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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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blood-brain+barrier/neurovascular+unit+in+health+and+disease.">The blood-brain barrier/neurovascular unit in health and disease.</a> Pharmacol Rev. 57 (2), 173-185 (2005).</li><li>Coisne, C., Mao, W., Engelhardt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+Natalizumab+blocks+adhesion+but+not+initial+contact+of+human+T+cells+to+the+blood-brain+barrier+in+vivo+in+an+animal+model+of+multiple+sclerosis.">Cutting edge: Natalizumab blocks adhesion but not initial contact of human T cells to the blood-brain barrier in vivo in an animal model of multiple sclerosis.</a> J Immunol. 182 (10), 5909-5913 (2009).</li><li>Andresen, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+intravital+microscopy.">High-resolution intravital microscopy.</a> PLoS One. 7 (12), e50915 (2012).</li><li>Bukilica, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress-induced+suppression+of+experimental+allergic+encephalomyelitis+in+the+rat.">Stress-induced suppression of experimental allergic encephalomyelitis in the rat.</a> Int J Neurosci. 59 (1-3), 167-175 (1991).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" width="1287">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">AngioSpark-680</td>
			<td style="width:267px;">Perkin Elmer, Inc., Waltham, USA</td>
			<td style="width:119px;">NEV10149</td>
			<td style="width:636px;">Imaging probe, pegylated nanoparticles, useful for imaging of blood brain barrier integrity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">PLP139-151/CFA emulsion&#160;</td>
			<td>Hooke Labs, St Lawrence, MA</td>
			<td>EK-0123</td>
			<td>EAE induction kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pertussis Toxin</td>
			<td>Hooke Labs, St Lawrence, MA</td>
			<td>EK-0123</td>
			<td>EAE induction kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IVIS Lumina Spectrum</td>
			<td>Perkin Elmer, Inc., Waltham, USA</td>
			<td style="width:119px;"></td>
			<td>Bioluminescence and Infrared Imaging System</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LivingImage 4.5 software&#160;</td>
			<td>Perkin Elmer, Inc., Waltham, USA</td>
			<td>CLS136334</td>
			<td>IVIS analysis software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Isoflurane</td>
			<td style="width:267px;">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="https://ecsource-jove-com.remotexs.ntu.edu.sg/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="https://ecsource-jove-com.remotexs.ntu.edu.sg/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="https://ecsource-jove-com.remotexs.ntu.edu.sg/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="https://ecsource-jove-com.remotexs.ntu.edu.sg/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

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading to an accumulation of lipid-laden intra-luminal plaque, smooth muscle cell proliferation and progressive narrowing of the vessel lumen. The formation of such vulnerable plaques prone to rupture underlies the majority of cases of acute myocardial infarction. The complex molecular and cellular inflammatory cascade is orchestrated by the recruitment of T lymphocytes and macrophages and their paracrine effects on endothelial and smooth muscle cells.1
Molecular imaging in atherosclerosis has evolved into an important clinical and research tool that allows in vivo visualization of inflammation and other biological processes. Several recent examples demonstrate the ability to detect high-risk plaques in patients, and assess the effects of pharmacotherapeutics in atherosclerosis.4 While a number of molecular imaging approaches (in particular MRI and PET) can image biological aspects of large vessels such as the carotid arteries, scant options exist for imaging of coronary arteries.2 The advent of high-resolution optical imaging strategies, in particular near-infrared fluorescence (NIRF), coupled with activatable fluorescent probes, have enhanced sensitivity and led to the development of new intravascular strategies to improve biological imaging of human coronary atherosclerosis.
Near infrared fluorescence (NIRF) molecular imaging utilizes excitation light with a defined band width (650-900 nm) as a source of photons that, when delivered to an optical contrast agent or fluorescent probe, emits fluorescence in the NIR window that can be detected using an appropriate emission filter and a high sensitivity charge-coupled camera. As opposed to visible light, NIR light penetrates deeply into tissue, is markedly less attenuated by endogenous photon absorbers such as hemoglobin, lipid and water, and enables high target-to-background ratios due to reduced autofluorescence in the NIR window. Imaging within the NIR 'window' can substantially improve the potential for in vivo imaging.2,5
Inflammatory cysteine proteases have been well studied using activatable NIRF probes10, and play important roles in atherogenesis. Via degradation of the extracellular matrix, cysteine proteases contribute importantly to the progression and complications of atherosclerosis8. In particular, the cysteine protease, cathepsin B, is highly expressed and colocalizes with macrophages in experimental murine, rabbit, and human atheromata.3,6,7 In addition, cathepsin B activity in plaques can be sensed in vivo utilizing a previously described 1-D intravascular near-infrared fluorescence technology6, in conjunction with an injectable nanosensor agent that consists of a poly-lysine polymer backbone derivatized with multiple NIR fluorochromes (VM110/Prosense750, ex/em 750/780nm, VisEn Medical, Woburn, MA) that results in strong intramolecular quenching at baseline.10 Following targeted enzymatic cleavage by cysteine proteases such as cathepsin B (known to colocalize with plaque macrophages), the fluorochromes separate, resulting in substantial amplification of the NIRF signal. Intravascular detection of NIR fluorescence signal by the utilized novel 2D intravascular NIRF catheter now enables high-resolution, geometrically accurate in vivo detection of cathepsin B activity in inflamed plaque.
In vivo molecular imaging of atherosclerosis using catheter-based 2D NIRF imaging, as opposed to a prior 1-D spectroscopic approach,6 is a novel and promising tool that utilizes augmented protease activity in macrophage-rich plaque to detect vascular inflammation.11,12 The following research protocol describes the use of an intravascular 2-dimensional NIRF catheter to image and characterize plaque structure utilizing key aspects of plaque biology. It is a translatable platform that when integrated with existing clinical imaging technologies including angiography and intravascular ultrasound (IVUS), offers a unique and novel integrated multimodal molecular imaging technique that distinguishes inflammatory atheromata, and allows detection of intravascular NIRF signals in human-sized coronary arteries.

In vivo Near Infrared Fluorescence NIRF Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

We detail a new near-infrared fluorescence (NIRF) catheter for 2-dimensional intravascular molecular imaging of plaque biology in vivo. The NIRF catheter can visualize key biological processes such as inflammation by reporting on the presence of plaque-avid activatable and targeted NIR fluorochromes. The catheter utilizes clinical engineering and power requirements and is targeted for application in human coronary arteries. The following research study describes a multimodal imaging strategy that utilizes a novel in vivo intravascular NIRF catheter to image and quantify inflammatory plaque in proteolytically active inflamed rabbit atheromata.

体内近红外荧光（NIRF）血管内分子成像炎性斑块，多式联运方针成像动脉粥样硬化

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading ...

科研

医学

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

在小鼠模型体内生物发光成像乳腺癌脑转移的肿瘤乏氧动力学

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis (ALS)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 years for the sporadic disease and around 50 years for the familial disease. Due to its progressive course, 50% of the patients die within 30 months of symptom onset. In order to evaluate novel treatment options for this disease, genetic mouse models of ALS have been generated based on human familial mutations in the SOD gene, such as the SOD1 (G93A) mutation. Most important aspects that have to be evaluated in the model are overall survival, clinical course and motor function. Here, we demonstrate the clinical evaluation, show the conduction of two behavioural motor tests and provide quantitative scoring systems for all parameters. Because an in depth analysis of the ALS mouse model usually requires an immunohistochemical examination of the spinal cord, we demonstrate its preparation in detail applying the dorsal laminectomy method. Exemplary histological findings are demonstrated. The comprehensive application of the depicted examination methods in studies on the mouse model of ALS will enable the researcher to reliably test future therapeutic options which can provide a basis for later human clinical trials.

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis ALS

A mouse model for amyotrophic lateral sclerosis (ALS) is examined clinically and behaviorally. As a prerequisite for an accompanying immunohistological analysis the preparation of the spinal cord is depicted in detail.

临床试验和脊髓去除在肌萎缩性侧索硬化症的小鼠模型（ALS）

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 ...

Dynamic Visual Tests to Identify and Quantify Visual Damage and Repair Following Demyelination in Optic Neuritis Patients

In order to follow optic neuritis patients and evaluate the effectiveness of their treatment, a handy, accurate and quantifiable tool is required to assess changes in myelination at the central nervous system (CNS). However, standard measurements, including routine visual tests and MRI scans, are not sensitive enough for this purpose. We present two visual tests addressing dynamic monocular and binocular functions which may closely associate with the extent of myelination along visual pathways. These include Object From Motion (OFM) extraction and Time-constrained stereo protocols. In the OFM test, an array of dots compose an object, by moving the dots within the image rightward while moving the dots outside the image leftward or vice versa. The dot pattern generates a camouflaged object that cannot be detected when the dots are stationary or moving as a whole. Importantly, object recognition is critically dependent on motion perception. In the Time-constrained Stereo protocol, spatially disparate images are presented for a limited length of time, challenging binocular 3-dimensional integration in time. Both tests are appropriate for clinical usage and provide a simple, yet powerful, way to identify and quantify processes of demyelination and remyelination along visual pathways. These protocols may be efficient to diagnose and follow optic neuritis and multiple sclerosis patients.
In the diagnostic process, these protocols may reveal visual deficits that cannot be identified via current standard visual measurements. Moreover, these protocols sensitively identify the basis of the currently unexplained continued visual complaints of patients following recovery of visual acuity. In the longitudinal follow up course, the protocols can be used as a sensitive marker of demyelinating and remyelinating processes along time. These protocols may therefore be used to evaluate the efficacy of current and evolving therapeutic strategies, targeting myelination of the CNS.

Object From Motion (OFM) and Time-constrained stereo protocols are sensitive tools to identify monocular and binocular dynamic visual function deficits, which are uniquely affected in optic neuritis patients. Furthermore, these tests may be used as quantitative noninvasive tools to assess the extent of myelination along visual pathways.

动态视觉测试，以确定和量化视觉损伤与修复继脱髓鞘视神经炎患者

In order to follow optic neuritis patients and evaluate the effectiveness of their treatment, a handy, accurate and quantifiable tool is required to ...

The Multiple Sclerosis Performance Test (MSPT): An iPad-Based Disability Assessment Tool

Precise measurement of neurological and neuropsychological impairment and disability in multiple sclerosis is challenging. We report a new test, the Multiple Sclerosis Performance Test (MSPT), which represents a new approach to quantifying MS related disability. The MSPT takes advantage of advances in computer technology, information technology, biomechanics, and clinical measurement science. The resulting MSPT represents a computer-based platform for precise, valid measurement of MS severity. Based on, but extending the Multiple Sclerosis Functional Composite (MSFC), the MSPT provides precise, quantitative data on walking speed, balance, manual dexterity, visual function, and cognitive processing speed. The MSPT was tested by 51 MS patients and 49 healthy controls (HC). MSPT scores were highly reproducible, correlated strongly with technician-administered test scores, discriminated MS from HC and severe from mild MS, and correlated with patient reported outcomes. Measures of reliability, sensitivity, and clinical meaning for MSPT scores were favorable compared with technician-based testing. The MSPT is a potentially transformative approach for collecting MS disability outcome data for patient care and research. Because the testing is computer-based, test performance can be analyzed in traditional or novel ways and data can be directly entered into research or clinical databases. The MSPT could be widely disseminated to clinicians in practice settings who are not connected to clinical trial performance sites or who are practicing in rural settings, drastically improving access to clinical trials for clinicians and patients. The MSPT could be adapted to out of clinic settings, like the patient&#8217;s home, thereby providing more meaningful real world data. The MSPT represents a new paradigm for neuroperformance testing. This method could have the same transformative effect on clinical care and research in MS as standardized computer-adapted testing has had in the education field, with clear potential to accelerate progress in clinical care and research.

The Multiple Sclerosis Performance Test MSPT: An iPad-Based Disability Assessment Tool

Precise measurement of neurological and neuropsychological impairment and disability in multiple sclerosis is challenging. We report methodologic details on a new test, the Multiple Sclerosis Performance Test (MSPT). This new approach to the objective of quantification of MS related disability provides a computer-based platform for precise, valid measurement of MS severity.

多发性硬化症性能测试（MSPT）:一个iPad为基础的残疾评估工具

Precise measurement of neurological and neuropsychological impairment and disability in multiple sclerosis is challenging. We report a new test, the ...

Th17 Inflammation Model of Oropharyngeal Candidiasis in Immunodeficient Mice

Oropharyngeal Candidiasis (OPC) disease is caused not only due to the lack of host immune resistance, but also the absence of appropriate regulation of infection-induced immunopathology. Although Th17 cells are implicated in antifungal defense, their role in immunopathology is unclear. This study presents a method for establishing oral Th17 immunopathology associated with oral candidal infection in immunodeficient mice. The method is based on reconstituting lymphopenic mice with in vitro cultured Th17 cells, followed by oral infection with Candida albicans (C. albicans). Results show that unrestrained Th17 cells result in inflammation and pathology, and is associated with several measurable read-outs including weight loss, pro-inflammatory cytokine production, tongue histopathology and mortality, showing that this model may be valuable in studying OPC immunopathology. Adoptive transfer of regulatory cells (Tregs) controls and reduces the inflammatory response, showing that this model can be used to test new strategies to counteract oral inflammation. This model may also be applicable in studying oral Th17 immunopathology in general in the context of other oral diseases.

Although Candida infection models are available to study host immune resistance, a model to study T cell mediated immunopathology in the context of Candida infection is absent. Here we describe a method to establish Th17 immunopathology associated with oral Candida infection in immunodeficient mice.

口咽念珠菌病的免疫缺陷小鼠Th17细胞炎症模型

Oropharyngeal Candidiasis (OPC) disease is caused not only due to the lack of host immune resistance, but also the absence of appropriate regulation of ...

A Method of Trigonometric Modelling of Seasonal Variation Demonstrated with Multiple Sclerosis Relapse Data

This report describes a novel Stata-based application of trigonometric regression modelling to 55 years of multiple sclerosis relapse data from 46 clinical centers across 20 countries located in both hemispheres. Central to the success of this method was the strategic use of plot analysis to guide and corroborate the statistical regression modelling. Initial plot analysis was necessary for establishing realistic hypotheses regarding the presence and structural form of seasonal and latitudinal influences on relapse probability and then testing the performance of the resultant models. Trigonometric regression was then necessary to quantify these relationships, adjust for important confounders and provide a measure of certainty as to how plausible these associations were. Synchronization of graphing techniques with regression modelling permitted a systematic refinement of models until best-fit convergence was achieved, enabling novel inferences to be made regarding the independent influence of both season and latitude in predicting relapse onset timing in MS. These methods have the potential for application across other complex disease and epidemiological phenomena suspected or known to vary systematically with season and/or geographic location.

Combining plot analysis with trigonometric regression is a robust method for exploring complex, cyclical phenomena such as relapse onset timing in multiple sclerosis (MS). This method enabled unbiased characterisation of seasonal trends in relapse onset permitting novel inferences around the influence of seasonal variation, ultraviolet radiation (UVR) and latitude.

季节变化的三角建模的方法证明与多发性硬化症复发数据

This report describes a novel Stata-based application of trigonometric regression modelling to  55 years of multiple sclerosis relapse data from 46 ...

A Protocol for the Use of Remotely-Supervised Transcranial Direct Current Stimulation (tDCS) in Multiple Sclerosis (MS)

Transcranial direct current stimulation (tDCS) is a noninvasive brain stimulation technique that uses low amplitude direct currents to alter cortical excitability. With well-established safety and tolerability, tDCS has been found to have the potential to ameliorate symptoms such as depression and pain in a range of conditions as well as to enhance outcomes of cognitive and physical training. However, effects are cumulative, requiring treatments that can span weeks or months and frequent, repeated visits to the clinic. The cost in terms of time and travel is often prohibitive for many participants, and ultimately limits real-world access. 
Following guidelines for remote tDCS application, we propose a protocol that would allow remote (in-home) participation that uses specially-designed devices for supervised use with materials modified for patient use, and real-time monitoring through a telemedicine video conferencing platform. We have developed structured training procedures and clear, detailed instructional materials to allow for self- or proxy-administration while supervised remotely in real-time. The protocol is designed to have a series of checkpoints, addressing attendance and tolerability of the session, to be met in order to continue to the next step. The feasibility of this protocol was then piloted for clinical use in an open label study of remotely-supervised tDCS in multiple sclerosis (MS). This protocol can be widely used for clinical study of tDCS.

A Protocol for the Use of Remotely-Supervised Transcranial Direct Current Stimulation tDCS in Multiple Sclerosis MS

The goal of this pilot study is to describe a protocol for the remotely-supervised delivery of transcranial direct current stimulation (tDCS) so that the procedure maintains standards of in-clinic practice, including safety, reproducibility, and tolerability. The feasibility of this protocol was tested in participants with multiple sclerosis (MS).

一个协议，用于远程监督经颅直流电刺激（TDCS）在多发性硬化症的使用（MS）

Transcranial direct current stimulation (tDCS) is a noninvasive brain stimulation technique that uses low amplitude direct currents to alter cortical ...

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of stroke than relying on indirect measurements, and will be a potent and robust vascular research tool. We use an optical imaging approach that labels thrombi with a molecular imaging thrombus marker&#160;&#8212; a Cy5.5 near-infrared fluorescent (NIRF) probe that is covalently linked to the fibrin strands of the thrombus by the fibrin-crosslinking enzymatic action of activated coagulation factor XIIIa during the process of clot maturation. A micro-computed tomography (microCT)-based approach uses thrombus-seeking gold nanoparticles (AuNPs) functionalized to target the major component of the clot: fibrin. This paper describes a detailed protocol for the combined in vivo microCT and ex vivo NIRF imaging of thromboemboli in a mouse model of embolic stroke. We show that in vivo microCT and fibrin-targeted glycol-chitosan AuNPs (fib-GC-AuNPs) can be used for visualizing both in situ thrombi and cerebral embolic thrombi. We also describe the use of in vivo microCT-based direct thrombus imaging to serially monitor the therapeutic effects of tissue plasminogen activator-mediated thrombolysis. After the last imaging session, we demonstrate by ex vivo NIRF imaging the extent and the distribution of residual thromboemboli in the brain. Finally, we describe quantitative image analyses of microCT and NIRF imaging data. The combined technique of direct thrombus imaging allows two independent methods of thrombus visualization to be compared: the area of thrombus-related fluorescent signal on ex vivo NIRF imaging vs. the volume of hyperdense microCT thrombi in vivo.

This protocol describes the application of combined near-infrared fluorescent (NIRF) imaging and micro-computed tomography (microCT) for visualizing cerebral thromboemboli. This technique allows the quantification of thrombus burden and evolution. The NIRF imaging technique visualizes fluorescently labeled thrombus in excised brain, while the microCT technique visualizes thrombus inside living animals using gold-nanoparticles.

结合近红外荧光成像和微计算机断层扫描直接可视化脑血栓

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of ...

Skeletal Muscle Neurovascular Coupling, Oxidative Capacity, and Microvascular Function with 'One Stop Shop' Near-infrared Spectroscopy

Exercise represents a major hemodynamic stress that demands a highly coordinated neurovascular response in order to match oxygen delivery to metabolic demand. Reactive hyperemia (in response to a brief period of tissue ischemia) is an independent predictor of cardiovascular events and provides important insight into vascular health and vasodilatory capacity. Skeletal muscle oxidative capacity is equally important in health and disease, as it determines the energy supply for myocellular processes. Here, we describe a simple, non-invasive approach using near-infrared spectroscopy to assess each of these major clinical endpoints (reactive hyperemia, neurovascular coupling, and muscle oxidative capacity) during a single clinic or laboratory visit. Unlike Doppler ultrasound, magnetic resonance images/spectroscopy, or invasive catheter-based flow measurements or muscle biopsies, our approach is less operator-dependent, low-cost, and completely non-invasive. Representative data from our lab taken together with summary data from previously published literature illustrate the utility of each of these end-points. Once this technique is mastered, application to clinical populations will provide important mechanistic insight into exercise intolerance and cardiovascular dysfunction.

Here, we describe a simple, non-invasive approach using near-infrared spectroscopy to assess reactive hyperemia, neurovascular coupling and skeletal muscle oxidative capacity in a single clinic or laboratory visit.

"一站式" 近红外光谱对骨骼肌神经血管的耦合、氧化能力及微血管功能的作用

Exercise represents a major hemodynamic stress that demands a highly coordinated neurovascular response in order to match oxygen delivery to metabolic ...