这项工作是由荷兰科学研究组织（NWO）（的Veni没有。916.86.125）授予研究Reneman，。出资者有没有作用的研究设计，数据收集和分析，发布决定，或准备的手稿。有没有利益冲突。

准备动物体内磁共振成像 1。手术插管<ol><li>麻醉与异氟醚（5％的诱导和维持麻醉减少1.5-2％，在动物准备和扫描的话）（21％的O 2，中行英国）医用空气中的大鼠（雄性Wistar大鼠，200-300克） 。确保该动物以​​及麻醉和展品脚趾掐无反应。股动脉和静脉插管血气和血压测量，并分别毒品挑战的管理。在手术过程中，动物的体温监测，并通过直肠探头和热毯（哈佛设备）的维护。 </li><li>麻醉动物的位置上背卧在解剖显微镜下的暖垫。刮大腿中部地区和皮肤用酒精擦拭。请2厘米的皮肤切口沿腹部和右大腿形成折痕。用于可视化的股动脉，静脉和股神经，内收肌钝性分离。仔细分离的船只。 </li><li>轻轻绑丝线结扎完全围绕该船只的末端，放置在近端站点外科结松散的一半与另一领带。应用牵引，以两个连字，在余下的中间部分的之间结扎暴露的船只闭塞血流。使约三分之一的船只围在这部分船只的小切口，以便插入一个PE-50导管（内部直径0.54毫米和0.96毫米外径在成年雄性大鼠的情况下，否则0.40 mm内径0.80毫米ED）进入血管。 </li><li>套管应被插入到几个毫米的船只（至少5个）。一旦进入管腔，通过船只冲洗少量的肝素生理盐水（15 UI /毫升），以避免任何血液凝块的形成。近端丝绸循环也结扎完全修复套管。 ŕEPEAT此过程中的第二艘。胶水的皮肤，使用Vetbond组织粘合剂（3M英国公司，英国布拉克内尔）当两个套管到位。套管的确切位置， 见图1。 </li><li>放置在MR兼容的立体床俯卧（美国M2M成像公司）的动物。通过插入耳酒吧和牙齿栏保持动物的头。在这一点上，动物可放置在磁共振成像扫描仪。动物仍然是麻醉和整个成像过程是自由呼吸。 </li></ol> 2。监测整个成像过程中，应不断监测几个生理反应，并尽可能保持恒定。这是至关重要的，因为这些反应可以大大超过同一时间窗口作为phMRI信号变化，也影响利率的信号。同样重要的是，由于该动物将被放置在马主干网络，并因此淡出人们的视线和不服从检查麻醉深度的标准（如脚趾捏），为确保有足够的麻醉深度。此外，由于许多药物，心血管参数的改变，如血压测量，这些是至关重要的，以确保可以考虑采取全球性的生理影响药物的行动在phMRI数据。还为基准值和预期的反应输液5毫克/公斤氟西汀4条。 <ol><li>体温保持在37±1.5°C的温暖空气加热系统（SA仪器，纽约，美国）。要知道，磁共振成像可以影响温度测量，检查自己的系统。 </li><li>监视和记录动物的呼吸频率，呼吸袖加上使用压力传感器（SA仪器，纽约，美国）。 </li><li>使用压力传感器（TSD104A，BIOPAC系统公司，美国）记录有创动脉血压，并定期撤回倒是在成像分析动脉血气样本（RapidLab，西门子诊断）监测动脉PCO 2和氧分压（PO 2），通过空心股动脉。 </li><li>作为主要的的药理挑战（氟西汀（氟西汀从Sigma-Aldrich公司，英国）的解决方案，5毫克/公斤，溶于生理盐水中）输液线，使用空心股静脉。 </li></ol> 在体内成像在图2给出了一个功能磁共振成像实验装置示意图。 3。成像参数<ol><li>一旦动物被放置在扫描仪，并继续呈现稳定的生理反应，成像可以启动。在我们的研究中，我们使用4.7 T小动物磁共振成像系统（安捷伦科技），圆柱正交发送/接收射频线圈内径72毫米（M2M影像公司，美国）。使三个平面球探图像正确带够在磁共振成像领域的观点中osition大脑和使用本地化的垫片校正（fastmap序列），以提高大脑中的磁场均匀性。 </li><li>对于每个动物，第一次获得T2加权解剖图像体积为登记和分割的目的。我们用回声列车长度= 8涡轮自旋回波序列;矩阵大小= 256×256;视野= 50×50毫米，厚度为1毫米，30个连续冠状切片交错收购中心8毫米尾鳍后缘嗅球;平均= 4; TR / TE = 5112/60毫秒。 </li><li>确保动物的生理反应常数开始前phMRI扫描。对于时间序列的收购，我们使用了回声火车的长度= 16同T2加权快速自旋回波序列;交错收购的20个连续片1毫米厚度集中在同一位置; TR / TE，矩阵的大小= 128×128;四九一五/ 60毫秒。我们总共获得32个时间点与Acquisition时间每158秒的时间序列量和总扫描时间84分钟。作为第一册“假人扫描”，以解决T1饱和效应，而不是用于数据分析。也可以使用其他的功能磁共振成像序列，如梯度回波或回波平面成像（EPI）序列。确保评估实验开始前您所选择的序列信号的稳定性。 </li><li>收购数量的基线量，管理挑战用药前。我们认为，在稳定的条件下，基线收购至少10分钟。正是所有动物的同时，开始输液。在我们的协议，我们就开始在开始的第9卷（约21分钟的基线扫描后）的管理。输液后，图像采集持续了60分钟（共32册）。确保输液后的时间足够长，以可视化的变化，并达到稳定状态或恢复的信号，这取决于你的研究questi和你选择药物的挑战。 </li><li>当图像采集完成后，从扫描仪中取出的动物。执行最后血气测量血气参数，以确保稳定，使药物作用的基本生理上的评价。 </li></ol>数据处理 4。生理反应预计到的挑战生理反应是依赖于所选择的药物。下面，普遍接受的基准值（雄性大鼠）和预期的反应，静脉注射5毫克/公斤氟西汀。 <ol><li>在45-75次/分钟，呼吸率应该是稳定的。氟西汀的药理挑战，引起呼吸率在短期上升（15-20％）。 </li><li>血压应该是常数与100-150毫米汞柱（BIOPAC Systems公司，Goweta，美国）。氟西汀的挑战，引起动脉血压在20％左右的短，但急剧下降。这应该在5-10分钟内恢复。这是如图3所示。 </li><li>血气值应该是稳定的（测量至少两次），并在以下范围：开始前phMRI扫描的pCO 2，35-45毫米汞柱; PO 2，80-130毫米汞柱; pH值7.35-7.45。经常检查这些值再次扫描后看到的动物，如果保持稳定，使基本的生理上的药物作用的评价。高的pCO 2值会诱发血管舒张，从而避免看到BOLD信号变化。 </li><li>确保动物麻醉下连续和稳定的水平（2±0.25％的较高水平，可能会导致抑郁症和脑血管反应性较低的麻醉和不足，因此运动）才开始的phMRI扫描和重要的，避免在任何调整这个功能的图像采集麻醉政权期间（如％异氟醚和/或气体的流量），还可能影响BOLD信号。 </li></ol>在这里，我们描述了在致辞数据的预处理几个步骤，以optimalize的数据进行统计分析。我们提到，在我们的实验室中使用的工具，然而有许多不同的工具。 5.1数据准备<ol><li>把MRI分析软件在正确的文件格式原始图像，您更喜欢使用（NIfTI1.1或Analyze7.5格式FSL的方案）。在网上提供一些免费的文件转换程序。取决于所使用的扫描仪，它可能需要先构建一个三维（解剖扫描）或4D（phMRI扫描）所有单独的二维切片的图像。这可以使用一个图像处理程序，如ImageJ的1。 </li><li>为了确保与分析算法设计与人类数据的使用（例如FSL的方案）的相容性，像素尺寸必须乘以10的一个因素（例如使用ImageJ的，这也可以做）。在我们的研究，这导致在像素尺寸为3.91 x 3.91 x 10毫米3。 </li><li>目视检查方向中的违规行为，手工艺品，和运动的图像。小心不要使用扫描您的分析中有明确的文物或过度运动，因为他们会扭曲你的结果。 </li><li>所有扫描的方向应该是同类之间的解剖和功能图像和使用的参考脑的一致性。在我们的研究中，我们使用施瓦茨14所描述的大鼠脑立体定向模板。 FSL的命令fslswapdim可用于重新调整。 </li></ol> 5.2运动校正<ol><li>为了纠正4D时间序列中的任何议案的文物中，我们使用的议案修正的工具McFlirt（运动校正，采用线性图像FMRIB的注册工具，FMRIB的软件库的一部分， <a href="http://www.fmrib.ox.ac.uk/fsl" target="_blank">www.fmrib.ox.ac.uk / FSL</a> ）。 MCFLIRT是一个模式内的议案校正工具DESigned使用功能磁共振成像时间序列和基于优化和使用登记在调情技术，为线性（仿射）多式联运脑图像配准的完全自动化的工具。经常检查之后，如果结果是令人满意的。 </li></ol> 5.3脑分割<ol><li>删除所有从整个头部图像四维3D解剖图像的时间序列的非脑组织。为此，我们使用FSL工具下注（脑提取工具诉2.1，FMRIB的软件库的一部分， <a href="http://www.fmrib.ox.ac.uk/fsl" target="_blank">www.fmrib.ox.ac.uk / FSL</a> ）。默认设置是与人类大脑的使用和开发，因而不理想的大鼠脑组织。我们使用以下参数：分数强度阈值，F = 1.0;和头部半径（毫米），R = 175为大多数动物在垂直梯度分数强度阈值，G = 0.1。如果有必要，可以优化每个科目的这些值。 </li></ol> 6。数据Analysi小号致辞数据统计分析的目标是确定的像素表现出额外的方差，由于药物在统计学上可靠的方式挑战。这可用各种方式方法，甚至可数的软件包。您所做的选择是依赖于在实验室的可用性，软件和知识/经验和您的具体研究问题。在这里，我们建议在我们的实验室中使用的方法。 6.1 <ol><li>前的磁共振成像数据的分析，确定一般线性模型（GLM）的数据将被安装。这可以是一个简单的方形开关模式（关闭前药物和对药物输注）或基于数据的一个特定的模型。我们已经使用刺激计划21，以确定数据为基础的GLM模型。 </li><li>执行两个样本的T检验与所有挑战后卷所有的基线量（例如，在刺激）。或者，离开ØUT第一册（S），在这期间面临的挑战是，因为那些可能并不代表稳定状态的成像量。随后，歧视，超过一定的变动率从基线的所有像素。我们使用所有的像素超过1％的变化。 </li><li>下一步，平均时间在所有这些像素当然，这已经给你一个模型的形状印象。在这种方式，它可以决定，如果挑战1）立即或延迟的影响，2），如果效果达到高原和/或峰和3是否或何时期间再次下降的时间当然效果）扫描。 图4A和4B所示的例子。 </li></ol> 6.2 然后，下一步就是统计测试每个动物原料4D时间对以前建立的GLM模型系列形象。为此，我们使用的固定利差贷款计划壮举（FMRI，专家分析工具，v5.98）17,24。然而，其他功能磁共振成像分析工具availaBLE以及。第一级分析，在分析工具，将设立。这需要以下步骤： <ol><li>确保每个动物使用相同的设置。你可以选择删除第一册（二）前分析，自稳态成像可能不会在这一点上尚未达成。设置的TR，这是每个连续卷的开始之间的时间（秒）。既然你是在寻找这可能会持续整个扫描时间的影响，有没有必要成立一个高或低通滤波器。 </li><li>为了降低噪声，提高信号的信噪比（SNR）的比例，空间平滑的数据。我们选择了一个半高宽8毫米的内核。 </li><li>现在运行所需的GLM模型对您的数据。这是你的主要解释变量（EV），即你正在测试你的数据，对波形。的GLM，决定根据我们自己的数据可以看出，在图4C。也有加入额外混淆EV如运动参数的可能性，低频噪音（扫描仪漂移）或生理参数，如血压，消除一般的生理药物效果。 内壮举：使用胶片白化。新增颞衍生。在对比与F检验标签上，成立了一个对比。一个Z统计图像转换成一个单一的EV，其对比度值设置为1。这使您可以在它的时间当然可以显着的GLM解释像素。将该值设置为-1，将给予负激活。 </li><li>开展初步统计测试后，所产生的统计图像需要门槛值显示在一个特别重要的意义激活的像素或像素集群。多重比较校正是必要的，由于大量测试的脑体素。 FSL程序了FEAT使用一个自动化的基于集群的多重比较校正根据GRF的理论（高斯随机场）25时。 </li><li>最后，数据应该是空间<html>标准化为参考图像，以执行组的统计信息。先注册到动物大脑中提取的解剖图像，然后参考图像的功能数据。我们用14施瓦茨描述作为参考脑立体定向大鼠脑模板。 </li><li>在此之后，所有动物的第一级的分析，可以在更高的水平（集团）统计分析相结合。这是高度依赖自己的研究设计和研究的问题。 </li></ol> 6.3 在此之后，所有动物的第一级的分析，可以在更高的水平（集团）统计分析相结合。这是高度依赖自己的研究设计和研究的问题。 6.4 可以加上生理对药物的反应，或相关的MR信号，如果需要的话。亦见增加混淆的EV的6.2.3节。 7。代表结果ove_content“&gt;具有挑战性的药物（5毫克/公斤IV氟西汀）当进入血管系统，明确的生理反应应该是可见的呼吸速率（上）和血压（下）。这些反应5-10分钟内平均正常化血压下降，在图3是清晰可见。 平均信号的时间当然应该表现出相对稳定的基线和挑战明显的效果。最好，应该是没有独立的漂移信号的挑战。平均信号的时间当然一个有代表性的例子可以看出，在图5A。文物，如呼吸抑制/故障或变化，在麻醉往往在信号清晰可见。呼吸抑制，会产生负面影响在整个大脑的信号。这可以看出，在图5B。 第一层分析后，激活模式预计将主要是积极与定位只有特定地区的TED（即皮层区，海马，下丘脑和丘脑; 见图6A）。如果整个大脑被停用，这往往是麻醉过深和/或在扫描过程中供氧不足的迹象。 图6B中可以看到这样一个例子。 <img alt="图1" src="/files/ftp_upload/3956/3956fig1.jpg" /> 图1。安置在股动脉和静脉插管的位置。 <img alt="图2" src="/files/ftp_upload/3956/3956fig2.jpg" /> 图2表示的MRI设置示意图;所有设备需要非铁磁性，并连接到一个模块系统允许从运动的干扰，避免因呼吸和/或心脏跳动的图像采集门。体温调节，通过加热模块，监视和控制动物的温度在成像<a href="http://www.jove.com/files/ftp_upload/3956/3956fig2large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图3" src="/files/ftp_upload/3956/3956fig3.jpg" /> 图3。血压数据的代表性的例子。有明显下降后开始输液（红色条）直接在血压可见。在10分钟内再次达到正常值。后的挑战管理。 <img alt="图4" src="/files/ftp_upload/3956/3956fig4.jpg" /> 图4 a）预期使用的磁共振成像分析程序刺激（红色是积极激活的激活模式，蓝色是负激活）。二）所有激活的像素，在所有的动物（从基线的变化≥1％）的平均时间的过程。 c）在FSL，/了FEAT GLM模型的范例。 <a href="http://www.jove.com/files/ftp_upload/3956/3956fig4large.jpg" target="_blank">氯</a>ICK这里查看大图。 <img alt="图5" src="/files/ftp_upload/3956/3956fig5.jpg" /> 图5。 <ol class="ualpha"><li>例如，积极激活。在激活体素（红色）的时间当然是继大约GLM模型的形状。药物输注时间8点左右开始。 </li><li>例如在整个大脑的负激活后，麻醉过深。时间过程中的负激活的像素（蓝色）显示在信号普遍下降的挑战没有影响是可见的。 </li></ol> <a href="http://www.jove.com/files/ftp_upload/3956/3956fig5large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图6" src="/files/ftp_upload/3956/3956fig6.jpg" /> 图6。 <ol class="ualpha"><li>预计第一层分析后激活模式。激活集群像素（红色为yellow），只有在特定的大脑区域。 </li><li>例如，&#39;坏&#39;的激活模式。全脑负激活（蓝色）在图5B相同的动物。 </li></ol>

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我们什么都没有透露。

5 -羟色胺phMRI是一种很有前途的工具，以评估在动物体内的神经递质功能。它可视化的5 - 羟色胺的挑战与功能磁共振成像的大脑反应。 MRI具有很大的优势，具有较高的空间分辨率和不需要注射造影剂或放射性标记的分子，从而避免重复暴露于电离辐射。这种技术是适用于人类和动物的科目，因此非常适合神经递质系统和精神疾病的转化研究。其应用当然不仅限于5 -羟色胺的途径，并已被广泛用于多巴胺能药物的效果，以评估这两种动物5,15和人类22。 然而，在小动物phMRI依然严峻，马丁和Sibson 11的管家20的评论文章中已经指出。这些挑战之一是维护f稳定的图像采集过程中的生理参数。大多数麻醉剂可以改变心血管功能，并鉴于phMRI是严重依赖心血管/血流动力学参数，它是必不可少的，以确保任何血流动力学变化完全是由于给定的药物挑战。因此，这是非常重要的，PCO 2水平仍然在基线收购不断。机械通气可帮助确保生理稳定性，并经常使用这种类型的实验。不过，我们选择使用自由呼吸的动物，离开的可能性进行纵向研究，在未来开放。相反，我们广泛的监测和改变呼吸速率和血气值，以确保在正常范围内的生理稳定性，功能扫描开始前，并以这种方式来维护稳定的血管反应性，从而与T2 * / T2信号。全身麻醉的影响脑血流和metabolis文学m是丰富的20本手稿的范围之外。我们选择使用气体麻醉，±2％异氟醚在这个特定的协议，因为吸入性麻醉剂，麻醉深度可以迅速和容易控制。这是在我们的设置重要的图像采集开始前，以确保正常的范围内保持稳定的pCO 2的水平。异氟醚是今天最常用的吸入麻醉，并允许快速诱导和恢复，这是纵向研究的重要。它也产生最小的心血管和呼吸抑制和诱导骨骼肌松弛。 其次，具有挑战性的药物静脉给药是更复杂，比人类的小动物。股动脉和静脉插管需要手术需要训练有素和经验丰富的员工。由于这些入侵程序，它主要用于终端程序的那一刻。然而，非侵入性的监测血液稳态和尾静脉注射，可用于23个纵向研究。 此外，还有一些更普遍的技术，是动物phMRI不具体限制。此外，正如马丁·和Sibson 11的了，所有的功能磁共振成像研究的潜在混淆的是，它假定诱发挑战大脑活动的变化反映在神经元的活动变化，而 ​​不是外围的系统性影响。尤其是在更深的大脑结构，神经血管耦合的神经元活动的变化和血流动力学变化之间的关系仍然相对贫困的理解。 Logothetis 10，以确定在大脑皮层的神经血管耦合的研究尚未在大脑的其他部分。因此，它是未知的BOLD信号等重要结构，增加纹状体或杏仁核是泰兰我们Ğ有关的神经元活动。最好的，我们可以说在这一刻，大脑区域的反应给定的挑战，并根据治疗和/或条件，我们可以监视大脑反应的显着变化。这可以在很大程度上得到验证的磁共振成像数据和生理反应。一般的脑激活模式应该是特定区域和限制与地区，在这种情况下，高5 - 羟色胺的神经支配，而不是一般的血管反应。此外，不同的血管和血流动力学变化之间的时空轮廓预计。而血压的变化在几分钟内返回他们的基线值，大胆的激活作用的药物是图像采集结束在氟西汀可见的情况下，直到这种药物的认识的药代动力学性质和对应。最后，所有动物的生理反应应该是相似的，以使跨学科的比较。 nonetheleSS，它是已知的5 -羟色胺的神经调节局部血流量存在4。因此，它不能被排除在外，当地的BOLD信号变化可能归因于由于5 - 羟色胺的释放船只靠近血管的变化。虽然这些影响都没有关联到地方的神经元激活，因此可以考虑为假阳性结果，它也是一个整体的5 -羟色胺系统的特定功能（另见3）的指数。 因此，这一技术的关键步骤是广泛监测的生理反应，并确保动物生理条件下是稳定的图像采集之前和期间。扫描仪的条件也应该尽可能稳定和每个动物完全一样。信号序列的稳定性实验开始前，应检查并证实。此外，确保始终有足够大的统计力量，甚至小题目石斑鱼PS。为实验，动物phMRI的考虑，一般漂亮的审查，见管家20，在大鼠和小鼠的药理功能磁共振成像实验协议的其他例子，看到法拉利5。 这里描述的技术可能的修改是多方面的。一个可能： <ol><li>使用不同的药物5 -羟色胺的挑战，如另一SSRIs或5 -羟色胺受体（蚂蚁）激动剂16,13,18,7甚至是一个双重挑战，为了揭示药物作用的底层机制6,19; </li><li>使用不同的实验装置，如麻醉制度不同，机械通气，血池造影剂代替血氧15，纵向研究（动物需要更让，所以没有侵入性血压/气体测量和/或机械通风是可能的），或者甚至组合与其他（侵入性），如神经元活动的记录使用兼容议员，元素的方法ctrodes 10或PET / SPECT检查4; </li><li>使用不同的磁共振成像数据分析方法，如“P-块方法麦基12或功能连接分析15。 </li></ol>这你在实验装置中作出的选择是高度依赖的可能性和/或在你的实验室和研究问题的类型，你想回答的经验。

<table border="1"><tbody><tr><td> 试剂/设备名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>异氟醚</td><td>雅培 <a href="http://www.abbott.co.uk/" target="_blank">雅培制药有限公司，英国梅登黑德，</a> </td><td>没有B506 </td><td>医用空气混合</td></tr><tr><td>医用空气</td><td> <a href="http://www.bochealthcare.co.uk/" target="_blank">中银医疗</a> </td><td></td><td></td></tr><tr><td>加热垫</td><td> <a href="http://www.harvardapparatus.co.uk/" target="_blank">哈佛仪器</a> </td><td> 507223F <a href="http://www.harvardapparatus.co.uk/webapp/wcs/stores/servlet/haisku3_10001_11555_39108_-1_HAUK_ProductDetail_N_37610_37611_37613" target="_blank">柔性探头，中等，230伏，50赫兹的完整Homeothermic毛毯系统</a> </td><td></td></tr><tr><td>丝绸升<html> igature </td><td> <a href="http://www.harvardapparatus.com/" target="_blank">http://www.harvardapparatus.com/</a> </td><td> <a href="http://www.harvardapparatus.com/webapp/wcs/stores/servlet/product_11051_10001_43051_-1_HAI_ProductDetail" target="_blank">2-0黑色编织丝绸非吸收性</a> 猫编号51-7631 </td><td></td></tr><tr><td>体育-50套管</td><td> <a href="http://www.scientificlabs.co.uk/" target="_blank">http://www.scientificlabs.co.uk/~~V</a> </td><td> <a href="http://www.scientificlabs.co.uk/product/TUB3662" target="_blank">portex油管体育0.58x0.96mm</a> </td><td> 0.96 0.58编号外径mm </td></tr><tr><td>肝素钠</td><td> <a href="http://www.leo-pharma.com/Home/Products/List-of-LEO-Pharma-products.aspx" target="_blank">利奥制药有限公司，雄鹿队，英国</a> </td><td>肝素钠1000IU/ml </td><td> 15 U / ml的</td></tr><tr><td> vetbond组织胶黏剂</td><td> <a href="http://solutions.3m.co.uk" target="_blank">3M</a> </td><td>TTP :/ / solutions.3m.co.uk/wps/portal/3M/en_GB/HealthCare/Home/ProdInfo/VetProducts的/“目标=”_blank“&gt; H Vetbond组织粘合剂</td><td></td></tr><tr><td>监控系统</td><td> <a href="http://www.i4sa.com/" target="_blank">SA的仪器</a> </td><td> <a href="http://www.i4sa.com" target="_blank">http://www.i4sa.com</a> <a href="http://69.20.42.253/web_app/main/defaultProduct.aspx?ID=16&amp;PT=3" target="_blank">型号1025L监测系统</a> </td><td>呼吸和温度监视器</td></tr><tr><td>压力传感器</td><td> <a href="http://www.biopac.com/" target="_blank">BIOPAC系统公司</a> </td><td> <a href="http://www.biopac.com/blood-pressure-transducer" target="_blank">血液压力传感器- TSD104A</a> <a href="http://www.biopac.com/data-acquisition-analysis-system-mp150-system-windows" target="_blank">MP150的数据采集系统-胜- MP150WSW</a> </td><td>监测血压</td></tr><tr><td> RapidLab血液气体分析仪</td><td> <a href="http://www.medical.siemens.com/" target="_blank">西门子诊断</a> </td><td> <a href="http://www.medical.siemens.com/webapp/wcs/stores/servlet/ProductDisplay~q_catalogId~e_-111~a_catTree~e_100001,1023069,1016115~a_langId~e_-111~a_productId~e_172940~a_storeId~e_10001.htm" target="_blank">RAPIDLab 248/348系统</a> </td><td></td></tr><tr><td> 4.7T动物扫描器</td><td>安捷伦科技（以前Magnex） 4.7T频率199.845兆赫</td><td></td><td></td></tr><tr><td>议员兼容立体床</td><td> <a href="http://www.m2mimaging.com/products/preclinical.html" target="_blank">M2M成像公司</a> </td><td>大鼠床：PA的多元素热源50-72-1003/100 </td><td></td></tr><tr><td>盘</td><td> <a href="http://www.m2mimaging.com/products/preclinical.html" target="_blank">M2M成像公司</a> </td><td>量TH /接收 RQD1 72/112 200 </td><td></td></tr><tr><td>盐酸氟西汀</td><td> Sigma-Aldrich公司</td><td>架F-132 </td><td>按5mg/kg生理盐水</td></tr></tbody></table>

the use of pharmacological-challenge fmri in pre-clinical research: application to the 5-ht system

药理MRI（phMRI），是一个新的和有前途的方法来研究最终可以被用来解开药物作用和神经递质有关的疾病，如抑郁症和多动症，背后潜在的神经生物学机制的物质对大脑功能的影响。最喜欢的成像方法（PET，SPECT显像，CT），它代表了一种脑部疾病的调查进展和相关功能的非侵入性的方式，在尊重整体的神经元连接的神经递质途径。此外，还提供翻译临床研究的理想工具。 MRI检查，同时仍然落后分子成像战略PET和SPECT相比，具有很大的优势，具有较高的空间分辨率和无需注射对比剂或放射性标记的分子，从而避免了重复暴露于电离辐射。功能磁共振成像（fMRI）技术被广泛用于研究和临床设置，它是generally结合与心理电机的任务。 phMRI是一个适应的功能磁共振成像使一个特定的神经递质系统，如五羟色胺（5-HT）的生理或病理条件下，通过一个特定的具有挑战性的药物管理活化后，进行调查。 这里描述的方法的目的是评估自由呼吸的动物的大脑5 - 羟色胺的功能。由5 - 羟色胺系统的挑战，同时采集功能磁共振图像随着时间的推移，大脑应对这一挑战，可以可视化。动物中的几项研究已经证明，如5 - 羟色胺（释放剂，选择性再摄取阻滞剂等）外的药物引起的增加引起血氧水平依赖（BOLD）的特定区域的变化（MRI信号由于信号通过增加供应氧气和g的血液供应发生在大脑激活的含氧/脱氧血红蛋白水平的变化lucose苛刻的神经元），提供神经递质功能的索引。它也被证明，这些影响可以通过治疗，减少5 -羟色胺的可用性16,13,18,7逆转。在成年大鼠，BOLD信号变化后急性SSRI的管理，已被描述几个5 -羟色胺相关的大脑区域，即皮层区，海马，下丘脑和丘脑9,16,15。的5 -羟色胺系统和应对这一挑战的刺激，从而可以被用来作为衡量其在动物和人类的2,11功能。

Watch this Scientific Journal Video about The Use of Pharmacological-challenge fMRI in Pre-clinical Research: Application to the 5-HT System at JoVE.com

The Use of Pharmacological-challenge fMRI in Pre-clinical Research: Application to the 5-HT System

这项技术的目的是评估5 - 羟色胺（5-HT）神经递质的功能，在生活和自由呼吸的动物药理磁共振成像（phMRI）和静脉注射一种选择性血清素再摄取抑制剂（SSRI），氟西汀挑战。

挑战的药理功能磁共振成像：在临床前研究使用的5  - 羟色胺系统中的应用

Pharmacological MRI (phMRI) is a new and promising method to study the effects of substances on brain function that can ultimately be used to unravel ...

the-use-pharmacological-challenge-fmri-pre-clinical-research

Research

JoVE Journal

Medicine

15.3K 次观看.  Academic Medical Center Amsterdam. 这项技术的目的是评估5 - 羟色胺（5-HT）神经递质的功能，在生活和自由呼吸的动物药理磁共振成像（phMRI）和静脉注射一种选择性血清素再摄取抑制剂（SSRI），氟西汀挑战。

视频: The Use of Pharmacological-challenge fMRI in Pre-clinical Research: Application to the 5-HT System

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP)1-3. CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture2. In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis3. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis1-3.
Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-&gamma;)4-6 and late proinflammatory mediators (e.g., HMGB1)7. Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality8,9, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

脓毒症动物模型，用于评估新型草药疗法

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

科研

医学

Handling of the Cotton Rat in Studies for the Pre-clinical Evaluation of Oncolytic Viruses

Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact. For this strategy to be successful, recent studies have shown that involvement of the host immune system is essential. Therefore, oncolytic virotherapy should be evaluated within the context of an immunocompetent model. Furthermore, the study of antitumor therapies in tolerized animal models may better recapitulate results seen in clinical trials. Cotton rats, commonly used to study respiratory viruses, are an attractive model to study oncolytic virotherapy as syngeneic models of mammary carcinoma and osteosarcoma are well established. However, there is a lack of published information on the proper handling procedure for these highly excitable rodents. The handling and capture approach outlined minimizes animal stress to facilitate experimentation. This technique hinges upon the ability of the researcher to keep calm during handling and perform procedures in a timely fashion. Finally, we describe how to prepare cotton rat mammary tumor cells for consistent subcutaneous tumor formation, and how to perform intratumoral and intraperitoneal injections. These methods can be applied to a wide range of studies furthering the development of the cotton rat as a relevant pre-clinical model to study antitumor therapy.

Cotton rats are extremely excitable and have a strong flight-or-fight response. A handling method optimized to reduce the stress of the animals is described which will make cotton rats more accessible as a preclinical model.

处理棉鼠的研究为溶瘤病毒的临床前评估

Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact.  For this strategy to be ...

Pre-clinical Orthotopic Murine Model of Human Prostate Cancer

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information about this disease. The human orthotopic prostate cancer xenograft mouse model provides a useful alternative approach for understanding the specific interactions between genetically and molecularly altered tumor cells, their organ microenvironment, and for evaluation of efficacy of therapeutic regimens. This is a well characterized model designed to study the molecular events of primary tumor development and it recapitulates the early events in the metastatic cascade prior to embolism and entry of tumor cells into the circulation. Thus it allows elucidation of molecular mechanisms underlying the initial phase of metastatic disease. In addition, this model can annotate drug targets of clinical relevance and is a valuable tool to study prostate cancer progression. In this manuscript we describe a detailed procedure to establish a human orthotopic prostate cancer xenograft mouse model.

Prostate cancer is the second most common cause of cancer-related deaths in the United States. An orthotopic cancer model provides a useful approach to understand the biology of prostate cancer and to evaluate the efficacy of therapeutic regimens. This protocol describes detailed steps necessary to establish an orthotopic prostate cancer mouse model.

人前列腺癌的临床前原位小鼠模型

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information ...

Use of a Low-flow Digital Anesthesia System for Mice and Rats

A traditional vaporizer depends on flowing gas and atmospheric pressure for passive anesthetic vaporization. Newly developed direct injection vaporizers utilize a syringe pump to directly administer volatile anesthetics into a gas stream. Unlike a traditional vaporizer, it can be used at very low flow rates, making it ideal for use on mice and rats. 
The equipment's capability to use low flow rates could result in a substantial cost savings due to the reduced need for anesthetic agents, compressed gas, and charcoal scavenging filters1. A lower flow rate means less waste of anesthetic gas and likely reduces the risk of anesthetic exposure to laboratory personnel. Thus, the high levels of precision and safety associated with direct injection vaporizers, along with a reduced need for anesthetic agents, compressed gas, and charcoal filters are beneficial for research requiring small animal anesthesia. 
The goal of this protocol is to demonstrate the use of a syringe-driven direct injection vaporizer as part of a digital, low-flow anesthesia system. The direct injection vaporizer is capable of accurately delivering anesthesia at very low flow rates compared to a traditional vaporizer, making it a promising alternative for controlled gas anesthetic delivery to rodents.

Here, we present a protocol to more safely and efficiently administer anesthetic gas to mice using a digital, low flow anesthesia system utilizing a syringe-driven direct injection vaporizer.

低流量数字麻醉系统的小鼠和大鼠的用途

A traditional vaporizer depends on flowing gas and atmospheric pressure for passive anesthetic vaporization. Newly developed direct injection vaporizers ...

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the subretinal space, and to evaluate toxicity and efficacy in vivo. HR-SD-OCT uses near infrared light (800-880 nm) and has optics specifically designed for the unique optics of the mouse eye with sub-2-micron axial resolution. Transgenic mouse models of outer retinal (photoreceptor) degeneration and controls were imaged to assess the disease progression. Pulled glass microneedles were used to deliver sub retinal injections of adeno-associated virus (AAV) or nanoparticles (NP) via a trans-scleral and trans-choroidal approach. Careful positioning of the needle into the subretinal space was required prior to a calibrated pressure injection, which delivers fluid into the sub retinal space. Real time subretinal surgery was conducted on our retinal imaging system (RIS). HR-SD-OCT demonstrated progressive uniform retinal degeneration due to expression of a toxic mutant human mutant rhodopsin (P347S) (RHOP347S) transgene in mice. HR-SD-OCT allows rigorous quantification of all the retinal layers. Outer nuclear layer (ONL) thickness and photoreceptor outer segment length (OSL) measurements correlate with photoreceptor vitality, degeneration, or rescue. The RIS delivery system allows real-time visualization of subretinal injections in neonatal (~P10-14) or adult mice, and HR-SD-OCT immediately determines success of delivery and maps areal extent. HR-SD-OCT is a powerful tool that can evaluate the success of subretinal surgery in mice, in addition to measuring vitality of photoreceptors in vivo. HR-SD-OCT can also be used to identify uniform animal cohorts to evaluate the extent of retinal degeneration, toxicity, and therapeutic rescue in preclinical gene therapy research studies.

Here we demonstrate a novel approach to using high resolution spectral-domain optical coherence tomography (HR-SD-OCT) to assist delivery of gene therapy agents into the subretinal space, assess its areal coverage, and characterize photoreceptor vitality.

超高分辨率小鼠光学相干层析成像辅助眼内注射在视网膜基因治疗中的应用

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the ...

A Pre-Clinical Porcine Model of Orthotopic Heart Transplantation

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart failure. Multiple small-animal models of heart transplantation have been used to study the acute and long-term effects of novel therapies. However, few are tested and demonstrated success in clinical trials. It is of critical importance to evaluate new therapies in a clinically relevant large-animal model for efficient and reliable translation of basic studies&#39;&#160;findings. Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and previously used to investigate novel cardioprotective strategies. This procedure focuses on acute ischemia-reperfusion injury and is a reliable method to investigate novel interventions which have been tested and validated in smaller experimental models, such as the murine model. We demonstrate its usefulness in assessing cardiac performance during the early post-transplantation period and other potential possibilities enabled by the model.

Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and utilized to investigate novel cardioprotective strategies.

正交心脏移植的临床前猪模型

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart ...

Pre-clinical Model of Cardiac Donation after Circulatory Death

Cardiac transplantation demand is on the rise; nevertheless, organ availability is limited due to a paucity of suitable donors. Organ donation after circulatory death (DCD) is a solution to address this limited availability, but due to a period of prolonged warm ischemia and the risk of tissue injury, its routine use in cardiac transplantation is seldom seen. In this manuscript we provide a detailed protocol closely mimicking current clinical practices in the context of DCD with continuous monitoring of heart function, allowing for the evaluation of novel cardioprotective strategies and interventions to decrease ischemia-reperfusion injury.
In this model, the DCD protocol is initiated in anesthetized Lewis rats by stopping ventilation to induce circulatory death. When systolic blood pressure drops below 30 mmHg, the warm ischemic time is initiated. After a pre-set warm ischemic period, hearts are flushed with a normothermic cardioplegic solution, procured, and mounted onto a Langendorff ex vivo heart perfusion system. Following 10 min of initial reperfusion and stabilization, cardiac reconditioning is continuously evaluated for 60 min using intraventricular pressure monitoring. A heart injury is assessed by measuring cardiac troponin T and the infarct size is quantified by histological staining. The warm ischemic time can be modulated and tailored to develop the desired amount of structural and functional damage. This simple protocol allows for the evaluation of different cardioprotective conditioning strategies introduced at the moment of cardioplegia, initial reperfusion and/or during ex vivo perfusion. Findings obtained from this protocol can be reproduced in large models, facilitating clinical translation.

This protocol shows a simple and flexible approach for the evaluation of new conditioning agents or strategies to increase the feasibility of cardiac donation after circulatory death.

循环死亡后心脏捐献的临床前模型

Cardiac transplantation demand is on the rise; nevertheless, organ availability is limited due to a paucity of suitable donors. Organ donation after ...

Invasive Hemodynamic Assessment for the Right Ventricular System and Hypoxia-Induced Pulmonary Arterial Hypertension in Mice

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. However, the evaluation of right ventricular pressure (RVP) and pulmonary artery pressure (PAP) remains technically challenging in mice. RVP and PAP are more difficult to measure than left ventricular pressure because of the anatomical differences between the left and right heart systems. In this paper, we describe a stable right heart hemodynamic measurement method and its validation using healthy and PAH mice. This method is based on open-chest surgery and mechanical ventilation support. It is a complicated procedure compared to closed chest procedures. While a well-trained surgeon is required for this surgery, the advantage of this procedure is that it can generate both RVP and PAP parameters at the same time, so it is a preferable procedure for the evaluation of PAH models.

Here, we present a protocol to perform an invasive hemodynamic assessment of the right ventricle and pulmonary artery in mice using an open-chest surgery approach.

小鼠右心室系统及缺氧诱发肺动脉高血压的侵入性血液动力学评估

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

重建显微外科缺血再灌注损伤研究的临床前大鼠模型

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...

Use of an Integrated Low-Flow Anesthetic Vaporizer, Ventilator, and Physiological Monitoring System for Rodents

Low-flow digital vaporizers commonly utilize a syringe pump to directly administer volatile anesthetics into a stream of carrier gas. Per animal welfare recommendations, animals are warmed and monitored during procedures requiring anesthesia. Common anesthesia and physiological monitoring equipment include gas tanks, anesthetic vaporizers and stands, warming controllers and pads, mechanical ventilators, and pulse oximeters. A computer is also necessary for data collection and to run equipment software. In smaller spaces or when performing field work, it can be challenging to configure all this equipment in limited space.
The goal of this protocol is to demonstrate best practices for use of a low-flow digital vaporizer using both compressed oxygen and room air, along with an integrated mechanical ventilator, pulse oximeter, and far infrared warming as an all-inclusive anesthesia and physiological monitoring suite ideal for rodents.

Here, we present a protocol to safely and effectively administer anesthetic gas to mice using a digital, low flow anesthesia system with integrated ventilator and physiological monitoring modules.

使用啮齿动物综合低流量麻醉蒸发器、呼吸机和生理监测系统

Low-flow digital vaporizers commonly utilize a syringe pump to directly administer volatile anesthetics into a stream of carrier gas. Per animal welfare ...