这项研究是由德意志研究联合会（CRC1039 A3）和研究资助计划黑森州，研究中心转化医学和药理学TMP的国家"Landesoffensive祖尔发展协会wissenschaftlich-ökonomischerExzellenz"（LOEWE）和否则克朗，费森尤斯基金会支持（EKFS），研究培训组转化研究创新 - 制药（TRIP）。

1. EAE诱导SJL / J小鼠<ol><li> 老鼠 <ol><li>使用11周龄的雌性SJL / J小鼠，并允许他们使其习惯的实验室进行约7天。使用每组N = 10只。 </li><li>对于药物影响的评估，通过饮用水或通过启动免疫后3或5天（每组n = 10）的食物颗粒连续地管理为对照组的药物和安慰剂。在疾病的高峰期，管理药物或安慰剂用牛奶或3％的糖水渍玉米片。 </li></ol></li><li> 免疫物质 <ol><li>在用完全弗氏佐剂（CFA，热杀死的结核分枝杆菌H37 Ra）和2小瓶（5微克每）的乳液使用EAE诱导试剂盒由抗原（蛋白脂质蛋白的肽，PLP139-151，1毫克/毫升的乳剂）冻干百日咳毒素（PTX）。 </li><li>溶解PTX（2微克/毫升）在1×磷酸盐缓冲盐水（PBS; 即&lt;/ em&gt;的加1.5毫升的PBS每个PTX管，拌匀，用相同的枪头删除，并添加1毫升的PBS在50毫升管）;拌匀。 </li></ol></li><li> 免疫接种 <ol><li>注入PLP / CFA皮下注射2份，每份100微升，同时在尾的基部。不注入颈后，因为在上背部或颈部的皮肤的任何免疫反应将干扰头部和脊髓的成像。 </li><li>注入100μL的PTX腹膜内（IP）的，每只小鼠两次，第1 - 免疫后2小时，第二个为24小时。 </li><li>对于对照小鼠，注射CFA而不PLP（100μL2份）加紫杉醇而不PLP。 </li></ol></li><li> 免疫接种后的鼠标处理 <ol><li>称量小鼠隔日达7天，然后每天的体重它们。 注:老鼠损失大约1 - EAE在进行二克的机身重量。下降标志着EAE的发作。 </li><li>从第7天，每天符合评估的临床症状ING的标准评分系统（即 0分:在运动功能无明显变化;得分0.5:尾部的远端瘫痪;比分1:完整的尾巴瘫痪;得分1.5:一个或两个后肢轻度麻痹; 2分:后腿严重麻痹;得分2.5:一只后腿完全瘫痪; 3分:两条后腿完全瘫痪;得分3.5:后腿和一个前腿麻痹完全瘫痪安乐死小鼠的3.5或更高的分数&gt; 12小时。 </li></ol></li><li> 当然EAE和成像的时间 <ol><li>在该疾病的发病执行第一成像，当小鼠达到分数&gt; 1，这将发生约10天 - 免疫后12。 </li><li> 3天 - 在高峰期，这将达到1或2天的初期症状发展后，将持续1执行第二成像。 注意:此后，小鼠将充分于7至10天恢复。间隔在成像过程中可能仍然显示血管泄漏，但炎症的指标应该是否定的。 </li></ol></li></ol> 2.生物发光和视神经炎及脑部炎症的近红外成像<ol><li> 成像系统的设置 <ol><li>执行的体内成像的任何设备，允许生物发光和近红外信号的分析。 </li><li>不时2鼠标 - 2.5％，异氟醚麻醉过程中的所有成像过程。 </li><li>位置的一个或使用中间的天然气供应在装置彼此旁边两只老鼠。在中心的脊柱上部位置。 </li><li>同时使用两只老鼠，每一组都有，对于药物影响的评价比较对。这对于生物发光成像重要。 </li><li>盾用黑布免疫的网站，并拍摄照片和基准图像来评估鼠标/小鼠的正确定位。使用B对焦一个6.5厘米的距离相机的所有图像。 </li></ol></li><li> 注射液和生物发光炎症探头成像 <ol><li>使用活体成像系统设置:外延BLI，EM过滤开，防爆过滤块，fstop 1，分档8，关注B =6.5厘米，广告曝光120秒;取基准图像。 </li><li>注入准备使用的化学发光试剂（40毫克/毫升）的100微升的ip。灌装注射器之前拌匀。 </li><li>捕捉生物发光图像5，10和注射后15分钟。生物发光峰值的时间过程的动物之间是不同的。 注:峰值将出现5 - 注射后10分钟;在15分钟的下降表明不需要进一步的图像。使用控制和治疗组小鼠对消除偏见未成年人由于不同的时间进程。 </li><li>填写相关的实验说明。观察一个对话框会自动弹出;它包括信息，如小鼠品系，性别，时间点，探针注射，组等的时间。保存文件中的所有邻NE文件夹;他们将有时间标签和所有描述。 </li></ol></li><li> 注入和成像BBB完整性近红外荧光纳米粒子 <ol><li>在B-焦（6.5公分距离）使用近红外成像落射荧光。聚乙二醇化的荧光纳米粒子的激发/发射最大值是690分之675纳米。捕获在不同的波长，在Ex640 / Em700和Ex675 / EM720两个图像;使用2-S曝光，分箱8和fstop 2.取一个基准图像。 </li><li>注入四70μL的聚乙二醇化的荧光红外线纳米颗粒的通过尾静脉和想象小鼠3小时，使用上述设置注射后24小时。灌装注射器之前搅拌溶解良好。 </li><li>在对照小鼠，这将需要进行评估的信号的特异性注入0.9％氯化钠。 </li></ol></li><li> 注射和MMP活性的近红外荧光探针的成像 <ol><li>剃须或脱毛头部和uPPER脊区采取基线映像之前谨慎一天。皮肤不能受伤。 </li><li>在B-焦（6.5公分距离）使用近红外成像落射荧光。基质金属蛋白酶活化探针的激发/发射最大值是700分之680纳米。捕获在不同的波长，在Ex640 / Em700和Ex675 / EM720两个图像;使用1-S曝光，分箱8和fstop 2.取一个基准图像。 </li><li>添加200μL的1×PBS中的至准备使用的解决方案（20纳摩尔/ 1.5毫升在1×PBS中），因此，这将是足够的10只小鼠的1.5毫升管;灌装注射器之前拌匀。 注意:提供体积不考虑某些容积注射器填充和注射过程中丢失的帐户。 </li><li>成像前注入探针四的150微升通过尾静脉24小时。只在对照动物注射的PBS，以评估信号的特异性。 </li><li>在注射后24小时，采取落射荧光图像至少在两个波长Ex640 / Em700和Ex 675 / EM720，与设置上述（1 - S的曝光，对焦B，分箱8，和fstop 2）进行说明。 注:使用两种波长的允许光谱分离减去非特异性信号。 </li></ol></li></ol> 3.图像分析<ol><li> 生物发光分析（BLI） <ol><li>双击软件来打开它。 </li><li>在菜单栏上，点击文件浏览器图标，进入实验的文件夹目录，然后选择它;这将在表中的文件夹中打开的所有文件。 </li><li>配置示出有关的实验的描述的列。 </li><li>对于质量控制，对非应答鼠标文件而不EAE和/或天真鼠标症状双击检查EAE信号的特异性。 注:应该有天真的小鼠和非应答小鼠最小的信号没有信号。 <ol><li>亲注射前检查基线图像对于每个小鼠作为信号的特异性的进一步控制;它应该是负的。 </li></ol></li><li>要选择一个图像，就先EAE小鼠的第一个文件，然后双击并检查力度生物发光（LUT条范围）和本地化。检查所有图像逐一。关闭与每个鼠标的最低强度的文件（ 即 ，保持2出3图像的每个鼠标）。 </li><li>图像调整和出口<ol><li>双击进行量化的第一个文件。观察一个新的窗口弹出。在"选项"（上一级菜单），自定义标签每个图像中显示。 </li><li>在右边的工具选项板，进入"图像调整"。缺省情况下，最小和最大强度被设置为"自动"和彩虹伪显示。选择"手动"，如果需要更改设置。 注:例如，所有导出的图像可能有相同的LUT条（相同的最小值和最大值）是易于比较。的最小值和最大值的调整对定量结果没有任何影响。 </li><li>点击图片导出，选择PNG，目录，图像名</li></ol></li><li>感兴趣区域的定量分析（ROI） <ol><li>转到工具选项板中的"投资回报率"的工具。选择的ROI方法（圆圈，矩形，汽车，或自由手）和感兴趣区域的数目。观察ROI窗口在图像窗口弹出。 </li><li>使用鼠标，调整大小和位置。如果使用自动ROI工具中使用相同的ROI阈值的所有图像。如果投资回报率大小和位置手动（ 例如，圆形的投资回报）定义使用相同的领域的所有图像。 </li><li>点击"衡量RO.I.观察一个新的窗口弹出，自定义的列（如文件名，动物数量，组，实验区，总计数，平均计数，计数SD，最大和最小数，面积，时间点，注射探针等 ）的时间。保存自定义设置。当再安以轩，选择所有（按Ctrl + A），复制并表粘贴到电子表格。 </li><li>导出图像作为替代的投资回报为PNG。保存并关闭图像文件。 </li></ol></li><li>重复该过程（步骤3.1.6 - 3.1.7）对需要进行量化的所有图像文件。所有的投资回报率quantifications复制到电子表格。 注:在这里，结果可以通过组，时间点等进行分类和统计分析。使用生物发光信号的总数在感兴趣区为统计分析。 </li></ol></li><li> 荧光纳米粒子的近红外（NIR）分析 <ol><li>在软件使用控制，调整图像的阈值。 注意:这对定量结果没有影响。 </li><li>目视比较在实施例/ EM七百二十零分之六百七十五和Ex / Em比值七百分之六百四十零捕获，以评估信号的特异性的图像。 </li><li>在使用前/ EM七百二十零分之六百七十五拍摄的图像进行定量分析（激发最大:680纳米）。定义的ROI，可以使用的量，自动ROI工具。调整自动ROI阈值和使用它的所有图像。量化的投资回报的总辐射效率（见步骤3.1）。 </li></ol></li><li> 蛋白酶敏感探针的近红外（NIR）分析 <ol><li>利用软件控制，调整图像的门槛。 注意:这对定量结果没有影响。执行自动荧光光谱分离。去混合工具在生活图像来实现。自去混合使用防爆/ EM七百分之六百四十零作为具体七百二十零分之六百七十五作为自体荧光图像。 </li><li>选择未混合图像和定义的ROI，如上所述。使用投资回报的总辐射效率的量化和统计分析。 </li></ol></li></ol>

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作者宣称，他们没有竞争的经济利益。

本视频显示了在 SJL / J小鼠EAE的体内成像生物发光和近红外荧光的技术。我们表明，使用炎症敏感探针生物发光成像主要表示视神经炎，以及量化与EAE严重程度的临床评估和药物的效果一致。但是，由于该信号被脊柱所吸收的生物发光成像方法是不能检测的腰脊髓，这是EAE表现17的原发部位的炎症，可能的。 近红外成像是更敏感，但在与自体荧光，它不与生物发光产生高干扰为代价的。近红外的曝光时间短得多 - 相比，BLI（1 2秒）（2 - 5分钟），这使得近红外的优选方法为快速变化的信号强度的时间序列。 关键步骤与在协议和技术的局限性免疫站点的信号干扰对EAE脊髓成像，但是这可以通过在尾巴，这是不逊色于推荐注射部位（颈部和尾部的碱）的底部定位两个注射部位被规避。尽管如此，有必要屏蔽用黑布注射部位。 对于生物发光成像，这是捕获的时间序列图像的关键，因为动力学动物之间不同。以避免由于动力学偏见，对控制（ 例如，车辆或野生型）和八角（ 例如，药物或转基因）的小鼠的成像是有利的。根据生产商，该信号应为30分钟是稳定的。然而，在EAE，我们观察到更快，更瞬态动力学，与5峰发生 - 10分钟和15分钟大幅下降。 对于近红外成像中，厂家专业cturer建议特异性探针防爆/ EM设置。然而，它是最初运行一个过滤器系列有用和两个激发/发射组合总是捕获至少图像，从而紧密地匹配所报告的实施例/ EM最大值和稍后可用于非特异性信号的光谱分离。 它是用白老鼠，像SJL / J重要的，因为光和荧光深受黑毛吸收。黑鼠需要谨慎剃光头，成像前回来一天。皮损必须避免，因为它们会出现炎症斑点和与脑或脊髓的摄像干涉。对于近红外成像，制造商建议所有小鼠，甚至白的小鼠的脱毛。即使剃须后，头部和背部导致黑鼠比用白老鼠（未显示）那么令人信服。在C57BL6小鼠初级渐进EAE模型，白色C57BL6小鼠，它是市售的，可以是一个ALTER当地人。无毛，免疫小鼠SKH1是近红外成像有用的，但不是EAE，因为这些老鼠有白化病的遗传背景和不可靠的发展EAE（最高得分:0.5 - 1）。使用在这些小鼠中的炎症探针生物发光成像显示在皮肤几种炎症斑（未示出），其中毛囊会丢失。 蛋白酶活性的近红外成像显示脑和脊髓炎，但信号被自动荧光叠加，需要定量分析之前光谱分离。因此，近红外成像比生物发光成像不太可靠的并且是更昂贵的。然而，使用蛋白酶活化探针可以是用于特异性靶向基质金属蛋白酶的药物的评估是有用的。 的优点和缺点不同的成像技术是免费的，解决具体问题。 biolumin的优点escent成像的承受能力（约20欧元/鼠标）;缺乏自动生物发光，这将干扰信号的;特异性高;方便腹腔注射和图像分析;和鲁棒性和可靠性。缺点是长时间曝光时间和信号吸收黑毛和骨头。 NIR的优点是近红外标记探针，易于定制的近红外标记，曝光时间短，灵敏度高的更广泛的可用性。缺点是成本高（50 - 100欧元/鼠标），吸收的皮毛，自动荧光强干扰和必要性分析之前，光谱分离和图像处理。 一些探针是可用的检测炎症部位，因为它们是由是在炎症（ 例如，过氧化物酶或金属蛋白酶）的位点上调由于免疫细胞的浸润促炎酶激活。一些这些探针的还将检测着性RS显示出免疫细胞浸润到肿瘤微环境或酶的由肿瘤本身的释放（ 如蛋白酶 ）。在组织积累由于毛细血管渗漏探针将检测中断在血脑屏障，而且在炎症和癌症的其他部位。 关于到现有/替代方法的技术意义 "影像加上临床疮"的组合明显优于"只分"为疾病状态评估和药物治疗效果的检测。眼睛周围的生物发光信号还同意与先前的组织病理学研究显示在视神经5髓鞘破坏和免疫细胞浸润。关于MS患者的2/3发展视神经炎发作。到目前为止，还没有可靠的，非侵入性的方法来量化视神经炎在活除了扩散磁共振成像（MRI）和运算的小鼠蒂卡尔相干断层扫描（OCT），这是技术要求高的18。 OCT已被引入的EAE作为表示EAE期间眼底改变和萎缩的方法，这表明在视神经19的自身免疫反应。 相比，在本手稿中描述的方法，OCT是小鼠更高的压力源，因为它需要深度麻醉。读出是不视神经19的直接可视化。 荧光纳米粒子的近红外成像是可视化的血脑屏障，这是EAE和MS的另一个标志的破坏是有用的。除了MRI 20,21，对于血脑屏障完整性的体内监测没有非侵入性的方法。这将是非常有用的，因为实验性药物和植物药物，特别是通过收紧屏障22行事， </suP&gt; 23，通常阻碍淋巴细胞募集过度进入CNS 24。通过血脑屏障阻止白细胞附件或轮回，并减少其泄漏是MS治疗25和纳米粒子成像的有效策略可能有助于评估候选药物的疗效。到目前为止，该颗粒是昂贵的。活体显微镜是用于可视化血脑屏障完整性26的另一种技术，但是它通常需要持久的，深度麻醉（ 例如，用氯胺酮和赛拉嗪），因为开颅和不允许重新唤醒小鼠，从而防止时间过程分析。然而，活体显微镜提供在细胞的高清晰度图像，以亚细胞水平，这是不与体内成像实现的。 掌握技术后，未来的应用方向或总之，成像德在本视频帮助呈现chniques来评估疾病的个体的课程和监测药物治疗的效果，这是在不通过单独临床得分显示部。该技术的同意更换，减少和细化在动物实验中的3"R"的原则，并附加在药物研究工具是有用的。

Multiple sclerosis is caused by the autoimmune-mediated attack and destruction of the myelin sheath in the brain and the spinal cord1. With an overall incidence of about 3.6 cases per 100,000 people a year in women and about 2.0 in men, MS is the second most common cause of neurological disability in young adults, after traumatic injuries2,3. The disease pathology is contributed to by genetic and environmental factors4 but is still not completely understood. Autoreactive T lymphocytes enter the central nervous system and trigger an inflammatory cascade that causes focal infiltrates in the white matter of the brain, spinal cord, and optic nerve. In most cases, these infiltrates are initially reversible, but persistence increases with the number of relapses. A number of rodent models have been developed to study the pathology of the disease. The relapsing-remitting EAE in SJL/J mice and the primary-progressive EAE in C57BL6 mice are the most popular models.
The clinical EAE scores, which describe the extent of the motor function deficits, and body weight are the gold standards to assess EAE severity. These clinical signs agree with the extent of immune cell infiltration and myelin destruction in the spinal cord and moderately predict drug treatment efficacy in humans5. However, these signs mainly reflect the destruction of the ventral fiber tracts in the spinal cord. Presently, there is no easy, non-invasive, reliable, and reproducible method to assess in vivo brain infiltration and optic neuritis in living mice. 
The in vivo imaging agrees with the 3 "R" principles of Russel and Burch (1959), which claim a Replacement, Reduction, and Refinement of animal experiments6, because imaging increases the readouts of one animal at several time points and allows for a reduction of the overall numbers. Presently, inflammation or myelin status is mainly assessed ex vivo via immunohistochemistry, FACS-analysis, or different molecular biological methods7, all requiring euthanized mice at specific time points. 
A number of in vivo imaging system probes have been developed to assess inflammation in the skin, joints, and vascular system. The techniques rely on the activation of bioluminescent or near-infrared fluorescent substrates by tissue peroxidases, including myeloperoxidase (MPO), matrix metalloproteinases (MMPs)8, and cathepsins9 or cyclooxygenase2. These probes have been mainly validated in models of arthritis or atherosclerosis9,10. A cathepsin-sensitive probe has also been used for fluorescence molecular tomographic imaging of EAE11. MMPs, particularly MMP2 and MMP9, contribute to the protease-mediated BBB disruption in EAE and are upregulated at sites of immune cell infiltration12, suggesting that these probes may be useful for EAE imaging. The same holds true for peroxidase or cathepsin-based probes. Technically, imaging of inflammation in the brain or spinal cord is substantially more challenging because the skull or spine absorb bioluminescent and near-infrared signals. 
In addition to inflammation indicators, fluorescent chemicals have been described, which specifically bind to myelin and may allow for quantification of myelination13. A near-infrared fluorescent probe, 3,3'-diethylthiatricarbocyanine iodide (DBT), was found to specifically bind to myelinated fibers and was validated as a quantitative tool in mouse models of primary myelination defects and in cuprizone-evoked demyelination14. In EAE, the DBT signal was rather increased, reflecting the inflammation of the myelin fibers5. 
An additional hallmark of EAE and MS is the BBB breakdown, resulting in increased vascular permeability and the extravasation of blood cells, extracellular fluid, and macromolecules into the CNS parenchyma. This can lead to edema, inflammation, oligodendrocyte damage, and, eventually, demyelination15,16. Hence, visualization of the BBB leak using fluorescent probes, such as fluorochrome-labeled bovine serum albumin5, which normally distribute very slowly from blood to tissue, may be useful to assess EAE.
In the present study, we have assessed the usefulness of different probes in EAE and show the procedure for the most reliable and robust bioluminescent technique. In addition, we discuss the pros and cons of near-infrared probes for MMP activity and BBB integrity.

<table><tbody><tr><td>AngioSpark-680</td><td>Perkin Elmer, Inc., Waltham, USA</td><td>NEV10149</td><td>Imaging probe, pegylated nanoparticles, useful for imaging of blood brain barrier integrity</td></tr><tr><td>MMP-sense 680</td><td>Perkin Elmer, Inc., Waltham, USA</td><td>NEV10126</td><td>Imaging probe, activatable by matrix metalloproteinases, useful for imaging of inflammation</td></tr><tr><td>XenoLight RediJect Inflammation Probe</td><td>Perkin Elmer, Inc., Waltham, USA</td><td>760535</td><td>Imaging probe, activatable by oxidases, useful for imaging of inflammation</td></tr><tr><td>PLP139-151/CFA emulsion&amp;nbsp;</td><td>Hooke Labs, St Lawrence, MA</td><td>EK-0123</td><td>EAE induction kit</td></tr><tr><td>Pertussis Toxin</td><td>Hooke Labs, St Lawrence, MA</td><td>EK-0123</td><td>EAE induction kit</td></tr><tr><td>IVIS Lumina Spectrum</td><td>Perkin Elmer, Inc., Waltham, USA</td><td></td><td>Bioluminescence and Infrared Imaging System</td></tr><tr><td>LivingImage 4.5 software&amp;nbsp;</td><td>Perkin Elmer, Inc., Waltham, USA</td><td>CLS136334</td><td>IVIS analysis software</td></tr><tr><td>Isoflurane</td><td>Abbott Labs, Illinois, USA</td><td>26675-46-7</td><td>Anaesthetic</td></tr></tbody></table>

bioluminescence and near-infrared imaging of optic neuritis and brain inflammation in the eae model of multiple sclerosis in mice

在SJL / J小鼠实验性自身免疫性脑脊髓炎（EAE）是复发缓解型多发性硬化症（RRMS）的模型。描述运动功能障碍的临床EAE分数是脊髓的免疫介导的炎症的基本读数。但是，得分和体重不允许大脑炎症和视神经炎的体内评估。后者是在MS患者的大约2/3的早期和频繁表现。这里，我们表明使用体内成像系统在活小鼠生物发光和近红外实时成像以评估EAE诱发视神经炎，脑炎症，和血-脑屏障（BBB）的破坏的方法。通过氧化酶激活的生物发光底物，主要表现为视神经炎。该信号是具体和允许的药物治疗效果和疾病时程可视化，从而平行临床得分。聚乙二醇化荧光纳米粒子仍然是vasculatur内e表示延长的时间段被用来评估血脑屏障完整性。近红外成像显示在疾病的峰的血脑屏障渗漏。该信号是眼睛周围的最强的。为基质金属蛋白酶近红外基材是用来评估EAE诱发炎症。自动荧光干扰信号，要求对定量光谱分离。总体而言，生物发光成像是评估EAE相关视神经炎和药物作用的可靠的方法，并在信号的特异性，鲁棒性，易于量化的，和成本方面优于近红外技术。

视神经炎的生物发光的时间进程 炎症探针的生物发光信号是眼睛周围的最强和只在EAE小鼠视神经炎的发生。发生在没有非EAE小鼠也不不与炎症探针注入的小鼠的信号。当小白鼠恢复的信号消失了。因此，该信号是特异于视神经炎，信号的峰值平行于临床EAE分数的峰。 图1示出了在EAE诱导后第10天14成像的SJL / J小鼠的两个例子。生物发光信号是最高的第10天消失，当小鼠开始复苏。临床评分的时间进程相匹配的生物发光信号（例如-1）或之前（例如-2）的分数下降的消失。 图1.视神经炎和EAS-SJL / J小鼠临床评分的时间过程。视神经炎的生物发光图像捕获的疾病的1 次光斑在炎症探针（100μLIP）注射后10分钟，在免疫后的不同时间点的两个SJL / J小鼠。生物发光图像（左图）进行免疫后10和14天捕获并表示为彩虹伪照片重叠。甩杆范围从蓝色（低），红色（高生物发光）。比例尺:1厘米。右图显示了在投资回报个人总生物发光计数的条形图（平均值±3的每个图像的SD）和临床EAE分数的全日制课程。红色箭头标记成像天。 <a href="http://ecsource.jove.com/files/ftp_upload/55321/55321fig1large.jpg" target="_blank">请点击此处查看大图已经</a>这个数字rsion。 疗效评估 炎症探头 用药（R-氟比洛芬5毫克/公斤/天）5的影响示于图2。各组中的生物发光图像的五个实例。车辆和治疗组中的得分分别为异质的，但在所有的小鼠中，在示出视神经炎症眼部生物发光信号是药物组（ 图 2A）中更低。在感兴趣区的总生物发光计数的定量证实显著治疗功效（ 图2B，总生物发光计数的箱形图，不成对的双尾t-检验，P &lt;0.05）。成像效果同意的治疗效果在临床评分和脊髓中的EAE的组织病理学表现和视神经5方面的药物的第<img alt="图2" src="/files/ftp_upload/55321/55321fig2.jpg" /> 图2.采用生物光学成像EAE-SJL / J小鼠用药的疗效。 SJL / J小鼠免疫后接受载体或药物（R-氟比洛芬5毫克/公斤/天）从第5天。图像在1 日 EAE高峰炎症探头（100微升IP）注射后被俘，每组n = 10。 EAE开发两组7/10，和EAE非应答者是没有信号。 A）活小鼠生物发光图像捕获5 - 100μL炎症探头的IP注射后15分钟显示为彩虹伪照片覆盖。 LUT酒吧范围从蓝色（低），红色（高强度）。比例尺:1厘米。个别高峰在感兴趣区的总生物发光计数的用于定量。感兴趣区被限制在头部。该PLP注射部位用黑布屏蔽。 B）的箱形图表示的ROI的总计数的定量。盒子表示四分位数间距，行是中值，并且晶须显示最小到最大。总计数组之间显著差异（双侧非配对t-检验，P &lt;0.05），显示了在接受药物治疗的小鼠的降低视神经炎和脑炎。 <a href="http://ecsource.jove.com/files/ftp_upload/55321/55321fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 聚乙二醇化荧光纳米粒子 近红外纳米粒子的表面荧光图像显示在眼睛周围，并在两个治疗GROU脑血管泄漏PS（ 图3A中 ，2的例子）。颗粒从血液分布非常缓慢的间隙，除了炎症，其中染料积累，允许BBB完整性的评估的网站。泄漏是显而易见的在3小时和纳米颗粒的注射后24小时，但它是在稍后的时间点更强。有非EAE小鼠或小鼠未用纳米颗粒（右面板）注入无特定信号。因此，该信号是特异性的。在投资回报发光效率（ 图3B）的定量分析没有发现治疗组（n = 3每组，不成对的双尾t检验，P &lt;0.05）显著差异。 <img alt="图3" src="/files/ftp_upload/55321/55321fig3.jpg" /> 图3.血脑屏障破坏聚乙二醇荧光纳米粒子的评价。 SJL / J小鼠接受载体或药物（R-flurbiprof烯免疫后5毫克/公斤/天）从第5天。图像被捕获的3小时和近红外标记的纳米颗粒的注射后24小时（70微升ⅳ）第1 EAE高峰期间，n = 3的每个组。 EAE非应答者是没有信号。自动ROI工具被用于的辐射效率的量化。感兴趣区被限制在头部。所述PLP注射部位屏蔽。 A）活小鼠的典型落射荧光图像，抓获3小时和纳米粒子注射后24小时。没有EAE或没有纳米颗粒的注射的小鼠的图像被用来作为成像对照。比例尺:1厘米。 LUT酒吧范围从暗红色（低），黄色（高强度）。 B）条形图显示的投资回报率的辐射效率的量化（平均值±SD）。治疗组没有显著差异（双侧非配对t检验）。 <a href="http://ecsource.jove.com/files/ftp_upload/55321/55321fig3large.jpg" target="_blank">请点击此处到v</a><html> IEW这个数字的放大版本。 基质金属蛋白酶敏感探头 减去自发荧光后，蛋白酶可活化的基质金属蛋白酶探针的图像显示在脑和在EAE小鼠的脊髓（ 图4A，每组4只小鼠的例子）炎症。有在不与探针注射的小鼠没有信号，和发生在一个EAE小鼠无临床症状（非应答）的弱信号。 图4图像显示在防爆/ EM七百分之六百四十零在防爆/ EM七百二十零分之六百七十五由图像减去的信号。在治疗之间的差异仅光谱分离后在自动的ROI的辐射效率的定量分析后发现（ 图4B，不成对的双尾t-检验中，n = 6和4，P &lt;0.05）。 尔斯/ ftp_upload / 55321 / 55321fig4.jpg"/&gt; 图4.金属蛋白酶活性的评估与近红外MMP-敏感成像探头。 SJL / J小鼠免疫后接受载体或药物（R-氟比洛芬5毫克/公斤/天）从第5天。图像探头的注射后捕获的24小时在第1次的EAE峰（150微升ⅳ）中，n = 6和4中的PLP注射部位屏蔽。 EAE非应答者和小鼠无MMP探针注射作为对照。每2图像是在防爆/ EM700分之640纳米（特定信号）和七百二十零分之六百七十五纳米（自动荧光）抓获。使用光谱分离工具，自发荧光减去，并随后，使用自动ROI工具来识别特定MMP活性的位点。用于定量的总辐射效率。 A）活小鼠典型的落射荧光图像捕获探针注射后24小时。这些图像光谱分离（UMX）的结果。比例尺:1厘米。 LUT酒吧从暗红色（低），黄色（高强度）■范围内。 B）箱形图显示的投资回报率的总辐射效率的量化。盒子表示四分位数间距，行是中值，并且晶须显示最小到最大。星号表示（2面非配对t-检验，P &lt;0.05）基团之间的显著差异。 <a href="http://ecsource.jove.com/files/ftp_upload/55321/55321fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Bioluminescence and Near-infrared Imaging of Optic Neuritis and Brain Inflammation in the EAE Model of Multiple Sclerosis in Mice at JoVE.com

Bioluminescence and Near-infrared Imaging of Optic Neuritis and Brain Inflammation in the EAE Model of Multiple Sclerosis in Mice

我们表明用于体内实时生物发光和近红外中在SJL / J小鼠多发性硬化的实验性自身免疫性脑脊髓炎（EAE）模型视神经炎和脑炎的成像技术。

多发性硬化症的小鼠模型EAE视神经炎及脑部炎症的生物发光和近红外成像

Experimental autoimmune encephalomyelitis (EAE) in SJL/J mice is a model for relapsing-remitting multiple sclerosis (RRMS). Clinical EAE scores describing ...

bioluminescence-near-infrared-imaging-optic-neuritis-brain

Nanyang Technological University

Research

JoVE Journal

Medicine

9.1K 次观看.  University Hospital Frankfurt. 我们表明用于体内实时生物发光和近红外中在SJL / J小鼠多发性硬化的实验性自身免疫性脑脊髓炎（EAE）模型视神经炎和脑炎的成像技术。 

视频: Bioluminescence and Near-infrared Imaging of Optic Neuritis and Brain Inflammation in the EAE Model of Multiple Sclerosis in Mice

Implanting Glass Spinal Cord Windows in Adult Mice with Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as multiple sclerosis. Here we present a low-cost and reproducible glass window implantation protocol that is suitable for intravital microscopy and studying the dynamics of spinal cord cytoarchitecture with subcellular resolution in live adult mice with EAE. Briefly, we surgically expose the vertebrae T12-L2 and construct a chamber around the exposed vertebrae using a combination of cyanoacrylate and dental cement. A laminectomy is performed from T13 to L1, and a thin layer of transparent silicone elastomer is applied to the dorsal surface of the exposed spinal cord. A modified glass cover slip is implanted over the exposed spinal cord taking care that the glass does not directly contact the spinal cord. To reduce the infiltration of inflammatory cells between the window and spinal cord, anti-inflammatory treatment is administered every 2 days (as recommended by ethics committee) for the first 10 days after implantation. EAE is induced only 2-3 weeks after the cessation of anti-inflammatory treatment.
Using this approach we successfully induced EAE in 87% of animals with implanted windows and, using Thy1-CFP-23 mice (blue axons in dorsal spinal cord), quantified axonal loss throughout EAE progression. Taken together, this protocol may be useful for studying the recruitment of various cell populations as well as their interaction dynamics, with subcellular resolution and for extended periods of time. This intravital imaging modality represents a valuable tool for developing therapeutic strategies to treat autoimmune demyelinating diseases such as EAE.

We describe a method for implanting and maintaining glass windows over the exposed spinal cords of adult mice for studying experimental autoimmune encephalomyelitis. These windows provide chronic optical access to the spinal cord for monitoring cell dynamics at the subcellular level in live animals using two-photon microscopy.

玻璃植入脊髓的Windows在成年小鼠实验性自身免疫性脑脊髓炎

Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as ...

科研

医学

Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) resulting in cognitive and motor impairment. Experimental autoimmune encephalomyelitis (EAE) is a useful animal model of MS, because it is also characterized by lesion formation in the CNS, motor impairment and is also driven by autoimmune and inflammatory reactions. One of the EAE models is induced with a peptide derived from the myelin oligodendrocyte protein (MOG)35-55 in mice. The EAE mice develop a progressive disease course. This course is divided into three phases: the preclinical phase (day 0 - 9), the disease onset (day 10 - 11) and the acute phase (day 12 - 14). MS and EAE are induced by autoreactive T cells that infiltrate the CNS. These T cells secrete chemokines and cytokines which lead to the recruitment of further immune cells. Therefore, the immune cell distribution in the spinal cord during the three disease phases was investigated. To highlight the time point of the disease at which the activation/proliferation/accumulation of T cells, B cells and monocytes starts, the immune cell distribution in lymph nodes, spleen and blood was also assessed. Furthermore, the levels of several cytokines (IL-1&#946;, IL-6, IL-23, TNF&#945;, IFN&#947;) in the three disease phases were determined, to gain insight into the inflammatory processes of the disease. In conclusion, the data provide an overview of the functional profile of immune cells during EAE pathology.

This manuscript describes the methods for induction and scoring of the experimental autoimmune encephalomyelitis (EAE) model, together with the assessment of immune cell distribution and mRNA cytokine levels in lymph nodes, spleen, blood and spinal cord using flow cytometry and quantitative PCR, respectively, at various disease phases.

在免疫细胞在不同组织与疾病相关的分布和小鼠实验评价性自身免疫性脑脊髓炎的诱导

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) ...

免疫与感染

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

细胞动力学在小鼠脊髓双光子成像

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

神经科学

Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein prionoids, which cause neuropathology within the central nervous system (CNS). Here we describe that intraglossal or intraperitoneal injection of alpha-synuclein fibrils into bigenic Tg(M83+/-:Gfap-luc+/-) mice, which overexpress human alpha-synuclein with the A53T mutation from the prion protein promoter and firefly luciferase from the promoter for glial fibrillary acidic protein (Gfap), is sufficient to induce neuropathologic disease. In comparison to homozygous Tg(M83+/+) mice that develop severe neurologic symptoms beginning at an age of 8 months, heterozygous Tg(M83+/-:Gfap-luc+/-) animals remain free of spontaneous disease until they reach an age of 22 months. Interestingly, injection of alpha-synuclein fibrils via the intraperitoneal route induced neurologic disease with paralysis in four of five Tg(M83+/-:Gfap-luc+/-) mice with a median incubation time of 229 &#177;17 days. Diseased animals showed severe deposits of phosphorylated alpha-synuclein in their brains and spinal cords. Accumulations of alpha-synuclein were sarkosyl-insoluble and colocalized with ubiquitin and p62, and were accompanied by an inflammatory response resulting in astrocytic gliosis and microgliosis. Surprisingly, inoculation of alpha-synuclein fibrils into the tongue was less effective in causing disease with only one of five injected animals showing alpha-synuclein pathology after 285 days. Our findings show that inoculation via the intraglossal route and more so via the intraperitoneal route is suitable to induce neurologic illness with relevant hallmarks of synucleinopathies in Tg(M83+/-:Gfap-luc+/-) mice. This provides a new model for studying prion-like pathogenesis induced by alpha-synuclein prionoids in greater detail.

Peripheral injection of alpha-synuclein fibrils into the peritoneum or tongue of Tg(M83+/-:Gfap-luc+/-) mice, which express human alpha-synuclein with the familial A53T mutation and firefly luciferase, can induce neuropathology, including neuroinflammation, in their central nervous system.

在转基因小鼠神经炎症的生物发光成像的α-共核蛋白纤丝的外围接种后

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein ...

Visualizing Impairment of the Endothelial and Glial Barriers of the Neurovascular Unit during Experimental Autoimmune Encephalomyelitis In Vivo

The neurovascular unit (NVU) is composed of microvascular endothelial cells forming the blood-brain barrier (BBB), an endothelial basement membrane with embedded pericytes, and the glia limitans composed by the parenchymal basement membrane and astrocytic end-feed embracing the abluminal aspect of central nervous system (CNS) microvessels. In addition to maintaining CNS homeostasis the NVU controls immune cell trafficking into the CNS. During immunosurveillance of the CNS low numbers of activated lymphocytes can cross the endothelial barrier without causing BBB dysfunction or clinical disease. In contrast, during neuroinflammation such as in multiple sclerosis or its animal model experimental autoimmune encephalomyelitis (EAE) a large number of immune cells can cross the BBB and subsequently the glia limitans eventually reaching the CNS parenchyma leading to clinical disease. 
Immune cell migration into the CNS parenchyma is thus a two-step process that involves a sequential migration across the endothelial and glial barrier of the NVU employing distinct molecular mechanisms. If following their passage across the endothelial barrier, T cells encounter their cognate antigen on perivascular antigen-presenting cells their local reactivation will initiate subsequent mechanisms leading to the focal activation of gelatinases, which will enable the T cells to cross the glial barrier and enter the CNS parenchyma. Thus, assessing both, BBB permeability and MMP activity in spatial correlation to immune cell accumulation in the CNS during EAE allows to specify loss of integrity of the endothelial and glial barriers of the NVU. 
We here show how to induce EAE in C57BL/6 mice by active immunization and how to subsequently analyze BBB permeability in vivo using a combination of exogenous fluorescent tracers. We further show, how to visualize and localize gelatinase activity in EAE brains by in situ zymogaphy coupled to immunofluorescent stainings of BBB basement membranes and CD45+ invading immune cells.

Here, we present protocols to investigate impairment of the neurovascular unit during experimental autoimmune encephalomyelitis in vivo. We specifically address how to determine blood-brain barrier permeability and gelatinase activity involved in leukocyte migration across the glia limitans.&#160;

体内自身免疫性脑髓炎实验性神经血管内障碍和胶质屏障的可视化

The neurovascular unit (NVU) is composed of microvascular endothelial cells forming the blood-brain barrier (BBB), an endothelial basement membrane with ...

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases1,2,3,4,5,6. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1gfp/gfp mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 &#181;m and 6 &#181;m, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies7, which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate8.

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

在体内图像的Cx3cr1gfp/gfp 记者光谱域光学相干层析成像和扫描激光眼底

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

脊髓组织学染色用于评估 EAE 疾病严重程度

Scoring Central Nervous System Inflammation, Demyelination, and Axon Injury in Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a common immune-based model of multiple sclerosis (MS). This disease can be induced in rodents by active immunization with protein components of the myelin sheath and Complete Freund&#39;s adjuvant (CFA) or by the transfer of myelin-specific T effector cells from rodents primed with myelin protein/CFA into na&#239;ve rodents. The severity of EAE is typically scored on a 5-point clinical scale that measures the degree of ascending paralysis, but this scale is not optimal for assessing the extent of recovery from EAE. For example, clinical scores remain high in some EAE models (e.g., myelin oligodendrocyte glycoprotein [MOG] peptide-induced model of EAE) despite the resolution of inflammation. Thus, it is important to complement clinical scoring with histological scoring of EAE, which also provides a means to study the underlying mechanisms of cellular injury in the central nervous system (CNS).
Here, a simple protocol is presented to prepare and stain spinal cord and brain sections from mice and to score inflammation, demyelination, and axonal injury in the spinal cord. The method for scoring leukocyte infiltration in the spinal cord can also be applied to score brain inflammation in EAE. A protocol for measuring soluble neurofilament light (sNF-L) in the serum of mice using a Small Molecule Assay (SIMOA) assay is also described, which provides&#160;feedback on the extent of overall CNS injury in live mice.

作者聚焦：揭示多发性硬化症中肥胖与自身免疫性炎症之间的通路 

Experimental autoimmune encephalomyelitis (EAE) serves as an animal model of multiple sclerosis. This article describes an approach for scoring spinal cord inflammation, demyelination, and axonal injury in EAE. Additionally, a method to quantify soluble neurofilament light levels in the mice serum is presented, facilitating the assessment of axonal injury in live mice.

对实验性自身免疫性脑脊髓炎中的中枢神经系统炎症、脱髓鞘和轴突损伤进行评分

Experimental autoimmune encephalomyelitis (EAE) is a common immune-based model of multiple sclerosis (MS). This disease can be induced in rodents by ...

Induction and Diverse Assessment Indicators of Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is a typical autoimmune disease of the central nervous system (CNS) characterized by inflammatory infiltration, demyelination, and axonal damage. Currently, there are no measures to cure MS completely, but multiple disease-modifying therapies (DMT) are available to control and mitigate disease progression. There are significant similarities between the CNS pathological features of experimental autoimmune encephalomyelitis (EAE) and MS patients. EAE has been widely used as a representative model to determine MS drugs' efficacy and explore the development of new therapies for MS disease. Active induction of EAE in mice has a stable and reproducible effect and is particularly suitable for studying the effects of drugs or genes on autoimmune neuroinflammation. The method of immunizing C57BL/6J mice with myelin oligodendrocyte glycoprotein (MOG35-55) and the daily assessment of disease symptoms using a clinical scoring system is mainly shared. Given the complex etiology of MS with diverse clinical manifestations, the existing clinical scoring system can't satisfy the assessment of disease treatment. To avoid the shortcomings of a single intervention, new indicators to assess EAE based on clinical manifestations of anxiety-like moods and osteoporosis in MS patients are created to provide a more comprehensive assessment of MS treatment.

The present protocol describes the induction of experimental autoimmune encephalomyelitis in a mouse model using myelin oligodendrocyte glycoprotein and monitoring the disease process using a clinical scoring system. Experimental autoimmune encephalomyelitis-related symptoms are analyzed using mouse femur micro-computed tomography analysis and open field test to assess the disease process comprehensively.

实验性自身免疫性脑脊髓炎的诱导及多样评价指标

Multiple sclerosis (MS) is a typical autoimmune disease of the central nervous system (CNS) characterized by inflammatory infiltration, demyelination, and ...

多发性硬化症的小鼠模型EAE视神经炎及脑部炎症的生物发光和近红外成像

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玻璃植入脊髓的Windows在成年小鼠实验性自身免疫性脑脊髓炎

在免疫细胞在不同组织与疾病相关的分布和小鼠实验评价性自身免疫性脑脊髓炎的诱导

细胞动力学在小鼠脊髓双光子成像

在转基因小鼠神经炎症的生物发光成像的α-共核蛋白纤丝的外围接种后

体内自身免疫性脑髓炎实验性神经血管内障碍和胶质屏障的可视化

在体内图像的Cx3cr1^gfp/gfp 记者光谱域光学相干层析成像和扫描激光眼底

对实验性自身免疫性脑脊髓炎中的中枢神经系统炎症、脱髓鞘和轴突损伤进行评分

对实验性自身免疫性脑脊髓炎中的中枢神经系统炎症、脱髓鞘和轴突损伤进行评分

对实验性自身免疫性脑脊髓炎中的中枢神经系统炎症、脱髓鞘和轴突损伤进行评分

实验性自身免疫性脑脊髓炎的诱导及多样评价指标