这项工作是支持公共卫生服务津贴（HL086513）从NHLBI私立教育机构，以及来自加州再生医学（RC1 - 00104），公共卫生服务津贴（HL085377）从NHLBI研究所的综合研究资助和礼品波林基金会HSB 资深大律师是一个加州再生医学的桥梁研究所的支持干细胞研究奖（TB1 - 01194）旧金山州立大学。

加州大学旧金山分校的机构动物护理和使用委员会（IACUC）批准该协议。 1。鼠标趾长伸肌 （EDL）肌肉的解剖<ol><li>按照机构准则执行的所有动物的程序。 </li><li>安乐死与200 mg / kg腹腔巴比妥/颈椎脱位，肌肉收获10前的动物。剥离应实行，使肌肉可收获和"安乐死"的15分钟内，安装在张力换能器。 </li><li>夹层纸盒和引脚腿到托盘上排列胴体仰卧。 </li><li>在解剖镜下，开放的皮肤，小心地打开筋膜（图1a），和剥离胫骨从脚踝向上暴露的EDL（图1b） 。使用滴乳酸林格氏液，以保持肌肉湿润和收获期间的缓冲。 </li><li>删除的EDL，保留尽可能多的肌腱上，每年年底，并把到一个Petri菜含有乳酸林格氏液。铁缝合肌腱（ 图1c）。重要的是，肌纤维不能触及，或在清扫不安。 </li></ol> 2。鼠标EDL的安装在肌肉地带肌动描记<ol><li>对于这些研究中，一个组织洗澡是需要的，固定的肌肉，同时允许它在恒温沐浴在生理溶液中连续充氧。洗澡加上一个力传感器的测量肌肉紧张。我们采用从丹麦MYO技术（DMT型号820MS）的综合肌肉地带肌动描记洗澡为此。此外，一个方形脉冲电刺激（草型号S48）和数据采集平台（ADInstruments PowerLab系统的数据采集系统和LabChart软件）引出，记录和分析MYO机械的反应，分别需要。大唐820MS集成到盒盖定位肌肉两侧肌肉地带中旬部分，铂电极。其他myographs可能需要特别注意电极放置。 </li><li>肌动描记浴填写5毫升克雷布斯Henseleit解决方案11。加热至25 ° C。泡泡O 2 / CO 2（95％/ 5％），通过洗澡前15分钟使用。 </li><li>使用缝线的EDL肌动描记夹具之间的延长和夹具之间的EDL肌筋（Fig.1D五 ）安全。要小心不要夹肌肉本身。 </li><li>肌动描记浴保持在25 ° C。 </li></ol> 3。肌的力学分析 A.抽搐紧张<ol><li>在洗澡时设置的初始长度，以便有没有肌肉松弛。 </li><li>确定通过调整电压，以获得最大的抽搐紧张的最大刺激（持续时间为0.5毫秒），然后设定在20％以上最大的刺激（实现超最大刺激）。在我们的研究，超最大的刺激，通常在40伏的输出实现。 </li><li>验证从使用示波器的刺激输出。 </li><li>确定最佳长度逐渐伸展的肌肉，直到没有抽搐的紧张局势进一步增加。 </li><li>允许3分钟的肌肉平衡。 </li><li>交付平方米超最大的刺激在最佳使用电子刺激S44草的长度（0.5毫秒），并记录输出。 </li><li>记录:抽搐张力曲线（P T随时间; 图2A）。 </li></ol> B.破伤风紧张<ol><li>让肌肉休息3分钟。 </li><li>应用最佳使用电子刺激S44草的长度在150赫兹的超最大刺激的列车为300毫秒，并记录输出。 </li><li>记录:破伤风张力曲线（P O随时间; 图2B）。 </li></ol> C.部队频率<ol><li>让肌肉休息3分钟。 </li><li>部队频率:适用于30，60，100，140和160赫兹的超最大刺激的列车，彼此之间的刺激（图3）休息3分钟。此外，火车可应用于15，25，35，45，55，65，75，100，140和160赫兹在较低的频率更高的分辨率，其中力变化显着。 </li><li>剧情:力频率的关系（％最大力量与刺激频率）。 </li></ol> D.疲劳<ol><li>应用短破伤风的火车:300毫秒（或调整，以产生50％的峰值力的频率），每10分钟3秒60赫兹。 10分钟，强直力下降的一个〜15％的初始值（ 图4）高原水平。 </li><li>剧情:低频疲劳（％最大力量与时间）。 </li></ol> E.在协议结束额外的数据收集<ol><li>在卸载肌肉的肌动描记之前，设置MUSC乐在最佳长度确定的步骤III.A.4，使用显微镜或一个眼，用游标卡尺测量其直径。计算截面积（2微米）。 </li><li>通过去除缝线，体重肌肉测量肌肉质量（毫克）。 </li><li>称重鼠标评估体重（GM）。 </li></ol> 4。计算<ol><li>肌肉:体重比= 肌肉质量/体质量</li><li>抽搐紧张，P T（MN）= 在抽搐产生的最大张力</li><li>具体抽搐张力（N /厘米2）= 抽搐张力（MN）/截面积（2微米）× 10 5 / MN•微米2 /厘米2 </li><li>到峰值张力时间（毫秒）= 从收缩发病时间最大张力</li><li>半弛豫时间（毫秒）= 从峰值紧张到峰值张力的50％ </li><li>强直紧张，P O（MN）= 在破伤风产生的最大张力</li><li>具体强直张力（N /厘米2）= 强直张力（MN）/截面积（2微米）× 10 5 / MN•微米2 /厘米2 </li><li>破伤风上升最大速率（N / S）= 期间，破伤风，即紧张，强直张力曲线的最大斜率（或DP的O / DT）上升的紧张局势加剧的最高税率</li><li>半松弛强直张力（MS）= 时间从停止刺激，在紧张刺激终止的50％ </li><li>抽搐紧张强直张力比，P - T / P O = 最大抽搐张力/最大强直张力</li><li>疲劳指数= 经过两分钟的低频疲劳等长张力最大的张力比</li></ol> 5。代表性的成果<img src="/files/ftp_upload/2582/2582fig1.jpg" alt="图1" /> 图1。剖析的EDL肌肉。 EDL的一个 muscles.TA，后肢胫前，B，EDL（趾长伸肌）肌曝光，曝光C，EDL的肌腱缝合的附件，D，张力传感器浴（从侧面看），E，安装在洗澡（从上面）。在肌肉未完全沉浸在缓冲区，仅供;在实践中，应完全沉浸肌肉，防止干燥。 <img src="/files/ftp_upload/2582/2582fig2.jpg" alt="图2" /> 图2。张力曲线的例子 。 的抽搐紧张说明抽搐最大张力（P T），收缩时间（CT）和半弛豫时间（HRT）的曲线，例如酒吧，1秒，B，强直张力曲线显示最大强直张力（P O）的例子，半松弛强直张力（HRTT）。酒吧，1S。 <img src="/files/ftp_upload/2582/2582fig3.jpg" alt="图3" /> 图3。力频率的关系分析的例子 。 ，增量刺激频率产生的紧张局势，B，例如，在30MHz的脉冲序列。酒吧，80毫秒，C，例如，在140MHz的脉冲序列。酒吧，80毫秒，D，从A 中显示的数据派生的力-频率曲线的例子。力 - 频率曲线的形状是肌肉力量的特点，可以比较来自不同动物的肌肉之间。 <img src="/files/ftp_upload/2582/2582fig4.jpg" alt="图4" /> 图4。低频疲劳分析的例子 。超过stimulation.Examples在指定的时间点（B，C，D）的低频脉冲串期间产生了递减的紧张局势图所示，E，从 A中显示的数据产生的低频疲劳曲线的范例。低频疲劳曲线的形状是肌肉力量的特点，可以比较来自不同动物的肌肉之间。

<ol>
	<li>Harcourt, L. J., Schertzer, J. D., Ryall, J. G., Lynch, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+dose+formoterol+administration+improves+muscle+function+in+dystrophic+mdx+mice+without+increasing+fatigue.">Low dose formoterol administration improves muscle function in dystrophic mdx mice without increasing fatigue.</a> Neuromuscul Disord. 17, 47-55 (2007).</li><li>Messina, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF+overexpression+via+adeno-associated+virus+gene+transfer+promotes+skeletal+muscle+regeneration+and+enhances+muscle+function+in+mdx+mice.">VEGF overexpression via adeno-associated virus gene transfer promotes skeletal muscle regeneration and enhances muscle function in mdx mice.</a> FASEB J. 21, 3737-3746 (2007).</li><li>Danieli-Betto, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sphingosine+1-phosphate+protects+mouse+extensor+digitorum+longus+skeletal+muscle+during+fatigue.">Sphingosine 1-phosphate protects mouse extensor digitorum longus skeletal muscle during fatigue.</a> Am J Physiol Cell Physiol. 288, C1367-C1373 (2005).</li><li>MacIntosh, B. R., Willis, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force-frequency+relationship+and+potentiation+in+mammalian+skeletal+muscle.">Force-frequency relationship and potentiation in mammalian skeletal muscle.</a> J Appl Physiol. 88, 2088-2096 (2000).</li><li>Hayes, A., Williams, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contractile+properties+of+clenbuterol-treated+mdx+muscle+are+enhanced+by+low-intensity+swimming.">Contractile properties of clenbuterol-treated mdx muscle are enhanced by low-intensity swimming.</a> J Appl Physiol. 82, 435-439 (1997).</li><li>Coulton, G. R., Curtin, N. A., Morgan, J. E., Partridge, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mdx+mouse+skeletal+muscle+myopathy:+II.+Contractile+properties.">The mdx mouse skeletal muscle myopathy: II. Contractile properties.</a> Neuropathol Appl Neurobiol. 14, 299-314 (1988).</li><li>Danieli-Betto, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deficiency+of+alpha-sarcoglycan+differently+affects+fast-+and+slow-twitch+skeletal+muscles.">Deficiency of alpha-sarcoglycan differently affects fast- and slow-twitch skeletal muscles.</a> Am J Physiol Regul Integr Comp Physiol. 289, R1328-R1337 (2005).</li><li>Chan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gene+for+speed:+contractile+properties+of+isolated+whole+EDL+muscle+from+an+alpha-actinin-3+knockout+mouse.">A gene for speed: contractile properties of isolated whole EDL muscle from an alpha-actinin-3 knockout mouse.</a> Am J Physiol Cell Physiol. 295, C897-C904 (2008).</li><li>Personius, 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=Grip+force,+EDL+contractile+properties,+and+voluntary+wheel+running+after+postdevelopmental+myostatin+depletion+in+mice.">Grip force, EDL contractile properties, and voluntary wheel running after postdevelopmental myostatin depletion in mice.</a> J Appl. , (2010).</li><li>Donovan, J., Brown, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Euthanasia.">Euthanasia.</a> Curr Protoc Immunol. Chap 1, (2006).</li><li>Beekley, M. D., Wetzel, P., Kubis, P., Gros, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contractile+properties+of+skeletal+muscle+fibre+bundles+from+mice+deficient+in+carbonic+anhydrase+II.">Contractile properties of skeletal muscle fibre bundles from mice deficient in carbonic anhydrase II.</a> Pflugers Arch. 452, 453-463 (2006).</li></ol>

我们描述了一个详细的评估，在explanted从小鼠后肢肌肉的肌机械性能的方法。 EDL的，而更难以解剖，因为其背后的胫前肌后的位置，比胫前，因为其突出的腱附件的脚踝和膝盖关节更容易评估。这些筋便于安装在肌肉地带肌动描记。相反，更容易访问的胫前在膝关节广泛，几乎atendinous附件，异常艰难的两个剖析不影响肌肉的前提下，并安装在肌动描记安全。我们还指出，迅速安装在生理缓冲液和温度中的含氧浴肌肉，保持肌肉的力学性能是必不可少的。我们发现，我们可以长达30分钟重复这种分析没有显着变化，在这种条件下肌肉反应。最后，它是必不可少的，肌纤维不被感动在解剖和安装程序，可以有肌肉功能上的不利影响，并导致低估肌机械力。遵循这些程序，这种分析提供了可靠的定量方法来评估的肌肉功能的基因改造6,7,8,9影响，以及在肌肉疾病的小鼠模型1,2,3,5治疗性干预之间的比较。

解决方案和媒体 乳酸林格氏液 <ul><li> 100毫米氯化钠</li><li> 30毫米CH3CH（OH）COONa（乳酸钠） </li><li> 4 mM的氯化钾</li><li> 1毫米氯化钙2 2H 2 O（二水氯化钙） <ul><li>调节pH值至6.75 </li></ul></li></ul> 克雷布斯Henseleit解决方案<ul><li> 118毫米氯化钠</li><li> 4.7 mM的氯化钾</li><li> 1.25毫米氯化钙2 </li><li> 1.2毫米氯化镁2 </li><li> 1.2毫米的KH 2 PO 4 </li><li> 25毫米碳酸氢钠3 </li><li> 11毫米葡萄糖<ul><li>与O 2 / CO 2（95％/ 5％）气体平衡，调整pH值到7.2-7.4 </li></ul></li></ul> 戊巴比妥 <ul><li> 5 mg / mL的工作液在无菌水</li></ul>

myo-mechanical analysis of isolated skeletal muscle

在体内治疗肌肉疾病1,2,3的治疗干预的影响评估，定量分析方法，测量力的产生和治疗肌肉疲劳性。我们描述了一个新鲜explanted从小鼠后肢肌肉中的肌机械性能评估的详细方法。我们描述了小鼠趾长伸肌肌肉的非创伤性的收获，安装的肌肉，在肌肉地带肌动描记器（型号820MS;丹麦MYO技术）和最大抽搐，强直张力，收缩时间，半弛豫时间的测量，使用方波脉冲刺激器（型号S48;草技术）。使用这些测量，我们展示了具体的抽搐和规范化肌肉的横截面积，抽搐，强直紧张的比例，力-频率关系曲线和低频疲劳曲线 4强直张力的计算。这种分析方法提供了一个为1,2,3,5肌肉疾病的小鼠模型，以及治疗干预的肌肉功能的基因改造6,7,8,9影响比较之间的定量比较。

Watch this Scientific Journal Video about Myo-mechanical Analysis of Isolated Skeletal Muscle at JoVE.com

Myo-mechanical Analysis of Isolated Skeletal Muscle

为了评估<em&gt;在体内</em&gt;肌肉疾病的治疗性干预的影响，方法是需要定量力量的产生和治疗肌肉疲劳性。我们详细评估explanted小鼠后肢肌肉的肌力学性能的方法。这种分析提供了一个强大的方法，以定量的影响肌肉功能的基因改造，以及在治疗肌肉疾病的小鼠模型的比较。

离体骨骼肌肌的力学分析

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force ...

myo-mechanical-analysis-of-isolated-skeletal-muscle

Research

JoVE Journal

Medicine

26.7K 次观看.  University of California San Francisco. 为了评估在体内肌肉疾病的治疗性干预的影响，方法是需要定量力量的产生和治疗肌肉疲劳性。我们详细评估explanted小鼠后肢肌肉的肌力学性能的方法。这种分析提供了一个强大的方法，以定量的影响肌肉功能的基因改造，以及在治疗肌肉疾病的小鼠模型的比较。

视频: Myo-mechanical Analysis of Isolated Skeletal Muscle

Skeletal Muscle Gender Dimorphism from Proteomics

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably adaptable to environmental factors1 such as hypertrophy by resistance exercise and atrophy by disuse. It thereby exhibits remodeling and adaptations to stressors (heat, ischemia, heavy metals, etc.)2,3. Damage can occur to muscle by a muscle exerting force while lengthening, the so-called eccentric contraction4. The contractile proteins can be damaged in such exertions and need to be repaired, degraded and/or resynthesized; these functions are not part of the contractile proteins, but of other much less abundant proteins in the cell. To determine what subset of proteins is involved in the amelioration of this type of damage, a global proteome must be established prior to exercise5 and then followed subsequent to the exercise to determine the differential protein expression and thereby highlight candidate proteins in the adaptations to damage and its repair. Furthermore, most studies of skeletal muscle have been conducted on the male of the species and hence may not be representative of female muscle. 
In this article we present a method for extracting proteins reproducibly from male and female muscles, and separating them by two-dimensional gel electrophoresis followed by high resolution digital imaging6. This provides a protocol for spots (and subsequently identified proteins) that show a statistically significant (p &lt; 0.05) two-fold increase or decrease, appear or disappear from the control state. These are then excised, digested with trypsin and separated by high-pressure liquid chromatography coupled to a mass spectrometer (LC/MS) for protein identification (LC/MS/MS)5. This methodology (Figure 1) can be used on many tissues with little to no modification (liver, brain, heart etc.).

A straight-forward set of methods to isolate and determine the identity of the most abundant proteins expressed in skeletal muscle. About 800 spots are discerned on a two-dimensional gel from 10 mg muscle; this allows for the determination of gender-specific protein expression. These methods will give equivalent results in most tissues.

骨骼肌蛋白质组学性别异形

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably ...

科研

医学

Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. Although originally developed and used extensively in the rat due to its larger size, the tibial nerve in mice is big enough that it can be easily manipulated with either crush or transection, leaving the peroneal and sural nerve branches of the sciatic nerve intact and thereby preserving their target muscles. Thus, this model offers the advantages of inducing less morbidity and impediment of ambulation than the sciatic nerve transection model and also allows investigators to study the physiologic, cellular and molecular biologic mechanisms regulating the process of muscle atrophy in genetically engineered mice. The tibial nerve supplies the gastrocnemius, soleus and plantaris muscles, so its transection permits the study of denervated skeletal muscle composed of fast twitch type II fibers and/or slow twitch type I fibers. Here we demonstrate the tibial nerve transection model in the C57Black6 mouse. We assess the atrophy of the gastrocnemius muscle, as a representative muscle, at 1, 2, and 4 weeks post-denervation by measuring muscle weights and fiber type specific cross-sectional area on paraffin-embedded histologic sections immunostained for fast twitch myosin.

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of skeletal muscle atrophy. The model surgical protocol is described and demonstrated in C57Black6 mice. &#160;

胫骨神经切断术 - 一个标准化的模型失神经诱导小鼠骨骼肌

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. ...

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergstr&#246;m needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergstr&#246;m needle consists of an outer cannula with a small opening (&#8216;window&#8217;) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90&#176;&#160;and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.

The purpose of this video is to present the modified Bergstr&#246;m skeletal muscle biopsy technique on human subjects.

人骨骼肌活检手术使用修改伯格斯坦技术

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

线粒体的制备及透气性评估，从骨骼肌组织通过穿刺活检获得的分离

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of living systems. Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using qMRI methods. The MRI acquisition protocol begins with localizer images (used to locate the position of the body and tissue of interest within the MRI system), quality control measurements of relevant magnetic field distributions, and structural imaging for general anatomical characterization. The qMRI portion of the protocol includes measurements of the longitudinal and transverse relaxation time constants (T1 and T2, respectively). Also acquired are diffusion-tensor MRI data, in which water diffusivity is measured and used to infer pathological processes such as edema. Quantitative magnetization transfer imaging is used to characterize the relative tissue content of macromolecular and free water protons. Lastly, fat-water MRI methods are used to characterize fibro-adipose tissue replacement of muscle. In addition to describing the data acquisition and analysis procedures, this paper also discusses the potential problems associated with these methods, the analysis and interpretation of the data, MRI safety, and strategies for artifact reduction and protocol optimization.

Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using non-invasive magnetic resonance imaging methods.

骨骼肌疾病的定量磁共振成像

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of ...

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow reserve (CFR) in humans. Reduced CFR is accompanied by marked intramyocardial and pericoronary fibrosis and is used as an indication of the severity of dysfunction. This study explores, step-by-step, the real-time changes measured in the coronary flow velocity, CFR and systolic to diastolic peak velocity (S/D) ratio in the setting of an aortic banding model in mice. By using a Doppler transthoracic imaging technique that yields reproducible and reliable data, the method assesses changes in flow in the septal coronary artery (SCA), for a period of over two weeks in mice, that previously either underwent aortic banding or thoracotomy. 
During imaging, hyperemia in all mice was induced by isoflurane, an anesthetic that increased coronary flow velocity when compared with resting flow. All images were acquired by a single imager. Two ratios, (1) CFR, the ratio between hyperemic and baseline flow velocities, and (2) systolic (S) to diastolic (D) flow were determined, using a proprietary software and by two independent observers. Importantly, the observed changes in coronary flow preceded LV dysfunction as evidenced by normal LV mass and fractional shortening (FS). 
The method was benchmarked against the current gold standard of coronary assessment, histopathology. The latter technique showed clear pathologic changes in the coronary artery in the form of peri-coronary fibrosis that correlated to the flow changes as assessed by echocardiography. 
The study underscores the value of using a non-invasive technique to monitor coronary circulation in mouse hearts. The method minimizes redundant use of research animals and demonstrates that advanced ultrasound-based indices, such as CFR and S/D ratios, can serve as viable diagnostic tools in a variety of investigational protocols including drug studies and the study of genetically modified strains.

Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

冠脉流量和冠状动脉血流储备用小鼠压力超负荷基于模型的超声评估

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

Histological evaluation of muscle biopsies has served as an indispensable tool in the understanding of the development and progression of pathology of neuromuscular disorders. However, in order to do so, proper care needs to be taken when excising and preserving tissues to achieve optimal staining. One method of tissue preservation involves fixing tissues in formaldehyde and then embedding them with paraffin wax. This method preserves morphology well and allows for long-term storage at RT but is cumbersome and requires handling of toxic chemicals. Further, formaldehyde fixation results in antigen cross-linking, which necessitates antigen retrieval protocols for effective immunostaining. On the contrary, frozen sectioning does not require fixation and thus retains biological antigen conformation. This method also provides a distinct advantage in quick turn around time, making it especially useful in situations needing quick histological evaluation like intraoperative surgical biopsies. Here we describe the most effective method of preparing muscle biopsies for visualization with different histological and immunological stains.

Here we demonstrate the most efficient methods for freezing, embedding, cryosectioning, and staining of muscle biopsies to avoid freezing artifacts.

该做什么和不该做什么的肌肉冰冻切片的准备组织学分析

Histological evaluation of muscle biopsies has served as an indispensable tool in the understanding of the development and progression of pathology of ...

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and well tolerated technique. The Weil-Blakesley conchotome has a sharp biting tip with a 4 - 6 mm wide hollow. It is inserted through a 5 - 10 mm skin incision and can be maneuvered for controlled tissue penetration. The tip is opened and closed within the tissue and then rotated through 90 -180&#176; to cut the muscle. The amount of muscle obtained following repeated sampling can vary from 20 mg to 290 mg which can be processed for both histology and molecular studies. The wound needs to be kept dry and vigorous physical activity kept to a minimum for approximately 72 hr although normal levels of activity can restart immediately following the procedure. This procedure is safe and effective when close attention is paid to the selection of subjects, full asepsis and post procedure care. &#160;Both right and left vastus lateralis are suitable for biopsy dependent on participant preference.

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

This video demonstrates the technique of percutaneous muscle biopsy of the human vastus lateralis using the Weil-Blakesley conchotome.

人的<em&gt;股外侧肌</em&gt;骨骼肌活检使用韦尔 - Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and ...

Induction and Assessment of Exertional Skeletal Muscle Damage in Humans

Contraction-induced muscle damage via voluntary eccentric (lengthening) contractions offers an excellent model for studying muscle adaptation and recovery in humans. Herein we discuss the design of an eccentric exercise protocol to induce damage in the quadriceps muscles, marked by changes in strength, soreness, and plasma creatine kinase levels. This method is simple, ethical, and widely applicable since it is performed in human participants and eliminates the interspecies translation of the results. Subjects perform 300 maximal eccentric contractions of the knee extensor muscles at a speed of 120&#176;/sec on an isokinetic dynamometer. The extent of the damage is measurable using relatively non-invasive isokinetic and isometric measures of strength loss, soreness, and plasma creatine kinase levels over several days following the exercise. Therefore, its application can be directed to specific populations in an attempt to identify mechanisms for muscle adaptation and regeneration.

This article describes a safe and reliable method to induce and quantify exertional skeletal muscle damage in human subjects.

诱导和人类劳力骨骼肌损伤评估

Contraction-induced muscle damage via voluntary eccentric (lengthening) contractions offers an excellent model for studying muscle adaptation and recovery ...

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.

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 ...