美国国立卫生研究院R21NS42516以TGH 克雷格H尼尔森基金会赠款83860到TGH NIDRR - RRTC。 "亚太贸易协定"的博士奖学金，以CKT

为了评估急性口服一种SSRI（10毫克草酸依地普仑，Lexapro，森林制药公司）的影响，电机与电机不完全脊髓损伤，双盲，随机，安慰剂对照的交叉设计是受雇于个人活动。作为入选标准的一部分，受试者将被要求接受一个14天的清洗期为所有抗抑郁药，抗痉挛药物和其他药物，与著名的SSRIs的相互作用。 两个小时需要为每个实验的协议，包括临床，定量的静态和定量动态评估。一个4.5小时的延迟是药物管理局（血浆峰浓度）7。科目重新测试以下用药管理，使用相同的实验设置和用药前测试期间雇用的范式。继前和测试后的第一次用药（SSRI药物或安慰剂），最低为7天分隔两个测试条件（剂的半衰期约27小时）。 第1部分:临床评估改良Ashworth 股四头肌和腿筋肌肉群痉挛是量化使用改良Ashworth量表（捣烂）。 <ol><li>个人置于仰卧于软垫垫。 </li><li>痉挛性反射分级在0-4整数组成，除了1 +"级的规模。分级标准是根据下表（表1）。 </li><li>评估股四头肌痉挛，病人的腿是提出的垫和扩展与对侧腿完全伸展大腿和踝关节的支持下，。舒展股四头肌提供有关轻快的被动伸屈膝关节。伸展的反应是量化，按照以下的规模。 </li><li>评估腿筋痉挛，病人的腿是提出的垫子，并充分支持下扩展与对侧腿的大腿和脚踝弯曲，。一气，提供轻快的膝关节被动扩展提供了腿筋。伸展的反应是量化，按照以下的规模。 </li><li>原始分数转换成一个有序的规模和总结双边获得综合得分。 </li></ol>脊髓痉挛反射的评估工具屈肌痉挛，伸肌痉挛和阵挛的持续时间或幅度量化使用脊髓痉挛反射的评估工具（SCATS） <ol><li>个人置于仰卧于软垫垫。 </li><li>痉挛反射分级在0-3采用全数字的规模。分级标准是根据下表（见表2）。 </li><li>为了评估屈肌痉挛，两条腿完全伸展与病人的鞋和删除袜子。一针挑破应用为主体的脚内侧足弓1秒。量化，按照以下规模的大脚趾，膝关节和髋关节的运动幅度。 </li><li>为了评估伸肌痉挛，腿部支持在90 °屈曲髋关节和膝关节屈曲110 °，与健侧腿延长。从这个位置的腿轻快地扩展，并安息在垫子上。可见股四头肌活动期间，上级髌骨位移指出，量化，按照以下的规模。 </li><li>为了评估阵挛，小腿略微提起从垫子上，脚踝是在中性至微跖，对侧腿延长。一个舒展的plantarflexors交付通过轻快的被动背屈踝关节。阵挛性爆破的时间是按照以下规模的量化。 </li><li>分数相加，在双腿之间，以获得综合得分。 </li></ol>下肢运动评分使用亚洲下肢运动分数（LEMS）评估个人能力volitionally合同按照myotomes肌肉。 <ol><li>个人置于仰卧于软垫垫。 </li><li>强度分级0-5采用全数字的规模。分级标准是根据下表（见表3）。 </li><li>让病人试图从仰卧位弯曲髋关节的髋部屈肌（L2肌节）评估，并提供必要的手动阻力。如果病人是无法完全克服重力移动，髋关节外旋，以减轻重力和髋关节屈曲的影响再次尝试。 </li><li>膝伸肌（L3肌节），让病人尝试延长膝关节从Flex位置，对考官前臂旋转，手动阻力是提供必要的评估。如果病人是无法完全克服重力移动，髋关节外旋，以减轻电子重力和伸膝ffects再次尝试。 </li><li>评估病人的尝试与腿的延伸，在必要时提供的手动阻力dorsiflex脚踝脚踝dorsiflexors（L4肌节）。如果病人是无法完全克服重力移动的脚踝，髋关节外旋，以减轻重力和踝关节背屈的影响再次尝试。 </li><li>大脚趾伸肌（L5的肌节）是病人企图延长腿部的大脚趾延伸和脚踝plantarflexed，手动阻力是提供必要的评估。如果病人是无法完全克服重力移动的脚趾，髋关节外旋，以减轻重力和大脚趾延伸的影响再次尝试。 </li><li>踝plantarflexiors（S1肌节）评估由手动阻力是提供必要的扩展腿的病人尝试plantarflex。如果病人是寸步难行通过全方位的阻力，踝关节，髋关节和膝关节弯曲放置在垫子上，病人的脚跖面是要求增加对他们的脚趾。如果病人是无法完全上升，对他们的脚趾，髋关节外旋，以减轻重力和踝关节跖的影响再次尝试。 </li><li>下肢运动评分的计算方法是将每个肌肉群的结果双边。 </li></ol>第2部分:定量静态评估速度依赖牵张反射用等速测功机，肌电图（EMG），可应用和一个精确的拉伸，一个定量的反应可以评估。 <ol><li>个人是坐在舒适的检测仪器（Biodex系统3，雪莉，纽约） </li><li>表面肌电信号的比目鱼肌和腓肠肌内侧。 </li><li>腿是连接到脚踏板加上一个6度自由称重传感器（ATX工业自动化，耳尖，NC）和踝关节为轴是称重传感器的中​​心对齐。 </li><li>与病人放松，单个或多个延伸到背屈（DF）或跖（PF）是适用于各种转速，其中包括5，30，60和120度/秒</li><li>为了量化的转矩响应，扭矩信号被过滤在200Hz，在​​1000赫兹的采样与肌电图数据同步。被动和引力的扭矩得到从慢伸展扰动（5度/秒），并从快速延伸减去，以计算反射反应。伸展分割为每个DF和PF游览，最小和最大扭矩确定，峰值扭矩定义为价值观的差异。 </li><li>要量化肌电图响应，肌电图信号被过滤在20 - 450Hz，在​​1000赫兹，与扭矩数据同步采样。整流和平滑的信号是，在10 Hz低通使用4阶递归巴特沃斯滤波器，平滑信号面积计算是所有游览激动剂和拮抗剂。对于扰动，肌电图往往存在以下的运动停止在最后举行的立场。综合肌电图面积计算，从进入保持阶段延长3秒的最后一次联合旋转开始。 </li></ol>等距的力量使用等速测功机，可精确量化的意志的力量。 <ol><li>个人是坐在舒适的检测仪器。 </li><li>表面肌电信号贴6每条腿的主要肌肉群包括股直肌，股外侧肌，胫骨前，比目鱼肌和腓肠肌内侧（Delsys 2.1，马萨诸塞州波士顿），内侧hamsting。站是连接到脚踏板，再加上到6度自由称重传感器和膝盖关节轴称重传感器的中​​心对齐。 </li><li>与病人的放松，病人的指示，以产生最大力量。为了确保最大的意志的力量生产，积极的口头鼓励病人提供实验者。当扭矩开始下降，超最大的刺激传递到通过3"× 5"不干胶，凝胶刺激板激动剂（10个脉冲，持续时间600微秒，100赫兹，135 V，S48草，西华威，扶轮社）电极（ConMed公司的Utica，NY）。扭矩和肌电图的收集和空调（步骤2）的速度取决于拉伸。 </li><li>离线</li><li>要量化最大转矩响应，平滑信号的峰值扭矩。最大扭矩定义为平均转矩产生± 50ms的峰值扭矩。 </li><li>为了定量肌电图的反应，最大的肌电图是计算的平均信号，目前0到峰值扭矩为100ms。 </li><li>为了量化意志激活的赤字，中央激活率计算公式1。在这个公式中的 T中华人民共和国推荐性是指自愿的扭矩产品uced前100 ms的电刺激和Telectrical是指峰值电引起的扭矩。 8 </li></ol><img src="/files/ftp_upload/2148/2148eq1.jpg" alt="公式1" /> 公式＃1 第3部分:定量的动态评估山顶跑步机的速度功能下地进行分级的跑步机测试，以获得跑步机的速度。 <ol><li>主题是安全的（Bertec，哥伦布，OH）在跑步机上使用的是架空的安全带，不限制运动，并没有提供支持身体重量。心率监测器贴。一个反光标志设置适用于使用修改后的克利夫兰诊所的模板（32个反光标记）。表面肌电信号贴6每条腿的主要肌肉群包括股直肌，hamsting内侧，股外侧肌，胫前，比目鱼和内侧gastrocnemious（Noraxon，亚利桑那州斯科茨代尔） </li><li>主体开始在跑步机上走在0.1米/秒。速度是速度增加0.1米/秒，每2分钟</li><li>测试是3个条件下终止<ol class="lalpha"><li>意志疲劳</li><li>步态不稳</li><li>根据美国运动医学学院（ACSM）的指引9 <ol class="iroman"><li>主观体力感觉等级= 20 </li><li>心率范围内的预测最高的10次</li><li>呼吸交换比率= 1.15 </li></ol></li></ol></li><li>最大速度为至少1分钟获得，跑步机速度峰值记录。 </li></ol>步态运动学在步行多个运动的措施，其中包括跑步机，峰值范围内的议案，峰值速度和可变性是使用运动捕捉系统（运动分析，圣罗莎，CA）进行评估。 intralimb之间的髋关节和膝关节协调的一致性量化计算的对应的平均系数（ACC 10 ）。行政协调会使用一个向量编码技术的矢状平面的髋关节和膝关节角度分析上的一个角角的阴谋。 <ol><li>每制造商的建议在每次使用前校准动作捕捉摄像头。在数据采集，数据采样从6台摄像机在100赫兹。 </li><li>脱机状态，动作捕捉数据是手动跟踪准确性和预任后SSRI的获得的禁食速度至少10步周期的时代，是孤立的进行分析。 </li><li>步态周期正常化％，从脚跟脚跟罢工的罢工和使用的髋关节和膝关节角度的三次样条插值。 </li><li>髋膝角角的情节构造（图3a）。 </li><li>平均余弦和正弦（X）髋关节和膝关节（Y）规范化向量的长度（L），分别为跨分离分析（公式1-2）的所有步骤计算每帧帧间隔。 </li></ol><img src="/files/ftp_upload/2148/2148eq2.jpg" alt="公式（2）" /> 公式＃2 <img src="/files/ftp_upload/2148/2148eq3.jpg" alt="公式3" /> 公式＃3 <ol start="6"><li>每帧到帧的时间间隔平均向量计算使用跨的平均帧到帧的正弦和余弦值分析（公式3）孤立的所有步骤。 </li></ol><img src="/files/ftp_upload/2148/2148eq4.jpg" alt="公式4" /> 公式＃4 <ol start="7"><li>平均向量（一），代表在帧到帧的时间间隔或多个步骤周期（公式4）之间的髋关节和膝关节的intralimb协调的一致性的变化不断变异。当A = 1周期值大于1表明逐步减少intralimb的步骤（图3b）之间的一致性的是完全一致的。 </li></ol><img src="/files/ftp_upload/2148/2148eq5.jpg" alt="公式（5）" /> 公式＃5 肌肉活动期间下地肌肉活动时步态是量化使用肌电信号。 <ol><li>为了收集肌电反应，肌电图信号硬件过滤在20 - 450Hz，采样1000赫兹，而运动捕获数据的同步。 </li><li>脱线，信号在30Hz高通滤波消除运动神器全波整流和低通滤波在20赫兹使用4 阶递推巴特沃斯创建一个线性的信封。平滑肌电信号正常化％步态周期和预任后SSRI的获得的禁食速度至少10步之间的平均值。 </li><li>肌肉活动的规范价值是建立一个健康对照组的数据库建立时间和关闭时间的规范。下表列出了整个步态CY 6肌肉的规范活动CLE。 </li><li>每块肌肉的时间和截止时间的规范，覆盖从题目中收集到的肌电信号。计算上的时间和关闭时间的综合区。 5月11日通过公式计算痉挛指数（SI）。代表结果显示在图3c。 </li></ol><img src="/files/ftp_upload/2148/2148eq6.jpg" alt="公式（6）" /> 公式＃6 下床活动时的代谢录音心肺/代谢能力评估使用跑步机上使用便携式一个K4b2代谢系统（康是美芝加哥（美国）公司，）在分级测试措施的峰值摄氧量（VO2 峰值 ;毫升/公斤/分钟）。 <ol><li>代谢系统是按照制造商的建议进行校准，并贴到病人。 </li><li> VO2的基线测量获得了至少2分钟，坐姿，然后再在一个至少2分钟的站立姿势。内部用户对代谢系统的控制标志是用来同步事件。 </li><li>在跑步机上行走时摄氧量是收集的速度增加（每2分钟）由实验者提供的手动标记。 </li><li>跑步机测试后，数据下载到个人电脑作进一步分析。 </li><li> VO2的数据分析，平均每2分钟的速度划时代的最后分钟，并绘制每个速度（图3d）。 </li></ol><img src="/files/ftp_upload/2148/2148fig1.jpg" alt="图1" /> 图1:在一个单独的SSRI药物与口服给药后电机不完全脊髓损伤有显着的改变是在电机活动的临床措施。