作者想对编辑评论者以及两位匿名评论者表示感谢。他们的建议和批评是有效的, 非常赞赏和大大改进了原稿。

本议定书坚持北卡罗莱纳州立大学的人类研究道德准则。
1. 机器准备
注意: 以下协议可广泛应用于任何全身、临床 DXA 和 BMD 扫描仪。
<ol><li>在扫描任何个人之前每天进行一次校准, 以确保质量控制。在系统软件启动后出现校准提示后, 扫描已知密度的腰椎幻像, 以确保正确读取 BMD 扫描仪。</li>
	<li>如果使用的扫描仪没有在软件中的质量控制功能, 比较腰椎脊柱的结果与那些记录在脊椎幻影, 以确保正确的测量。 注意: 脊柱的幻影, 应放置在中心的扫描台和腰椎应选择的质量控制。</li>
	<li>根据需要执行附加测试 (e. g, 射线照相一致性)。每十次扫描均执行射线照相一致性, 以确保扫描仪检测到整个扫描表面。</li>
	<li>如果使用的扫描仪在 "质量控制" 菜单中没有射线一致性测试, 请选择 "全身扫描" 以确保扫描仪可以读取整个扫描表面。 注意: 在考试前, 要始终把试卷放在质量控制的中心。</li>
</ol>2. 执行考试
<ol><li>创建患者配置文件<ol>
<li>为每个被扫描的新个人创建新的患者配置文件, 以保持保管链, 并确保扫描与个人遗骸正确关联。如果被扫描的个体被辨认, 继续步2.1.2。如果个体是不明的, 在扫描之前建立生物外形使用最准确的数据库参考。</li>
		<li>输入人口统计信息到患者的外形包括估计的身材, 如果未知。确保为所调查的遗骸选择最合适的方程式。</li>
		<li>选择扫描类型。对于步骤 2.2, 选择前后 (AP) 腰椎。对于步骤 2.3, 选择左或右髋关节扫描。</li>
	</ol>
</li>
	<li>AP 腰椎扫描 注意: 需要腰椎 (L) 一到四。<ol>
<li>选择执行考试 | 选择患者 |选择扫描类型 |AP 腰椎 |下一个。选择一个打开的容器, 至少与 L1-L4 的铰接段一样大。 注: 本研究使用的是 48.26L x 26.85W x 8.89D 厘米 (19 英寸。L X 10.57 英寸W X 3.5 英寸D)。</li>
		<li>用大米作为软组织的代表来填满容器的底部。 注意: 任何种类的大米都可以作为软组织的代表。</li>
		<li>安置 L1-L4 在解剖学位置 (棘过程应该朝向向下) 在米与大约 0.7 cm (0.28 英寸) 在每个椎体之间如显示在图 1A。确保上级和下级关节的事实是明确的, 但椎体没有相互接触。</li>
		<li>中心的扫描表和地方的容器与 L1 是面向顶端 (头部) 的扫描表和 L4 被放置在1厘米优于交叉十字准线。垂直激光线应平分所有四椎骨的脊椎动物体 (图 1B)。</li>
		<li>用大米覆盖裸露的骨头。</li>
		<li>选择开始扫描。</li>
		<li>如果正确扫描 (图 2), 则继续进行分析 (步骤 3.1)。重复扫描, 如果不是所有的椎骨被抓获。</li>
	</ol>
</li>
	<li>左或右髋关节扫描 注意:图 3来自左髋关节检查, 如果执行正确的臀部检查, 定位是镜像的。<ol>
<li>选择执行考试 | 选择患者 |选择扫描类型 |左髋关节 (或右臀部) |下一个。选择一个打开的容器, 至少与被扫描的关节系统髋和股骨一样大。 注: 本研究使用的是 88.5L x 41.5W x 13.9D 厘米 (34.85 英寸。L X 16.35 英寸W X 5.47 英寸D)。</li>
		<li>用大米填满容器的底部 (任何一种大米都可以作为软组织的代表)。</li>
		<li>将髋臼和闭孔与耻骨骨内侧侧向侧面。将坐骨节在股骨头下方的位置与髋臼 (图 3A) 明确表达。 注: 坐骨节的定位是最重要的, 因为如果它在股骨颈以下延伸, 它将会膨胀 BMD 的估计。</li>
		<li>将股骨与股骨头在髋臼中, 并与大转子和股骨头平行的扫描表 (i. e, 在同一平面)。确保股骨轴是内侧旋转的远端髁突旋转内侧和略高于内侧髁 (图 3B)。</li>
		<li>中心扫描表, 然后移动扫描臂和表的位置, 直到激光十字瞄准, 使中心是直接高于股骨的下面积与垂直线平分股骨上半轴 (图 3A)。在定位后不要移动遗体。移动表格确保骨骼保持在适当的解剖位置。</li>
		<li>用大米覆盖股骨-髋臼关节的剩余可见部分。</li>
		<li>选择开始扫描。</li>
		<li>如果正确扫描 (图 4), 请在步骤3.2 中进行分析。 注意: 扫描应捕捉关节的对准, 使股骨近端的中线在一平面上。中线应从股骨头中心到大转子的正下方。</li>
	</ol>
</li>
</ol>3. 分析考试
<ol><li>分析 AP 腰椎扫描<ol>
<li>扫描后, 将出现一个退出考试提示框。选择分析扫描。 注意: 软件将每个椎骨分成各自的区域, 以便在正确扫描时评估单个元素和总 BMD, 如图 5所示。</li>
		<li>在扫描分析窗口中选择结果。选择椎线, 如果椎骨没有适当的分离, 为小的调整或直接重新定位椎骨的扫描。</li>
		<li>在执行少年 bmd 扫描时, 获得年龄匹配和填充特定的 bmd 参考措施, 以计算z分数。</li>
		<li>收集结果图以显示个体相对于参考种群的可视性。 注意:图 6显示了31岁女性的 AP 腰椎的扫描结果。</li>
	</ol>
</li>
	<li>分析臀部扫描。<ol>
<li>扫描后, 将出现一个退出考试提示框。选择分析扫描。 注意: 如果正确扫描, 软件将自动捕获股骨颈、病房的三角形和转子区域, 如图 7所示。</li>
		<li>选择骨骼图工具, 以添加或删除不属于股骨颈和转子地区的区域时, 由于轻微错位的软件不准确阅读。通过选择颈部工具并重新定位中线, 使中线在扫描时直接调整。</li>
		<li>如果这些小的调整不允许在图 7中显示正确的对齐方式, 请重新定位和重新扫描。</li>
		<li>在扫描分析窗口中选择结果。对成人股骨颈、转子区和股骨粗隆间区的参考数据进行比较。</li>
		<li>比较结果与适当年龄和人口匹配的参考时, 评估青少年。</li>
		<li>对成年人使用t评分, 因为这是最合适的差异性评估病理条件。 注意:图 8显示了31岁女性左髋关节分析的理想扫描结果。</li>
	</ol>
</li>
</ol>

作者声明没有竞争的金融利益。

本文的研究结果说明了 BMD 测量方法在法医学中的适用性。如图 6和图 8所示, 活体个体对临床 BMD 扫描的扫描位置是可重现的骨骼遗骸, 但必须注意确保正确定位。这是特别关键的髋关节检查, 确定股骨颈中线需要正确的角度股骨和高估 BMD 可能发生, 如果髂节没有正确定位内侧 acetabulo 股关节.对于在案例研究 1中讨论的成年男性, BMD 指标可以为个?...

法医人类学分析的目的是依靠从业者对骨骼的理解, 因为它是一个具有多重单位和变异的复杂组织。骨是一种分层的复合组织, 具有有机和无机成分组织成基质的胶原和碳酸化磷灰石1,2,3,4。无机成分, 或骨矿物是组织在一个纳米晶体结构提供的刚度和框架的有机部分1,2,5。矿物方面包括大约65% 的骨重量和它的质量受遗传和环境因素的影响1,2,4,6。由于骨矿物质占三维空间, 它可以被测量为骨矿物质密度 (BMD), 或质量的功能和体积占用7。骨矿物质的体积密度随着年龄的增长而变化8,9,10,11,12 , 并在临床设置中广泛使用, 作为骨质疏松和骨折危险指标4,13,14,15,16,17,18。双能量 X 射线密度 (DXA) 自1987年推出以来, 一直是评估骨骼健康的广泛工具, 特别是在腰椎和臀部区域进行的扫描11,13,19.在调查 BMD13、19、20、21、22时, DXA 扫描的验证已显示为金标准.随后, 世界卫生组织 (WHO) 制定了规范性标准, 包括t-和z-青少年和成人腰椎 (L1-L4) 和臀部的评分定义, 因为这些区域很容易被捕获 volumetrically11 ,13,19,24。
越来越多地依赖法医人类学在法医学个案中, 鼓励调查新的技术, 以更好地评估骨骼遗骸在各种情况下。这些潜在的技术之一是应用 DXA 扫描, 以评估骨密度作为一个指标的骨骼质量的情况下, 涉及致命饥饿和忽视在青少年25,26, 代谢性骨疾病的鉴定,埋藏研究中骨骼元素的生存性估计7,27。
在2015美国卫生和人类服务部虐待儿童的报告中, 75.3% 的被报告的虐待儿童案件是某种形式的疏忽, 造成1670人死亡, 其中49州的死亡人数为28人, 而这些人员被忽视。大多数被忽视的青少年受害者没有表现出外在身体虐待的迹象, 但是在所有情况下都可以看到失败--茁壮成长29,30。失败--茁壮成长定义为支持增长和发展的营养摄入不足。这些可能有不同的因素, 其中之一是由于营养缺乏造成的疏忽25,31 (请参见罗斯和阿贝尔32以获得更全面的审阅)。蓄意的饥饿导致儿童或婴儿死亡的情况更少见, 被认为是最极端的虐待形式25,33,34。这些营养缺陷对骨骼生长, 特别是儿童的纵向生长有很大影响, 这是营养不良的直接后果35。骨骼生长和矿化主要依赖维生素 D 和钙, 其补充已与增加骨密度25,35,36。
这是非常困难的, 以确定或起诉这些情况下, 即使在完整的解剖31,37,38 , 并特别考虑到使用的方法必须用。因此, 在怀疑有致命饥饿或营养不良的情况下, 需要采取多学科的办法, 特别是在涉及残留在先进的分解状态26的情况下。当骨骼遗骸参与, 骨密度是一个有用的工具与其他骨骼指标, 如牙科发展, 测量的基底的头骨, 和长骨长度26。如果不使用上述对婴幼儿的骨骼指标, 就不可能辨别低 BMD 是否是先天代谢紊乱、营养不良或埋藏过程的结果。另一个问题是对婴儿或少年骨骼遗骸的体型大小 (体重和身高) 的估计。大多数规范性数据集需要关于身高或体重的信息来进行比较, 因为儿童的骨骼生长是大小和年龄依赖于12。当被评估的遗骸不明时, 应采用估算方法。对于一岁以下的婴儿, 规范性 DXA 数据只有年龄匹配。在1岁以上的青少年中, 为了估计骨骼残骸中的体型, 建议使用39或考吉尔40 , 因为它们基于丹佛生长研究样本, 包括年龄 1-1739,40。当年龄和身体大小的估计, 置信区间的变化, 并与疾病控制中心 (CDC) 产生的生长曲线的平均值的比较41应包括在报告中, 以及估计的身体尺寸的置信区间。重要的是要注意, 在大多数情况下, 有关血统和性别的信息不能从少年骨骼遗骸确定青春期前, 这是特别重要的青少年, 因为祖先和性别是已知的显著影响 BMD成人.在这种情况下, DXA 方法可能不适用。在确定的情况下, 应在分析之前获得有关血统、性别和体型的生物信息。
随着规范性数据的发展, 在儿科的骨密度增加了42,43 , DXA 是最广泛使用的技术44。营养不良的儿童在 BMD 方面的水平明显低于健康儿童, 与营养不良的严重程度相关45。DXA 扫描的腰椎和臀部是最合适的领域, 以评估青少年根据美国放射学院46。在整个生长期47期间, 儿童的脊柱、整个髋关节和全身都有重复性表现。然而, 腰椎是首选的, 因为它主要是由小梁骨, 这是更敏感的代谢变化, 在成长过程中发现更精确的比全髋关节评估25,47, 48. 使用 DXA 扫描在儿科评估中很常见。但是, 由于 DXA 是二维的, 它不捕获真实的体积, 并根据骨面积13生成 BMD。在儿童中, 这是一个重要的区别, 因为身体和骨骼的大小在儿童的年龄组内和之间变化12。大多数可用的规范性数据是与 DXA 测量进行比较, 但应谨慎选择适当的引用填充 (请参见 Binkovitz 和 Henwood13以获取常用的 DXA 规范数据库的列表)。
在扫描之后, 将使用年龄匹配和填充特定的参考示例来计算z分数。Z-分数更适合青少年, 因为t-分数比较测量的 BMD 与年轻成人样本12。一个z-介于-2 到2之间的分数表示按年代年龄的正常 bmd, 而任何低于-2 的分数表示按年代年龄的低 bmd49。t-和z-分数的-2 到2范围表示两个标准偏差。显然, 如果一个测量的 BMD 分数是在两个标准偏差以上或低于他们的参考人口的意思, 他们被认为是临床正常的。
法医人类学家对形态变异的依赖来自于许多来源。其中之一是从疾病过程中产生的骨骼变异, 包括代谢性骨骼疾病50。在骨骼遗骸中识别特定疾病的能力有两个优势: 1) 向生物剖面中添加信息, 使其更加健壮, 2) 确定骨折是否是病理性的, 或造成创伤的结果。有多种代谢性骨紊乱51,52,53, 但最相关的 BMD 措施的当代遗骸是骨质疏松症。骨质疏松症的发病率在骨密度损失大于皮质骨丢失率的情况下发展为53,54,55。小梁骨丢失与骨折的危险性增加有关, 特别是在有更大的骨的骨骼内容 (例如, os 髋关节)4,55。
大量的骨质疏松症和骨密度的研究已经进行了考古组合使用 DXA56,57,58,59和其他方法60,61,62. 然而, 当评估成人骨骼中的骨质疏松症的考古背景, 从业者无视诊断骨质疏松症临床需要的平均值的年轻参考样品同期与个人被评估55,63,64。这不是一个问题在法医人类学背景下, 因为个体是年龄-和性-匹配的现代人口与开发的参考样品为臀部和腰椎, 虽然 BMD 的变动通过成岩作用应该考虑法医遗骸然而, 埋藏是可能的因素影响的能力获得合法的 BMD 措施从考古标本。这也是法医方面的一个考虑因素, 那里的遗体从埋葬条件中恢复过来, 可能在几个月后死亡。尽管仍有法医的兴趣, 但对于从这些情况中发现的遗骸的骨密度得分, 还是有足够的怀疑。
骨质疏松症是临床评估使用t-分数的 bmd 措施, 从个人的 bmd 措施, 在髋关节或腰椎相对于一个年轻的成人参考样品使用 DXA65,66,67 ,68。此参考样品可用于鉴定骨骼中骨质疏松症的发生。在法医方面, 这是有用的两个原因: 1) 区分之间的骨折有关的虐待造成的创伤老年人和那些从增加骨质疏松症的个人69, 和 2) 作为一个可能的个人标识功能50。
骨密度一直被认为是一个指标, 反映了动物的活动和营养70,71。最近有人指出, 骨密度, 作为骨骼的内在属性, 在埋藏过程中会影响其生存能力7。 分解的结果是骨骼元素的差异生存能力 (即, 骨骼的离散, 解剖完整的单位) 和骨密度可以作为生存能力的预测因子, 或骨强度7,70,71,72,73,74,75. 这在法医背景以及考古和古生物环境中都很重要, 因为它影响到从业者充分运用方法估计生物剖面 (或年龄、性别、身高和血统) 的能力。仅某些骨骼元素代表。
体积密度 (包含在测量中的孔隙空间的骨密度) 是在这种情况下的适当测量, 考虑到它正是骨骼的多孔结构, 影响其对埋藏过程的敏感性7。许多评估骨密度的方法包括单束光子密度测量27,75, 计算机断层扫描76,77,78, photodensitometry72 ,79和 DXA80,81,82。DXA 扫描可能比其他方法更可取, 因为它是相对便宜的, 全身扫描可以执行, 个别骨骼元素可以单独评估或在分析期间一起。在埋藏研究之前和之后使用 BMD 扫描, 提供了关于不同埋藏因素和环境导致的骨骼存活性的有用信息82。
本文概述了获取骨骼遗骸的 DXA 扫描的协议。该方法使用的普通, 临床定位的个人时, 执行腰椎和髋关节扫描。这使得从业者可以将骨骼遗骸与适当的规范性标准进行比较。所概述的议定书适用于青少年和成人遗骸, 并在以后讨论的限制。

<table border="0" cellpadding="0" cellspacing="0" width="869">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">QDR Discovery 4500W system</td>
			<td style="width:71px;">Hologic</td>
			<td style="width:119px;">Discovery W</td>
			<td style="width:463px;">All inclusive DXA whole body scanner that includes APEX software for visualization and analysis of scans. Incorporates FRAX reference data developed by WHO to provide both t- and z- scores.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">APEX 3.2</td>
			<td style="width:71px;">Hologic</td>
			<td style="width:119px;">APEX</td>
			<td>Software used by the DXA PC connected to the bone desitometer (QDR Discovery 4500W system) to acquire the BMD data and analyze results.</td>
		</tr>
	</tbody>
</table>

scanning skeletal remains for bone mineral density in forensic contexts

本文的目的是介绍一种有前途的, 新的方法, 以帮助评估骨质量的法医相关的骨骼遗骸。骨密度是骨骼营养状况的重要组成部分, 在青少年和成人的骨骼残留中, 它可以提供骨骼质量的信息。对于成人遗骸, 它能提供关于病理条件或当骨头不足可能发生的信息。在青少年中, 它提供了一个有用的指标来阐明严重饥饿或忽视的情况, 这通常难以确定。本文提供了一个协议的解剖定位和分析的骨骼遗骸扫描通过双能 x 射线密度 (DXA)。三例研究表明, 当 DXA 扫描可以向法医医生提供信息。第一个案例研究提出了一个个人与观察到的纵向骨折的负重骨和 DXA 是用来评估骨不足。BMD 被发现是正常的建议的另一种病因的骨折模式目前。第二个案例研究采用 DXA 来调查疑似慢性营养不良。骨密度的结果与长骨长度的结果一致, 并表明青少年患有慢性营养不良。最后的案例研究提供了一个例子, 在一个十四月的婴儿中, 有致命的饥饿被怀疑, 这支持尸检结果的致命饥饿。DXA 扫描显示低骨密度的年龄, 并证实了传统的婴儿健康评估。然而, 在处理骨骼遗骸时, 应考虑埋藏的改变, 然后再应用此方法。

这里提出的方法是常用的在活的患者和考虑它的新颖性对已故的个体应该注意。图 6和图 8分别显示 AP 腰椎和左髋关节扫描的结果。在这些扫描评估的个人是一个已故的白人, 女性, 31 岁, 被关押在北卡罗莱纳州立大学法医分析实验室。该个体的总 BMD 评分为0.944 克/cm2 , 具有相应的t评分 (-0.9), 用于祖先和性?...

Watch this Scientific Journal Video about Scanning Skeletal Remains for Bone Mineral Density in Forensic Contexts at JoVE.com

在法医环境中扫描骨骼遗骸以获取骨矿物质密度 |医学|视频

Scanning Skeletal Remains for Bone Mineral Density in Forensic Contexts

骨密度 (BMD) 是了解营养摄入的重要因素。对于人类骨骼遗骸, 这是一个有用的指标, 以评估青少年和成人的生活质量, 特别是在致命的饥饿和忽视的情况。本文提供了为法医目的扫描人体骨骼遗骸的指南。

扫描骨骼遗骸的骨骼矿物密度在法医学背景下

The purpose of this paper is to introduce a promising, novel method to aid in the assessment of bone quality in forensically relevant skeletal remains. ...

scanning-skeletal-remains-for-bone-mineral-density-forensic

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17.4K 次观看.  North Carolina State University. 骨密度 (BMD) 是了解营养摄入的重要因素。对于人类骨骼遗骸, 这是一个有用的指标, 以评估青少年和成人的生活质量, 特别是在致命的饥饿和忽视的情况。本文提供了为法医目的扫描人体骨骼遗骸的指南。

视频: Scanning Skeletal Remains for Bone Mineral Density in Forensic Contexts

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

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

骨转移模型

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

Photothrombotic Ischemia: A Minimally Invasive and Reproducible Photochemical Cortical Lesion Model for Mouse Stroke Studies

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected light-sensitive dye. Following illumination, the dye is activated and produces singlet oxygen that damages components of endothelial cell membranes, with subsequent platelet aggregation and thrombi formation, which eventually determines the interruption of local blood flow. This approach, initially proposed by Rosenblum and El-Sabban in 1977, was later improved by Watson in 1985 in rat brain and set the basis of the current model. Also, the increased availability of transgenic mouse lines further contributed to raise the interest on the photothrombosis model. Briefly, a photosensitive dye (Rose Bengal) is injected intraperitoneally and enters the blood stream. When illuminated by a cold light source, the dye becomes activated and induces endothelial damage with platelet activation and thrombosis, resulting in local blood flow interruption. The light source can be applied on the intact skull with no need of craniotomy, which allows targeting of any cortical area of interest in a reproducible and non-invasive way. The mouse is then sutured and allowed to wake up. The evaluation of ischemic damage can be quickly accomplished by triphenyl-tetrazolium chloride or cresyl violet staining. This technique produces infarction of small size and well-delimited boundaries, which is highly advantageous for precise cell characterization or functional studies. Furthermore, it is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

Photothrombosis is a quick, minimally-invasive technique for inducing small and well-delimited infarction in areas of interest in highly reproducible manner. It is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

光化学缺血:一种微创和可重复性的光化学皮质病变模型小鼠中风研究

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected ...

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. ...

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.

Receptor tyrosine kinases are ectopically expressed in many cancers and have been identified as therapeutic targets in acute leukemia. This manuscript describes an efficient strategy for pre-clinical evaluation of tyrosine kinase inhibitors for the treatment of acute leukemia.

酪氨酸激酶抑制剂的临床前评价急性白血病的治疗

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are ...

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 ...

Using a Knee Arthrometer to Evaluate Tissue-specific Contributions to Knee Flexion Contracture in the Rat

Normal knee range of motion (ROM) is critical to well-being and allows one to perform basic activities such as walking, climbing stairs and sitting. Lost ROM is called a joint contracture and results in increased morbidity. Due to the difficulty of reversing established knee contractures, early detection is important, and hence, knowing risk factors for their development is essential. The rat represents a good model with which the effect of an intervention can be studied due to the similarity of rat knee anatomy to that of humans, the rat's ability to tolerate long durations of knee immobilization in flexion, and because mechanical data can be correlated with histologic and biochemical analysis of knee tissue.
Using an automated arthrometer, we demonstrate a validated, precise, reproducible, user-independent method of measuring the extension ROM of the rat knee joint at specific torques. This arthrometer can be used to determine the effects of interventions on knee joint ROM in the rat.

The goal of the protocol is to measure the extension range of motion of the rat knee. The effects of various diseases that increase the stiffness of the knee joint and the effectiveness of treatments can be quantified.

用膝关节关节仪评价大鼠膝关节屈曲收缩的特定组织贡献

Normal knee range of motion (ROM) is critical to well-being and allows one to perform basic activities such as walking, climbing stairs and sitting. Lost ...

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. Bone is a dynamic tissue undergoing continuous remodeling with the competing activity of osteoblasts, which produce the new bone matrix, and osteoclasts, whose function is to eliminate mineralized bone. [18F]-NaF is a positron emission tomography (PET) radiotracer that enables visualization of bone metabolism. [18F]-NaF is chemically absorbed into hydroxyapatite in the bone matrix by osteoblasts and can thus noninvasively detect osteoblastic activity, which is occult to conventional imaging techniques. Kinetic modeling of dynamic [18F]-NaF-PET data provides detailed quantitative measures of bone metabolism. Conventional semi-quantitative PET data, which utilizes standardized uptake values (SUVs) as a measure of radiotracer activity, is referred to as a static technique due to its snapshot of tracer uptake in time.&#160; Kinetic modeling, however, utilizes dynamic image data where tracer levels are continuously acquired providing tracer uptake temporal resolution. From the kinetic modeling of dynamic data, quantitative values like blood flow and metabolic rate (i.e., potentially informative metrics of tracer dynamics) can be extracted, all with respect to the measured activity in the image data. When combined with dual modality PET-MRI, region-specific kinetic data can be correlated with anatomically registered high-resolution structural and pathologic information afforded by MRI. The goal of this methodological manuscript is to outline detailed techniques for performing and analyzing dynamic [18F]-NaF-PET-MRI data. The lumbar facet joint is a common site of degenerative arthritis disease and a common cause for axial low back pain.&#160; Recent studies suggest [18F]-NaF-PET may serve as a useful biomarker of painful facetogenic disease.&#160; The human lumbar facet joint will, therefore, be used as a prototypical region of interest for dynamic [18F]-NaF-PET-MRI analysis in this manuscript.

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. We present detailed methodologies for performing and analyzing dynamic [18F]-NaF-PET-MRI data in a patient with facetogenic low back pain using the lumbar facet joints as a prototypical region of interest.

定量 =18F_-Naf-PET-MRI 分析,用于评估面部腰痛患者的动态骨质周转率

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. Bone is a dynamic tissue undergoing ...

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent evidence indicates a potential inflammatory component of the disease, with both T-cells and monocytes/macrophages potentially associated with the pathogenesis of OA. Further studies postulated an important role for subsets of both inflammatory cell lineages, such as Th1, Th2, Th17, and T-regulatory lymphocytes, and M1, M2, and synovium-tissue-resident macrophages. However, the interaction between the local synovial and systemic inflammatory cellular response and the structural changes in the joint is unknown. To fully understand how T-cells and monocytes/macrophages contribute towards OA, it is important to be able to quantitively identify these cells and their subsets simultaneously in synovial tissue, secondary lymphatic organs and systemically (the spleen and bone marrow). Nowadays, the different inflammatory cell subsets can be identified by a combination of cell-surface markers making multi-color flow cytometry a powerful technique in investigating these cellular processes. In this protocol, we describe detailed steps regarding the harvest of synovial tissue and secondary lymphatic organs as well as generation of single cell suspensions. Furthermore, we present both an extracellular staining assay to identify monocytes/macrophages and their subsets as well as an extra- and intra-cellular staining assay to identify T-cells and their subsets within the murine spleen, bone marrow, lymph nodes and synovial tissue. Each step of this protocol was optimized and tested, resulting in a highly reproducible assay that can be utilized for other surgical and non-surgical OA mouse models.

Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis.

骨关节炎模型中骨髓、骨髓、淋巴结和合成组织内免疫细胞子集的流动细胞测量分析

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent ...

Evaluation of Amino Acid Consumption in Cultured Bone Cells and Isolated Bone Shafts

Bone development and homeostasis is dependent upon the differentiation and activity of bone forming osteoblasts. Osteoblast differentiation is sequentially characterized by proliferation followed by protein synthesis and ultimately bone matrix secretion. Proliferation and protein synthesis require a constant supply of amino acids. Despite this, very little is known about amino acid consumption in osteoblasts. Here we describe a very sensitive protocol that is designed to measure amino acid consumption using radiolabeled amino acids. This method is optimized to quantify changes in amino acid uptake that are associated with osteoblast proliferation or differentiation, drug or growth factor treatments, or various genetic manipulations. Importantly, this method can be used interchangeably to quantify amino acid consumption in cultured cell lines or primary cells in vitro or in isolated bone shafts ex vivo. Finally, our method can be easily adapted to measure the transport of any of the amino acids as well as glucose and other radiolabeled nutrients.

This protocol presents a radiolabeled amino acid uptake assay, which is useful for evaluating amino acid consumption either in primary cells or in isolated bones.

扫描骨骼遗骸的骨骼矿物密度在法医学背景下

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