We would like to thank everyone at Shriners Hospital Pediatric Research Center, in particular Dr. Mickey Seltzer, of whom without his support, this work would not have been funded. In addition, we would like to thank Isha Srivastava, who contributed to early data collection and Amy He, who helped with the figures. This study was funded by Shriners Hospitals for Children. No funding source played a role in experimental design or decision to submit the paper for publication.

注:所有动物手术均按照坦普尔大学的ULAR部门和IACUC政策和程序进行。 C57BL / 6水坝和公牛从Charles River实验室购买并饲养在笼子里饲养了12小时的光/暗周期（灯亮7:00 - 晚上7:00），免费获得食物和水。对繁殖产生5之间产羔 - 10幼崽。 1.脑瘫手术诱导<ol><li>注:脑瘫使用产后天（PND）6小鼠的幼崽引起的，如前面所述8,9（https://www-jove-com.remotexs.ntu.edu.sg/video/1951/mouse-models-of-periventricular-leukomalacia）。 </li><li>放置在小狗在冰玻璃碗里用实验室擦拭，以保护小狗的皮肤。步行捏和缺乏运动检查适当的麻醉平面。移动小狗到填充冰袋进行手术。 </li><li>消毒用70％的乙醇的小狗的皮肤上。干燥时，可使用11＃无菌手术BLA德和使在颈部1厘米的切口。 </li><li>使用立体手术显微镜，隔离用小钩的右颈总动脉，并用便携式手持cauterizer烧灼。目视确认该动脉闭塞。假手术包括可视化和颈总动脉的隔离而不烧灼。 </li><li>重新调整皮肤和使用紧密缝合胶（正氰基丙烯酸正丁酯）。 </li><li>放置在34℃下加热垫的小狗30分钟监视自主呼吸和正常运行。 </li><li>返回小狗（S）大坝30分钟。 </li><li>放置幼仔在加热垫或其他加热装置低氧室35分钟设定在6％的氧气内部设定在34℃下。氧被氮置换。密切监察一致的伤害结果室内氧气含量和温度。 </li><li>从缺氧室中取出幼崽，并将其返回加热垫。 </li><li>腹腔注射脂多糖糖在无菌盐水中以1微克/千克稀释，小狗返回大坝。深水注射是唯一的盐水注射。 </li></ol> 2.新生儿电机测试注:在PND 8，48小时CP诱导后，小鼠的幼崽是为神经行为发育测试。幼仔一个4小时块内中午前，以消除行为的差异一天时间的考验。幼仔从坝为不超过15分钟，在一个时间，以防止体热和饥饿/分离问题迅速丧失除去。此外，幼崽被允许测试之间的休息，这样最大的努力将在每个测试被引出。新生儿马达试验的基础是一种适于使用的测试10,11-福克斯的电池和福克斯的测试12 Wahlsten的适应性，以及款待-NMD和其他行为的出版物（如文中对于每个测试说明）。测试福克斯的电池适用于PND 2 - 21福克斯的测试，面糊Ÿ在座的包括:翻正反射，抓握反射，负趋地（称为狐狸的电池竖屏测试）和四个肢体握力（从福克斯和Wahlsten的屏幕爬坡测试修改）。在这里，步行，前下肢肌力，及后肢力量也被测试来区分虚假和CP的乳鼠之间的反身性的运动行为。为了消除在测试由于学习改进，试验仅限于指出最多3试验。所有其他测试过每个动物只有一个审判。 <ol><li> 行走 （ 图1）（改编自大鼠协议13）: 注:抓取是在PND 0之间的鼠标小狗早期开发的行为- 5，在该点的小鼠开始过渡到步行，从5 - - 10日龄14。在PND 8，下地测试需要这个过渡时间过程的优势。下地可以，但是，在整个鼠标的寿命得分，可以在任何年龄来确定。至于存在用于学习没有潜在的行走试验可作为通过实验的过程中根据需要重复多次。 <ol><li>在透明外壳地方小鼠，其中小鼠是可见从顶部以及侧。通过触摸小狗的尾巴用轻微的针刺激励小狗走路。 </li><li>分数下地使用下列标准3分:0 =无动作，1 =非对称肢体运动爬行，2 =缓慢爬行，但对称的肢体运动，而3 =快速爬行/行走。 注意:在此，对称肢体运动被描述，其中后爪在每个步骤中满足frontpaws，并且每个步骤顺利过渡到下一步骤。鼠标显示不对称肢体运动具有不稳定的爪子安置和过渡从一步到下的并不顺利。 </li></ol></li></ol><img alt="图1" src="/files/ftp_upload/53569/53569fig1.jpg" /> 图1.从转变爬行到散步可以通过观察后爪，以及在头部和尾部进行区分:（A）在爬行，整个背部爪，从脚趾到脚跟，走动时接触地面，并标示为（*）。一个成年人的步行模式观察时只的脚趾和前部的后爪接触地面（脚跟升高时，由[**] deonoted）。（B）中的头部和一爬行小鼠的尾部低到地面。头开始从爬行到行走在过渡期间上升。过渡完成后，无论是头部和尾部升高，只有后肢的前部接触地面。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53569/53569fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="2"><li> 后肢足角 （ 图2） 注:在后肢姿势作为谅解备忘录的明显变化发展E从爬行到行走，其中该后肢被身下行走和后肢之间的角度小于在爬行看到的角度定位时的成熟。即使后肢足角随时间变化，同样的年龄不同伤害或疾病的小鼠的幼崽可以比拟的。类似于步行测试（3.1），对于学习的可能。因此，如通过实验的过程中所需要的后肢脚角度测试可以重复多次。 <ol><li>无论是在明确公开字段框或封闭区域，从下方或上方，分别安装摄像机，记录小狗，因为它绕着字段。通过触摸小狗的尾巴用轻微的针刺激励小狗走路。记录两分钟。 </li><li>使用录像，通过从脚后跟/胫最长（中）脚尖的端画线测量幼崽的脚角度。只有当小狗正在执行一个完整的步幅进行测量在一条直线上并且两个脚平放在地面上。而小狗是固定的，不要进行测量或在小狗正在转向。 </li><li>测量三个五套脚的角度，计算所测试的各小狗平均角度。 </li></ol></li></ol><img alt="图2" src="/files/ftp_upload/53569/53569fig2.jpg" /> 图2.后肢足角度可用于确定步态异常，脚的角度可以通过中间的（最长）数字绘画从脚跟中间线来衡量。相比普通（见代表性的成果，脚角度）时，受伤的动物有更大的脚的角度。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53569/53569fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="3"><li> 面翻正 （ 图3）: 注:翻正反射是运动能力<html>鼠标小狗，以便能够从仰卧位置翻转到它的脚。为正位反射出现在啮齿动物的平均年龄是PND 5从PND 1的范围内- 10 15本试验是一种反射，没有学习组件，它可以在整个实验期间进行重复。 <ol><li>放置在棉片或板凳垫在背上的幼崽，并保持在适当位置，持续5秒。 </li><li>释放幼仔并记录所花费的小狗返回到俯卧位置的时间，以及扶正的方向（左或右）。共1分钟，给出了每个试验中，如果需要的话。 </li><li>重复一共有三个试验。 </li></ol></li></ol><img alt="图3" src="/files/ftp_upload/53569/53569fig3.jpg" /> 图3.表面扶正。这个测试需要躯干控制，并可能测试姿势不平衡。人类CP患者可能有自己的核心赤字。e.com/files/ftp_upload/53569/53569fig3large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <ol start="4"><li> 负趋地性 （ 图4） 注:平均年龄为负趋地反射出现在啮齿动物是PND 7与PND 3的范围- 15 15负趋地试验评估了年轻小鼠运动协调。小鼠置于朝下的斜面，并且由于重力的前庭线索，幼仔转向面对的斜坡。应对刺激，或出租车，是一种与生俱来的行为。 <ol><li>将小狗与它的头在45 度斜面朝下，并保持5秒。 </li><li>松开小狗并记录小狗转向朝上的时间和方向。总测试时间为2分钟。 </li><li>重复一共有三个试验。那倒下的倾斜或无法开机的小鼠可以是重新测试，淘汰，或给予零分。 注:这一决定为lEFT考官，因为偶尔会幼仔斜面滚下因嗜睡而不是弱点。一旦决定如何得分的倒下倾斜幼仔提出，应该在方法要注意，应该在所有科目的测试是一致的。 </li></ol></li></ol><img alt="图4" src="/files/ftp_upload/53569/53569fig4.jpg" /> 图4.负趋地，电机和前庭输入所需的鼠标来识别一个斜坡的方向和转身。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53569/53569fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="5"><li> 前悬挂肢 （ 图5）16;改编自17，18: 注:前悬挂肢体幼仔测试，包括手臂和爪强度的前肢力量。 ŧ不推荐他的测试比幼仔PND 10 15岁。幼仔把握不准跨越一个稳定的对象串成一个电线挂到与两个前爪电线。测试面积超过软垫放置区。该测试可以检测左/右侧的实力差异。学习和没有负筋可导致增加的非参与。小鼠释放或当放置在电线未能把握时立即降指示不参与。 <ol><li>人体握紧幼仔，使他们与两个前爪抓住电线。 </li><li>松开小狗。使用定时器或秒表，记录的总时间下跌，以及爪子的弱点。 注意:如果小狗始终从一只爪子电线落在之前，其他而不是在同一时间两个爪子拉线释放爪子弱点是确定的。 </li><li>重复试验，共三次。 </li></ol></li></ol> 图5.前肢悬挂。这悬浮试验引起前肢紧张，直到肌肉疲劳。通过这种方法，在前肢基线强度成立。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53569/53569fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="6"><li> 后肢悬架 （ 图6）: 注:本悬架测试确定后肢力量。它是专为新生儿设计了一个试验，最初PND 2之间使用动物- 12 19,20，但可以适用于小鼠高达PND 14这个测试可检测左/右后肢强度差异以及神经肌肉功能。标准的50毫升锥形被使用，实验室抹布填充。类似于前肢悬浮试验，该试验中可以得知，尤其罪CE没有负面后果下降。因此，增加了非参与，一旦落小鼠为视为释放或不留时放置在管的边缘，可以注意到。 <ol><li>用50毫升的锥形，地点小狗轻轻面朝下放入管后腿挂在篮筐。 </li><li>松开小狗。观察后肢的姿势。 </li><li>按照下列标准得分的姿势。 注:4分表示尾部提出后肢正常分离; 3得分手段的弱点是明显，后肢更接近在一起，但他们很少互相接触; 2分表示后肢接近，彼此经常接触; 1比分显示的弱点是明显，后肢几乎总是与提出的尾部紧握位置; 0分表示与降低尾部或未能守住管的任何一段时间的后肢不断扣紧。 </li><li>如有必要，伯爵拉。一拉就是资格赛当指明分数小狗尝试使用它的后肢提起它的身体，而在锥形管的一侧暂停。 </li><li>使用定时器或秒表，记录潜伏期下降。 </li><li>重复一式三份整个测试。 </li></ol></li></ol><img alt="图6" src="/files/ftp_upload/53569/53569fig6.jpg" /> 图6.后肢暂停。（A）。此悬浮试验导致后肢紧张，直至肌肉疲劳。在后肢基线强度和姿势的建立。（B）。计分。注意代表的小鼠表现出可能的姿势评分上面的数字。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53569/53569fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="7"><li> 握力 （ 图7）: 注意:此测试将检查爪子小号同时四只爪子的trength。一个16×18的屏幕玻璃纤维丝被使用。对啮齿动物能够掌握一个横屏的平均年龄是PND 8的范围内的PND 5 - 15 15 福克斯使用的四种肢横屏试验从PND 2 - 21 10本试验是从标准的水平改性屏幕测试;这里的画面从水平到垂直位置，缓慢转动，以挑战所有四肢21的把持;改编自尔蒂第16如果当反转180°鼠标保持到网筛，记录延迟下降。另外，还要注意体重。一辆挂冲动可以计算为[重量（g）×潜伏期下降（SEC）反映抗拒地心引力所需要的力量。 <ol><li>使用一块金属丝网，将小狗在屏幕上。让小狗适应这样的环境，时间约为5秒。 </li><li>缓慢倒置屏幕180度。记录的大致ANGL屏幕的E当小狗脱落。 </li><li>重复一共有三个试验和平均的审判。 </li></ol></li></ol><img alt="图7" src="/files/ftp_upload/53569/53569fig7.jpg" /> 图7.握力。小鼠需要维持四肢的引力增大肌肉紧张。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53569/53569fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="8"><li> 把持反射 （ 图8） 注:抓握反射通常在PND 7显示在啮齿动物中与来自PND 3的范围- 15 15每个爪被单独测试，从而测试可以揭示前置式或后肢的问题，以及片面性的问题。由于它是一种反射，这种测试可以重复，直到出现反射。这是不容易学习。作为一个重要的警告，该测试<html>不区分抓实力，只有能力，而且必须在幼年小鼠开始掌握因害怕应对15日龄前进行测试。 <ol><li>它的脖子颈背，类似于鼠标小狗被大坝携带的方式握住鼠标。这使得持有小狗成为本能地不动，宽松，允许便于测试。 </li><li>中风与直率的小狗爪子每一个，圆刀片的一侧。 </li><li>单独测试每个爪子抓记录的存在或不存在和得分每个爪子1点与鼠标抓手。 注:右爪偏好的评分是右爪偏爱100％， - - -100％左爪子偏好，两个爪子50％的把握，而对于没有爪子抓0％。以确定这些数字的公式为[（右爪 - 左爪）/（右爪+左爪子+两者爪子）]×100％。 </li></ol></li></ol>/53569/53569fig8.jpg"/&gt; 图8.抓握反射。由于新生小鼠没有强烈的恐惧反应，本次测试严格确定跖/手掌反射。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53569/53569fig8large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="9"><li> 崖厌恶 （ 图9）: 注:崖厌恶测试迷宫反射，以及强度和协调，并可以用来从PND 1-14 22测试幼仔。预香味中（其中至少5只小鼠的已被允许一个框来自由漫游）用平升高的壁架的使用量和小狗被置于与数字只前爪和定位在边缘的口鼻部。得分是通过计算花费的小狗把从悬崖路程，移动它的爪子和鼻子远离边缘的总时间进行。如果没有响应30秒钟后看到的那样，试验被终止。如果小狗脱落的边缘，可以执行单个附加试验。 <ol><li>使用的侧视图，放置在预香味框的边缘的小狗，确保前脚掌，数字和吻是在边缘的唯一零件。 </li><li>放开小狗，并开始计时。 </li><li>一旦两个鼻子和爪子已经从边缘去除，停止计时和记录时间。 </li><li>重复试验，共进行3次。如果小狗没有在30秒内摆脱悬崖而去，没有得分给出。 注:小狗是一个非参与者对受损的是留给考官的自由裁量权的决心。悬崖的高度可以为年龄小狗，以保证小狗的安全性进行调整。一个较小的高度可以与黑"地板"用于模拟一个更高的高度。 </li></ol></li></ol><img alt="图9" src="/files/ftp_upload/53569/53569fig9.jpg" /> FIGURË9.悬崖厌恶。前庭失衡正在使用的悬崖厌恶测试测量。在这里，小狗的眼睛仍然关闭，怕是没把从悬崖边走的驱动因素。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53569/53569fig9large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 3.统计学意义<ol><li>使用统计软件分析结果。表示该数据为平均值（SEM）的平均值±标准误差。测试参数，因此，检查用t检验分析数据。 注:实验的目的不是测试性别差异。差异被认为是当p &lt;0.05统计学显著。 </li></ol>

