我们亲切地感谢杠杆实验室的其他成员谁促成了数据采集（安德里斯·费雷拉，Danarae阿莱曼，亚历克西斯莫，凯特林·弗林，伊丽莎白Bearce和马坦Kadosh）和审稿（安德里斯·费雷拉，丽贝卡施耐德和凯特·罗宾斯）。我们也承认罗德里克Schlotzhauer和埃德温Honse从MU物理机店为他们设计的输入在这项研究中所用的啮齿动物观察管和制造。我们特别感谢Malea月库克尔（放射科主管在兽医和密苏里大学外科系 - 兽医学院）和扬艾维（研究动物导管室在密苏里大学管理器 - 医学院）为展示不断的耐心和动力，同时经营高能荧光镜为我们开发了小鼠VFSS协议。资金来源为这项研究包括NIH / NIDCD（TE杆），NIH / NINDS（GK Pavlath），Otolaryngo呆呆 - 头颈外科启动资金（TE杆），MU PRIME基金（TE杆），密苏里大学优势（TE杆），和MU中心老龄（TE杆）。

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市售研究人类疾病数百鼠（小鼠和大鼠）模型。然而，只有三个鼠疾病模型已专门研究了相对于吞咽困难：帕金森氏病12,15-17和中风18的ALS 13,14的小鼠模型和大鼠模型。这些初步的研究中使用不同的方法来评估吞咽困难，使其不可能获得物种和疾病之间的有意义的比较。这一重大限制可能利用新开发的小鼠VFSS协议，允许众多的燕子参数自饲养动物客观量化来克服在未来的研究。 成功VFSS结果取决于三个关键部分组成：1）测试室，允许自进，而在有限的空间站在奔放，2）食谱掩盖市售口服造影AG的厌恶味道/气味需求测试，和3）一步一步测试协议，允许吞咽生理学的量化。集聚效应产生舒适，低应力，自我喂养检查，唤起典型的喂养和吞咽行为的环境。消除其中的一个或多个部件，不会对研究的结果有不利的影响。负面结果的例子包括无法维持动物中的，从饮用，厌恶分散于口服造影剂图，不希望的行为的透视字段，以及无法量化由于饮用发作不足吞参数。 获得最佳VFSS结果的一个主要挑战是设计一个合适的试验室。许多我们的原型设计的修改最终在观察室中充分保持的小鼠中的视场，并防止其从饮用分散行为。该室用铣床获得日统一尺寸制作Ë管和端盖，从而确保部件的互换性为相同直径的若干观察室。的内部尺寸（直径和长度）进行匹配，以比一个成年小鼠的身体尺寸，这导致了一个窄的测试室，其足以允许走在一条直线上且扭转稍大。的窄边设计，与壶嘴和PEG-碗只有年底的战略定位相结合，坚持沿着室的长度一边喝着对准老鼠的头部和身体。一旦在饮酒，小鼠保持显着的自稳定在管口或碗几秒钟的时间，从而导致最小的运动伪影干扰的测试。因此，有可能获得无失真，特写观察/视频记录和小鼠的同时在横向和背腹平面饮用videofluoroscopic成像。 小鼠（和其他小型啮齿类动物）的自然倾向于看ķ住所在狭小的空间。其结果是，他们可以自由进入测试室（其一端已经关闭由端帽）时，它被放置在笼中，从而消除了由处理（ 即，手动拾取动物将其置于应力/焦虑在腔室）。一旦鼠标进入腔室，另一端则通过附加一个第二端盖封闭。这种设计可以防止逃跑创造了低焦虑试验室老鼠自由探索的同时。 腔室的正方形形状提供了内置的运动稳定，允许它在一个独立的方式使用，从而消除了一个标准的啮齿动物笼中，需要进行测试。整个装置的重量轻，携带方便，可堆叠，储存的目的，坚固，易于清洗，可高压灭菌。而室最初设计为与透视使用，它们也都与点片放射摄影术，神经影像（ 例如，MRI，PET，CT），和visua兼容升观察/各种行为的视频录制。 克服第二个重大挑战是掩蔽口服造影剂（ 即硫酸钡和碘海醇）的厌恶味道/气味。鉴于味道灵敏度差别很大之间小鼠品系19-21和或许与22,23年龄，这是必要的，以确定单一的测试解决方案，是适口的所有小鼠，无论应变和年龄。这个结果是至关重要，以允许吞咽功能/跨菌株和年龄功能障碍的直接比较，同时消除了混杂的结果，由于在流变（ 例如，粘度，密度等 ）和试验溶液的化学性质的差异。为了达到这个目的，我们开发了一种简单，快速适口筛选方法来确定优选的风味增强剂期间鼠VFSS掩盖口服造影剂的厌恶味道/气味。方法简单暴露试验，这需要一个舔后仿照ometer（ 即，舔传感器）水调节期（ 即代扣水过夜）诱导口渴24,25后的第2分钟时记录舔率。一个舔食测试器是不适用于这项研究;因此，优选由行为观察，以及标准的视频记录方法，对于已经在我们的实验室13,14被预先验证的舔率评估。使用这种适口筛选方法，巧克力被确定为优选的风味增强剂通过C57和C57 / SJL株。具体来说，小鼠在每个笼子100％容易喝巧克力味的解决方案，在30秒的曝光，多个小鼠同时在饮水嘴。然而，添加钡的导致仅简要狂饮大多数小鼠中，无论钡或巧克力浓度。 另一种钡是碘海醇，是最近才被确认为一种专门的碘造影剂表替代硫酸人类VFSS 10钡;因此，还没有被标准化用于此目的。几种不同浓度的巧克力香料碘海醇被提供给小鼠。含有至多库存碘海醇（每毫升350毫克碘）的50％的溶液配方易于被大多数小鼠的过夜水调整期间后饮用。较高浓度导致避税行为。 50％的碘海醇（每毫升350毫克碘）的解决方案产生足够的放射密度，同时被吞噬小鼠，而低浓度均明显不太明显，阻碍燕子生理量化。因此，对于VFSS最优试验溶液用小鼠，鉴定为用巧克力香料50％碘海醇溶液加入。重复适口测试没有导致回避行为或不良事件。 第三个挑战，克服是防止老鼠转动/倾斜他们的头一边喝，这掩盖了可视化VFSS的过程中吞咽机制。从PEG-碗饮用正上方在室的一端解决此问题的地板上。有利用PEG-碗，而不是一个吸管瓶的几个额外的优点。例如，液体的校准体积可以通过在端盖观察管的通风孔被吸移到栓柱碗。这种方法允许的短暂VFSS测试时间过程中消耗的测试解决方案的每分钟通气量进行​​定量。此外，在钉碗试液的增加的表面积，相对于小的吸管口，可以提供增加的嗅觉刺激，以进一步激励饮用。 PEG-碗可能更适合用于研究年轻或更小的应变的小鼠，因为碗高度是从地板的标准化距离。与此相反，吸管长度必须进行调整，以适应不同大小的小鼠，这又增加了潜在的混杂变量需要考虑。此外，鼠标模式神经系统疾病的LS可能难以达到吸管奶瓶，由于肢体运动障碍，而他们可以很容易达到一个挂碗。小鼠舌和/或颚功能障碍可能无法充分地按球在喷口访问液体;采用PEG-碗可以消除这一变乱。由于这些原因，在吸管瓶的使用钉碗是鼠VFSS测试的首选方法。然而，在观察室被设计成根据需要，以适应喷口饮用。要考虑的一个重要的条件是舔率被称为壶嘴和碗喝13,26之间的不同。因此，无论是壶嘴或PEG-碗的选择VFSS必须在与实验一致。 第四个挑战是，以确定老鼠是媲美人类研究的研究和临床实践中常用的VFSS参数量化的燕子参数。我们的初步调查结果显示，透视系统的类型决定了吞参数可以在小鼠进行调查。大多数研究中心和医疗机构具有高能量（75-95千伏，1-5毫安）荧光镜设计用于人和大型动物，测试老鼠等小动物时，其导致的图像质量非常差的使用。作为一个例子，使用高能量透视老鼠最近的一项研究能够确定只4量化的吞参数12，并且我们能够对小鼠确定在这一本研究中只有7吞参数。为了克服这一主要限制，我们最近获得所谓的LabScope（格兰布鲁克Technologies）的低能量X线系统。该系统是一个微型透视生成一个连续的锥形束X射线15和40千伏和0.2mA的峰值管电流（8 W的最大功率）之间的光子能量。该系统的较低的能量水平更好的薄骨小鼠和软组织衰减，从而提供解释第1条比传统的（ 即高能量）R对比分辨率荧光镜。该LabScope的X射线束被引导为5厘米直径的图像增强器，其显着地比常规荧光镜的15-57厘米直径图像增强小。该LabScope的最小源 - 增强器的距离（SID）是〜6厘米（对比于约30厘米的常规荧光镜），它提供增加的放大能力。此外，该LabScope采用专利技术，数字化将图像放大至实时40倍，而不会改变SID。其结果是在本质上的X射线显微镜，能够放大和缩小在实时查看感兴趣的小区域，如鼠标的吞咽机制。 这种低能量X线透视系统的主要优点是改善辐射安全。除了动物接受较低的辐射剂量与该LabScope，使用该系统的研究人员暴露于显著莱的照射下散射。辐射照射直接在单元的控制面板前方是10.3 MR /小时。在设备前方的距离1M，暴露降至580μR/小时。在房间里其他大部分地区都低于10μR/小时极低的接触。尽管这样的改善，我们已经采取了额外措施，以改善辐射安全。例如，含铅丙烯酸屏蔽层已被周围的LabScope加入到阻止散射X射线光子，这使得研究人员进行小鼠VFSS测试无需佩戴个人防护（ 如铅围裙，甲状腺盾牌，和眼镜）。此外，透明的丙烯酸允许鼠标的可视距离的距离。进一步辐射安全是由机动剪刀升降台，这是由研究者远程控制提供。从远处可达3米的透视，研究人员可以使用远程控制装置向X射线BEA内调整观察室的垂直和水平位置米。其结果是，感兴趣的解剖区域可以保持视透视字段内，而鼠标自由观察室中移动。虽然剪叉式升降机被设计用于LabScope使用，它也是使用与传统荧光镜，以提高辐射安全研究人员兼容。最后一步，以改善辐射安全期间鼠VFSS要求使用的注射器输送系统的液体。这个系统包括一个3-4英尺（或更长的时间，如果需要的话）的PE管，它允许快速和有效地提供液体到钉碗从远处的长度。用于液体此注射器输送系统，在与观察室组合，也可与常规荧光镜。 使用该LabScope前期工作，在与新的鼠VFSS协议结合，表明了比传统的系统的一个主要优点是：可以可靠地量化I的吞参数的数量年近一倍。然而，小鼠的吞咽机制（ 例如，舌，软腭，咽后壁，会厌和）的软组织结构不容易看到使用低或高能量X线透视系统时。因此，我们针对量化推流的措施，而不是吞咽的生物力学。我们主要感兴趣的是可以基于时间，面积，距离，体积等 ，而不是使用李克特式量表措施为单位进行量化的参数。众多丸药流参数满足这个要求已在人类VFSS文献中描述，如口服途时间27-29，咽过境时间27-33和食管过境时间34-36，仅举几例。通过口腔丸药传输不在小鼠中容易看到，有可能是由于在自发饮用小丸剂的大小。但是，我们能够可靠地量化咽及食管过境时间，以及一切有关推流和清理其他一些措施。额外的翻译吞参数辨识有望为我们优化的LabScope的能力。 这项研究的结果表明，小鼠取每吞几个节奏舔期间自发饮酒，与每个小丸药液体依次触发咽吞之前填充vallecular空间。这种行为，这是典型的哺乳动物使用舔作为液体摄入37-40的主要手段，类似于人类婴儿的吞咽和一般所有哺乳动物的婴儿有节奏的吸吮吞咽模式。婴幼儿吞咽生理的特点是由几个有节奏吮吸之后反身咽吞，通常被描述为吸吞周期37,41-43。因此，涉及的小鼠的摄食舔行为的节奏舌头和下颚运动可能更媲美摄食嗡的抽吸行为一个婴儿而不是杯饮用由儿童和成人。因此，我们一直在量化小鼠舔率和舔吞咽比例为将来的比较与人类婴儿的吮吸率和吸吞比例。也许鼠VFSS研究将提供洞察发展吞咽障碍。 正如任何新的研究方法，需要改进的地方已被确定。例如，鼠VFSS协议是仅使用的C57和C57 / SJL小鼠品系开发;它尚未进行过测试大鼠。观察室将需要被放大的尺寸（直径和长度），以适应大鼠的较大的身体尺寸。此外，它是未知的，如果巧克力香料碘海醇是适合作为一种通用的鼠VFSS试验溶液。因此，较大规模的试验用小鼠和大鼠的多株是必要用于此目的。此外，使用钡作为鼠VFSS造影剂不应该被排除。小鼠显然倾向于iohex醇配方在钡;然而，更严格和系统的企图掩盖钡的厌恶味道/气味可以提供可口的替代碘海醇。口味偏好进行比较的碘海醇与硫酸钡的影响未来的研究（以及其他潜在口服造影剂）和吞咽生理学小鼠和大鼠无疑提供直接相关及平移人类VFSS重要信息。 VFSS与人类包括食品和液体数的一致性，和吞咽薄液体和干燥的固体食物时，44,45吞咽困难是最明显的。小鼠VFSS协议因此被扩大到包括额外的一致性，可能有利于吞咽困难的检测和定量的疾病模型。它也将是必要的，以进行液体配方鼠VFSS的粘度测试，以调节粘度，以匹配人类VFSS期间使用。解决这些限制ations将有助于确定吞咽困难的平移VFSS生物标志物，可以只小鼠，大鼠和人之间进行直接比较。 鼠VFSS的效用可通过注入不透射线标记成吞咽机制的软组织结构在其他方面不可见，从而允许吞咽的生物力学的调查来显著改善。这种方法已被成功地使用了许多年，以研究在吞咽婴儿猪，用金属夹和导线37,42的各种各样的生物力学。我们预计在小鼠中使用的相似，但较小，标记将允许若干额外吞参数与较大的哺乳动物，包括人类在内的比较定量。我们目前正在开发的方法用于植入的不透射线标记物插入舌头，软腭，咽，喉，和小鼠的近端食道检验这一假设。 视频recordin的LabScope和常规荧光镜克帧速率限制为每秒（fps）的30帧。然而，我们的初步结果表明，对于吞咽健康小鼠的整个咽阶段发生在小于66毫秒（ 即，2个帧），这是比人类快大约10倍。因此，吞咽在小鼠的咽阶段发生如此之快，细节不是明显具有每秒30帧的摄像头。较高的帧速率（可能&gt; 100 fps）时，有必要充分地可视化和量化吞咽在小鼠和其它啮齿动物的咽阶段的极为迅速的和复杂的动作。在具有较高帧速率的同时，掺入双平面技术用于3D荧光透视成像肯定扩大效用鼠VFSS。因此，未来的设计考虑因素应该包括更高的帧速率的摄像头和双平面成像能力。 最后，低剂量辐射已被证明可导致不育雌性C57小鼠，导致卵巢刺激激素可能混淆寿命研究46物质的改变。具体涉及与VFSS测试相关反复的低剂量辐射暴露的影响的结果还没有被研究在小鼠中，其他的动物或人类。然而，卵巢功能障碍（不相关的辐射暴露）的人类女性已与胃肠运动障碍，并且具体地涉及吞咽困难在某些情况下47，这提供了另一种警告设计未来VFSS研究，包括雌性（动物和人类时，需要考虑）。应避免排斥女性，如在吞咽功能显著的性别差异已报道的人48,49和将是重要的，以检测和表征在动物疾病模型中也是如此。因此，从在小鼠和大鼠两种性别的纵向VFSS研究成果具有巨大潜力的平移人类相至dysphagia，以及低剂量的辐射暴露的与重复VFSS测试相关的风险。

吞咽困难（吞咽障碍）是影响所有年龄的人很多医疗条件的常见症状。实例包括中风，帕金森氏病，阿尔茨海默氏病，脑性麻痹，进行性肌营养不良，肌萎缩性侧索硬化症（ALS），Batten病，头颈癌，早产，以及先进的老化。吞咽困难是高度相关的死亡率，通常为严重营养不良或肺炎，开发，当细菌载货食品/液/唾液被吸入肺部1-4的结果。这衰弱和威胁生命的医疗条件影响，每年在美国超过1500万的人仅3。尽管高患病率和相关的负面结果，对吞咽困难目前的治疗方案是有限的姑息性（而非治疗）方法，如饮食修改（ 比如，避免特定的食品/液体稠度），体位变化（ 如 tucki纳克吞咽时供给下巴），马 ​​达的方法（ 例如，行使在口腔癌，咽癌靶向肌肉，和喉），感觉的方法（ 例如，实施味道，温度，和/或机械刺激）和管（ 例如，营养和水化通过鼻饲（NG）管或经皮内镜下胃造瘘术（PEG）管）进行管理。这些治疗方法仅仅作为对症治疗，而不是针对问题的根本原因。事实上，一个主要障碍新颖，有效的治疗方法发现的吞咽困难是负责任的病理机制，这对于每种疾病有可能不同的有限的科学知识。 吞咽困难诊断用放射线照相过程称为videofluoroscopic吞咽研究（VFSS），也称为改性吞钡研究主要制成。在过去的30多年里，这个诊断测试一直被认为是黄金标准的EValuating吞咽功能5-7。此测试要采取具有病人坐在或站在一个透视机器的X射线束的路径内而自愿摄入的食物和液体的一致性与口服造影剂混合，通常硫酸钡8,9或碘海醇10。作为患者吞咽，食品和含有造影剂的液体，可以实时地经由计算机监视器，而从嘴到胃行进看见。软组织结构也都可见，并且可以相对于结构和功能进行评估。病人被要求执行每种食物和液体稠度的几个燕子，所有这一切都是记录以便随后观看和帧接一帧的分析来量化吞咽困难的存在和程度的视频。吞咽的众多生理组分一般分析，如解剖触发点咽吞的，丸剂过境时间通过laryn的咽和食管，程度和持续时间geal标高，位置和后吞咽残留量，以及发生的生理原因抽吸7,11。 人类VFSS协议方面最近改编为研究自由，大鼠的行为;然而，结果是有限的，因为老鼠没有留在视videofluoroscopic场测试12期间。 VFSS以前未曾尝试用小鼠。成功适应人类VFSS协议与小鼠和大鼠的使用将提供一种新的研究方法，调查了数百现有鼠（小鼠和大鼠）的已知会引起吞咽困难在人类疾病模型。因此，这种新的方法（以下称为鼠VFSS）将加速辨识吞咽困难的小鼠模型适合于调查肌肉，神经和脑组织内的是病态的底层神经生理机制和促进吞咽困难和验证我ñ人类。此外，鼠类VFSS将允许识别燕子功能/功能障碍可能与人类直接进行比较的客观指标（生物标记物）。这些跨物种videofluoroscopic生物标志物便可以作为新的观察指标进行量化与小鼠和大鼠的临床前试验，这将更好地转化为与人的临床试验疗效。 为此，鼠VFSS协议，使用100〜小鼠任一性别的确立。所有小鼠要么C57或C57混合动力/ SJL株。在C57小鼠均未遗传改变，而C57 / SJL是背景菌株的转基因SOD1-G93A（或SOD1）小鼠的ALS的最广泛使用的动物模型的一个菌落。在SOD1殖民地是一个近似的50-50组合的转基因（ 即 ALS影响）小鼠和非转基因（ 即不受影响）窝的。 小鼠VFSS协议由三部分组成： <ol><l我&gt;自定义设计的观察室，允许自愿喂养和吞咽而透视机内站立奔放在密闭的空间， </li><li>食谱掩盖口服造影剂的厌恶味道/气味并产生足够的放射密度，以允许吞咽的适当的可视化， </li><li>一个一步一步的测试协议，最大限度地提高动物的合规性，最大限度地减少总测试时间和辐射暴露，并允许几个燕子参数量化吞咽（ 即，口腔，咽，食管）的每个阶段。 </li></ol>的共同作用产生一个舒适，低应力，自我喂养检查环境允许的典型进料和小鼠的吞咽行为评估。

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Name of Material/ Equipment</td>
			<td >Company</td>
			<td >Catalog Number</td>
			<td >Comments/Description</td>
		</tr>
		<tr >
			<td >Polycarbonate tubing for observation chambers</td>
			<td >McMaster-Carr</td>
			<td >3161T41</td>
			<td>Body of observation tubes, 2&#34;X2&#34; diameter, 0.080&#34; thick wall</td>
		</tr>
		<tr >
			<td >Polycarbonate sheet&#160; for observation chambers</td>
			<td >McMaster-Carr</td>
			<td >9115K71</td>
			<td>End-caps for observation tubes, 2&#34;x12&#34;x3/4&#34;</td>
		</tr>
		<tr >
			<td >Polycarbonate sheet&#160; for observation chambers</td>
			<td >McMaster-Carr</td>
			<td >8574K281</td>
			<td>Peg-bowls for observation tubes</td>
		</tr>
		<tr >
			<td >Silicone O-rings&#160; for end-caps of observation chambers</td>
			<td >McMaster-Carr</td>
			<td >9396K108</td>
			<td>S1138 AS568-029, pack of 25 
			http://www.mcmaster.com/#o-rings/=t0wt5r&#160;</td>
		</tr>
		<tr >
			<td >Silicone stoppers for observation chambers</td>
			<td >McMaster-Carr</td>
			<td >2903K22</td>
			<td>Package of 10 stoppers to plug the oval opening in the top of the observation chamber when using a peg-bowl 
			http://www.mcmaster.com/#catalog/120/3803/=t0y5at</td>
		</tr>
		<tr >
			<td >Centrifuge tubes for sipper tube bottles</td>
			<td >Evergreen Scientific</td>
			<td >222-3530-G80</td>
			<td>30 ml freestanding centrifuge tubes, with caps, sterile 
			https://www.evergreensci.com/labware-catalog/tubes-and-vials/30-and-50-ml-centrifuge-tubes/&#160;</td>
		</tr>
		<tr >
			<td >Silcone stoppers for sipper tube bottles</td>
			<td >Saint-Gobain Performance Plastics</td>
			<td >DX263031-10&#160;</td>
			<td>Number 31D, size: 26 mm bottom, 32 mm top, 30 mm high; 10 pack;&#160; 
			http://www.labpure.com/en/Products.asp?ID=179&#38;PageBrand=STOPPERS</td>
		</tr>
		<tr >
			<td >Stopper borers for sipper tube bottles</td>
			<td >Thomas Scientific</td>
			<td >3276G40</td>
			<td>Cork Borer Set that ranges from 3/16-15/16 inch&#160; 
			http://www.thomassci.com/Supplies/Corks/_/CORK-BORER-SET-316-1516-IN?q=Humboldt</td>
		</tr>
		<tr >
			<td >Drinking tubes for sipper tube bottles</td>
			<td >Ancare</td>
			<td >TD-100&#160;</td>
			<td>2 1/2&#8221; long drinking tubes with 5/16&#8221; opening, straight ball-spout 
			http://www.ancare.com/products/watering-equipment/open-drinking-tubes/straight-tubes-ball-point&#160;</td>
		</tr>
		<tr >
			<td >Iohexol for making oral contrast agent solution</td>
			<td >GE Healthcare</td>
			<td ></td>
			<td>350 mg iodine per ml 
			http://www3.gehealthcare.com/en/products/categories/contrast_media/omnipaque&#160;</td>
		</tr>
		<tr >
			<td >Chocolate syrup for flavoring oral contrast agent</td>
			<td >Herseys</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >10 ml syringe for syringe delivery system</td>
			<td >Becton, Dickinson and Company</td>
			<td >309604</td>
			<td>Luer lock tip syringe without needle, 100 per box 
			http://www.bd.com/hypodermic/products/syringeswithoutneedles.asp</td>
		</tr>
		<tr >
			<td >Catheter tubing for syringe delivery system</td>
			<td >Becton, Dickinson and Company</td>
			<td >427451</td>
			<td>Polyethylene Tubing (Non-Sterile) (PE 240) 100&#39; 
			http://www.bd.com/ds/productCenter/427451.asp&#160;</td>
		</tr>
		<tr >
			<td >Needle for syringe delivery system</td>
			<td >Becton, Dickinson and Company</td>
			<td >427560</td>
			<td>15-gauge needle, fits into PE 240 catheter tubing 
			http://www.bd.com/ds/productCenter/427560.asp&#160;</td>
		</tr>
		<tr >
			<td >Delrin acetal resin rod for syringe delivery system</td>
			<td >McMaster-Carr</td>
			<td >8576K15</td>
			<td>1/2 inch diameter, black 
			http://www.mcmaster.com/#catalog/120/3609/=t0wvaf&#160;</td>
		</tr>
		<tr >
			<td >Acrylic sheeting for scissor lift</td>
			<td >Ponoko</td>
			<td ></td>
			<td>Laser cut 
			http://www.ponoko.com&#160;</td>
		</tr>
		<tr >
			<td >3D printed ABS frame</td>
			<td >Engineering Rapid Prototyping Facility, University of Missouri</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Brass rods for scissor lift</td>
			<td >Amazon</td>
			<td >TTRB-03-12-03</td>
			<td>made into axles 
			http://www.amazon.com/Brass-Seamless-Round-Tubing-Length/dp/B000FN898M</td>
		</tr>
		<tr >
			<td >Drawer slide for scissor lift</td>
			<td >Richelieu</td>
			<td >10292G116</td>
			<td>Attaches to base of scissor lift 
			http://www.lowes.com/pd_380986-93052-T35072G16_0__?productId=50041754</td>
		</tr>
		<tr >
			<td >28BYJ-48 stepper motor for scissor lift</td>
			<td ></td>
			<td ></td>
			<td>2 each</td>
		</tr>
		<tr >
			<td >ULN2003 Darlington transistor array for scissor lift</td>
			<td >Toshiba</td>
			<td >ULN2003APG</td>
			<td>Used as stepper drivers (2 each)</td>
		</tr>
		<tr >
			<td >ATTINY85 microcontroller for scissor lift</td>
			<td >Atmel</td>
			<td >ATTINY85-20PU</td>
			<td>2 each 
			http://www.taydaelectronics.com/attiny85-attiny85-20pu-8-bit-20mhz-microcontroller-ic.html</td>
		</tr>
		<tr >
			<td >Nylon spur gear</td>
			<td >McMaster-Carr</td>
			<td >57655K34</td>
			<td>2 each 
			http://www.mcmaster.com/#57655k34/=t0yaqz</td>
		</tr>
		<tr >
			<td >Nylon spur gear rack</td>
			<td >McMaster-Carr</td>
			<td >57655K62</td>
			<td>2 each 
			http://www.mcmaster.com/#57655k62/=t0ybh9</td>
		</tr>
		<tr >
			<td >4-40 nylon machine screws</td>
			<td >McMaster-Carr</td>
			<td >95133A315</td>
			<td>Lift assembly 
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			<td>Lift assembly 
			http://www.mcmaster.com/#94812a200/=t0ye29</td>
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			<td >Buna-N O-Ring AS568A Dash No. 104</td>
			<td >McMaster-Carr</td>
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adapting human videofluoroscopic swallow study methods to detect and characterize dysphagia in murine disease models

这项研究适应人类videofluoroscopic吞咽研究（VFSS）方法用于小鼠疾病模型用于促进转化研究吞咽困难的目的。成功的结果取决于三个关键组成部分：试验室，允许自进，而在有限的空间站在奔放，掩盖市售的口服造影剂的厌恶味道/气味的食谱，并一步一步的测试协议允许燕子生理量化。消除其中的一个或多个部件，不会对研究的结果有不利的影响。此外，透视系统的能量水平的能力将决定哪些吞参数可以进行调查。大多数研究中心有专为人们和更大的动物，测试老鼠等小老鼠时，这会导致图像质量非常差的高能量荧光镜。尽管有此限制，我们已经确定了七个VFS的S参数组合使用高能量透视用新的鼠VFSS协议时是在小鼠中始终可量化的。最近，我们获得了低能量X光透视系统被设计用于小鼠和其他小型啮齿类动物使用极高的成像分辨率和放大功能。使用这种新的系统与新的鼠VFSS协议前期工作的结合使用，已确定了13吞参数是在小鼠中始终如一地量化的，这是近两倍使用常规（即，高能量）中获得的数字荧光镜。额外的燕子参数辨识预计为我们优化这个新系统的功能。结果迄今为止证明使用低能量X线透视系统来检测和量化使用高能荧光镜来调查鼠疾病模型时可能被忽视在吞咽生理学微妙变化的效用。

我们已经成功地设计了一种新的和可复制的鼠特异性VFSS协议，其包括测试室，其允许自进，食谱调味口服造影剂，并在步一步的测试协议，允许吞咽生理学的量化。的透视系统的能量水平的能力来确定该吞服参数可在小鼠中进行研究。我们最初用于设计用于与人及较大的动物使用高能量荧光镜（ 例如，通用电气公司Advantx，GE OEC 9600，和欧米茄心脏蛋白酶的CS-25，各以每秒30帧）。然而，这些系统具有放大能力不足，用于测试的小鼠，导致动物填充的视场中的一小部分 （ 图9）。作为结果，图像质量是格外差，使得它不可能以可视化的吞咽机制的最结构。尽管有此限制，我们确定了7客观VFSS吞组合使用常规的（ 即，高能量）透视与新的鼠VFSS协议（ 表3）当在始终量化的小鼠的参数。此外，我们确定了vallecular空间作为解剖触发点吞咽在健康成年小鼠（3-17个月的年龄），以及小鼠的高龄（&gt; 18个月）和末期的ALS的条件。 <img alt="图9" src="/files/ftp_upload/52319/52319fig9highres.jpg" width="650" /> 图9：高能透视系统左：使用高能量（ 即传统的）透视系统获得鼠标的形象代表。需要注意的是鼠标填充视透视字段的仅一小部分，从而表明对于成像啮齿类常规荧光镜的不足倍率能力。右：同样的图像放大后captu重新使用视频编辑软件程序。黑色箭头：吞触发点（valleculae）。白色箭头：推在食管远端前夕，通过GE结（白星号） <a href="https://www.jove.com/files/ftp_upload/52319/52319fig9highres.jpg" target="_blank">，请点击此处查看该图的放大版本。</a> <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>燕子参数</td><td style="width:140px">高能源系统</td><td style="width:140px">低能源系统</td></tr><tr><td>间燕子间隔（ISI） </td><td> X </td><td> X </td></tr><tr><td>颚游览率（利克汇率折合） </td><td> X </td><td> X </td></tr><tr><td>颚漂移距离</td><td> X </td><td> X </td></tr><tr><td>舔，燕子比</td><td> X </td><td> X </td></tr><tr><td>燕子率</td><td> X </td><td> X </td></tr><tr><td> PharyngEAL通过时间（PTT） </td><td></td><td> X </td></tr><tr><td>通过咽丸速度</td><td></td><td> X </td></tr><tr><td>食管通过时间（ETT） </td><td></td><td> X </td></tr><tr><td>通过食管丸速度</td><td></td><td> X </td></tr><tr><td>推注速度通过咽食管和</td><td> X </td><td> X </td></tr><tr><td>丸区</td><td></td><td> X </td></tr><tr><td>咽部残留区</td><td></td><td> X </td></tr><tr><td>液体量消耗</td><td> X </td><td> X </td></tr></tbody></table> 表3：燕子参数量化使用高与低能源透视系统。 最近，我们获得了低能量放大透视系统，称为LabScope（格兰布鲁克技术，兰多夫，新泽西州），这是专门为我们的实验室设计与应用老鼠等小型啮齿动物（ 图10）。然而，这种系统的显着更大的放大倍率水平呈现无法查看的视图中的单个字段的鼠标整个吞咽的机制。相反，两个测试位置是必需的， 如图11。整个头部的位置1许可的可视化和近侧胸部区域。这一立场是必要的评估吞咽的口腔和咽部阶段。位置2允许可视化从燕子的触发点（ 即，valleculae）的胃（GE）交界处。这一立场是必要的评估吞咽的食管阶段。组合使用的LabScope用新的鼠VFSS协议初步工作已经确定了13个目标吞参数是在小鼠中始终如一地量化的，这是近两倍使用高能（ 即，常规）荧光镜（ 表3）中得到的数目。这Ø<html> utcome归因于该LabScope，其允许许多解剖结构的可视化（ 图12）使用常规系统时基本上不可见的先进倍率功能： 例如，舌骨，气管，和颈椎。其结果是，我们还能够分析视频用于喉渗透和抽吸证据。无论是普及率也不愿望观察到在这项研究中任何鼠标，无论健康或疾病状态。 <img alt="图10" src="/files/ftp_upload/52319/52319fig10highres.jpg" width="650" /> 图10：本LabScope左：LabScope执行作为桌面透视小动物。右：特写观点与标记组件LabScope。剪刀升降台的定位来看的透视领域内的观察室。<html> tp_upload / 52319 / 52319fig10highres.jpg“目标=”_空白“&gt;点击此处查看该图的放大版本。 <img alt="图11" src="/files/ftp_upload/52319/52319fig11highres.jpg" width="650" /> 图11：使用低能量荧光系统所获得的小鼠低能透视系统映像。需要注意的是高倍率能力防止视透视字段内的整个吞咽机构的可视化。左：位置1 - 允许在整个头部和胸部近端区域的可视化。燕子触发点（黑箭头）基本上视野内居中。右图：2的位置 - 从燕子触发点（黑色箭头）允许可视化的GE交界处（白星号）。注意推穿过食管远端（白色箭头）。<html> g11highres.jpg“目标=”_空白“&gt;点击此处查看该图的放大版本。 <img alt="图12" src="/files/ftp_upload/52319/52319fig12highres.jpg" width="650" /> 图12：解剖结构可见使用低能量X线透视系统即使在最低的放大倍率设置（左），鼠标的头部和颈部的骨骼结构清晰可见用我们的低能量X线透视系统（ 即 LabScope）。黑色正方形内的解剖结构被示出（且标）在更高的放大倍数在右边。提高BONEY结构的可视化允许一些额外的燕子参数是不可能使用高能荧光镜分析定量。 <a href="https://www.jove.com/files/ftp_upload/52319/52319fig12highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 吨“&gt;吞率和跨吞咽间隔是可以使用低或高能量透视系统结合了新的鼠VFSS协议来量化代表VFSS参数这两个吞参数进行量化为三组小鼠：SOD1-G93A （SOD1）的转基因小鼠（即 ALS的模型），在疾病的终末期4-5个月的年龄，年龄C57小鼠（18-24个月的年龄）之间，和健康年轻（4-8个月的对照组C57小鼠和来自SOD1菌落非转基因同窝出生的年龄）。所有的数据属于只喷出饮用，使用一个低或高能量透视系统。年轻C57小鼠和非转基因幼（对照）的小鼠的SOD1之间没有发现差异显著相对于这两个燕子参数的殖民地;因此，数据合并为与年龄C57小鼠和终末期SOD1小鼠相比一般的“控制”一群年轻健康的老鼠吞率（ 即，燕子在连续2秒不间断饮用的）的数目是显著慢于SOD1小鼠相比老年C57小鼠和控制。间燕子间隔（即两个连续的燕子之间的时间）是不是群体之间显著的不同。这些发现支持这一概念吞咽困难型材很可能是对每个疾病状态（ 图13）明显不同。 <img alt="图13" src="/files/ftp_upload/52319/52319fig13highres.jpg" width="650" /> 图13：初步结果此图显示了两个VFSS燕子参数代表的初步调查结果用鼠VFSS协议量化：吞率（左）和跨燕子间隔（右）。燕子率显著慢的SOD1小鼠相比，年龄C57小鼠和控制。被确定为跨燕子跨不显著的组间差异VAL。线在酒吧的顶部显示组间统计学差异显著（P &lt;0.05），使用邦费罗尼两两比较鉴定。误差线代表±1 SEM。 <a href="https://www.jove.com/files/ftp_upload/52319/52319fig13highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Adapting Human Videofluoroscopic Swallow Study Methods to Detect and Characterize Dysphagia in Murine Disease Models at JoVE.com

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

这项研究成功地适应人类videofluoroscopic吞咽研究（VFSS）方法用于小鼠疾病模型用于促进转化研究吞咽困难的目的。

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

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

adapting-human-videofluoroscopic-swallow-study-methods-to-detect

Research

JoVE Journal

Medicine

20.9K 次观看.  University of Missouri. 这项研究成功地适应人类videofluoroscopic吞咽研究（VFSS）方法用于小鼠疾病模型用于促进转化研究吞咽困难的目的。 

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

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

Spinal cord injury (SCI) is a debilitating disorder, which produces profound deficits in volitional motor control. Following medical stabilization, recovery from SCI typically involves long term rehabilitation. While recovery of walking ability is a primary goal in many patients early after injury, those with a motor incomplete SCI, indicating partial preservation of volitional control, may have the sufficient residual descending pathways necessary to attain this goal. However, despite physical interventions, motor impairments including weakness, and the manifestation of abnormal involuntary reflex activity, called spasticity or spasms, are thought to contribute to reduced walking recovery. Doctrinaire thought suggests that remediation of this abnormal motor reflexes associated with SCI will produce functional benefits to the patient. For example, physicians and therapists will provide specific pharmacological or physical interventions directed towards reducing spasticity or spasms, although there continues to be little empirical data suggesting that these strategies improve walking ability.
In the past few decades, accumulating data has suggested that specific neuromodulatory agents, including agents which mimic or facilitate the actions of the monoamines, including serotonin (5HT) and norepinephrine (NE), can initiate or augment walking behaviors in animal models of SCI. Interestingly, many of these agents, particularly 5HTergic agonists, can markedly increase spinal excitability, which in turn also increases reflex activity in these animals. Counterintuitive to traditional theories of recovery following human SCI, the empirical evidence from basic science experiments suggest that this reflex hyper excitability and generation of locomotor behaviors are driven in parallel by neuromodulatory inputs (5HT) and may be necessary for functional recovery following SCI. 
The application of this novel concept derived from basic scientific studies to promote recovery following human SCI would appear to be seamless, although the direct translation of the findings can be extremely challenging. Specifically, in the animal models, an implanted catheter facilitates delivery of very specific 5HT agonist compounds directly onto the spinal circuitry. The translation of this technique to humans is hindered by the lack of specific surgical techniques or available pharmacological agents directed towards 5HT receptor subtypes that are safe and effective for human clinical trials. However, oral administration of commonly available 5HTergic agents, such as selective serotonin reuptake inhibitors (SSRIs), may be a viable option to increase central 5HT concentrations in order to facilitate walking recovery in humans. Systematic quantification of how these SSRIs modulate human motor behaviors following SCI, with a specific focus on strength, reflexes, and the recovery of walking ability, are missing.
This video demonstration is a progressive attempt to systematically and quantitatively assess the modulation of reflex activity, volitional strength and ambulation following the acute oral administration of an SSRI in human SCI. Agents are applied on single days to assess the immediate effects on motor function in this patient population, with long-term studies involving repeated drug administration combined with intensive physical interventions.

This video demonstrates modulation of reflex activity, volitional strength and ambulation through clinical and quantitative assessments in individuals with motor incomplete SCI as a result of acute oral administration of a serotonin reuptake inhibitor (SSRI).

量化人类不完全脊髓损伤运动功能的药理学诱导改建的方法

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

科研

医学

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed 1, 2. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.
SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope 3, 4. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies 5, 6. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes 7-10.
The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (<a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a>).
SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia 11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models 14. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).

Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.

光谱核型多囊肾病的研究在人类和小鼠的染色体异常

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease (PD) pathogenesis are less understood. Gene-environment interaction models have utility in unmasking the impact of specific cellular pathways in toxicity that may not be observed using a solely genetic or toxicant disease model alone.&#160;To evaluate if distinct LOX isozymes selectively contribute to PD-related neurodegeneration, transgenic (i.e. 5-LOX and 12/15-LOX deficient) mice can be challenged with a toxin that mimics cell injury and death in the disorder.&#160;Here we describe the use of a neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces a nigrostriatal lesion to elucidate the distinct contributions of LOX isozymes to neurodegeneration related to PD. The use of MPTP in mouse, and nonhuman primate, is well-established to recapitulate the nigrostriatal damage in PD.&#160;The extent of MPTP-induced lesioning is measured by HPLC analysis of dopamine and its metabolites and semi-quantitative Western blot analysis of striatum for tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine. To assess inflammatory markers, which may demonstrate LOX isozyme-selective sensitivity, glial fibrillary acidic protein (GFAP) and Iba-1 immunohistochemistry are performed on brain sections containing substantia nigra, and GFAP Western blot analysis is performed on striatal homogenates.&#160;This experimental approach can provide novel insights into gene-environment interactions underlying nigrostriatal degeneration and PD.

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson&#039;s Disease

Lipoxygenase (LOX) isozymes can generate products that may increase or decrease neuroinflammation and neurodegeneration.&#160;A gene-environment interaction study could identify LOX isozyme-specific effects. Using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of nigrostriatal damage in two LOX isozyme-deficient transgenic lines allows for comparison of the contribution of LOX isozymes on dopaminergic integrity and inflammation.

基因-环境相互作用模型揭开帕金森病的隐形易感机制

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease ...

Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of care consisting of surgical debulking and combination chemotherapy consisting of platinum and taxane compounds, almost 90% of patients recur within a few years. In these patients the development of chemoresistant disease limits the efficacy of currently available chemotherapy agents and therefore contributes to the high mortality. To discover novel therapy options that can target recurrent disease, appropriate animal models that closely mimic the clinical profile of patients with recurrent ovarian cancer are required. The challenge in monitoring intra-peritoneal (i.p.) disease limits the use of i.p. models and thus most xenografts are established subcutaneously. We have developed a sensitive optical imaging platform that allows the detection and anatomical location of i.p. tumor mass. The platform includes the use of optical reporters that extend from the visible light range to near infrared, which in combination with 2-dimensional X-ray co-registration can provide anatomical location of molecular signals. Detection is significantly improved by the use of a rotation system that drives the animal to multiple angular positions for 360 degree imaging, allowing the identification of tumors that are not visible in single orientation. This platform provides a unique model to non-invasively monitor tumor growth and evaluate the efficacy of new therapies for the prevention or treatment of recurrent ovarian cancer.

We describe a non-invasive animal imaging platform that allows the detection, quantification, and monitoring of ovarian cancer growth and recurrence. This intra-peritoneal xenograft model mimics the clinical profile of patients with ovarian cancer.

小鼠模型的非侵入性成像检测和监测卵巢癌复发

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of ...

Isolation and Excision of Murine Aorta; A Versatile Technique in the Study of Cardiovascular Disease

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated blood is the aorta. The aorta may become affected in diseases such as atherosclerosis and aneurysm. Researchers investigating these diseases would benefit from direct observation of the aorta to characterize disease progression as well as to evaluate efficacy of potential therapeutics. The goal of this protocol is to describe proper isolation and excision of the aorta to aid investigators researching cardiovascular disease. Isolation and excision of the aorta allows investigators to look at gross morphometric changes as wells as allowing them to preserve and stain the tissue to look at histologic changes if desired. The aorta may be used for molecular studies to evaluate protein and gene expression to discover targets of interest and mechanisms of action. This technique is superior to imaging modalities as they have inherent limitations in technology and cost. Additionally, primary isolated cells from a freshly isolated and excised aorta can allowing researchers to perform further in situ and in vitro assays. The isolation and excision of the aorta has the limitation of having to sacrifice the animal however, in this case the benefits outweigh the harm as it is the most versatile technique in the study of aortic disease.

Pathology of the aorta can lead to severe morbidity and mortality, therefore research of disease progression and potential therapies is warranted. Here, we present a protocol to isolate and excise the murine aorta to aid researchers in their investigation of cardiovascular disease.

隔离和小鼠主动脉切除;心血管疾病研究的一个多功能的技术

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated ...

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling M&#252;ller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5&#160;has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label M&#252;ller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

使用腺相关病毒作为一种工具来研究疾病中的视网膜障碍

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little is known about breast cancer cell properties and behaviors within the native microenvironment of human bone tissue.We have developed approaches to track, quantify and modulate human breast cancer cells within the microenvironment of cultured human bone tissue fragments isolated from discarded femoral heads following total hip replacement surgeries. Using breast cancer cells engineered for luciferase and enhanced green fluorescent protein (EGFP) expression, we are able to reproducibly quantitate migration and proliferation patterns using bioluminescence imaging (BLI), track cell interactions within the bone fragments using fluorescence microscopy, and evaluate breast cells after colonization with flow cytometry. The key advantages of this model include: 1) a native, architecturally intact tissue microenvironment that includes relevant human cell types, and 2) direct access to the microenvironment, which facilitates rapid quantitative and qualitative monitoring and perturbation of breast and bone cell properties, behaviors and interactions. A primary limitation, at present, is the finite viability of the tissue fragments, which confines the window of study to short-term culture. Applications of the model system include studying the basic biology of&#160;breast cancer and other bone-seeking malignancies within the metastatic niche, and developing therapeutic strategies to effectively target breast cancer cells in bone tissues.

Protocols are described for studying breast cancer cell migration, proliferation and colonization in a human bone tissue explant model system.

方法培养人股骨组织外植体，究其利基转移性乳腺癌细胞定居

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little ...

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying tissue compartments, restricting the transport of smaller molecules across the epithelium and almost completely prohibiting epithelial macromolecular transport. This selectivity is determined by the mucous gel layer, which limits the transport of lipophilic molecules and both the apical receptors and tight junctional protein complexes of the epithelium. In vitro cell culture models of the epithelium are convenient, but as a model, they lack the complexity of interactions between the microbiota, mucous-gel, epithelium and immune system. On the other hand, in vivo assessment of intestinal absorption or permeability may be performed, but these assays measure overall gastrointestinal absorption, with no indication of site specificity. Ex vivo permeability assays using &#34;intestinal sacs&#34; are a rapid and sensitive method of measuring either overall intestinal integrity or comparative transport of a specific molecule, with the added advantage of intestinal site specificity. Here we describe the preparation of intestinal sacs for permeability studies and the calculation of the apparent permeability (Papp) of a molecule across the intestinal barrier. This technique may be used as a method of assessing drug absorption, or to examine regional epithelial barrier dysfunction in animal models of gastrointestinal disease.

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

This protocol describes the use of excised intestinal tissue preparations or "intestinal sacs" as an ex vivo model of intestinal barrier function. This model may be used to assess integrity of both the epithelial barrier and the mucous gel layer at specific intestinal sites in animal models of digestive disease.

体外肠囊评估黏膜通透性在胃肠道疾病模型

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying ...

Methods for Acute and Subacute Murine Hindlimb Ischemia

Peripheral artery disease (PAD) is a leading cause of cardiovascular morbidity and mortality in developed countries, and animal models that reliably reproduce the human disease are necessary to develop new therapies for this disease. The mouse hindlimb ischemia model has been widely used for this purpose, but the standard practice of inducing acute limb ischemia by ligation of the femoral artery can result in substantial tissue necrosis, compromising investigators&#39; ability to study the vascular and skeletal muscle tissue responses to ischemia. An alternative approach to femoral artery ligation is the induction of gradual femoral artery occlusion through the use of ameroid constrictors. When placed around the femoral artery in the same or different locations as the sites of femoral artery ligation, these devices occlude the artery over 1 - 3 days, resulting in more gradual, subacute ischemia. This results in less substantial skeletal muscle tissue necrosis, which may more closely mimic the responses seen in human PAD. Because genetic background influences outcomes in both the acute and subacute ischemia models, consideration of the mouse strain being studied is important in choosing the best model. This paper describes the proper procedure and anatomical placement of ligatures or ameroid constrictors on the mouse femoral artery to induce subacute or acute hindlimb ischemia in the mouse.

Surgical induction of hindlimb ischemia in the mouse is useful to examine angiogenesis, however this is compromised in certain inbred mouse strains that display marked ischemia-induced tissue necrosis. Methods are described to induce subacute limb ischemia using ameroid constrictors to circumvent this problem through the induction of gradual arterial occlusion.

对急性和亚急性鼠下肢缺血的方法

Peripheral artery disease (PAD) is a leading cause of cardiovascular morbidity and mortality in developed countries, and animal models that reliably ...

Ultrasound-guided Intracardiac Injection of Human Mesenchymal Stem Cells to Increase Homing to the Intestine for Use in Murine Models of Experimental Inflammatory Bowel Diseases

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have immunosuppressive potential and have been shown to suppress inflammation in mouse models of intestinal inflammation, even though the route of administration can limit their homing and effectiveness 1,3,4,5. Local application of MSCs to colonic injury models has shown greater efficacy at ameliorating inflammation in the colon. However, there is paucity of data on techniques to enhance the localization of human bone marrow-derived MSCs (hMSCs) to the small intestine, the site of inflammation in the SAMP-1/YitFc (SAMP) model of experimental Crohn&#39;s disease. This work describes a novel technique for the ultrasound-guided intracardiac injection of hMSCs in SAMP mice, a well-characterized spontaneous model of chronic intestinal inflammation. Sex- and age-matched, inflammation-free AKR/J (AKR) mice were used as controls. To analyze the biodistribution and the localization, hMSCs were transduced with a lentivirus containing a triple reporter. The triple reporter consisted of firefly luciferase (fl), for bioluminescent imaging; monomeric red fluorescent protein (mrfp), for cell sorting; and truncated herpes simplex virus thymidine kinase (ttk), for positron emission tomography (PET) imaging. The results of this study show that 24 h after the intracardiac administration, hMSCs localize in the small intestine of SAMP mice as opposed to inflammation-free AKR mice. This novel, ultrasound-guided injection of hMSCs in the left ventricle of SAMP mice ensures a high success rate of cell delivery, allowing for the rapid recovery of mice with minimal morbidity and mortality. This technique could be a useful method for the enhanced localization of MSCs in other models of small-intestinal inflammation, such as TNF&#916;RE6. Future studies will determine if the increased localization of hMSCs by intra-arterial delivery can lead to increased therapeutic efficacy.

Murine studies in models of colonic inflammation have demonstrated that a small percentage (1 - 5%) of mesenchymal stem cells (MSC) injected intravenously or intraperitoneally home to the inflamed colon1,2. This study shows that ultrasound-guided intracardiac injections of MSCs result in increased localization to the intestine.

超声引导下人骨髓间充质干细胞在实验性肠炎小鼠模型中的应用

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have ...