This work was supported in part by a Davis Heart and Lung Research Institute Trifit Award, as well as by the Intramural Research Program of the NIH National Institute on Aging.

该协议由认可并遵循美国俄亥俄州立大学伦理委员会对人体科研的准则。这是非常重要的，涉及MR设备全部过程由训练有素的人员秉承MR安全59的最高标准执行。 1.材料和准备<ol><li>确保所有必要的材料的实验之前可用（ 图2）。 </li><li>塞31 P线圈插入在表线圈连接在最靠近孔中的考试表的末尾。将一个大三角形泡沫垫的MR检查表头附近，但不是直接在31 P线圈。放置一个头部枕在磁共振检查台，从孔最远的另一端，对受试者的舒适性。 </li></ol> 2.除定位（图3a） <ol><li>指示受仰卧，双脚首先在MR表。将膝盖下一个泡沫垫，以支持部分弯曲位置的腿。 </li><li>为了尽可能接近居中左大腿到磁体的等角点尽可能靠近表（被检者的右侧）的右侧的主体定位，从而保证下检查在大腿肌肉最佳B0均匀性。提供耳塞和/或耳机的主题。 </li><li>在大约髌骨和股骨头之间的中点的左四头肌的31 P RF线圈定位，并用带子固定到腿。放置线圈在腿部的侧部，股外侧上方。 </li><li>固定婴儿油与用于将线圈固定到腿部相同绑带大腿内侧面。这有利于扫描定位。 </li><li>放置在线圈下方，膝盖以上的带子绑定对象的双腿并拢。安全主体的腿额外的STRA MR表PS，一个膝盖以上和膝盖和脚踝之间的一个中间。 </li><li>使用导激光划定线圈的中心和移动表以使用此定心里程碑式的磁体的等角点。 </li></ol> 3.运动方案<ol><li>解释到，这次演习的协议包括三个阶段的主题:一个初始，基础阶段;短暂，剧烈的运动阶段;和复苏阶段。 </li><li>指示受试者对躺着和放松光谱获取的基线和恢复阶段期间的腿部肌肉，以便尽量减少运动伪影。 </li><li>提供指示运动的开始倒计时受试者。在这一点上，具有主体发起伸膝/屈曲作为有力和尽可能快地对条带的阻力。 注:股四头肌被用来上下移动左小腿，直到指示停止。 </li><li> 30％的下跌后终止运动在PCR峰高度。 <ol><li>观察在收购查看器窗口在PCR峰高，并且还查看了运动序列结束后。 注:一般原则是，在肌酸峰高大约30％，降对应于丕峰是在PCR峰高度的50％。如果肌酸枯竭没有发生足够迅速地实现在考试的运动阶段下降30％，激励对象踢更难或运动时速度更快。 注:运动停止是通过监测在PCR峰高度和锻炼的持续时间来确定。这可能会导致在锻炼中不同患者的稍有不同的持续时间和可占在分析中。 </li></ol></li></ol> 4.扫描协议<ol><li>收购三平面定位，以验证正确的主题定位和识别31 P线圈的位置。 注:定位序列自动开始，并在印度语中心使用导激光ated位置（步骤2.9） </li><li>获得第二个三平面定位。 <ol><li>打开第一个三平面定位图像切片视图。 注:此过程可以针对不同的软件和硬件系统的不同。 </li><li>中心和旋转左键点击并按住在片组切片方向。旋转片组。确保切片的最终取向与婴儿油的位置相匹配。 </li><li>在序列例程窗口，增加的片数以覆盖在轴向和矢状图像（ 图3b）中的整个腿。 </li></ol></li><li> 31 P光谱序列: <ol><li>使用下面非本地化脉冲采集序列参数:TR:1,000毫秒; TE:0.34毫秒;谱宽:2000赫兹;翻转角:90度;采集的数据点:1024; 4平均值导致1光谱每6秒的时间分辨率。 </li></ol></li><li> 31 P垫片博点¯x位置: <ol><li>使用鼠标，拖动第二个三平面定位图像到查看窗口在屏幕的顶部。在光谱序列拖到协议窗口，然后双击打开。 </li><li>使用位置工具条显示垫片体素（选择与水平线的黑色矩形）。选择此选项后，观察其对定位图像的绿色方块。 注意:这是垫片像素。 </li><li>左键点击移动体素并保持在该中心的体素。改变大小和旋转通过左击体素的方位和在盒子的角保持体素。放置垫片框，以确保直接在线圈下方B 0场的均匀性和平行于股四头肌的平面。 注意:这是为了确保线圈，这是组织正下方的线圈的中心的体积下的敏感区域内的正确垫补。 </li><li>使用三平面定位图像以识别sensitiv线圈电子区域，调整垫片框股四头肌中包括这一地区。 注:垫片框可以比在表面线圈的真正范围较大，以保证数据采集的体素（ 图3c）内的B0均匀性。 </li><li> 31 P测试采集: <ol><li>打开浏览器收购窗口，选择在获取工具栏上的图标头。这将允许观看实时光谱获取。 </li><li>在31P垫片体素的位置后，运行该程序通过点击在协议窗口顶部的"运行"按钮来获得一个频谱。 </li><li>审查B0匀场的质量。观察采集窗口所产生的光谱。观察在0ppm为中心的突出的肌酸峰和无显著噪声（ 图4a，左）。 注:故障排除:如果频谱显示噪声，确保垫片框放在肌肉内。广告只是大小和垫片框的位置，提高了信噪比。根据需要重复试验的收购。 </li><li>为了看到在PCR峰高，在光谱工具打开频谱（"应用程序"→"光谱"）。打开患者的文件夹（文件夹树图标），选择适当的扫描，然后双击加载的频谱。 </li></ol></li></ol></li><li>运动前T1图像: <ol><li>在线圈的中心获得的单切片轴向T1加权图像。 </li></ol></li><li> 31 P运动前采集: <ol><li>复制从步骤4.4（即产生最佳光谱质量）通过左击，并在协议窗口中拖动序列的序列。使用该序列的所有后续测量。 </li><li>在常规序列窗口，增加测量次数从1到10中选择运行获得10次测量，而主题是静止的。 </li></ol></li><li> 31 &lt;/ SUP&gt;点锻炼收购: 注:请仔细记录的开始和结束运动时间，因为这将是分析的重要。 <ol><li>休息:从以前的扫描应用垫片设置，并设置以获得20次测量的序列。指示的主题，开始倒计时后踢。指示须保持静止2测量。 </li><li>练习:让拍摄对象进行伸膝练习〜30秒（或实现在PCR峰值下降30％所需的时间）。标的达到足够的肌酸消耗之后，让他们休息。 </li></ol></li><li> 31 P运动后的收购: <ol><li>在休息获得一个额外的20次测量。确保运动后收购立即开始下面的练习顺序，没有停顿或匀场（ 图4a，右）。 注:此恢复期的细分成两个独立的收购允许i的分析采集第二20动态光谱的期间nitial 20动态光谱，使操作者能够避免采集完整恢复期间，如果练习需要重复。 </li></ol></li><li>确保实行质量: <ol><li>比较在开始和运动结束在PCR峰高。高品质的锻炼期间导致在PCR浓度的〜30％的下降。 </li><li>验证在PCR峰高度是静止的开始和在恢复端相同（通常&lt;10％差被需要）。这保证了有采集期间场均匀性可忽略不计的损失。 注:如PCR细分不足，或者出现了磁场均匀性的损失，然后重复考试的行使/恢复部分（注意避免疲劳），确保了线圈和带连接牢固，并延长锻炼和/或持续时间鼓励更多的剧烈运动（ 图4b）。 不E:在步骤6和11允许额外的质量控制步骤中获得的图像的比较来可视化大腿和线圈的任何位移，由于运动，从而确保该协议，这可能显著影响所获取的数据的过程中出现最小运动。 </li></ol></li><li>下面的练习后的T1成像，重复运动前轴向使用相同的采集参数T1成像（步骤4.5）。 <ol><li>除了PCR法充分耗尽，测量结束锻炼的pH，以确保运动没有引起的肌肉酸中毒。 </li><li>通过测量Pi和PCR（δPⅰ）之间的化学位移和使用以下方程60执行这样的: pH值= 6.77 +日志[（ΔP 我 -3.29）/（5.68-δP 我 ）] 注:pH值应保持大于6.8 61。如果在PCR击穿是足够的，但是在pH值过低，重复练习回合一个短器的持续时间和/或具有降低的强度。 </li></ol></li><li>保存数据: <ol><li>保存所有取得的频谱为DICOM文件，并使用JMRUI导出进行处理。 </li><li>如果使用扫描仪，在"导航器"窗口中选择所有的光谱收购。 </li><li>在"应用程序"，选择"DICOM的工具"→"导出磁共振波谱"和DICOM（* .dcm）文件保存到C:/用户/ MEDCOM /温度/ CDROFFLINE （该工具会自动选择这个位置）。 </li><li>在"转移"，选择"导出到脱机"。保存到所需的位置。 </li></ol></li></ol> 5.数据处理与分析62 <ol><li>具有可自由可用JMRUI软件（; http://www.jmrui.eu/ 5.2版）分析MR光谱。 </li><li>变迹和相移的频谱，以确保在所有采集时间点（ 图5）的均匀性。在PCR高峰将集中一个T 0的PPM的光谱。 </li><li>使用内置的阿马里什算法量化肌酸峰的幅度在每一个获得的光谱。峰值幅度代表了PCR的表面线圈的感应区域内的特定时间点的浓度。 </li><li>在计算软件，绘制肌酸浓度的采集时间的函数。使用内置的计算软件曲线拟合工具，适合在PCR恢复期数据下面的方程52，63: <img alt="式（1）" src="/files/ftp_upload/54977/54977eq1.jpg" /></li><li>记录基线的PCR值（ <img alt="公式（2）" src="/files/ftp_upload/54977/54977eq2.jpg" /> ），最低的PCR（ <img alt="公式3" src="/files/ftp_upload/54977/54977eq3.jpg" /> ），以及恢复时间（ <img alt="公式4" src="/files/ftp_upload/54977/54977eq4.jpg" /> 。 </li><li>确保适当的条件简体字课程期间会晤通过计算在PCR耗尽，基线PCR和最低肌酸之间的百分差值CISE会话。理想的运动时间导致20 - 50％的损耗。 注意:曲线拟合的质量可以通过验证的R 2值是大于0.75来保证。 R 2值是由拟合软件自动计算。 </li></ol>

The authors have nothing to disclose.

本文描述为能提供串行和无创体内骨骼肌线粒体功能的测量31 PMRS检测的标准协议。考虑到调查针对代谢综合征的日益沉重的负担及其导致的发病率和死亡率的广度时，协议持有相当大的吸引力。这31 PMRS协议要求的扫描仪最短时间内，可以在用市售的MRS设施的中心并入综合代谢的研究科目。 该议定书中的关键步骤 <stro...

这项工作的目的是概述可再现的方法来在体内骨骼肌线粒体功能非侵入性测量具有大范围的能力的个体。异常线粒体损伤是多种代谢综合征和遗传性疾病的一个标志，从常见的情况，如老龄化和糖尿病罕见疾病，如弗里德共济失调。 代谢综合征和线粒体功能障碍 代谢综合征已经显示出干扰线粒体功能，抑制骨骼肌OXPHOS，并导致异位脂肪储存在骨骼肌1,2。调节代谢和能量稳态的关键的细胞器，线粒体有牵连的肥胖3,4的病理生理学，胰岛素抵抗5 </sup&gt;，2型糖尿病（T2DM）6，7，与糖尿病有关的微8，9，10，11和大血管并发症12，13，以及非酒精性脂肪肝病（NAFLD）14，15，16，除其他.Insulin性的特点是骨骼肌线粒体活性深刻的变化，其中包括减少线粒体三羧酸（TCA）通量率，ATP合成率和柠檬酸合成酶和NADH:O 2氧化还原酶活性5。一种假设是，这些改变可能是由于游离脂肪酸（FFA）的代谢物在肌肉的积累，这是肥胖等肥胖-R中显着增强兴高采烈的疾病2,17。肌肉的高架游离脂肪酸和脂质中间体曝光可降低基因在脂质氧化途径的表达，以及TCA循环和电子传输链（ETC）18。这种减少在脂质过载的设置线粒体骨骼肌OXPHOS容量是通过在定量（线粒体含量和生物合成）的下降伴随着19和骨骼肌线粒体20的定性作用。曝光骨骼肌和肌细胞对游离脂肪酸导致严重的胰岛素抗性，并增加了在肌肉FFA摄取用在人类和啮齿类动物21胰岛素抗性相关。脂质中间体酰胺和二酰基甘油（DAG）已经显示出，通过改变激酶，例如蛋白激酶C和PROT的活性直接抑制胰岛素信号传导途径EIN激酶B 21。因此，脂质衍生分子似乎发挥骨骼肌胰岛素抵抗和2型糖尿病的发展显着的作用。然而，在线粒体容量的变化是否是一个原因或胰岛素抵抗22的结果目前还不清楚。 弗里德里希共济失调和线粒体功能障碍 下降OXPHOS也可以从基因缺陷引起的。弗里德里希共济失调（FA），遗传性共济失调的最常见的形式，是一种遗传性疾病引起的frataxin的突变（FXN）基因，产生帧内线粒体的铁蓄积，活性氧的产生，和氧化磷酸23的异常， 24，25，26。这一重大发现已导致靶向治疗的发展，whicħ目的是提高在亚细胞水平的线粒体功能。尽管有这样的认识，出现了体内发育受限，为FA临床研究可重复的生物标志物。事实上，在FA靶向治疗的有效评价的关键障碍是不能跟踪线粒体功能的变化。当前功能的措施，例如，可以识别心输出量减少;然而，它们不能确定在哪些功能障碍发生水平（ 图1）的。线粒体功能的可靠标记物可用于鉴定和评估弗里德里希共济失调疾病进展的发展是至关重要的，以评估靶向疗法的相关机械冲击。 受损的氧化磷酸化和心功能不全 异常线粒体功能，无论是后天或遗传性，有助于CARDI的发展或进展AC功能障碍。下压力超负荷和心脏衰竭的条件下，从FFA初级能量底物优先开关为葡萄糖。这是通过ETC活性降低和氧化磷酸化27相关联。在心脏功能障碍的线粒体生物能量学的病理生理学可根据线粒体缺陷的主要起源不同。糖尿病和心肌线粒体异常，如受损的生物合成和脂肪酸代谢，从而导致降低的衬底灵活性，能效，并且最终，舒张功能障碍28,29代谢综合征的结果。在FA，另一方面，一个frataxin缺乏导致心肌30,31显著线粒体的铁蓄积。铁积累通过芬顿反应32 &lt;导致产生的自由基/ SUP&gt;和增加的自由基诱导的心肌损伤的机会。内线粒体的铁蓄积也与氧化应激的增加的灵敏度和减小的氧化能力30,31相关联。铁的积累和随后的异常线粒体功能，由于frataxin缺乏，因此可负责在FA 33,34观察到受损心脏能量和心肌病。这也是有趣的是，在骨骼肌线粒体减少氧化能力的平行运动耐受力，减少心脏衰竭（HF）35代谢能力。骨骼肌OXPHOS容量测量，如本文详述，很容易实现的和健壮;再加HF骨骼肌氧化磷酸化的意义，这些特性使其成为一个有吸引力的生物标志物在听到的全面研究ŧ疾病36。 受损的氧化磷酸化和伴随的心脏功能障碍并不是代谢和线粒体疾病的无关紧要的方面。与糖尿病和代谢性疾病科是在患心血管疾病的风险较高，并有心肌梗死（MI）37，38，39，40，41后的超额死亡;在FA科目有一半心肌病和心律失常或心脏衰竭42多人死亡。因此，降低的OXPHOS量化不仅可以允许早期检测和治疗的心脏功能障碍，但它也可以减轻这些疾病的主要临床负担。 靶向疗法直接增加OXPHOS容量是提高的受试者治疗中的有希望的领域，磨片疗法的代谢功能障碍的原因是遗传性或获得。目前，新的发展靶向药物，无论是缓解异常线粒体功能43或纠正的主要遗传缺陷44可以提高FA的疯狂生物能的特点。在所获取的线粒体功能障碍的情况下，增加体力活动可改善线粒体功能45，46，47。 31磷磁共振波谱作为线粒体功能的无创生物标志物 不管测试的治疗，综合骨骼肌生物能的体内评估是评估有针对性的干预措施的影响的一个重要工具，尤其是在严重的运动耐受或无法接受常规科目太保LIC测试。调谐到磷（31 PMRS），整个身体细胞内的各种高能量底物中发现的内源性核的磁共振光谱，已被用于使用各种方法，包括量化线粒体氧化容量在磁铁运动恢复协议和肌肉刺激方案48。在运动恢复协议依赖于各种设备的范围从调节和衡量工作量，皮带和垫允许突发型电阻和准静态运动的简单配置，MRI兼容的测力计的复杂性。之一的任何这些协议的主要目标是产生的量为三磷酸腺苷（ATP）的需求是通过磷酸的酶分解（PCR）通过肌酸激酶反应49最初遇到的能量不平衡。一旦运动停止，ATP生成率是由氧化河粉为主sphorylation并代表在线粒体50的体内容量最大。此外，运动后恢复期间OXPHOS可以通过一阶速率反应51进行说明。肌酸的锻炼后的恢复可以因此通过一个指数时间常数（τPCR），与表示对氧化ATP合成更大的能力τ 肌酸的较小的值的嵌合来定量。显著已作出努力，以验证对体外 31 PMRS和氧化磷酸化的更直接的措施，并演示了这种技术52，53，54，55的潜在的临床应用。 值得注意的是，在这项工作中所描述的协议可在临床上可用的扫描仪来实现，并且它已被广泛地验证为无创生物标记ö˚F线粒体功能56。然而，对于应用程序优化，个体神经肌肉功能障碍或行动不便的严重程度不同的锻炼31 PMRS协议尚未很好地建立57。定义良好的，广泛适用的运动方案和31 PMRS技术是在与线粒体功能异常的基础疾病的评价特别有用。 几个先前的研究已经探索的非侵入性技术应用在受试者量化线粒体功能。例如，这些技术已显示在2型糖尿病36对受损OXPHOS。洛等。首先测试的PMRS技术的可行性与FA受试者，发现1）在FA的基本遗传缺陷损害的骨骼肌OXPHOS和2）GAA三联体的数目重复成反比骨骼肌öXPHOS 33。最近，Nachbauer 等。二手PMRS作为在FA药物试验有7例次要转归指标。肌酸恢复时间在受试者显著不再与对照组相比，重申洛的早期工作，并指示可导致线粒体容量下降可检测使用PMRS技术58异常frataxin表达的FA的影响。 可靠的方法，以充分体内骨骼肌功能限定在一个可行的，经济有效的，并且可重复的方式是改善受试者的结果的范围内，影响线粒体功能的疾病的关键。 这项工作概述了使用 31 PMRS骨骼肌体内最大的氧化能力获得一个强大的过程。内的磁铁练习协议已被个人涉及广泛的物理和泛函容忍升的能力，并让使用廉价且广泛可用的设备简化学科设置。

<table><tbody><tr><td>1.5 T MR Scanner</td><td>Siemens</td><td></td><td>manufacturer will not affect results</td></tr><tr><td>10 cm 31P transmit-receive coil, 1.5T compatible</td><td>PulseTeq</td><td></td><td>manufacturer will not affect results</td></tr><tr><td>3 fl oz Baby Oil</td><td>Johnson &amp;amp; Johnson</td><td></td><td>manufacturer will not affect results</td></tr><tr><td>Foam triangle cushion (Knee)</td><td>Siemens</td><td></td><td>manufacturer will not affect results</td></tr><tr><td>(3) plastic buckle resistive straps; table to table</td><td>Siemens</td><td></td><td>manufacturer will not affect results</td></tr><tr><td>(1) plastic buckle resistive strap; self-connecting</td><td>Siemens</td><td></td><td></td></tr></tbody></table>

phosphorus-31 magnetic resonance spectroscopy: a tool for measuring in vivo mitochondrial oxidative phosphorylation capacity in human skeletal muscle

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo and noninvasively in humans via phosphorus-31 magnetic resonance spectroscopy (31PMRS). However, the approach has not been widely adopted in translational and clinical research, with variations in methodology and limited guidance from the literature. Increased optimization, standardization, and dissemination of methods for in vivo 31PMRS would facilitate the development of targeted therapies to improve OXPHOS capacity and could ultimately favorably impact cardiovascular health. 31PMRS produces a noninvasive, in vivo measure of OXPHOS capacity in human skeletal muscle, as opposed to alternative measures obtained from explanted and potentially altered mitochondria via muscle biopsy. It relies upon only modest additional instrumentation beyond what is already in place on magnetic resonance scanners available for clinical and translational research at most institutions. In this work, we outline a method to measure in vivo skeletal muscle OXPHOS. The technique is demonstrated using a 1.5 Tesla whole-body MR scanner equipped with the suitable hardware and software for 31PMRS, and we explain a simple and robust protocol for in-magnet resistive exercise to rapidly fatigue the quadriceps muscle. Reproducibility and feasibility are demonstrated in volunteers as well as subjects over a wide range of functional capacities.

重复性研究 6名志愿者（4男2女，平均年龄24.5±6.2岁），没有自报的心脏，代谢或线粒体疾病进行1星期内2不同的日子里所描述的31 PMRS锻炼和成像技术的会议，以评估技术再现性（ 图6a）。对正常的志愿者进行的研究证实了线粒体功能的定量31 PMRS研究的再现性。进行PCR恢复时间奥特曼...

Watch this Scientific Journal Video about Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle at JoVE.

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (31PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

磷31磁共振波谱分析:一种测量工具<em&gt;在体内</em&gt;线粒体氧化磷酸化能力人骨骼肌

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...

phosphorus-31-magnetic-resonance-spectroscopy-tool-for-measuring-vivo

Research

JoVE Journal

Medicine

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

11.6K 次观看.  The Ohio State University. This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (31PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle. 

视频: Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

使用分离线粒体从小鼠骨骼肌进行高通量微孔板呼吸测量最小数量

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

科研

发育生物学

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

Muscle strains are one of the most common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and physical exam alone, however the clinical presentation can vary greatly depending on the extent of injury, the patient's pain tolerance, etc. In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Biological markers, such as serum creatine kinase levels, are typically elevated with muscle injury, but their levels do not always correlate with the loss of force production. This is even true of histological findings from animals, which provide a "direct measure" of damage, but do not account for all the loss of function. Some have argued that the most comprehensive measure of the overall health of the muscle in contractile force. Because muscle injury is a random event that occurs under a variety of biomechanical conditions, it is difficult to study. Here, we describe an in vivo animal model to measure torque and to produce a reliable muscle injury. We also describe our model for measurement of force from an isolated muscle in situ. Furthermore, we describe our small animal MRI procedure.

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

An in vivo animal model of injury is described. The method takes advantage of the subcutaneous position of the fibular nerve. Velocity, timing of muscle activation, and arc of motion are all pre-determined and synchronized using commercial software. Post injury changes are monitored in vivo using MR imaging/spectroscopy.

一个在体内收缩引起的损伤，非侵入性的监测恢复啮齿动物模型

Muscle strains are one of the most  common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and  physical ...

医学

Assessing Mitochondrial Function in Sciatic Nerve by High-Resolution Respirometry

Mitochondrial dysfunction in peripheral nerves accompanies several diseases associated with peripheral neuropathy, which can be triggered by multiple causes, including autoimmune diseases, diabetes, infections, inherited disorders, and tumors. Assessing mitochondrial function in mouse peripheral nerves can be challenging due to the small sample size, a limited number of mitochondria present in the tissue, and the presence of a myelin sheath. The technique described in this work minimizes these challenges by using a unique permeabilization protocol adapted from one used for muscle fibers, to assess sciatic nerve mitochondrial function instead of isolating the mitochondria from the tissue. By measuring fluorimetric reactive species production with Amplex Red/Peroxidase and comparing different mitochondrial substrates and inhibitors in saponin-permeabilized nerves, it was possible to detect mitochondrial respiratory states, reactive oxygen species (ROS), and the activity of mitochondrial complexes simultaneously. Therefore, the method presented here offers advantages compared to the assessment of mitochondrial function by other techniques.

High-resolution respirometry coupled to fluorescence sensors determines mitochondrial oxygen consumption and reactive oxygen species (ROS) generation. The present protocol describes a technique to assess mitochondrial respiratory rates and ROS production in the permeabilized sciatic nerve.

通过高分辨率呼吸测量法评估坐骨神经的线粒体功能

Mitochondrial dysfunction in peripheral nerves accompanies several diseases associated with peripheral neuropathy, which can be triggered by multiple ...

生物化学

Mitochondrial Functional Assessment in Brush Lizard Liver Mitochondria

Assessment of Mitochondrial Oxygen Consumption Using a Plate Reader-based Fluorescent Assay

Mitochondria serve many important functions, including cellular respiration, ATP production, controlling apoptosis, and acting as a central hub of metabolic pathways. Therefore, experimentally assessing mitochondrial functionality can provide insight into variations among different populations or disease states. Additionally, it is valuable to assess whether isolated mitochondria are healthy enough to proceed with experiments. One characteristic often used to compare mitochondrial function in different samples is the rate of oxygen consumption. Oxygen consumption and subsequent calculation of the respiratory control ratio in either intact cells or mitochondria isolated from tissue can serve all three purposes. Using mitochondria isolated from the livers of brush lizards in conjunction with a phosphorescent probe that is sensitive to the fluctuations in oxygen concentration of a solution, we measured oxygen consumption using a fluorescent plate reader. This method is not only quick and efficient but also can be conducted with a small amount of mitochondria and without the need for specialized equipment. The step-by-step protocol described here increases the accessibility of mitochondrial functional assessment to researchers.

Assessment of oxygen consumption provides integral information about mitochondrial function. Using a phosphorescent probe with a fluorescent plate reader, accurate and reproducible data can be obtained easily without specialized equipment. This assay enables any lab to measure the oxygen consumption of isolated mitochondria and calculate respiratory control ratios.

Mitochondria serve many important functions, including cellular respiration, ATP production, controlling apoptosis, and acting as a central hub of ...

Skeletal Muscle Neurovascular Coupling, Oxidative Capacity, and Microvascular Function with 'One Stop Shop' Near-infrared Spectroscopy

Exercise represents a major hemodynamic stress that demands a highly coordinated neurovascular response in order to match oxygen delivery to metabolic demand. Reactive hyperemia (in response to a brief period of tissue ischemia) is an independent predictor of cardiovascular events and provides important insight into vascular health and vasodilatory capacity. Skeletal muscle oxidative capacity is equally important in health and disease, as it determines the energy supply for myocellular processes. Here, we describe a simple, non-invasive approach using near-infrared spectroscopy to assess each of these major clinical endpoints (reactive hyperemia, neurovascular coupling, and muscle oxidative capacity) during a single clinic or laboratory visit. Unlike Doppler ultrasound, magnetic resonance images/spectroscopy, or invasive catheter-based flow measurements or muscle biopsies, our approach is less operator-dependent, low-cost, and completely non-invasive. Representative data from our lab taken together with summary data from previously published literature illustrate the utility of each of these end-points. Once this technique is mastered, application to clinical populations will provide important mechanistic insight into exercise intolerance and cardiovascular dysfunction.

Here, we describe a simple, non-invasive approach using near-infrared spectroscopy to assess reactive hyperemia, neurovascular coupling and skeletal muscle oxidative capacity in a single clinic or laboratory visit.

"一站式" 近红外光谱对骨骼肌神经血管的耦合、氧化能力及微血管功能的作用

Exercise represents a major hemodynamic stress that demands a highly coordinated neurovascular response in order to match oxygen delivery to metabolic ...

High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in biological samples (intact and permeabilized cells, tissues or isolated mitochondria). The high-resolution oxygraph device is equipped with two chambers and uses polarographic oxygen sensors to measure oxygen concentration and calculate oxygen consumption within each chamber. Oxygen consumption rates are calculated using software and expressed as picomoles per second per number of cells. Each high-resolution oxygraph chamber contains a stopper with injection ports, which makes it ideal for substrate-uncoupler-inhibitor titrations or detergent titration protocols for determining effective and optimum concentrations for plasma membrane permeabilization. The technique can be applied to measure respiration in a wide range of cell types and also provides information on mitochondrial quality and integrity, and maximal mitochondrial respiratory electron transport system capacity.

High-resolution respirometry is used to determine mitochondrial oxygen consumption. This is a straightforward technique to determine mitochondrial respiratory chain complexes&#39; (I-IV) respiratory rates, maximal mitochondrial electron transport system capacity, and mitochondrial outer membrane integrity.

高分辨率呼​​吸测量，以评估线粒体功能的通透和完整细胞

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in ...

生物学

Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the mitochondrial oxidative phosphorylation (OXPHOS) system, which is regulated in response to cellular demands. The lipid molecule Coenzyme Q (CoQ) is essential in this process by transferring electrons to complex III in the electron transport chain (ETC) through constant oxidation/reduction cycles. Mitochondria status and, ultimately, cellular health can be assessed by measuring ETC oxygen consumption using respirometric assays. These studies are typically performed in established or primary cell lines that have been cultured for several days. In both cases, the respiration parameters obtained may have deviated from normal physiological conditions in&#160;any given organ or tissue.
Additionally, the intrinsic characteristics of cultured single fibers isolated from skeletal muscle impede this type of analysis. This paper presents an updated and detailed protocol for the analysis of respiration in freshly isolated mitochondria from mouse skeletal muscle. We also provide solutions to potential problems that could arise at any step of the process. The method presented here could be applied to compare oxygen consumption rates in diverse transgenic mouse models and study the mitochondrial response to drug treatments or other factors such as aging or sex. This is a feasible method to respond to crucial questions about mitochondrial bioenergetics metabolism and regulation.

Here, we describe a detailed method for mitochondria isolation from mouse skeletal muscle and the subsequent analysis of respiration by Oxygen Consumption Rate (OCR) using microplate-based respirometric assays. This pipeline can be applied to study the effects of multiple environmental or genetic interventions on mitochondrial metabolism.

从小鼠骨骼肌中分离线粒体用于呼吸测定

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the ...

High-Resolution Fluoro-Respirometry of Equine Skeletal Muscle

Mitochondrial function-oxidative phosphorylation and the generation of reactive oxygen species-is critical in both health and disease. Thus, measuring mitochondrial function is fundamental in biomedical research. Skeletal muscle is a robust source of mitochondria, particularly in animals with a very high aerobic capacity, such as horses, making them ideal subjects for studying mitochondrial physiology. This article demonstrates the use of high-resolution respirometry with concurrent fluorometry, with freshly harvested skeletal muscle mitochondria, to quantify the capacity to oxidize substrates under different mitochondrial states and determine the relative capacities of distinct elements of mitochondrial respiration. Tetramethylrhodamine methylester is used to demonstrate the production of mitochondrial membrane potential resulting from substrate oxidation, including calculation of the relative efficiency of the mitochondria by calculating the relative membrane potential generated per unit of concurrent oxygen flux. The conversion of ADP to ATP results in a change in the concentration of magnesium in the reaction chamber, due to differing affinities of the adenylates for magnesium. Therefore, magnesium green can be used to measure the rate of ATP synthesis, allowing the further calculation of the oxidative phosphorylation efficiency (ratio of phosphorylation to oxidation [P/O]). Finally, the use of Amplex UltraRed, which produces a fluorescent product (resorufin) when combined with hydrogen peroxide, allows the quantification of reactive oxygen species production during mitochondrial respiration, as well as the relationship between ROS production and concurrent respiration. These techniques allow the robust quantification of mitochondrial physiology under a variety of different simulated conditions, thus shedding light on the contribution of this critical cellular component to both health and disease.

Horses have an exceptional aerobic exercise capacity, making equine skeletal muscle an important tissue for both the study of equine exercise physiology as well as mammalian mitochondrial physiology. This article describes techniques for the comprehensive assessment of mitochondrial function in equine skeletal muscle.

马骨骼肌的高分辨率氟呼吸测定法

Mitochondrial function-oxidative phosphorylation and the generation of reactive oxygen species-is critical in both health and disease. Thus, measuring ...

Investigating Cardiac Metabolism in the Isolated Perfused Mouse Heart with Hyperpolarized [1-13C]Pyruvate and 13C/31P NMR Spectroscopy

Metabolism is the basis of important processes in cellular life. Characterizing how metabolic networks function in living tissues provides crucial information for understanding the mechanism of diseases and designing treatments. In this work, we describe procedures and methodologies for studying in-cell metabolic activity in a retrogradely perfused mouse heart in real-time. The heart was isolated in situ,&#160;in conjunction with cardiac arrest to minimize the myocardial ischemia and was perfused inside a nuclear magnetic resonance (NMR) spectrometer. While in the spectrometer and under continuous perfusion, hyperpolarized [1-13C]pyruvate was administered to the heart, and the subsequent hyperpolarized [1-13C]lactate and [13C]bicarbonate production rates served to determine, in real-time, the rates of lactate dehydrogenase and pyruvate dehydrogenase production. This metabolic activity of hyperpolarized [1-13C]pyruvate was quantified with NMR spectroscopy in a model free-manner using the product selective saturating-excitations acquisition approach. 31P spectroscopy was applied in between the hyperpolarized acquisitions to monitor the cardiac energetics and pH. This system is uniquely useful for studying metabolic activity in the healthy and diseased mouse heart.

Investigating Cardiac Metabolism in the Isolated Perfused Mouse Heart with Hyperpolarized [1-13C]Pyruvate and 13C/31P NMR Spectroscopy

We describe an experimental setup for administrating hyperpolarized 13C-labeled metabolites in continuous perfusion mode to an isolated perfused mouse heart. A dedicated 13C-NMR acquisition approach enabled the quantification of metabolic enzyme activity in real-time, and a multiparametric 31P-NMR analysis enabled the determination of the tissue ATP content and pH.

用超极化[1-13C]丙酮酸和 13C / 31P NMR波谱研究分离灌注小鼠心脏中的心脏代谢

Metabolism is the basis of important processes in cellular life. Characterizing how metabolic networks function in living tissues provides crucial ...

人 PBMC 中线粒体功能的高分辨率分析

High-Resolution Fluorespirometry to Assess Dynamic Changes in Mitochondrial Membrane Potential in Human Immune Cells

Peripheral mononuclear cells (PBMCs) exhibit robust changes in mitochondrial respiratory capacity in response to health and disease. While these changes&#160;do not always reflect what occurs in other tissues, such as skeletal muscle, these cells are an accessible and valuable source of viable mitochondria from human subjects. PBMCs are exposed to systemic signals that impact their bioenergetic state. Thus, expanding our tools to interrogate mitochondrial metabolism in this population will elucidate mechanisms related to disease progression. Functional assays of mitochondria are often limited to using respiratory outputs following maximal substrate, inhibitor, and uncoupler concentrations to determine the full range of respiratory capacity, which may not be achievable in vivo. The conversion of adenosine diphosphate (ADP) to adenosine triphosphate (ATP) by ATP-synthase results in a decrease in mitochondrial membrane potential (mMP) and an increase in oxygen consumption. To provide a more integrated analysis of mitochondrial dynamics, this article describes the use of high-resolution fluorespirometry to measure the simultaneous response of oxygen consumption and mitochondrial membrane potential (mMP) to physiologically relevant concentrations of ADP. This technique uses tetramethylrhodamine methylester (TMRM) to measure mMP polarization in response to ADP titrations following maximal hyperpolarization with complex I and II substrates. This technique can be used to quantify how changes in health status, such as aging and metabolic disease, affect the sensitivity of mitochondrial response to energy demand in PBMCs, T-cells, and monocytes from human subjects.

作者聚焦：使用荧光螺线测定法对 PBMC 线粒体反应的新见解

Methods for studying mitochondrial bioenergetics under physiologically relevant substrate concentrations in immune cells are limited. We provide a detailed protocol that uses high-resolution fluorespirometry to assess changes in the response of the mitochondrial membrane potential to energy demand in human T-cells, monocytes, and peripheral mononuclear cells.

磷31磁共振波谱分析:一种测量工具

摘要

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此视频中的章节

使用分离线粒体从小鼠骨骼肌进行高通量微孔板呼吸测量最小数量

一个在体内收缩引起的损伤，非侵入性的监测恢复啮齿动物模型

通过高分辨率呼吸测量法评估坐骨神经的线粒体功能

Assessment of Mitochondrial Oxygen Consumption Using a Plate Reader-based Fluorescent Assay

"一站式" 近红外光谱对骨骼肌神经血管的耦合、氧化能力及微血管功能的作用

高分辨率呼吸测量，以评估线粒体功能的通透和完整细胞

从小鼠骨骼肌中分离线粒体用于呼吸测定

马骨骼肌的高分辨率氟呼吸测定法

用超极化[^1-13C]丙酮酸和 ¹³C / ³¹P NMR波谱研究分离灌注小鼠心脏中的心脏代谢

高分辨率荧光肺活测定法评估人免疫细胞中线粒体膜电位的动态变化

磷31磁共振波谱分析:一种测量工具

摘要

探索更多视频

此视频中的章节

使用分离线粒体从小鼠骨骼肌进行高通量微孔板呼吸测量最小数量

一个在体内收缩引起的损伤，非侵入性的监测恢复啮齿动物模型

通过高分辨率呼吸测量法评估坐骨神经的线粒体功能

Assessment of Mitochondrial Oxygen Consumption Using a Plate Reader-based Fluorescent Assay

"一站式" 近红外光谱对骨骼肌神经血管的耦合、氧化能力及微血管功能的作用

高分辨率呼​​吸测量，以评估线粒体功能的通透和完整细胞

从小鼠骨骼肌中分离线粒体用于呼吸测定

马骨骼肌的高分辨率氟呼吸测定法

用超极化[1-13C]丙酮酸和 13C / 31P NMR波谱研究分离灌注小鼠心脏中的心脏代谢

高分辨率荧光肺活测定法评估人免疫细胞中线粒体膜电位的动态变化

高分辨率呼吸测量，以评估线粒体功能的通透和完整细胞

用超极化[^1-13C]丙酮酸和 ¹³C / ³¹P NMR波谱研究分离灌注小鼠心脏中的心脏代谢