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Development+of+Social+Behavior+Patterns+in+the+Mouse,+in+Relation+to+Natural+Periods.">The Development of Social Behavior Patterns in the Mouse, in Relation to Natural Periods.</a> Behaviour. 6 (1), 35-65 (1954).</li><li>Heyser, C. J., Crawley, J. Q., 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=Assessment+of+developmental+milestones+in+rodents.">Assessment of developmental milestones in rodents.</a> Current protocols in neuroscience. Chapter 8, (2004).</li><li>Corti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Grip strength TREAT-NMD: Experimental protocols for SMA animal models. , (2014).</li><li>Corti, S., Nizzardo, M., 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=Neural+stem+cell+transplantation+can+ameliorate+the+phenotype+of+a+mouse+model+of+spinal+muscular+atrophy.">Neural stem cell transplantation can ameliorate the phenotype of a mouse model of spinal muscular atrophy.</a> J Clin Invest. 118 (10), 3316-3330 (2008).</li><li>Grondard, C., Biondi, O., 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=Regular+exercise+prolongs+survival+in+a+type+2+spinal+muscular+atrophy+model+mouse.">Regular exercise prolongs survival in a type 2 spinal muscular atrophy model mouse.</a> J Neurosci. 25 (33), 7615-7622 (2005).</li><li>El-Khodor, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+Phenotyping+for+Neonates.">Behavioral Phenotyping for Neonates.</a> Experimental Protocols for SMA animal models. , (2011).</li><li>El-Khodor, B. F., Edgar, N., 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=Identification+of+a+battery+of+tests+for+drug+candidate+evaluation+in+the+SMNDelta7+neonate+model+of+spinal+muscular+atrophy.">Identification of a battery of tests for drug candidate evaluation in the SMNDelta7 neonate model of spinal muscular atrophy.</a> Exp Neurol. 212 (1), 29-43 (2008).</li><li>Venerosi, A., Ricceri, L., Scattoni, M. L., Calamandrei, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prenatal+chlorpyrifos+exposure+alters+motor+behavior+and+ultrasonic+vocalization+in+CD-1+mouse+pups.">Prenatal chlorpyrifos exposure alters motor behavior and ultrasonic vocalization in CD-1 mouse pups.</a> Environ Health. 8 (12), (2009).</li><li>Hill, J. M., Lim, M. A., Stone, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+milestones+in+the+newborn+mouse.">Developmental milestones in the newborn mouse.</a> Neuromethods 39: Neuropeptide Techniques. , (2008).</li><li>Yu, Y., Keshner, A. E., Tucker, C. A., Thompson, E. D., Lauer, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+dependence+influences+postural+responses+to+continuous+visual+perturbation+in+adults+with+spastic+cerebral+palsy.">Visual dependence influences postural responses to continuous visual perturbation in adults with spastic cerebral palsy.</a> , (2016).</li><li>Carlson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+four+limb+hanging+tests+to+monitor+muscle+strength+and+condition+over+time.">The use of four limb hanging tests to monitor muscle strength and condition over time.</a> Experimental Protocols for SMA animal models. , (2011).</li><li>Gordon, A. M., Duff, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relation+between+clinical+measures+and+fine+manipulative+control+in+children+with+hemiplegic+cerebral+palsy.">Relation between clinical measures and fine manipulative control in children with hemiplegic cerebral palsy.</a> Dev Med Child Neurol. 41 (9), 586-591 (1999).</li><li>Futagi, Y., Toribe, Y., Suzuki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+grasp+reflex+and+moro+reflex+in+infants:+hierarchy+of+primitive+reflex+responses.">The grasp reflex and moro reflex in infants: hierarchy of primitive reflex responses.</a> Int J Pediat. , (2012).</li></ol>

The authors declare that they have no competing financial interests.

使用动物模型来研究人类疾病是否存在人类和啮齿动物之间，并且所执行的行为测试有直接关系到人类的症状的细胞和分子响应之间的重叠是唯一相关。之一的儿科疾病的研究的主要问题是，许多研究者使用成年啮齿动物创建模型，以及成年啮齿动物行为的评价，在不考虑发育差异可能对疾病过程重要。由于这些问题，重要的是在儿科疾病使用研究不仅适当调整的发育时间点（ 例如，人的中枢神经系统发育，在28 - 51周内，相当于一个产后第2天-第7天的啮齿动物）7，而且行为测试，将审查适当的运动，感觉或反射性发育行为。因此，因为每个新的新生儿疾病模型开发，它必须经过严格的测试，以确保蜂窝和行为反应将提供啮齿类动物和人类之间最合适的翻译资料。 脑瘫是一种运动障碍，它持续到成年。许多当今脑瘫模型的一个问题是缺乏可重复的，标准化的电机测试，可以在儿科患者看到的赤字相关的。在这种新的模式，它结合缺氧，缺血和炎症的新生小鼠，运动行为，用具体的新生小鼠试验电池进行评估。为了减小主体和增加定量报告，几个测试已被修改，以包括非常具体的，但容易评估，可以标准化的措施。此外，前端和后肢评价可以单独执行，并且左/右差异可被确定。测试该电池是特异于新生小鼠最多两个周龄。 这种CP模型演示行走困难（行走，后肢足角），以及肢体特异性无力（4肢悬浮，后肢悬挂），和赤字发育反射（抓握反射）。尽管在本研究中检测只有一个时间点，这些缺陷可以随时间被跟踪。 有测试其他电池可以在新生儿中使用，如测试的福克斯的电池或发育里程碑15的Heyser的评价。然而，这些测试的新生儿进行比较的成人，其响应可能是不一样的，因为新生儿仍在发展。福克斯的电池和Heyser的Assement测试依赖于观测主观信息用二分（是或否）的评估，而不是客观的数据（角度，姿势的基础上强度等 ）。由于这些测试的主观性，许多科学家已经适应，添加或删除的标准，从而使他们的结果无比给他人，并限制ING在对于特定疾病或病症建立基线赤字方面的数据的有用性。通过建立一组标准化电机测试是定性和专为测试新生儿，从单个研究小组结果能够精确和可靠地报告和比较。

开发儿童受伤或利用啮齿类动物疾病的新车型往往是困难的，因为大鼠和小鼠的惊人的能力，迅速从神经损伤恢复。因此，为了验证任何新的儿科疾病模型，深入研究细胞和分子的变化必须去手牵手与行为的结果。在许多方面，功能行为恢复可能比在治疗或翻译相关性方面潜在的细胞的变化更为重要。随着研究人员更多地了解在成人和新生儿损伤，很显然，他们的反应是非常不同的，两者不能外推。例如，新生小鼠显示不同水平的神经生长因子，脑源性神经营养因子，神经营养因子-3和脊髓损伤后1,2胶质细胞源性神经营养因子。此外，有新生儿中风后3，D显著血脑屏障泄漏emonstrate皮层神经元重排周围神经损伤后4，并有脊髓损伤和缺血缺氧5,6延迟或减缓星形胶质细胞增生。因此，平移儿科研究使用发育等效模型和这些模型是为两个蜂窝/分子变化和年龄适当行为测试评价是很重要的。 脑瘫（CP）是一种运动障碍，影响3:1000名活产儿每年（NIH）。带CP的儿童显示出一系列的症状和共病的条件下，根据疾病的严重程度。难度与运动和协调是最常见的迹象，在达到马达发育标志延迟沿。其他症状包括肌张力异常（增加或减少），降低的精细动作技能，行走困难，流口水过多和吞咽，和语音延迟（NIH）。 CP的根本原因被认为是在芽前或围产期期间，或最多一年产后缺乏氧和/或血液流向脑部的。此外，炎症现在认为是在CP的发展的重要组成部分。 大多数CP案件与周围的脑室，被称为脑室周围白质软化症（PVL）脑白质损伤有关。这种神经标志表明，最初的侮辱导致CP期间大脑发育的时期，少突胶质细胞最容易受到侮辱发生。快速少突胶质细胞的生长在一个人的期间，也期间时少突胶质细胞是最容易受到伤害，是24之间 - 周32妊娠。在啮齿动物，等效周期是产后天7月2日至7日，并且是当CP在这个模型中被诱导。 这是用于进行此处所述的测试CP的新生小鼠模型结合缺氧和局部缺血与炎症创建一个注射URY，更好地模仿神经退行性疾病在人类CP看到。该模型解决一些在CP的其他动物模型中观察到的大的缺点，缺乏鲜明运动缺陷类似于人类CP患者，以及不同的白质损伤。通过使用相同的模型协作者以前的研究已经表明，在加入炎症增强脑白质损伤，从而更好地模仿在儿童看到具有CP 8的PVL。在以前的数据的基础上，提出了以评估在运动行为变化动物年龄的新生儿电机测试的综合电池。

<table border="0" cellpadding="0" cellspacing="0" width="1354">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:271px;">C57BL/6 mice</td>
			<td style="width:239px;">Charles River Laboratories</td>
			<td style="width:103px;">STRAIN CODE: 027&#160;</td>
			<td style="width:741px;">C57BL/6NCrl is the exact strain we use</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:271px;">Anesthesia Dish, PYREX&#8482; Crystallizing Dish</td>
			<td style="width:239px;">Corning Life Sciences Glass&#160;</td>
			<td style="width:103px;">3140125</td>
			<td style="width:741px;">Capacity: 25.03 oz. (740mL); Dia. x H: 4.92 x 2.55 in. (125 x 65mm). However, any small round glass container will work. A 2 cup capacity pyrex food storage bowl with flat bottom will also work and is much cheaper (Pyrex model number: 6017399).</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:271px;">Covered lead ring</td>
			<td style="width:239px;">Fisher Scientific</td>
			<td style="width:103px;">S90139C</td>
			<td style="width:741px;">Lead ring for stablizing flasks in a water bath. It is used inside the anesthesia dish.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:271px;">Scalpel Blade #11</td>
			<td style="width:239px;">World Precision Instrucments, Inc.</td>
			<td style="width:103px;">500240</td>
			<td style="width:741px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:271px;">Small Vessel Cauterizer</td>
			<td style="width:239px;">Fine Science Tools</td>
			<td style="width:103px;">18000-00</td>
			<td style="width:741px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:271px;">Micro Hook</td>
			<td style="width:239px;">Fine Science Tools</td>
			<td style="width:103px;">10064-14</td>
			<td style="width:741px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:271px;">Vetbond Suture Glue</td>
			<td style="width:239px;">3M</td>
			<td style="width:103px;">1469SB</td>
			<td style="width:741px;">n-butyl cyanoacrylate adhesive</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:271px;">Lipopolysaccharide</td>
			<td style="width:239px;">Sigma Life Science</td>
			<td style="width:103px;">L4391</td>
			<td style="width:741px;">Lipopolysaccaride from e.coli 0111:B4, gamma irradiated</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:271px;">12x12 inch opaque box</td>
			<td style="width:239px;">Acrylic Display Manufacturing: A division of Piasa Plastics</td>
			<td style="width:103px;">C4022</td>
			<td>Colored Acrylic 5-Sided Cube, 3/16&#34; Colored Acrylic, 12&#34;W x 12&#34;D x 12&#34;H;&#160; http://www.acrylicdisplaymfg.com/html/cubes_19.html</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:271px;">Camera/camcorder</td>
			<td style="width:239px;">JVC</td>
			<td style="width:103px;">GC-PX100BUS</td>
			<td style="width:741px;">Any camcorder that works well in low light and can be imported and edited. We use the JVC GC-PX100 Full HD Everio Camcorder.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:271px;">Covidien Tendersorb&#8482; Underpads</td>
			<td style="width:239px;">Kendall Healthcare Products Co</td>
			<td style="width:103px;">7174</td>
			<td style="width:741px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:271px;">WypAll L40</td>
			<td style="width:239px;">Kimberly-Clark Professional</td>
			<td style="width:103px;">5600</td>
			<td style="width:741px;">Any surface with moderate grip will do</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:271px;">Surface at 45 degree incline</td>
			<td style="width:239px;"></td>
			<td style="width:103px;"></td>
			<td style="width:741px;">We use a cardboard box.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:271px;">Thin wire from a pipe cleaner</td>
			<td style="width:239px;">Creatology</td>
			<td style="width:103px;">M10314420</td>
			<td style="width:741px;">Any pipe cleaner from any craft store will work.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:271px;">50mL conical tube</td>
			<td style="width:239px;">Falcon</td>
			<td style="width:103px;">352070</td>
			<td style="width:741px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:271px;">Fiberglass Screen Wire</td>
			<td style="width:239px;">New York Wire&#160; www.lowes.com</td>
			<td style="width:103px;">14436</td>
			<td style="width:741px;">Any supplier can be used as long as their screen is 16x16 or 18x16</td>
		</tr>
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			<td height="20" style="height:20px;width:271px;">Razor blade</td>
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			<td style="width:741px;">A wooden stick applicator or wooden part of a cotton-tipped swab will also work.</td>
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			<td style="width:741px;">Clear acrylic (1/8&#34; thick) with sides and a top to limit exploration. We bought a sheet of acrylic from a local hardware store and had them cut it to size. (2) 2&#34;x2&#34;; (3) 2&#34;x 18&#34;; (1) 2&#34;x15.5&#34;; (1) 2&#34;x3&#34;. Using clear tape, tape all sides together, with the 15.5&#34; piece on top. Tape the 3&#34; piece to the end of the 15.5&#34; piece to create a flap/entryway for the mice. Alternatively, part or all of the walkway can be glued together, and only taping on the top pieces. This design will allow for the walkway to be opened for easy cleaning.</td>
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			<td height="20" style="height:20px;width:271px;">Protractor</td>
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</table>

a battery of motor tests in a neonatal mouse model of cerebral palsy

As the sheer number of transgenic mice strains grow and rodent models of pediatric disease increase, there is an expanding need for a comprehensive, standardized battery of neonatal mouse motor tests. These tests can validate injury or disease models, determine treatment efficacy and/or assess motor behaviors in new transgenic strains. This paper presents a series of neonatal motor tests to evaluate general motor function, including ambulation, hindlimb foot angle, surface righting, negative geotaxis, front- and hindlimb suspension, grasping reflex, four limb grip strength and cliff aversion. Mice between the ages of post-natal day 2 to 14 can be used. In addition, these tests can be used for a wide range of neurological and neuromuscular pathologies, including cerebral palsy, hypoxic-ischemic encephalopathy, traumatic brain injury, spinal cord injury, neurodegenerative diseases, and neuromuscular disorders. These tests can also be used to determine the effects of pharmacological agents, as well as other types of therapeutic interventions. In this paper, motor deficits were evaluated in a novel neonatal mouse model of cerebral palsy that combines hypoxia, ischemia and inflammation. Forty-eight hours after injury, five tests out of the nine showed significant motor deficits: ambulation, hindlimb angle, hindlimb suspension, four limb grip strength, and grasping reflex. These tests revealed weakness in the hindlimbs, as well as fine motor skills such as grasping, which are similar to the motor deficits seen in human cerebral palsy patients.

小鼠用不同的小鼠每个时间点从P7（24小时以下手术）到P13（1周手术后），进行测试，以便学习测试范例不是混杂变量。 P8被选为代表性结果，小鼠显示出最大的缺陷在该时间点。 从爬行到步行转型CP新生小鼠被延迟 人类CP患者有步态异常，从脚趾走路到步态剪下。由于这种模式CP步态显示与人类相似的赤字，下床活动进行了评估。小鼠在一条直线走了得分步态对称性和肢体，爪子运动。在48小时以下的手术（PND 8），CP小鼠少对称的肢体运动和"爬行"步态相比，他们的同行假的（平均得分下床活动:CP 1.083±0.6337，N = 12 VS假1.639±0.4859，N = 9; P &lt;0.05， 图10）。一个星期，两个CP和假小鼠已经转变到正（数据未示出）。 <img alt="图10" src="/files/ftp_upload/53569/53569fig10.jpg" /> 图10. CP的乳鼠不走动以及沙姆斯。深水小鼠（黑条）有1.639±0.4859（9例），这意味着它们的动态发展不对称的肢体运动和缓慢爬行介于一个平均分。 CP小鼠（灰色条）收到的1.083±0.6337（N = 12）的平均分，这意味着他们行走欠发达，而且往往有不对称的肢体运动。数据表示为平均值±SEM; *为P &lt;0.05。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53569/53569fig10large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 轩dlimb脚角度以增加CP 除了下地活动，后肢足角度进行了评估。八天的假乳鼠走他们的后爪朝前，相比HIL老鼠，走在一条直线（ 图2时，谁也张开后爪;平均角度:CP 77.48±9.848，N = 9，与对照组分别54.54± 8.043，N = 11; p &lt;0.0001， 图11）。这增加了角步态不稳相关，在幼崽需要增加他们的后爪的角度，以稳定他们的步态和平衡和协调帮助。 <img alt="图11" src="/files/ftp_upload/53569/53569fig11.jpg" /> 图11. CP的乳鼠张开他们的后爪行走时，CP鼠（黑网吧）有77.48±8.043（N = 11），它们的后肢之间的平均角度，而假小鼠（灰色B<html> ARS）有54.54±9.848（平均角N = 9）。数据表示为平均值±SEM; ****为p &lt;0.0001。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53569/53569fig11large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> CP小鼠没有表现出赤字时，表面正位 被列入表面扶正试验一些CP患者受损主干管（Heyrman 等 ，2013）。此外，前庭系统是必要的，以检测用于扶正的必要性，并有在一些CP患者23前庭赤字。相比假控件扶正时的CP小鼠不显示显著缺陷（数据未显示）。 CP小鼠执行相同的深水中负趋地性测试 T"FO:保together.within页="1"&gt;负趋地是用来测试在年轻的幼崽运动协调小鼠被质疑正对倾斜表面面对下坡的地方延迟或未能东方上坡可能表明赤字。在协调性，平衡，或前庭输入。当与相比，假小鼠（数据未示出）的负趋地性攻击的CP小鼠显示无缺陷。此外，CP小鼠没有表现出偏爱转向一侧与另一当重新定向。 前悬挂肢体测试适用于小鼠超过10天以上 CP患者的精细动作技能，如抓肌张力下降和赤字。为了测试在此小鼠模型中的弱点，我们使用了一个前肢悬浮试验。此外，这款机型采用单方面的缺血性损伤和片面的烦躁可能使用这种悬挂试验确定。该测试对于超过10天15老年小鼠更好。在过去8天，损伤后的两天，有CP和假小鼠之间无显著差异（数据未显示）。 后肢力量在CP减少小鼠 人类CP患者常需要大括号或辅助行走装置，由于缺乏电机控制和力量。为了比较的啮齿动物的CP模型对人类，后肢强度用后肢悬浮试验评估。当从锥形管的侧悬挂，CP小鼠显示后肢无力，如通过减少在吊得分证实（后肢悬挂得分:CP 3.468±0.5561，每组13，与假3.891±0.1329，每组13; P &lt; 0.05， 图 12）。在后肢悬挂时间没有观察到差异（数据未显示）。因此，类似于人类CP患者，CP小鼠展示出hindlIMB（腿）的弱点。 <img alt="图12" src="/files/ftp_upload/53569/53569fig12.jpg" /> 图12.深水小鼠轻微，但在他们的后肢比CP小鼠明显强。在平均得分挂的3.891±0.1329（N = 13），假鼠（黑条）表现出更多的后肢分离，因此，更强的姿态后肢，平均挂得分3.468±0.5561（N = 13）挂在管比CP小鼠（灰色条）边缘的时候。数据表示为平均值±SEM; *为P &lt;0.05。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53569/53569fig12large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 握力降低的关注CP损伤 用四只爪子抓是用于在啮齿动物重要攀爬并在不平的地面上运行的条款。握要求显著持续强劲，而不是灵巧或线性力，主要是在数字和爪子24。小鼠必须持有一个倒置的丝网屏幕上自己的体重。 CP小鼠无法维持他们的抓地力和这些小鼠落到了实处显著较低的角度（4肢平均角度:CP 75.627±24.48，N = 11，与对照组分别96.57±10.836，N = 9，P &lt;0.05， 图13）。该数据表明，有在CP小鼠握力一个显著赤字。 <img alt="图13" src="/files/ftp_upload/53569/53569fig13.jpg" /> 图13. CP小鼠比沙姆斯较弱的抓地力。深水小鼠（黑条）可以把握的平均倒角96.57±10.836（N = 9）。 CP小鼠（灰色条）只能达到75.627±24.48（N = 11）倒角度。数据表示为平均值±SEM; *为p0; 0.05。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53569/53569fig13large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 抓握反射赤字在CP表观小鼠 随着粗大运动障碍，精细动作的动作也受损CP患者25,26。在人类的抓握反射是在出生时并消失约5 - 6个月。然而，变化的抓握反射，比如夸张的速度和抓力，未能把握，或6个月的年龄后，抓握反射的再度出现，都表明神经系统的损害。为了比较在CP模式抓，抓反身赤字进行了测定。 在伤后48小时，CP小鼠展示抓握反射减少（平均爪子抓住在48小时:CP 2.429±0.9376，N = 14，与对照组分别3.214±0.8018，N = 14; P &lt;0.05， 图14A）。有在前爪右爪偏好轻微，但不显著增加（数据未显示）。有一个在后爪一个显著右爪偏好（CP 75.0±42.74，N = 14，与对照组分别17.86±54.09，N = 14，P &lt;0.005， 图14B）。伤后一周，CP小鼠显示抓赤字（平均爪子抓住了在第1周:CP 2.75±1.035，N = 8，与对照组分别3.80±0.6325，N = 10，P &lt;0.05， 图14C），没有显着的爪子偏好。 <img alt="图14" src="/files/ftp_upload/53569/53569fig14.jpg" /> 图14. CP小鼠有把握赤字，在后爪，对侧脑损伤区域。 （A）48小时以下的损伤（PND 8），CP小鼠（灰色条）抓住了棍子，平均比假灵魂爪子少LS（黑条）。（B）CP小鼠（灰色条）显示的右后爪（对侧受伤），而不是使用左后肢（同侧伤害）抓的偏好。深水小鼠（黑条）不显示此右爪偏好。右爪子偏好的计算公式为（[右爪子-左爪子] / [右爪子+左爪子+两个爪子] * 100）（C）一周损伤后，CP小鼠（灰色条）仍然显示抓赤字相比。沙姆斯（黑条）。数据表示为平均值±SEM; *为p &lt;0.05，**是P &lt;0.005。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53569/53569fig14large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> CP小鼠转远离边缘在悬崖厌恶 悬崖厌恶测试依赖于小鼠转AWA的内在恐惧y坐标从对安全陡峭的山崖和头部。虽然一些CP患者有前庭的困难，以及受损马达控制，在CP小鼠没有显示在此测试的任何缺陷。

Watch this Scientific Journal Video about A Battery of Motor Tests in a Neonatal Mouse Model of Cerebral Palsy at JoVE.com

A Battery of Motor Tests in a Neonatal Mouse Model of Cerebral Palsy

Presented is a concise battery of mouse neonatal motor tests. Using these tests, neonatal motor deficits can be demonstrated in a variety of neonatal motor disorders. By having a standardized set of tests, results from different studies can be compared, allowing for better and accurate reporting between groups.

汽车测试的脑瘫新生儿小鼠模型电池

As the sheer number of transgenic mice strains grow and rodent models of pediatric disease increase, there is an expanding need for a comprehensive, ...

a-battery-of-motor-tests-in-a-neonatal-mouse-model-of-cerebral-palsy

Nanyang Technological University

Research

JoVE Journal

Medicine

24.0K 次观看.  Lewiz Katz School of Medicine at Temple University. Presented is a concise battery of mouse neonatal motor tests. Using these tests, neonatal motor deficits can be demonstrated in a variety of neonatal motor disorders. By having a standardized set of tests, results from different studies can be compared, allowing for better and accurate reporting between groups.

视频: A Battery of Motor Tests in a Neonatal Mouse Model of Cerebral Palsy

Making Sense of Listening: The IMAP Test Battery

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the abilities to extract and use the information encoded in the auditory signal. We refer to these as listening skills (or auditory processing AP). Deficits in these skills are associated with delayed language and literacy development, though the nature of the relevant deficits and their causal connection with these delays is hotly debated.
When a child is referred to a health professional with normal hearing and unexplained difficulties in listening, or associated delays in language or literacy development, they should ideally be assessed with a combination of psychoacoustic (AP) tests, suitable for children and for use in a clinic, together with cognitive tests to measure attention, working memory, IQ, and language skills. Such a detailed examination needs to be relatively short and within the technical capability of any suitably qualified professional. Current tests for the presence of AP deficits tend to be poorly constructed and inadequately validated within the normal population. They have little or no reference to the presenting symptoms of the child, and typically include a linguistic component. Poor performance may thus reflect problems with language rather than with AP. To assist in the assessment of children with listening difficulties, pediatric audiologists need a single, standardized child-appropriate test battery based on the use of language-free stimuli.
We present the IMAP test battery which was developed at the MRC Institute of Hearing Research to supplement tests currently used to investigate cases of suspected AP deficits. IMAP assesses a range of relevant auditory and cognitive skills and takes about one hour to complete. It has been standardized in 1500 normally-hearing children from across the UK, aged 6-11 years. Since its development, it has been successfully used in a number of large scale studies both in the UK and the USA. IMAP provides measures for separating out sensory from cognitive contributions to hearing. It further limits confounds due to procedural effects by presenting tests in a child-friendly game-format. Stimulus-generation, management of test protocols and control of test presentation is mediated by the IHR-STAR software platform. This provides a standardized methodology for a range of applications and ensures replicable procedures across testers. IHR-STAR provides a flexible, user-programmable environment that currently has additional applications for hearing screening, mapping cochlear implant electrodes, and academic research or teaching.

A test battery (IMAP) for performing an in-depth assessment of auditory and cognitive abilities contributing to listening skills is described. It is quick to administer, child-friendly and free from linguistic confounds. Stimulus generation and protocol management are controlled via a software platform (IHR-STAR) to ensure replicable procedures.

听力:IMAP测试电池的决策意识

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the ...

科研

医学

Mouse Model of Middle Cerebral Artery Occlusion

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the brain (ischemic stroke) 2. Since most ischemic strokes (~80%) occur in the territory of middle cerebral artery (MCA) 3, many animal stroke models that have been developed have focused on this artery. The intraluminal monofilament model of middle cerebral artery occlusion (MCAO) involves the insertion of a surgical filament into the external carotid artery and threading it forward into the internal carotid artery (ICA) until the tip occludes the origin of the MCA, resulting in a cessation of blood flow and subsequent brain infarction in the MCA territory 4. The technique can be used to model permanent or transient occlusion 5. If the suture is removed after a certain interval (30 min, 1 h, or 2 h), reperfusion is achieved (transient MCAO); if the filament is left in place (24 h) the procedure is suitable as a model of permanent MCAO. This technique does not require craniectomy, a neurosurgical procedure to remove a portion of skull, which may affect intracranial pressure and temperature 6. It has become the most frequently used method to mimic permanent and transient focal cerebral ischemia in rats and mice 7,8. To evaluate the extent of cerebral infarction, we stain brain slices with 2,3,5-triphenyltetrazolium chloride (TTC) to identify ischemic brain tissue 9. In this video, we demonstrate the MCAO method and the determination of infarct size by TTC staining.

We demonstrate in the video a method for producing a middle cerebral artery occlusion in adult mice using an intraluminal monofilament. We also show how to evaluate the extent of cerebral infarction by 2,3,5-triphenyltetrazolium chloride (TTC) staining.

大脑中动脉闭塞的小鼠模型

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the ...

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

多光子成像在口腔鳞状细胞癌原位小鼠模型的肿瘤细胞的侵袭

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

A Swine Model of Neonatal Asphyxia

Annually more than 1 million neonates die worldwide as related to asphyxia. Asphyxiated neonates commonly have multi-organ failure including hypotension, perfusion deficit, hypoxic-ischemic encephalopathy, pulmonary hypertension, vasculopathic enterocolitis, renal failure and thrombo-embolic complications. Animal models are developed to help us understand the patho-physiology and pharmacology of neonatal asphyxia. In comparison to rodents and newborn lambs, the newborn piglet has been proven to be a valuable model. The newborn piglet has several advantages including similar development as that of 36-38 weeks human fetus with comparable body systems, large body size (&tilde;1.5-2 kg at birth) that allows the instrumentation and monitoring of the animal and controls the confounding variables of hypoxia and hemodynamic derangements.
We here describe an experimental protocol to simulate neonatal asphyxia and allow us to examine the systemic and regional hemodynamic changes during the asphyxiating and reoxygenation process as well as the respective effects of interventions. Further, the model has the advantage of studying multi-organ failure or dysfunction simultaneously and the interaction with various body systems. The experimental model is a non-survival procedure that involves the surgical instrumentation of newborn piglets (1-3 day-old and 1.5-2.5 kg weight, mixed breed) to allow the establishment of mechanical ventilation, vascular (arterial and central venous) access and the placement of catheters and flow probes (Transonic Inc.) for the continuously monitoring of intra-vascular pressure and blood flow across different arteries including main pulmonary, common carotid, superior mesenteric and left renal arteries. Using these surgically instrumented piglets, after stabilization for 30-60 minutes as defined by Z&lt;10% variation in hemodynamic parameters and normal blood gases, we commence an experimental protocol of severe hypoxemia which is induced via normocapnic alveolar hypoxia. The piglet is ventilated with 10-15% oxygen by increasing the inhaled concentration of nitrogen gas for 2h, aiming for arterial oxygen saturations of 30-40%. This degree of hypoxemia will produce clinical asphyxia with severe metabolic acidosis, systemic hypotension and cardiogenic shock with hypoperfusion to vital organs. The hypoxia is followed by reoxygenation with 100% oxygen for 0.5h and then 21% oxygen for 3.5h. Pharmacologic interventions can be introduced in due course and their effects investigated in a blinded, block-randomized fashion.

Large animal models have good translational values in the examination of physiology and pharmacology of neonatal asphyxia. Using newborn piglets, we develop an experimental protocol to simulate neonatal asphyxia which has advantages of studying the systemic and regional hemodynamics, oxygen transport with biochemical and pathologic pathways and correlations.

新生儿窒息了猪模型

Annually more than 1 million neonates die worldwide as related to asphyxia. Asphyxiated neonates commonly have multi-organ failure including hypotension, ...

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

婴幼儿的脑代谢和血流动力学的非侵入式光学测量

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world&#8217;s 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials1,2. The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (&#60; 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy.

新生儿缺氧缺血性脑病的小猪模型

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the ...

Assessment of Perigenital Sensitivity and Prostatic Mast Cell Activation in a Mouse Model of Neonatal Maternal Separation

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has a lifetime prevalence of 14% and is the most common urological diagnosis for men under the age of 50, yet it is the least understood and studied chronic pelvic pain disorder. A significant subset of patients with chronic pelvic pain report having experienced early life stress or abuse, which can markedly affect the functioning and regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Mast cell activation, which has been shown to be increased in both urine and expressed prostatic secretions of CP/CPPS patients, is partially regulated by downstream activation of the HPA axis. Neonatal maternal separation (NMS) has been used for over two decades to study the outcomes of early life stress in rodent models, including changes in the HPA axis and visceral sensitivity. Here we provide a detailed protocol for using NMS as a preclinical model of CP/CPPS in male C57BL/6 mice. We describe the methodology for performing NMS, assessing perigenital mechanical allodynia, and histological evidence of mast cell activation. We also provide evidence that early psychological stress can have long-lasting effects on the male urogenital system in mice.

We are measuring perigenital mechanical sensitivity and mast cell activation in the prostate of male C57BL/6 mice that underwent an early life stress paradigm &#8211; neonatal maternal separation, in order to induce a preclinical model of chronic prostatitis/chronic pelvic pain syndrome.

的Perigenital敏感性和前列腺肥大细胞活化新生母婴分离的小鼠模型评估

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has a lifetime prevalence of 14% and is the most common urological diagnosis for men under the ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

新生儿小鼠脊髓压迫损伤模型

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and regeneration, and to define new targets for therapeutics. For decades, models of complete heart regeneration existed in amphibians and fish, but a mammalian counterpart was not available. The recent discovery of a postnatal window during which mice possess regenerative capabilities has led to the establishment of a mammalian model of cardiac regeneration. A surgical model of mammalian cardiac regeneration in the neonatal mouse is presented herein. Briefly, postnatal day 1 (P1) mice are anesthetized by isoflurane and placed on an ice pad to induce hypothermia. After the chest is opened, and the left anterior descending coronary artery (LAD) is visualized, a suture is placed around the LAD to inflict myocardial ischemia in the left ventricle. The surgical procedure takes 10-15 min. Visualizing the coronary artery is crucial for accurate suture placement and reproducibility. Myocardial infarction and cardiac dysfunction are confirmed by triphenyl-tetrazolium chloride (TTC) staining and echocardiography, respectively. Complete regeneration 21 days post myocardial infarction is verified by histology. This protocol can be used to as a tool to elucidate mechanisms of mammalian cardiac regeneration after myocardial infarction.

This protocol describes a highly reproducible model of cardiac regeneration by surgical induction of myocardial infarction in the left ventricle of postnatal day 1 mice. The method involves induction of hypothermic anesthesia and ligation of the left anterior descending coronary artery.

心肌梗死新生小鼠，心肌再生的模型

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and ...

Transient Middle Cerebral Artery Occlusion Model of Neonatal Stroke in P10 Rats

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature and mature brain. Stroke occurs commonly in the perinatal period in humans, and transient ischemia-reperfusion is the most common form of stroke in neonates. The reperfusion phase is a critical component of injury progression, which occurs over a period of days to weeks, and of the endogenous response to injury. This postnatal day 10 (p10) rat model of transient middle cerebral artery occlusion (tMCAO) creates a unilateral, non-hemorrhagic focal ischemia-reperfusion injury that can be utilized to study the mechanisms of focal injury and repair in the full-term-equivalent brain. The injury pattern that is produced by tMCAO is consistent and highly reproducible and can be confirmed with MRI or histological analyses. The severity of injury can be manipulated through changes in occlusion time and other methods that will be discussed.

Neonatal stroke is a significant cause of early brain injury requiring a translational model with consistent focal injury patterns and high reproducibility in order to enable study. This study describes the detailed surgical procedure for creating a non-hemorrhagic, unilateral focal ischemia-reperfusion injury in full-term-equivalent rodents.

在P10大鼠新生儿中风的短暂大脑中动脉栓塞模型

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature ...