红色，绿色）或双向（蓝色）方式通过长对准的轴突束连接。作为一个例子，微型TENN可以重建皮质丘脑和黑质纹状体途径或从内嗅皮质到海马的穿孔途径中的丢失的连接（改编自Struzyna 等 ，2015） 1 。 （ B ）单向图l和双向微型TENN（分别为红色和蓝色）与主机电路（紫色）进行突触式积分，以用作病变两端之间的功能继电器。 （ C ）单向微-TENN（绿色）的轴突区的示意图，作为用于轴突促进再生主轴突（紫色）朝向与微TENN相互作用的靶标的指导。 （ D ）使用光生物活性微TENNS作为神经调节剂的概念图，利用与兴奋性或抑制性神经元的突触整合（底部）。 <a href="http://ecsource.jove.com/files/ftp_upload/55609/55609fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 目前的手稿详细介绍了使用胚胎衍生的大脑皮质神经元制造微型TENN的方法。值得注意的是，微TENN可以用其他类型的神经细胞制造。例如充足的，成功的微型TENN发展的初步报告是背根神经节（DRG）神经元32 。可以通过将液体琼脂糖加入到定制的激光切割的圆柱形通道阵列或毛细管中（均包含对准的针灸针）来产生水凝胶微柱（ 图2A ）。针形成内腔并确定微柱的内径（ID），而激光切割装置中的毛细管ID和圆柱体的直径决定了构造体的外径（OD）。 OD和ID可以根据所需的应用选择，分别选择器件/毛细管和针灸针的不同直径。微柱的长度也可以变化;到目前为止，我们已经报道了长达10毫米的微型TENN的建设10 ，并且正在积极追求更长的长度。琼脂糖凝胶和针灸后eedles被去除，通常由I型胶原和层粘连蛋白组成的ECM溶液被添加到构建体的内腔（ 图2C ）。 ECM核心提供支架，以支持神经细胞粘附和轴突生长。最初，使用解离的细胞悬浮液10,31,32，将原代大鼠皮质神经元电镀在微量柱中。然而，这种方法在所有情况下都没有产生靶细胞结构，其被定义为限于微柱末端的神经细胞体，其中心腔由纯对准的轴突区组成。从那时起，使用强制神经元聚集方法（基于Ungrin 等人改编的协议）使得具有理想结构的微TENN更加可靠和一致的制造（ 图2B ） 44 。除了描述当前方法论，本文将展示微型TENNs的代表性相位对比和共聚焦图像，证明随着时间的推移形成轴突束，以及完成的目标细胞结构。该手稿还将扩大协议的重要方面，还将展示微型TENN技术的未来挑战和未来发展方向。 <img alt="图2" src="/files/ftp_upload/55609/55609fig2.jpg" /> 图2:三阶段微型TENN制造工艺的示意图。 （ A ）琼脂糖水凝胶的开发:（i）最初，将小针灸针（ 例如 ，直径为180-350μm）插入定制的激光切割模具或毛细管的圆柱形通道中（ 例如 ，直径380-700μm）。在下一步中，将DPBS中的液体琼脂糖引入圆柱形通道或毛细管中。 （ii）琼脂糖凝胶后，取出针头将模具拆开以产生中空的琼脂糖微柱。 （iii）然后将这些构建体灭菌并储存在DPBS中。 （ B ）主要神经元培养和聚集方法:（i）神经元聚集在适合于12孔培养板的孔中的3D印刷模具铸造的金字塔微孔阵列中进行。 （ii）微TENN包括从胚胎-18天大鼠的胎脑分离的原代大鼠神经元。在用胰蛋白酶-EDTA和DNA酶I组织解离之后，制备密度为1.0-2.0×10 6个细胞/ mL的细胞溶液。 （iii）将12μL该溶液转移到锥体微孔阵列中的每个孔中。将含有这些微孔的板离心以产生细胞聚集体。 （iv）然后将它们在电镀之前在微柱中孵育过夜。 （ C ）ECM核心制备和细胞接种:（i）细胞播种前，将含有1mg / mL I型胶原和1mg / mL的ECM溶液层粘连蛋白转移到微TENN的内部并使其聚合。 （ii）根据单向或双向微型TENN是否正在制造，聚集体分别放置在微柱的一个或两个极端。 （iii）经过一段时间的温育以促进粘附，将微型TENN在充满补充的胚胎神经元基础培养基的培养皿中培养。 （iv）在培养3-5天后，最终的微TENN结构应在微柱的极端处展示细胞聚集体，其轴突束跨越其长度。 <a href="http://ecsource.jove.com/files/ftp_upload/55609/55609fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

<table border="0" cellpadding="0" cellspacing="0" width="1571">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Laser cutter</td>
			<td style="width:195px;">Universal Laser Systems</td>
			<td style="width:119px;">PLS4.75</td>
			<td style="width:944px;">Used to fabricate the laser-cut micro-channel mold.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Laser-cut micro-column fabrication device</td>
			<td style="width:195px;">Custom-made</td>
			<td style="width:119px;">--------------</td>
			<td>Contact our research group if interested. Dimensions and blueprints provided in the manuscript.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Screws</td>
			<td style="width:195px;">--------------</td>
			<td style="width:119px;">--------------</td>
			<td>#4-40 with a thread diameter of 3.05 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Nuts</td>
			<td style="width:195px;">--------------</td>
			<td style="width:119px;">--------------</td>
			<td>#4-40 with a thread diameter of 3.05 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Acupuncture needle (180 &#181;m diameter)</td>
			<td style="width:195px;">Lhasa Medical</td>
			<td>sj.16X40</td>
			<td>The diameter may be varied according to the desired size for the micro-column lumen.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Petri dish</td>
			<td style="width:195px;">Fisher</td>
			<td>08772B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Dulbecco&#39;s phosphate buffered saline (DPBS)</td>
			<td style="width:195px;">Invitrogen</td>
			<td>14200075</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Polystyrene disposable serological pipet</td>
			<td style="width:195px;">Fisher</td>
			<td>13-678-11D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Agarose</td>
			<td>Sigma</td>
			<td>A9539-50G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Capillary tube (398 &#181;m diameter)</td>
			<td style="width:195px;">Fisher</td>
			<td>21170D</td>
			<td>The diameter may be varied according to the desired size for the micro-column shell.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Hot plate</td>
			<td style="width:195px;">Fisher</td>
			<td>SP88857200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Magnetic bar</td>
			<td style="width:195px;">Fisher</td>
			<td>1451352</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Micropipette</td>
			<td style="width:195px;">Sigma</td>
			<td>Z683884-1EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">25 mm gauge needle</td>
			<td style="width:195px;">Fisher</td>
			<td style="width:119px;">14-826-49</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Microscalpel</td>
			<td style="width:195px;">Roboz Surgical</td>
			<td>RS-6270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Scissors</td>
			<td style="width:195px;">Fine Science Tools</td>
			<td>14081-09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Forceps</td>
			<td>World Precision Instruments</td>
			<td>501985</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Hot bead sterilizer</td>
			<td style="width:195px;">Sigma</td>
			<td>Z378550-1EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Stereoscope</td>
			<td style="width:195px;">Nikon</td>
			<td>SMZ800N</td>
			<td>Used for all dissection steps and for micro-TENN fabrication.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Rat tail type I collagen</td>
			<td style="width:195px;">Corning</td>
			<td>354236</td>
			<td>Maintain at 4 &#186;C and remove only when needed.&#160; Use ice to preserve its temperature when in use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Microcentrifuge tube</td>
			<td style="width:195px;">Fisher</td>
			<td>02-681-256</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Mouse laminin</td>
			<td style="width:195px;">Corning</td>
			<td>354232</td>
			<td>Maintain at 4 &#186;C and remove only when needed.&#160; Use ice to preserve its temperature when in use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Neurobasal medium</td>
			<td style="width:195px;">Invitrogen</td>
			<td>21103049</td>
			<td>Basal medium for the culture of pre-natal and embryonic neuronal cells. Store at 4&#186;C and warm at 37 &#186;C before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Sodium hydroxide (NaOH)</td>
			<td style="width:195px;">Fisher</td>
			<td>SS2661</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Hydrochloric acid (HCl)</td>
			<td style="width:195px;">Fisher</td>
			<td style="width:119px;">SA48-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Litmus paper</td>
			<td style="width:195px;">Fisher</td>
			<td>09-876-18</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Hank&#39;s balanced salt solution (HBSS)</td>
			<td style="width:195px;">Invitrogen</td>
			<td>14170112</td>
			<td>Store at 4 &#186;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">0.25% Trypsin-EDTA</td>
			<td style="width:195px;">Invitrogen</td>
			<td>25200056</td>
			<td>Store at -20 &#186;C and warm at 37 &#186;C before use.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Bovine pancreatic deoxyribonuclease (DNase) I</td>
			<td style="width:195px;">Sigma</td>
			<td>10104159001</td>
			<td>Store at -20 &#186;C and warm at 37 &#186;C before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">B-27 Supplement</td>
			<td style="width:195px;">Invitrogen</td>
			<td>12587010</td>
			<td>Supplement added to Neurobasal medium for the culture of hippocampal and cortical neurons. Store at -20 &#186;C and warm at 37 &#186;C before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">L-glutamine&#160;</td>
