Name: using multi-fluorinated bile acids and in vivo magnetic resonance imaging to measure bile acid transport
Uploaded: 2016-11-27T14:00:00.000+00:00
Duration: 8 min 42 s
Description: Watch this Scientific Journal Video about Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport at JoVE.com

这项工作是由美国国立卫生研究院，糖尿病和消化研究所和肾脏疾病（资助号R21 DK093406和T32 DK067872到JP.R.）和VA优异奖（授权号1BX002129到JP.R.）的支持。

以下协议遵循由机构动物护理和使用委员会（IACUC）在医学马里兰大学大学（IACUC协议＃0415011，批准2015年6月18日）批准的指南。 1. Gavaging小鼠19架F-标记的胆汁酸<ol><li>管饲小鼠150毫克/公斤体重的19 F标记的胆汁酸。填充1-ml注射器用19 F-标记的胆汁酸储备溶液[胆酸三氟乙酰赖氨酸必要量（CA-LYS-TFA;在1:1的聚乙二醇400:Dulbecco氏磷酸盐缓冲盐水）或cholylsarcosine-三氟-N-甲基 - 乙酰胺（CA-SAR-TFMA;在60％的聚乙二醇400和40％的Dulbecco磷酸盐缓冲盐水]和附加一个20号1.5英寸的弯曲灯泡尖灌胃针确保管饲针头。在食道上插入针的毂当足够长以达到鼠标的xyphoid软骨水平</li><li>坚决摹锉刀由松弛的皮肤的动物在颈部的拇指和食指之间的背部，并使用其余手指把握皮肤上的下背部和尾巴。 </li><li>竖直握住鼠标，并通过沿侧灌胃针口的屋顶进入食道，下到肚子里。如果阻力是在咽部遇到，重新定位针，直到动物&#39;燕子&#39;吧 - 不要反对抵抗。 </li><li>如果需要对管饲法麻醉，将鼠标在含有5ml异氟醚和靠近钟罩。当鼠标落在其一侧，等待7秒，取出鼠标进行灌胃。为了保护人员免受麻醉剂蒸气，使用钟罩只在通风橱中。 </li><li>观察动物从异氟烷在几分钟恢复。 注意:由于异氟醚是由肝脏代谢，从完整的药物或其代谢物发出的信号氟排入胆道系统和胆囊可以混淆FLUO海相从19 F标记的胆汁酸15的信号。另一种方法是使用氯胺酮加甲苯噻嗪（见剂量3.1​​节）。 </li></ol> 2.收获胆囊，肝脏和血液的胆汁酸测定液相色谱/质谱<ol><li>收获器官之前达到最大的胆囊充盈，快速老鼠至少6小时。制备氯胺酮和赛拉嗪在磷酸盐缓冲盐水（100微升氯胺酮，62.5微升赛拉嗪，840微升PBS中）。 </li><li>用1毫升无菌注射器，注入器官收获前给小鼠皮下注射1小时与15微升/ g体重的氯胺酮/甲苯噻嗪溶液（150毫克氯胺酮和每18毫克甲苯噻嗪kg体重）。 </li><li>管理氯胺酮/赛拉嗪后的一个小时，确认通过脚趾捏足够的麻醉，然后将鼠标麻醉仰卧。 </li><li>用5或6英寸剪刀使从耻骨中线腹部皮肤切口到xyphoid和音响NE剪刀（4英寸）的削减腹膜衬里和暴露腹部器官 - 不要刺破隔膜。 </li><li>抓住xyphoid进程与5英寸的夹子，将后部交叉于胸前，露出上腹腔。使用镊子和一把钝刀剖析，并预留移动肝脏，露出了胆囊。 注意:不要划破肝脏或触摸胆囊因为前者会导致严重出血，而后者可能会刺激胆囊收缩和排空。 </li><li>放置横跨胆总管一个4英寸的夹具（ 图3中，虚线箭头）。切韧带附着胆囊在隔膜的上磁极和轻轻胆囊移动到腹部的右侧。 </li></ol><img alt="图3" src="/files/ftp_upload/54597/54597fig3.jpg" /> 图3:鼠标胆囊的解剖与质子MRI意见。 左侧面板显示暴露的谅解备忘录Ë胆囊中线腹部切口后左侧。钳位掌握xyphoid过程。胆汁填充禁食胆囊由大箭头和夹紧胆总管由虚线箭头指示。 [ 插图:离体与胆总管完好胆囊夹紧。统治者被标记为毫米（mm）]右图显示了空腹胆囊鼠（箭头）的高分辨率质子密度加权MRI图像。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54597/54597fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="7"><li>前切除胆囊进行心脏穿刺，收获血，抽血动物核实安乐死。 注意:首先收获胆囊可能划破肝脏造成心血管塌陷和未能获得足够的血液样品（≥200微升）。 </li><li>露出左隔板的下侧和确定心脏跳动表面。在最大心搏动的点刺穿隔膜和心脏与连接到一个1-ml注射器一个23号针头。 <ol><li>吸气的同时慢慢退出注射器。当血液开始填充注射器，停止计提和维护吸收集0.2 - 0.6毫升血。轻轻转动针或撤销稍微如果不再可能重新建立流动。 </li><li>血液转移到1.5毫升的肝素化管中并离心以2000 xg离心15分钟。沉淀四份乙腈和离心机的等离子体以12,000 xg离心10分钟。分析通过液相色谱/质谱法（LC / MS / MS）11-1312-1412-1412-1412-14上清液。如果有必要，在-80℃下分析之前储存血浆。 </li></ol></li><li>使用钝性分离，不受肝脏胆囊。横切夹下面的胆总管，删除，重胆囊，并将其放置在一个1.5毫升microcentrifuge管。收获肝脏。 </li><li>均质化约100毫克的肝脏，并在尺寸-21玻璃组织匀浆器在冰上整个胆囊。用75％乙腈和25％水（800微升为肝脏，300微升用于胆囊）和离心机提取在12,000 xg离心10分钟。稀释提取物作为必要和量化用LC / MS / MS 11-13胆汁酸含量。 </li></ol> 3.活体动物的质子（1 H）和氟（19 F），磁共振成像<ol><li>为了达到最大的胆囊充盈，快速的小鼠成像前至少6小时。用氯胺酮和赛拉嗪，麻醉小鼠，以防止在MRI扫描仪的运动。在磷酸盐缓冲盐水（130微升氯胺酮，42.5微升赛拉嗪，827微升PBS中）制备氯胺酮加赛拉嗪的贮备液。磁共振成像前一小时，用1毫升无菌注射器与5微升/ g体重的该溶液（65毫克氯胺酮和4.25毫克xylaz注入小鼠皮下注射每公斤体重INE）。为了防止麻醉下干燥适用兽医软膏动物的眼睛。 </li><li>如上诱导氯胺酮/赛拉嗪后，夹在鼠标左下半部分1.5厘米的面积2采用腹部40＃或更细的电动更换刀片。毛皮移除之后，预习8的面积 - 12％稀碘手术擦洗溶液并用70％乙醇冲洗 - 重复这两个步骤。将0.75寸毫针/导管和皮下隧道24号进入腹腔。确保导管不是在盲肠或其他腹部器官通过向后拉柱塞 - 应在导管没有血液或粪便物质。 </li><li>取出针，留下了腹腔导管。将鼠标在MRI扫描器动物室的温度控制的导热垫。 </li><li>制备含有氯胺酮和赛拉嗪在磷酸盐缓冲盐水1毫升无菌注射器（1,000微升氯胺酮，300微升甲苯噻嗪，6700微升的PBS）和填充72英寸无菌管道的期望长度。腹腔导管连接到预充式无菌管道，并延长它的核磁共振成像扫描仪了。维持麻醉注射该溶液的50微升每20分钟，如果鼠标的生命体征是稳定的。 注:影像之前，请一定没有金属是核磁共振成像扫描仪附近。 </li><li>使用19 F / 1小时的双调谐线性体积MRI线圈，以在300.283兆赫1 H和282.524兆赫19 F核发射和接收射频信号。 <ol><li>执行系统校准11,13和动物的定位与使用快速低角度拍摄序列（FLASH），三片（轴向，正中矢状，和冠状）侦察图像。要启动实验，请点击在软件控制台上的扫描控制窗口中的"红绿灯"按钮。 </li><li>利用快速采集弛豫增强多层获得1小时的MR图像定（RARE）序列中的样品或具有重复时间2200毫秒的动物的身体，回波时间8.9毫秒的横视图，稀有因子8，视4×4 平方厘米字段，切片厚度为1.0毫米，矩阵大小266点¯x266，平面分辨率150×150微米2，和平均值6.要开始实验的数量，点击软件控制台上的扫描控制窗口中的"红绿灯"按钮。 </li><li>获得使用闪存序列19女性形象在1小时MRI的同一区域有重复时间220毫秒，翻转角= 30°，回波时间3.078毫秒，矩阵大小为32×32，在平面1.25 mm的分辨率1.25×2，层厚4.0毫米，以及平均768人数开始实验，点击"共和党"（G邻ØN- P ipeline）在软件控制台上的光谱仪控制工具窗口按钮。 </li></ol></li><li> MRI后，安乐死与腹腔injecti鼠标上15微升/克体重氯胺酮/后跟放血心脏穿刺甲苯噻嗪溶液（每公斤体重150mg的氯胺酮/ 18毫克甲苯噻嗪）。 </li><li>要恢复从麻醉鼠标，除去腹腔导管，但直到它重新获得足够的意识，以保持胸骨斜卧不要让动物无人值守。 </li><li>为了测量从器官收获19 F-标记的胆汁酸浓度11-13，如上所述维持用氯胺酮加赛拉嗪麻醉。 </li></ol>

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作者什么都没有透露。

CA-LYS-TFA和CA-SAR-TFMA和使用表达ASBT和人胚肾细胞中表达钠/牛磺胆共输送多肽稳定转染的Madin-Darby犬肾细胞其运输的体外分析的合成（NTCP）在别的地方13,14详细说明。这里的重点是关于离子烫口服灌胃到活的动物，随后胆囊，肝和血液离子烫内容的分析的收获，和，特别是，在由活的动物的MRI胆囊成像离子烫。关键的步骤包括避免基于氟的麻醉剂可产生竞争的19 F信号，避免钳位胆总管这可能导致胆囊的收缩和排空器官切除之前前胆囊操纵，并避免MRI扫描器附近的金属的位置。 由于这里和其他地方13,14详细的，离子烫-MRI具有很大的潜在的T直径:成功地分析在正常和异常的生理条件下在体内胆汁酸运输。离子烫-MRI的主要优势是，它不涉及电离辐射（均未离子烫标记天然存在的，非放射性氟也不MRI发射电离辐射）和离子烫口服给药，而不需要静脉穿刺（不同于小鼠，大多数人将不要求非肠道给药麻醉剂或​​镇静剂，以防止在MRI扫描器运动伪影）。用合成胆汁酸水解抗性由肠道菌群13,14可水解胆汁酸的取代，可以克服关于在肠道的离子烫的体内稳定性问题。初始原型离子烫涉及每胆酸的分子加入三个氟原子，即在肠道通过梭菌水解天然存在的人的胆汁酸水解酶13,14。一种新型的三氟化胆汁酸，CA-SAR-TFMA的基础上，肌氨酸主链是通过细菌酶抗水解规避此限制，延伸离子烫13,14的半衰期。 最后，一个潜在竞争的方法，使用N-甲基- [11 C] cholylsarcosine用于PET / CT，是由从两个放射性标记的胆汁酸和CT，以及需要制备，船舶，并存储放射性辐射暴露限制胆汁酸17。 关于修改和故障排除这种新颖的方法，可以考虑，未能检测从胆囊发出可能是一种MRI信号由于与19 F-标记的胆汁酸或其生物利用度，图像采集的定时，或填充不足的问题胆囊。如上所讨论的，我们通过自己的能力进行测试这些试剂的结构完整性，以在体外通过表达适当的胆汁酸转运的细胞系进行运输。我们允许至少1.5小时r表示图像采集，但在某些情况下，此持续时间可能需要扩展。我们发现，空腹小鼠预先MRI，以确保最大的胆囊填充是重要的。 检测的19架F-MRI信号的限制要求在90动物成像- 120分钟足够信号采集;这很可能是太长了实用的临床试验 - 患者将不得不躺在依然在核磁共振成像扫描仪为持续时间。此外，当前，临床MRI主要依赖于质子（1H）成像;氟的应用程序（19 F）成像，需要在硬件投资（19 F盘）和软件，至少每核磁共振成像扫描仪一$ 250,000个投资。直到其他氟系的MRI应用程序使用离子烫-MRI来评估患者对胆汁酸吸收障碍开发是不太可能是划算的。 离子烫-MRI提供了一种有前途的替代测量75 SeHCAT保留，等离子体7α羟基-4-<html> -cholesten -3-酮（4），和血浆FGF19，或执行使用胆汁酸结合剂的治疗试验。在美国，75 SeHCAT测试，它涉及辐射暴露，没有FDA批准。 C4和FGF19测试有弱阳性 - 预测值与测试目前只能通过专门的实验室提供。此外，无论是C4和FGF19水平反映胆汁酸肝脏合成。因此，不像离子烫-MRI，这些测试不能检测表达改变或回肠胆汁酸转运或独立于增加胆汁酸的合成胆汁酸吸收不良的其他机制的突变。的胆汁酸结合剂治疗试验可能是有益的，但具有不确定性的预测值，并且不能提供胆汁酸吸收不良5的一个明确的诊断。最后，虽然粪胆汁酸直接测量是貌似合理这个被拒绝年前过于繁琐，不切实际的，不可靠的常规临床应用5。 ENT"&gt;其他挑战必须克服，使这一创新做法可以转化为在诊所体内胆汁酸转运测量（ 例如 ，胆汁酸吸收障碍的诊断为慢性腹泻的原因）。一个主要的挑战是最大限度地提高通过增加每个胆汁酸分子的19 F原子的数量，而不破坏离子烫的表现得像天然胆汁酸，也就是，胆汁酸分子是太笨重，因为加入的19 F原子不会被任一被运送的能力信号强度ASBT或胆汁酸转运在肝脏。新颖离子烫的体外待运输的能力可以在细胞系中测试表达关键人胆汁酸转运13,14和应该预测体内交通工具。随着增加的灵敏度，离子烫-MRI具有用于改进整个胆道树（ 例如，胆总管及其分支）的成像电势;在这方面，能力吨O映像在一些小鼠胆总管出现看好（ 图4，左图）。 虽然通过离子烫-MRI胆汁酸检测的当前限制允许其可视化只在胆囊，假设该技术的灵敏度可以得到改善，我们预计离子烫-MRI可用于测量体内胆汁酸运输和分配，从而提供全面的空间在实时信息解剖学;一个有利的技术，将推动分子成像。离子烫-MRI也将提供一种新的技术来评估基因多态性，并且削弱胆汁酸转运功能的药物的效果。离子烫-MRI具有平移潜力，帮助医生筛选胆汁酸的吸收障碍，从而识别和管理增加粪便胆汁酸（ 如腹泻，模仿肠易激综合症），导致疾病。最后，这应该成为技术临床上有很大的亲濑提供的能力，以确定与结肠肿瘤或其他条件可以通过粪便胆汁酸的变化影响的人改变肠道胆汁酸的吸收，加速精密医学的进步。

连同它们作为促进从肠道吸收脂肪洗涤剂古典作用，胆汁酸已成为影响除了与其肠肝循环1,2-相关的那些多器官效信号分子。除了控制其自身的新陈代谢，胆汁酸调节胃肠生理（ 例如 ，肠能动性和肠降血糖素激素的产生，结肠生理学和癌症的易感性）的若干方面，并且对血管张力，葡萄糖和脂质代谢，能量利用全身效应。虽然其中一些效果在肠道被介导，其它的是由于在全身胆汁酸水平餐后变化，如在肥胖患者或胃旁路手术后指出。为了阐明的胆汁酸，需要新技术的复杂的代谢作用，其允许在不同的解剖隔间胆汁酸水平的同时监测，在胃肠道中和元栓塞性组织（肝，胰，骨骼肌和脂肪）。获得这样的时间和空间信息需要的创新技术- 活体成像使用这里描述的这样一种新的方法新的胆汁酸示踪剂。 在解剖车厢胆汁酸的成分和分布是由调节其肝脏合成和回肠的吸收，包括饮食，手术，抗生素的使用和肠道菌群的变化因素调控。肠胆汁酸的摄取其肠肝循环3（ 图1）的一个关键调节器是回肠心尖钠依赖性胆汁酸转运（ASBT; SLC10A2）。虽然在整个肠发生被动吸收，ASBT介导的肠胆汁酸的95％的吸收，使通常存在的胆汁酸进入粪便有限溢出。 ASBT缺陷（Slc10a2 - / - ）小鼠也增加粪便胆汁酸和减弱胆汁ACIð池4。 <img alt="图1" src="/files/ftp_upload/54597/54597fig1.jpg" /> 图1:胆汁酸的肠肝循环。 肝肠循环，从而胆汁酸在肝脏中合成，排泄到胆道，胆囊储存的插图，释放到近端小肠与膳食，并通过在ASBT回肠末端积极采纳。而胆汁酸少量在整个肠被动吸收的肠胆汁酸约95％是由导致在此通过的新胆汁酸合成的类似量补偿大便最小（约5％）损失ASBT主动转运肝脏，从而保持稳态胆汁酸池。在右边的箭头识别可能影响天然和氟标记的胆汁酸稳定性因素，包括胃酸，胰腺癌和肠粘膜酶，和，最importantl通过殖民远端小肠和结肠梭菌种释放Y，水解酶。 （修改许可16） <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54597/54597fig1large.jpg" target="_blank">点击此处查看该图的放大版本。</a> 胆汁酸吸收障碍可分为三种类型，其中每一个增加粪便羟基胆汁酸，从而使间歇性或慢性腹泻。键入从总回肠病变（如切除，克罗恩病）5 1的结果。键入从胆囊切除术，迷走神经，腹腔疾病，细菌过度生长，和胰腺功能不全3的结果。与此相反，"主要"（类型2）胆汁酸吸收障碍的人带来巨大的诊断的挑战，因为它们缺乏这种前提条件，不具备在回肠病理学证据。因此，初级胆汁酸吸收障碍通常是误诊为痢疾-Predominant肠易激综合征（IBS-D），也许对胃肠相关的门诊访问的最常见原因。据估计，IBS患者-D-三分之一具有初级胆汁酸吸收障碍;在美国，这可以表示几百万人5。最近的见解显示，主要由BAM肠道成纤维细胞生长因子19（FGF19）肝脏胆汁酸合成的反馈抑制受损的派生，而不是表达降低或ASBT的功能。 在主要的胆汁酸吸收不良，FGF19的低血浆水平未能关闭肝胆汁酸合成-在肠胆汁酸所得增加饱和胆汁酸转运，包括ASBT，和胆汁酸增加泄漏到粪便引起腹泻6 （ 图2）。在小鼠FGF15（鼠FGF19）有缺陷的扩大胆汁酸池，并增加粪便胆汁酸7。 图2:肠道胆汁酸吸收不良的机制。 通常，如图面板 A，肠胆汁酸约95％是由主动转运在经由ASBT回肠末端吸收。当ASBT表达或活性被降低（ 图B），在胆汁酸溢出进入结肠受损肠胆汁酸吸收的结果。受损FGF19信号（C组 ），缺乏在肠道胆汁酸压倒与胆汁酸进入结肠溢出ASBT运力浓度升高的肝胆汁酸合成结果反馈抑制。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54597/54597fig2large.jpg" target="_blank">请点击此处查看大图这个数字。</a> 从长期来看，在粪胆ACI慢性抬高DS可促进大肠肿瘤。结肠肿瘤起因于体细胞基因突变，但环境因素，增加粪便胆汁酸可以加速并扩大此过程相关进步胃黏膜不典型增生。在啮齿类动物中，增加粪便胆汁酸无论是作为外源性或ASBT不足的后果来促进肠道发育异常和肿瘤形成8-10。 值得注意的是，挑衅性的调查结果表明，美国食品和药物管理局（FDA）批准的常用药物通过ASBT 体外 11强效抑制胆汁酸转运。如果这些药物减少体内小肠的胆汁酸转运，增加粪便胆汁酸水平，结肠病变的潜在影响是非常令人担忧。即使在结肠病理少量增加归因于使用这样的药物可以有一个主要的健康影响。它可以评估在体外的研究结果和流行病学OB的这些合理性的工具包servations会刺激更多的研究，包括上市后安全性研究。 尽管需要，实际检测来确定人的胆汁酸吸收不良所缺乏的。粪便胆汁酸直接测量被拒绝年前累赘，不切实际的，和不可靠的5。替代方法包括测量放射性硒标记胆酸衍生物（75 SeHCAT）和7α羟基-4-胆甾烯-3-酮（4），或胆汁酸结合剂的治疗试验的血浆水平保持75 SeHCAT测试有从诊断测试在欧洲有限，不FDA批准或可在美国使用。此外，即使是少量的辐射照射（0.26毫希/ 75 SeHCAT测试）引起了关注，并且细菌过度生长和先进的肝病可能混淆75 SeHCAT结果。 C4测试是潜在的有吸引力的，因为只有需要等离子体，但它具有低阳性预测VALUE和测试不够普及。测量FGF19血清水平也有类似的限制。频繁的临床医生诉诸的胆汁酸多价螯合剂治疗试验中，但这种方法不能提供胆汁酸吸收不良5的一个明确的诊断。 由于这些原因，一个新颖的MRI方法被设想使用创新的多氟化的胆汁酸（离子烫-MRI）来测量体内胆汁酸运输和分配。含氟（19 F），100％的天然丰度的稳定同位素的三个原子离子烫，类似地运送到天然胆汁酸12，并可以用于可视化胆汁酸运输与质子（1 H）和氟（的组合19 F）MRI，无电离辐射13,14敏感，安全的方法。

<table><tbody><tr><td>Duall size-21 all glass tissue grinder</td><td>Kimble Chase Life Science, Vineland, NJ</td><td>885351-0022</td><td></td></tr><tr><td>Bruker BioSpec 70/30USR Avance III 7T horizontal bore MR Scanner</td><td>Bruker Biospin MRI GmbH, Germany</td><td></td><td>Use companion Paravision Version 5.1 software (see step 3.5)</td></tr><tr><td>Bruker 40 mm &lt;sup&gt;19&lt;/sup&gt;F/&lt;sup&gt;1&lt;/sup&gt;H dual-tuned linerar volume coil</td><td>Bruker Biospin MRI GmbH, Germany</td><td></td><td>Use companion Paravision Version 5.1 software (see step 3.5)</td></tr><tr><td>Waters Acquity UPLC System with Quadrupole Detector</td><td>Waters Corporation, Milford, MA</td><td></td><td></td></tr><tr><td>Waters Acquity UPLC ethylene bridged hybrid C8 1.7 &amp;mu;m 2.1 x 50 mm column</td><td>Waters Corporation, Milford, MA</td><td></td><td></td></tr><tr><td>Gavage Needle</td><td>Braintree Scientific, INC.</td><td>N-010</td><td>20 G-1.5&quot; curved 2.25 mm ball</td></tr><tr><td>2 Stainless Steel Hemostats&amp;nbsp;</td><td>VWR</td><td>10755-018</td><td>4 and 5 inch, straight</td></tr><tr><td>Ketamine</td><td>MWI Veterinary Supply</td><td>501090</td><td>Ketamin zetamine 100 mg/ml</td></tr><tr><td>Xylazine</td><td>Akorn, Inc.</td><td></td><td>20 mg/ml</td></tr><tr><td>Intraperitoneal Catheter</td><td>Abbott</td><td>AbbocathTM-T.I.V. G720-A01 4535-42</td><td>24-G x 0.75&quot;</td></tr></tbody></table>

using multi-fluorinated bile acids and in vivo magnetic resonance imaging to measure bile acid transport

随着他们作为促进脂肪的吸收洗涤剂的传统角色，新兴的文献表明，胆汁酸是影响多个器官强有力的信号分子;它们调节肠能动性和激素的产生，以及改变血管张力，糖代谢，脂质代谢和能量利用率。在粪便胆汁酸的变化可能会改变肠道微生物，促进结肠的病理包括cholerrheic腹泻和结肠癌。粪便胆汁酸成分的主要监管机构小肠心尖钠依赖性胆汁酸转运体（ASBT）和成纤维细胞生长因子19（FGF19）。 ASBT减少表达和功能降低肠内胆酸向上走。此外， 体外数据表明，一些FDA批准的药物抑制ASBT功能。缺乏FGF19释放增加肝脏胆汁酸的合成和释放进入肠道，以压倒ASBT水平。无论是ASBT功能障碍或缺失FGF19增加˚FECAL胆汁酸，并可能导致慢性腹泻，促进结肠肿瘤。遗憾的是，工具测量胆汁酸吸收不良及药物对胆汁酸转运体内的行动是有限的。理解的胆汁酸的复杂的动作，技术要求允许在肠道和代谢组织胆汁酸同时监测。这使我们设想一种创新的方法来测量在活的动物胆汁酸运输用质子（1 H）和氟（19 F），磁共振成像（MRI）的组合。为氟（19 F）的新颖的示踪剂为基础的活的动物的MRI创建和测试，无论是在体外和体内 。这种方法的优点包括缺乏暴露于电离辐射和潜力转化为临床研究和实践。

实时用于体内 MRI使用离子烫的"看见"胆汁酸运输具有用于研究和临床应用的巨大潜力。此外，这里描述为胆囊切除和利用液相色谱法和质谱法及其内容生化分析的方法提供了确定的成像效果的手段。然而，这些方法的有效性需要精确的定量给料，测定的定时，和胆囊用于成像或手术切除的定位。对于后者，这是很重要的，该小鼠的腹部器官心烦尽可能少;甚至胆囊温柔的操纵可以刺激收缩和排空。因此，以捕获它的全部内容，关键是要尽快放置横跨胆总管钳。 采用手术方法建议在这里，胆囊应当容易识别在腹部的右上象限中的肝脏的右前叶（ 图3）的后面。移动肝脏一边，而不触及胆囊暴露于夹紧胆总管。一旦此夹紧到位，应该没有困难的议定书所述去除胆囊完好的其它步骤进行。 表1中的数据在胆囊13,14揭示离子烫的高度选择性浓度。离子烫的器官（肝和胆囊）浓度通过假定1微克/毫升13,14的密度来计算。内口服给药7小时后，在胆囊离子烫的平均积累幅度（1000倍），在任一肝或血液13,14观察到高几个数量级;在胆囊与肝脏和血液中（ 表1微摩尔水平观察毫摩尔含量&lt;/ STRONG&gt;）。 1.4微米的血液，14.5 - - 78.8毫米，肝脏，18.4 - 27.0毫米胆囊平均浓度离子烫从0.4不等。这些发现与离子烫一致被处理相同生理胆汁酸;口服给药后，离子烫正在积极通过ASBT在胆囊输送到肠肝循环，由此它们是由钠/牛磺胆共转运多肽（NTCP）携带至肝脏用于主动运输到肝细胞，并排泄到胆管树为浓度（ 图1）。 图4示出在胆囊与离子烫灌胃后19 F信号的依赖于时间的积累。使用的分析方法的更详细的时间过程（LC / MS / MS）表明，在4个的范围内，观察到7小时离子烫的峰胆囊浓度口服给药12，与生理动力学的发现一致后的胆汁酸的肠肝循环。 图4中的左侧面板中，MRI图像获得的两个和一个半与离子烫口灌胃经过四小时，发现从似乎是胆总管（虚线箭头）和发出一个更强大的信号发出19 F信号胆囊（箭头）;由7至8.5小时，胆总管信号不再检测和胆囊19 F信号上升到相同的强度为从相邻的离子烫幻象（ 图4，右侧面板，箭头）发出。 我们使用小鼠ASBT的缺陷表达来测试离子烫-MRI来检测的胆汁酸降低肠道吸收的能力。如在图5A中，在离子烫的浓度的约22倍的降低在胆囊从ASBT缺陷小鼠示出通过LC测定/ MS 13,14;基于这些发现，预期第在这些动物中的离子烫的19 F磁共振信号将低于检测限。的确， 如图5B所示 ，而在野生型小鼠中检测到从胆囊发出一个健壮19 F信号，有一个在ASBT缺陷小鼠13,14没有相应的19 F信号。这些发现证实，离子烫类似处理，以生理胆汁酸和离子烫-MRI可用于检测的胆汁酸的受损肠摄取。 <img alt="图4" src="/files/ftp_upload/54597/54597fig4.jpg" /> 图4:离子烫代表-MRI图像。 在活体小鼠的实验结果显示，19 F重建叠加和1小时的图像表明19 F信号从鼠胆（大箭头）发出。包含19 F标记的胆汁酸已知浓度的参考幻影放置在MRI扫描器沿着鼠标（左箭头）。在左边的面板中，虚线箭头表示从胆总管发出19 F标记的胆汁酸信号。记录在每个面板上方是19 F-标记的胆汁酸灌胃该磁共振成像开始到图像采集完成的时间（1.5小时）后的时间。正如预期的那样，胆囊与时间的19 F标记的胆汁酸升高填写。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54597/54597fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54597/54597fig5.jpg" /> 图5:减毒离子烫-MRI信号从ASBT缺陷小鼠的胆囊。 （A）的条形图说明离子烫的通过LC测定的浓度/ MS / MS是约22倍的ASBT缺陷型小鼠比较低d，来对照小鼠。 （B）从ASBT缺陷小鼠的胆囊缺席离子烫-MRI信号。箭头表明19 F-标记的胆汁酸幻影（左图）和从体模19 F磁共振信号（右图）。箭头，右上图表示从胆囊发出19 F标记的胆汁酸信号;有一个在ASBT缺陷小鼠（下右图）没有相应的19 F标记的胆汁酸信号。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54597/54597fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td rowspan="2"></td><td rowspan="2"> 胆囊重量（mg） </td><td colspan="3"> 离子烫浓度 </td></tr><tr><td> 胆囊（MM） </td><td> 肝（μM） </s仲&gt; </td><td> 等离子体（μM） </td></tr><tr><td> CA-LYS-TFA </td><td> 25.2±3.2 </td><td> 27.0±2.4 </td><td> 78.8±35.1 </td><td> 0.4±0.2 </td></tr><tr><td> CA-SAR-TFMA </td><td> 29.2±2.4 </td><td> 18.4±1.6 </td><td> 14.5±0.6 </td><td> 1.4±0.1 </td></tr></tbody></table> 表1:19 F标记的胆汁酸的胆囊，肝脏和血浆浓度代表值。 平均值±标准误差（SE）值示为胆囊重量和指示离子烫的胆囊，肝和血浆12,13中的浓度。所示的值，测定5至7小时，以150毫克/公斤体重的指示离子烫的gavaging小鼠后。注离子烫的毫摩尔浓度在胆囊相比微摩尔浓度在肝脏和血浆。 N = 3英里每个值CE为CA-LYS-TFA，每个价值5只小鼠CA-SAR-TFMA。

Watch this Scientific Journal Video about Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport at JoVE.com

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

工具来诊断胆汁酸的吸收不良，并测量在体内胆汁酸转运的限制。活体动物一个创新的方法，描述了利用质子结合（1小时）加氟（19 F）磁共振成像;这种新颖的方法具有平移潜力筛选在临床实践中的胆汁酸吸收不良。

使用多氟化胆汁酸和<em&gt;在体内</em&gt;磁共振成像来衡量胆汁酸运输

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling ...

using-multi-fluorinated-bile-acids-vivo-magnetic-resonance-imaging-to

Universidad San Sebastian

Research

JoVE Journal

Medicine

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

11.1K 次观看.  University of Maryland School of Medicine. 工具来诊断胆汁酸的吸收不良，并测量在体内胆汁酸转运的限制。活体动物一个创新的方法，描述了利用质子结合（1小时）加氟（19 F）磁共振成像;这种新颖的方法具有平移潜力筛选在临床实践中的胆汁酸吸收不良。 

视频: Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Magnetic Resonance Imaging Assessment of Carcinogen-induced Murine Bladder Tumors

Murine bladder tumor models are critical for the evaluation of new therapeutic options. Bladder tumors induced with the N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) carcinogen are advantageous over cell line-based models because they closely replicate the genomic profiles of human tumors, and, unlike cell models and xenografts, they provide a good opportunity for the study of immunotherapies. However, bladder tumor generation is heterogeneous; therefore, an accurate assessment of tumor burden is needed before randomization to experimental treatment. Described here is a BBN mouse model and protocol to evaluate bladder cancer tumor burden in vivo using a fast and reliable magnetic resonance (MR) sequence (true FISP). This method is simple and reliable because, unlike ultrasound, MR is operator-independent and allows for the straightforward post-acquisition image processing and review. Using axial images of the bladder, analysis of regions of interest along the bladder wall and tumor allow for the calculation of bladder wall and tumor area. This measurement correlates with ex vivo bladder weight (rs= 0.37, p = 0.009) and tumor stage (p = 0.0003). In conclusion, BBN generates heterogeneous tumors that are ideal for evaluation of immunotherapies, and MRI can quickly and reliably assess tumor burden prior to randomization to experimental treatment arms.

Murine bladder tumors are induced with the N-butyl-N-(4-hydroxybutyl) nitrosamine carcinogen (BBN). Bladder tumor generation is heterogeneous; therefore, an accurate assessment of tumor burden is needed before randomization to experimental treatment. Here we present a fast, reliable MRI protocol to assess tumor size and stage.

癌地致小鼠膀胱肿瘤的磁共振成像评价

Murine bladder tumor models are critical for the evaluation of new therapeutic options. Bladder tumors induced with the N-butyl-N-(4-hydroxybutyl) ...

科研

癌症研究

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

监测树突状细胞迁移<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H磁共振成像

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

免疫与感染

Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. Biosensors relying on the principle of FRET have enabled real-time visualization of subcellular signaling events in live cells with high temporal and spatial resolution. Here, we describe the application of a genetically encoded Bile Acid Sensor (BAS) that consists of two fluorophores fused to the farnesoid X receptor ligand binding domain (FXR-LBD), thereby forming a bile acid sensor that can be activated by a large number of bile acids species and other (synthetic) FXR ligands. This sensor can be targeted to different cellular compartments including the nucleus (NucleoBAS) and cytosol (CytoBAS) to measure bile acid concentrations locally. It allows rapid and simple quantitation of cellular bile acid influx, efflux and subcellular distribution of endogenous bile acids without the need for labeling with fluorescent tags or radionuclei. Furthermore, the BAS FRET sensors can be useful for monitoring FXR ligand binding. Finally, we show that this FRET biosensor can be combined with imaging of other spectrally distinct fluorophores. This allows for combined analysis of intracellular bile acid dynamics and i) localization and/or abundance of proteins of interest, or ii) intracellular signaling in a single cell.

We provide a detailed protocol to study bile acid dynamics in living cells using a genetically encoded BAS FRET sensor. This Bile Acid Sensor represents a unique tool to study (regulation of) bile acid transport and FXR activation in a wide range of cell types.

细胞内胆汁酸动态实时监控使用遗传编码的FRET为基础的胆汁酸传感器

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. ...

生物工程

Determining Bile Duct Density in the Mouse Liver

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques are used, including serum chemistry, histological analysis, and immunostaining for specific markers. Although these techniques can provide important information about the biliary system, they often do not present a full picture of bile duct (BD) developmental defects across the whole liver. This is in part due to the robust ability of the mouse liver to drain the bile even in animals with significant impairment in biliary development. Here we present a simple method to calculate the average number of BDs associated with each portal vein (PV) in sections covering all lobes of mutant/transgenic mice. In this method, livers are mounted and sectioned in a stereotypic manner to facilitate comparison among various genotypes and experimental conditions. BDs are identified via light microscopy of cytokeratin-stained cholangiocytes, and then counted and divided by the total number of PVs present in liver section. As an example, we show how this method can clearly distinguish between wild-type mice and a mouse model of Alagille syndrome. The method presented here cannot substitute for techniques that visualize the three-dimensional structure of the biliary tree. However, it offers an easy and direct way to quantitatively assess BD development and the degree of ductular reaction formation in mice.

We present a rather simple and sensitive method for accurate quantification of bile duct density in the mouse liver. This method can aid in determining the effects of genetic and environmental modifiers and the effectiveness of potential therapies in mouse models of biliary diseases.

确定小鼠肝脏中的胆汁密度

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques ...

发育生物学

Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, a significant stress response is induced during gastrointestinal transit and upon bile exposure, a normal component of human digestion. To overcome the bactericidal effects of bile, many enteric pathogens form a biofilm hypothesized to permit survival when transiting through the small intestine. Here we present methodologies to define biofilm formation through solid-phase adherence assays as well as extracellular polymeric substance (EPS) matrix detection and visualization. Furthermore, biofilm dispersion assessment is presented to mimic the analysis of events triggering release of bacteria during the infection process. Crystal violet staining is used to detect adherent bacteria in a high-throughput 96-well plate adherence assay. EPS production assessment is determined by two assays, namely microscopy staining of the EPS matrix and semi-quantitative analysis with a fluorescently-conjugated polysaccharide binding lectin. Finally, biofilm dispersion is measured through colony counts and plating. Positive data from multiple assays support the characterization of biofilms and can be utilized to identify bile salt-induced biofilm formation in other bacterial strains.

This protocol enables the reader to analyze bile salt-induced biofilm formation in enteric pathogens using a multifaceted approach to capture the dynamic nature of bacterial biofilms by assessing adherence, extracellular polymeric substance matrix formation, and dispersion.

胆汁盐诱导肠道病原菌生物膜形成的鉴别和定量技术

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, ...

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not routinely employed in animal models. Conventional experimental readouts often rely on post mortem and ex vivo analyses, which prevent intra-individual follow-up examinations and increase the number of study animals needed. Fluorescence-mediated tomography enables the non-invasive, repetitive, quantitative, three-dimensional assessment of fluorescent probes. It is highly sensitive and permits the use of molecular makers, which allows for the specific detection and characterization of distinct molecular targets. In particular, targeted probes represent an innovative tool for analyzing gene activation and protein expression in inflammation, autoimmune disease, infection, vascular disease, cell migration, tumorigenesis, etc. In this article, we provide step-by-step instructions on this sophisticated imaging technology for the in vivo detection and characterization of inflammation (i.e., F4/80-positive macrophage infiltration) in a widely used murine model of intestinal inflammation. This technique might also be used in other research areas, such as immune cell or stem cell tracking.

Target-specific probes represent an innovative tool for analyzing molecular mechanisms, such as protein expression in various types of disease (e.g., inflammation, infection, and tumorigenesis). In this study, we describe a quantitative three-dimensional tomographic assessment of intestinal macrophage infiltration in a murine model of colitis using F4/80-specific fluorescence-mediated tomography.

荧光介导的断层扫描对巨噬细胞相关小鼠肠道炎症的检测和定量

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not ...

医学

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity is disturbed in gastrointestinal, cardiovascular and metabolic diseases, which may result in easier access of biologically active compounds, such as gut bacterial metabolites, to the bloodstream. Thus, the permeability of the GBB may be a marker of both intestinal and extraintestinal diseases. Furthermore, the increased penetration of bacterial metabolites may affect the functioning of the entire organism.
Commonly used methods for studying the GBB permeability are performed ex vivo. The accuracy of those methods is limited, because the functioning of the GBB depends on intestinal blood flow. On the other hand, commonly used in vivo methods may be biased by liver and kidney performance, as those methods are based on evaluation of urine or/and peripheral blood concentrations of exogenous markers. Here, we present a direct measurement of GBB permeability in rats using an in vivo method based on portal blood sampling, which preserves intestinal blood flow and is virtually not affected by the liver and kidney function. 
Polyurethane catheters are inserted into the portal vein and inferior vena cava just above the hepatic veins confluence. Blood is sampled at baseline and after administration of a selected marker into a desired part of the gastrointestinal tract. Here, we present several applications of the method including (1) evaluation of the colon permeability to TMA, a gut bacterial metabolite, (2) evaluation of liver clearance of TMA, and (3) evaluation of a gut-portal blood-liver-peripheral blood pathway of gut bacteria-derived short-chain fatty acids. Furthermore, the protocol may also be used for tracking intestinal absorption and liver metabolism of drugs or for measurements of portal blood pressure.

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

The access of nutrients, microbiota metabolites and medicines to the circulation is controlled by the gut-blood barrier (GBB). We describe a direct method for measuring the GBB permeability in vivo, which, in contrast to commonly used indirect methods, is virtually not affected by liver and kidney functions.

微生物肠道血屏障及肝脏代谢的体内评价方法

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity ...

Studying Murine Small Bowel Mechanosensing of Luminal Particulates

Gastrointestinal (GI) motility is critical for normal digestion and absorption. In the small bowel, which absorbs nutrients, motility optimizes digestion and absorption. For this reason, some of the motility patterns in the small bowel include segmentation for mixing of luminal contents and peristalsis for their propulsion. Physical properties of luminal contents modulate the patterns of small bowel motility. The mechanical stimulation of GI mechanosensory circuits by transiting luminal contents and underlying gut motility initiate and modulate complex GI motor patterns. Yet, the mechanosensory mechanisms that drive this process remain poorly understood. This is primarily due to a lack of tools to dissect how the small bowel handles materials of different physical properties. To study how the small bowel handles particulates of varying sizes, we have modified an established in vivo method to determine small bowel transit. We gavage live mice with fluorescent liquid or tiny fluorescent beads. After 30 minutes, we dissect out the bowels to image the distribution of fluorescent contents across the entirety of the GI tract. In addition to high-resolution measurements of the geometric center, we use variable size binning and spectral analysis to determine how different materials affect small bowel transit. We have explored how a recently discovered "gut touch" mechanism affects small bowel motility using this approach.

To study how the small bowel handles particulates of varying sizes, we have modified an established in vivo method to determine small bowel transit.

研究小鼠小肠对管腔颗粒的机械传感

Gastrointestinal (GI) motility is critical for normal digestion and absorption. In the small bowel, which absorbs nutrients, motility optimizes digestion ...

使用 Dixon MRI 的综合 3D 肝脏脂肪功能分布模型

A 3D Quantification Technique for Liver Fat Fraction Distribution Analysis Using Dixon Magnetic Resonance Imaging

This study presents a 3D quantification methodology for the distribution of liver fat fraction (LFF) through the utilization of Dixon MRI image analysis. The central aim is to offer a highly accurate and non-invasive means of evaluating liver fat content. The process involves the acquisition of In-phase and Water-phase images from a Dixon sequence. LFF maps are then meticulously computed voxel by voxel by dividing the Lipid Phase images by the In-phase images. Simultaneously, 3D liver contours are extracted from the In-phase images. These crucial components are seamlessly integrated to construct a comprehensive 3D-LFF distribution model. This technique is not limited to healthy livers but extends to those afflicted by hepatic steatosis. The results obtained demonstrate the remarkable effectiveness of this approach in both visualizing and quantifying liver fat content. It distinctly discerns patterns that differentiate between normal and steatotic livers. By harnessing Dixon MRI to extract the 3D structure of the liver, this method offers precise LFF assessments spanning the entirety of the organ, thereby holding great promise for the diagnosis of hepatic steatosis with remarkable effectiveness.

作者聚焦：一种使用 3D Dixon MRI 评估和区分肝脏脂肪模式的非侵入性工具 

This study introduces a unique 3D quantification method for liver fat fraction (LFF) distribution using Dixon Magnetic Resonance Imaging (Dixon MRI). LFF maps, derived from in-phase and water-phase images, are integrated with 3D liver contours to differentiate LFF patterns between normal and steatotic livers, enabling precise assessment of liver fat content.

使用Dixon磁共振成像分析肝脏脂肪分数分布的3D定量技术

This study presents a 3D quantification methodology for the distribution of liver fat fraction (LFF) through the utilization of Dixon MRI image analysis. ...

使用多氟化胆汁酸和

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此视频中的章节

癌地致小鼠膀胱肿瘤的磁共振成像评价

监测树突状细胞迁移

细胞内胆汁酸动态实时监控使用遗传编码的FRET为基础的胆汁酸传感器

确定小鼠肝脏中的胆汁密度

胆汁盐诱导肠道病原菌生物膜形成的鉴别和定量技术

荧光介导的断层扫描对巨噬细胞相关小鼠肠道炎症的检测和定量

微生物肠道血屏障及肝脏代谢的体内评价方法

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