我们要感谢的材料和结构试验核心设施梅奥诊所寻求技术支持。此外，我们要感谢遵义伯格伦德，布兰特纽曼，乔恩运书房Buijs，博士，在研究过程中他们的帮助。这项研究是由财政从格兰杰基金会格兰杰创新基金的支持。

注:本协议提出的所有研究是由梅奥诊所的机构审查委员会（IRB）的批准。尸骨了来自不同组织的一个为期6年获得。所有标本72小时死亡的内收集的，包裹在盐水饱和毛巾，并储存于-20℃，直至准备。 1.使用DXA测量骨密度<ol><li>除去保持在-20℃下冷冻样品在室温下解冻约24小时标本并不需要从原始包装中删除，如果最软组织已被删除。 </li><li>大米使用两个5磅袋占了软组织。封面上DXA表两种大米袋用塑料袋，以防止污染。的米袋将模拟周边扫描期间的软组织（体内 ）， 如图1。 </li><li>保护DXA扫描器表面衬塑纸，并放置2个塑料包装里CE袋放在扫描仪表（ 图1A）。 </li><li>莱在的大米包装袋顶部2股骨（左，右），使得近端（包括股骨头）集中在包包和后侧下跌（ 图1B）。这模仿患者仰卧在他们的后面。 </li><li>覆盖前/暴露股骨近端年底与另外两个5磅的大米包装袋（ 图1C）。 </li><li>机头跟在股骨近端，并根据病人的骨密度测量（ 图1C）的标准机构的程序扫描股骨。按照具体DXA制造商的说明。 <ol><li>从DXA机软件界面执行正常股骨扫描。选择股骨考试，通过相应地按压向左或向右箭头上DXA手臂上的尸体股骨顶部的DXA手臂的位置，并通过点击"开始"按钮，开始考试。通过点击"分析"执行BMD分析。 不E:从扫描得到的自动T分数分类骨为正常，骨质减少或骨质疏松性（ 图1D）。按照具体DXA制造商的说明。 </li></ol></li></ol> 2.清洗，切割和钻孔骨的骨远端<ol><li>通过仔细地除去从骨的任何剩余的软组织清洁股骨的最基端为300 mm。此步骤是必须允许PMMA期间在机械测试准备制陶过程联系骨头。骨头不需要解冻到室温这个过程。 </li><li>消毒用70％异丙醇的工作区，并覆盖在一侧（ 图2A）与塑料薄膜吸收纸垫的表。设置在桌子上（ 图2B）的开始清洗，切割（ 图2A至2H）的整个过程全股骨。穿戴个人防护装备（PPE），包括手套和保护眼睛。 </li><li>刮去多余的骨膜和用刮刀和手术刀（ 图2C，2H）切去多余的组织。 </li><li>放置骨中的定制切割夹具如图2D与抗夹具的丙烯酸板股骨的头部。 </li><li>对准并靠住上切割夹具（ 图2D）的两个插针的骨干。 </li><li>固定骨头拧紧向下骨干开槽板的夹具;如果骨不平躺在夹具上，挂前端关闭桌面并且还根据需要握住标本就位（ 图2D）任选拉链扎股骨颈。 </li><li>切割通过开槽板作为引导用铸件切割器（ 图2E）的股骨的远端轴;持有用干抹布/毛巾骨头更好的抓地力。 </li><li>除去从夹具的骨;后切正常骨长度为255毫米（ 图2F）。 </li><li>用刮匙约25毫米深清洁骨髓的骨髓腔。然后，将纱布海绵吸干的内表面。取出纱布刚刚之前放置在模具中的股骨的远端。把手用干毛巾/抹布骨和钻10毫米的孔，通过所述远端在从试样的近端切割端约25毫米。注:这是使PMMA穿透运河和牢固地固定骨头。 </li></ol> 3.灌封骨<ol><li>设计和制造封装容器。灌封容器形成5毫米厚的丙烯酸系片材，并具有以下外部尺寸50毫米乘50毫米的方形横截面和100毫米高（ 图3A）。 </li><li>适当的骨骼识别标签封装容器（ 图3A和3B -看到亚克力盒标签）。 </li><li>调整嵌入夹具适当取向（左腿或右腿; 例如 15°或30°内旋转）。 </li><li>放置灌封容器在灌封容器（ 图3B）中嵌入夹具，代替骨的基部和与指针在夹具（ 图3C）对齐颈部到骨的内部的旋转角度调整到所需的值。 </li><li>测量60克PMMA粉，并在通风橱用30克液体树脂混合，直到粉末溶解。该混合物应该是可流动的。使用一次性纸杯这一进程。 </li><li>将混合物倒入带有通风橱（ 图3D）下骨灌封容器，允许以固化约10 - 15分钟，直到PMMA是透明和硬的。这应该只填写〜PMMA的灌封容器的1/2。小心包装在盐水饱和毛巾骨，以防止由于聚甲基丙烯酸甲酯聚合（ 图3E）中的发热组织干。 </li><li>定期检查股骨，以确保它保持在对齐在固化过程中的容器。 </li><li>从灌封夹具除去股骨并用盐水浸泡纸巾（ 图3E）包裹。 </li><li>在通风橱下制备90克PMMA的如在步骤3.5解释和完全填充灌封容器。治愈PMMA约10 - 15分钟，直到它变得坚硬。 </li><li>树脂固化后，紧紧包裹在盐水浸湿的纸巾/重新包骨，在冷冻室-20℃的塑料袋和存储样本覆盖。 </li></ol> 4.成像的X射线骨 （ 注意！使用机器时，与X射线辐射的适当的照顾工作） <ol><li>如果使用用于X射线的薄膜，由旋钮顺时针旋转（在显影剂室）打开X射线显影剂上至少20分钟（按照制造商说明书）扫描前。 </li><li>确保有之前的X射线照相暗盒未曝光的胶片;卡带只能在一个黑暗的房间打开。 </li><li>打开机器上解锁并延长机器头上。 </li><li>放置一个购物的光束的路径下和放置一个盒上的光束（ 图4A）下的车。 </li><li>地点和录像带上（ 图4B）股骨定位;两个方向将被捕获:内侧 - 外侧视图和前 - 后视图。相应地标出标本的图像。 </li><li>第一次曝光后，交换铅和骨骼的位置。 </li><li>覆盖已露出的一半与铅和在未曝光侧的第二取向露出骨。这允许用户使用一个X射线胶片用于在两个取向（ 图4C-D）的一个单一的骨。 </li><li>更换磁带，每个股骨旋转到第二方位。 </li><li>在一个单一的股骨的情况下，覆盖引线盖的磁带盒的一半，以避免整个膜的X射线的初始曝光。 </li><li>移动个人防护铅衬便携式墙后面，使用触发器揭露骨头。 </li><li>完成后，返回X射线头锁定和存储位置，转动钥匙到X射线机上关闭并删除曝光X光片。 </li><li>开发该膜，得到使用普通膜显影剂的X射线图像（ 图4E）。打开白色的光在房间里，找到了电影的开发。打开红灯打开磁带之前关闭的白色光，并与电影发展过程中进行。打开纸盒，并通过开发商放电影。打开白色光，并且一旦膜已经开发关掉红光。 </li></ol> 5.骨骼CT扫描<ol><li>从冰箱扫描约24小时前，剔除骨头。骨骼必须在扫描之前完全解冻。 </li><li>确保骨头被包裹在塑料袋进行扫描，以尽量减少清理底。 </li><li>放置并固定股骨和在CT扫描灯具（ 图5A-B）的校准幻像。在Fixture保持校准体模（ 图5C），也保持在一个方向（ 图5D-E）的具有期望用于随后机械测试的取向相同的股骨。这种交叉登记需要在QCT / FEA建模过程（在不同的协议描述）使用从CT扫描（ 图5F）中的数据。 注:夹具被设计为使得它暴露作CT扫描股骨而不妨碍股骨近端（股骨头，颈，大转子，和近侧部轴）。 </li><li>确保与在CT扫描器股骨沿固定装置使用系统激光作为导向件（ 图5D-E）的正确对准。重新检查与CT造影长轴激光夹具的取向（按激光的开/关按钮）。假想不需要，因为它被固定在夹具要与激光对准。按零按钮操零从机器的控制面板的表中的位置N（→0←）在控制面板上。 </li><li>继CT标准作业程序，在120 kVp的，216毫安秒，1秒旋转时间，操作CT机，并使用超高分辨率模式（晌礼）的1间距。这给出了0.4毫米的切片厚度，以及0.30的像素大小 - 取决于视场（FOV）字段的为0.45毫米。 </li><li>前机械测试检查的CT扫描数据，以确保感兴趣的图像被捕获并保存。重新冻结在-20℃的骨，直到实验当天。 </li></ol>

<ol>
	<li>Cristofolini, L., Schileo, E., Juszczyk, M., Taddei, F., Martelli, S., Viceconti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+testing+of+bones:+the+positive+synergy+of+finite-element+models+and+in+vitro+experiments.">Mechanical testing of bones: the positive synergy of finite-element models and in vitro experiments.</a> Philos Trans A Math Phys Eng Sci. 368, 2725-2763 (2010).</li><li>Cartner, J. L., Hartsell, Z. M., Ricci, W. M., Tornetta, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+we+trust+ex+vivo+mechanical+testing+of+fresh-frozen+cadaveric+specimens?+The+effect+of+postfreezing+delays.">Can we trust ex vivo mechanical testing of fresh-frozen cadaveric specimens? The effect of postfreezing delays.</a> J Orthop Trauma. 25 (8), 459-461 (2011).</li><li>An, Y. H., Draughn, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Mechanical testing of bone and the bone-implant interface. , (1999).</li><li>van Haaren, E. H., van der Zwaard, B. C., van der Veen, A. J., Heyligers, I. C., Wuisman, P. I., Smit, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+long-term+preservation+on+the+mechanical+properties+of+cortical+bone+in+goats.">Effect of long-term preservation on the mechanical properties of cortical bone in goats.</a> Acta Orthop. 79, 708-716 (2008).</li><li>Shaw, J. M., Hunter, S. A., Gayton, J. C., Boivin, G. P., Prayson, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeated+freeze-thaw+cycles+do+not+alter+the+biomechanical+properties+of+fibular+allograft+bone.">Repeated freeze-thaw cycles do not alter the biomechanical properties of fibular allograft bone.</a> Clin Orthop Relat Res. 470 (3), 937-943 (2012).</li><li>Topp, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embalmed+and+fresh+frozen+human+bones+in+orthopedic+cadaveric+studies:+which+bone+is+authentic+and+feasible?.">Embalmed and fresh frozen human bones in orthopedic cadaveric studies: which bone is authentic and feasible?.</a> Acta Orthop. 83 (5), 543-547 (2012).</li><li>Manske, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+and+trabecular+bone+in+the+femoral+neck+both+contribute+to+proximal+femur+failure+load+prediction.">Cortical and trabecular bone in the femoral neck both contribute to proximal femur failure load prediction.</a> Osteoporos Int. 20 (3), 445-453 (2009).</li><li>Rezaei, A., Dragomir-Daescu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Femoral+Strength+Changes+Faster+With+Age+Than+BMD+in+Both+Women+and+Men:+A+Biomechanical+Study.">Femoral Strength Changes Faster With Age Than BMD in Both Women and Men: A Biomechanical Study.</a> J Bone Miner Res. 30, 2200-2206 (2015).</li><li>Cristofolini, L., McNamara, B., Freddi, A., Viceconti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+measured+strains+in+the+loaded+femur:+quantification+of+experimental+error.">In vitro measured strains in the loaded femur: quantification of experimental error.</a> J Strain Anal Eng Des. 32, 193-200 (1997).</li><li>Dragomir-Daescu, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+QCT/FEA+models+of+proximal+femur+stiffness+and+fracture+load+during+a+sideways+fall+on+the+hip.">Robust QCT/FEA models of proximal femur stiffness and fracture load during a sideways fall on the hip.</a> Ann Biomed Eng. 39, 742-755 (2011).</li></ol>

作者有没有相关的披露。

我们提出了确保臀部配置横盘下跌股力量力学测试和QCT / FEA建模一个强大的骨骼准备协议。这种方法成了我们的标准的内部协议。在6年的过程中，有不同的人员，成功制备出约200股骨下此协议。该协议的成果包括分类使用DXA骨条件下，排除了转移性疾病，先前的骨折，或利用X射线的植入物，并获得矿物分布和利用CT用于随后QCT / FEA建模的3D几何形状。切割，灌封，并且扫描装置被设计成容纳左和?...

