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本出版物中详述的方法由Hilltop实验室动物机构动物护理和使用委员会（IACUC）审查，以确保适当的照顾，使用和人道对待动物，遵守适用的联邦，州和当地法律法规，例如作为联邦动物福利条例，或AWR（CFR 1985）和"人体护理和使用实验动物公共卫生服务政策"（PHS政策）（PHS，1996）。 1.所有行为评估总则<ol><li>在任何动物处理之前，使用新的笼子卡盖住现有的笼子卡，仅显示唯一的，盲人的动物标识符。 注意:在常规化合物/安慰剂给药期间处理小鼠的调查员不得处理小鼠进行行为评估。 </li><li>点亮或关闭头顶灯，调整照明，使得竞技场或迷宫地板的照明为30-35勒克斯。 </li><li> F或研究跨越几个星期，每周记录体重作为总体健康的间接测量。 注意:如果需要进行更健壮的身体检查，可以包括外套质量，姿势，步态和自发运动的额外的笼侧检查。 </li></ol> 2.调查小鼠处理小鼠<ol><li>在任何行为测试前两天，将老鼠习惯于处理。将笼子从笼架上取下，放在水平面上。 </li><li>从笼子上取下盖子。正如处理即将到来的行为测试时所处理的一样，处理鼠标。将鼠标放在家中笼子上的杯状手中。 </li><li>测量潜伏期从研究者的手跳回到家庭笼中。握住鼠标最多5秒。 注意:延迟≥2秒的小鼠被视为"习惯"。在第一次试验期间显示延迟&lt;2秒的小鼠经历2次额外的习惯那天的n个会议。 </li><li>让老鼠连续2天处理习惯。注意在第二天结束时没有习惯的任何鼠标。 </li></ol>通过测量Y型迷宫中的自发交替来评估空间工作记忆8 <ol><li>在初次使用之前，用无味的漂白杀菌擦拭物，70％EtOH，然后用dH 2 O 9彻底清洁Y-迷宫。明确将迷宫的手臂指定为"A"，"B"和"C"或其他相似的唯一标识符。 </li><li>在测试开始之前，设置数据采集系统或摄像机，并在迷宫中设置适当的鼠标跟踪。使用已知长度的标尺或其他物体的捕获视频图像校准迷宫中的距离。 注意:此过程中的行为方法将适用于各种数据采集系统，作者假设anyo执行此过程精通使用他们选择的数据采集系统。功率分析表明，β≤0.2需要每组10-15只小鼠的样本量。 </li><li>从机架上取下保持架，轻轻将其放置在靠近Y型迷宫的桌子上。将鼠标从其家中的笼子中取出，轻轻地将其放置在Y-迷宫的一个臂上，面向中心。让调查员远离迷宫，使鼠标看不到调查员。 </li><li>将鼠标放入迷宫后立即激活数据/视频采集系统。 </li><li>按播放并记录每只小鼠的自发行为10分钟。会话完成后，将鼠标轻轻放回家中的笼子并将笼子返回到机架。 </li><li>在每次会话之间彻底清洁迷宫，使用无味的漂白杀菌擦拭剂，70％EtOH，然后加入dH 2 O.重复步骤3.4，评估所有麦克风即</li><li>一旦所有小鼠完成了Y迷宫的探索，分析采集系统的数据或手动对会议视频进行评分。当鼠标的所有4只爪穿过中央区域的阈值并进入臂并且动物的鼻孔朝向手臂的端部时，发生臂进入。 注意:要分析的端点包括:迷宫中的总距离，每个臂（包括中央区域）中行进的总距离，每个臂（包括中央区域）的总时间，手臂总数，进入每个手臂的条目，以及输入的武器序列列表，以评估所做的更改次数。 </li><li>当三只连续的手臂条目中的每一只小鼠进入迷宫的不同臂时，发生自发的交替。然后用以下公式计算自发交替％。 <img alt="公式1" src="/files/ftp_upload/55523/55523eq1.jpg" /> 注意:例如，胳膊入门的顺序是:ABCCBABCABC，调查员总共得到6次自发的交替（按照ABC，CBA，ABC，BCA，CAB，ABC）。共有11个手臂入口，自发交替率为67％。 </li><li> 进行以下质量控制检查，以确保数据代表对自发交替的无偏估计。 <ol><li>执行Pearson的自发交替百分比与总行进距离和所进行的手臂数量的相关性。 注意:如果自发交替％与任一参数存在显着相关性，则由于超动力运动对明显认知终点的潜在影响，应进一步检查数据。 </li><li>使用单向方差分析测试分析进入每个手臂的条目数。 注意:如果这个分析是重要的，那么这将表明在环境中存在吸引小鼠到特定区域的线索在迷宫上。 </li></ol></li></ol> 4.通过测量新对象识别来评估中期识别记忆11,12,13 <ol><li>对于该测试的每个阶段，在初始使用之前，用无味的漂白杀菌擦拭剂，70％EtOH，然后用dH 2 O彻底清洁开放场地。 </li><li> 在暴露对象前一天，将老鼠习惯于野外野外。 <ol><li>在习惯开始之前，设置数据采集系统或摄像机，并确认迷宫中鼠标的正确跟踪。使用已知长度的标尺或其他物体的捕获视频图像来校准舞台上的距离。在软件中标记竞技场的角落，以允许评分位置偏差。 注意:此过程中的行为方法将适用于各种数据采集系统茎和作者假设执行此过程的任何人精通使用他们选择的数据采集系统。功率分析表明，β≤0.2需要每组15-20只小鼠的样本量。 </li><li>从机架上取下笼子，轻轻地放在靠近竞技场的桌子上。 </li><li>从家笼中取出鼠标，轻轻将鼠标放在竞技场的中心。将鼠标放入竞技场后立即打开跟踪软件和/或录像系统。 </li><li>允许老鼠自由探索竞技场30分钟。 注意:在此期间，调查员不会打扰小鼠。 </li><li>习惯性会话后，将小鼠放回自己的家笼内，用无味的漂白杀菌擦拭剂70％EtOH，然后用dH 2 O彻底清洗。 </li><li>从步骤4.2.2重复，直到所有的老鼠都习惯于竞技场。 </li><li>所有的老鼠都习惯了分享到舞台，分析视频。 注意:要分析的终点包括竞技场中的总距离和每个角落附近花费的时间。如果与小鼠模型相关，这些分析中包括刻板行为（ 即 ，肌阵挛角跳，盘旋等 ）。在竞技场的特定地区表现出偏袒的小鼠被排除在进一步的实验之外，因为这将影响对象探索。 注意:新颖的对象识别的第一阶段涉及熟悉一个对象的小鼠。在这里，新的对象识别过程的这一部分将被称为样本阶段。 </li><li>在采样阶段开始之前，将物体放置在竞技场中，并将其固定在地板上，并安装腻子，以使动物无法移动物体。将两个相同的物体对准一个墙壁和物体之间的足够距离的特定墙壁，以便老鼠可以从所有角度自由探索物体LES。 </li><li>设置数据采集系统或摄像机，并确认迷宫中鼠标和对象的正确跟踪。使用已知长度的标尺或其他物体的捕获的视频图像校准竞技场中的距离。 </li><li>在软件中标记竞技场的角落，以允许评分位置偏差。在软件中标记对象并分别跟踪每个对象的探索行为（ 即 "对象A"和"对象B"）。 </li><li>从机架上取下机架，轻轻将其放在靠近舞台的桌子上。 </li><li>从家笼中取出鼠标，轻轻将其放入竞技场的中心，面对物体。 </li><li>让鼠标自由探索物体15分钟。在此期间不要打扰老鼠。 </li><li>在会话结束时，将鼠标轻轻放回家中的笼子中。用70％EtOH和dH 2 O清洁竞技场和物体。将这些物品放回e竞技场</li><li>重复步骤4.2.11，直到所有小鼠熟悉一个对象。 </li><li>一旦所有的老鼠熟悉一个对象，分析视频。 注意:一旦符合以下条件，对象探索将被计数:鼠标面向对象，鼻子在对象的2厘米以内，动物身体的中点距离对象超过2厘米，并且以前的标准已经达到至少1秒。另外，如果一只动物已经满足了勘探标准，但是表现为10秒以上的不动，那么这个探索性的回合就被认为是完成的。 </li><li>计算每个鼠标的对象偏差得分如下。 <img alt="公式2" src="/files/ftp_upload/55523/55523eq2.jpg" /> 注意:显示目标偏差得分低于20％或高于80％的小鼠被排除在进一步的实验之外。 </li></ol></li><li> 新的对象识别的最后阶段涉及评估探索行为指向环境中的一个新颖和熟悉的对象，这里被称为测试阶段。此阶段在样品相完成2-3小时后进行。 <ol><li>在测试阶段会话开始之前，将对象放入竞技场，并将其固定在地板上，以使动物无法移动对象。 <ol><li>将物体放在相对于样品阶段13的竞技场中的相同位置。 </li><li>平衡基因型和治疗组中新颖和熟悉的对象的相对位置。 </li><li>确保墙壁和物体之间有足够的距离，以便老鼠可以从各个角度自由探索物体。 </li></ol></li><li>设置数据采集系统和/或摄像机。确认在迷宫中正确追踪小鼠和物体。使用已知长度的标尺或其他物体的捕获视频图像校准竞技场中的距离。 </li><li>在软体中标记竞技场的角落是允许得分的位置偏见。用软件标记对象，并对每个对象（ 即 "小说"和"熟悉"）追踪探索性行为。 </li><li>从机架上取下机架，轻轻将其放在靠近舞台的桌子上。 </li><li>轻轻地将动物放在竞技场的中心，面对物体。记录小鼠自由探索物体10分钟。 </li><li>在测试结束时，将小鼠从竞技场中取出，将小鼠放回家中。每个会话之后，彻底清洁竞技场和物体，含有无味的漂白杀菌擦拭剂，70％EtOH和dH 2 O。 </li><li>从步骤4.4.3重复，直到所有动物进行了评估。 </li><li>一旦对所有小鼠进行物体探查，就分析视频。 注意:满足以下条件后，对象探索进行计数:鼠标面向对象，鼻子位于对象的2厘米以内，中点动物的身体距离物体超过2厘米，并且以前的标准已经达到至少1秒。另外，如果一只动物已经满足了勘探标准，但是表现为10秒以上的不动，那么这个探索性的回合就被认为是完成的。 </li><li>通过比较探索小说与熟悉对象的时间来评估新颖的对象识别。文献中通常报道了三种方法。 <ol><li>分析使用重复测量测试探索新颖和熟悉的对象的原始时间。当基因型和/或治疗不影响总的探索时间时，最好使用这种方法。 </li><li>使用以下公式计算新颖性偏好: <img alt="公式3" src="/files/ftp_upload/55523/55523eq3.jpg" /> 注意:这提供了探索新对象相对于探索对象的总时间所花费的时间百分比。价值范围从0％（没有探索新物体）到100％（仅探索小说object），值为50％，表示探索小说和熟悉对象的时间相等。 </li><li>计算辨别指数11 ，使用下式: <img alt="公式4" src="/files/ftp_upload/55523/55523eq4.jpg" /> 注意:这产生了相对于探索对象的总时间来探索小说和熟悉对象的时间差异。值范围从-1（只有熟悉的对象的探索）到+1（仅探索新颖的对象，值为0表示等待时间花费在探索小说和熟悉的对象上。 </li></ol></li><li>从动物运动或其他刻板印象中删除不参加测试的动物，从考虑11 。 注意:用于去除的标准必须客观和先验确定小鼠模型（ 即 ，总探查时间的第 5百分位数，&gt; 100平均值测试期间转弯角度或呈现肌阵挛角跳跃的第 50百分位数时间）。 </li></ol></li></ol> 5.用肢体夹住评估小鼠皮质脊髓功能14 <ol><li>视频记录整个会话。使用便携式手持设备（ 即 ，智能手机或等效物体）录制视频。 注意:功率分析表明，β≤0.2需要每组10-15只小鼠的样本量。 </li><li>将家用笼子从机架上取下并放在桌子上。在下一步之前记录视频中的动物ID。 </li><li>轻轻地将鼠标从笼子中取出并由尾巴悬挂5-10秒。视频必须在暂停时记录动物的后爪和前爪。 </li><li>拍摄至少5秒的视频后，将鼠标放回家中的笼子，然后将笼子放回机架。 </li><li>清理桌子。从步骤5.2重复，直到所有的小鼠都被记录<html>天。 </li><li> 评分肢体从小尾巴悬挂的小鼠的视频上按0-4的比例（见表1的评分描述）。检查暂停的小鼠的视频，然后根据以下标准分配分数。 <ol><li>没有肢体紧握。正常逃生扩展。一个后肢显示不完全的显示和流动性的丧失。脚趾表现正常。 </li><li>两肢后肢表现不均匀，流动性下降。脚趾表现正常。 </li><li>两个后肢都表现出蜷缩的脚趾和不动。 </li><li>前肢和后肢表现出扣合，交叉，卷曲的脚趾和不动。 </li></ol></li><li> 所有小鼠均由2名独立研究者评分。任何一个2分的差异超过1分的鼠标再次被打破。 <ol><li>不同的得分平均。 </li></ol></li></ol>p_upload / 55523 / 55523table1.jpg"/&gt; 表1:肢体分离得分的描述。

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+characterization+of+the+Tg2576+transgenic+model+of+Alzheimer's+disease+through+19+months.">Behavioral characterization of the Tg2576 transgenic model of Alzheimer's disease through 19 months.</a> Physiol Behav. 75 (5), 627-642 (2002).</li><li>Lalonde, R., Lewis, T. L., Strazielle, C., Kim, H., Fukuchi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+mice+expressing+the+betaAPP695SWE+mutation:+effects+on+exploratory+activity,+anxiety,+and+motor+coordination.">Transgenic mice expressing the betaAPP695SWE mutation: effects on exploratory activity, anxiety, and motor coordination.</a> Brain Res. 977 (1), 38-45 (2003).</li><li>Lewis, J., 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=Neurofibrillary+tangles,+amyotrophy+and+progressive+motor+disturbance+in+mice+expressing+mutant+(P301L)+tau+protein.">Neurofibrillary tangles, amyotrophy and progressive motor disturbance in mice expressing mutant (P301L) tau protein.</a> Nat Genet. 25 (4), 402-405 (2000).</li><li>Spittaels, K., 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=Prominent+axonopathy+in+the+brain+and+spinal+cord+of+transgenic+mice+overexpressing+four-repeat+human+tau+protein.">Prominent axonopathy in the brain and spinal cord of transgenic mice overexpressing four-repeat human tau protein.</a> Am J Pathol. 155 (6), 2153-2165 (1999).</li><li>Terwel, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changed+conformation+of+mutant+Tau-P301L+underlies+the+moribund+tauopathy,+absent+in+progressive,+nonlethal+axonopathy+of+Tau-4R/2N+transgenic+mice.">Changed conformation of mutant Tau-P301L underlies the moribund tauopathy, absent in progressive, nonlethal axonopathy of Tau-4R/2N transgenic mice.</a> J Biol Chem. 280 (5), 3963-3973 (2005).</li><li>Merrow, M., Spoelstra, K., Roenneberg, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+circadian+cycle:+daily+rhythms+from+behaviour+to+genes.">The circadian cycle: daily rhythms from behaviour to genes.</a> EMBO Rep. 6 (10), 930-935 (2005).</li><li>Smarr, B. L., Jennings, K. J., Driscoll, J. R., Kriegsfeld, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+time+to+remember:+the+role+of+circadian+clocks+in+learning+and+memory.">A time to remember: the role of circadian clocks in learning and memory.</a> Behav Neurosci. 128 (3), 283-303 (2014).</li><li>Kandel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+biology+of+memory+storage:+a+dialogue+between+genes+and+synapses.">The molecular biology of memory storage: a dialogue between genes and synapses.</a> Science. 294 (5544), 1030-1038 (2001).</li><li>Stough, S., Shobe, J. L., Carew, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intermediate-term+processes+in+memory+formation.">Intermediate-term processes in memory formation.</a> Curr Opin Neurobiol. 16 (6), 672-678 (2006).</li><li>Stevens, L. M., Brown, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reference+and+working+memory+deficits+in+the+3xTg-AD+mouse+between+2+and+15-months+of+age:+a+cross-sectional+study.">Reference and working memory deficits in the 3xTg-AD mouse between 2 and 15-months of age: a cross-sectional study.</a> Behav Brain Res. 278, 496-505 (2015).</li><li>Yue, X., 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=Brain+estrogen+deficiency+accelerates+Abeta+plaque+formation+in+an+Alzheimer's+disease+animal+model.">Brain estrogen deficiency accelerates Abeta plaque formation in an Alzheimer's disease animal model.</a> Proc Natl Acad Sci U S A. 102 (52), 19198-19203 (2005).</li><li>McAllister, C., 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=Genetic+targeting+aromatase+in+male+amyloid+precursor+protein+transgenic+mice+down-regulates+beta-secretase+(BACE1)+and+prevents+Alzheimer-like+pathology+and+cognitive+impairment.">Genetic targeting aromatase in male amyloid precursor protein transgenic mice down-regulates beta-secretase (BACE1) and prevents Alzheimer-like pathology and cognitive impairment.</a> J Neurosci. 30 (21), 7326-7334 (2010).</li><li>Blaney, C. E., Gunn, R. K., Stover, K. R., Brown, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+genotype+influences+behavioral+development+of+3xTg-AD+mouse+pups.">Maternal genotype influences behavioral development of 3xTg-AD mouse pups.</a> Behav Brain Res. , 40-48 (2013).</li></ol>

JM Levenson由Proclara Biosciences，Inc.雇用.Miedel，J.Partton，A.Miedel和E.Miedel由Hilltop Laboratory Animals雇用。

技术对现有方法的重要意义 该方法设计用于筛选化合物在β-淀粉样变性和tau蛋白病的转基因小鼠模型中的体内活性。这里使用的分期方法确保检测与AD 3相关的认知领域中的有效化合物。此外，此处详细介绍的方法使用具有明确定义的端点的行为测试，易于实施的质量控制检查，可以以适度的吞吐量格式运行，并且几乎不需要调查员的干预。这些特征导致在动物和跨群体中表现出良好的再现性的测定法，其导致低的体内和分析间差异和效应大小（2≤f≤6），其足够强大以支持药物发现环境中的行为分析。 Pr。中的关键步骤otocol 用于AD药物发现的许多小鼠模型表现出与高度焦虑和侵略相一致的行为。这使得处理习惯对于执行此处描述的任何行为测试至关重要。由于这些测试依赖于无动力的行为，由于过度活跃和焦虑或侵略性的小鼠，研究者的粗暴处理可能会显着影响性能。焦虑增加可能导致执行任务失败，降低了测试的整体力量。此外，竞技场的光线水平对于促进每次测试所需的自发运动至关重要。明亮的光线往往会增加焦虑，抑制啮齿动物的运动，因此应注意调整环境光线水平至30-35勒克斯。 该过程的另一个重要方面是最小化强烈的环境线索，这将妨碍动物执行任务的能力。清洁竞技场和运动之间的对象是必不可少的，因为小鼠被吸引到环境中的新鲜气味。未能彻底清洁竞技场和物体可能会导致鼠标的自发活动倾斜，并掩盖真正的认知表现。调查员也应尽量减少在执行这些程序时使用个人卫生用品和古龙水/香水。最后，啮齿动物在许多公开行为20中表现出强大的昼夜和昼夜变化，包括学习和记忆21 。因此，为了尽可能减少由于基础行为和认知表现的昼夜节律引起的差异，所有的测试都应该在同一时间进行队列和研究。 此外，具体地关于新颖的对象识别，样本和测试阶段之间的延迟间隔以及对象在环境中的选择和放置是关键参数。内存存在3种不同的形式:短期记忆ory（STM），中期记忆（ITM）和长期记忆（LTM） 22,23 。将样本和测试阶段之间的间隔从分钟（STM）更改为小时（ITM）或天数（LTM）将会更改程序12测试的内存类型。此外，在运行新颖的对象识别测试之前，应该在小鼠的测试队列中筛选许多对象，用于潜在的探索偏倚。在评估新颖的对象识别时，不能使用对测试队列过于吸引或排斥的对象。理想情况下，在测试中使用的所有对象，当放置在竞技场中时，将从幼稚的小鼠中引出相同的探索时间。物体的测试和优化不足可以显着降低新物体识别的力量。 修改和故障排除 有几个因素可能会增加这里描述的认知测试中的表观变异性。 AD的许多小鼠模型表现出超动力运动3 ，其可以掩盖或改变作为认知终点测量的行为。此外，越来越多的证据表明， 24,25,26甚至母亲基因型27可以影响AD小鼠模型中神经病理学和认知表型的发展和进展。意外的变化或未能实施行为任务可能是由于这些因素中的任何一个。当首次实施特定行为测试时，结果应始终按性别，年龄和母亲基因型分类。此外，应始终执行本程序中概述的质量检查，以确保多动症或其他定型行为不会干扰认知终点的量化。 信封熨烫也可以影响啮齿动物的自发探索行为。对研究人员无法察觉的气味或声音可能会吸引或排斥小鼠，从而偏离依赖于自发行为的认知测试结果。当初始建立Y迷宫或新颖的物体识别时，执行控制措施以确保在物体和/或环境的勘探中没有位置偏差是至关重要的。如果观察到位置偏差，调查员必须彻底审查环境，并可能调整照明，竞技场位置，测试室相对于设施中其他房间的位置（ 即 ，不靠近高流量区域或重型设备）和竞技场清洁程序。 测试环境的习惯是在新颖的对象识别测试中实现最佳性能的关键。例如，总勘探时间较短可能是由于习惯不适。作为proc的替代方法本文概述的处理（第2节）和竞技场（第4.2节）习惯，习惯处理和测试环境可以每天3,5分钟的时间进行连续2天。 技术的局限性 与任何程序一样，这些行为测试有局限性。已经使用这些程序，因为它们测试各种皮层区域和海马的功能。如果小鼠模型在这些测试探测到的脑区域中没有显示功能缺陷，那么这些技术将不会有用。此外，我们选择了探索短期记忆的认知测试。如果化合物临床前评估的作用机制不会影响短期记忆，则应相应修改这些程序（ 即增加样本 - 测试相间隔以测试长期记忆）。最后，这些测试使用没有动机的行为。因此，如果鼠标模型是exces活跃的活动或显示其他刻板行为，阻止对环境的探索，那么这些程序可能不是最佳的。作为替代方案，可以使用Tg2576或其他β-淀粉样变性小鼠模型的恐惧调理，或者用于rTg4510或其他小鼠模型的tau蛋白病3的空间水迷宫。 未来的应用 一旦这些程序已经在实验室中成功采用，可以进行几项修改或扩展，以评估额外的认知和功能运动措施。例如，改变新颖的对象识别任务以确定鼠标是否可以识别对象13的位置的改变。或者，不是使用对象，可以使用其他鼠标并实施社会识别测试。关于肢体夹紧和运动功能，可以用线悬挂和/或抓力测试来补充该测试。测试在这种方法中详细描述形成一个坚实的基础来筛选在 AD的翻译小鼠模型中具有体内功效的化合物，并且可以以许多方式进行调整或修饰以最好地询问特定的小鼠模型或满足独特药物发现程序的需要。

阿尔茨海默病（AD）是进行性神经变性疾病，导致认知衰退衰弱，影响全球约4400万人。目前尚无AD治疗可以进行疾病修复，强调迫切需要临床前发现这种疾病的新型治疗策略。已经创建了许多不同的转基因小鼠模型，其重述了AD1,2的各个方面，包括在患者中破坏的认知结构域的缺陷3 。这些小鼠模型代表有助于在体内有效筛选的工具。 当评估化合物潜在的体内功效时，必须采取分阶段的方法，筛选适当的认知领域的功效，还可以监测可能影响特定终点的行为ssess认知AD的许多转基因小鼠模型表现出多动症和其他可能干扰特定认知测试的行为，并禁止其用于药物筛选4 。此外，对于在药物筛选环境中实施的方法，所使用的特定测试应至少保持适度的产量，具有明确定义的终点，以及调查人员最少干预的程序。使用这些标准，可以实施行为屏幕，其显示复合筛选所需的再现性，低的测定内和测定间差异以及效果尺寸。这里详细描述了我们用于筛选有效减轻β-淀粉样变性和tau蛋白病转基因小鼠模型中存在的认知和运动表型的化合物的方法5,6 。所描述的方法适用于l中报告的常用行为范例迭代7 ，具有特定的优化和质量控制检查，以便它们可以用于与AD相关的转基因小鼠模型。该协议可用于各种数据采集和分析系统，并假设调查员具有相关软件的工作知识。

<table border="0" cellpadding="0" cellspacing="0" width="1375">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Topscan Lite-High Throughput</td>
			<td style="width:211px;">Cleversys</td>
			<td style="width:136px;"></td>
			<td style="width:812px;">Automated behavioral analysis. Includes cameras and video acquisition system, laptop.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">ObjectScan</td>
			<td style="width:211px;">Cleversys</td>
			<td style="width:136px;"></td>
			<td>Software module for accurate object exploration quantification</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Open field for mouse</td>
			<td style="width:211px;">Cleversys</td>
			<td style="width:136px;">CSI-OF-M</td>
			<td>Arena for novel object recognition</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Y-maze for mouse</td>
			<td style="width:211px;">Custom</td>
			<td style="width:136px;"></td>
			<td>Arms: 30cm long, 10 cm wide, 20cm high walls, placed 120deg apart.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Camera mount for open field</td>
			<td style="width:211px;">Custom</td>
			<td style="width:136px;">Custom</td>
			<td>76 cm tall, 115 cm wide, cameras mounted @ 30cm in from either side.&#160; Two mounts, each covers two boxes.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Camera mount for Y-maze</td>
			<td style="width:211px;">Custom</td>
			<td style="width:136px;">Custom</td>
			<td>76 cm tall, 115 cm wide, cameras mounted @ 30cm in from either side.&#160; One mount covers two mazes.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Marbles</td>
			<td style="width:211px;">Inperial Toy</td>
			<td>8565</td>
			<td>Standard (15.5mm Dia) glass marbles.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Dice</td>
			<td style="width:211px;">Cardinal Industries</td>
			<td>770</td>
			<td>Standard (0.650 inch) white dice with black dots.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">LOCTITE Fun-Tak</td>
			<td style="width:211px;">Henkel</td>
			<td>B018A3AG0W</td>
			<td>Standard blue sticky tak</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">EtOH</td>
			<td style="width:211px;">Nexeo Solutions</td>
			<td style="width:136px;">82452</td>
			<td>100% Ethanol Diluted to 70% using distilled Water</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">dH2O</td>
			<td style="width:211px;">Tulpenhocken Spring Water Co.</td>
			<td style="width:136px;">-</td>
			<td>PA D.E.P. #31, NJ D.O.H. #0049, NYSHD Cert. #320</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Paper towels</td>
			<td style="width:211px;">Procter &#38; Gamble</td>
			<td>B019DM86LA</td>
			<td>Bounty, White</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Handheld video camera</td>
			<td style="width:211px;">Apple, Inc.</td>
			<td style="width:136px;">MKV92LL/A</td>
			<td>Acquisition of Limb clasping video, Iphone 6S Plus (or functional equivalent).</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Gloves</td>
			<td style="width:211px;">SafePOINT, L.L.C.</td>
			<td style="width:136px;">GL640-2</td>
			<td>Standard, Powder free Latex Gloves, Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Light meter</td>
			<td style="width:211px;">Dr. Meter</td>
			<td style="width:136px;">LX1330B</td>
			<td>Lighting @ the bottom of Open Field= 35 LUX, Lighting @ bottom of Y-Maze= 32 LUX</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bleach germicidal wipes</td>
			<td style="width:211px;">Clorox</td>
			<td style="width:136px;"></td>
			<td>Sterilization of equipment during &#38; after use</td>
		</tr>
	</tbody>
</table>

assessment of spontaneous alternation, novel object recognition and limb clasping in transgenic mouse models of amyloid-&#946; and tau neuropathology

在这里，我们描述了一种分阶段的行为测试方法，可用于筛选在β淀粉样变性和tau蛋白病的转基因小鼠模型中对体内功效的认知和功能运动行为的化合物的筛选。范式包括Y迷宫中自发交替的测试，新颖的对象识别和肢体扣紧。选择这些测试是因为:1）询问认知或运动领域的功能以及与人类疾病状态相关的相关神经电路，2）具有明确界定的终点3）可以轻松实施质量控制检查4）中等吞吐量格式，以及5）调查员几乎不需要干预。这些方法设计用于寻找筛选化合物用于短期和工作记忆任务或与阿尔茨海默病小鼠模型相关的功能运动行为的研究者。这里描述的方法使用行为测试年龄不同的大脑区域包括海马和各种皮层区域。要求认知测试专门评估由单个脑区介导的认知的研究者可以使用这些技术补充其他行为测试。

老化的Tg2576小鼠表现出在Y-迷宫15,16内发生的自发变化的强大缺陷，可以使用本文详述的方法复制表型（ 图1A ）。虽然在这些小鼠中观察到增加的臂进入的趋势（ 图1B ），但是在该小鼠系列中观察到的多动症并不影响自发的交替率（ 图1C ）。相比之下，老化的rTg4510小鼠在放置在Y型迷宫中似乎表现出增加的自发交替（ 图1D ）。这是由于极度多动（ 图1E ）和定型10 ，这显着干扰了自发交替的测量（ 图1F ）。最初在这项任务中评估小鼠时，确保手臂输入和/或行驶距离不是至关重要的与自发交替率显着相关。 在评估新颖的对象识别之前，小鼠习惯于进行测试的竞技场。在习惯中，可以评估与小鼠模型相关的多动症（ 图2A ）和其他定型行为。在样本阶段，单独测量每个对象的检测是至关重要的，因此在进一步的评估中可以排除在探索行为中显示出显着偏差的小鼠（ 图2B ，空心圆圈）。通过比较一个熟悉和新颖的对象的探索来评估新的对象识别，并以三种不同的方式进行分析。如果总体探索时间在基因型和/或治疗组之间是可比较的，则可以使用探索每个对象的原始时间和适当的重复测量测试来确定新物体r是否存在差异识别（ 图2C ）。如果特定的小鼠品系在总探索时间上表现出差异，则可以使用新颖性偏好（ 图2D ）或鉴别指数（ 图2E ）来评估新颖的对象识别。 肢体扣紧是量化皮质脊髓功能缺陷的功能运动测试。在几种转基因tau小鼠模型6,17,18,19中观察到肢体扣紧，其不是认知措施，并且概括了晚期AD患者中观察到的一些功能性运动障碍。由尾巴悬挂小鼠引起逃生反应（ 图3A ，"0"）。根据他们的严重程度，从0-4开始，打破后肢和伸展脚趾的能力下降得分（ 图3A）。使用本文概述的程序，可以观察到在rTg4510小鼠中显着的肢体夹紧（ 图3B ）。 <img alt="图1" src="/files/ftp_upload/55523/55523fig1.jpg" /> 图1:Y迷宫中的自发交替。 （ A ）当放置在Y型迷宫中时，小鼠采用失调搜索策略，导致探索模式，每三臂进入一次，每只手臂只进行一次探索。老化的Tg2576小鼠表现出自发交替的显着缺陷。使用该方法中概述的程序，在用专有化合物处理后观察到自发交替的显着恢复。使用单因素方差分析数据，并使用Dunnett检验进行与Tg-PBS的事后比较。 ** p &lt;0.01。误差条表示SEM。 （ B ）任何一个手臂条目的数量没有显着差异在本实验中监测的组。使用单因素方差分析测试数据。误差条表示SEM。 （ C ）自发交替与所进行的手臂数量之间无相关性，表明自发运动活动中的任何差异不影响自发交替的量化。使用Pearson相关分析进行相关性检验。 （ D ）当置于Y型迷宫中时，相对于同窝鼠WT小鼠，rTg4510小鼠（6个月）似乎显示出显着更多的自发交替。通过单因素方差分析数据，并使用Dunnett检验进行与Tg-PBS的事后比较。 ** p &lt;0.01。误差条表示SEM。 （ E ）rTg4510小鼠由于极端的高动力运动而显着增加了手臂入口。通过单因素方差分析数据，并使用Dunnett检验进行与Tg-PBS的事后比较。 *** p &lt;0.001。错误栏表示SE（ F ）自发交替行为与手臂条目显着相关，表明超动力运动表型掩盖了真实的自发交替。使用Pearson相关分析进行相关性检验（r = 0.7，p &lt;0.0001）。 <a href="http://ecsource.jove.com/files/ftp_upload/55523/55523fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" src="/files/ftp_upload/55523/55523fig2.jpg" /> 图2:新对象识别。 （ A ）竞技场习惯允许测量与特定小鼠模型相关的自发运动和其他定型行为。在这里，老年Tg2576小鼠（22个月）相对于同窝鼠WT小鼠显示出显着更多的自发运动。数据通过单因素方差分析进行分析，并使用Dunnett&#39;的测试。 ** p &lt;0.01。误差条表示SEM。 （ B ）在采样阶段，分别追踪两个相同物体的探测。从测试阶段排除显示对探测两个物体（空心圆）之一的大偏差的小鼠。 （ C - E ）通过测量一个新颖和熟悉的对象的探索来评估新的对象识别。使用（ C ）原始探索时间，（ D ）新颖性偏好或（ E ）辨别指数来评估新的对象识别。使用重复测量的双向ANOVA分析C组中的数据，并使用Sidak检验进行成对比较。使用单因素方差分析进行面板DE中的数据，并使用Dunnett检验进行与Tg-Veh的事后比较。 * p &lt;0.05，** p &lt;0.01。误差条表示SEM。 <a href="http://ecsource.jove.com/files/ftp_upload/55523/55523fig2large.jpg" target="_blank">请点击</a>这里可以查看这个数字的较大版本。 <img alt="图3" src="/files/ftp_upload/55523/55523fig3.jpg" /> 图3:肢体扣紧。 （ A ）如表1所示，表现出各种程度的肢体紧握的小鼠的代表性图像。（ B ）rTg4510小鼠（6个月）相对于同窝小鼠WT小鼠显示出显着的肢体扣紧，使用这些方法评分。使用单因素方差分析数据，并使用Dunnett检验进行与Tg-PBS的事后比较。 *** p &lt;0.001，**** p &lt;0.0001。误差条表示SEM。 <a href="http://ecsource.jove.com/files/ftp_upload/55523/55523fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-Î² and Tau Neuropathology at JoVE.co

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-&#946; and Tau Neuropathology

这里描述的是一种分阶段的行为筛选方法，可用于筛选在β淀粉样变性和tau蛋白病的转基因小鼠模型中对体内功效的认知功能运动行为进行筛选的化合物。这些方法被优化以筛选化合物用于短期和工作记忆任务中的活动。

自发交替评估，淀粉样蛋白β和脑神经病理学转基因小鼠模型中的新物体识别和肢体紧扣

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional ...

assessment-spontaneous-alternation-novel-object-recognition-limb

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

26.5K 次观看.  Hilltop Laboratory Animals. 这里描述的是一种分阶段的行为筛选方法，可用于筛选在β淀粉样变性和tau蛋白病的转基因小鼠模型中对体内功效的认知功能运动行为进行筛选的化合物。这些方法被优化以筛选化合物用于短期和工作记忆任务中的活动。 

视频: Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-β and Tau Neuropathology

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher frequency of fractures 1. As these fractures often occur in regions that have lost normal sensory function, the patient is at a greater risk of fracture-dependent pathologies, including death. SCI-dependent loss in both bone mineral density (BMD, grams/cm2) and bone mineral content (BMC, grams) has been attributed to mechanical disuse 2, aberrant neuronal signaling 3 and hormonal changes 4. The use of rodent models of SCI-induced osteoporosis can provide invaluable information regarding the mechanisms underlying the development of osteoporosis following SCI as well as a test environment for the generation of new therapies 5-7 (and reviewed in 8). Mouse models of SCI are of great interest as they permit a reductionist approach to mechanism-based assessment through the use of null and transgenic mice. While such models have provided important data, there is still a need for minimally-invasive, reliable, reproducible, and quantifiable methods in determining the extent of bone loss following SCI, particularly over time and within the same cohort of experimental animals, to improve diagnosis, treatment methods, and/or prevention of SCI-induced osteoporosis.
An ideal method for measuring bone density in rodents would allow multiple, sequential (over time) exposures to low-levels of X-ray radiation. This study describes the use of a new whole-animal scanner, the IVIS Lumina XR (Caliper Instruments) that can be used to provide low-energy (1-3 milligray (mGy)) high-resolution, high-magnification X-ray images of mouse hind limb bones over time following SCI. Significant bone density loss was seen in the tibiae of mice by 10 days post-spinal transection when compared to uninjured, age-matched control (na&iuml;ve) mice (13% decrease, p&lt;0.0005). Loss of bone density in the distal femur was also detectable by day 10 post-SCI, while a loss of density in the proximal femur was not detectable until 40 days post injury (7% decrease, p&lt;0.05). SCI-dependent loss of mouse femur density was confirmed post-mortem through the use of Dual-energy X-ray Absorptiometry (DXA), the current "gold standard" for bone density measurements. We detect a 12% loss of BMC in the femurs of mice at 40 days post-SCI using the IVIS Lumina XR. This compares favorably with a previously reported BMC loss of 13.5% by Picard and colleagues who used DXA analysis on mouse femurs post-mortem 30 days post-SCI 9. Our results suggest that the IVIS Lumina XR provides a novel, high-resolution/high-magnification method for performing long-term, longitudinal measurements of hind limb bone density in the mouse following SCI.

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

A longitudinal examination of bone loss in the femurs and tibiae of adult mice was performed following spinal cord injury using sequential low-dose X-ray scans. Tibia bone loss was detected throughout the study, while bone loss in the femur was not detected until 40 days post injury.

鼠标使用新型脊髓损伤的后肢骨质流失的纵向评价，在体内，方法论

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher ...

科研

医学

A Novel Application of Musculoskeletal Ultrasound Imaging

Ultrasound is an attractive modality for imaging muscle and tendon motion during dynamic tasks and can provide a complementary methodological approach for biomechanical studies in a clinical or laboratory setting. Towards this goal, methods for quantification of muscle kinematics from ultrasound imagery are being developed based on image processing. The temporal resolution of these methods is typically not sufficient for highly dynamic tasks, such as drop-landing. We propose a new approach that utilizes a Doppler method for quantifying muscle kinematics. We have developed a novel vector tissue Doppler imaging (vTDI) technique that can be used to measure musculoskeletal contraction velocity, strain and strain rate with sub-millisecond temporal resolution during dynamic activities using ultrasound. The goal of this preliminary study was to investigate the repeatability and potential applicability of the vTDI technique in measuring musculoskeletal velocities during a drop-landing task, in healthy subjects. The vTDI measurements can be performed concurrently with other biomechanical techniques, such as 3D motion capture for joint kinematics and kinetics, electromyography for timing of muscle activation and force plates for ground reaction force. Integration of these complementary techniques could lead to a better understanding of dynamic muscle function and dysfunction underlying the pathogenesis and pathophysiology of musculoskeletal disorders.

We describe a new ultrasound-based vector tissue Doppler imaging technique to measure muscle contraction velocity, strain and strain rate with sub-millisecond temporal resolution during dynamic activities. This approach provides complementary measurements of dynamic muscle function and could lead to a better understanding of mechanisms underlying musculoskeletal disorders.

肌肉骨骼超声成像的一种新应用

Ultrasound is an attractive modality for imaging muscle and tendon  motion during dynamic tasks and can provide a complementary methodological  approach ...

MRI and PET in Mouse Models of Myocardial Infarction

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ideal model for testing novel therapeutic strategies.
In vivo magnetic resonance imaging (MRI) gives excellent views of the heart noninvasively with clear anatomical detail, which can be used for accurate functional assessment. Contrast agents can provide basic measures of tissue viability but these are nonspecific. Positron emission tomography (PET) is a complementary technique that is highly specific for molecular imaging, but lacks the anatomical detail of MRI. Used together, these techniques offer a sensitive, specific and quantitative tool for the assessment of the heart in disease and recovery following treatment.
In this paper we explain how these methods are carried out in mouse models of acute myocardial infarction. The procedures described here were designed for the assessment of putative protective drug treatments. We used MRI to measure systolic function and infarct size with late gadolinium enhancement, and PET with fluorodeoxyglucose (FDG) to assess metabolic function in the infarcted region. The paper focuses on practical aspects such as slice planning, accurate gating, drug delivery, segmentation of images, and multimodal coregistration. The methods presented here achieve good repeatability and accuracy maintaining a high throughput.

We describe how to perform MRI and PET imaging of the mouse heart. The protocol is tailored to assess treatment efficacy in models of myocardial infarction and heart failure.

MRI和PET心肌梗死小鼠模型

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

乳腺癌的一种新的转基因小鼠模型:转移性乳腺癌通过瞄准导管上皮与腺病毒Cre重组酶的启动

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal systems. HFUS has the ability to detect structures as small as 30 &#181;m, a property that has been utilized for visualizing superficial lymph nodes in rodents in brightness (B)-mode. Combining power Doppler with B-mode imaging allows for measuring circulatory blood flow within lymph nodes and other organs. While HFUS has been utilized for lymph node imaging in a number of mouse&#160; model systems, a detailed protocol describing HFUS imaging and characterization of the cervical lymph nodes in mice has not been reported. Here, we show that HFUS can be adapted to detect and characterize cervical lymph nodes in mice. Combined B-mode and power Doppler imaging can be used to detect increases in blood flow in immunologically-enlarged cervical nodes. We also describe the use of B-mode imaging to conduct fine needle biopsies of cervical lymph nodes to retrieve lymph tissue for histological&#160; analysis. Finally, software-aided steps are described to calculate changes in lymph node volume and to visualize changes in lymph node morphology following image reconstruction. The ability to visually monitor changes in cervical lymph node biology over time provides a simple and powerful technique for the non-invasive monitoring of cervical lymph node alterations in preclinical mouse models of oral cavity disease.

This protocol describes the application of high-frequency ultrasound (HFUS) for imaging mouse cervical lymph nodes. This technique optimizes visualization and quantification of cervical lymph node morphology, volume and blood flow. Image-guided biopsy of cervical lymph nodes and processing of lymph tissue for histological evaluation is also demonstrated.

鼠标颈淋巴结高频超声成像

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal ...

Motor and Hippocampal Dependent Spatial Learning and Reference Memory Assessment in a Transgenic Rat Model of Alzheimer's Disease with Stroke

Alzheimer&#39;s disease (AD) is a debilitating neurodegenerative disease that results in neurodegeneration and memory loss. While age is a major risk factor for AD, stroke has also been implicated as a risk factor and an exacerbating factor. The co-morbidity of stroke and AD results in worsened stroke-related motor control and AD-related cognitive deficits when compared to each condition alone. To model the combined condition of stroke and AD, a novel transgenic rat model of AD, with a mutated form of amyloid precursor protein (a key protein involved in the development of AD) incorporated into its DNA, is given a small unilateral striatal stroke.
For a model with the combination of both stroke and AD, behavioral tests that assess stroke-related motor control, locomotion and AD-related cognitive function must be implemented. The cylinder task involves a cost-efficient, multipurpose apparatus that assesses spontaneous forelimb motor use. In this task, a rat is placed in a cylindrical apparatus, where the rat will spontaneously rear and contact the wall of the cylinder with its forelimbs. These contacts are considered forelimb motor use and quantified during video analysis after testing. Another cost-efficient motor task implemented is the beam-walk task, which assesses forelimb control, hindlimb control and locomotion. This task involves a rat walking across a wooden beam allowing for the assessment of limb motor control through analysis of forelimb slips, hindlimb slips and falls. Assessment of learning and memory is completed with Morris water maze for this behavioral paradigm. The protocol starts with spatial learning, whereby the rat locates a stationary hidden platform. After spatial learning, the platform is removed and both short-term and long-term spatial reference memory is assessed. All three of these tasks are sensitive to behavioral differences and completed within 28 days for this model, making this paradigm time-efficient and cost-efficient.

To investigate the co-morbid Alzheimer&#39;s disease (AD) and stroke condition in a novel model, three behavior tasks are described that assess both motor control and cognitive behaviors. These tasks include the beam-walk task, cylinder task and Morris water maze.

汽车和海马依赖空间学习和记忆参考评估与中风阿尔茨海默氏病的转基因大鼠模型

Alzheimer&#39;s disease (AD) is a debilitating neurodegenerative disease that results in neurodegeneration and memory loss. While age is a major risk ...

Orthotopic Hind Limb Transplantation in the Mouse

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and translational research in the field of transplantation medicine. A vast array of reagents is available exclusively in this setting, including mono- and polyclonal antibodies for both diagnostic and interventional applications. In addition, a vast number of genotyped, inbred, transgenic, and knock out strains allow detailed investigation of the individual contributions of humoral and cellular components to the complex interplay of an immune response and make the mouse the gold standard for immunological research. 
Vascularized Composite Allotransplantation (VCA) delineates a novel field of transplantation using allografts to replace "like with like" in patients suffering traumatic or congenital tissue loss. This surgical methodological protocol shows the use of a non-suture cuff technique for super-microvascular anastomosis in an orthotopic mouse hind limb transplantation model. The model specifically allows for comparison between established paradigms in solid organ transplantation with a novel form of transplants consisting of various different tissue components. Uniquely, this model allows for the transplantation of a viable vascularized bone marrow compartment and niche that have the potential to exert a beneficial effect on the balance of immune acceptance and rejection. This technique provides a tool to investigate alloantigen recognition and allograft rejection and acceptance, as well as enables the pursuit of functional nerve regeneration studies to further advance this novel field of transplantation.

This novel model for orthotopic hind limb transplantation in the mouse, applying a non-suture cuff technique for super-microvascular anastomosis, provides a powerful tool for in&#160;vivo mechanistic immunological research related to vascularized composite allotransplantation (VCA).

原位后肢移植中的鼠标

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and ...

An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer metastasis, is largely unknown and is a focus in cancer research. Various breast cancer spontaneous metastasis mouse models have been established. Here, we report a simplified procedure to establish orthotopic transplanted breast cancer primary tumor and resultant spontaneous metastasis that mimic human breast cancer metastasis. Combined with the bioluminescence live tumor imaging, this mouse model allows tumor growth and progression kinetics to be monitored and quantified. In this model, a low dose (1 x 104 cells) of 4T1-Luc breast cancer cells was injected into BALB/c mouse mammary fat pad using a tuberculin syringe. Mice were injected with luciferin and imaged at various time points using a bioluminescent imaging system. When the primary tumors grew to the size limit as in the IACUC-approved protocol (approximately 30 days), mice were anesthetized under constant flow of 2% isoflurane and oxygen. The tumor area was sterilized with 70% ethanol. The mouse skin around the tumor was excised to expose the tumor which was removed with a pair of sterile scissors. Removal of the primary tumor extends the survival of the 4T-1 tumor-bearing mice for one month. The mice were then repeatedly imaged for metastatic tumor spreading to distant organs. Therapeutic agents can be administered to suppress tumor metastasis at this point. This model is simple and yet sensitive in quantifying breast cancer cell growth in the primary site and progression kinetics to distant organs, and thus is an excellent model for studying breast cancer growth and progression, and for testing anti-metastasis therapeutic and immunotherapeutic agents in vivo.

An orthotopic breast cancer primary tumor model and surgical removal of primary tumor to extend mouse life to generate spontaneous metastasis are described. The tumor growth and progression are monitored and quantified by luciferase fluorescence imaging.

自发性乳腺癌转移的原位小鼠模型

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

小鼠肾小球疾病模型肾脏功能的评价

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

Preclinical Model of Hind Limb Ischemia in Diabetic Rabbits

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular disease is the development of ischemia. In severe cases, patients can develop critical limb ischemia in which they experience constant pain and an increased risk of limb amputation. Current therapies for peripheral ischemia include bypass surgery or percutaneous interventions such as angioplasty with stenting or atherectomy to restore blood flow. However, these treatments often fail to the continued progression of vascular disease or restenosis or are contraindicated due to the overall poor health of the patient. A promising potential approach to treat peripheral ischemia involves the induction of therapeutic neovascularization to allow the patient to develop collateral vasculature. This newly formed network alleviates peripheral ischemia by restoring perfusion to the affected area. The most frequently employed preclinical model for peripheral ischemia utilizes the creation of hind limb ischemia in healthy rabbits through femoral artery ligation. In the past, however, there has been a strong disconnect between the success of preclinical studies and the failure of clinical trials regarding treatments for peripheral ischemia. Healthy animals typically have robust vascular regeneration in response to surgically induced ischemia, in contrast to the reduced vascularity and regeneration in patients with chronic peripheral ischemia. Here, we describe an optimized animal model for peripheral ischemia in rabbits that includes hyperlipidemia and diabetes. This model has reduced collateral formation and blood pressure recovery in comparison to a model with a higher cholesterol diet. Thus, the model may provide better correlation with human patients with compromised angiogenesis from the common co-morbidities that accompany peripheral vascular disease.

We describe a surgical procedure used to induce peripheral ischemia in rabbits with hyperlipidemia and diabetes. This surgery acts as a preclinical model for conditions experienced in peripheral artery disease in patients. Angiography is also described as a means to measure the extent of introduced ischemia and recovery of perfusion.

糖尿病家兔欣德肢体缺血的临床前模型

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular ...