Bioluminescence et proche infrarouge Imaging de névrite optique et inflammation du cerveau dans le modèle EAE de la sclérose en plaques chez des souris
Summary
Nous montrons une technique de bioluminescence direct in vivo et l' imagerie dans l'infrarouge proche de la névrite optique et l' encéphalite dans le modèle expérimental d' encéphalomyélite auto – immune (EAE) pour la sclérose en plaques chez les souris SJL / J.
Abstract
Experimental encéphalomyélite auto-immune (EAE) dans SJL / J souris est un modèle pour la sclérose en plaques récurrente-rémittente (RRMS). scores EAE cliniques décrivant moteur déficits de fonction sont des lectures de base de l'inflammation de la médiation immunitaire de la moelle épinière. Toutefois, les scores et le poids corporel ne permettent pas une évaluation in vivo de l' inflammation du cerveau et de la névrite optique. Ce dernier est une manifestation précoce et fréquente dans environ 2/3 des patients atteints de sclérose en plaques. Ici, nous montrons des méthodes de bioluminescence et de l' imagerie en direct proche infrarouge pour évaluer EAE a évoqué la névrite optique, une inflammation du cerveau, et barrière hémato-encéphalique (BHE) perturbation chez les souris vivant en utilisant un système d'imagerie in vivo dans. Un substrat de bioluminescence activée par des oxydases a montré principalement la névrite optique. Le signal était spécifique et a permis la visualisation des effets des médicaments et des cours de temps de la maladie, qui en parallèle les scores cliniques. nanoparticules fluorescentes pégylés qui sont restés dans le vasculature pendant de longues périodes de temps ont été utilisés pour évaluer l'intégrité du Bureau. Proche infrarouge imagerie a révélé une fuite BBB au sommet de la maladie. Le signal est le plus fort autour des yeux. Un substrat proche de l'infrarouge pour métalloprotéinases de matrice a été utilisé pour évaluer l'inflammation de l'EAE évoquée. Auto-fluorescence interféré avec le signal, ce qui nécessite déconvolution spectrale pour la quantification. Dans l'ensemble, l'imagerie par bioluminescence est une méthode fiable pour évaluer la névrite optique et de médicaments effets EAE-associé et était supérieur aux techniques du proche infrarouge en termes de spécificité du signal, la robustesse, la facilité de quantification, et le coût.
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
La présente vidéo montre les techniques de la bioluminescence et la fluorescence dans le proche infrarouge dans l' imagerie in vivo de l' EAE chez les souris SJL / J. Nous montrons que l'imagerie par bioluminescence à l'aide d'une sonde sensible à l'inflammation montre principalement la névrite optique et la quantification en accord avec l'évaluation clinique de l'EAE et la sévérité des effets des médicaments. Cependant, le procédé d'imagerie par biolumin…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Cette recherche a été soutenue par la Deutsche Forschungsgemeinschaft (CRC1039 A3) et le programme de financement de la recherche "Landesoffensive zur Entwicklung Wissenschaftlich-ökonomischer Exzellenz" (LOEWE) de l'Etat de Hesse, Centre de recherche pour la médecine translationnelle et Pharmacologie TMP et Else Kröner-Fresenius Foundation (EKFS), Groupe de formation en recherche translationnelle innovation Research – Pharma (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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