Determining the true cadaveric proximal femoral fracture strength with mechanical testing is a destructive method that requires a rigorous testing approach for accurate measurements. In particular, proper bone preparation methods are necessary to maintain near in vivo integrity of the bones prior to mechanical fracture testing1. This is achieved by proper bone storage and minimizing handling at room temperature. This test data is extensively used to validate QCT/FEA models of femoral fracture which have the potential to be used clinically to understand the fracture risk, especially in osteoporotic patients. Unfortunately, there is no current standard procedure to prepare proximal femur samples for mechanical testing. A good testing procedure should ensure repeatability and reproducibility of the preparation process. Therefore, fixtures required for sample preparation need to be carefully designed and fabricated to minimize the likelihood of various testing errors. We also need to minimize the preparation time for which bone tissue is at room temperature and thus in danger of degradation with irreversible changes in mechanical properties. 
To this end, we have developed a procedure that preserves bone tissue across multiple preparation steps. This is important to ensure minimal exposure time at room temperature while also minimizing the number of freeze/thaw cycles which can affect tissue physical properties2. The entire procedure is long and nontrivial as the steps occurred over multiple weeks and required scheduling for scanning procedures and personnel availability. The steps included thawing bone samples, screening the samples using DXA scanning to obtain bone mineral density (BMD) values, X-ray to rule out any diseased specimens, and finally CT scanning to estimate distribution of bone mineral and femoral geometry. All the specimens were prepared for testing by removing extraneous soft tissues from the bone surface, cutting the femur to a length required for testing, and potting the femur in a desired orientation for simulating a sideways fall on the hip during subsequent testing. It is essential to keep the time period for all these operations as short as possible. A robust protocol is thus mandatory for consistent specimen preparation, tissue preservation between steps, and for reducing the overall preparation time. 
The aim of this paper is to present in detail the procedures involved in the preparation of femoral samples for subsequent mechanical testing under various conditions. Preservation of the bone tissue is crucial in this process and we achieved it by keeping specimens frozen between steps and keeping them carefully wrapped in saline saturated towels at all times except when scanning and mechanically testing the bones. Femora were also kept wrapped in saline wet towels during the steps involving PMMA curing to prevent dryness of the bone tissue.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CT potting container and scanning fixture</td>
			<td>Internally manufactured</td>
			<td>N/A</td>
			<td>Custom designed and manufactured</td>
		</tr>
		<tr>
			<td>CT scanner</td>
			<td>Siemens</td>
			<td>Somatom Definition scanner (Siemens, Malvern, PA)</td>
			<td>CT scanning equipment</td>
		</tr>
		<tr>
			<td>Quantitative CT Phantom</td>
			<td>Midways Inc, San Francisco, CA</td>
			<td>Model 3 CT calibration Phantom</td>
			<td>Used for obtaining BMD values from Hounsfield units in the CT image</td>
		</tr>
		<tr>
			<td>Dual Energy X-ray Absorptiometry scanner</td>
			<td>General Electric</td>
			<td>N/A</td>
			<td>GE Lunar iDXA scanner for bone health or any similar BMD scanners</td>
		</tr>
		<tr>
			<td>Hygenic Orhodontic Resin (PMMA)</td>
			<td>Patterson Dental Supply</td>
			<td>H02252</td>
			<td>Controlled substance and can be purchased with proper approval</td>
		</tr>
		<tr>
			<td>Freezer</td>
			<td>Kenmore</td>
			<td>N/A</td>
			<td>This is a -20oC storage for bones</td>
		</tr>
		<tr>
			<td>X-ray scanner</td>
			<td>General Electric</td>
			<td>&#160;46-270615P1</td>
			<td>X-ray imaging equipment.&#160;&#160;</td>
		</tr>
		<tr>
			<td>X-ray films</td>
			<td>Kodak</td>
			<td>N/A</td>
			<td>Used to display x-ray images</td>
		</tr>
		<tr>
			<td>X-ray developer</td>
			<td>Kodak X-Omatic</td>
			<td>M35A X-OMAT&#160;</td>
			<td>Used for developing X-ray images</td>
		</tr>
		<tr>
			<td>X-ray Cassette</td>
			<td>Kodak X-Omatic</td>
			<td>N/A</td>
			<td>Used for holding x-ray films</td>
		</tr>
		<tr>
			<td>5-pound Rice Bags</td>
			<td>Great Value</td>
			<td>N/A&#160;</td>
			<td>Used for mimicking soft tissue during the DXA scanning process</td>
		</tr>
		<tr>
			<td>Physiologic Saline (0.9% Sodium Chloride)</td>
			<td>Baxter</td>
			<td>NDC 0338-0048-04</td>
			<td>Used for keeping samples hydrated</td>
		</tr>
		<tr>
			<td>Scalpels and scrapers</td>
			<td>Bard-Parker</td>
			<td>N/A</td>
			<td>Used to clean the bone from soft tissue</td>
		</tr>
		<tr>
			<td>Cast cutter</td>
			<td>Stryker</td>
			<td>810-BD001</td>
			<td>Used to cut femoral shaft</td>
		</tr>
		<tr>
			<td>Drilling machine</td>
			<td>Bosch</td>
			<td>N/A</td>
			<td>Used to drill the femoral shaft</td>
		</tr>
		<tr>
			<td>Fume Hood</td>
			<td>Hamilton</td>
			<td>70532</td>
			<td>Used for ventilation when using making PMMA</td>
		</tr>
	</tbody>
</table>

proximal cadaveric femur preparation for fracture strength testing and quantitative ct-based finite element analysis

尸体断裂测试通常用来了解影响股骨近端强度的因素。由于体外生物组织很容易随着时间的推移失去其机械性能，样品必须仔细进行实验测试的准备，以获得在体内条件代表可靠的结果。出于这个原因，我们设计了一个协议和一套夹具以制备股骨标本，使得它们的机械性能经历最小的变化。在准备步骤和机械测试除股骨保持在冷冻状态。有临床双能X线骨密度仪（DXA）骨密度仪获得了全髋关节和股骨颈骨密度（BMD）的相关临床措施，并利用CT与校准幻影，获得三维几何和骨矿物质的分布基于所述灰度值的定量估计。任何可能的骨骼疾病，骨折，或植入物或影响骨结构的工件的情况下，被排除用X射线扫描。对于制备，所有的骨头被仔细清洗过量的软组织，并切断与盆栽在感兴趣的内部旋转角度。切割夹具允许骨的远端被切断而使股骨近端在所希望的长度。以允许以后的CT扫描和机械测试期间在规定的角度股骨颈定位，股骨近端的轴是在聚甲基丙烯酸甲酯（PMMA），使用专为所需的方向而设计的夹具盆栽。然后从我们的实验中收集的数据被用于定量计算机断层扫描（QCT）的验证为基础的有限元分析（FEA），如在不同的协议描述。在这个手稿中，我们提出了力学性能试验和随后的QCT / FEA建模精确的骨骼制备的协议。目前的协议已成功应用于准备约200 CADaveric股骨超过6年的时间。

该股骨尸体被运冷冻并保持在-20°C，直到准备开始了。使用DXA扫描仪测量每个样品（ 图1）全髋和颈部的BMD以及T分数进行骨密度扫描。 T-分数是所测量骨密度的标准偏差相比，年轻健康受试者的平均值的数量。它的范围可以从-2.5或更低骨质疏松筋骨，-1和-2.5之间的骨质疏松的骨骼和高于-1正常的骨头。一旦完成后，骨头被清洗多余的组织，切成使用一个内部?...

Watch this Scientific Journal Video about Proximal Cadaveric Femur Preparation for Fracture Strength Testing and Quantitative CT-based Finite Element Analysis at JoVE.com

Proximal Cadaveric Femur Preparation for Fracture Strength Testing and Quantitative CT-based Finite Element Analysis

我们提出了如何妥善保存，并准备测试断裂和定量计算机断层成像尸体股骨一个强大的协议。该方法提供了对用于确定骨矿物密度，断裂强度之间的关系，并限定有限元模型的几何形状和性能的目的输入条件的精确控制。

近端股骨尸制备基于CT的断裂强度测试和定量的有限元分析

Cadaveric fracture testing is routinely used to understand factors that affect proximal femur strength.  Because ex vivo biological tissues are prone to ...

proximal-cadaveric-femur-preparation-for-fracture-strength-testing

Research

JoVE Journal

Medicine

9.2K 次观看.  Mayo Clinic. 我们提出了如何妥善保存，并准备测试断裂和定量计算机断层成像尸体股骨一个强大的协议。该方法提供了对用于确定骨矿物密度，断裂强度之间的关系，并限定有限元模型的几何形状和性能的目的输入条件的精确控制。 

视频: Proximal Cadaveric Femur Preparation for Fracture Strength Testing and Quantitative CT-based Finite Element Analysis

Quantitative Autonomic Testing

Disorders associated with dysfunction of autonomic nervous system are quite common yet frequently unrecognized. Quantitative autonomic testing can be invaluable tool for evaluation of these disorders, both in clinic and research. There are number of autonomic tests, however, only few were validated clinically or are quantitative. Here, fully quantitative and clinically validated protocol for testing of autonomic functions is presented. As a bare minimum the clinical autonomic laboratory should have a tilt table, ECG monitor, continuous noninvasive blood pressure monitor, respiratory monitor and a mean for evaluation of sudomotor domain. The software for recording and evaluation of autonomic tests is critical for correct evaluation of data. The presented protocol evaluates 3 major autonomic domains: cardiovagal, adrenergic and sudomotor. The tests include deep breathing, Valsalva maneuver, head-up tilt, and quantitative sudomotor axon test (QSART). The severity and distribution of dysautonomia is quantitated using Composite Autonomic Severity Scores (CASS). Detailed protocol is provided highlighting essential aspects of testing with emphasis on proper data acquisition, obtaining the relevant parameters and unbiased evaluation of autonomic signals. The normative data and CASS algorithm for interpretation of results are provided as well.

Standardized, comprehensive and fully quantitative testing of autonomic functions is described. The autonomic tests consist of evaluation of all three major autonomic domains including cardiovagal, adrenergic and sudomotor. The severity and distribution of dysautonomia is quantitated using Composite Autonomic Severity Scores.

定量自主测试

Disorders associated with dysfunction of autonomic nervous system are quite common yet frequently unrecognized. Quantitative autonomic testing can be ...

科研

医学

Manual Muscle Testing:  A Method of Measuring Extremity Muscle Strength Applied to Critically Ill Patients

Survivors of acute respiratory distress syndrome (ARDS) and other causes of critical illness often have generalized weakness, reduced exercise tolerance, and persistent nerve and muscle impairments after hospital discharge.1-6 Using an explicit protocol with a structured approach to training and quality assurance of research staff, manual muscle testing (MMT) is a highly reliable method for assessing strength, using a standardized clinical examination, for patients following ARDS, and can be completed with mechanically ventilated patients who can tolerate sitting upright in bed and are able to follow two-step commands. 7, 8 
This video demonstrates a protocol for MMT, which has been taught to &ge;43 research staff who have performed &gt;800 assessments on &gt;280 ARDS survivors. Modifications for the bedridden patient are included. Each muscle is tested with specific techniques for positioning, stabilization, resistance, and palpation for each score of the 6-point ordinal Medical Research Council scale.7,9-11 Three upper and three lower extremity muscles are graded in this protocol: shoulder abduction, elbow flexion, wrist extension, hip flexion, knee extension, and ankle dorsiflexion. These muscles were chosen based on the standard approach for evaluating patients for ICU-acquired weakness used in prior publications. 1,2.

Survivors of acute respiratory distress syndrome (ARDS) and critical illness frequently develop long-lasting muscle weakness. Manual muscle testing (MMT) is a standardized clinical examination commonly used to measure strength of peripheral skeletal muscle groups. This video demonstrates MMT using the 6-point Medical Research Council scale.

危重病人中的应用手册肌力测试:下肢肌力测量方法

Survivors of acute respiratory distress syndrome (ARDS) and other causes of critical illness often have generalized weakness, reduced exercise tolerance, ...

Quantitative Analysis of Chromatin Proteomes in Disease

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due to the high percentage of binucleated cells,1 the predominantly heterochromatic state of the DNA, and the non-dividing nature of the cardiomyocyte which renders adult nuclei in a permanent state of interphase.2 Transcriptional regulation during development and disease have been well studied in this organ,3-5 but what remains relatively unexplored is the role played by the nuclear proteins responsible for DNA packaging and expression, and how these proteins control changes in transcriptional programs that occur during disease.6 In the developed world, heart disease is the number one cause of mortality for both men and women.7 Insight on how nuclear proteins cooperate to regulate the progression of this disease is critical for advancing the current treatment options.
Mass spectrometry is the ideal tool for addressing these questions as it allows for an unbiased annotation of the nuclear proteome and relative quantification for how the abundance of these proteins changes with disease. While there have been several proteomic studies for mammalian nuclear protein complexes,8-13 until recently14 there has been only one study examining the cardiac nuclear proteome, and it considered the entire nucleus, rather than exploring the proteome at the level of nuclear sub compartments.15 In large part, this shortage of work is due to the difficulty of isolating cardiac nuclei. Cardiac nuclei occur within a rigid and dense actin-myosin apparatus to which they are connected via multiple extensions from the endoplasmic reticulum, to the extent that myocyte contraction alters their overall shape.16 Additionally, cardiomyocytes are 40% mitochondria by volume17 which necessitates enrichment of the nucleus apart from the other organelles. Here we describe a protocol for cardiac nuclear enrichment and further fractionation into biologically-relevant compartments. Furthermore, we detail methods for label-free quantitative mass spectrometric dissection of these fractions-techniques amenable to in vivo experimentation in various animal models and organ systems where metabolic labeling is not feasible.

Advances in mass spectrometry have allowed the high throughput analysis of protein expression and modification in a host of tissues. Combined with subcellular fractionation and disease models, quantitative mass spectrometry and bioinformatics can reveal new properties in biological systems. The method described herein analyzes chromatin-associated proteins in the setting of heart disease and is readily applicable to other in vivo models of human disease.

在疾病的染色质蛋白质组的定量分析

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due ...

Quantitative Multispectral Analysis Following Fluorescent Tissue Transplant for Visualization of Cell Origins, Types, and Interactions

With the desire to understand the contributions of multiple cellular elements to the development of a complex tissue; such as the numerous cell types that participate in regenerating tissue, tumor formation, or vasculogenesis, we devised a multi-colored cellular transplant model of tumor development in which cell populations originate from different fluorescently colored reporter gene mice and are transplanted, engrafted or injected in and around a developing tumor. These colored cells are then recruited and incorporated into the tumor stroma. In order to quantitatively assess bone marrow derived tumor stromal cells, we transplanted GFP expressing transgenic whole bone marrow into lethally irradiated RFP expressing mice as approved by IACUC. 0ovarian tumors that were orthotopically injected into the transplanted mice were excised 6-8 weeks post engraftment and analyzed for bone marrow marker of origin (GFP) as well as antibody markers to detect tumor associated stroma using multispectral imaging techniques. We then adapted a methodology we call MIMicc- Multispectral Interrogation of Multiplexed cellular compositions, using multispectral unmixing of fluoroprobes to quantitatively assess which labeled cell came from which starting populations (based on original reporter gene labels), and as our ability to unmix 4, 5, 6 or more spectra per slide increases, we&#39;ve added additional immunohistochemistry associated with cell lineages or differentiation to increase precision. Utilizing software to detect co-localized multiplexed-fluorescent signals, tumor stromal populations can be traced, enumerated and characterized based on marker staining.1

Complex tissue masses, from organs to tumors, are composed of various cellular elements. We elucidated the contribution of cellular phenotypes within a tissue utilizing multi-labeled fluorescent transgenic mice in combination with multiparameter immunofluorescent staining followed by spectral unmixing to decipher cell origin as well as cell characteristics based on protein expression.

多光谱定量分析继荧光组织移植的细胞起源，类型和相互作用的可视化

With the desire to understand the contributions of multiple cellular elements to the development of a complex tissue; such as the numerous cell types that ...

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

顺序<em&gt;在体内</em&gt;成骨干/祖细胞在骨折修复的影像学

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

Adjustable Stiffness, External Fixator for the Rat Femur Osteotomy and Segmental Bone Defect Models

The mechanical environment around the healing of broken bone is very important as it determines the way the fracture will heal. Over the past decade there has been great clinical interest in improving bone healing by altering the mechanical environment through the fixation stability around the lesion. One constraint of preclinical animal research in this area is the lack of experimental control over the local mechanical environment within a large segmental defect as well as osteotomies as they heal. In this paper we report on the design and use of an external fixator to study the healing of large segmental bone defects or osteotomies. This device not only allows for controlled axial stiffness on the bone lesion as it heals, but it also enables the change of stiffness during the healing process in vivo. The conducted experiments have shown that the fixators were able to maintain a 5 mm femoral defect gap in rats in vivo during unrestricted cage activity for at least 8 weeks. Likewise, we observed no distortion or infections, including pin infections during the entire healing period. These results demonstrate that our newly developed external fixator was able to achieve reproducible and standardized stabilization, and the alteration of the mechanical environment of in vivo rat large bone defects and various size osteotomies. This confirms that the external fixation device is well suited for preclinical research investigations using a rat model in the field of bone regeneration and repair.

One constraint of preclinical research in the field of bone repair is the lack of experimental control over the local mechanical environment within a healing bone lesion. We report the design and use of an external fixator for bone repair with the ability to change fixator stiffness in vivo.

可调节刚度，外固定支架的大鼠股骨截骨和节段性骨缺损模型

The mechanical environment around the healing of broken bone is very important as it determines the way the fracture will heal. Over the past decade there ...

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 ...

Adapted Resistance Training Improves Strength in Eight Weeks in Individuals with Multiple Sclerosis

Hip weakness is a common symptom affecting walking ability in people with multiple sclerosis (MS). It is known that resistance strength training (RST) can improve strength in individuals with MS, however; it remains unclear the duration of RST that is needed to make strength gains and how to adapt hip strengthening exercises for individuals of varying strength using only resistance bands. This paper describes the methodology to set up and implement an adapted resistance strength training program, using resistance bands, for individuals with MS. Directions for pre- and post-strength tests to evaluate efficacy of the strength-training program are included. Safety features and detailed instructions outline the weekly program content and progression. Current evidence is presented showing that significant strength gains can be made within 8 weeks of starting a RST program. Evidence is also presented showing that resistance strength training can be successfully adapted for individuals with MS of varying strength with little equipment.

Hip weakness is a common symptom affecting walking ability in people with multiple sclerosis. Isolated muscle strengthening is a useful method to target specific weaknesses. This protocol describes a progressive resistance-training program using exercise bands to increase hip muscle strength.

改编阻力训练提高力量在八周内的个人与多发性硬化症

Hip weakness is a common symptom affecting walking ability in people with multiple sclerosis (MS). It is known that resistance strength training (RST) can ...

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The amount of the different tissues in the callus provides important information on the fracture healing progress. Available in vivo techniques to longitudinally monitor the callus tissue development in preclinical fracture-healing studies using small animals include digital radiography and &#181;CT imaging. However, both techniques are only able to distinguish between mineralized and non-mineralized tissue. Consequently, it is impossible to discriminate cartilage from fibrous tissue. In contrast, magnetic resonance imaging (MRI) visualizes anatomical structures based on their water content and might therefore be able to noninvasively identify soft tissue and cartilage in the fracture callus. Here, we report the use of an MRI-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice. The experiments demonstrated that the fixator and a custom-made mounting device allow repetitive MRI scans, thus enabling longitudinal analysis of fracture-callus tissue development.

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

The evaluation of tissue development in the fracture callus during endochondral bone healing is essential to monitor the healing process. Here, we report the use of a magnetic resonance imaging (MRI)-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice.

在体内MRI 对小鼠股骨骨愈合过程中骨折愈伤组织发育的评价

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The ...

Muscle Imbalances: Testing and Training Functional Eccentric Hamstring Strength in Athletic Populations

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite of these eccentric hamstring actions are concentric quadriceps actions, where the larger and likely stronger quadriceps straighten the knee. Therefore, to stabilize the lower limbs during movement, the hamstrings must eccentrically combat against the strong knee-straightening torque of the quadriceps. As such, eccentric hamstring strength expressed relative to concentric quadricep strength is commonly referred to as the &#34;functional ratio&#34; as most movements in sports require simultaneous concentric knee extension and eccentric knee flexion. To increase the strength, resiliency, and functional performance of the hamstrings, it is necessary to test and train the hamstrings at different eccentric speeds. The main purpose of this work is to provide instructions for measuring and interpreting eccentric hamstring strength. Techniques for measuring the functional ratio using isokinetic dynamometry are provided and sample data will be compared. Additionally, we briefly describe how to address hamstring strength deficiencies or unilateral strength differences using exercises that specifically focus on increasing eccentric hamstring strength.

The hamstrings are a group of muscles that are sometimes problematic for athletes, resulting in soft tissue injury in the lower limbs. To prevent such injuries, functional training of the hamstrings requires intensive eccentric contractions. Additionally, hamstring function should be tested in relation to quadricep function at different contraction speeds.

肌肉失衡: 运动人群功能性偏心肌筋强度的检测与训练

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite ...