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
Summary
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。
Abstract
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。
Introduction
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.
Protocol
Representative Results
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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）。
Materials
| AngioSpark-680 | Perkin Elmer, Inc., Waltham, USA | NEV10149 | Imaging probe, pegylated nanoparticles, useful for imaging of blood brain barrier integrity |
| MMP-sense 680 | Perkin Elmer, Inc., Waltham, USA | NEV10126 | Imaging probe, activatable by matrix metalloproteinases, useful for imaging of inflammation |
| XenoLight RediJect Inflammation Probe | Perkin Elmer, Inc., Waltham, USA | 760535 | Imaging probe, activatable by oxidases, useful for imaging of inflammation |
| PLP139-151/CFA emulsion | Hooke Labs, St Lawrence, MA | EK-0123 | EAE induction kit |
| Pertussis Toxin | Hooke Labs, St Lawrence, MA | EK-0123 | EAE induction kit |
| IVIS Lumina Spectrum | Perkin Elmer, Inc., Waltham, USA | Bioluminescence and Infrared Imaging System | |
| LivingImage 4.5 software | Perkin Elmer, Inc., Waltham, USA | CLS136334 | IVIS analysis software |
| Isoflurane | Abbott Labs, Illinois, USA | 26675-46-7 | Anaesthetic |
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