我们要感谢英国皇家军团爆炸伤研究中心对医管局的财政支持, 以及医学研究委员会 (M01858X/1) 的资助。

1. 细胞培养和样品制备 <ol> <li> 获取适当的细胞线 (例如, 真皮乳突细胞)。
	注意: 目前的协议已被设计为黏附细胞, 与大鼠真皮乳成纤维细胞分离的触须用作模型. </li>
	<li> 培养含有最低必需培养基 (记忆) 和 #945 的 T-75 烧瓶中的细胞; 辅以10% 胎牛血清和1% 青霉素链霉素。保持细胞在标准培养条件下37和 #176; C 和 5% CO 2 在湿化环境中, 直到它们达到80% 汇合. </li>
	<li> 吸入培养基并在 Dulbecco 和 #39 中两次冲洗细胞; 磷酸缓冲盐 (PBS)。在标准培养条件下, 在2毫升的胰蛋白酶/EDTA 中, 将细胞从烧瓶中分离出来5分钟. 简要检查, 以确保细胞已成功地分离出瓶使用的光/相对比显微镜 (10X 物镜).</li>
	<li> 添加4毫升培养基以中和 trypsinization 反应。将电池悬浮液转移到15毫升离心管上. </li>
	<li> 离心机在 200 x g 为4分钟的颗粒细胞。慢慢地吸取上清液, 小心不要把小球取出。在5毫升介质中重新悬浮颗粒. </li>
	<li> 传输20和 #181; 将分的细胞悬浮例, 用装有10X 物镜的显微镜对细胞进行计数. </li>
	<li> 将三 35 mm 的盘子放在 90 mm 的盘子里, 准备爆炸的细胞。适当地给它们贴上标签。每道菜加2毫升培养基至每35毫米碟, 种子 5 x 10 4 细胞, 在盘子周围均匀分布细胞。在标准培养条件下一夜孵化实验样品, 在冲击波照射前允许足够的细胞附着. 
	注意: 对于荧光成像, 细胞必须在无菌玻璃片上播种. </li>
</ol> 2。激波管总成 <img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55618/55618fig2.jpg"/> 图 2 : 祥钻机和激波管总成。( A ) 祥钻机的图像。( B ) 激波管装置示意图。激波管尺寸包括内径59毫米和总长度, 包括祥钻机, 4.13 米。驱动管和祥钻机的长度共计 2.71 m. <a href="http://ecsource.jove.com/files/ftp_upload/55618/55618fig2large.jpg" target="_blank"> 请单击此处查看此图的较大版本. </a> 
<ol> <li> 在装配过程中穿钢趾工作靴和实验室工作服. </li>
	<li> 通过在出口法兰的水平平面上找到的两个螺栓孔插入两个对齐条。在对齐条的一端放置一个丁腈橡胶垫片, 使其位于插座法兰和 前体 器官培养 (祥) 平台之间. 
	注: 祥钻机是一种定制设计的夹具, 适用于 35 mm 的培养皿 ( 图 2A ). </li>
	<li> 将祥钻机连接到激波管的出口法兰上, 通过将夹具滑动到对齐条上, 确保其朝向正确, 以便保持培养皿的截面位于其底座上. </li>
	<li> 将四 M24 螺栓和垫圈插入剩余的孔中。放置螺母使其接触法兰. </li>
	<li> 以对角对称的方式紧扣螺母和螺栓, 同时检查祥钻机和激波管是否已成功对准. </li>
	<li> 将压力传感器连接到祥钻机, 并使用传感器电缆将其连接到当前源。然后, 使用 BNC 电缆将电流源连接到示波器. </li>
	<li> 确保所有释放阀和激波管上的流量控制都关闭。打开内置的外部压缩空气线, 并手动将压力调节器顺时针旋转, 将螺线管充电至 2.5 bar. </li>
	<li> 逆时针旋转180和 #176 打开压缩气瓶上的安全阀. </li>
	<li> 慢慢转动压力调节器, 位于气瓶顶部, 顺时针方向将压力增加到大约 15 bar. 
	注: 该调节器的设置点应略高于最厚膜片的最高爆破压力, 这将在实验中使用. </li>
	<li> 通过将0.125 毫米厚的塑料板切割成 10 cm x 10 cm 正方形来准备横膈膜. 
	注: 膜片的厚度将与爆破压力相关, 从而改变冲击波的峰值压力 ( 即, 较厚的膜片会导致具有较大峰值压力的冲击波; 表 1 ). </li>
</ol> <table fo:keep-together.within-page="1"> <tbody> <tr> <td> 聚酯薄膜尺寸 (cm) </td> <td> 压力 (bar) </td> <td> 传感器3压力范围 (人民军) </td> </tr> <tr> <td> 10 x 10 x0.023 </td> <td> 2 和 #177; 0.2 </td> <td> 47 和 #177; 7.5 </td> </tr> <tr> <td> 10 x 10 x 0.050 </td> <td> 4 & #177; 0.2 </td> <td> 72 和 #177; 7.5 </td> </tr> <tr> 10 x 10 x 0.125 <td> </td> 9.5 和 #177; 0.5 <td> </td> 127 和 #177; 12.5 <td> </td> </tr>/t能够 >> 表 1: 与膜片厚度和峰值压力相对应的破裂压力. 
<ol start="11"> <li> 从磁带中创建手柄, 将其固定在膜片的顶部和底部, 并使用它们仔细地将膜片放在前面的小法兰旁边, 由后膛室放置。在关闭臀位前, 确保隔膜是直的和正确定位的. </li>
	<li> 使用四 M24 螺栓和螺母将其固定。紧固螺母和螺栓按对角线对称的方式. </li>
</ol> 3。在冲击波曝光前制备细胞 <ol> <li> 通过执行紫外线灭菌准备组织层流罩。用消毒剂溶液和70% 乙醇清洗。收集处理细胞之前, post-shock 波曝光和放置在无菌罩内所需的所有项目. 
	注: 这包括新鲜的培养基, 粘接透气膜, 无菌剪刀, 管和吸管提示. </li>
	<li> 将一个 90 mm 的培养皿中的三实验样品从孵化箱转移到无菌罩. </li>
	<li> 将粘接透气的薄膜片 (参见 材料表 ) 切成六平方, 这样每个正方形就足以覆盖一个 35 mm Petri 盘的顶部. </li>
	<li> 从 35 mm 的培养皿中吸取培养基, 将盖子放在一侧, 并用两个粘附透气膜部分盖住, 确保膜与盘边缘之间有紧密的密封. </li>
	<li> 将样品转移到祥钻机, 并将其固定在夹具的底座上, 使盘的顶部与激波管的内部底座对齐. </li>
</ol> 4。激波管操作 <ol> <li> 在加压激波管系统时, 戴上耳后卫、护目镜、安全靴和实验室大衣. </li>
	<li> 顺时针旋转调节器上的流量控制旋钮, 允许气体流入软管, 将压缩空气钢瓶连接到激波管. </li>
	<li> 在控制面板的底部, 选择 #34;D 双的臀位和 #34; 短期冲击 wav 的入口e 或 #34;D 河管和 #34; 入口为增加的波浪持续时间. </li>
	<li> 在直流电源和示波器上切换以获取波形数据。设置示波器采样率为50.0 毫秒/秒, 记录长度为1米点。设置一个范围为 50 mv 射击在2酒吧和 100 mv 以上4酒吧. </li>
	<li> 使用控制面板对面墙上的开关打开安全灯 
	注: 这告诉其他研究人员在电击管充电时不要进入房间. </li>
	<li> 打开电磁控制盒上的开关, 关闭螺线管. 
	注: 此安全功能关闭位于双膛室的阀门, 允许系统充电. </li>
	<li> 在控制面板上, 通过逆时针旋转旋钮慢慢打开流量控制, 逐渐增加压缩腔内的压力。使用控制面板显示器显示压力. </li>
	<li> 一旦膜片爆裂压力达到, 膜片将破裂, 并 #34; 砰和 #34; 将被听到。通过转动旋钮快速关闭流量控制. 
	注: 冲击波将沿着管状的细胞样本和管道的末端移动. </li>
	<li> 打开螺线管, 关闭电磁控制盒上的开关, 关掉安全灯 (使用控制面板对面墙上的开关). </li>
	<li> 将电池样品转移到无菌罩上, 取出粘接透气膜, 用2毫升的新鲜培养基填充该盘。如步骤5所述, 这些细胞可立即用于评估, 或返回 longer-term 研究的孵化器. </li>
</ol> 5。细胞存活性 <ol> <li> 使用氧化还原指标测定和活体和死细胞的荧光成像相结合的方法评估冲击波照射后的细胞活力. 
	注意: 对于荧光成像, 细胞必须在1节: 细胞培养和样品制备过程中, 在无菌玻璃片上播种。这些可以放在35毫米的培养皿中. </li>
	<li> 传输200和 #181; L 氧化还原指标试剂 (参见 材料表 ) 对每一个实验菜的直接填充后2毫升中 post-shock 波曝光。在标准培养条件下孵育 4 h. </li>
	<li> 对于每道菜, 转移 (在一个黑暗的无菌罩) 2 x 100 和 #181; L 等分到黑色或透明的 96-井板, 这取决于是否会使用荧光或吸光度来评估生存能力. </li>
	<li> 将96个井板转印到合适的平板读卡器上, 并使用激发带530前/590 em 进行荧光或 570 nm, 并结合 600 nm 的吸光度参考。导出和保存数据. </li>
	<li> 分析数据以识别冲击波暴露的样本和医护控件之间的差异, 因为这可能意味着细胞活力下降. 
	注: 实验设计中还应包括负控制。此外, 标准曲线的插值可以用来计算单元数. </li>
	<li> 吸入含有氧化还原指示剂的培养基。将其替换为2毫升的新鲜培养基, 并在标准培养条件下孵育24小时的细胞. </li>
	<li> 根据需要重复上述步骤以获得第二个生存时间点. </li>
	<li> 用于活体和死细胞的荧光成像, 在1毫升 PBS 中吸入培养基并冲洗两次细胞。传输100和 #181; 在成像套件中找到的完整试剂 (请参见 材料表 ) 到每个样品, 并在室温下孵育 15 min. </li>
	<li> 用1毫升 PBS 吸出试剂并冲洗一次。使用细点钳, 小心地去除片从35毫米菜和地方它朝到玻璃显微镜幻灯片. </li>
	<li> 使用荧光显微镜 (10X 物镜、0.50 数字孔径) 为每个样品获取图像, 并配有正确的励磁和发射过滤器 (前/em 488 nm/515 nm 和 570 nm/602 nm). 
	注意: 活细胞会发出强烈、均匀、绿色的荧光。死亡或垂死的细胞与一个受损的细胞膜将摄取死亡套件组件, 导致红色荧光的发射, 发现主要在细胞核区域的细胞. </li>
	<li> 使用图像分析软件手动或自动计算每个图像中的活和死单元格数. </li>
</ol>

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+ossification+after+extremity+blast+amputation+in+a+Sprague-Dawley+rat+animal+model.">Heterotopic ossification after extremity blast amputation in a Sprague-Dawley rat animal model.</a> J Orthop Trauma. 25 (8), 506-510 (2011).</li><li>Chen, Y. C., Smith, D. H., Meaney, D. F. <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 (6), 861-876 (2009).</li><li>Nguyen, T. T. N., Wilgeroth, J. M., Proud, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+blast+wave+generation+in+a+shock+tube+for+biological+applications.">Controlling blast wave generation in a shock tube for biological applications.</a> JPCS. 500 (14), 142025 (2014).</li></ol>

作者没有相互竞争的金融利益。

从接触爆炸事件中获得的主要伤害尚未完全了解。识别和理解触发损伤的机制, 如外伤性颅脑损伤3,4和异位骨化6,7, 是开发有效的预防方法。为了帮助实现这一目标, 已经开发了一些实验系统来复制爆炸事件曝光11,12,13,14,18,,19.这里所描述的技术使用的冲击波管设备 (图 2) 能够在整个身体 (即,啮齿动物), 组织, 或细胞样本的压力范围内发射冲击波。加载单个单元格类型而不是整个组织的能力赋予了解析不同的蜂窝响应的能力, 因为通过一系列机制1,2可以同时发生破坏。例如, 对创伤性脑损伤的模型, 对单个细胞类型的评估, 如神经元和星形细胞, 可以识别细胞特异性损伤。此外, 整个器官的反应可以用脑组织来评估。个体细胞类型和组织标本都有其价值, 可以提供不同的信息。也可以通过选择 double-breech 或驱动管入口来改变加压产生冲击的空气量。这控制了冲击波的持续时间。另一种可能性是改变隔膜材料和厚度来改变峰值压力25。
另一个考虑因素是, 当样品壳体位于激波管出口附近时, 可能存在干扰端效应, 如在本系统所描述的祥钻机上所发现的。钱德拉et al.研究了在压缩驱动激波管的不同位置上发现的冲击波剖面, 发现弗里德兰德波形在激波管15深处的位置表现最好。库利亚科塞et al.还研究了样品的二次加载, 发现在激波管末端的端板位置能够消除不需要的反射波16。考虑到这些出版物中的数据15,16, 将来改进本文所描述的激波管系统的修改可能涉及将祥钻机放置在驱动管内的较深位置, 或或者, 在激波管上加入一个端板。所述方法的局限性可能包括样本的相对较低的吞吐量。一个用户可以安全地操作激波管每小时大约6-8 样品的输出。目前, 该系统是围绕使用单 35 mm 培养皿而设计的。因此, 包含多个组和生物复制的较大实验可能难以实现。
这一方法的文章显示了如何在一个单一的冲击波接触的皮肤乳突细胞的生存能力。与控制 (图 3和图 4) 相比, ≤72人民军的短期冲击波 (和 #60; 10 毫秒) 没有影响生存能力。相比之下, 127 人民军的冲击波刺激了 24 h 后的生存能力显著下降, 如氧化还原指标测定 (图 3) 和荧光图像分析 (图 4) 所示。米勒et al.报告了类似的减少大鼠器官海马切片培养细胞的活力, 当细胞暴露在一个147帕或278帕斯卡冲击波使用一个开放的, 氦驱动的激波管14。相比之下, VandeVord et al.报告说, 对短期超压的大鼠星形胶质细胞的生存能力没有影响; 200 人民军, 虽然使用了 barochamber, 而不是电击管18。应该指出的是, 外部压力依赖于冲击波, 虽然这在体内产生了复杂的应力波, 因此使负载的性质高度依赖于组织或细胞的机械特性。需要对爆炸事件的细胞反应进行额外的特性研究。此外, 通过评估冲击波暴露在细胞水平, 如这项技术所示, 从损伤触发的生物反应, 如信号通路的扰动或后生变化, 可以识别和进一步探索。
总之, 这项工作描述了使用不锈钢激波管和改良的祥钻机, 以纳入原细胞培养。冲击波在一定范围内的压力可以产生和传播的活细胞, 以复制的影响, 发生的冲击波。该协议演示了如何评估细胞的生存能力, 但也可以研究 longer-term 对单个细胞类型的改变。展望未来, 我们计划评估复杂冲击波在不同细胞类型中所产生的差异效应, 目的是进一步了解爆炸诱发的主要损伤。

随着最近在现代战争中使用简易爆炸装置和对平民采取恐怖主义行动的趋势, 了解爆炸性事件对人体的影响是非常重要的。通过接触爆炸事件获得的伤害可能是致命的和致命的, 伤害的物理过程分为四类。主要损伤起因于直接暴露在冲击波, 在局部地与身体相互作用以压缩和随后膨胀的方式, 导致细胞膜和软的组织的破坏1。继发性损伤包括钝挫伤或由冲击波推动的质量物体撞击造成的穿透伤。三级损伤发生在冲击波有足够的能量投掷物体的高质量或个人反对对象。最后, 第四纪爆炸伤害的定义是其他一些不适合其他类别的伤害, 如闪光烧伤2。在接触此类爆炸事件后, 主要损伤包括外伤性脑损伤3、4、5、异位骨化6、7、炸肺损伤8、听力丧失9和其他10。
一个通常观察到的波形来自爆炸事件是弗里德兰德波, 代表一个自由, 而不是封闭空间, 爆炸。波形由一个爆炸锋组成, 可以定义为正压的急剧和快速上升。紧随其后的是气流在高速移动时的爆炸风, 以及释放波, 使压力降至低于大气的水平。部分真空留在初始爆炸区域, 导致空气缓慢回流。波浪的正面和反面阶段 (图 1A) 导致冲击波的推挽运动1。为了帮助阐明主要的爆炸伤害的机制, 建立了实验模型, 以产生波形, 如弗里德兰德波, 细胞和组织将面对真正的爆炸事件。目前在文献中列出的系统包括激波管11,12,13,14,15,16,17,barochambers18,19, Kolsky 条20, 高级爆炸模拟器21, 拆分霍普金森压力条22, 以及在受控环境中使用可选爆炸事件的娱乐季戊四醇硝酸23。尽管可用的模型范围很广, 但许多变量影响了从冲击波中获得的损伤, 包括在评估24下的单个细胞类型或组织的预应力应用和力学性能。虽然组织或器官的研究可以揭示组织变形和总形态学的变化持续的爆炸事件的结果, 在细胞水平的分析可以发现转录和表观变化受冲击波的影响。
此方法文章描述了一种技术, 以传播冲击波的压力范围内的活细胞在单层。这使得细胞活力的直接表征, 阐明了冲击波的潜在损伤阈值。此外, 可行的细胞可以返回到标准的培养条件, 和长期的生物效应, 从爆炸事件可以评估。下面的协议描述了两种可用于培养细胞的细胞活力技术。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55618/55618fig1.jpg"> 
图 1:弗里德兰德波浪的近似值.(a) 在激波管上的传感器3上观察到的弗里德兰德波的近似值。(B) 具有代表性的数据, 显示在传感器1、2和3上观察到的不同压力剖面。<a href="http://ecsource.jove.com/files/ftp_upload/55618/55618fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

<table><tbody><tr><td>MEM &amp;alpha;, nucleosides</td><td>ThermoFisher</td><td>22571020</td><td></td></tr><tr><td>Fetal Bovine Serum, certified, US origin</td><td>ThermoFisher</td><td>16000044</td><td>Supplement to create complete growth media.</td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline</td><td>Sigma Aldrich</td><td>D8537</td><td></td></tr><tr><td>Penicillin-Streptomycin&amp;nbsp;</td><td>ThermoFisher</td><td>15070-063</td><td>Supplement to create complete growth media.</td></tr><tr><td>Trypsin-EDTA (0.5%), no phenol red</td><td>ThermoFisher</td><td>15400-054</td><td>Dilute 1 in 10 before use.</td></tr><tr><td>CytoOne T-75 Flask, TC-Treated, vented</td><td>Starlab</td><td>CC7682-4875</td><td></td></tr><tr><td>TC Dish (PS) 35 mm, 8.5 cm&lt;sup&gt;2&lt;/sup&gt;</td><td>Triple Red</td><td>TCD010035</td><td></td></tr><tr><td>Petri dish (PS) 90 x 14.2 mm no vent</td><td>VWR UK</td><td>391-0453&amp;nbsp;</td><td></td></tr><tr><td>Gas Permeable Adhesive Plate Seals</td><td>ThermoFisher</td><td>AB-0718</td><td></td></tr><tr><td>LIVE/DEAD Cell Imaging Kit (488/570)</td><td>ThermoFisher</td><td>R37601</td><td></td></tr><tr><td>Alamarblue cell viability reagent</td><td>Fisher Scientific</td><td>13494309</td><td></td></tr><tr><td>Virkon tablets</td><td>VWR UK</td><td>115-0020</td><td>Use to create 2% solution as viability control reagent.</td></tr><tr><td>Dumont forceps</td><td>SurgicalTools</td><td>11295-10</td><td>Use to remove coverslips from petri dish.</td></tr><tr><td>Cover glass, square</td><td>VWR UK</td><td>631-0125</td><td></td></tr><tr><td>Microscope slides</td><td>VWR UK</td><td>631-1553</td><td></td></tr><tr><td>96 Well plate, solid black</td><td>AppletonWoods</td><td>CC760</td><td>Plate to be used for fluorescence measurements.</td></tr><tr><td>96 Well plate, clear, (PS)</td><td>VWR UK</td><td>734-1799</td><td>Plate to be used for absorbance measurments.</td></tr><tr><td>Leica DMi1 Camera stand outfit</td><td>Leica Microsystems</td><td></td><td>Optical microscope used for cell culture.</td></tr><tr><td>Zeiss PALM MicroBeam Laser Capture Microdisseciton</td><td>Zeiss</td><td></td><td>Fluorescence microsope used for LIVE/DEAD imaging.</td></tr><tr><td>EnVision Multilabel Reader</td><td>PerkinElmer</td><td>2104-0010A</td><td>Plate reader to be used for fluorescence/absorbance readings.</td></tr><tr><td>Mylar Electrical &amp;amp; Chemical Insulating Film, 304 mm x 200 mm x 0.023 mm&amp;nbsp;</td><td>RS Components</td><td>785-0782</td><td>Use to create shock tube diaphragm.</td></tr><tr><td>Mylar Electrical &amp;amp; Chemical Insulating Film, 304 mm x 200 mm x 0.05 mm&amp;nbsp;</td><td>RS Components</td><td>785-0786</td><td>Use to creatw shock tube diaphragm.</td></tr><tr><td>Mylar Electrical &amp;amp; Chemical Insulating Film, 304 mm x 200 mm x 0.125 mm&amp;nbsp;</td><td>RS Components</td><td>785-0798</td><td>Use to create shock tube diaphragm.</td></tr><tr><td>Current source power unit</td><td>Dytran Intruments Inc.</td><td>4103C</td><td>Power source for 2300V1 sensor.</td></tr><tr><td>IEPE Pressure Sensor</td><td>Dytran Intruments Inc.</td><td>2300V1</td><td>Pressure sensor located on shock tube.</td></tr><tr><td>Digital Phosphor Oscilloscope</td><td>Tektronix</td><td>DPO 4104B</td><td>Use to gather and save sensor 2300V1 data.</td></tr></tbody></table>

evaluating primary blast effects in vitro

暴露在爆炸事件中会对诸如肺部、耳朵和大脑等重要器官造成严重的创伤。考虑到最近在现代战争和与恐怖主义有关的事件中使用炸药的趋势, 了解这种爆炸造成的伤害的机制非常重要。为了充分理解爆炸引起的损伤, 我们必须首先能够使用可重现的方法在受控环境中复制此类爆炸事件。在这项技术使用激波管设备, 冲击波的压力范围内可以传播的活细胞生长在 2D, 和细胞活力的标志可以立即分析使用氧化还原指标检测和活体和死细胞的荧光成像。这种方法表明, 将峰值冲击波超压提高到127帕, 与未经处理的控制相比, 可以刺激细胞活力的显著下降。测试样本不限于附着细胞, 但可以包括细胞悬浮, 全身和组织样本, 通过对休克管设置的轻微修改。复制的确切条件, 组织和细胞的经验, 当暴露于一个真正的爆炸事件是困难的。如本文所介绍的技术可以帮助定义损伤阈值, 并确定在冲击波照射引起的细胞内的转录和表观变化。

使用上述方法, 在单层中生长的细胞被暴露在一式三, 冲击波产生使用激波管 (图 2B)。评估细胞活力的标记。通过使用氧化还原指标测定, 发现127人民军冲击波的应用能显著降低真皮乳突细胞的存活能力, 与24小时后的对照组比较 (图 3)。冲击波≤72人民军的应用并没有减少其生存能力。为了支持这些观察, 采用了一种荧光图像检测方法, 可以分别使用绿色或红色荧光荧光标记活细胞或死单元。从每一个生物复制的13荧光图像中活细胞和死单元的定量显示, 与对照组相比, 在127人民军冲击波照射下的细胞存活率降低 (图 4)。统计分析是使用 one-way 方差检验, 其次是 Tukey 的多重比较试验, 与 p 和 #60; 0.05 认为统计学意义。每组样品的粒度总计为氧化还原指标测定值的 9, 39 用于定量荧光成像分析。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55618/55618fig3.jpg"> 
图 3: 氧化还原指标测定数据显示冲击波照射后细胞活力的时间过程.与未处理的对照相比, 127-帕斯卡冲击波暴露组24小时后观察到的细胞明显减少。与所有其他组相比, 由 30-2% 消毒剂 (见材料表) 组成的负控制显示, T0 和24小时的细胞明显减少。每个小节代表平均±1标准偏差 (SD);生物复制, N = 3;技术复制, n = 3。= p & #60; 0.0001。* = p 和 #60; 0.05。<a href="http://ecsource.jove.com/files/ftp_upload/55618/55618fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55618/55618fig4.jpg"> 
图 4: 荧光成像.(A) 通过荧光显微镜在 24 post-shock 波照射下采集的活体和死细胞的荧光图像收集的定量数据。与47人民军 (平均 = 98.18)、72人民军 (平均 = 98.87) 和对照组 (平均值 = 99.10) 相比, 127 帕斯卡 (平均 = 93.42) 冲击波暴露的样本中观察到的存活细胞明显减少。阴性对照组在 24 h (平均值 = 0) 后未观察到可行细胞。(B) 具有代表性的荧光图像。绿色显示活细胞, 而红色显示死细胞。每个方块图显示的上部和下部四与 Tukey 胡须;生物复制, N = 3;技术复制, n = 13。= p & #60; 0.0001。缩放栏 = 300 µm.<a href="http://ecsource.jove.com/files/ftp_upload/55618/55618fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Evaluating Primary Blast Effects In Vitro at JoVE.com

Evaluating Primary Blast Effects In Vitro

了解如何通过接触冲击波来调节细胞, 有助于识别爆炸事件引发的损伤机制。该协议使用 custom-built 激波管设备, 应用冲击波在一系列的压力, 以细胞单分子膜和确定后的影响细胞的生存能力。

评估主要爆炸效果体外

Exposure to blast events can cause severe trauma to vital organs such as the lungs, ears, and brain. Understanding the mechanisms behind such ...

evaluating-primary-blast-effects-in-vitro

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

Evaluating Primary Blast Effects In Vitro

7.9K 次观看.  Imperial College London. 了解如何通过接触冲击波来调节细胞, 有助于识别爆炸事件引发的损伤机制。该协议使用 custom-built 激波管设备, 应用冲击波在一系列的压力, 以细胞单分子膜和确定后的影响细胞的生存能力。

视频: Evaluating Primary Blast Effects In Vitro

Blast Quantification Using Hopkinson Pressure Bars

Near-field blast load measurement presents an issue to many sensor types as they must endure very aggressive environments and be able to measure pressures up to many hundreds of megapascals. In this respect the simplicity of the Hopkinson pressure bar has a major advantage in that while the measurement end of the Hopkinson bar can endure and be exposed to harsh conditions, the strain gauge mounted to the bar can be affixed some distance away. This allows protective housings to be utilized which protect the strain gauge but do not interfere with the measurement acquisition. The use of an array of pressure bars allows the pressure-time histories at discrete known points to be measured. This article also describes the interpolation routine used to derive pressure-time histories at un-instrumented locations on the plane of interest. Currently the technique has been used to measure loading from high explosives in free air and buried shallowly in various soils.

This protocol details the use of Hopkinson pressure bars to measure reflected blast loading from near-field explosive events. It is capable of interpolating a pressure-time history at any point on a reflective boundary and as such can be used to fully characterize the spatial and temporal variations in loading produced.

高炉利用定量霍普金森杆

Near-field blast load measurement presents an issue to many sensor types as they must endure very aggressive environments and be able to measure pressures ...

科研

工程学

A New Portable In Vitro Exposure Cassette for Aerosol Sampling

This protocol introduces a new in vitro exposure system, capable of being worn, including its characterization and performance. Air-liquid interface (ALI) in vitro exposure systems are often large and bulky, making transport to the field and operation at the source of emission or within the breathing zone difficult. Through miniaturization of these systems, the lab can be brought to the field, expediting processing time and providing a more appropriate exposure method that does not alter the aerosol prior to contacting the cells. The Portable In vitro Exposure Cassette (PIVEC) adapts a 37 mm filter cassette to allow for in vitro toxicity testing outside of a traditional laboratory setting. The PIVEC was characterized using three sizes of copper nanoparticles to determine deposition efficiency based on gravimetric and particle number concentration analysis. Initial cytotoxicity experiments were performed with exposed lung cells to determine the ability of the system to deposit particles while maintaining cell viability. The PIVEC provides a similar or increased deposition efficiency when comparing to available perpendicular flow in vitro exposure devices. Despite the lower sample throughput, the small size gives some advantages to the current in vitro ALI exposure systems. These include the ability to be worn for personal monitoring, mobility from the laboratory to the source of emission, and the option to set-up multiple systems for spatial resolution while maintaining a lower user cost. The PIVEC is a system capable of collecting aerosols in the field and within the breathing zone onto an air-interfaced, in vitro model.

A New Portable In Vitro Exposure Cassette for Aerosol Sampling

Here, we present a protocol to perform portable cellular aerosol exposures and measure cellular response. The method uses cells, grown at the air-liquid interface, mimicking in vivo physiology. Cellular response to copper nanoparticle aerosols was observed as oxidative stress through reactive oxygen species generation and cytotoxicity as lactate dehydrogenase release.

一种用于气溶胶取样的新型便携式体外曝光盒

This protocol introduces a new in vitro exposure system, capable of being worn, including its characterization and performance. Air-liquid interface (ALI) ...

环境科学

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same pathway, are likely to fail. Emphasis must be placed upon the inhibition of pathways that are responsible for intrinsic and/or adaptive resistance to therapy. An active field of investigation is the development and testing of DNA repair inhibitors that promote the action of, and prevent resistance to, commonly used chemotherapy and radiotherapy. We used a novel protocol to evaluate the effectiveness of BRCA2 inhibition as a means to sensitize tumor cells to the DNA damaging drug cisplatin. Tumor cell metabolism (acidification and respiration) was monitored in real-time for a period of 72 hr to delineate treatment effectiveness on a minute by minute basis. In combination, we performed an assessment of metastatic frequency using a chicken embryo chorioallantoic membrane (CAM) model of extravasation and invasion. This protocol addresses some of the weaknesses of commonly used in vitro and in vivo methods to evaluate novel cancer therapy regimens. It can be used in addition to common methods such as cell proliferation assays, cell death assays, and in vivo murine xenograft studies, to more closely discriminate amongst candidate targets and agents, and select only the most promising candidates for further development.

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Here, real-time monitoring of tumor cell metabolism, combined with an in vivo chicken embryo chorio-allantoic membrane (CAM) model of metastasis, are used to evaluate novel anti-cancer targets/agents for their ability to sensitize tumor cells to DNA damaging chemotherapeutics.

评估癌症药物过敏的有效性<em&gt;在体外</em&gt;和<em&gt;在体内</em

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same ...

医学

Protocol for Human Blastoids Modeling Blastocyst Development and Implantation

A model of the human blastocyst formed from stem cells (blastoid) would support scientific and medical advances. However, its predictive power will depend on its ability to efficiently, timely, and faithfully recapitulate the sequences of blastocyst development (morphogenesis, specification, patterning), and to form cells reflecting the blastocyst stage. Here we show that na&#239;ve human pluripotent stem cells cultured in PXGL conditions and then triply inhibited for the Hippo, transforming growth factor- &#946;, and extracellular signal-regulated kinase pathways efficiently undergo morphogenesis to form blastoids (&gt;70%). Matching with developmental timing (~4 days), blastoids unroll the blastocyst sequence of specification by producing analogs of the trophoblast and epiblast, followed by the formation of analogs of the primitive endoderm and the polar trophoblasts. This results in the formation of cells transcriptionally similar to the blastocyst (&gt;96%) and a minority of post-implantation analogs. Blastoids efficiently pattern by forming the embryonic-abembryonic axis marked by the maturation of the polar region (NR2F2+), which acquires the specific potential to directionally attach to hormonally stimulated endometrial cells, as in utero. Such a human blastoid is a scalable, versatile, and ethical model to study human development and implantation in vitro.

A protocol outlining the formation of human blastoids that efficiently, timely, and sequentially generate blastocyst-like cells.

人爆破模拟胚发育和植入的协议

A model of the human blastocyst formed from stem cells (blastoid) would support scientific and medical advances. However, its predictive power will depend ...

发育生物学

Rapid Quantification of Mitogen-induced Blastogenesis in T Lymphocytes for Identifying Immunomodulatory Drugs

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., immunosuppressive or immunostimulatory) chemical compounds and biologics. One of the earliest steps during mitogenesis is cell enlargement or blastogenic transformation, whereupon the cell volume increases before division. It is usually detectable in the first several hours of T-lymphocyte stimulation. Here, we describe a rapid method to quantify blastogenesis in T lymphocytes isolated from mouse spleens and human peripheral blood mononuclear cells (PBMCs) using an automated cell counter. Various commonly used proliferation assays for the most part are laborious and only reflect the overall population effect rather than individual cellular effects within a population. In contrast, the presented automated cell counter assay provides rapid, direct, and precise measurements of cell diameters that can be used for assessing the effectiveness of various mitogens and immunomodulatory drugs in vitro.

T-lymphocyte mitogenesis is accompanied by blastogenic transformation, whereupon the cell volume enlarges before cell division. Here, we describe a method to quantify blastogenesis in T lymphocytes using an automated cell counter with the capability of measuring cell diameters.

T淋巴细胞有丝分裂原诱导芽生的快速定量辨识免疫调节药物

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., ...

免疫与感染

A Novel In Vitro Model of Blast Traumatic Brain Injury

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the detonation of explosive devices, however, the mechanisms that underlie the brain damage resulting from blast overpressure exposure are not entirely understood and are believed to be unique to this type of brain injury. Preclinical models are crucial tools that contribute to better understand blast-induced brain injury. A novel in vitro blast TBI model was developed using an open-ended shock tube to simulate real-life open-field blast waves modelled by the Friedlander waveform. C57BL/6N mouse organotypic hippocampal slice cultures were exposed to single shock waves and the development of injury was characterized up to 72 h using propidium iodide, a well-established fluorescent marker of cell damage that only penetrates cells with compromised cellular membranes. Propidium iodide fluorescence was significantly higher in the slices exposed to a blast wave when compared with sham slices throughout the duration of the protocol. The brain tissue injury is very reproducible and proportional to the peak overpressure of the shock wave applied.

A Novel In Vitro Model of Blast Traumatic Brain Injury

This paper describes a novel model of primary blast traumatic brain injury. A compressed-air driven shock tube is used to expose in vitro mouse hippocampal slice cultures to a single shock wave. This is a simple and rapid protocol generating a reproducible brain tissue injury with a high throughput.

一种新的脑外伤体损伤模型

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the ...

神经科学

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

爆炸性神经损伤对压生啮鼠中脑动脉的影响

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...

Low-intensity Blast Wave Model for Preclinical Assessment of Closed-head Mild Traumatic Brain Injury in Rodents

Traumatic brain injury (TBI) is a large-scale public health problem. Mild TBI is the most prevalent form of neurotrauma and accounts for a large number of medical visits in the United States. There are currently no FDA-approved treatments available for TBI. The increased incidence of military-related, blast-induced TBI further accentuates the urgent need for effective TBI treatments. Therefore, new preclinical TBI animal models that recapitulate aspects of human blast-related TBI will greatly advance the research efforts into the neurobiological and pathophysiological processes underlying mild to moderate TBI as well as the development of novel therapeutic strategies for TBI.
Here we present a reliable, reproducible model for the investigation of the molecular, cellular, and behavioral effects of mild to moderate blast-induced TBI. We describe a step-by-step protocol for closed-head, blast-induced mild TBI in rodents using a bench-top setup consisting of a gas-driven shock tube equipped with piezoelectric pressure sensors to ensure consistent test conditions. The benefits of the setup that we have established are its relative low-cost, ease of installation, ease of use and high-throughput capacity. Further advantages of this non-invasive TBI model include the scalability of the blast peak overpressure and the generation of controlled reproducible outcomes. The reproducibility and relevance of this TBI model has been evaluated in a number of downstream applications, including neurobiological, neuropathological, neurophysiological and behavioral analyses, supporting the use of this model for the characterization of processes underlying the etiology of mild to moderate TBI.

We present here a protocol of a blast wave model for rodents to investigate neurobiological and pathophysiological effects of mild to moderate traumatic brain injury. We established a gas-driven, bench-top setup equipped with pressure sensors allowing for reliable and reproducible generation of blast-induced mild to moderate traumatic brain injury.

低强度冲击波模型用于啮齿动物闭头轻度创伤性脑损伤的临床前评估

Traumatic brain injury (TBI) is a large-scale public health problem. Mild TBI is the most prevalent form of neurotrauma and accounts for a large number of ...

Assessment of the Acute Inhalation Toxicity of Airborne Particles by Exposing Cultivated Human Lung Cells at the Air-Liquid Interface

Here, we present a specially designed modular in vitro exposure system that enables the homogenous exposure of cultivated human lung cells at the ALI to gases, particles or complex atmospheres (e.g., cigarette smoke), thus providing realistic physiological exposure of the apical surface of the human alveolar region to air. In contrast to sequential exposure models with linear aerosol guidance, the modular design of the radial flow system meets all requirements for the continuous generation and transport of the test atmosphere to the cells, a homogenous distribution and deposition of the particles and the continuous removal of the atmosphere. This exposure method is primarily designed for the exposure of cells to airborne particles, but can be adapted to the exposure of liquid aerosols and highly toxic and aggressive gases depending on the aerosol generation method and the material of the exposure modules.
Within the framework of a recently completed validation study, this exposure system was proven as a transferable, reproducible and predictive screening method for the qualitative assessment of the acute pulmonary cytotoxicity of airborne particles, thereby potentially reducing or replacing animal experiments that would normally provide this toxicological assessment.

We present a robust, transferable and predictive in vitro exposure system for the screening and monitoring of airborne particles concerning their acute pulmonary cytotoxicity by exposing cultivated human lung cells at the air-liquid interface (ALI).

通过在空气-液体界面中暴露培养的人类肺细胞，评估空气中颗粒的急性吸入毒性

Here, we present a specially designed modular in vitro exposure system that enables the homogenous exposure of cultivated human lung cells at the ALI to ...

Shocktube 方法模拟小鼠高度重复的低水平爆炸

Modeling Highly Repetitive Low-level Blast Exposure in Mice

Exposure to explosive blasts is a significant risk factor for brain trauma among exposed persons. Although the effects of large blasts on the brain are well understood, the effects of smaller blasts such as those that occur during military training are less understood. This small, low-level blast exposure also varies highly according to military occupation and training tempo, with some units experiencing few exposures over the course of several years whereas others experience hundreds within a few weeks. Animal models are an important tool in identifying both the injury mechanisms and long-term clinical health risks following low-level blast exposure. Models capable of recapitulating this wide range of exposures are necessary to inform acute and chronic injury outcomes across these disparate risk profiles.
Although outcomes following a few low-level blast exposures are easily modeled for mechanistic study, chronic exposures that occur over a career may be better modeled by blast injury paradigms with repeated exposures that occur frequently over weeks and months. Shown here are methods for modeling highly repetitive low-level blast exposure in mice. The procedures are based on established and widely used pneumatic shocktube models of open-field blast exposure that can be scaled to adjust the overpressure parameters and the number or interval of the exposures. These methods can then be used to either enable mechanistic investigations or recapitulate the routine blast exposures of clinical groups under study.

作者聚焦:破译小鼠低水平爆炸暴露的长期影响

Presented here are methods for producing repeated low-intensity blast exposures using mice.

对小鼠高度重复的低水平爆炸暴露进行建模

Exposure to explosive blasts is a significant risk factor for brain trauma among exposed persons. Although the effects of large blasts on the brain are ...