作者想感谢的资金来源:陆军UNL的创伤力学中心（美国国防部/ ARO，W911NF-11-1-0033，PI:适合本国的缓解钱德拉博士），UNL的门外汉奖（26-1110-0033 -001 PI:LIM）。

1。神经元细胞培养<ol><li>可以使用包括神经细胞，星形胶质细胞，和它们的共培养的脑细胞作为细胞模型。作为一个可行性论证，细胞系的神经细胞的脉冲增压。 </li><li>直径为18毫米盖玻片上培养SH-SY5Y人神经母细胞瘤细胞（ATCC CRL-2266）。细胞接种密度为3×10 3细胞/ cm 2使用组成的DMEM生长培养基补充有10％胎牛血清和1％青霉素-链霉素。细胞培养玻片保持在5％CO 2培养箱中，在37℃下的</li><li>要获得的SH-SY5Y细胞的神经元细胞的分化，细胞进一步补充用10μM - 视黄酸（RA）为7天，与媒体的媒体处理改变每两天。在第7天，细胞是准备被加压。 </li></ol> 2。加压设备:Kolsky酒吧<ol><li> Kolsky酒吧，于1949年1 Kolsky开发的，已用于测量材料的力学性能在一个非常高的负荷率。该装置由两个线条之间接触放置在与样品的酒吧。事件栏上创建，传播的应力波，波分裂成一个反射和透射波的样品。样品中的应力是正比于发射波。在这项研究中，我们利用一个单元格被放置的加压室之间的两个杆的Kolsky设置。 </li><li>通过对齐的黄铜轴承的两个铝合金棒（每6米长）悬浮，和一个被夹持在体外细胞加压室之间的横杠。上游酒吧是酒吧，一端夹到了130磅的质量事件。一种摩擦夹紧装置被放置的位置处，将得到所需的脉冲持续时间。从事钳，拧紧剪式千斤顶的质量和一个加载的支持，钳质量部分的事件栏前Compressed到所需的水平。强制锁定夹具带有液压系统突然断裂的切口螺栓迅速释放预先存储的压缩，从而产生一个压力脉冲。这通过传播事件栏，驱动测试室的活塞，加压液和细胞内室的冲动。反过来加压室启动压力脉冲在传输的酒吧下游传播。该菌株的压力脉冲在酒吧与复合高电阻应变计被激发到45伏。计信号与数字示波器在1 MHz的频率作为一个加载历史记录。 </li><li>在体外细胞加压室的活塞缸组成。缸具有2.6厘米内径和3.8厘​​米外径，并有一个小孔，在腔体的基础上抽头。孔作为在细胞样品安装过程中的空气和多余的液体通气孔。活塞是7.5厘米长的并且是由相同的棒材。活塞是包裹着两到三层的水喉匠（聚四氟乙烯）磁带作为低摩擦密封。端盖是32毫米长，有两个小不锈钢螺丝固定在玻片上（细胞培养），在加载。 </li></ol> 3。神经细胞的脉冲增压<ol><li>全部加压室部使用高压釜进行灭菌，并保持在UV光下。的组件的腔室的内部进行的细胞培养罩。腔室的排气螺钉是松散从事的通气孔中在基地的空腔。预先温热的（37℃）新鲜的生长培养基被吸移到未做气泡室。然后，将细胞培养载玻片被拾起，被放置在所述活塞帽（面朝外的细胞）。小的固定螺丝拧紧在幻灯片上举行的地方。用培养细胞的载玻片上的活塞被插入了一个小i置身于腔的腔室，并且装配倾斜时，在最高点与通气。除去排气螺钉和活塞推入空腔首先迫使气泡，然后过量的流体。一个引用标记或夹具是用来为指导，以确保相同的培养基体积为每一个测试。腔室中的介质的轴向尺寸是约6毫米。消毒通风的螺丝和更换，使室内的水紧。腔室不应该泄漏此静载荷下，如果这样做，聚四氟乙烯包裹需要更换或加强。 </li><li>在这一点上，的Kolsky杆系统复位。沉重的质量被移回原来的位置，并且一个新的锁紧螺栓替换所使用的（破碎）1。约200磅的液压搞新的锁定螺栓。用剪刀插孔压缩预装载部分的入射杆的压力略高于所需的值，然后回到它关闭，以充分的摩擦接合杰克的螺丝。现在手持数据采集。 </li><li>组装的细胞加压室被安装到系统中。它被放置在一个V形块由小型实验室剪刀插孔支持，并与两个杆对齐。润滑脂每个接口的光油脂层和摩擦的对接面，以消除空气之间的差距。 </li><li>现在已经准备好进行测试。夹锁紧螺栓强制打破，迅速泵送液压钳驱动程序。钳将分离和应显示的数据采集的结果。从发送巴（表压）的测量值确定的细胞加压历史。如果传输的酒吧是足够长的时间，这应该只从第一干扰的测量结果表明负压。所发送的脉冲的持续时间可能会比入射脉冲短，如果气泡被困住。的幅度可能不到达该入射脉冲，但它应该有足够离子克起卸前的高原。否则，无论是一个大的气泡，有可能发生在密封腔或错位之间的装配和酒吧。 </li><li>然后除去细胞加压室内部的细胞培养罩和拆卸。除去排气螺钉和活塞被拉到免费腔室。取自所述活塞的端部的细胞培养的载玻片。 </li></ol> 4。评估增压细胞生物学特性<ol><li>在加压后，细胞可以立即检查，或进一步培养，以便以后检查。通过适当的无菌操作过程中，更长期的培养是可能的。 </li><li>加压的细胞可以检查所有的分子和细胞生物学技术。特别是神经细胞增压，调查的细胞和分子生理学在TBI条件，在突起生长的压力引起的变化，微管细胞骨架的变化，神经基因expre的分析评估裂变，细胞凋亡，等等，都可以被执行。作为对照细胞样品，培养的细胞相同，保持在相同的时间期间内的加压室，但不被加压的使用（所谓的，腔室控制）。 </li><li>为了评估在突起生长的变化中，加压室的控制细胞后立即加压和1和24小时的孵育后用光学显微镜检查。神经元细胞的图像的一个例子示出在&#39;代表结果"部分（图2）。可以量化的肌动蛋白免疫荧光染色和图像分析突起长度的变化。固定细胞，用4％w / v的多聚甲醛溶液，用0.05％体积/体积的Tween-20洗涤缓冲液漂洗，并用0.1％体积/体积的Triton X-100溶液，透。封闭后用1％w / v的牛血清白蛋白溶液，四甲基罗丹明异硫氰酸酯（TRITC）共轭的鬼笔环肽的细胞孵育。采取的细胞的免疫荧光图像使用荧光显微镜和加压和控制细胞的突起的长度使用ImageJ软件（NIH）的量化。 </li><li>可以被认定为轴突或树突的突起。为了评估轴突或树突中的形态变化，上述实验可以重复通过检测每个结构的抗体，即，神经丝（neurofilament，NF）抗体轴突和微管相关蛋白（MAP2）抗体树突。 </li><li>微管是细胞骨架的主要成分的神经元之一，和与微管 ​​的破坏已被用作一个标记的神经元损伤的2微管免疫荧光可以可视化使用的β-微管蛋白抗体。加压后0和24小时后，固定细胞与β-微管蛋白抗体染色，并通过荧光显微镜观察到的。 </li><li>为了评估脉冲增压对神经元和神经细胞凋亡基因表达的影响，压力和控制细胞的总RNA提取。根E的表达进行定量RT-PCR和实时RT-PCR，我们公布的协议，可以检查。 </li></ol> 5。代表性的成果: <img src="/files/ftp_upload/2723/2723fig1.jpg" alt="图1"/> 图1，一种压力更新的例子适用于细胞内的加压室。 Kolsky酒吧设备成功地产生2 MPa级，持续时间约为0.7毫秒，单脉冲型脉冲增压。 <img src="/files/ftp_upload/2723/2723fig2.jpg" alt="图2"/> 图2。神经元细胞的一个例子，响应于脉冲加压。在2兆帕显示出明显的神经轴突/轴突击穿相对的腔体控制细胞SH-SY5Y细胞暴露于冲动增压。 6。难点与对策<ol><li>腔室的密封是要克服的主要障碍之一。这是fo&gt;公司&gt;，与O形圈的宽磁盘有一个刨等增加的摩擦问题。为了消除摩擦的影响，以及防止欺诈特富龙水喉匠的活塞录音磁带，一个简单的方法是使用。这解决了问题，并产生所需的压力水平和持续时间。 </li><li>加压神经元细胞的行为，尤其是在评估二次的TBI机制或长期的影响，应进行检查，经过了漫长的孵化时间。含细胞的腔室暴露于潜在的未消毒的条件，移动的同时的Kolsky栏，加压，和移动回到细胞培养罩。随着预灭菌的腔室部件和正确的操作，在细胞培养的滑动和腔室内部的细胞培养罩，组装/拆卸较长期的孵育后是可能长达数周。 </li><li>的数据的可再现性是一个重要的参数，在蜂窝的机械性刺激实验。我们的设备，reproducibility在神经细胞的反应确定所需的脉冲增压个人资料如何，在这两个压力的程度及持续时间，重复获得。由于我们记录每个升压得到的压力分布，细胞的反应不必要的压力分布数据，可以手动排除之后。 </li><li>整个加压步骤，包括室组件，转让和安装在Kolsky酒吧，增压，调回，和室拆卸，用了不到10分钟。加压下一个细胞培养幻灯片可以紧接前一后的加压。因此，足够数量的加压试验，可以有效地完成。我们的设置采用了直径为18毫米盖玻片。一个加压实验并不能提供足够的蛋白质，Western免疫印迹由于基板尺寸（也有一定的压力，因为有些细胞都死了）。关于三个重复的的加压实验​​提供immunobl所需的蛋白量otting。同样地，再现性检查每次用的压力轮廓。 </li></ol>

<ol>
	<li>Kolsky, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+investigation+of+mechanical+properties+of+materials+at+very+high+rates+of+loading.">An investigation of mechanical properties of materials at very high rates of loading.</a> Prec. Phys. Soc. London. 62, 676-700 (1949).</li><li>Tang-Schomer, M. D., Patel, A. R., Baas, P. 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=Mechanical+breaking+of+microtubules+in+axons+during+dynamic+stretch+injury+underlies+delayed+elasticity,+microtubule+disassembly,+and+axon+degeneration.">Mechanical breaking of microtubules in axons during dynamic stretch injury underlies delayed elasticity, microtubule disassembly, and axon degeneration.</a> FASEB. J. 24, 1401-1410 (2010).</li><li>Lim, J. Y., Taylor, A. F., Li, Z., Vogler, E. A., Donahue, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin+expression+and+osteopontin+regulation+in+human+fetal+osteoblastic+cells+mediated+by+substratum+surface+characteristics.">Integrin expression and osteopontin regulation in human fetal osteoblastic cells mediated by substratum surface characteristics.</a> Tissue. Eng. 11, 19-29 (2005).</li><li>Chen, Y. C., Smith, D. H., Meaney, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+approaches+for+studying+blast-induced+traumatic+brain+injury.">In vitro approaches for studying blast-induced traumatic brain injury.</a> J. Neurotrauma. 26, 861-876 (2009).</li><li>Ling, G., Bandak, F., Armonda, R., Grant, G., Ecklund, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Explosive+blast+neurotrauma.">Explosive blast neurotrauma.</a> J. Neurotrauma. 26, 815-825 (2009).</li><li>Shepard, S. R., Ghajar, J. B. G., Giannuzzi, R., Kupferman, S., Hariri, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluid+percussion+barotrauma+chamber:+a+new+in+vitro+model+for+traumatic+brain+injury.">Fluid percussion barotrauma chamber: a new in vitro model for traumatic brain injury.</a> J. Surg. Res. 51, 417-424 (1991).</li><li>VandeVord, P. J., Leung, L. Y., Hardy, W., Mason, M., Yang, K. H., King, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Up-regulation+of+reactivity+and+survival+genes+in+astrocytes+after+exposure+to+short+duration+overpressure.">Up-regulation of reactivity and survival genes in astrocytes after exposure to short duration overpressure.</a> Neurosci. Lett. 434, 247-252 (2008).</li><li>Huang, H., Feng, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+study+of+the+dynamic+tribological+response+of+closed+fracture+surface+pairs+by+Kolsky-bar+compression-shear+experiment.">A study of the dynamic tribological response of closed fracture surface pairs by Kolsky-bar compression-shear experiment.</a> Int. J. Solids. Struct. 41, 2821-2835 (2004).</li><li>Song, B., Chen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Split+Hopkinson+pressure+bar+techniques+for+characterizing+soft+materials.">Split Hopkinson pressure bar techniques for characterizing soft materials.</a> Lat. Am. J. Solids. Struct. 2, 113-152 (2005).</li></ol>

没有利益冲突的声明。

已经尝试了许多在体外实验技术研究脑细胞损伤TBI条件。这些措施包括神经元/胶质细胞拉伸，流引起的剪切应力，体重下降，手写笔裂伤，激光切断术，碎石等4,5它已被认为持续时间较短的超压主要物理因素TBI尤其是6,7 TBI的爆炸冲击波有一个脉冲持续时间为毫秒级。基于这种假设和观察，气压计室的瞬时增压已经制定和评估脑细胞行为在TBI的条件下使用。例如，在流体中的打击乐器气压伤室20-30毫秒的持续时间和长的峰值压力高达0.5MPa的压力脉冲被用来研究人类的神经胶质细胞的行为下TBI 6最近，VandeVord等7中使用的金属击球的晴雨表室检查的星形胶质细胞根据TBI的行为，但金属球惊人的多重压力脉冲。在这项研究中，我们已经开发了一种新的细胞产生单脉冲型脉冲加压的加压装置。装置的基础上的Kolsky杆设置，它已被用于作为材料试验装置，在我们的8和其它的研究开发。1,9的Kolsky杆材料测试能力已被修改，以便能够在非常高的负荷率应用单脉冲型超压的培养细胞内的加压室（图1）。我们可以成功地控制的幅度和持续时间的脉冲的超压。作为一个代表性的细胞反应，我们表明，在2 MPa压力下SH-SY5Y神经细胞显示显着的神经轴突和轴突损失相比，在毫无压力的腔体控制细胞。 总之，我们制定了一个新的冲动细胞加压装置，采用特殊设计的Kolsky在TBI条件研究神经细胞的反应，酒吧设备，并展示了其潜在的使用。我们还注意到，这种技术是非常灵活，可以接触到任何类型的细胞，在不同的程度和持续时间的冲动的压力。因此，该设备可以被用来研究的脉冲压力诱导的细胞的机械力行为，不仅对受损的细胞，但也为积极刺激细胞在它们的功能和命运决定。

<ul><li> SH-SY5Y（ATCC CRL-2266）人类神经母细胞瘤</li><li>直径为18毫米的玻璃盖玻片</li><li>细胞培养基:DMEM，10％胎牛血清，1％青霉素 - 链霉素</li><li>视黄酸（10μM）诱导神经发生</li><li> Kolsky酒吧脉冲增压装置</li><li>活塞缸细胞加压室</li><li>不锈钢螺钉固定在活塞上的细胞培养盖玻片</li><li>润滑脂之间的细胞室和Kolsky的酒吧接口的</li><li>一般分子生物学用品为评估细胞反应增压（固色剂，抗体，荧光染料，PCR耗材等） </li></ul>

impulsive pressurization of neuronal cells for traumatic brain injury study

一种新型的冲动的细胞加压实验已开发使用的Kolsky酒吧的移动设备以调查爆炸引起的创伤性脑损伤（TBI）。我们展现在爆炸TBI相关的脉冲增压视频文章如何适用于神经元细胞在体外。这是通过使用良好控制的压力脉冲创建由一个专门Kolsky的杆装置，与完整的压力历史记录细胞内的加压室。加压神经细胞加压后立即检查，或继续培养，研究长期影响的冲动加压在神经突起/轴突生长，神经元的基因表达，细胞凋亡，等我们观察到，冲动增压约2兆帕引起不同的神经轴突损失相对在毫无压力的细胞。我们的技术提供了一种新的方法来研究分子/细胞机制的爆炸TBI，，通过脉冲增压脑细胞控制的公关essure的幅度和持续时间。

Watch this Scientific Journal Video about Impulsive Pressurization of Neuronal Cells for Traumatic Brain Injury Study at JoVE.com

Impulsive Pressurization of Neuronal Cells for Traumatic Brain Injury Study

一种新型的冲动的细胞加压实验已开发使用Kolsky酒吧的移动设备以调查爆炸引起的外伤性脑损伤的分子/细胞的机制。

创伤性脑损伤研究神经细胞的脉冲增压

A novel impulsive cell pressurization experiment has been developed using a Kolsky bar device to investigate blast-induced traumatic brain injury (TBI). ...

impulsive-pressurization-neuronal-cells-for-traumatic-brain-injury

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10.1K 次观看.  University of Nebraska-Lincoln. 一种新型的冲动的细胞加压实验已开发使用Kolsky酒吧的移动设备以调查爆炸引起的外伤性脑损伤的分子/细胞的机制。

视频: Impulsive Pressurization of Neuronal Cells for Traumatic Brain Injury Study

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

Micropatterned表面透明质酸与肿瘤细胞相互作用的研究

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

科研

生物工程

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

在肌肉再生的老鼠模型中移植诱导多能干细胞衍生的中生代细胞样肌原体

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

一种新型三维流室装置研究生理流量条件下的趋化因子的细胞循环外渗

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

使用微流体芯片实时成像和损伤研究的响应<em&gt;果蝇</em&gt;幼虫

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

荧光共振能量转移研究构象变化的膜蛋白在哺乳动物细胞中表达

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Training Persons with Spinal Cord Injury to Ambulate Using a Powered Exoskeleton

Powered exoskeletons have become available for overground ambulation in persons with paralyses due to spinal cord injury (SCI) who have intact upper extremity function and are able to maintain upright balance using forearm crutches. To ambulate in an exoskeleton, the user must acquire the ability to maintain balance while standing, sitting and appropriate weight shifting with each step. This can be a challenging task for those with deficits in sensation and proprioception in their lower extremities. This manuscript describes screening criteria and a training program developed at the James J. Peters VA Medical Center, Bronx, NY to teach users the skills needed to utilize these devices in institutional, home or community environments. Before training can begin, potential users are screened for appropriate range of motion of the hip, knee and ankle joints. Persons with SCI are at an increased risk of sustaining lower extremity fractures, even with minimal strain or trauma, therefore a bone mineral density assessment is performed to reduce the risk of fracture. Also, as part of screening, a physical examination is performed in order to identify additional health-related contraindications. 
Once the person has successfully passed all screening requirements, they are cleared to begin the training program. The device is properly adjusted to fit the user. A series of static and dynamic balance tasks are taught and performed by the user before learning to walk. The person is taught to ambulate in various environments ranging from indoor level surfaces to outdoors over uneven or changing surfaces. Once skilled enough to be a candidate for home use with the exoskeleton, the user is then required to designate a companion-walker who will train alongside them. Together, the pair must demonstrate the ability to perform various advanced tasks in order to be permitted to use the exoskeleton in their home/community environment.

Training a person with paralysis to ambulate using a powered exoskeleton may present challenges. The goals are to present the candidate selection criteria and the training procedures for exoskeletal-assisted walking and other mobility skills that can be progressed as the participant's skill level improves.

训练脊髓损伤者走动使用动力服

Powered exoskeletons have become available for overground ambulation in persons with paralyses due to spinal cord injury (SCI) who have intact upper ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

三维组织工程化排列的星形胶质细胞网络, 以概括发展机制和促进神经系统再生

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

Targeting Neuronal Fiber Tracts for Deep Brain Stimulation Therapy Using Interactive, Patient-Specific Models

Deep brain stimulation (DBS), which involves insertion of an electrode to deliver stimulation to a localized brain region, is an established therapy for movement disorders and is being applied to a growing number of disorders. Computational modeling has been successfully used to predict the clinical effects of DBS; however, there is a need for novel modeling techniques to keep pace with the growing complexity of DBS devices. These models also need to generate predictions quickly and accurately. The goal of this project is to develop an image processing pipeline to incorporate structural magnetic resonance imaging (MRI) and diffusion weighted imaging (DWI) into an interactive, patient specific model to simulate the effects of DBS. A virtual DBS lead can be placed inside of the patient model, along with active contacts and stimulation settings, where changes in lead position or orientation generate a new finite element mesh and solution of the bioelectric field problem in near real-time, a timespan of approximately 10 seconds. This system also enables the simulation of multiple leads in close proximity to allow for current steering by varying anodes and cathodes on different leads. The techniques presented in this paper reduce the burden of generating and using computational models while providing meaningful feedback about the effects of electrode position, electrode design, and stimulation configurations to researchers or clinicians who may not be modeling experts.

The goal of this project is to develop an interactive, patient-specific modeling pipeline to simulate the effects of deep brain stimulation in near real-time and provide meaningful feedback as to how these devices influence neural activity in the brain.

应用交互式、患者特异性模型对神经纤维束进行深部脑刺激治疗的靶向性

Deep brain stimulation (DBS), which involves insertion of an electrode to deliver stimulation to a localized brain region, is an established therapy for ...

Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular matrix (ECM) of the unique brain environment, such as hyaluronic acid (HA), facilitates GBM progression and invasion. However, most in vitro culture models include GBM cells outside of the context of an ECM. Murine xenografts of GBM cells are used commonly as well. However, in vivo models make it difficult to isolate the contributions of individual features of the complex tumor microenvironment to tumor behavior. Here, we describe an HA hydrogel-based, three-dimensional (3D) culture platform that allows researchers to independently alter HA concentration and stiffness. High molecular weight HA and polyethylene glycol (PEG) comprise hydrogels, which are crosslinked via Michael-type addition in the presence of live cells. 3D hydrogel cultures of patient-derived GBM cells exhibit viability and proliferation rates as good as, or better than, when cultured as standard gliomaspheres. The hydrogel system also enables incorporation of ECM-mimetic peptides to isolate effects of specific cell-ECM interactions. Hydrogels are optically transparent so that live cells can be imaged in 3D culture. Finally, HA hydrogel cultures are compatible with standard techniques for molecular and cellular analyses, including PCR, Western blotting and cryosectioning followed by immunofluorescence staining.

Here, we present a protocol for three-dimensional culture of patient-derived glioblastoma cells within orthogonally tunable biomaterials designed to mimic the brain matrix. This approach provides an in vitro, experimental platform that maintains many characteristics of in vivo glioblastoma cells typically lost in culture.

透明质酸基水凝胶对3维患者源性胶质瘤细胞培养的研究

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular ...

Mouse Model of Pressure Ulcers After Spinal Cord Injury

Pressure ulcers (PUs) are common debilitating complications of traumatic spinal cord injury (SCI) and tend to occur in soft tissues around bony prominences. There is, however, little known about the impact of SCI on skin wound healing in the context of animal models in controlled experimental settings. In this study, a simple, non-invasive, reproducible and clinically relevant mouse model of PUs in the context of complete SCI is presented. Adult male mice (Balb/c, 10 weeks old) were shaved and depilated. Post-depilation (24 h), mice were subjected to laminectomy followed by complete spinal cord transection (T9-T10 vertebrae). Immediately after, a skin fold on the back of the mice was lifted and sandwiched between two magnetic discs held in place for next 12 h, thus creating an ischemic area that developed into a PU over the following days. The wounded areas demonstrated tissue edema and epidermal disappearance by day 3 post-magnet application. PUs spontaneously developed and healed. Healing was, however, slower in the SCI mice compared to control non-SCI mice when the wound was created below the level of SCI. Conversely, no difference in healing was seen between SCI and control non-SCI mice when the wound was created above the level of SCI. This model is a potentially useful tool to study the dynamics of skin PU development and healing after SCI, as well as to test therapeutic approaches that may help heal such wounds.

Here, we describe a simple method to induce clinically relevant skin pressure ulcers (PUs) in a mouse model of spinal cord injury (SCI). This model can be used in pre-clinical studies to screen for different therapeutics for healing PUs in SCI patients.