			<td style="width:195px;">Invitrogen</td>
			<td>35050061</td>
			<td>Store at -20 &#186;C and warm at 37 &#186;C before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Sprague Dawley embryonic day 18 rats</td>
			<td style="width:195px;">Charles River</td>
			<td style="width:119px;">Strain 001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Pasteur pipette</td>
			<td style="width:195px;">Fisher</td>
			<td style="width:119px;">22-042816</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">15 mL centrifuge tube</td>
			<td style="width:195px;">EMESCO</td>
			<td>1194-352099</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Vortex</td>
			<td style="width:195px;">Fisher</td>
			<td>02-215-414</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Centrifuge</td>
			<td style="width:195px;">Fisher</td>
			<td>05-413-115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Hemocytometer</td>
			<td style="width:195px;">Fisher</td>
			<td>02-671-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Objet30 3D-Printer</td>
			<td style="width:195px;">Stratasys&#160;</td>
			<td>--------------</td>
			<td>Used to fabricate the pyramidal micro-well molds.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">3D-printed pyramidal well mold</td>
			<td style="width:195px;">Custom-made</td>
			<td style="width:119px;">--------------</td>
			<td>Contact our research group if interested. Dimensions and blueprints provided in the manuscript.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Polydimethylsiloxane (PDMS) and curing agent</td>
			<td style="width:195px;">Fisher</td>
			<td>NC9285739</td>
			<td>Comes as kit with elastomer and curing agent. Use inside a chemical fume hood.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Funnel</td>
			<td style="width:195px;">Fisher</td>
			<td>10-348C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">1 ml pipette bulb</td>
			<td style="width:195px;">Sigma</td>
			<td>Z509035</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Micro-spatula</td>
			<td style="width:195px;">Fisher</td>
			<td>S50821</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">12-well culture plate</td>
			<td style="width:195px;">EMESCO</td>
			<td>1194-353043</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Oven</td>
			<td style="width:195px;">Fisher</td>
			<td>11-475-154</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Incubator</td>
			<td style="width:195px;">Fisher</td>
			<td>13 998 076</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AAV1.Syn.GCaMP6f.WPRE.SV40</td>
			<td style="width:195px;">UPenn Vector Core</td>
			<td style="width:119px;">36373</td>
			<td>Store at -80&#186;C. Commercially available adeno-associated virus (AAV) with the GCaMP6f calcium indicator.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Formaldehyde 40%</td>
			<td style="width:195px;">Fisher</td>
			<td style="width:119px;">F77P-4</td>
			<td>Formaldehyde is a toxic compound known to be carcinogenic, and must be disposed of in a separate container.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Triton X-100</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">T8787</td>
			<td>Non-ionic surfactant used to permeabilize cell membranes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Horse serum</td>
			<td style="width:195px;">Gibco</td>
			<td style="width:119px;">16050-122</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Mouse anti-Tuj-1/beta-III tubulin primary antibody</td>
			<td style="width:195px;">Sigma</td>
			<td>T8578-200UL</td>
			<td>Store at -20&#186;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Rabbit anti-synapsin 1 primary antibody</td>
			<td>Synaptic Systems</td>
			<td>106-001</td>
			<td>Store at -20&#186;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Donkey anti-mouse 568 secondary antibody</td>
			<td style="width:195px;">Invitrogen</td>
			<td style="width:119px;">A10037</td>
			<td>Store at 4&#186;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Donkey anti-rabbit 488 secondary antibody</td>
			<td>Invitrogen</td>
			<td>A21206</td>
			<td>Store at 4&#186;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33342, Trihydrochloride</td>
			<td>Invitrogen</td>
			<td>H3570</td>
			<td>Store at 4&#186;C.&#160; Hoechst is a known mutagen that should be treated as a carcinogen.&#160; Therefore, it must be disposed of in a separate container.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A1RSI Laser Scanning Confocal Microscope&#160;</td>
			<td style="width:195px;">Nikon</td>
			<td style="width:119px;">--------------</td>
			<td>Used for taking the confocal reconstructions of immunolabeled constructs.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eclipse Ti-S Microscope&#160;</td>
			<td>Nikon</td>
			<td style="width:119px;">--------------</td>
			<td>Used for taking the phase-contrast images.&#160; With digital image acquisition using a QiClick camera interfaced with Nikon Elements Basic Research software (4.10.01).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">High-speed Fluorescence Microscope</td>
			<td style="width:195px;">Nikon</td>
			<td style="width:119px;">--------------</td>
			<td>Nikon Eclipse Ti microscope paired with an ANDOR Neo/Zyla camera for calcium imaging.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NIS Elements AR 4.50.00 Software</td>
			<td style="width:195px;">Nikon Instruments</td>
			<td style="width:119px;">--------------</td>
			<td>Used to identify calcium transients from the recordings taken with the high-speed fluorescence microscope.&#160;</td>
		</tr>
	</tbody>
</table>

anatomically inspired three-dimensional micro-tissue engineered neural networks for nervous system reconstruction, modulation, and modeling

由于抑制环境和神经发生能力有限，功能恢复很少发生在中枢神经系统（CNS）中的损伤或疾病诱导的变性之后。我们正在开发一种同时解决损伤的CNS内的神经元和轴突途径损失的策略。该手稿提出了微组织工程神经网络（微型TENN）的制造方案，由神经元组成的可植入构造物和跨越细胞外基质（ECM）腔的对准轴突束，其预先形成的直径为数百微米的水凝胶圆柱体可以延伸厘米长度。神经元聚集体被划分为三维包围的极端，并被轴突突起所跨越。微型TENN作为CNS重建的策略是独一无二的，模拟了脑连接体细胞结构的方面，并为潜在的网络替代提供了手段。神奈轮状聚集体可能与宿主组织突触形成新的功能性继电器，以恢复和/或调节丢失或损坏的电路。这些构建体也可以作为能够利用细胞迁移和轴索寻路的发育机制的再生"活支架"，基于再生状态提供协同的结构和可溶性线索。将微型TENN通过将液体水凝胶倒入含有纵向中心针的圆柱形模具中来制造。一旦水凝胶凝胶化，就去除针头，留下中空微柱。将ECM溶液加入腔内以提供适合于神经元粘附和轴突生长的环境。机械地聚集离解的神经元用于在微柱的一端或两端精确播种。该方法可靠地产生具有长突出的轴突束的自足的微型构造，其可以重现脑神经解剖学的特征。突触动物非标记和遗传编码的钙指标表明微TENN具有广泛的突触分布和内在的电活动。因此，微型TENN代表脑途径靶向神经外科重建的有希望的策略，也可作为生物学模型应用于体外研究神经生物学现象 。

使用相差显微镜监测微TENNs，以评估其细胞结构和轴突生长（ 图4 ）。在单向，2mm长的微TENNs中，神经元聚集体被限制在微柱的一端并且通过内核投射一束轴突。轴突将体外的整个长度跨越5天（DIV）（ 图4A ）。双向2 mm长的微型TENN中的初始轴突生长率较高，因为轴突通过3 DIV跨越整个微柱（ 图4B ）。通过5 DIV，双向微TENNs特征是?...

Watch this Scientific Journal Video about Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling at JoVE.com

Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling

该手稿详细描述了微组织工程神经网络的制造:三维微米大小的构建体，包括跨越聚集的神经元群体的长比例的轴突区域，其包裹在管状水凝胶中。这些生活支架可以作为功能性继电器来重建或调节神经回路，或者作为模拟灰白色神经解剖学的生物学检验床。

解剖学启发的三维微组织工程神经网络用于神经系统重建，调制和建模

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory ...

anatomically-inspired-three-dimensional-micro-tissue-engineered

Research

JoVE Journal

Neuroscience

12.6K 次观看.  University of Pennsylvania. 该手稿详细描述了微组织工程神经网络的制造:三维微米大小的构建体，包括跨越聚集的神经元群体的长比例的轴突区域，其包裹在管状水凝胶中。这些生活支架可以作为功能性继电器来重建或调节神经回路，或者作为模拟灰白色神经解剖学的生物学检验床。 

视频: Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling

Designing and Implementing Nervous System Simulations on LEGO Robots

We present a method to use the commercially available LEGO Mindstorms NXT robotics platform to test systems level neuroscience hypotheses. The first step of the method is to develop a nervous system simulation of specific reflexive behaviors of an appropriate model organism; here we use the American Lobster. Exteroceptive reflexes mediated by decussating (crossing) neural connections can explain an animal's taxis towards or away from a stimulus as described by Braitenberg and are particularly well suited for investigation using the NXT platform.1 The nervous system simulation is programmed using LabVIEW software on the LEGO Mindstorms platform. Once the nervous system is tuned properly, behavioral experiments are run on the robot and on the animal under identical environmental conditions. By controlling the sensory milieu experienced by the specimens, differences in behavioral outputs can be observed. These differences may point to specific deficiencies in the nervous system model and serve to inform the iteration of the model for the particular behavior under study. This method allows for the experimental manipulation of electronic nervous systems and serves as a way to explore neuroscience hypotheses specifically regarding the neurophysiological basis of simple innate reflexive behaviors. The LEGO Mindstorms NXT kit provides an affordable and efficient platform on which to test preliminary biomimetic robot control schemes. The approach is also well suited for the high school classroom to serve as the foundation for a hands-on inquiry-based biorobotics curriculum.

An approach to neural network modeling on the LEGO Mindstorms robotics platform is presented. The method provides a simulation tool for invertebrate neuroscience research in both the research lab and the classroom. This technique enables the investigation of biomimetic robot control principles.

LEGO机器人神经系统仿真设计和实现

We present a method to use the commercially available LEGO Mindstorms NXT robotics platform to test systems level neuroscience hypotheses. The first step ...

科研

神经科学

Neural Circuit Recording from an Intact Cockroach Nervous System

The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological effects of insecticides. This article describes the scope of cockroach sensory modalities that can be used to assay how an insect nervous system responds to environmental perturbations. Emphasis here is on the escape behavior mediated by cerci to giant fiber transmission in Periplaneta americana. This in situ preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of neuronal activity. Peptides or other chemical reagents can then be applied directly to the nervous system in solution with the physiological saline. Insecticides could also be administered prior to dissection and the escape circuit can serve as a proxy for the excitable state of the central nervous system. In this context the assays described herein would also be useful to researchers interested in limb regeneration and the evolution of nervous system development for which P. americana is an established model organism.

This article describes the cockroach ventral nerve cord dissection and extracellular recordings from the cercal nerve and connectives. Evoked responses are generated by electrical stimulation of the cercal nerve or direct mechanical stimulation of the cerci.

神经电路记录从一个完整的蟑螂神经系统

The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

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

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

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

在啮齿动物中枢神经系统的体内光遗传学刺激

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

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

建模星形细胞瘤发病机制<em&gt;在体外</em&gt;和<em&gt;体内</em&gt;使用星形胶质细胞或条件，遗传工程小鼠神经干细胞

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify their functional and structural properties, a condition called reactive astrogliosis. Here, a protocol for assessment of the morphological properties of astrocytes is presented. This protocol includes quantification of 12 different parameters i.e. the surface area and volume of the tissue covered by an astrocyte (astrocyte territory), the entire astrocyte including branches, cell body, and nucleus, as well as total length and number of branches, the intensity of fluorescence immunoreactivity of antibodies used for astrocyte detection, and astrocyte density (number/1,000 &#181;m2). For this purpose three-dimensional (3D) confocal microscopic images were created, and 3D image analysis software such as Volocity 6.3 was used for measurements. Rat brain tissue exposed to amyloid beta1-40 (A&#946;1-40) with or without a therapeutic&#160;intervention was used to present the method. This protocol can also be used for 3D morphometric analysis of other cells from either in vivo or in vitro conditions.

Astrocytes in the CNS change their functional and structural properties in response to harmful stimuli. This report presents a protocol for assessment of three-dimensional astrocyte morphology in diseased conditions or after therapeutic interventions.

协议星形胶质细胞的三维共聚焦形态分析

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify ...

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory Sensory Neuron (OSN) is a bipolar cell with a single dendrite and a single unbranched axon. An OSN expresses only one Olfactory Receptor (OR) gene, OSNs expressing a given type of OR converge their axons to a few sets of invariant glomeruli in the Olfactory Bulb (OB). A remarkable feature of OSN projection is that the expressed ORs play instructive roles in axonal projection. ORs regulate the expression of multiple axon-sorting molecules and generate the combinatorial molecular code of axon-sorting molecules at the OSN axon termini. Thus, to understand the molecular mechanisms of OR-specific axon guidance mechanisms, it is vital to characterize their expression profiles at the OSN axon termini within the same glomerulus. The aim of this article was to introduce methods for collecting as many glomeruli as possible on a single OB section and for performing immunostaining using multiple antibodies. This would allow the comparison and analysis of the expression patterns of axon-sorting molecules without staining variation between OB sections.

Olfactory sensory neurons express a wide variety of axon-sorting molecules to establish proper neural circuitry. This protocol describes an immunohistochemical staining method to visualize combinatorial expressions of axon-sorting molecules at the axon termini of olfactory sensory neurons.

嗅球的四次免疫染色用于嗅觉感觉轴突分子识别码的可视化

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory ...

Simultaneous Recordings of Cortical Local Field Potentials, Electrocardiogram, Electromyogram, and Breathing Rhythm from a Freely Moving Rat

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls body functions and internal organ rhythms when animals are exposed to emotional challenges and changes in their living environments. In general experiments, signals from different organs, such as the brain and the heart, are recorded by independent recording systems that require multiple recording devices and different procedures for processing the data files. This study describes a new method that can simultaneously monitor electrical biosignals, including tens of local field potentials in multiple brain regions, electrocardiograms that represent the cardiac rhythm, electromyograms that represent awake/sleep-related muscle contraction, and breathing signals, in a freely moving rat. The recording configuration of this method is based on a conventional micro-drive array for cortical local field potential recordings in which tens of electrodes are accommodated, and the signals obtained from these electrodes are integrated into a single electrical board mounted on the animal's head. Here, this recording system was improved so that signals from the peripheral organs are also transferred to an electrical interface board. In a single surgery, electrodes are first separately implanted into the appropriate body parts and the target brain areas. The open ends of all of these electrodes are then soldered to individual channels of the electrical board above the animal's head so that all of the signals can be integrated into the single electrical board. Connecting this board to a recording device allows for the collection of all of the signals into a single device, which reduces experimental costs and simplifies data processing, because all data can be handled in the same data file. This technique will aid the understanding of the neurophysiological correlates of the associations between central and peripheral organs.

This study introduces a method for the simultaneous recording of local field potentials in the brain, electrocardiograms, electromyograms, and breathing signals of a freely moving rat. This technique, which reduces experimental costs and simplifies data analysis, will contribute to the understanding of the interactions between the brain and peripheral organs.

自由运动大鼠皮质局部电位、心电图、肌电图和呼吸节律的同步记录

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls ...

Three-Dimensional Shape Modeling and Analysis of Brain Structures

Statistical shape analysis of brain structures has been used to investigate the association between their structural changes and pathological processes. We have developed a software package for accurate and robust shape modeling and group-wise analysis. Here, we introduce an pipeline for the shape analysis, from individual 3D shape modeling to quantitative group shape analysis. We also describe the pre-processing and segmentation steps using open software packages. This practical guide would help researchers save time and effort in 3D shape analysis on brain structures.

We introduce a semi-automatic protocol for shape analysis on brain structures, including image segmentation using open software, and further group-wise shape analysis using an automated modeling package. Here, we demonstrate each step of the 3D shape analysis protocol with hippocampal segmentation from brain MR images.

脑结构三维形状建模与分析

Statistical shape analysis of brain structures has been used to investigate the association between their structural changes and pathological processes. ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of &#947;-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -&#160;MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice.

将化学遗传工程皮质内神经内生前生移植到早期产后小鼠大脑

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

Three-Dimensional Motor Nerve Organoid Generation

A fascicle of axons is one of the major structural motifs observed in the nervous system. Disruption of axon fascicles could cause developmental and neurodegenerative diseases. Although numerous studies of axons have been conducted, our understanding of formation and dysfunction of axon fascicles is still limited due to the lack of robust three-dimensional in vitro models. Here, we describe a step-by-step protocol for the rapid generation of a motor nerve organoid (MNO) from human induced pluripotent stem (iPS) cells in a microfluidic-based tissue culture chip. First, fabrication of chips used for the method is described. From human iPS cells, a motor neuron spheroid (MNS) is formed. Next, the differentiated MNS is transferred into the chip. Thereafter, axons spontaneously grow out of the spheroid and assemble into a fascicle within a microchannel equipped in the chip, which generates an MNO tissue carrying a bundle of axons extended from the spheroid. For the downstream analysis, MNOs can be taken out of the chip to be fixed for morphological analyses or dissected for biochemical analyses, as well as calcium imaging and multi-electrode array recordings. MNOs generated with this protocol can facilitate drug testing and screening and can contribute to understanding of mechanisms underlying development and diseases of axon fascicles.

This protocol provides a comprehensive procedure to fabricate human iPS cell-derived motor nerve organoid through spontaneous assembly of a robust bundle of axons extended from a spheroid in a tissue culture chip.

三维运动神经器官生成

A fascicle of axons is one of the major structural motifs observed in the nervous system. Disruption of axon fascicles could cause developmental and ...