非自愿的反射活动这两项措施，混搭（A）和SCATS（二）不同程度地增加，这表明这两个临床量表可捕捉增强非自愿活动的不同方面。 （三）此外，在临床检测的意志的力量变化观察以下SSRI的管理。 <img src="/files/ftp_upload/2148/2148fig2.jpg" alt="图2" /> 图2:在一个单独的SSRI药物与口服给药后电机不完全脊髓损伤有显着的改变是在运动活动的定量静态措施。 （一）增强和延长踝关节跖屈力矩和肌电图观察以下SSRI的管理plantarflexors延伸。等距扭矩增加SSRI的管理。有趣的是，激活赤字（更大的车），SSRI的管理，说明个人能够更充分地激活SSRI的管理过程中一个最大的意志收缩他们的肌肉。 <img src="/files/ftp_upload/2148/2148fig3.jpg" alt="图3" /> 图3:在一个单独的SSRI药物与口服给药后电机不完全脊髓损伤有在电机活动的定量动态措施改变。髋关节和膝关节运动的变异分析（一）在下地使用ACC（二）显示SSRI类药物有增加的一致性，前，后的措施都在0.7米/秒。 （三）分析下肢肌电图在下床活动，揭示了一个在增加肌肉活动不恰当的时机，SSRI类药物，前，后的措施都在0.7米/秒。 （四）下床活动时的代谢参数的分析，揭示了在跑步机测试，分级SSRI的管理适度增加氧气消耗量。 <table border="1"><tbody><tr><td> 级 </td><td> （序比分） </td><td> 标准 </td></tr><tr><td> 0 </td><td> （0） </td><td>肌张力没有增加</td></tr><tr><td> 1 </td><td> （1） </td><td>略有增加肌肉张力，表现在运动范围由渔获物和释放或最小阻力是在前屈或扩建移动时，受影响的部分（S） </td></tr><tr><td> 1 + </td><td> （2） </td><td>略有增加肌张力，表现一个陷阱，整个剩余的ROM（少于一半）的最小阻力</td></tr><tr><td> 2 </td><td> （3） </td><td>更为显着增加肌张力多数通过的ROM，但受影响的部分（S）容易被感动</td></tr><tr><td> 3 </td><td> （4） </td><td>大量增加肌肉张力，被动运动困难</td></tr><tr><td> 4 </td><td> （5） </td><tD&gt;受影响的部分（S）的刚性在屈曲或延伸</td></tr></tbody></table> 表1:捣烂得分 ，从12修改<table border="1"><tbody><tr><td rowspan="2"> 级 </td><td colspan="3" style="text-align:center;"> 标准 </td></tr><tr><td> 屈指 </td><td> 伸肌 </td><td> 阵挛 </td></tr><tr><td> 0 </td><td>没有反应</td><td>没有反应</td><td>没有反应</td></tr><tr><td> 1 </td><td>漂移小于10 °屈曲，膝关节和髋关节或扩建的大脚趾</td><td>活动维持了不到3秒</td><td>阵挛是维持不到3秒</td></tr><tr><td> 2 </td><td> 10 °〜30 °屈曲，膝关节和髋关节</td><td>活动仍然存在3至10秒</td><td>阵挛仍然存在3至10秒</td></tr><tr><td> 3 </td><td> 30 °或更大的膝盖和髋关节屈曲</td><td>活动持续超过10秒</td><td>阵挛仍然存在超过10秒</td></tr></tbody></table> 表二:SCATS得分 ，从13修改<table border="1"><tbody><tr><td> 级 </td><td> 标准 </td></tr><tr><td> 0 </td><td>无可见或可触及的肌肉收缩指出</td></tr><tr><td> 1 </td><td>指出任何可见或可触及的肌肉</td></tr><tr><td> 2 </td><td>肌肉是可以移动，至少一次，它是通过一个全方位的议案，在重力被淘汰的位置插入下肢部分</td></tr><tr><td> 3 </td><td>肌肉是可以移动，至少一次，下肢的一部分，它是通过全方位的运动必须克服重力的位置插入</td></tr><tr><td> 4 </td><td>能够执行3级描述的功能和肌肉是对考官的努力，能够提供一些阻力</td></tr><tr><td> 5 </td><td>肌肉是能发挥，在考官的判断，正常量的抵抗考官的努力</td></tr></tbody></table> 表3:LEMS得分 <table border="1"><tbody><tr><td rowspan="2"> 肌肉 </td><td colspan="2" style="text-align:center;">步态周期百分比</td></tr><tr><td></td><td>在</td><td>关</td></tr><tr><td> 射频 </td><td> 0-30和55-75 </td><td> 30-55和75-100 </td></tr><tr><td> VL </td><td> 0-30和55-75和95-100 </td><td> 30-55和75-95 </td></tr><tr><td> 氢 </td><td> 0-25和65-100 </td><td> 25-65 </td></tr><tr><td> 爵 </td><td> 5-55 </td><td> 0-5和55-100 </td></tr><tr><td> 溶胶 </td><td> 5-55 </td><td> 0-5和55-100 </td></tr><tr><td> 电讯管理局局长 </td><td> 0-15 </td><td> 15-100 </td></tr></tbody></table> 表4:开启和关闭的LE肌肉在步态规范

<ol>
	<li>Miller, J. F., Paul, K. D., Rymer, W. Z., Heckman, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-HT1B/1D+agonist+CGS-12066B+attenuates+clasp+knife+reflex+in+the+cat.">5-HT1B/1D agonist CGS-12066B attenuates clasp knife reflex in the cat.</a> J Neurophysiol. 74, 453-456 (1995).</li><li>Gimenez y Ribotta, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinematic+analysis+of+recovered+locomotor+movements+of+the+hindlimbs+in+paraplegic+rats+transplanted+with+monoaminergic+embryonic+neurons.">Kinematic analysis of recovered locomotor movements of the hindlimbs in paraplegic rats transplanted with monoaminergic embryonic neurons.</a> Ann N Y Acad Sci. 860, 521-523 (1998).</li><li>Landry, E. S., Guertin, P. 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此视频演示突出的方法来评估反射活动的变化，意志的力量和下地SSRIs类药物的急性口服人类SCI。这种电池的评估表明，尽管所谓的异常反射活动，下面的一个SSRI急性口服增加，个人与SCI的运动能力只有轻微的变化表现出意志的力量改善，的。增强痉挛的影响，在这些人不出现有损于日间功能。此外，在行走能力的改善可能是在个人与急性病变更为显着。继续研究使用这些类似的定量和临床评估，以确定联合SSRIs和密集的物理干预的疗效，已受聘在脊髓损伤动物模型。有了一个良好的理论框架基础上的长期和相对较新的的基础科学的研究，这些方法可以帮助翻译等信息的临床人口，可能会挑战一些传统的协议，在临床实践中使用，以增加个人与SCI功能恢复。

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<td>Isokinetic Dynamometer</td>
<td>&nbsp;</td>
<td>Biodex</td>
<td>System 3 with Rehab Toolkit</td>
<td>&nbsp;</td>
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<td>EMG system</td>
<td>&nbsp;</td>
<td>Delysis</td>
<td>Bagnoli 2.1</td>
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<td>EMG system</td>
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<td>Noraxon</td>
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methods to quantify pharmacologically induced alterations in motor function in human incomplete sci

脊髓损伤（SCI）是一种使人衰弱的疾病，从而产生深刻的意志电机控制赤字。从SCI恢复医疗稳定，通常涉及长期的康复治疗。虽然恢复行走能力的主要目标是在许多患者伤后早期，电机不完全脊髓损伤，说明意志控制的部分保存，可有足够的剩余降实现这一目标的必要途径。然而，尽管物理干预，电机的障碍，包括弱点，表现异常的不自主反射活动，称为痉挛或痉挛，被认为有助于减少行走恢复。教条主义的思想表明，整治电机与SCI的这种不正常的反射，会产生对病人功能的好处。例如，医生和治疗师会提供特定的药物或物理干预措施，努力减少痉挛或痉挛，虽然仍然是很少的经验数据表明，这些策略提高行走能力。 在过去的几十年，积累数据，建议，具体neuromodulatory的代理，包括代理模仿或便利的单胺的行动，包括5 - 羟色胺（5HT）和去甲肾上腺素（NE），可以启动或扩大步行在脊髓损伤动物模型的行为。有趣的是，许多这些药物，尤其是5HTergic激动剂，可以显着提高脊髓兴奋性，这反过来也增加了这些动物的反射活动。有悖常理恢复的传统理论，人类脊髓损伤，从基本的科学实验的经验证据表明，这种反射超兴奋性和产生的运动行为并行驱动的neuromodulatory输入（5HT），可能是以下脊髓损伤功能恢复的必要。 应用从基础科学研究产生这种新颖的概念，促进人类脊髓损伤的恢复，似乎是无缝的，虽然直接翻译的结果可以是非常具有挑战性。具体来说，在动物模型，植入的导管有利于提供非常具体的，直接到脊髓电路5HT激动剂化合物。这项技术对人类的翻译是缺乏具体的手术技巧，或可针对人体临床试验的安全和有效的5HT受体亚型的药理代理人的阻碍。然而，口服常用5HTergic剂，如选择性5 - 羟色胺再摄取抑制剂（SSRIs），可能是一种可行的选择，以增加中央5HT浓度，以便走在人类的恢复。这些SSRIs的是如何调节人体动作行为SCI与强度，反射，恢复行走能力的具体重点，系统量化，缺少。 此视频演示是一个渐进的的尝试，以系统，定量评估调制反射活动，意志的力量和下地下列人类脊髓损伤的SSRI的急性口服。适用于单一天的代理，评估，长期的，涉及反复药品监督管理局结合密集的物理干预研究的，在这个病患族群运动功能的直接影响。

Watch this Scientific Journal Video about Methods to Quantify Pharmacologically Induced Alterations in Motor Function in Human Incomplete SCI at JoVE.com

Methods to Quantify Pharmacologically Induced Alterations in Motor Function in Human Incomplete SCI

本视频演示了一个5 - 羟色胺再摄取抑制剂（SSRI）的急性口服调制反射活动，电机不完全脊髓损伤的个人意志的力量，并通过临床和定量评估下地。

量化人类不完全脊髓损伤运动功能的药理学诱导改建的方法

Spinal cord injury (SCI) is a debilitating disorder, which produces profound deficits in volitional motor control. Following medical stabilization, ...

methods-to-quantify-pharmacologically-induced-alterations-motor

Nanyang Technological University

Research

JoVE Journal

Medicine

13.7K 次观看.  Rehabilitation Institute of Chicago. 本视频演示了一个5 - 羟色胺再摄取抑制剂（SSRI）的急性口服调制反射活动，电机不完全脊髓损伤的个人意志的力量，并通过临床和定量评估下地。

视频: Methods to Quantify Pharmacologically Induced Alterations in Motor Function in Human Incomplete SCI

Technique to Collect Fungiform (Taste) Papillae from Human Tongue

The sense of taste is critical for human life. It informs the body about the quality of food that will be potentially ingested and stimulates metabolic processes that prepare the alimentary canal for digestion. Steady progress is being made towards understanding the early biochemical and molecular events underlying taste transduction (for a review, Breslin and Spector, 20081). However, progress to date has largely resulted from animal models. Yet, since marked differences in receptor specificity and receptor density vary among species, human taste transduction will only be understood by using human taste tissue. Here we describe a biopsy technique to collect human fungiform papillae, visible as rounded pink anterior structures, about 0.5 mm in diameter that contain taste buds. These biopsied papillae are used for several purposes including the isolation of viable taste bud cells, in situ hybridization, immunohistochemistry and, through techniques of molecular biology, the identification of taste-specific novel proteins.

Technique to Collect Fungiform Taste Papillae from Human Tongue

Knowledge of molecular mechanisms underlying gustatory transduction has recently enjoyed significant advances, largely due to using animal models. However, the wide diversity in taste sensitivity and specificity among mammals warrants studies in human tissue. We describe a biopsy technique to collect living taste cells from the papillae on human tongue.

技术收集人舌菌状（味道）乳头

The sense of taste is critical for human life. It informs the body about the quality of food that will be potentially ingested and stimulates metabolic ...

科研

医学

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

一项战略，以确定在常见的疾病，如自闭症和精神分裂症的新突变

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
contractions. The pelvic limb of an anesthetized dog is immobilized in a stereotactic frame to align the tibia at a right angle to the femur. Adhesive wrap affixes the 
paw to a pedal mounted on the shaft of a servomotor to measure torque. Percutaneous nerve stimulation activates pelvic limb muscles of the paw to either push (extend) or 
pull (flex) against the pedal to generate isometric torque. Percutaneous tibial nerve stimulation activates tibiotarsal extensor muscles. Repeated eccentric (lengthening) 
contractions are induced in the tibiotarsal flexor muscles by percutaneous peroneal nerve stimulation. The eccentric protocol consists of an initial isometric contraction 
followed by a forced stretch imposed by the servomotor. The rotation effectively lengthens the muscle while it contracts, e.g., an eccentric contraction. During 
stimulation flexor muscles are subjected to an 800 msec isometric and 200 msec eccentric contraction. This procedure is repeated every 5 sec. To avoid fatigue, 4 min rest 
follows every 10 contractions with a total of 30 contractions performed.

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and painless method to measure canine hindlimb muscle strength and muscle response to repeated eccentric contractions.

在犬体内的肌肉功能测定

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
...

Methods for ECG Evaluation of Indicators of Cardiac Risk, and Susceptibility to Aconitine-induced Arrhythmias in Rats Following Status Epilepticus

Lethal cardiac arrhythmias contribute to mortality in a number of pathological conditions. Several parameters obtained from a 
non-invasive, easily obtained electrocardiogram (ECG) are established, well-validated prognostic indicators of cardiac risk in patients suffering 
from a number of cardiomyopathies. Increased heart rate, decreased heart rate variability (HRV), and increased duration and variability of cardiac 
ventricular electrical activity (QT interval) are all indicative of enhanced cardiac risk 1-4. In animal models, it is valuable to compare 
these ECG-derived variables and susceptibility to experimentally induced arrhythmias. Intravenous infusion of the arrhythmogenic agent aconitine has
 been widely used to evaluate susceptibility to arrhythmias in a range of experimental conditions, including animal models of depression 5 and 
 hypertension 6, following exercise 7 and exposure to air pollutants 8, as well as determination of the antiarrhythmic efficacy of pharmacological 
 agents 9,10. 
It should be noted that QT dispersion in humans is a measure of QT interval variation across the full set of leads from a 
standard 12-lead ECG. Consequently, the measure of QT dispersion from the 2-lead ECG in the rat described in this protocol is different than that 
calculated from human ECG records. This represents a limitation in the translation of the data obtained from rodents to human clinical medicine.
Status epilepticus (SE) is a single seizure or series of continuously recurring seizures lasting more than 30 min 
11,12 11,12, and results in mortality in 20% of cases 13. Many individuals survive the SE, but die within 30 days 14,15.
 The mechanism(s) of this delayed mortality is not fully understood. It has been suggested that lethal ventricular arrhythmias contribute to many of these 
 deaths 14-17. In addition to SE, patients experiencing spontaneously recurring seizures, i.e. epilepsy, are at risk of premature 
 sudden and unexpected death associated with epilepsy (SUDEP) 18. As with SE, the precise mechanisms mediating SUDEP are not known. 
 It has been proposed that ventricular abnormalities and resulting arrhythmias make a significant contribution 18-22.
 To investigate the mechanisms of seizure-related cardiac death, and the efficacy of cardioprotective therapies, it is 
 necessary to obtain both ECG-derived indicators of risk and evaluate susceptibility to cardiac arrhythmias in animal models of seizure disorders 
 23-25. Here we describe methods for implanting ECG electrodes in the Sprague-Dawley laboratory rat (Rattus norvegicus), following SE,
 collection and analysis of ECG recordings, and induction of arrhythmias during iv infusion of aconitine.
 These procedures can be used to directly determine the relationships between ECG-derived measures of cardiac electrical 
 activity and susceptibility to ventricular arrhythmias in rat models of seizure disorders, or any pathology associated with increased risk of sudden 
 cardiac death.

Techniques for measurement of electrical activity of the heart by electrocardiogram (ECG), and analysis of cardiac risk factors and susceptibility to arrhythmias following status epilepticus (SE) in the rat are described.

心电图心脏风险评价指标和乌头碱诱发大鼠心律失常的易感性继癫痫持续状态的方法

Lethal cardiac arrhythmias contribute to mortality in a number of pathological conditions. Several parameters obtained from a 
non-invasive, easily ...

Dynamic Visual Tests to Identify and Quantify Visual Damage and Repair Following Demyelination in Optic Neuritis Patients

In order to follow optic neuritis patients and evaluate the effectiveness of their treatment, a handy, accurate and quantifiable tool is required to assess changes in myelination at the central nervous system (CNS). However, standard measurements, including routine visual tests and MRI scans, are not sensitive enough for this purpose. We present two visual tests addressing dynamic monocular and binocular functions which may closely associate with the extent of myelination along visual pathways. These include Object From Motion (OFM) extraction and Time-constrained stereo protocols. In the OFM test, an array of dots compose an object, by moving the dots within the image rightward while moving the dots outside the image leftward or vice versa. The dot pattern generates a camouflaged object that cannot be detected when the dots are stationary or moving as a whole. Importantly, object recognition is critically dependent on motion perception. In the Time-constrained Stereo protocol, spatially disparate images are presented for a limited length of time, challenging binocular 3-dimensional integration in time. Both tests are appropriate for clinical usage and provide a simple, yet powerful, way to identify and quantify processes of demyelination and remyelination along visual pathways. These protocols may be efficient to diagnose and follow optic neuritis and multiple sclerosis patients.
In the diagnostic process, these protocols may reveal visual deficits that cannot be identified via current standard visual measurements. Moreover, these protocols sensitively identify the basis of the currently unexplained continued visual complaints of patients following recovery of visual acuity. In the longitudinal follow up course, the protocols can be used as a sensitive marker of demyelinating and remyelinating processes along time. These protocols may therefore be used to evaluate the efficacy of current and evolving therapeutic strategies, targeting myelination of the CNS.

Object From Motion (OFM) and Time-constrained stereo protocols are sensitive tools to identify monocular and binocular dynamic visual function deficits, which are uniquely affected in optic neuritis patients. Furthermore, these tests may be used as quantitative noninvasive tools to assess the extent of myelination along visual pathways.

动态视觉测试，以确定和量化视觉损伤与修复继脱髓鞘视神经炎患者

In order to follow optic neuritis patients and evaluate the effectiveness of their treatment, a handy, accurate and quantifiable tool is required to ...

Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which disrupt normal brain function. Despite treatment with currently available antiepileptic drugs targeting neuronal functions, one third of patients with epilepsy are pharmacoresistant. In this condition, surgical resection of the brain area generating seizures remains the only alternative treatment. Studying human epileptic tissues has contributed to understand new epileptogenic mechanisms during the last 10 years. Indeed, these tissues generate spontaneous interictal epileptic discharges as well as pharmacologically-induced ictal events which can be recorded with classical electrophysiology techniques. Remarkably, multi-electrode arrays (MEAs), which are microfabricated devices embedding an array of spatially arranged microelectrodes, provide the unique opportunity to simultaneously stimulate and record field potentials, as well as action potentials of multiple neurons from different areas of the tissue. Thus MEAs recordings offer an excellent approach to study the spatio-temporal patterns of spontaneous interictal and evoked seizure-like events and the mechanisms underlying seizure onset and propagation. Here we describe how to prepare human cortical slices from surgically resected tissue and to record with MEAs interictal and ictal-like events ex vivo.

We here describe how to perform multi-electrode array recordings of human epileptic cortical tissue. Epileptic tissue resection, slice preparation and multi-electrode array recordings of interictal and ictal events are demonstrated in detail.

人类癫痫术后皮层组织的多电极阵列录音

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which ...

Adapting Human Videofluoroscopic Swallow Study Methods to Detect and Characterize Dysphagia in Murine Disease Models

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research. Successful outcomes are dependent upon three critical components: test chambers that permit self-feeding while standing unrestrained in a confined space, recipes that mask the aversive taste/odor of commercially-available oral contrast agents, and a step-by-step test protocol that permits quantification of swallow physiology. Elimination of one or more of these components will have a detrimental impact on the study results. Moreover, the energy level capability of the fluoroscopy system will determine which swallow parameters can be investigated. Most research centers have high energy fluoroscopes designed for use with people and larger animals, which results in exceptionally poor image quality when testing mice and other small rodents. Despite this limitation, we have identified seven VFSS parameters that are consistently quantifiable in mice when using a high energy fluoroscope in combination with the new murine VFSS protocol. We recently obtained a low energy fluoroscopy system with exceptionally high imaging resolution and magnification capabilities that was designed for use with mice and other small rodents. Preliminary work using this new system, in combination with the new murine VFSS protocol, has identified 13 swallow parameters that are consistently quantifiable in mice, which is nearly double the number obtained using conventional (i.e., high energy) fluoroscopes. Identification of additional swallow parameters is expected as we optimize the capabilities of this new system. Results thus far demonstrate the utility of using a low energy fluoroscopy system to detect and quantify subtle changes in swallow physiology that may otherwise be overlooked when using high energy fluoroscopes to investigate murine disease models.

This study successfully adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research.

适应人类Videofluoroscopic燕子研究的方法来检测和检定吞咽困难的小鼠疾病模型

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating ...

Measurements of Motor Function and Other Clinical Outcome Parameters in Ambulant Children with Duchenne Muscular Dystrophy

While the number of new treatment options tested in patients with Duchenne muscular dystrophy (DMD) is increasing, there is still no defining of the most reliable assessments regarding therapeutic efficacy. We present clinical and radiological outcome measures used in ambulatory patients participating in our trial &#34;Treatment with L-citrulline and metformin in Duchenne muscular dystrophy&#34;. The motor function measure is a validated test in patients with neuromuscular disorders that consists of 32 items and assesses all three dimensions of motor performance including standing and transfer (D1 subscore), axial and proximal motor function (D2 subscore), and distal motor function (D3 subscore). The test shows high intra- and inter-rater variability but only when strictly following guidelines of the materials, examination steps, and calculation of scores. The 6-minute walk test, timed 10-meter walk/run test, and supine-up time are commonly used timed functional tests that also sufficiently monitor changes in muscle function; however, they strongly depend on patient collaboration. Quantitative MRI is an objective and sensitive biomarker to detect subclinical changes, though the examination costs may be a reason for its limited use. In this study, a high correlation between all clinical assessments and quantitative MRI scans was found. The combinational use of these methods provides a better understanding about disease progression; however, longitudinal studies are needed to validate their reliability.

The aim of this study is to present the most reliable clinical outcome measures and their correlations with quantitative muscle MRI in ambulant patients with Duchenne muscular dystrophy.

治疗杜氏肌营养不良患儿运动功能及其他临床结果参数的测量

While the number of new treatment options tested in patients with Duchenne muscular dystrophy (DMD) is increasing, there is still no defining of the most ...

Using a Chemical Biopsy for Graft Quality Assessment

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated with an increased survival rate and greater quality of patient&#8217;s life when compared to conventional dialysis. Regrettably, transplantology suffers from a lack of reliable methods for organ quality assessment. Standard diagnostic techniques are limited to macroscopic appearance inspection or invasive tissue biopsy, which do not provide comprehensive information about the graft. The proposed protocol aims to introduce solid phase microextraction (SPME) as an ideal analytical method for comprehensive metabolomics and lipidomic analysis of all low molecular compounds present in kidneys allocated for transplantation. The small size of the SPME probe enables performance of a chemical biopsy, which enables extraction of metabolites directly from the organ without any tissue collection. The minimum invasiveness of the method permits execution of multiple analyses over time: directly after organ harvesting, during its preservation, and immediately after revascularization at the recipient&#8217;s body. It is hypothesized that the combination of this novel sampling method with a high-resolution mass spectrometer will allow for discrimination of a set of characteristic compounds that could serve as biological markers of graft quality and indicators of possible development of organ dysfunction.

The protocol presents utilization of the chemical biopsy approach followed by comprehensive metabolomic and lipidomic analysis for quality assessment of kidney grafts allocated for transplantation.

使用化学活检进行移植质量评估

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated ...

A Non-Invasive Method for Generating the Cyclic Loading-Induced Intra-Articular Cartilage Lesion Model of the Rat Knee

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely correlated with a history of articular cartilage damage and ligament avulsion. Surgical animal models of OA of the knee play an important role in understanding the onset and progression of post-traumatic OA and aid in the development of novel therapies for this disease. However, non-surgical models have been recently considered to avoid traumatic inflammation that could affect the evaluation of the intervention. 
In this study, an intra-articular cartilage lesion rat model induced by in vivo cyclic compressive loading was developed, which allowed researchers to (1) determine the optimal magnitude, speed, and duration of load that could cause focal cartilage damage; (2) assess post-traumatic spatiotemporal pathological changes in chondrocyte vitality; and (3) evaluate the histological expression of destructive or protective molecules that are involved in the adaptation and repair mechanisms against joint compressive loads. This report describes the experimental protocol for this novel cartilage lesion in a rat model.

Here, we present the cyclic loading-induced intra-articular cartilage lesion model of the rat knee, generated by 60 cyclic compressions over 20 N, resulting in damage to the femoral condylar cartilage in rats.

一种生成周期负荷诱导的大鼠膝关节内软骨病变模型的非侵入性方法

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely ...