Name: automated gait analysis in mice with chronic constriction injury
Uploaded: 2017-10-17T14:00:00.000+00:00
Duration: 6 min 49 s
Description: Watch this Scientific Journal Video about Automated Gait Analysis in Mice with Chronic Constriction Injury at JoVE.com

这项研究得到了南国立大学、韩国东方医学研究所 (KIOM) 的支持, 并通过韩国卫生产业发展研究所 (KHIDI) 资助了韩国卫生技术研发 #38;D 项目, 由卫生部提供资金。和 #38; 大韩民国福利 (赠款号: HI15C0007)。

所有实验都是按照国际疼痛研究协会的道德准则进行的, 并得到了南国立大学机构动物保育和使用委员会的批准 (韩国大田). 
 1. 在坐骨神经上的诱导作用 <ol> <li> 家庭男性民事代表小鼠在受控室温下的 12 h 光照/暗循环下重 20-25 克 (维持在 20-25 和 #176; C) 和湿度 (40-60%), 可免费获得食物和水.在手术前至少1周, 允许动物保育室的小鼠适应时间. 
	<ol> <li> 在外壳期间, 观察小鼠的外观和行为, 不要使用显示异常运动活动的小鼠. 
		注意: 在前一天的手术, 测量机械灵敏度的后腿爪通过应用冯弗雷丝和执行自动步态分析, 以获得正常的基线值。然后, 随机分配给小鼠控制和实验组. </li>
	</ol> </li> <li> 在手术当天, 麻醉小鼠注射 22, 2-tribromoethanol (250 毫克/千克, 腹腔 (IP)). 
	注: 穿戴个人防护设备, 如手术礼服、手套和口罩。
	<ol> <li> 重2.5 克, 22, 2-tribromoethanol, 并添加 2-甲基-2-丁醇到最后的体积5毫升。使解决方案远离光 ( 例如 , 使用深色容器或用箔包装). </li>
		<li> 允许试剂在40和 #176 加热完全溶解; C 和搅拌10-30 分钟. </li>
		<li> 将蒸馏水加入200毫升的最后一卷, 搅拌至混合好. </li>
		<li> 存储等分在4和 #176; C, 并保持在黑暗中。两周后, 应该用新的等分代替麻醉剂. </li>
		<li> 注入20和 #181 的体积; L 每1克体重。例如, 如果鼠标的体重是25克, 给500和 #181; L 的解决方案。这种麻醉的典型持续时间为1小时. 
		注意: 此溶液在暴露于光和/或热时会变得有毒, 因此要小心避免暴露在光和热中。强烈建议使用 IP 注入路由. </li>
	</ol> </li> <li> 当鼠标进入深层麻醉状态时, 将它放在手术台上, 背侧向上 ( 即 , 俯卧位置), 并用70% 的乙醇拭子对右侧的外大腿区域进行消毒. 
	注: 通过检查对后爪或尾部的挤压或压力刺激缺乏反应来确认深层麻醉. </li>
	<li> 诱导鼠标右坐骨神经上的小鼠. 
	<ol> <li> 用手术刀刀片在 1.0-1.5 厘米长的大腿区域周围的皮肤上进行切口. </li>
		<li> 用微蚊子止血直接解剖大腿肌肉以暴露坐骨神经. </li>
		<li> 用4-0 个铬线缝合线结扎暴露的坐骨神经3次, 间隔 1.0-1.5 毫米. 
		注意: 通过结扎坐骨神经使松结扎, 直到同侧后爪出现轻度晃动。假组小鼠在类似条件下接受相同的手术切口, 除非没有神经结扎. </li>
	</ol> </li> <li> 关闭 3-4 简单中断的缝合线, 使用5-0 丝, 用聚维酮碘消毒, 用于外科手术的外区. </li>
	<li> 手术后, 把鼠标放在一个清洁的笼子上的暖气垫。当动物从麻醉中恢复过来时, 把它们送回自己的笼子里. 
	注意: 在这项研究中, 没有使用抗生素。该公司 + gbp 集团收到的 IP 丁 (gbp) 每天一次剂量为50毫克/千克作为一个积极的控制. </li>
</ol> 2。机械痛测量 (冯弗雷测试) 注意: 通过使用1克弗雷灯丝对同侧后爪的足底表面, 评估对机械刺激的撤回反应的频率. 
<ol> <li> 在每个测试日, 将鼠标带到行为测试室, 并在测试前至少30分钟将鼠标置于自己的主笼中. 
	注: 穿戴个人防护设备, 如手术礼服、手套和口罩. </li>
	<li> 将鼠标放在金属网格地板上的透明亚克力盒中, 并使鼠标适应30分钟. </li>
	<li> 轻轻地将1克的弗雷灯丝应用到后爪的足底表面, 直到灯丝弯曲. </li>
	<li> 将灯丝刺激应用于同侧后爪10次, 至少十年代间隔, 并记录结果. 
	注意: 在这项研究中, 10 试验的爪戒反应的次数显示为爪子退频率的百分比 (PWF,%)。每两天进行一次手术. </li>
</ol> 3。执行自动步态分析 注意: 步态分析系统在动物行走时将每个爪印形象化, 并自动分析各种步态参数, 如爪印、爪子强度、步幅、姿态相位, 步进顺序, 摆动, 摆动速度, 等 在本研究中, 我们在把数据转换成对侧左对同侧右后爪的百分比变化后, 显示了爪印面积和单姿态。因此, 50% 的结果表明, 爪印的大小和爪面积与地板接触的时间, 左右两侧是相同的。此外, 较低的数据值接近0% 表明, 与对侧相比, 同侧后爪的大小和持续时间都减少了 (请参见 图 2 的面板 B 和 图 3 ). 
<ol> <li> 为适应, 将小鼠的步态分析设备保持在10分钟, 每天开始前5天的手术. 
	注意: 步态分析测试包括适应应在黑暗中进行. </li>
	<li> 在测试前至少30分钟将鼠标带到测试室进行步态分析并适应其家庭保持架. 
	<ol> <li> 在 "程序" 菜单的 "设置" 选项卡中, 设置和 #34; 人行道长度和 #34; 设置和 #34; 最大运行持续时间和 #34; 到 5 s 和 #34; 最大运行变化和 #34; 到 50%. </li>
		<li> 从 #39 中选择已注册的照相机; #39;p rogram 和 #39; 菜单中的设置和 #39; 选项卡. </li>
		<li> 选择和 #34; 从和 #39 获取和 #39; #39;p rogram 和 #39 的选项卡; 菜单. </li>
		<li> 在状态消息之后, 单击 "#39"; 对齐背景和 #39; 按钮可采取空的背景图片 ( 即 空白) 通道. </li>
	</ol> </li> <li> 单击并 #39; 启动购置和 #39; 按钮, 然后将鼠标放在人行道上; 录制根据鼠标的移动自动启动. 
	注意: 当鼠标清晰地穿过走道时, 程序会自动将此移动分类为和 #34; 兼容的运行和 #34; 绿色图标。如果软件没有检测到脚印, 实验者将看到一个红色的图标, 必须重复录音。分析需要至少五成功的兼容运行. </li>
	<li> 测试后, 选择和 #39; 从 #39 的 "获取" 选项卡;p rogram 和 #39; 菜单中分类运行和 #39; </li>
	<li> 选择要分析的运行, 然后单击 #39; 自动分类和 #39; 按钮. 
	注: 分类后, 所有统计参数自动保存, 实验者可以通过点击和 #34 找到结果; 查看运行统计信息和 #34; 在 "分析" 菜单上 </li>
</ol>

<ol>
	<li>Loeser, J. D., Treede, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Kyoto+protocol+of+IASP+Basic+Pain+Terminology.">The Kyoto protocol of IASP Basic Pain Terminology.</a> Pain. 137 (3), 473-477 (2008).</li><li>Colloca, L., 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=Neuropathic+pain.">Neuropathic pain.</a> Nat Rev Dis Primers. 3, 17002 (2017).</li><li>Vranken, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+and+treatment+of+neuropathic+pain.">Mechanisms and treatment of neuropathic pain.</a> Cent Nerv Syst Agents Med Chem. 9 (1), 71-78 (2009).</li><li>DeLeo, J. A., 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=Characterization+of+a+neuropathic+pain+model:+sciatic+cryoneurolysis+in+the+rat.">Characterization of a neuropathic pain model: sciatic cryoneurolysis in the rat.</a> Pain. 56 (1), 9-16 (1994).</li><li>Kim, S. H., Chung, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+experimental+model+for+peripheral+neuropathy+produced+by+segmental+spinal+nerve+ligation+in+the+rat.">An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat.</a> Pain. 50 (3), 355-363 (1992).</li><li>Seltzer, Z., Dubner, R., Shir, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+behavioral+model+of+neuropathic+pain+disorders+produced+in+rats+by+partial+sciatic+nerve+injury.">A novel behavioral model of neuropathic pain disorders produced in rats by partial sciatic nerve injury.</a> Pain. 43 (2), 205-218 (1990).</li><li>Bennett, G. J., Xie, Y. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+peripheral+mononeuropathy+in+rat+that+produces+disorders+of+pain+sensation+like+those+seen+in+man.">A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man.</a> Pain. 33 (1), 87-107 (1988).</li><li>Dixon, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+analysis+of+experimental+observations.">Efficient analysis of experimental observations.</a> Annu Rev Pharmacol Toxicol. 20, 441-462 (1980).</li><li>Bonin, R. P., Bories, C., De Koninck, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+up-down+method+(SUDO)+for+measuring+mechanical+nociception+in+rodents+using+von+Frey+filaments.">A simplified up-down method (SUDO) for measuring mechanical nociception in rodents using von Frey filaments.</a> Mol Pain. 10, 26 (2014).</li><li>Chaplan, S. R., Bach, F. W., Pogrel, J. W., Chung, J. M., Yaksh, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+tactile+allodynia+in+the+rat+paw.">Quantitative assessment of tactile allodynia in the rat paw.</a> J Neurosci Methods. 53 (1), 55-63 (1994).</li><li>Chen, H., Du, J., Zhang, Y., Barnes, K., Jia, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+a+Reliable+Gait+Evaluation+Method+for+Rodent+Studies.">Establishing a Reliable Gait Evaluation Method for Rodent Studies.</a> J Neurosci Methods. , (2017).</li><li>Kang, D. W., 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=Antinociceptive+Profile+of+Levo-tetrahydropalmatine+in+Acute+and+Chronic+Pain+Mice+Models:+Role+of+spinal+sigma-1+receptor.">Antinociceptive Profile of Levo-tetrahydropalmatine in Acute and Chronic Pain Mice Models: Role of spinal sigma-1 receptor.</a> Sci Rep. 6, 37850 (2016).</li><li>Huehnchen, P., Boehmerle, W., Endres, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+paclitaxel+induced+sensory+polyneuropathy+with+&amp;#34;Catwalk&amp;#34;+automated+gait+analysis+in+mice.">Assessment of paclitaxel induced sensory polyneuropathy with &amp;#34;Catwalk&amp;#34; automated gait analysis in mice.</a> PLoS One. 8 (10), e76772 (2013).</li><li>Vrinten, D. H., Hamers, F. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term='CatWalk'+automated+quantitative+gait+analysis+as+a+novel+method+to+assess+mechanical+allodynia+in+the+rat;+a+comparison+with+von+Frey+testing.">'CatWalk' automated quantitative gait analysis as a novel method to assess mechanical allodynia in the rat; a comparison with von Frey testing.</a> Pain. 102 (1-2), 203-209 (2003).</li><li>Martinov, T., Mack, M., Sykes, A., Chatterjea, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+changes+in+tactile+sensitivity+in+the+hind+paw+of+mice+using+an+electronic+von+Frey+apparatus.">Measuring changes in tactile sensitivity in the hind paw of mice using an electronic von Frey apparatus.</a> J Vis Exp. (82), e51212 (2013).</li><li>Ferland, C. E., Laverty, S., Beaudry, F., Vachon, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gait+analysis+and+pain+response+of+two+rodent+models+of+osteoarthritis.">Gait analysis and pain response of two rodent models of osteoarthritis.</a> Pharmacol Biochem Behav. 97 (3), 603-610 (2011).</li><li>Mogil, J. 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=Hypolocomotion,+asymmetrically+directed+behaviors+(licking,+lifting,+flinching,+and+shaking)+and+dynamic+weight+bearing+(gait)+changes+are+not+measures+of+neuropathic+pain+in+mice.">Hypolocomotion, asymmetrically directed behaviors (licking, lifting, flinching, and shaking) and dynamic weight bearing (gait) changes are not measures of neuropathic pain in mice.</a> Mol Pain. 6, 34 (2010).</li><li>Ferreira-Gomes, J., Adaes, S., Castro-Lopes, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+movement-evoked+pain+in+osteoarthritis+by+the+knee-bend+and+CatWalk+tests:+a+clinically+relevant+study.">Assessment of movement-evoked pain in osteoarthritis by the knee-bend and CatWalk tests: a clinically relevant study.</a> J Pain. 9 (10), 945-954 (2008).</li></ol>

作者没有什么可透露的。

目前, 在疼痛动物模型中, 用痛纤维测量机械性的方法是最常用的方式来表现触觉过敏。随着神经性疼痛动物模型的不断发展, 感官功能评估的方法也得到了改善8,9,10,15。在这些报告中, 有人建议这些修改可以为旧方法提供更准确、快速和 user-friendly 的替换。然而, 这些方法的数据仍然可能受到其主观性和执行方法的技能或经验的要求的限制。此外, 在进行弗雷试验时, 不能避免抑制动物, 因此动物的克制相关压力可能影响疼痛反应结果。
自动化步态分析系统自动测量动物行走的各种参数 (例如, 爪子接触到地板的总面积, 爪子印的角度, 立场时间,等), 因此实验者可以使用更多的客观数据分析12,13,14,16,17,18。此外, 这种方法可以进行, 没有任何刺激;因此, 它是少侵入性和数据从自由移动的啮齿动物可以得到。如本研究结果所示, 与同侧后爪的足印尺寸和姿态时间通过手术减少, 并通过 GBP 治疗恢复;类似的结果也被发现在冯弗雷测试。与先前的观察结果14一致, 神经病理性疼痛动物模型的步态分析与冯弗雷试验有很高程度的正相关, 表明与之相关的变化参数可能反映了机械痛, 是神经病理性疼痛最恼人的症状之一。
在这里, 我们提供了一个如何操作自动化步态分析系统的方法, 并简要比较了数据与经典的冯弗雷试验的神经病理性小鼠。总之, 自动步态分析可能是神经性疼痛的最有效的研究工具, 因为它是 user-friendly、客观、敏感的。

创伤、代谢功能障碍、炎症、感染、缺血或自身免疫性疾病引起的神经系统病理改变, 有时会导致神经病理性疼痛, 这被定义为损害或疾病的直接后果所引起的疼痛。影响体感系统1。神经病理性疼痛通常是无法忍受的, 不幸的是, 常规镇痛药一般不会产生足够的疼痛缓解2。神经病理性疼痛的一个主要特点是自发的和刺激诱发的 (即, 痛和痛觉过敏) 疼痛。痛是一种伤害反应, 发生在通常非刺激, 如轻触或温暖刺激。痛觉过敏表示对有害的机械和/或热刺激的疼痛反应增强3。尽管这两种症状都严重损害了患者的生活质量, 但轻柔的触觉刺激所诱发的机械痛是最严重的症状, 因为在日常生活中很难避免接触。
为了探讨治疗神经病理性疼痛的基本机制和新的镇痛药, 对疼痛反应的精确测量是必不可少的。许多神经病理性疼痛动物模型已经发展到后爪区的伤害反应, 因为它的高可及性4,5,6,7。因此, 大多数疼痛反应评估已经执行的脚底或背部后爪表面的应用机械刺激使用特殊仪器, 如弗雷丝。最常用的方法之一是狄克逊8和以后修改后的版本910中描述的向上和向下的方法。然而, 非常熟练, 经验丰富的实验者需要执行冯弗雷测试, 结果可能是主观的。
自动步态分析系统可以通过测量在自由移动的啮齿动物中行走的各种参数来研究神经和神经肌肉紊乱。在各种神经损伤动物模型中, 伤害的程度和几种治疗方法的镇痛效果可以在不增加疼痛刺激的情况下进行评估11,12,13,14. 该分析系统可检测静态和动态步态参数, 如: 爪印区域 (与地板接触的完整爪印区域)、爪子强度 (接触爪面积的平均强度)、步幅长度 (同一个爪子的连续安置之间的距离), 姿态阶段 (地面接触的持续时间为一只后腿), 步顺序 (命令四爪被安置在地板上), 摇摆 (摇摆阶段的期间) 和摇摆速度 (计算从步长和摆动持续时间, 以每秒的像素表示)。本文介绍了使用一个分析系统, 并提供了一个简单的数据与冯弗雷试验使用慢性收缩损伤 (局) 神经病理性小鼠。

<table><tbody><tr><td>0.9% Saline</td><td>JW Pharmaceutical</td><td>N/A</td><td>Vehicle for drugs</td></tr><tr><td>1ml syringe</td><td>BD Plastipak</td><td>300013</td><td>Injecting device</td></tr><tr><td>2, 2, 2-tribromoethanol (97% purity)</td><td>Sigma</td><td>T48402</td><td>Anesthetic</td></tr><tr><td>2-methyl-2-buthanol (99% purity)</td><td>Sigma</td><td>152463</td><td>Solvent for 2, 2, 2-tribromoethanol</td></tr><tr><td>Catwalk Automated gait analysis system</td><td>Noldus</td><td>N/A</td><td>Automatic analysis software of aniaml gait</td></tr><tr><td>Chromic catgut (4-0 thickness)</td><td>AILEE</td><td>C442</td><td>Ligature to make chronic constriction injury on the sciatic nerve</td></tr><tr><td>Gabapentin</td><td>Sigma</td><td>Y0001280</td><td>Analgeisc, Used as a positive control drug in this study</td></tr><tr><td>Graefe Forceps</td><td>F.S.T</td><td>11051-10</td><td>Surgical instrument</td></tr><tr><td>Heating Pad</td><td>DAESHIN ELECTRONICS</td><td>M-303AT</td><td>Regulation of body temperature</td></tr><tr><td>ICR Mouse</td><td>Samtaco</td><td>N/A</td><td>Experimental animal</td></tr><tr><td>Mersilk (3-0 thickness)</td><td>ETHICON</td><td>W598H</td><td>Suture material for surgical closure of skin</td></tr><tr><td>Micro-Mosquito</td><td>F.S.T</td><td>13010-12</td><td>Surgical instrument</td></tr><tr><td>Micro-scissors</td><td>F.S.T</td><td>14090-09</td><td>Surgical instrument</td></tr><tr><td>Needle holder</td><td>F.S.T</td><td>12002-12</td><td>Surgical instrument</td></tr><tr><td>Povidone Iodine</td><td>Firson</td><td>N/A</td><td>Disinfectant to prevent infection after surgery</td></tr><tr><td>Scalpel blade</td><td>F.S.T</td><td>10010-00 (#10)</td><td>Surgical instrument to make an incision</td></tr><tr><td>Scalpel handle</td><td>F.S.T</td><td>10003-12 (#3)</td><td>Surgical instrument to make an incision</td></tr><tr><td>Von-Frey filaments</td><td>North Coast</td><td>NC12775-99</td><td>Measurement device to test sensory function for mechanical stimulation</td></tr></tbody></table>

automated gait analysis in mice with chronic constriction injury

弗雷试验是一种被广泛用于检测神经病理性疼痛动物感官功能的经典方法。然而, 它也有一些缺点, 如主观数据和要求的熟练, 经验丰富的实验者。到目前为止, 各种修改改进了冯弗雷方法, 但它仍然有一些限制。最近的报告表明, 步态分析产生更准确和客观的数据从神经病动物。该协议演示了如何执行自动步态分析, 以确定的神经病理性疼痛的小鼠。经过几天的驯化, 老鼠被允许在玻璃地板上自由行走以照亮脚印。然后, 通过对各种步行参数的自动分析, 如爪印面积、摆动时间、爪角、等, 对脚印和步态进行量化。
本研究的主要目的是描述自动步态分析的方法, 并将其与经典感官试验中的数据进行简单比较。

我们已经执行了冯弗雷测试和自动步态分析在会展小鼠, 直到10天后的手术。对于统计分析, two-way 方差分析的重复测量方法 (方差) 决定了总体效果, 并对 Dunnett 的事后进行了分析, 以确定实验组之间的 p 值。
图 1所示的结果表明了经典的冯弗雷测试数据的时间过程。与假组相比, PWF (%) 小鼠在术后2天内增加, 持续10天。在 "积极控制组" 中, GBP (50 毫克/千克, IP, 每日10天, 每天1天开始手术后) 显著减轻了 PWF。在小组 A 的假小组, PWF (%) 是8.00 ±5.83 在 timepoint 前 (前), 6.00 ±2.45 在天 2, 4.00 ±2.45 在天 4, 2.00 ±2.00 在天 6, 4.00 ±22.45 在天 8, 和6.00 ±2.45 在天10。在 PWF (%) 是2.86 ±1.84 在前, 61.43 ±5.95 在天 2, 68.57 ±4.59 在天 4, 72.86 ±5.22 在天 6, 75.71 ±6.49 在天 8, 和75.71 ±3.69 在天10。在工商会 + GBP 组, PWF (%) 是2.50 ±1.43 在前, 57.50 ±5.22 在天 2, 50.00 ±4.04 在天 4, 45.00 ±4.81 在天 6, 46.25 ±5.65 在天 8, 和47.50 ±4.88 在天10。在面板 B 中, 曲线 (联合自卫队) 的面积为23.00 ± 6.00, 在假, 317.86 ±17.04 在工商委员会, 和223.75 ±17.05 在该集团 + GBP 组。
在图 2中显示的结果表明, 在进行了手术后, 爪印区域的时间过程发生了变化。数据显示的百分比变化的爪子打印区域之间的左, 右后爪。如果在正常情况下, 在左侧和右侧的爪印区域相同, 则数据将为50%。在手术后2天内, 在同侧后肢爪的爪印面积明显减少, 并维持10天。英镑明显恢复了减少爪打印面积。在小组 B 的假小组, 爪子打印区域 (%) 是50.13 ±2.13 在前, 50.30 ±1.45 在天 2, 53.24 ±0.80 在天 4, 50.30 ±1.68 在天 6, 52.08 ±1.79 在天 8, 并且49.66 个2.24 在天10。在该集团, 爪打印面积 (%) 是50.71 ±3.17 在前, 0 ±0天 2, 0 ±0在天 4, 8.86 ±3.27 在天 6, 6.6 ±3.20 在天 8, 和10.30 ±5.60 在天10。在工商会 + GBP 组, 爪子打印区域 (%) 是55.59 ±2.01 在前, 4.65±4.17 在天 2, 15.18±5.57 在天 4, 20.20 ±4.00 在天 6, 26.01 ±5.53 在天8和28.40 ±6.04 在天10。
在图 3中显示的结果表明, 在进行了手术后, 单个姿势的时间变化。计算数据并显示为百分比变化 (类似于爪印区域数据)。该公司减少了同侧一方的单一立场, 英镑明显恢复。在小组 B 的假小组, 唯一立场 (%) 是49.31 ±2.15 在前, 50.71 ±0.67 在天 2, 50.76 ±0.44 在天 4, 50.60 ±1.11 在天 6, 51.50 ±0.96 在天 8, 和49.00 个±2.35 在天10。在这一组中, 单姿态 (%) 是50.36 ±3.17 在前, 0 ±0在天 2, 0 ±0在天 4, 11.5 ±3.25 在天 6, 13.61 ±5.04 在天8和12.94 个±6.40 在天10。在工商会 + GBP 小组, 唯一立场 (%) 是52.35 ±0.91 在前, 5.44 ±4.87 在天 2, 18.66 ±4.33 在天 4, 25.48 ±4.10 在天 6, 30.26 ±2.17 在天 8, 和32.24 个±4.95 在天10。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56402/56402fig1.jpg"> 
图 1: 由弗雷试验评估的机械痛.(A) 在 PWF 的实验中, 2 天后, 与假组相比, 在10天内, 在弗雷试验中增加了爪子的提取频率 (%)。丁 (GBP, 50 毫克/千克, IP) 显着逆转了该机构引起的机械痛。(B) PWF 的累计数据显示为 "曲线" 下的区域。** 和 #60; 0.01 和 *** 和 #60; 0.001 与集团。误差线表示平均±.<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56402/56402fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56402/56402fig2.jpg"> 
图 2: 在小鼠的自动步态分析中, 爪印区域.(A) 通过分析软件捕获来自假冒、工商会和丁 (50 毫克/千克、IP) 的组的后爪图像。白色长方形是分析系统检测后爪的指示器。注意到最小的爪大小的工商会和这种减少部分恢复了英镑的待遇。(B) 爪印区域的时间过程 (%)。数据被计算为左和右后爪之间打印区域的百分比变化 (例如, 50% 表示左侧和右侧的爪印区域相同)。* p & #60; 0.05 和 ** 和 #60; 0.01 与集团。误差线指示平均值± SD。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56402/56402fig3.jpg"> 
图 3: 在会展小鼠中采用自动步态分析的单姿态.(A) 捕获和转换每个爪子的不同颜色突出显示的单一姿态的图像。请注意, 与同侧后爪的粉红色痕迹在该组中缩短, 丁 (GBP, 50 毫克/千克, IP) 在相当程度上同样地恢复到了假水平。(B) 单姿态的时间过程 (%)。在计算左、右后爪之间单姿态的百分比变化 (例如, 50% 表示左侧和右侧相同的单姿态) 后, 将数据汇总为折线图。* p & #60; 0.05, ** 和 #60; 0.01 和 *** 和 #60; 0.001 与集团。误差线表示平均±.<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56402/56402fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Automated Gait Analysis in Mice with Chronic Constriction Injury at JoVE.com

Automated Gait Analysis in Mice with Chronic Constriction Injury

准确评估神经病理性动物模型的疼痛反应, 对于研究疼痛疾病的病理生理学和开发新的镇痛药是至关重要的。我们提出了一个敏感和客观的方法, 以确定的感官功能的啮齿动物后爪的自动步态分析系统。

慢性压迫性损伤小鼠步态的自动分析

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some ...

automated-gait-analysis-in-mice-with-chronic-constriction-injury

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

10.0K 次观看.  Chungnam National University. 准确评估神经病理性动物模型的疼痛反应, 对于研究疼痛疾病的病理生理学和开发新的镇痛药是至关重要的。我们提出了一个敏感和客观的方法, 以确定的感官功能的啮齿动物后爪的自动步态分析系统。

视频: Automated Gait Analysis in Mice with Chronic Constriction Injury

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3. Specifically, it affects the ability of neurons to receive inputs from other cells2 and contributes to the propagation of action potentials4,5. The morphology of the neurites also affects how information is processed. The diversity of dendrite morphologies facilitate local and long range signaling and allow individual neurons or groups of neurons to carry out specialized functions within the neuronal network6,7. Alterations in dendrite morphology, including fragmentation of dendrites and changes in branching patterns, have been observed in a number of disease states, including Alzheimer's disease8, schizophrenia9,10, and mental retardation11. The ability to both understand the factors that shape dendrite morphologies and to identify changes in dendrite morphologies is essential in the understanding of nervous system function and dysfunction.
Neurite morphology is often analyzed by Sholl analysis and by counting the number of neurites and the number of branch tips. This analysis is generally applied to dendrites, but it can also be applied to axons. Performing this analysis by hand is both time consuming and inevitably introduces variability due to experimenter bias and inconsistency. The Bonfire program is a semi-automated approach to the analysis of dendrite and axon morphology that builds upon available open-source morphological analysis tools. Our program enables the detection of local changes in dendrite and axon branching behaviors by performing Sholl analysis on subregions of the neuritic arbor. For example, Sholl analysis is performed on both the neuron as a whole as well as on each subset of processes (primary, secondary, terminal, root, etc.) Dendrite and axon patterning is influenced by a number of intracellular and extracellular factors, many acting locally. Thus, the resulting arbor morphology is a result of specific processes acting on specific neurites, making it necessary to perform morphological analysis on a smaller scale in order to observe these local variations12.
The Bonfire program requires the use of two open-source analysis tools, the NeuronJ plugin to ImageJ and NeuronStudio. Neurons are traced in ImageJ, and NeuronStudio is used to define the connectivity between neurites. Bonfire contains a number of custom scripts written in MATLAB (MathWorks) that are used to convert the data into the appropriate format for further analysis, check for user errors, and ultimately perform Sholl analysis. Finally, data are exported into Excel for statistical analysis. A flow chart of the Bonfire program is shown in Figure 1.

We have developed a computer program to analyze neuronal morphology. In combination with two existing open source analysis tools, our program performs Sholl analysis and determines the number of neurites, branch points, and neurite tips. The analyses are performed so that local changes in neurite morphology can be observed.

肖尔自动数字化的神经元形态分析，多尺度

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

科研

神经科学

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes 1,2. Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement 3,4. The resulting hematomas and lacerations cause a vascular response 3,5, and the morphological and functional damage of the white matter leads to diffuse axonal injury 6-8. Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure 9. Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals 10-12, which ultimately result in long-term neurological disabilities 13,14. Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair.
Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration 1. The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue 1,15. Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure 16,17. The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull 18. Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury 19. It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation 20.
We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP.

Lateral fluid percussion (LFP), an established model of traumatic brain injury in mice, is demonstrated. LFP fulfills three major criteria for animal models: validity, reliability and clinical relevance. The procedure, consisting of surgical craniotomy, fixation of hub followed by induction of injury, resulting in focal and diffuse injuries, is described.

侧向液压冲击：创伤性脑损伤小鼠模型

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and ...

3D Kinematic Gait Analysis for Preclinical Studies in Rodents

The utility of three-dimensional (3D) kinematic motion analysis systems is limited in rodents. Part of the reason for this inadequacy is the use of complex algorithms and mathematical modeling that accompany 3D data collection and analysis procedures. This work provides a simple, user-friendly, step-by-step detailed methodology for 3D kinematic gait analysis during treadmill locomotion in healthy and neurotraumatic rats using a six-camera motion capture system. Also provided are details on 1) calibration of the system in an experimental set-up customized for quadrupedal locomotion, 2) data collection for treadmill locomotion in adult rats using markers positioned on all four limbs, 3) options available for video tracking and processing, and 4) basic 3D kinematic data generation and visualization and quantification of data using the built-in data collection software. Finally, it is suggested that the utility of this motion capture system be expanded to studying a variety of motor behaviors before and after neurotrauma.

Presented here is a protocol to collect and analyze three-dimensional kinematics of quadrupedal locomotion in rodents for preclinical studies.

3D运动性步态分析,用于啮齿动物的临床前研究

The utility of three-dimensional (3D) kinematic motion analysis systems is limited in rodents. Part of the reason for this inadequacy is the use of ...

行为学

Gait Analysis of Age-dependent Motor Impairments in Mice with Neurodegeneration

Motor behavior tests are commonly used to determine the functional relevance of a rodent model and to test newly developed treatments in these animals. Specifically, gait analysis allows recapturing disease relevant phenotypes that are observed in human patients, especially in neurodegenerative diseases that affect motor abilities such as Parkinson&#39;s disease (PD), Alzheimer&#39;s disease (AD), amyotrophic lateral sclerosis (ALS), and others. In early studies along this line, the measurement of gait parameters was laborious and depended on factors that were hard to control (e.g., running speed, continuous running). The development of ventral plane imaging (VPI) systems made it feasible to perform gait analysis at a large scale, making this method a useful tool for the assessment of motor behavior in rodents. Here, we present an in-depth protocol of how to use kinematic gait analysis to examine the age-dependent progression of motor deficits in mouse models of neurodegeneration; mouse lines with decreased levels of endophilin, in which neurodegenerative damage progressively increases with age, are used as an example.

In this study, we demonstrate the use of kinematic gait analysis based on ventral plane imaging to monitor the subtle changes in motor coordination as well as the progression of neurodegeneration with advancing age in mouse models (e.g., endophilin mutant mouse lines).

变性小鼠年龄依赖性运动障碍的步态分析

Motor behavior tests are commonly used to determine the functional relevance of a rodent model and to test newly developed treatments in these animals. ...

发育生物学

Low-Cost Gait Analysis for Behavioral Phenotyping of Mouse Models of Neuromuscular Disease

Measurement of animal locomotion is a common behavioral tool used to describe the phenotype of a given disease, injury, or drug model. The low-cost method of gait analysis demonstrated here is a simple but effective measure of gait abnormalities in murine models. Footprints are analyzed by painting a mouse&#8217;s feet with non-toxic washable paint and allowing the subject to walk through a tunnel on a sheet of paper. The design of the testing tunnel takes advantage of natural mouse behavior and their affinity for small dark places. The stride length, stride width, and toe spread of each mouse is easily measured using a ruler and a pencil. This is a well-established and reliable method, and it generates several metrics that are analogous to digital systems. This approach is sensitive enough to detect changes in stride early in phenotype presentation, and due to its non-invasive approach, it allows for testing of groups across life-span or phenotypic presentation.

Footprint analysis is a low-cost alternative to digitized gait analysis programs for researchers quantifying movement abnormalities in mice. Because of its speed, simplicity, and longitudinal potential, it is ideal for behavioral phenotyping of mouse models.

神经肌肉疾病小鼠模型行为花型的低成本盖特分析

Measurement of animal locomotion is a common behavioral tool used to describe the phenotype of a given disease, injury, or drug model. The low-cost method ...

Sagittal Plane Kinematic Gait Analysis in C57BL/6 Mice Subjected to MOG35-55 Induced Experimental Autoimmune Encephalomyelitis

Kinematic gait analysis in the sagittal plane has frequently been used to characterize motor deficits in multiple sclerosis (MS). We describe the application of these techniques to identify gait deficits in a mouse model of MS, known as experimental autoimmune encephalomyelitis (EAE). Paralysis and motor deficits in mice subjected to EAE are typically assessed using a clinical scoring scale. However, this scale yields only ordinal data that provides little information about the precise nature of the motor deficits. EAE disease severity has also been assessed by rotarod performance, which provides a measure of general motor coordination. By contrast, kinematic gait analysis of the hind limb in the sagittal plane generates highly precise information about how movement is impaired. To perform this procedure, reflective markers are placed on a hind limb to detect joint movement while a mouse is walking on a treadmill. Motion analysis software is used to measure movement of the markers during walking. Kinematic gait parameters are then derived from the resultant data. We show how these gait parameters can be used to quantify impaired movements of the hip, knee, and ankle joints in EAE. These techniques may be used to better understand disease mechanisms and identify potential treatments for MS and other neurodegenerative disorders that impair mobility.

Kinematic gait analysis in the sagittal plane yields highly precise information about how movement is executed. We describe the application of these techniques to identify gait deficits for mice subjected to autoimmune-mediated demyelination. These methods may also be used to characterize gait deficits for other mouse models featuring impaired locomotion.

MOG35-55 诱发自身免疫性脑脊髓炎 C57BL/6 小鼠矢状面运动步态分析

Kinematic gait analysis in the sagittal plane has frequently been used to characterize motor deficits in multiple sclerosis (MS). We describe the ...

Automated Gait Analysis to Assess Functional Recovery in Rodents with Peripheral Nerve or Spinal Cord Contusion Injury

Peripheral and central nerve injuries are mostly studied in rodents, especially rats, given the fact that these animal models are both cost-effective and a lot of comparative data has been published in the literature. This includes a multitude of assessment methods to study functional recovery following nerve injury and repair. Besides evaluation of nerve regeneration by means of histology, electrophysiology, and other in vivo and in vitro assessment techniques, functional recovery is the most important criterion to determine the degree of neural regeneration. Automated gait analysis allows recording of a vast quantity of gait-related parameters such as Paw Print Area and Paw Swing Speed as well as measures of inter-limb coordination. Additionally, the method provides digital data of the rats&#8217; paws after neuronal damage and during nerve regeneration, adding to our understanding of how peripheral and central nervous injuries affect their locomotor behavior. Besides the predominantly used sciatic nerve injury model, other models of peripheral nerve injury such as the femoral nerve can be studied by means of this method. In addition to injuries of the peripheral nervous systems, lesions of the central nervous system, e.g., spinal cord contusion can be evaluated. Valid and reproducible data assessment is strongly dependent on meticulous adjustment of the hard- and software settings prior to data acquisition. Additionally, proper training of the experimental animals is of crucial importance. This work aims to illustrate the use of computerized automated gait analysis to assess functional recovery in different animal models of peripheral nerve injury as well as spinal cord contusion injury. It also emphasizes the method&#8217;s limitations, e.g., evaluation of nerve regeneration in rats with sciatic nerve neurotmesis due to limited functional recovery. Therefore, this protocol is thought to help researchers interested in peripheral and central nervous injuries to assess functional recovery in rodent models.

Automated gait analysis is a feasible tool to evaluate functional recovery in rodent models of peripheral nerve injury and spinal cord contusion injury. While it requires only one setup to assess locomotor function in various experimental models, meticulous hard- and soft-ware adjustment and training of the animals is highly important.

自动步态分析，评估带外周神经或脊髓挫伤的啮齿动物的功能恢复

Peripheral and central nerve injuries are mostly studied in rodents, especially rats, given the fact that these animal models are both cost-effective and ...

Multifactorial Assessment of Motor Behavior in Rats after Unilateral Sciatic Nerve Crush Injury

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In addition, in the research field of movement disorders, sciatic crush injury has been employed to trigger a dystonia-like phenotype in genetically predisposed DYT-TOR1A rodent models of dystonia. To achieve consistent, reproducible and comparable results after a sciatic nerve crush injury, a standardized method for inducing the nerve crush is essential, in addition to a standardized phenotypical characterization. Attention must be paid not only to the specific assortment of behavioral tests, but also to the technical requirements, the correct execution and consecutive data analysis. This protocol describes in detail how to perform a sciatic nerve crush injury and provides a behavioral test battery for the assessment of motor deficits in rats that includes the open field test, the CatWalk XT gait analysis, the beam walking task, and the ladder rung walking task.

We provide a protocol for the assessment of motor behavior via a behavioral test battery in rats after sciatic nerve crush injury.

单边坐骨神经粉碎损伤后大鼠运动行为的多因素评估

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In ...

Comprehensive Understanding of Inactivity-Induced Gait Alteration in Rodents

It is well known that disuse affects neural systems and that joint motions become altered; however, which outcomes properly exhibit these characteristics is still unclear. The present study describes a motion analysis approach that utilizes three-dimensional (3D) reconstruction from video captures. Using this technology, disuse-evoked alterations of walking performances were observed in rodents exposed to a simulated microgravity environment by unloading their hindlimb by their tail. After 2 weeks of unloading, the rats walked on a treadmill, and their gait motions were captured with four charge-coupled device (CCD) cameras. 3D motion profiles were reconstructed and compared to those of control subjects using the image processing software. The reconstructed outcome measures successfully portrayed distinct aspects of distorted gait motion: hyperextension of the knee and ankle joints and higher position of the hip joints during the stance phase. Motion analysis is useful for several reasons. First, it enables quantitative behavioral evaluations instead of subjective observations (e.g., pass/fail in certain tasks). Second, multiple parameters can be extracted to fit specific needs once the fundamental datasets are obtained. Despite hurdles for broader application, the disadvantages of this method, including labor intensity and cost, may be alleviated by determining comprehensive measurements and experimental procedures.

The present protocol describes three-dimensional motion tracking/evaluation to depict gait motion alteration of rats after exposure to a simulated disuse environment.

全面了解啮齿动物不活动引起的步态改变

It is well known that disuse affects neural systems and that joint motions become altered; however, which outcomes properly exhibit these characteristics ...

使用 MouseWalker 量化脊髓损伤小鼠的运动功能障碍

Using the MouseWalker to Quantify Locomotor Dysfunction in a Mouse Model of Spinal Cord Injury

The execution of complex and highly coordinated motor programs, such as walking and running, is dependent on the rhythmic activation of spinal and supra-spinal circuits. After a thoracic spinal cord injury, communication with upstream circuits is impaired. This, in turn, leads to a loss of coordination, with limited recovery potential. Hence, to better evaluate the degree of recovery after the administration of drugs or therapies, there is a necessity for new, more detailed, and accurate tools to quantify gait, limb coordination, and other fine aspects of locomotor behavior in animal models of spinal cord injury. Several assays have been developed over the years to quantitatively assess free-walking behavior in rodents; however, they usually lack direct measurements related to stepping gait strategies, footprint patterns, and coordination. To address these shortcomings, an updated version of the MouseWalker, which combines a frustrated total internal reflection (fTIR) walkway with tracking and quantification software, is provided. This open-source system has been adapted to extract several graphical outputs and kinematic parameters, and a set of post-quantification tools can be to analyze the output data provided. This manuscript also demonstrates how this method, allied with already established behavioral tests, quantitatively describes locomotor deficits following spinal cord injury.

作者聚焦:使用 MouseWalker 量化脊髓损伤小鼠模型中的运动功能障碍  

An experimental pipeline to quantitatively describe the locomotor pattern of freely walking mice using the MouseWalker (MW) toolbox is provided, ranging from initial video recordings and tracking to post-quantification analysis. A spinal cord contusion injury model in mice is employed to demonstrate the usefulness of the MW system.