作者感谢海军研究办公室(格兰特#12166253)的资助。此外,作者衷心感谢动物护理人员、兽医以及堪萨斯州立大学和弗吉尼亚理工大学的学生在动物工作期间给予的支持。作者还要感谢娜迪格·克雷布斯的技术援助,感谢谢尔比·楼梯、莎拉·格林威和米凯拉·戈林的技术援助和额外的动物护理。

每个地点(堪萨斯州立大学和弗吉尼亚理工大学)的单独 IACUC 都批准了设施和程序的使用。
1. 相机和笔的设置和常规的建立
<ol><li>在将动物放入笔中之前,请将相机以 90° 角固定在每支笔上(请参阅建议的相机系统的材料表)。</li>
	<li>在研究期间或仅在测试期间以 30 帧/秒 (fps) 连续记录动物。</li>
	<li>用螺栓和链子固定碗、水器、垫子和玩具。</li>
	<li>将连续播放白色或粉红色噪音(例如瀑布声)的音响机放置在猪舍内。 注:外部突然噪音(例如,门打开和关闭)在会议23、24期间会导致突然反射。</li>
	<li>在设施内用笔随机化或分层处理。</li>
	<li>
建立既定的畜牧业常规。这将有助于猪知道什么时候期望人类清洁笔,饲料和处理它们,并执行测试。
	<ol>
<li>使用熟悉的治疗,让猪与人类联系在一起的奖励。</li>
		<li>在奖励期间使用点击器,允许猪将点击声音与奖励相关联。不要使用发声和视觉信号来使猪熟悉奖励(见材料表)。</li>
	</ol>
</li>
	<li>在早饭前或为喂猪投放新饲料之前,进行会议。</li>
</ol>2. 猪的识别
<ol><li>使主题在视频源上可识别,即使它们是单一存储的。</li>
	<li>确保观察者对治疗保持盲目,并在时间戳期间使用与治疗无关的标记系统保持公正。使用粘附在特定颜色、圆形和图案的胶带条上的医疗级胶带(见材料表)。</li>
	<li>使用一个圆形补丁来标记猪的顶部和每侧一个向下(参见图1,绿色和蓝色标记)。</li>
	<li>在胶带角上涂抹粘附水泥(小于 0.35 g),以帮助延长粘附寿命。 注:太多的标签水泥不会很快干燥,导致胶带过早脱落。</li>
	<li>在适应期间对标记策略进行故障排除和定制,以便高效执行官方测试,而不会给猪带来额外的压力。</li>
	<li>使用数据收集表跟踪标记和主题标识(有关示例数据表,请参阅补充文件1)。 注:要取出胶带,请勿撕下胶带,因为如果拉出任何头发,会导致疼痛。它要么可以自己脱落,水性润滑剂可以帮助将其驱离,也可以用剪子把它剪掉。如果磁带在不需要的时间脱落,请准备额外的标记胶带,并在猪吃一顿饭时重新涂抹,而不是限制猪。</li>
</ol>3. HAT 会话
<ol><li>每次进行训练时,都要让测试人员戴同一顶帽子、帽子、靴子、气味等。</li>
	<li>每天进行治疗,至少3次,然后,然后,在治疗后每天进行。</li>
	<li>在开始会话之前,猪可以在笔中的任何区域。要开始会话,请测试者将款待放在碗中或笔前,然后单击单击 3x。</li>
	<li>测试者需要把手从猪的视线中伸出来,在测试过程中保持静止。让另一位研究人员在数据表上标记会话的开始,然后启动计时器。120后,研究人员默默地向测试人发出信号,要求进入下一个科目并重新启动测试。</li>
</ol>4. 建立HAT软件图
<ol><li>使用专用软件在一个项目下构建内周图(参见图1和图2以及补充视频1)。 注:空间行为是动物相对于人类的位置。在透像图中,空间关系必须根据动物的笔进行定制,并在每次使用新颖的笔组时发布(图1)。在此类别中,行为被视为互斥。空间分为四个区域,方法级别不同(图1)。这些区域是跨住房类型的标准化区域。最近或攀爬(Cl)意味着猪可以爬上栅栏,以接近人类;因此,攀岩被认为是一种空间行为,表明猪最积极地寻求人类接触。关闭(Co)表示人类61厘米以内的区域。中 (M) 是距离人类 61-122 厘米以内的区域。远 (F) 是 123 厘米或以上的面积从人类。</li>
	<li>
结构行为侧重于整个身体或身体部位的方向(图2)。创建两个结构类别:1) 猪鼻位置和 2) 猪的活动状态。使用图2A识别方向性(方向1表示猪的鼻子朝向人类;方向2表示猪将鼻子指向人类)。</li>
	<li>将活动行为(图 2B)划分为三种互斥的方法状态:NNOB、在没有 NNOB 的情况下站立或行走,在没有 NNOB 的情况下休息(请参阅视频中的内周图中的颜色编码)。 注:NNOB 用于描述猪何时用嘴、鼻涕或面部来舔、嗅、咀嚼、咬、摩擦或根上非营养性物体,以寻找熟悉或新颖的机会。因此,当它处于这种活动状态时,它对人类感兴趣,并且处于接近状态。如果以 90° 角使用架空摄像头,则猪的鼻子位置和头部移动是 NNOB 的指示器。偶尔,猪会咬人或打假;鼻子可以看到,但头部上下移动。在没有NNOB的情况下站立或行走用于描述当猪处于直立位置时,头部静止不动,鼻子没有接触基板或乔打,这意味着它处于接近状态较小。没有NNOB的休息用于描述猪通过躺着或坐着休息时,这是这类行为下最不接近的状态。</li>
</ol>5. 视频的时戳,提高效率和可靠性
<ol><li>根据数据收集器录制的开始时间,将素材编辑成精确的 3 分钟会话。时间戳方法每会话需要 9 分钟。</li>
	<li>最多使用两个经过训练的观察人员来标记视频。 注:如果使用两个观察者,则在观察者对相同的样本视频进行时间戳后,应量化、评估、调整观察者内部变异,然后报告为 Pearson 相关系数(有关方法,请参阅 Martin 和贝特森25。</li>
	<li>将播放速度设置为 1 倍常规速度(即 30 fps)。不要逐帧暂停、倒带或时间戳。</li>
	<li>
分别对每一类互斥行为进行时间戳。
	<ol>
<li>时间戳空间行为。重新启动视频。</li>
		<li>时间戳结构行为。重新启动视频。</li>
		<li>结构/鼻子位置行为的时间戳。</li>
	</ol>
</li>
	<li>使用每个行为结果的持续时间(请参阅补充视频 2和3) 来汇总数据。持续时间测量需要转换为每个类别的时间百分比。</li>
</ol>6. 方法索引
<ol><li>应用公式(图3),使每个结构和空间行为相结合,以创建一个AI(图3,图4)。除了以数字(图5)或表格形式分别报告行为和类别外,AI 还用于使用 AI。 注:有关软件详细信息,请参阅材料表。在每个类别中,行为持续时间首先转换为百分比(行为持续时间除以测试会话的总持续时间)。每个行为都根据方法级别进行加权 (图 3)。回避行为(在远区域,休息,鼻子转远的时间百分比乘以0。中等方法行为(中间区域的时间百分比,转向人类,静止不动)乘以 1。方法行为的最大级别(近部和最近区域中的时间百分比以及显示 NNOB 的时间百分比)乘以 2。然后,每个类别分别对空间、鼻子方向和活动进行 3、2 和 1 的加权。应用常量 (0.10) 以创建百分比刻度。例如,如果猪面对人类,位于接近/最近区域,并在整个测试会话期间执行 NNOB,请注意 AI 为 100%(图 3中的红色猪 )。相反,如果猪在遥远的区域,不面对人类,并在整个测试过程中保持静止位置,则 AI 为 0%(图 3中的黑猪)。封闭区域的猪,如果远离人类,仍然站立着头(图3中的橙色猪),则可以与在中间区域执行NNOB的猪具有相同的AI。</li>
</ol>

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作者没有什么可透露的。

轻度损伤的大脑,不导致显着的解剖学和结构变化,可检测与最先进的成像可能难以识别和治疗28。然而,mTBI患者特别容易受到额外的侮辱,这些侮辱会对大脑造成重大损害,因此,识别这一人群非常重要。在mTBI的迷你猪模型中开发的行为测试与人类mTBI患者特别相关,因为猪的生理与人类相似,并且表达类似的情感状态,如8、9、10 ,20.在这里,我们开发了一个非侵入性的笔内行为测试(HAT),并表明它足够敏感,能够区分mTBI猪和假猪。此外,为在 HAT 期间观察到的行为开发了加权索引(AI),这些行为在住房和猪类型中普遍存在。
修改和故障排除:
HAT 的方法基于环境学指南25和若干试错策略建立,以提高测试6的可靠性、可重复性和有效性。可靠性度量有助于确定测试的优点和局限性。可靠性定义了测量的可重复性和一致性,并且没有随机误差的程度28,29。我们之前已经报告了 HAT 的内观察者和观察者间的可靠性,并且随着额外的结构内型,在持续时间6中,可靠性同样高(皮尔森的 R2 > 0.90)。频率和延迟测量需要经过训练的观察者,而持续时间测量较少依赖于观察者,因此,在实验室30中更可靠。
实验室内的可靠性和实验室之间的可重复性取决于方法。在我们的实验室中,视频系统不断录制,文件最初存储为 5 分钟文件,一些 HAT 会话发生在两个文件中。当使用数据表的精确时间来剪辑和合并视频时,错误更少。在开发内周图之前,允许观察者暂停、停止和倒带视频素材,以标记整个内周图中的所有行为。该方法不仅造成每个样本的时间戳变化,从3分钟到20分钟不等,而且对于大多数行为来说,观察者之间的和观察者内的可靠性也较差。因此,我们设置播放速度,并让观察器一次对每个类别进行时间戳。因此,当可靠性仅属于一个类别时,观察者在同时查阅定义和素材后,会独立地重新标记该类别,而不是整个内周图。设置的播放和类别方法允许一致预测每个样本需要时间来加时。对于超过一个月的项目,对编码视频和观察者内可靠性进行例行审查非常重要。
降低可靠性和可重复性的另一个因素是视频设置。最初,使用手持相机和三脚架,从笔移到笔。当使用这种方法时,猪需要在HAT之前被引入到三脚架和相机中;否则,猪似乎对三脚架和运动的反应比测试人多。此外,非顶置摄像机角度限制了在时间戳期间观察的观察者的视角,空间深度感知增加了空间行为测量中观察者内部和之间的变化;因此,我们开发了固定摄像头的协议。使用此方法时,需要格外小心,以确保在每次测试之前正确放置摄像机,并且需要在每个 Pig 会话之间的设置中留出更多时间。但是,我们了解到,连续视频开销系统需要在第一个 HAT 之前至少 24 小时午夜开始初始录制。许多视频系统的时间戳显示不准确,并且与帧同步;因此,我们不再依赖显示时间。午夜开始允许进行精确的帧捕获和视频编辑,并且未使用时间戳显示。
此外,在排除此测试的故障时,猪的适应和设置例程非常重要。在猪的录像中,它们不适应他们的环境,在HAT期间观察到起搏。这是一个指标,猪可能处于激动状态31,而不是在探索状态32。三周或更长时间的适应期可能会减少实验中速度的猪的数量。但是,如果在整个采样期间持续起搏,则可能需要调整此内周图,以包括行走和静止不动。
有效性是度量表示所提问问题的预期范围的程度25。刚开发HAT时,我们只使用了空间内周图。空间内周图行为的定义准确而具体地描述了与人类主体的接近程度,它们直接告诉观察者猪在它们自己和人类之间留下了多少空间。然而,一旦这些方法需要应用于新的实验室设置,我们认识到空间内周图是实验室特有的。笔尺寸和其他物体的位置会影响空间内周图的结果;因此,如果笔设置之前未报告,则需要发布包含笔的测量值和细节的图表。除了报告笔环境外,结构行为还添加到了内周图中。与空间行为不同,结构行为在实验室中可以更容易地进行评估;这些行为之所以有效,是因为它们具体地描述了猪的活动状态水平。当猪休息时,它很可能没有接近的动机,并且无法改变立场,以像站立的猪一样快速接近。类似地,显示NNOB的猪处于探索状态,但站立时头部静止的猪更有可能处于催化状态。鼻子定向有助于有效,因为鼻子、耳朵和眼睛是猪用来收集人类信息的东西。
技术的限制:
这种技术的一个潜在问题是猪对测试人类的反应的变异性。此外,猪会看着测试人的手,这可能导致这个人无意的提示。因此,通过实验测试1)猪对熟悉的人类和不熟悉的人的反应,以及2)标准化,在颗粒被丢弃后,测试人静止不动,并把他们的手从猪的视线。数据显示,在HAT期间没有治疗或治疗x时差(表2),表明HAT可以由熟悉或不熟悉的人管理。其他研究人员建议,猪倾向于根据以前的相互作用11,12,13来概括人类;因此,猪以前与人类的经历需要是积极的。这一挑战也可以通过警惕的实验设计来补救;对于每个区块,需要为每次利息处理提供足够的实验单元。
在这项研究中,虽然只有两个有经验的观察者对所有三种住房类型的所有视频进行时间戳,但特定行为结果的住房类型之间存在差异(表1)。例如,住房类型 B 中的猪比住房类型 A 和 C 中的猪更频繁地进入最近区域。这可能是由于笔材料的差异;在B型住房中,笔的前部是一个链状门,带有水平条,允许猪在HAT期间爬上大门。另一方面,A型和C型的外壳有垂直条形,水平表面较少,供猪攀爬。这种可变性可以通过在接近和最近区域的持续时间花费,然后比较不同住房类型的持续时间来补救(表1;P > 0.10)。然而,C类房屋中的猪在较远地区逗留的时间,较A类及B类房屋为多(表1;P < 0.05),这可能是由于将水器放在笔的后面,而不是笔的前面。如果实验室选择标准化水器、碗和玩具的放置,并确保它们固定,以便猪不会将物体移到其他区域,则可以纠正这一限制。
此测试对于所有类型的实验室都有很大的可访问性,但是,如前所述,手动盖章的空间内周图和测量在不同实验室之间将变化更大。尽管如此,身体和头部结构的中长线图无处不在。能够对猪进行经过验证的自动跟踪的实验室可能会通过自动跟踪空间内周图而不是手动跟踪而受益,因为移动的距离和移动速率可能是行为测量的附加结果。帽子。通过调整 AI 公式,可以弥补笔内设置和传统技术(而不是测试区域和自动跟踪技术)的限制。AI 提供标准化的测量和术语,用于个体猪如何使用笔空间并表达对人类的兴趣。这种计算,派生自常见的行为测量,是敏感的猪模型的亚结节mTBI,并可能,其他状态的亚临床损伤或疾病。此外,AI 可减少实验过程中的随机变化,并且与依赖更多特定于实验的测量方法相比,在实验和实验室之间可能更容易进行比较。结构行为为此公式提供了基础,因为这些行为是跨处理的标准测量,而空间行为取决于笔设置、笔中的猪数和跟踪系统。例如,我们观察到,当两只健康的猪在一支笔中测试时,它们会通过一起接近来执行类似的空间行为,但跟随第一只的猪可能更多地将鼻子朝向笔伴侣,而不是向人类方向,并表达更多的NNOB,因为猪谁领导作为哨兵。尽管如此,AI 有助于减少这种变化,即使从配对行为。
尽管人工智能是跨实验室标准化测试的优秀工具集,但研究人员可能仍希望检查实验室或实验中的测试特定行为结果,特别是如果它们有足够的能力(即实验单元和重复测试)。)。因此,此处包含表 1,其中包含所有行为结果、方差、分布以及每个特定行为的动物数量的计算检验。例如,如果研究人员有笔环境,允许猪在HAT期间持续攀爬,并且他们知道他们的治疗导致超过75%的攀爬行为差异,那么他们就可以根据测得的方差来证明动物数量的合理性。如果新行为被添加到内周图中,科学家将需要证明哪些行为是方法或退出的指示,然后再将它们纳入索引。例如,如果大多数动物在实验步速沿笔壁(即,thigmotaxis)32,这种行为的持续时间可以纳入身体结构内周图类别。行为可以在详细的堆叠条形图(即图5)或表格形式中表示,然后,在应用索引计算之前,可以采用静止状态进行总结。因此,AI 可以表示在实验室中普遍存在的行为,但其他独特的行为仍可以单独表示。
相对于现有方法的重要性:
现有的HAT方法为商业农场的猪建立了评估动物福利的方法。在这里,实验室猪已经建立了一个协议,可以帮助研究人员评估动物福利,并区分mTBI猪和假处理猪。另一种传统测试可能是使用开放字段测试。这个测试以前用来评估猪的情绪和福利33。露天测试最初旨在通过测量啮齿动物对开放空间和光线的自然厌恶来测试它们的情感状态。相反,健康的猪可能认为同样的刺激,在生病,受伤,或压力治疗后,他们可能表达恐惧。这种测试需要更多的实验室空间,并且需要猪适应被处理和放置在一个露天竞技场。如果实验室有处理猪的空间和协议到位,重复的HAT会话,除了一个露天测试,可能有助于进一步区分处理动物和假处理的动物。
《议定书》内的关键步骤:
协议中的前三个步骤对于 HAT 措施的成功最为关键。每头猪的会话只需 3 分钟;然而,充分的准备将有助于使这个测试可靠。如上所述,摄像机位置和录制设置对于清晰度和复制至关重要。相机角度不当会限制观察者的视野,从而增加测量误差。另一个经常被忽视的步骤是固定笔中的对象。猪会移动未固定的物体,这可能会影响它接近人类的动机。设置和管理系统很重要,因为猪需要适应他们的环境,然后才能持续执行测试。猪,如果不太适应他们的家庭笔或日常或遇到压力,将排便在其他地区,而不是在笔34的背面。排便区可能会影响他们接近的动机。从摄像机的视角来看,观察者应该能够识别单个猪;然而,重要的是,标记方案没有提供有关动物治疗的信息,因为这将偏向观察者25。
识别猪对于获得正确的猪的正确行为数据非常重要,即使它们是单一饲养的。猪经常被移动进行治疗,并且标记使观察者重新确认,在猪被移除并放回笔中后,他们正在观察同一头猪。猪可以成对安置,如在住房类型C,因此,它变得非常重要,以确定猪。牲畜标记油漆和标记需要日常应用;因此,该协议要求使用医疗级胶带和标记水泥的涂抹。胶带最适合长头发的猪。短发和皮肤干燥的猪比头发较长的猪更频繁地从胶带上脱落。
未来应用:
总之,此处描述的非侵入性笔内 HAT 测试足够敏感,能够检测 mTBI 后猪的轻度和时间相关变化。此外,我们开发了一个加权指数,称为AI,以评估不同笔型和不同类型猪的猪的变化。虽然 HAT 已用于检测暴露于 mTBI 的猪的变化,但此行为测试可用于检测经历压力或病理前状况的动物的可测量行为变化。

尽管大脑中没有全球结构变化或明显的水肿,但人类mTBI通常被功能缺陷所定义。事实上,在一些mTBI患者中,这种损伤的特征是,在没有任何神经解剖学变化的情况下,他们的心理状态发生了变化。我们使用了mTBI6的猪模型,因为猪的大脑在解剖学和生理上都比啮齿动物7更接近人类,而相应的测量可以提供一组与人类相关的共同数据元素。
近年来,猪模型引起了神经创伤科学家和mTBI利益相关者对临床前研究的兴趣;然而,与TBI的啮齿动物模型不同,只有一些标准化的行为测试发布,允许评估实验室猪的情感状态(即心理状态)7,8,9, 10.我们的实验室的一个长期目标是开发几种互补的行为工具,这些工具足够敏感,能够测量猪何时经历亚临床疾病或动物处于病理前压力相关状态。
重复的行为测试,测量在实验室猪的情感状态的变化可能是区分具有前病理状况的动物和健康动物的良好候选者。例如,在商业生猪生产中使用笔内HAT,帮助农民选择性情良好的健康猪,或修改管理和住房策略,造成痛苦、伤害和疾病11、12。这些测试用于量化一头猪或一组猪的动机和整体情感状态。
我们的实验室和其他研究人员通过量化三类行为来测量猪的动机:1)通过非营养性口腔行为(NNOB)表达的探索性状态,即猪用嘴、鼻涕或面部嗅觉、舔、咀嚼、根基材,或无基板14、15的基板;2) 猪与物体的空间关系或16;3)鼻子方向,这是使用代替眼睛接触,因为猪有单目17,但近视视力,他们优先考虑他们的嗅觉比视觉18。如果一个健康的猪将人类与有益的刺激联系在一起,它们就会表达出高频率的NNOB,将鼻子指向人类,并寻求更接近人类11,16。然而,在生病、受伤或痛苦的经历之后,寻求甚至愉悦刺激的动机就会减少,因此,这些可衡量的行为可能会减少19。猪行为研究人员指出,在猪的家庭环境中,缺乏体验愉悦刺激的动机,在猪体内是可识别的,也是可测量的。因此,重复的HATs(治疗前后)可以作为一种敏感措施,区分用亚结对mTBI治疗的实验室猪与假治疗(仅麻醉)受试者。安赫多尼亚是一个情感状态,TBI患者可能经历21。此处使用的 HAT 具有帮助简化行为发现从动物模型到临床工作的翻译的潜力。在实验过程中,每天可以施用HAT,这也有助于规范实验室猪的护理,优化动物福利和畜牧业。
在这里,使用HAT,对迷你猪中mTBI产生的行为差异进行了探讨。通过利用HAT的非侵入性措施,让猪适应他们的家庭笔,日常管理和日常治疗,可以最大限度地减少行为变异。传统上,测试竞技场用于测量行为(例如,开场测试)。在空间有限的实验室中,笔内测试非常有用。在测试区域移动和处理猪可能会导致压力响应(痛苦或压力),并可能增加对测试响应的变化。笔内测试可移除该处理组件,因此可能会减少处理应力17的变异。出于上述原因,我们为此 mTBI 模型开发了每日笔内 HAT。
正确界定动物情感状态的标准化和量化措施是开发新的行为测试的重要方面。此外,测试应在多个实验室中重复。在这里,为了开发该协议,HAT在三个实验室的不同外壳系统中进行了测试。创建了三个子线图,以从示例视频中标记特定行为。接下来,创建了一个加权公式,以合并三个内型图,并允许在多个实验室中使用HAT。虽然此测试是专门开发和用于用亚结节 mTBI 治疗的迷你猪,但此处开发的方法和协议将具有区分亚临床受伤/生病或受困猪与健康的猪。
行为结果可能受单一与组住房、自由空间允许、使用的地板类型、使用的围栏类型、进给和水的位置、排便区域和环境富集位置的影响。因此,对三种住房类型进行了检查(图1):A类住房在堪萨斯州立大学(堪萨斯州曼哈顿);住房类型B和C在弗吉尼亚理工大学(阿灵顿,弗吉尼亚州)。每个地点的个别机构动物护理和使用委员会(IACUC)都批准了设施和程序的使用。
用于开发住房类型 A 的内周图 (图 1A),明尼苏达州交叉迷你猪 (野猪 = 7, 镀金 = 1;国家生猪研究资源中心,哥伦比亚,MO;年龄 = 25.6 = 3.66 [平均 = 标准偏差 (SD)] 周)被安置在室内,采用单笔,地板适合动物(IACUC #3881)。用于此协议的猪健康状况良好,没有应用治疗方法。为了开发B型住房协议,尤卡坦迷你猪(年龄= 25.3 × 2.80周[平均= SD]在弗吉尼亚理工大学设施(IACUC #15-060)是单体住宅(图1B)。动物治疗在其他地方描述29,包括诱导亚结节mTBI使用爆炸波过压或假控制(仅麻醉)。为了开发住房C型的协议,五只雌性哥廷根迷你猪(年龄=23.7~1.18周[平均[SD])在弗吉尼亚理工大学用一支大笔成对安置(图1C;IACUC #15-060)。前两个住房环境是典型的实验室住房或包含单一住房的猪。C型住房是一种非典型住房环境,可以容纳两头或两只以上的猪,而且可能比标准实验室住房更丰富。如果遵循以下方法,则可以跨外壳类型使用此协议。

<table><tbody><tr><td>Dome 3.0 Megapixel Cameras with 2.8-12 mm lens set between 2.8-3.2 mm</td><td>Points North Surveillance, Auburn, ME</td><td>CDL7233S</td><td>Lower mm lenses are needed for low-profile pens</td></tr><tr><td>Manfrotto 244 friction arm kit</td><td>B&amp;amp;H Photo</td><td>B&amp;amp;H # MA244; MFR # 244</td><td>To mount and secure cameras at a 90&amp;deg; angle</td></tr><tr><td>Video Recording System</td><td>Points North Surveillance, Auburn, ME</td><td>NVR-RACK64</td><td>NVR is customized</td></tr><tr><td>Colored and patterned duct tape attached to a double-sided medical grade tape</td><td>&amp;nbsp;MBK Tape Solutions, Chatsworth, CA</td><td>3M 1522H</td><td>Sustainable marking of pigs</td></tr><tr><td>Approach Index Formula generator</td><td>Dinasym, Manhattan, KS</td><td>Approach Formula</td><td>Company will customize macros for specific lab needs</td></tr><tr><td>Geovision Software</td><td>Points North Surveillance, Auburn, ME</td><td>Geovision</td><td>Software to edit video time into 180 s clips</td></tr><tr><td>Clicker</td><td>Petco</td><td>Good2Go Dog Training Clicker</td><td></td></tr><tr><td>Reward treat (feed pellet, carob chip, raisin, marshmallow)</td><td>Variable</td><td>N/A</td><td>Depending on previous exposure, adult pigs are very&amp;nbsp; neophobic when new food is introduced. Limit-fed pigs can be fed a few pellets of feed.&amp;nbsp;</td></tr><tr><td>Statistical Analysis System (SAS)</td><td>SAS Institute, Cary, North Carolina</td><td>SAS 9.0</td><td>Our laboratories preference for analyzing mixed models and repeated measures</td></tr><tr><td>Observer 11.5 software</td><td>Noldus Information Technology, Leesburg, VA</td><td>Observer 11.5</td><td>Software to manually timestamp video clips</td></tr></tbody></table>

noninvasive, in-pen approach test for laboratory-housed pigs

创伤性脑损伤(TBI)在平民和军人中都有所增加,许多研究人员正在采用一种猪模型来治疗TBI。与TBI的啮齿动物模型不同,很少有行为测试被标准化。比啮齿动物在试验区里,较大的动物需要更多的侵入性处理,这可能会给动物的反应增加压力和变化。在这里,描述了人类方法测试(HAT),这是开发在实验室猪的家笔前进行的。它是非侵入性的,但足够灵活,它允许在住房设置的差异。
在HAT期间,开发了三个行为内周图,然后应用了一个公式来创建方法索引(AI)。结果表明,HAT及其指数AI足够敏感,能够检测猪在轻度TBI(mTBI)后行为的轻度和暂时性变化。此外,尽管特定的行为结果取决于房屋,但使用 AI 可减少变异,并允许跨实验室进行一致的测量。此测试可靠且有效;HAT 可用于许多实验室和不同类型的伤害、疾病和危难的猪模型。此测试是为优化的手动时间戳方法开发的,以便观察者在每个样本上的花费始终不超过 9 分钟。

Watch this Scientific Journal Video about Noninvasive, In-pen Approach Test for Laboratory-housed Pigs at JoVE.com

Noninvasive, In-pen Approach Test for Laboratory-housed Pigs

该协议描述了一种新的行为测试——猪家笔中的人类方法测试——用于检测亚脑震荡创伤性脑损伤后实验室猪的功能缺陷。

实验室安置猪的非侵入性、笔内方法测试

Traumatic brain injury (TBI) incidences have increased in both civilian and military populations, and many researchers are adopting a porcine model for ...

这种方法可以帮助回答神经科学领域的关键问题，特别是神经创伤。这种技术的主要优点是，它不需要一个竞技场或额外的处理的主题。可以看到我的研究团队演示了整个协议中的程序。这包括爱德华达 · 博尔托鲁齐、米凯拉 · 戈林、卢克 · 鲁伊斯、贾里德 · 穆姆和摩根 · 考芬。

noninvasive-in-pen-approach-test-for-laboratory-housed-pigs

Universidad de Cartagena UDEC

Research

JoVE Journal

Behavior

8.3K 次观看.  Kansas State University. 这种方法可以帮助回答神经科学领域的关键问题，特别是神经创伤。这种技术的主要优点是，它不需要一个竞技场或额外的处理的主题。可以看到我的研究团队演示了整个协议中的程序。这包括爱德华达 · 博尔托鲁齐、米凯拉 · 戈林、卢克 · 鲁伊斯、贾里德 · 穆姆和摩根 · 考芬。

视频: Noninvasive, In-pen Approach Test for Laboratory-housed Pigs

Basic Surgical Techniques in the G&ouml;ttingen Minipig: Intubation, Bladder Catheterization, Femoral Vessel Catheterization, and Transcardial Perfusion

The emergence of the G&ouml;ttingen minipig in research of topics such as neuroscience, toxicology, diabetes, obesity, and experimental surgery reflects the close resemblance of these animals to human anatomy and physiology 1-6.The size of the G&ouml;ttingen minipig permits the use of surgical equipment and advanced imaging modalities similar to those used in humans 6-8. The aim of this instructional video is to increase the awareness on the value of minipigs in biomedical research, by demonstrating how to perform tracheal intubation, transurethral bladder catheterization, femoral artery and vein catheterization, as well as transcardial perfusion. 
Endotracheal Intubation should be performed whenever a minipig undergoes general anesthesia, because it maintains a patent airway, permits assisted ventilation and protects the airways from aspirates. Transurethral bladder catheterization can provide useful information about about hydration state as well as renal and cardiovascular function during long surgical procedures. Furthermore, urinary catheterization can prevent contamination of delicate medico-technical equipment and painful bladder extension which may harm the animal and unnecessarily influence the experiment due to increased vagal tone and altered physiological parameters. Arterial and venous catheterization is useful for obtaining repeated blood samples and monitoring various physiological parameters. Catheterization of femoral vessels is preferable to catheterization of the neck vessels for ease of access, when performing experiments involving frame-based stereotaxic neurosurgery and brain imaging. When performing vessel catheterization in survival studies, strict aseptic technique must be employed to avoid infections6. Transcardial perfusion is the most effective fixation method, and yields preeminent results when preparing minipig organs for histology and histochemistry2,9. For more information about anesthesia, surgery and experimental techniques in swine in general we refer to Swindle 2007. Supplementary information about premedication and induction of anesthesia, assisted ventilation, analgesia, pre- and postoperative care of G&ouml;ttingen minipigs are available via the internet at <a href="http://www.minipigs.com">http://www.minipigs.com</a>10. For extensive information about porcine anatomy we refer to Nickel et al. Vol. 1-511.

Basic Surgical Techniques in the G&#246;ttingen Minipig: Intubation, Bladder Catheterization, Femoral Vessel Catheterization, and Transcardial Perfusion

The use of domestic and miniature pigs in science has increased significantly in recent years. By demonstrating how to perform intubation, transurethral bladder catheterization, femoral artery and vein catheterization, as well as transcardial perfusion, we aim to further increase the value of G&ouml;ttingen minipigs in biomedical research.

基本外科技术，在哥廷根微型猪：气管插管，膀胱导尿，股血管的导管和Transcardial灌注

The emergence of the G&ouml;ttingen minipig in research of topics such as neuroscience, toxicology, diabetes, obesity, and experimental surgery reflects ...

科研

医学

Transition of Farm Pigs to Research Pigs using a Designated Checklist followed by Initiation of Clicker Training - a Refinement Initiative

Implementation of 3R initiatives is important. To refine the life of laboratory pigs, we focused on the time period prior to inclusion of animals in experiments. We improved the checklist during the development. By using the checklist each individual pig&#39;s progress was documented. This aids the caretakers so they remain focused on the actions necessary to socialize the animals and limit the animal&#39;s stress and anxiety level. During this phase, the pigs become calm and ready to commence additional clicker training to further limit stress during the experimental period. The pigs will readily allow basic outcome measures to be collected without sedation or fixation.
The protocol is divided in two. First the transition phase that begins as the pigs arrive at the research facility. Introducing treats and gradually closer contact to the caretakers are the first steps. Then, the caretaker introduces actions to be able to touch the head and hind, to mimic e.g. manipulating the ear to take a blood sample or the hind to obtain a rectal temperature. A strong foundation is established in the animal-caretaker relationship and this swiftly minimizes the stress the pigs express after arrival. Secondly, the positive reinforcement training commences to teach the animal how to carry out more complex tasks without becoming stressed. A clicker is introduced as a secondary reinforcer.
Time, structure, education and thorough communication seem to be the most important criteria to succeed in the application of this protocol. In addition to having calm and compliant pigs, the caretakers report that they enjoy the focus they are allowed to give each individual pig, adding to their overall job satisfaction. Having caretakers with a strong commitment, and a focus on the transition progress and welfare of each pig, additionally strengthens the department&#39;s Culture of Care.

Refinement of porcine studies is achieved by introducing a standardized checklist and positive reinforcement training using a clicker. This work supports the collection of samples and the conduct of daily chores related to the animals.

农场猪向研究猪的过渡使用指定的清单，然后启动点击培训 - 改进计划

Implementation of 3R initiatives is important. To refine the life of laboratory pigs, we focused on the time period prior to inclusion of animals in ...

行为学

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

鼻湿巾从猪流感病毒检测和​​隔离

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

免疫与感染

Chronic Thromboembolic Pulmonary Hypertension and Assessment of Right Ventricular Function in the Piglet

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. Pulmonary Hypertension (PH) was induced in 3-week-old piglets by a progressive obstruction of the pulmonary vascular bed. A ligation of the left Pulmonary Artery (PA) was performed first through a mini-thoracotomy. Second, weekly embolizations of the right lower pulmonary lobe were done under fluoroscopic guidance with n-butyl-2-cyanoacrylate during 5 weeks. Mean Pulmonary Arterial Pressure (mPAP) measured by ritght heart catheterism, increased progressively, as well as Right Atrial pressure and Pulmonary Vascular Resistances (PVR) after 5 weeks compared to sham animals. Right Ventricular (RV) structural and functional remodeling were assessed by transthoracic echocardiography (RV diameters, RV wall thickness, RV systolic function). RV elastance and RV-pulmonary coupling were assessed by Pressure-Volume Loops (PVL) analysis with conductance method. Histologic study of the lung and the right ventricle were also performed. Molecular analyses on RV fresh tissues could be performed through repeated transcutaneous endomyocardial biopsies. Pulmonary microvascular disease in obstructed and unobstructed territories was studied from lung biopsies using molecular analyses and pathology. Furthermore, the reliability and the reproducibility was associated with a range of PH severity in animals. Most aspects of the human CTEPH disease were reproduced in this model, which allows new perspectives for the understanding of the underlying mechanisms (mitochondria, inflammation) and new therapeutic approaches (targeted, cellular or gene therapies) of the overloaded right ventricle but also pulmonary microvascular disease.

Chronic Thromboembolic Pulmonary Hypertension (CTEPH) and Right Ventricular (RV) dysfunction were induced in piglets by progressive obstruction of the pulmonary arteries. Consequences were remarkably similar to those observed in CTEPH patients. This animal model would be a very useful tool for pathophysiology and therapeutic experiments on CTEPH and RV failure.

慢性血栓栓塞性肺动脉高压和右心室功能的仔猪评估

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. ...

Methods for In Vivo Biomechanical Testing on Brachial Plexus in Neonatal Piglets

Neonatal brachial plexus palsy (NBPP) is a stretch injury that occurs during the birthing process in nerve complexes located in the neck and shoulder regions, collectively referred to as the brachial plexus (BP). Despite recent advances in obstetrical care, the problem of NBPP continues to be a global health burden with an incidence of 1.5 cases per 1,000 live births. More severe types of this injury can cause permanent paralysis of the arm from the shoulder down. Prevention and treatment of NBPP warrants an understanding of the biomechanical and physiological responses of newborn BP nerves when subjected to stretch. Current knowledge of the newborn BP is extrapolated from adult animal or cadaveric BP tissue instead of in vivo neonatal BP tissue. This study describes an in vivo mechanical testing device and procedure to conduct in vivo biomechanical testing in neonatal piglets. The device consists of a clamp, actuator, load cell, and camera system that apply and monitor in vivo strains and loads until failure. The camera system also allows monitoring of the failure location during rupture. Overall, the presented method allows for a detailed biomechanical characterization of neonatal BP when subjected to stretch.

Methods for In Vivo Biomechanical Testing on Brachial Plexus in Neonatal Piglets

Presented here are methods to perform in vivo biomechanical testing on brachial plexus in a neonatal piglet model.

新生儿小猪体内胸腺菌的活体力学试验方法

Neonatal brachial plexus palsy (NBPP) is a stretch injury that occurs during the birthing process in nerve complexes located in the neck and shoulder ...

生物工程

Adaptation of Microelectrode Array Technology for the Study of Anesthesia-induced Neurotoxicity in the Intact Piglet Brain

Every year, millions of children undergo anesthesia for a multitude of procedures. However, studies in both animals and humans have called into question the safety of anesthesia in children, implicating anesthetics as potentially toxic to the brain in development. To date, no studies have successfully elucidated the mechanism(s) by which anesthesia may be neurotoxic. Animal studies allow investigation of such mechanisms, and neonatal piglets represent an excellent model to study these effects due to their striking developmental similarities to the human brain.
This protocol adapts the use of enzyme-based microelectrode array (MEA) technology as a novel way to study the mechanism(s) of anesthesia-induced neurotoxicity (AIN). MEAs enable real-time monitoring of in vivo neurotransmitter activity and offer exceptional temporal and spatial resolution. It is hypothesized that anesthetic neurotoxicity is caused in part by glutamate dysregulation and MEAs offer a method to measure glutamate. The novel implementation of MEA technology in a piglet model presents a unique opportunity for the study of AIN.

This study explores the novel use of enzyme-based microelectrode array (MEA) technology to monitor in vivo neurotransmitter activity in piglets. The hypothesis was that glutamate dysregulation contributes to the mechanism of anesthetic neurotoxicity. Here, we present a protocol to adapt MEA technology to study the mechanism of anesthesia-induced neurotoxicity.

微电极阵列技术在完整仔猪脑麻醉诱导神经毒性研究中的应用

Every year, millions of children undergo anesthesia for a multitude of procedures. However, studies in both animals and humans have called into question ...

神经科学

Non-Terminal Blood Sampling Techniques in Guinea Pigs

Guinea pigs possess several biological similarities to humans and are validated experimental animal models1-3. However, the use of guinea pigs currently represents a relatively narrow area of research and descriptive data on specific methodology is correspondingly scarce. The anatomical features of guinea pigs are slightly different from other rodent models, hence modulation of sampling techniques to accommodate for species-specific differences, e.g., compared to mice and rats, are necessary to obtain sufficient and high quality samples. As both long and short term in vivo studies often require repeated blood sampling the choice of technique should be well considered in order to reduce stress and discomfort in the animals but also to ensure survival as well as compliance with requirements of sample size and accessibility. Venous blood samples can be obtained at a number of sites in guinea pigs e.g., the saphenous and jugular veins, each technique containing both advantages and disadvantages4,5. Here, we present four different blood sampling techniques for either conscious or anaesthetized guinea pigs. The procedures are all non-terminal procedures provided that sample volumes and number of samples do not exceed guidelines for blood collection in laboratory animals6. All the described methods have been thoroughly tested and applied for repeated in vivo blood sampling in studies within our research facility.

Though a known model, the guinea pig currently represents a niche in experimental animal sciences and limited data is available on the execution of most procedures. Here we present four different approaches to non-terminal in vivo blood sampling techniques in either conscious or anaesthetized guinea pigs.

豚鼠非终端采血技术

Guinea pigs possess several biological similarities to humans and are validated experimental animal models1-3. However, the use of guinea pigs currently ...

生物学

Endotracheal Intubation Using a Flexible Intubation Endoscope as a Standardized Model for Safe Airway Management in Swine

Endotracheal intubation is often a basic requirement for translational research in porcine models for various interventions that require a secured airway or high ventilation pressures. Endotracheal intubation is a challenging skill,&#160;requiring a minimum number of successful endotracheal intubations to achieve a high success rate under optimal conditions, which is often unachievable for non-anaesthesiology researchers. Due to the specific porcine airway anatomy, a difficult airway can usually be assumed. The impossibility of establishing a secure airway can result in injury, adverse events, or death of the laboratory animal. Using a prospective, randomized, controlled evaluation approach, it has been shown that fiberoptic-assisted endotracheal intubation takes longer but has a higher first-pass success rate than conventional intubation without causing clinically relevant drops in oxygen saturation. This model presents a standardized regimen for endoscopically guided endotracheal intubation, providing a secured airway, especially for researchers who are inexperienced in the technique of endotracheal intubation via direct laryngoscopy. This procedure is expected to minimize animal suffering and unnecessary animal losses.

The use of pigs in research has increased in recent years. Nevertheless, pigs are characterized by difficult airway anatomy. By demonstrating how to perform endoscopically guided endotracheal intubation, the present protocol aims to further increase laboratory animals' safety to avoid animal suffering and unnecessary death.

使用柔性插管内窥镜作为猪安全气道管理的标准化模型的气管插管

Endotracheal intubation is often a basic requirement for translational research in porcine models for various interventions that require a secured airway ...

Transdermal Measurement of Glomerular Filtration Rate in Mechanically Ventilated Piglets

Transdermal measurement of glomerular filtration rate (GFR) has been used to evaluate kidney function in conscious animals. This technique is well established in rodents to study acute kidney injury and chronic kidney disease. However, GFR measurement using the transdermal system has not been validated in pigs, a species with a similar renal system to humans. Hence, we investigated the effect of sepsis on transdermal GFR in anesthetized and mechanically ventilated neonatal pigs. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP). The transdermal GFR measurement system consisting of a miniaturized fluorescence sensor was attached to the pig's shaved skin to determine the clearance of fluorescein-isothiocyanate (FITC) conjugated sinistrin, an intravenously injected GFR tracer. Our results show that at 12 h post-CLP, serum creatinine increased with a decrease in GFR. This study demonstrates, for the first time, the utility of the transdermal GFR approach in determining renal function in mechanically ventilated, neonatal pigs.

Glomerular filtration rate (GFR) is the ideal marker for assessing kidney function. However, the standard measurement method using inulin injection with serial blood and urine analysis is impractical. This article delineates a practical method to measure GFR transdermally in piglets.

机械通气仔猪肾小球滤过率的透皮测量

Transdermal measurement of glomerular filtration rate (GFR) has been used to evaluate kidney function in conscious animals. This technique is well ...

Echocardiography Recording in Awake Miniature Pigs

Echocardiography uses ultrasonic waves to non-invasively assess cardiac structure and function and is the standard of care for cardiac assessment and monitoring. The miniature pig, or minipig, is increasingly being used as a model of cardiac disease in medical research. Pigs are notoriously difficult to restrain and handle safely, and, therefore, research echocardiography in this species is almost always performed under anesthesia or heavy sedation. Anesthetics and sedatives universally affect cardiovascular function and may cause the depression of cardiac output and blood pressure, increases or decreases in heart rate and systemic vascular resistance, changes in the electrical rhythm, and altered coronary blood flow. Therefore, sedated or anesthetized echocardiography may not accurately depict the progression of cardiac disease in large animal models, thereby limiting the translational value of these important studies. This paper describes a novel device that allows for standing awake echocardiography in minipigs. In addition, training techniques used to teach pigs to tolerate this painless and non-invasive procedure without the need for hemodynamic-altering anesthetics are described. Standing awake echocardiography represents a safe and feasible way to perform the most common cardiac monitoring test in minipigs for cardiovascular research.

A simple cart construct, built to perform research echocardiography in standing awake minipigs, is described, along with building considerations, training techniques, and representative ultrasound images.

清醒微型猪的超声心动图记录

Echocardiography uses ultrasonic waves to non-invasively assess cardiac structure and function and is the standard of care for cardiac assessment and ...

实验室安置猪的非侵入性、笔内方法测试

副本

摘要

探索更多视频

此视频中的章节

基本外科技术，在哥廷根微型猪：气管插管，膀胱导尿，股血管的导管和Transcardial灌注

农场猪向研究猪的过渡使用指定的清单，然后启动点击培训 - 改进计划

鼻湿巾从猪流感病毒检测和隔离

慢性血栓栓塞性肺动脉高压和右心室功能的仔猪评估

新生儿小猪体内胸腺菌的活体力学试验方法

微电极阵列技术在完整仔猪脑麻醉诱导神经毒性研究中的应用

豚鼠非终端采血技术

使用柔性插管内窥镜作为猪安全气道管理的标准化模型的气管插管

机械通气仔猪肾小球滤过率的透皮测量

清醒微型猪的超声心动图记录

实验室安置猪的非侵入性、笔内方法测试

副本

摘要

探索更多视频

此视频中的章节

基本外科技术，在哥廷根微型猪：气管插管，膀胱导尿，股血管的导管和Transcardial灌注

农场猪向研究猪的过渡使用指定的清单，然后启动点击培训 - 改进计划

鼻湿巾从猪流感病毒检测和​​隔离

慢性血栓栓塞性肺动脉高压和右心室功能的仔猪评估

新生儿小猪体内胸腺菌的活体力学试验方法

微电极阵列技术在完整仔猪脑麻醉诱导神经毒性研究中的应用

豚鼠非终端采血技术

使用柔性插管内窥镜作为猪安全气道管理的标准化模型的气管插管

机械通气仔猪肾小球滤过率的透皮测量

清醒微型猪的超声心动图记录

鼻湿巾从猪流感病毒检测和隔离