科研经费是通过研究所收到的神经疾病和中风（NS049177和NS087834）的。作者要感谢已故的保罗博士巴斯和他的同事对应变计的原设计的知识贡献;和卡罗尔Tollefsrud为应变片的制造和销售，直到生产在2010年和她的见地通信停止。

所有程序遵循健康指引全国学院和医学院的宾夕法尼亚州立大学贺喜批准的机构动物护理和使用委员会。大鼠用普通动物饲养规范安置。注意:该协议采用雄性Wistar大鼠≥8周龄和初始体重175 - 200克 1，程序应变仪的研制<ol><li>大多数工具和部件仍可以从原始或后继公司和总结于表1中 。 </li><li>准备和两个单元素应变计粘接<ol><li>处理应变元素（EA-06-031-350）小心地用干净的杜蒙＃5镊子。限制元件的不希望的运动，可以使用一个小的，清洁的，自粘一张纸与粘接面朝上，以确保元件的工作表面，而没有污染或过度密合性的危险。 </li><li>债券两个单应变计内容背到后面，形成一个双元件。清洗各元件薄膜的背面与异丙醇，并允许干燥，蒸发（用纱布干燥常常引入纤维污染物是很难除去）。体视显微镜下指导（1 - 3倍），并用干净的艺术家刷（10-0驼绒），适用于环氧 - 酚醛粘合剂的薄膜，以一个元件的背面，并立即放置在接触相对的背面的第二元件的并对准箔网格（ 图1A）。 </li><li>将在50粘合元素 - 60℃的烘箱中的O / N至完全固化的环氧树脂。 注意:不要用镊子保税元素，因为过多的环氧树脂可渗入到元件和压力可能会造成电网的错位。该两部分环氧具有混合后可用冷藏寿命只有6个星期。债券和治疗元素的充足供应一次，并将它们存储在一个干净，无灰尘，环境，供以后使用。 </li></ol></li><li>选型和布线双晶应变计<ol><li>修剪结合双元素的3×3mm的最终大小与＃11手术刀或单刃刀片。延迟修剪此时的双元件的最高部分，以便有一个区域，用于安全地处理元件（ 图1A）。 </li><li>每个元件都需要从三芯，粘合制成的四芯线，铁氟龙绝缘电线（P / N:336-FTE）。拆卸的三芯线之一30厘米编织股线分成三个构成线。 </li><li>为了让一个四线电缆，配对所产生的单线与包含三芯线的二三十厘米长的范围内相同颜色的线之一。在下面的步骤中，这些匹配的彩色导线会在末端被接合以形成一个共同的电线进行最后的应变仪（ 图1B）。 </li><li>从每根电线与热剥线机的两端除去1mm左右聚四氟乙烯绝缘的。采用活性松香溶胶dering通量和低温焊料（熔点183℃）用锡焊的铅笔线结束。 </li><li>在接下来的阶段中，microsoldering尖端仍需以形成多个离散的焊点，以防止热损坏的元件（ 图1C）的薄膜层。为了制造更小的microsoldering前端，包住一小块铜丝（〜0.250毫米直径）绕一圈的标准焊头，确保铜导线延伸超出标准焊头的长度。 </li><li>磁通只在粘接的双元件的一侧的焊剂焊盘用清洁刷10-0和焊料单一导线和一对公共引线的焊接垫（ 图1D）之一。剩余磁通之后可以用干净毛刷蘸树脂的溶剂清除。 </li><li>重复的相反侧的过程中，以确保剩余的公共引线焊接到焊盘相对原来的共用导线。 </li></ol></li><li>测试第二epoxying双元件应变计<ol><li>焊接金插座连接器（E363 / 0）到引线的自由端。在这一点上，应变计连接到一记录放大器（在下面描述），以测试双元件组件的完整性。 </li><li>衡量一个良好的质量电压电阻计的电阻。元素登记约350Ω的电阻。在这一点上与新鲜焊料重焊不充分的连接。 </li><li>如果焊接连接和双素体被认为是令人满意的，修剪掉任何剩余的元素影片。 </li><li>绝缘上的元件的焊盘的焊点有一层薄薄的两部分组成的，用于硅橡胶的环氧树脂（P / N:E211）。为获得最佳效果，部分地固化该树脂为20 - 30分钟之前，应用（ 图1E）。 </li></ol></li><li>封装在硅双晶应变计<ol><li>切成三片0.5毫米厚的硅树脂片材（P / N:20-20）到15mm 2和c瘦用蒸馏水硅氧烷。切割一块硅薄片的成U字形，以便适应最终的双元件装配而不会变形包封硅氧烷（ 图1F）。 </li><li>涂层的缺口无硅酮片与透明的硅粘合剂的内表面上。 </li><li>夹在中间的双元件组件的槽口和对准的外板内，然后轻轻地压出任何过量的有机硅，以及气泡，从中心向外。小心夹住的金属棒材两个块之间的密封组件，放置24小时，以确保均匀的厚度，并且没有变形发生。 </li><li>允许过量的硅保持沿装配和固化的边界。当纸张硅酮修剪成所需的最终尺寸（; 图1G常用6毫米×8 mm）该过量将被删除。 </li></ol></li><li>导线连接器和校准完成<ol><li>加强焊点对个人端子线黄金插座连接器的引线，3毫米（1/8英寸），热 ​​缩管，并在一个塑料电极座（MS363， 图1H）对齐。固定电极底座和电线0.125和0.25英寸直径的热缩管，以防止实验（ 图1I）在断开连接。 </li><li>应变计信号通过一个高增益放大器，桥接（P / N:AMP-01-SG）的处理。连接用电缆配套插头应变计到放大器（363 - SL / 6）来匹配电极座。螺纹瓶盖提供了额外的安全性，在实验过程中保持不间断的信号。 </li><li>调整放大器上的桥，平衡和增益设置，按制造商的指示专门的应变计。粘贴应变片的结尾，其中的电线横退出，以坚硬夹和放置在另一端一条1G的静载荷校准最初由Pascaud和他的同事描述2，16，17。 </li></ol></li></ol> 2，外科手术应变计急性植入<ol><li>动物护理和准备工作: <ol><li>夜食物剥夺试验动物手术植入前（水可随意提供）。 </li><li>深深地麻醉的动物。 Thiobutabarbital（100 - 150毫克/公斤; ip进行大鼠），优选为终端应变植入和实验，由于在大鼠10持续的麻醉效果和胃的反射最小的改动（ 即 ，非存活）。测试没有爪子捏反射来判断麻醉深度。 </li><li>准备大鼠无菌手术的复杂程度取决于实验设计和批准IACUC准则，包括消毒外科手术刀具，刮脸切口部位，应用审核眼膏和消毒所有的外科手术领域。 注意:如果应变计适于长期使用，消毒应变计只与环氧乙烷（气体）灭菌。利用热或化学灭菌技术可能会损坏应变计。 </li></ol></li><li>气管插管终端的实验: <ol><li>对于持续时间长，末端，实验插管的大鼠气管套管，以保持气道畅通。使1 - 从下颌到胸骨切迹下缘的颈部腹侧2厘米正中切口。 </li><li>单独使用钝性分离，在正中线，显露气管底层肩带肌肉。从底层隔离食道气管和地方的气管和食管之间3-0 ethilon缝合循环，形成一个连字。 </li><li>通过一个小incison在膜的两个气管软骨环只是远离甲状腺之间向前方打开气管。将一小片的聚乙烯管（P / N:PE-270），在5mm的长度（和斜角的一端）的进气管并固定到位结扎。 </li><li>把带状肌放回原处，并缝合皮肤覆以3-0 ethilon。 </li></ol></li><li>应变计仪器仪表胃肠道面: <ol><li>主题四个角应变计与4 - 4-0使用在手术前＃14锥度点3/8圆针5厘米长度，或更小的，无菌的丝线。丝缝合线提供的灵活性高的水平，并且更不容易损坏的硅氧烷密封应变计元件。 注:丝线是可以接受的非手术生存和内部应用程序，其中的细菌穿过上皮屏障排汗不是一个风险。在需要存活手术应用中，Prolene线缝合是必要的，以减少感染所固有的丝缝合线的编织布料的纤维的风险。 </li><li>通过切开沿中线腹部皮肤进行剖腹手术。部分腹直肌肌沿连接白线（缺血性），以防止出血。然后做一个非常肤浅的正中切口，在腹膜壁层，以免划破底层腹腔脏器。 </li><li> Exteriorize用生理盐水浸湿棉签的帮助胃。保持胃的位置小心地将它放在盐水浸泡纱布垫在腹部切口的尾端。 </li><li>对准封装应变平行于环形平滑肌纤维的网格。使用先前螺纹缝线（步骤2.3.1），附加计的角落，用＃14锥度点3/8圆针胃体的腹侧浆膜面。为了减少组织损伤和潜在的出血，不使用切削刃的针和不穿孔任何浅表血管的胃的表面上。 </li><li>开始计的缝线图案沿着附近的眼底/语料库边界胃的大弯，然后继续下沿眼底/胼边界朝向小弯。应变计底层浆膜应该既不是松弛也不过分拉伸，以便获得最好的结果。 </li><li>小心用盐水浸泡过的棉签肚子返回到它的解剖位置。 </li><li>在急性模型，exteriorize应变计导致的正中切口的腹壁切口闭合前的尾端。为了操纵该动物或端线连接器的过程中提供应变消除固定的自由电线的动物（ 例如 ，后足）。单独关闭腹直肌肌和腹部皮肤用3-0尼龙线。在慢性模型，皮下固定引线沿大鼠背侧和exteriorize他们的头骨18以上。 </li><li>手术器械后，将动物在立体定位框架，用于支撑头部和提高上部躯干。后者步骤有助于降低再录制过程中抽出鼻唇神器。监测肛温，并在37±1°C的使用反馈控制的加热垫维持。 </li><li>在利用thiobutabarbital麻醉终端实验结束时，动物必须被安乐死与安乐死美国兽医协会（AVMA）指南的方式。 </li></ol></li><li>胃动力录音: <ol><li>放大用任何市售的直流桥式放大器的应变计信号。 </li><li>记录使用任何市售的数据采集系统的图表记录功能的计算机上的DC输出信号。 注:放大器输出的硬拷贝可以通过测谎图表记录产生。 </li></ol></li></ol>胃收缩代表性的测量跟踪脑干刺激<ol><li>曝光脑干及第四脑室<ol><li>手术器械和placem后在立体框架的Thiobutabarbital麻醉动物耳鼻喉科，做一个1.5 - 2厘米中线切开皮肤，从枕骨向颈部的基地。 </li><li>分离结缔组织沿中线加盟底层颈部肌肉的双侧腹部肌肉（肌肉从浅到深的提耳长肌颅部，提耳长肌尾部分，和颈阔肌颅部分）。 </li><li>从枕骨分离提耳后长肌的中线是明确和公开。 </li><li>小心使用钝性分离分离从根本硬膜颈阔肌暴露颅骨的尾部区域。 </li><li>使用新的地下25针仔细分离跟着枕髁两侧延伸枕骨大孔硬膜。 </li><li>使用＃5杜蒙钳去除PIA和脑膜蛛网膜覆第四脑室和揭露脑干。 </li></ol></li><li>管理第四脑室​​促甲状腺激素释放激素或静脉注射硝普钠<ol><li>称重和溶解促甲状腺激素释放激素（TRH），在无菌盐水中，达到50μM的TRH的最终浓度。 </li><li>称重和溶解硝普钠（SNP）中的无菌盐水以达到150μM的SNP的最终浓度。 </li><li>用10微升注射器，辖2微升的TRH（最终剂量等于100皮摩尔）脑干第四脑室的背水面，以方便记录胃收缩。 </li><li>使用无菌注射器和27G的针头，辖150微摩尔/千克的SNP通过尾静脉，以方便记录胃放松。 </li></ol></li></ol>

<ol>
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Liver Physiol. 290 (1), G74-G82 (2005).</li><li>Bass, P., Wiley, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contractile+force+transducer+for+recording+muscle+activity+in+unanesthetized+animals.">Contractile force transducer for recording muscle activity in unanesthetized animals.</a> J. Appl. Physiol. 32 (4), 567-570 (1972).</li><li>Holmes, G. M., Browning, K. N., Tong, M., Qualls-Creekmore, E., Travagli, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vagally+mediated+effects+of+glucagon-like+peptide+1:+in+vitro+and+in+vivo+gastric+actions.">Vagally mediated effects of glucagon-like peptide 1: in vitro and in vivo gastric actions.</a> J. Physiol. 587 (19), 4749-4759 (2009).</li><li>Tong, M., Qualls-Creekmore, E., Browning, K. N., Travagli, R. A., Holmes, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+spinal+cord+injury+in+rats+diminishes+vagally-mediated+gastric+responses+to+cholecystokinin-8s.">Experimental spinal cord injury in rats diminishes vagally-mediated gastric responses to cholecystokinin-8s.</a> Neurogastroenterol. Motil. 23 (2), e69-e79 (2011).</li><li>Miyano, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+vagus+nerve+in+the+migrating+motor+complex+and+ghrelin-+and+motilin-induced+gastric+contraction+in+suncus.">The role of the vagus nerve in the migrating motor complex and ghrelin- and motilin-induced gastric contraction in suncus.</a> PLoS ONE. 8 (5), e64777 (2013).</li><li>Holmes, G. M., Rogers, R. C., Bresnahan, J. C., Beattie, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thyrotropin-releasing+hormone+(TRH)+and+CNS+regulation+of+anorectal+motility+in+the+rat.">Thyrotropin-releasing hormone (TRH) and CNS regulation of anorectal motility in the rat.</a> J Auton. Nerv. Syst. 56, 8-14 (1995).</li><li>Ormsbee, H. S., Bass, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastroduodenal+motor+gradients+in+the+dog+after+pyloroplasty.">Gastroduodenal motor gradients in the dog after pyloroplasty.</a> Am. J. Physiol. 230, 389-397 (1976).</li><li>Fukuda, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+gastric+motor+activity+after+abdominal+surgery+in+rats.">Impaired gastric motor activity after abdominal surgery in rats.</a> Neurogastroenterol. Motil. 17 (2), 245-250 (2005).</li><li>Browning, K. N., Babic, T., Holmes, G. M., Swartz, E., Travagli, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+re-evaluation+of+the+specificity+of+action+of+perivagal+capsaicin.">A critical re-evaluation of the specificity of action of perivagal capsaicin.</a> J. Physiol. 591 (6), 1563-1580 (2013).</li></ol>

The authors declare that they have no competing financial interests. The suppliers listed in this manuscript are provided for reference only.

这里介绍的方法允许单个实验室制造灵敏微型应变计用于生物应用，包括但不限于，胃肠道蠕动在小的实验室动物。由于这些应变片的商业化生产已经停止，实验室研究胃肠功能仅限于可能不允许全方位的实验应用可用的其他技术。这个报告对先前所描述的技术15的更新和更详细的描述。文本和相应的视频专门解决方案，使我们的发展制造工艺的掌握和认识过程中常见的陷阱。 <p class="...

在体内胃肠道（GI）在多个实验条件蠕动的记录仍然是一个强大的工具，了解必要的营养平衡的基本正常和病理生理过程的实验研究。传统上，无数的实验方法，用一些相似的临床实践1，已经采用直接量化的改变胃肠道的收缩率2-5，腔内压力6，7，或不可吸收标志物8的胃肠道转运，发现9或稳定同位素10-12。每种技术都有其独特的优点和缺点，这在文献中先前已经解决。例如，气囊测压的效用量化的压力变化受到质疑由于气囊材料的固有遵守而不可吸收的标记物胃肠恢复需要安乐死试验阴性特质l对于单个数据点。最近，一个微型动脉压导管的应用和验证有报道称，提供了在大鼠和小鼠3监测胃收缩的非手术方法。而一个orogastrically放置压力传感器有效地避免侵入性手术过程中消除了对胃肠功能混杂变量，这种方法只适用于麻醉制剂。此外，由于缺乏视觉引导不允许胃的特定区域内的换能器的相一致的位置。因此，本申请中限定为胃或结肠因为可视化，再加上相对较硬的传感器导线，十二指肠内或回肠不是一个选项。 同样，生物磁交流电biosusceptometry（ACB）技术已被证实为胃肠道收缩分析4。而ACB技术提供了一种非侵入性的APproach测定胃肠道收缩，ACB患有中，类似的限制，利用摄入磁检测介质中，没有允许在胃肠道的特定区域的精确记录。这一限制可以通过对磁性标记的手术植入来克服。尽管如此，ACB技术的必要条件是该动物被麻醉，进行数据收集。 Ultrasonomicrometry已经用于一些GI，以便采取尺寸小，空间的优势研究13，第14，和压电晶体发射器/接收器的时间的优点。胃平滑肌收缩波都没有的高频事件和发生在大约3的速率 - 5个周期/分钟。因此，声纳微测量的时间的优点可能是不必要的，以证明费用。此外，尽管线性运动被准确地测量与声纳微测量，限制已经提出关于准确胃肠数据解释可能导致植入晶体14的数量不足的。 基于Bass和同事2,15的原创设计该协议的可视化更充分地记载了一步一步的制造和试验应用缩影，双晶应变计是具有高灵敏度和灵活性，在整个胃肠记录平滑肌收缩道。应变计元件的尺寸适合于任何啮齿类动物的应用，因为灵敏度成品应变计和尺寸是最依赖于硅薄片封装的元件。这些应变片很容易适应的麻醉和自由活动的实验动物模型急性和慢性的应用程序，从而提供量化平滑肌收缩单一技术。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Strain gage element</td>
			<td>Micro-Measurements (Vishay Product Group)</td>
			<td>EA-06-031-350</td>
			<td>&#160;Linear pattern, foil, stress analysis strain gage (2 required) 
			<a href="http://www.vishaypg.com/micro-measurements/">www.vishaypg.com/micro-measurements/</a> 
			or 
			<a href="http://www.vishaypg.com/docs/11070/031ce.pdf">http://www.vishaypg.com/docs/11070/031ce.pdf</a></td>
		</tr>
		<tr>
			<td>epoxy-phenolic adhesive</td>
			<td>M-bond 610</td>
			<td>General purpose adhesive for bonding strain gage elements</td>
			<td><a href="http://www.vishaypg.com/docs/11024/wirecable.pdf">http://www.vishaypg.com/docs/11024/wirecable.pdf</a></td>
		</tr>
		<tr>
			<td>3 conductor insulated wire</td>
			<td>336-FTE</td>
			<td>Fine gage, flexible general purpose wire</td>
			<td><a href="http://www.vishaypg.com/docs/11024/wirecable.pdf">http://www.vishaypg.com/docs/11024/wirecable.pdf</a></td>
		</tr>
		<tr>
			<td>Flux and rosin solvent kit</td>
			<td>FAR-2 M-Flux AR kit</td>
			<td>Liquid solder flux</td>
			<td><a href="http://www.vishaypg.com/docs/11023/soldacce.pdf">http://www.vishaypg.com/docs/11023/soldacce.pdf</a></td>
		</tr>
		<tr>
			<td>Solder</td>
			<td>361A-20R-25</td>
			<td>Optimized and recommended for strain gage applications</td>
			<td><a href="http://www.vishaypg.com/docs/11023/soldacce.pdf">http://www.vishaypg.com/docs/11023/soldacce.pdf</a></td>
		</tr>
		<tr>
			<td>Gold socket connector</td>
			<td>PlasticsOne</td>
			<td>E363/0</td>
			<td>Socket contact for electrode pedestal 
			<a href="http://www.plastics1.com/PCR/Catalog/Item.php?item=407">http://www.plastics1.com/PCR/Catalog/Item.php?item=407</a></td>
		</tr>
		<tr>
			<td>Electrode pedestal</td>
			<td>MS363</td>
			<td>Secure platform for wire contacts</td>
			<td><a href="http://www.plastics1.com/PCR/Catalog/Item.php?item=499">http://www.plastics1.com/PCR/Catalog/Item.php?item=499</a></td>
		</tr>
		<tr>
			<td>6-wire cable</td>
			<td>363 PLUG W/VINYL SL/6</td>
			<td>Pre-fabricated vinyl-coated cable (in customized lengths) with plug adaptor to match electrode pedestal and tinned solder lugs on terminal end</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone rubber casting compound</td>
			<td>EIS electrical products</td>
			<td>Elan Tron E211</td>
			<td>Potting medium for gage/wire solder joints 
			<a href="http://www.eis-inc.com/">http://www.eis-inc.com</a></td>
		</tr>
		<tr>
			<td>HOTweezers</td>
			<td>Meisei Corporation</td>
			<td>Model 4B</td>
			<td>Wire insulation strippers 
			<a href="http://www.impexron.us/">http://www.impexron.us</a></td>
		</tr>
		<tr>
			<td>Soldering station</td>
			<td>Weller (Apex Tool Group)</td>
			<td>WES 51</td>
			<td>High quality soldering equipment 
			<a href="http://www.apexhandtools.com/weller/index.cfm">http://www.apexhandtools.com/weller/index.cfm</a></td>
		</tr>
		<tr>
			<td>Available through http://www.eis-inc.com or http://www.amazon.com</td>
		</tr>
		<tr>
			<td>Silicone sheet</td>
			<td>Trelleborg Sealing Solutions Northborough-Life Sciences</td>
			<td>Pharmelast 20-20</td>
			<td>Encapsulating strain gauge elements 
			10 B Forbes Road Northborough, MA 01532 (800) 634-2000</td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>Experimetria Ltd</td>
			<td>AMP-01-SG</td>
			<td> 
			<a href="http://experimetria.com/Biological_amplifiers.php">http://experimetria.com/Biological_amplifiers.php</a></td>
		</tr>
	</tbody>
</table>

fabrication and implantation of miniature dual-element strain gages for measuring in vivo gastrointestinal contractions in rodents.

Gastrointestinal dysfunction remains a major cause of morbidity and mortality. Indeed, gastrointestinal (GI) motility in health and disease remains an area of productive research with over 1,400 published animal studies in just the last 5 years. Numerous techniques have been developed for quantifying smooth muscle activity of the stomach, small intestine, and colon. In vitro and ex vivo techniques offer powerful tools for mechanistic studies of GI function, but outside the context of the integrated systems inherent to an intact organism. Typically, measuring in vivo smooth muscle contractions of the stomach has involved an anesthetized preparation coupled with the introduction of a surgically placed pressure sensor, a static pressure load such as a mildly inflated balloon or by distending the stomach with fluid under barostatically-controlled feedback. Yet many of these approaches present unique disadvantages regarding both the interpretation of results as well as applicability for in vivo use in conscious experimental animal models. The use of dual element strain gages that have been affixed to the serosal surface of the GI tract has offered numerous experimental advantages, which may continue to outweigh the disadvantages. Since these gages are not commercially available, this video presentation provides a detailed, step-by-step guide to the fabrication of the current design of these gages. The strain gage described in this protocol is a design for recording gastric motility in rats. This design has been modified for recording smooth muscle activity along the entire GI tract and requires only subtle variation in the overall fabrication. Representative data from the entire GI tract are included as well as discussion of analysis methods, data interpretation and presentation.

从Thiobutabarbital-麻醉大鼠的代表性数据示于图2中的顶部曲线表示的促甲状腺激素释放激素脑干给药（TRH，100皮摩尔），一种已知的能动性增强肽3,19期间从大鼠胃体收缩。它示出了现有的增加，相位胃平滑肌活性的基线收缩。注:在胃的收缩这些峰分析跟踪方法由安庶庇和低音20原配方运动指数=（N + 1×1）+（N 2×2）+（N 3×4）+（N 4个 x8） <...

Watch this Scientific Journal Video about Fabrication and Implantation of Miniature Dual-element Strain Gages for Measuring In Vivo Gastrointestinal Contractions in Rodents. at JoVE.com

Fabrication and Implantation of Miniature Dual-element Strain Gages for Measuring In Vivo Gastrointestinal Contractions in Rodents.

The in vivo measurement of smooth muscle contractions along the gastrointestinal tract of laboratory animals remains a powerful, though underutilized, technique. Flexible, dual element strain gages are not commercially available and require fabrication. This protocol describes the construction of reliable, inexpensive strain gages for acute or chronic implantation in rodents.

的微型双元应变计测量加工和植入<em&gt;体内</em&gt;胃肠收缩鼠害。

Gastrointestinal dysfunction remains a major cause of morbidity and mortality. Indeed, gastrointestinal (GI) motility in health and disease remains an ...

fabrication-implantation-miniature-dual-element-strain-gages-for

Research

JoVE Journal

Bioengineering

Fabrication and Implantation of Miniature Dual-element Strain Gages for Measuring In Vivo Gastrointestinal Contractions in Rodents.

10.3K 次观看.  Penn State University College of Medicine. The in vivo measurement of smooth muscle contractions along the gastrointestinal tract of laboratory animals remains a powerful, though underutilized, technique. Flexible, dual element strain gages are not commercially available and require fabrication. This protocol describes the construction of reliable, inexpensive strain gages for acute or chronic implantation in rodents.

视频: Fabrication and Implantation of Miniature Dual-element Strain Gages for Measuring In Vivo Gastrointestinal Contractions in Rodents.

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

无试剂检测核酸，蛋白质和小分子的电化学DNA生物传感器的制备

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

科研

生物工程

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

蛋白质和细胞坐月子创建二维图案衬底

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

全球舒张功能通过静脉流的通过参数化舒张形式主义运动学建模为基础的定量分析

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

A step-by-step protocol for the inter-positional placement of Tissue Engineered Vessels (TEVs) into the carotid artery of a sheep using end-to-end anastomosis and real-time digital assessment in vivo until animal sacrifice.

手术技术组织工程血管移植和后续的植入<em&gt;在体内</em&gt;监控

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

Gradient Strain Chip for Stimulating Cellular Behaviors in Cell-laden Hydrogel

Artificial guidance for cellular alignment is a hot topic in the field of tissue engineering. Most of the previous research has investigated single strain-induced cellular alignment on a cell-laden hydrogel by using complex experimental processes and mass controlling systems, which are usually associated with contamination issues. Thus, in this article, we propose a simple approach to building a gradient static strain using a fluidic chip with a plastic PDMS cover and a UV transparent glass substrate for the stimulation of cellular behavior in a 3D hydrogel. Overloading photo-patternable cell prepolymer in the fluidic chamber can generate a convex curved PDMS membrane on the cover. After UV crosslinking, through a concentric circular micropattern under the curved PDMS membrane, and buffer washing, a microenvironment for investigating cell behaviors under a variety of gradient strains is self-established in a single fluidic chip, without external instruments. NIH3T3 cells were demonstrated after observing the change in the cellular alignment trend under geometry guidance, in cooperation with strain stimulation, which varied from 15 - 65% on hydrogels. After a 3-day incubation, the hydrogel geometry dominated the cell alignment under low compressive strain, where cells aligned along the hydrogel elongation direction under high compressive strain. Between these, the cells showed random alignment due to the dissipation of the radical guidance of hydrogel elongation and the geometry guidance of the patterned hydrogel.

This article introduces a simple approach to providing non-continuous gradient static strains on a concentric cell-laden hydrogel to regulate cell alignment for tissue engineering.

在单元格拉丹水凝胶中刺激细胞行为的应变梯度芯片

Artificial guidance for cellular alignment is a hot topic in the field of tissue engineering. Most of the previous research has investigated single ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

一个<em&gt;体外</em鼠类的椎间盘&gt;器官培养模型

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

用微计算机断层扫描法对无节段3D 组织学进行固定染色、全尺寸、毫米度试样的刚性嵌入

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

Fabrication of Ti3C2 MXene Microelectrode Arrays for In Vivo Neural Recording

Implantable microelectrode technologies have been widely used to elucidate neural dynamics at the microscale to gain a deeper understanding of the neural underpinnings of brain disease and injury. As electrodes are miniaturized to the scale of individual cells, a corresponding rise in the interface impedance limits the quality of recorded signals. Additionally, conventional electrode materials are stiff, resulting in a significant mechanical mismatch between the electrode and the surrounding brain tissue, which elicits an inflammatory response that eventually leads to a degradation of the device performance. To address these challenges, we have developed a process to fabricate flexible microelectrodes based on Ti3C2 MXene, a recently discovered nanomaterial that possesses remarkably high volumetric capacitance, electrical conductivity, surface functionality, and processability in aqueous dispersions. Flexible arrays of Ti3C2 MXene microelectrodes have remarkably low impedance due to the high conductivity and high specific surface area of the Ti3C2 MXene films, and they have proven to be exquisitely sensitive for recording neuronal activity. In this protocol, we describe a novel method for micropatterning Ti3C2 MXene into microelectrode arrays on flexible polymeric substrates and outline their use for in vivo micro-electrocorticography recording. This method can easily be extended to create MXene electrode arrays of arbitrary size or geometry for a range of other applications in bioelectronics and it can also be adapted for use with other conductive inks besides Ti3C2 MXene. This protocol enables simple and scalable fabrication of microelectrodes from solution-based conductive inks, and specifically allows harnessing the unique properties of hydrophilic Ti3C2 MXene to overcome many of the barriers that have long hindered the widespread adoption of carbon-based nanomaterials for high-fidelity neural microelectrodes.

Fabrication of Ti3C2 MXene Microelectrode Arrays for In Vivo Neural Recording

We describe here a method for fabricating Ti3C2 MXene microelectrode arrays and utilizing them for in vivo neural recording.

用于Vivo神经记录的 Ti3C2 MXene 微电极阵列的制造

Implantable microelectrode technologies have been widely used to elucidate neural dynamics at the microscale to gain a deeper understanding of the neural ...

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct libraries of millions of genetically distinct strains, screening for a desired phenotype remains a significant obstacle. With existing screening techniques, there is a tradeoff between information output and throughput, with high-throughput screening typically being performed on one product of interest. Therefore, we present an approach to accelerate strain screening by adapting single cell RNA sequencing to isogenic picoliter colonies of genetically engineered yeast strains. To address the unique challenges of performing RNA sequencing on yeast cells, we culture isogenic yeast colonies within hydrogels and spheroplast prior to performing RNA sequencing. The RNA sequencing data can be used to infer yeast phenotypes and sort out engineered pathways. The scalability of our method addresses a critical obstruction in microbial engineering.

A bottleneck in the &#8216;design-build-test&#8217; cycle of microbial engineering is the speed at which we can perform functional screens of strains. We describe a high-throughput method for strain screening applied to hundreds to thousands of yeast cells per experiment that utilizes droplet-based RNA sequencing.

高通量酵母菌株，带基于滴滴的RNA测序

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct ...

Measuring 3D In-vivo Shoulder Kinematics using Biplanar Videoradiography

The shoulder is one of the human body's most complex joint systems, with motion occurring through the coordinated actions of four individual joints, multiple ligaments, and approximately 20 muscles. Unfortunately, shoulder pathologies (e.g., rotator cuff tears, joint dislocations, arthritis) are common, resulting in substantial pain, disability, and decreased quality of life. The specific etiology for many of these pathologic conditions is not fully understood, but it is generally accepted that shoulder pathology is often associated with altered joint motion. Unfortunately, measuring shoulder motion with the necessary level of accuracy to investigate motion-based hypotheses is not trivial. However, radiographic-based motion measurement techniques have provided the advancement necessary to investigate motion-based hypotheses and provide a mechanistic understanding of shoulder function. Thus, the purpose of this article is to describe the approaches for measuring shoulder motion using a custom biplanar videoradiography system. The specific objectives of this article are to describe the protocols to acquire biplanar videoradiographic images of the shoulder complex, acquire CT scans, develop 3D bone models, locate anatomical landmarks, track the position and orientation of the humerus, scapula, and torso from the biplanar radiographic images, and calculate the kinematic outcome measures. In addition, the article will describe special considerations unique to the shoulder when measuring joint kinematics using this approach.

Biplane videoradiography can quantify shoulder kinematics with a high degree of accuracy. The protocol described herein was specifically designed to track the scapula, humerus, and the ribs during planar humeral elevation, and outlines the procedures for data collection, processing, and analysis. Unique considerations for data collection are also described.

使用双平面影像学测量 3D 体内肩部运动学

The shoulder is one of the human body's most complex joint systems, with motion occurring through the coordinated actions of four individual joints, ...