这项工作是由比利时联邦政府（IUAP P6/28），研究基金会，富兰德（FWO）（G.0565.07和G.0686.09），安斯泰来欧洲基金会奖2009年和鲁汶大学研究理事会的拨款，支持（，EF/95/010 GOA 2009/07和PFV/10/006）。 PU，我们正在研究基金会龙龙（FWO）的博士后研究员。 MB是居里夫人研究员。 DDR的基本临床研究员的FWO。

1。实验动物<ol><li>动物（小鼠，大鼠）被安置在一个专门的动物设施，12个小时光暗周期和自由采食获得水和食物颗粒标准。这两种动物的年龄和性别，是重要的参数，根据需要，应该标准化。我们通常进行膀胱内压在10 - 12周龄雌性动物5,6。 </li><li>所有的动物进行了实验，根据与欧洲联盟共同体理事会准则和当地的伦理委员会的批准。 </li></ol> 2。麻醉<ol><li>异氟醚麻醉用于执行小的外科手术，因为它很容易的剂量，由于其短的半衰期。适当的废气清除，必要时使用异氟醚。 <ol class="lalpha"><li>动物被放置在一个封闭的盒子，用异氟烷在纯氧气中的5％（1升/分钟）毒气。 </li><li>诱导后，麻醉在适当的升埃维尔是由一个小的掩模（切口10毫升注射器）由0.8 - 1升/分钟氧气与1 - 1.5％的异氟烷​​维持。 </li></ol></li><li>对于小鼠，氨基甲酸乙酯麻醉过程中使用功能的录音（见“红外线）。乌拉坦麻醉期间使用的功能的录音，因为，在大多数其他麻醉药相反，排尿反射不抑制该麻醉剂4。氨基甲酸乙酯1.2克/公斤体重，皮下给药（SC）的前60分钟开始的录音。如果有必要，以获得适当的麻醉所需的剂量的氨基甲酸乙酯可通过施用额外的剂量（0.1克/公斤）皮下滴定。然而，应当指出，该额外的麻醉可能会改变排尿效率，进而排尿频率7,8。因此，应密切监测膀胱录音和观察到显着偏离的控制行为便会放弃。麻醉药不应该被管理，而测试化合物应用的数据，以避免误解。 </li></ol> 3。外科手术 - 膀胱导管植入术<ol><li>外科器械在使用前在高压釜中灭菌。剃须动物的腹部，用70％乙醇消毒，并执行低中线剖腹手术以暴露膀胱。 </li><li>在手术显微镜下，放置在膀胱圆顶使用非吸收性的，单纤维缝合线（尺寸6-0）的荷包缝合。 </li><li>执行钱袋内的一个小膀胱造瘘（我们优先使用一个地下18针）和PE 50聚乙烯导管插入，用一个小袖结束时（通过仔细加热管），通过此孔。在我们的手中，更薄管（PE）有一个更高，更可变电阻。管子可以通过将其在乙醇中70％的溶液中，或在高压釜中灭菌消毒。在注入之前，管子应用生理盐水冲洗。 </li><li>保护钱包海峡的安全ING管周围的外科医生的结。拉出导管轻轻向外直到钟形尖端的定位下缝合。按对缝合了一块18 G导管，以防止泄漏。 </li><li>一个小体积的生理盐水（100 - 200μL）注入轻轻地检查是​​否有泄漏。如果存在泄漏，额外的缝合线应放置的位置。 </li><li>关闭腹肌使用单丝缝合线，离开的通道的导管的上部中的切口。 </li><li>隧道的肩胛间的区域使用的中空的金属棒的管，从而防止动物咬在实验过程中的管。 </li><li>在皮肤上使用非吸收性，单丝缝线关闭。植入的导管，用生理盐水冲洗，以检查其透气性。 </li><li>在动物唤醒，止痛的皮下注射（丁丙诺啡，0.05毫克/千克）。 </li></ol> 4。安装和膀胱测压<ol><li>根据实验questi（种），膀胱内压（在清醒的约束或放纵）或氨基甲酸乙酯麻醉的动物。而，往往一种内敛的环境中保持冷静清醒大鼠膀胱测压是完全可行的，我们通常进行性膀胱测压时，在乌拉坦麻醉（1.2毫克/公斤），皮下利用老鼠3,5,6,9。 </li><li>在麻醉的小鼠中，不断地监测和体温保持在36.5℃，±0.2°C的温度探针和加热灯通过。 </li><li>每次实验前根据制造商的说明进行校准设置。 </li><li>植入的导管连接的路厄氏存根了3路抽头的一侧上和其他（哈佛器具）的输液泵，其被连接到所述压力传感器（Edwards Lifesciences公司）。通过一个放大器（78534c显示器，惠普）的数据采集系统（DI-730-USB，DATAQ文书）和一个共同的压力换能器被连接柴红斌。 Windaq /精简版软件可用于记录。 </li><li>开始的输液泵，使在例如生理盐水或PBS输注，以100μl/分钟（大鼠）或20μl/ min的（小鼠）。作为这种膀胱将被填充以恒定的速率，而记录膀胱内压。 </li><li> 图5示出了典型的压迹：输液过程中，有在膀胱是一个缓慢的压力累积，直到达到一定的体积/压力，排尿阈值。一旦达到这个阈值，膀胱会收缩，随后尿道括约肌会打开，允许通过尿液通过尿道。因此，被清空，膀胱收缩将停止和压力将再次下降的“基底”电平。 </li><li>由于大鼠置于代谢笼及以上的数字平衡，可以同时测量排尿量，提供空隙体积有关下列内容的信息。由于小排尿量英里CE，它可以是非常具有挑战性的使用该系统。因此，排尿量用小滤纸称重前，排尿后确定。 </li><li>通常情况下，我们让系统平衡30分钟，然后30分钟内由记录。然后，药物可以全身给药或膀胱内注射和另一个30分钟记录。 </li><li>记录的数据可以导出为csv文件，可以导入到一个专门的软件进行定量分析。我们通常使用地（OriginLab公司，MA，USA）。 </li><li>实验结束后，处死动物通过颈椎脱臼（小鼠）或CO 2的中毒（大鼠）。 </li></ol> 5。代表性的成果<img alt="图1" src="/files/ftp_upload/3869/3869fig1.jpg" /> 图1。剖腹探查术概述。 A）将大鼠仰卧位。 B）刮胡子，防腐准备手术部位。 C）切开皮肤。 D）切口Øf的腹部肌肉，膀胱曝光。 <img alt="图2" src="/files/ftp_upload/3869/3869fig2.jpg" /> 图2：荷包缝合。 <img alt="图3" src="/files/ftp_upload/3869/3869fig3.jpg" /> 图3。导管植入术。 <img alt="图4" src="/files/ftp_upload/3869/3869fig4.jpg" /> 图4的隧道的导管。 在有意识的大鼠和麻醉的小鼠的膀胱内灌注生理盐水期间获得的压力测量值的实施例示于图5。多个参数可以从压力信号（ 例如，在intercontractile的时间间隔，基准压力和临界压力）萃取。对于这些参数的完整描述，请参阅安德森等人。（参考文献4）和吉山等人。（<html>文献10）。 最近，我们已使用大鼠膀胱内压，以确定的分子靶标，芥子油（MO），高活性的化合物，已经长期使用在实验模型中的炎症和痛觉过敏的内脏器官，如膀胱11,12。膀胱内输注的10mM MO诱导强烈增加在排尿频率（减少在intercontractile间隔）在野生型小鼠（ 图6A，B）和空隙体积6减少。有趣的是，MO诱导小鼠的MO受体TRPA1类似的变化。与此相反，MO诱发弱得多TRPV1基因敲除小鼠较野生型小鼠膀胱参数的变化，并在Trpa1/Trpv1基因敲除小鼠没有任何影响。降钙素基因相关肽（CGRP）释放的测量，这些数据表明表明，TRPV1在内脏刺激引起的MO可能发挥了关键的作用。 ve_content“&gt; <img alt="图5" src="/files/ftp_upload/3869/3869fig5.jpg" /> 图5代表记录在有意识的雌性大鼠（A）和在麻醉的雌性小鼠（B）的膀胱内压的痕迹。被定义为“基线压力”（红色箭头）的最低压力。填充阶段的结尾处的压力被标记为与蓝色箭头。这些点之间的压力差除以注入流体的体积就可以计算出遵守的膀胱壁（合规=注入量/（阈值压力 - 基准压力）。的“intercontractile的间隔”（ICI）之间的时间两个排尿收缩。 <img alt="图6" src="/files/ftp_upload/3869/3869fig6.jpg" /> 图6。膀胱内应用的芥子油的膀胱内压在野生型和基因敲除小鼠TRPA1，TRPV1受体和Trpa1/Trpv1模式。（A）记录在WT，TRPA1 KO，TRPV1 KO和Trpa1/Trpv1的的基因敲除小鼠在输注生理盐水和10毫米MO膀胱内压力变化的代表性的例子。 （B）的时间过程和膀胱内输注MO期间前的平均瞬时排尿频率。对于所有的小鼠中，数据被归一化到盐水输注期间获得的平均频率。这些数据是适应从Everaerts 等。（参考文献6），爱思唯尔的许可。

<ol>
	<li>Everaerts, W., Gevaert, T., Nilius, B., De Ridder, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+origin+of+bladder+sensing:+Tr(i)ps+in+urology.">On the origin of bladder sensing: Tr(i)ps in urology.</a> Neurourol. Urodyn. 27, 264-2673 (2008).</li><li>Fry, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+and+their+use+in+understanding+lower+urinary+tract+dysfunction.">Animal models and their use in understanding lower urinary tract dysfunction.</a> Neurourol. Urodyn. 29, 603-608 (2010).</li><li>Gevaert, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+the+transient+receptor+potential+cation+channel+TRPV4+impairs+murine+bladder+voiding.">Deletion of the transient receptor potential cation channel TRPV4 impairs murine bladder voiding.</a> J. Clin. Invest. 117, 3453-3462 (2007).</li><li>Andersson, K. E., Soler, R., Fullhase, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+for+urodynamic+investigation.">Rodent models for urodynamic investigation.</a> Neurourol. Urodyn. 30, 636-646 (2011).</li><li>Everaerts, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+the+cation+channel+TRPV4+improves+bladder+function+in+mice+and+rats+with+cyclophosphamide-induced+cystitis.">Inhibition of the cation channel TRPV4 improves bladder function in mice and rats with cyclophosphamide-induced cystitis.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 19084-19089 (2010).</li><li>Everaerts, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+capsaicin+receptor+TRPV1+is+a+crucial+mediator+of+the+noxious+effects+of+mustard+oil.">The capsaicin receptor TRPV1 is a crucial mediator of the noxious effects of mustard oil.</a> Curr. Biol. 21, 316-321 (2011).</li><li>Yoshiyama, M., Roppolo, J. R., Thor, K. B., de Groat, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+LY274614,+a+competitive+NMDA+receptor+antagonist,+on+the+micturition+reflex+in+the+urethane-anaesthetized+rat.">Effects of LY274614, a competitive NMDA receptor antagonist, on the micturition reflex in the urethane-anaesthetized rat.</a> Br. J. Pharmacol. 110, 77-86 (1993).</li><li>Yoshiyama, M., Roppolo, J. R., de Groat, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+MK-801+on+the+micturition+reflex+in+the+rat--possible+sites+of+action.">Effects of MK-801 on the micturition reflex in the rat--possible sites of action.</a> J. Pharmacol. Exp. Ther. 265, 844-850 (1993).</li><li>Boudes, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Characterization+of+a+Chronic+Cyclophosphamide-Induced+Overactive+Bladder+Model+in+mice.">Functional Characterization of a Chronic Cyclophosphamide-Induced Overactive Bladder Model in mice.</a> Neurourol. Urodyn. , (2011).</li><li>Yoshiyama, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex-related+differences+in+activity+of+lower+urinary+tract+in+response+to+intravesical+acid+irritation+in+decerebrate+unanesthetized+mice.">Sex-related differences in activity of lower urinary tract in response to intravesical acid irritation in decerebrate unanesthetized mice.</a> Am. J. Physiol. Regul. Integr. Comp. Physiol. 295, R954-R960 (2008).</li><li>McMahon, S. B., Abel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+for+the+study+of+visceral+pain+states:+chronic+inflammation+of+the+chronic+decerebrate+rat+urinary+bladder+by+irritant+chemicals.">A model for the study of visceral pain states: chronic inflammation of the chronic decerebrate rat urinary bladder by irritant chemicals.</a> Pain. 28, 109-127 (1987).</li><li>Du, S., Araki, I., Yoshiyama, M., Nomura, T., Takeda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+receptor+potential+channel+A1+involved+in+sensory+transduction+of+rat+urinary+bladder+through+C-fiber+pathway.">Transient receptor potential channel A1 involved in sensory transduction of rat urinary bladder through C-fiber pathway.</a> Urology. 70, 826-831 (2007).</li><li>Streng, T., Santti, R., Talo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Similarities+and+differences+in+female+and+male+rat+voiding.">Similarities and differences in female and male rat voiding.</a> Neurourol. Urodyn. 21, 136-141 (2002).</li><li>Igawa, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystometric+findings+in+mice+lacking+muscarinic+M2+or+M3+receptors.">Cystometric findings in mice lacking muscarinic M2 or M3 receptors.</a> J. Urol. 172, 2460-2464 (2004).</li><li>Schroder, A., Newgreen, D., Andersson, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detrusor+responses+to+prostaglandin+E2+and+bladder+outlet+obstruction+in+wild-type+and+Ep1+receptor+knockout+mice.">Detrusor responses to prostaglandin E2 and bladder outlet obstruction in wild-type and Ep1 receptor knockout mice.</a> J. Urol. 172, 1166-1170 (2004).</li><li>Chen, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+the+lower+urinary+tract+in+mice+lacking+alpha1d-adrenoceptor.">Function of the lower urinary tract in mice lacking alpha1d-adrenoceptor.</a> J. Urol. 174, 370-374 (2005).</li><li>May, V., Vizzard, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bladder+dysfunction+and+altered+somatic+sensitivity+in+PACAP-/-+mice.">Bladder dysfunction and altered somatic sensitivity in PACAP-/- mice.</a> J. Urol. 183, 772-779 (2010).</li><li>Thorneloe, K. S., Meredith, A. L., Knorn, A. M., Aldrich, R. W., Nelson, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urodynamic+properties+and+neurotransmitter+dependence+of+urinary+bladder+contractility+in+the+BK+channel+deletion+model+of+overactive+bladder.">Urodynamic properties and neurotransmitter dependence of urinary bladder contractility in the BK channel deletion model of overactive bladder.</a> Am. J. Physiol. Renal. Physiol. 289, 604-610 (2005).</li></ol>

没有利益冲突的声明。

这里介绍的允许性膀胱测压技术在动物模型体内测量膀胱功能执行。老鼠可能是最常用的动物模型。的小鼠更难以处理，但提供的优势，利用基因操纵的动物。由于技术难度使用意识的小鼠，这往往是非常活跃的松动植入导管在腹腔内的压力，可能会影响膀胱内压力的变化导致的，我们建议在全膀胱压力，让他们用氨基甲酸乙酯麻醉协议。当然，有镇静小鼠的好处，对麻醉药的影响进行权?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 企业名称，公司， </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>聚氨酯</td><td>聚氨酯，Sigma-Aldrich公司</td><td> 315419 </td><td> 2B组致癌物质</td></tr><tr><td>异氟醚</td><td> Isoba，先灵葆雅动物保健</td><td></td><td></td></tr><tr><td>聚乙烯导管</td><td> Intramedic聚乙烯管材PE50，Becton Dickinson公司</td><td> 427411 </td><td></td></tr><tr><td>手术显微镜</td><td> OP-MI 6，卡尔·蔡司</td><td> OP-MI 6 </td><td></td></tr><tr><td>荷包缝合</td><td>普理灵，爱惜康6/0 </td><td> 8610H </td><td></td></tr><tr><td>筋膜和皮肤缝合</td><td> Ethilon 4/0或5/0，爱惜康</td><td> 662G或661G </td><td></td></tr><tr><td>术后镇痛</td><td> Temgesic，先灵葆雅动物保健</td><td></td><td>大鼠0.05毫克/千克的剂量</td></tr><tr><td>扩音器</td><td> 78534c显示器，惠普</td><td></td><td></td></tr><tr><td>分析天平和平衡的数据采集软件</td><td> FZ 300I，A＆D </td><td> FZ-300I </td><td></td></tr><tr><td>输液泵</td><td>泵33，哈佛装置</td><td> HA33 </td><td></td></tr><tr><td>膀胱测压记录系统</td><td> DATAQ仪器，DI-730的系列和Windaq /精简版</td><td> DI-730-USB Windaq /精简版</td><td></td></tr><tr><td>温度注册</td><td>福禄克52 KJ温度计</td><td> 52 KJ </td><td></td></tr><tr><td>压力传感器</td><td>爱德华生命科学，压力监测组</td><td> T322247A </td><td></td></tr></tbody></table>

the use of cystometry in small rodents: a study of bladder chemosensation

<p class="jove_content"&gt;下尿路（LUT）的功能，作为一个动态的水库，是能够存储尿液和有效地排出，在方便的时候。然而，在存储尿液，膀胱废旧产品长时间暴露。通过作为紧障碍，上皮衬里的LUT，尿路上皮，避免了有害物质的再吸收。此外，有毒化学品伤害性刺激膀胱的神经支配，并开始排尿收缩，排出膀胱的内容。有趣的是，膀胱的敏感性，有毒化学品已被成功地应用于临床实践中，通过膀胱内灌注TRPV1受体激动剂辣椒素来治疗神经源性膀胱过度活动<sup&gt; 1</sup&gt;。这突出的观察膀胱化学感受器官的优势和进一步的临床研究提示。然而，道德问题，严重限制了执行的可能性，在人体所必需解开的分子基础的LUT临床药理学，侵入性测量。一种方法来克服这种限制是使用的几种动物模型<sup&gt; 2</sup&gt;。在这里，我们描述了在小鼠和大鼠膀胱内压，一种技术，使测量控制膀胱灌注膀胱内压力的条件下实现的。</p&gt;<p class="jove_content"剖腹手术后，被植入的导管在膀胱的圆顶和隧道的肩胛间区域皮下。然后膀胱可填充以控制的速率，而尿道自由为排尿。在灌装和排尿的重复周期的期间，膀胱内的压力，可以测量通过植入的导管。因此，压力的变化可以被量化，并进行了分析。此外，空隙体积的同时测量从非排尿收缩允许区分排尿收缩<sup&gt; 3</sup&gt;。</p&gt;

Watch this Scientific Journal Video about The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation at JoVE.com

The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation

膀胱测压是一种有效的技术来测量小动物的膀胱功能<em在体内</em&gt;。膀胱是连续输注速率控制，而通过膀胱内灌注导管用于排尿的尿道自由。这允许重复填充和排空的膀胱，记录膀胱内压和排尿量。

的的膀胱测压小老鼠：A膀胱Chemosensation研究的使用

	The lower urinary tract (LUT) functions as a  dynamic reservoir that is able to store urine and to efficiently expel it at a  convenient time. While ...

the-use-cystometry-small-rodents-study-bladder

Research

JoVE Journal

Medicine

22.3K 次观看.  KU Leuven, Belgium. 膀胱测压是一种有效的技术来测量小动物的膀胱功能

视频: The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation

Mouse Bladder Wall Injection

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. Before starting injections, the surgical area must be cleaned with soap and water and antiseptic solution. Surgical equipment must be sterilized before use and between each animal. Each mouse is placed under inhaled isoflurane anesthesia (2-5% for induction, 1-3% for maintenance) and its bladder exposed by making a midline abdominal incision with scissors. If the bladder is full, it is partially decompressed by gentle squeezing between two fingers. The cell suspension of interest is intramurally injected into the wall of the bladder dome using a 29 or 30 gauge needle and 1 cc or smaller syringe. The wound is then closed using wound clips and the mouse allowed to recover on a warming pad. Bladder wall injection is a delicate microsurgical technique that can be mastered with practice.

Mouse bladder wall injection is a useful approach to orthotopically study bladder stem cell and cancer biology. This delicate microsurgical method can be mastered with careful technique and practice.

鼠标膀胱壁注射液

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. ...

科研

医学

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

在动物模型揭示神经，血液，创伤后应激障碍和行为相关因素的生物标志物

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

人类神经内分泌肿瘤细胞株为研究人类神经内分泌肿瘤治疗的三维模型

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized by increased urgency and frequency of urination, as well as nocturia and general pelvic pain, especially upon bladder filling or voiding. Despite years of research, the cause of IC/BPS remains elusive and treatment strategies are unable to provide complete relief to patients. In order to study nervous system contributions to the condition, many animal models have been developed to mimic the pain and symptoms associated with IC/BPS. One such murine model is urinary bladder distension (UBD). In this model, compressed air of a specific pressure is delivered to the bladder of a lightly anesthetized animal over a set period of time. Throughout the procedure, wires in the superior oblique abdominal muscles record electrical activity from the muscle. This activity is known as the visceromotor response (VMR) and is a reliable and reproducible measure of nociception. Here, we describe the steps necessary to perform this technique in mice including surgical manipulations, physiological recording, and data analysis. With the use of this model, the coordination between primary sensory neurons, spinal cord secondary afferents, and higher central nervous system areas involved in bladder pain can be unraveled. This basic science knowledge can then be clinically translated to treat patients suffering from IC/BPS.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized in part by pelvic pain. In order to study nervous system contributions to the condition, a physiological model of urinary bladder pain is used in mice and rats.

膀胱胀诱发内脏运动反应为膀胱疼痛小鼠模型

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition ...

Remote Limb Ischemic Preconditioning:  A Neuroprotective Technique in Rodents

Sublethal ischemia protects tissues against subsequent, more severe ischemia through the upregulation of endogenous mechanisms in the affected tissue. Sublethal ischemia has also been shown to upregulate protective mechanisms in remote tissues. A brief period of ischemia (5-10 min) in the hind limb of mammals induces self-protective responses in the brain, lung, heart and retina. The effect is known as remote ischemic preconditioning (RIP). It is a therapeutically promising way of protecting vital organs, and is already under clinical trials for heart and brain injuries. This publication demonstrates a controlled, minimally invasive method of making a limb &#8211; specifically the hind limb of a rat &#8211; ischemic. A blood pressure cuff developed for use in human neonates is connected to a manual sphygmomanometer and used to apply 160 mmHg pressure around the upper part of the hind limb. A probe designed to detect skin temperature is used to verify the ischemia, by recording the drop in skin temperature caused by pressure-induced occlusion of the leg arteries, and the rise in temperature which follows release of the cuff. This method of RIP affords protection to the rat retina against bright light-induced damage and degeneration.

Remote ischemic preconditioning (RIP) is a method of conditioning tissues against damaging stress. We have established a method of remote ischemia at the hind limb, by inflating a sphygmomanometer cuff for 5-10 min. The neuroprotective capabilities of RIP have been demonstrated in a model of retinal degeneration in rodents.

远程肢体缺血预适应：在啮齿动物的保护技术

Sublethal ischemia protects tissues against subsequent, more severe ischemia through the upregulation of endogenous mechanisms in the affected tissue. ...

Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment epithelium (RPE) cells. RPE provide critical support for photoreceptors and death or dysfunction of RPE cells is characteristic of age-related macular degeneration (AMD), the leading cause of permanent vision loss in people age 55 and older. While no cure for AMD has been identified, implantation of healthy RPE in diseased eyes may prove to be an effective treatment, and large numbers of RPE cells can be readily generated from pluripotent stem cells. Several interesting questions regarding the safety and efficacy of RPE cell delivery can still be examined in animal models, and well-accepted protocols used to inject RPE have been developed. The technique described here has been used by multiple groups in various studies and involves first creating a hole in the eye with a sharp needle. Then a syringe with a blunt needle loaded with cells is inserted through the hole and passed through the vitreous until it gently touches the RPE. Using this injection method, which is relatively simple and requires minimal equipment, we achieve consistent and efficient integration of stem cell-derived RPE cells in between the host RPE that prevents significant amount of photoreceptor degeneration in animal models. While not part of the actual protocol, we also describe how to determine the extent of the trauma induced by the injection, and how to verify that the cells were injected into the subretinal space using in vivo imaging modalities. Finally, the use of this protocol is not limited to RPE cells; it may be used to inject any compound or cell into the subretinal space.

Here we present a community accepted protocol in multimedia format for subretinally injecting a bolus of RPE cells in rats and mice. This approach can be used for determining rescue potentials, safety profiles, and survival capacities of grafted RPE cells upon implantation in animal models of retinal degeneration.

视网膜下进行注射的鼠害提供视网膜色素上皮细胞悬浮

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment ...

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold standard treatment for the high-grade non-muscle invasive bladder cancer (NMIBC). However, since the treatment-related side effects are relevant, newer biological response modifiers with a better benefit/side effects ratio are needed.
The tumour microenvironment can influence both tumour development and therapy efficacy. In order to obtain a good model, it is desirable to implant tumour cells in the organ from which the cancer originates.
In this protocol, we describe a method for establishing a tumour in the bladder cavity of female mice and subsequent delivery of therapeutic agents; the latter are exemplified by our use of Helicobacter pylori neutrophil activating protein (HP-NAP). A preliminary chemical burn of the mucosa, followed by the injection of mouse urothelial carcinoma cell line MB49 via urethral catheterization, enables the cells to attach to the bladder mucosa. After a period, required to allow an initial proliferation of the cells, mice are treated with HP-NAP, administrated again via catheterization. The anti-tumour activity of HP-NAP is evaluated comparing the tumour volume, the extent of necrosis and the degree of vascularization between vehicle- and HP-NAP-treated animals.

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Here the method to establish a syngeneic mouse model of orthotopic bladder tumour to evaluate the anti-tumour efficacy of the bacterial protein HP-NAP is described.

评价疗效<em&gt;小时。幽门螺杆菌</em&gt;蛋白HP-NAP为膀胱癌的治疗在原位小鼠模型的治疗工具

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold ...

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular mechanisms responsible for the disease, and are very convenient for rapid drug screening and testing of promising therapies. A limiting factor in these studies is often the lack of efficient non-invasive methods for sequentially analyzing the anatomical and functional changes in the kidney. Magnetic resonance imaging (MRI) is the current gold standard imaging technique to follow autosomal dominant polycystic kidney disease (ADPKD) patients, providing excellent soft tissue contrast and anatomic detail and allowing Total Kidney Volume (TKV) measurements.A major advantage of MRI in rodent models of PKD is the possibility for in vivo imaging allowing for longitudinal studies that use the same animal and therefore reducing the total number of animals required. In this manuscript, we will focus on using Ultra-high field (UHF) MRI to non-invasively acquire in vivo images of rodent models for PKD. The main goal of this work is to introduce the use of MRI as a tool for in vivo phenotypical characterization and drug monitoring in rodent models for PKD.

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

在多囊肾小型啮齿动物模型采用超高场磁共振成像<em&gt;在体内</em&gt;表型和药物监测

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...

Use of a Piglet Model for the Study of Anesthetic-induced Developmental Neurotoxicity (AIDN): A Translational Neuroscience Approach

Anesthesia cannot be avoided in many cases when surgery is required, particularly in children. Recent investigations in animals have raised concerns that anesthesia exposure may lead to neuronal apoptosis, known as anesthesia-induced developmental neurotoxicity (AIDN). Furthermore, some clinical studies in children have suggested that anesthesia exposure may lead to neurodevelopmental deficits later in life. Nonetheless, an ideal animal model for preclinical study has yet to be developed. The neonatal piglet represents a valuable model for preclinical study, as they share a striking number of developmental similarities with humans.
The anatomy and physiology of piglets allow for implementation of rigorous human perioperative conditions in both survival and non-survival procedures. Femoral artery catheterization allows for close monitoring, thus enabling prompt correction of any deviation of the piglet's vital signs and chemistries. In addition, there are multiple developmental similarities between piglets and human neonates. The techniques required to use piglets for experimentation will require experience to master. A pediatric anesthesiologist is a critical member of the investigative team. We describe, in a general sense, the appropriate use of a piglet model for neurodevelopmental study.

Use of a Piglet Model for the Study of Anesthetic-induced Developmental Neurotoxicity AIDN: A Translational Neuroscience Approach

Anesthesia-induced developmental neurotoxicity (AIDN) research has focused on rodents, which are not broadly applicable to humans. Non-human primate models are more relevant, but are cost-prohibitive and difficult to use for experimentation. The piglet, in contrast, is a clinically relevant, practical animal model ideal for the study of anesthetic neurotoxicity.

使用仔猪模型研究麻醉诱发的发育神经毒性（AIDN）：一种转化神经科学方法

Anesthesia cannot be avoided in many cases when surgery is required, particularly in children.  Recent investigations in animals have raised concerns that ...

Nerve-sparing Mid-urethral Obstruction (NeMO) in Female Small Rodents

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal model to investigate bladder remodeling in pBOO are female rodents undergoing partial obstruction at the proximal urethra. Variability in the degree of obstruction and animal mortality are major concerns with proximal obstruction. Furthermore, dissecting around the proximal urethra and bladder neck jeopardizes bladder innervation.
We developed a nerve-sparing mid-urethral obstruction (NeMO) model for pBOO avoiding the disadvantages of the traditional model. We approached the urethra just inferior to the pubic symphysis, which obviated the need for laparotomy as well as for dissection in this area; also, the striated urethral sphincter remained untouched. We performed NeMO in female Sprague-Dawley rats (12 obstructions, 6 sham animals) as well as in female C57/bl6 mice (20 obstructions, 18 sham animals). After two weeks, we evaluated bladder function, bladder mass, and body mass.
We had no mortalities among obstructed- or sham-operated female rats; as described for the traditional proximal pBOO-method, we tied the suture around the proximal urethra and a temporarily placed 0.9 mm metal rod. NeMO induced an 85% increase in bladder mass after two weeks, average residual urine volume was 0.4 mL in partially obstructed rats while only 0.03 mL in sham animals. In mice, we tested 3 sizes of cannulas that we placed along the urethra when tying the suture. We found that using a 27-gauge cannula resulted in over 50% animal mortality; placing the 25-gauge cannula did not yield the desired response in increasing bladder mass; utilizing a 26-gauge cannula yielded favorable results with minimal animal mortality (1/8) yet a significant 2-fold increase in bladder mass.

Nerve-sparing Mid-urethral Obstruction NeMO in Female Small Rodents

Traditional modeling of partial bladder outlet obstruction in rodents is fraught with animal mortality. A denervation injury from dissection around the proximal urethra and bladder neck is also of major concern. We developed and evaluated a safe and reliable mid-urethral obstruction model, avoiding the shortcomings of the traditional model.

在女小鼠害保留神经的中期尿道梗阻（NEMO）

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal ...