La bioluminiscencia e infrarrojo cercano de imagen de la neuritis óptica y la inflamación cerebral en el modelo de EAE de esclerosis múltiple en ratones
Summary
Mostramos una técnica para la bioluminiscencia in vivo en vivo y en el infrarrojo cercano de formación de imágenes de la neuritis óptica y la encefalitis en el modelo de encefalomielitis autoinmune experimental (EAE) para la esclerosis múltiple en ratones SJL / J.
Abstract
La encefalomielitis autoinmune experimental (EAE) en ratones SJL / J es un modelo para la esclerosis múltiple recurrente-remitente (EMRR). puntuaciones de EAE clínicos que describen déficit de la función de motor son lecturas básicas de la inflamación inmune mediada de la médula espinal. Sin embargo, las puntuaciones y el peso corporal no permiten una evaluación in vivo de la inflamación del cerebro y la neuritis óptica. Este último es una manifestación temprana y frecuente en aproximadamente 2/3 de los pacientes con EM. Aquí, se muestra métodos para la bioluminiscencia y de formación de imágenes en vivo en el infrarrojo cercano para evaluar EAE evocado neuritis óptica, inflamación del cerebro, y alteración de la barrera hematoencefálica (BBB) en ratones vivos utilizando un sistema de imagen in vivo. Un sustrato bioluminiscente activado por oxidasas mostró principalmente la neuritis óptica. La señal era específico y permite la visualización de los efectos de la medicación y cursos de tiempo de la enfermedad, que en paralelo las puntuaciones clínicas. nanopartículas fluorescentes pegilada que permanecían dentro del vasculature durante largos períodos de tiempo se utilizaron para evaluar la integridad BBB. Infrarrojo cercano de imágenes revela una fuga de acreditación en el pico de la enfermedad. La señal fue el más fuerte alrededor de los ojos. Un sustrato de infrarrojo cercano para metaloproteinasas de la matriz se utilizó para evaluar la inflamación EAE-evocado. Auto-fluorescencia interfirió con la señal, lo que requiere desmezcla espectral para la cuantificación. En general, imágenes de bioluminiscencia era un método fiable para evaluar la neuritis óptica y la medicación efectos de EAE asociado y fue superior a las técnicas de infrarrojo cercano en términos de especificidad de la señal, robustez, facilidad de cuantificación, y el costo.
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 presente video muestra técnicas para la bioluminiscencia y fluorescencia en el infrarrojo cercano de imágenes in vivo de la EAE en ratones SJL / J. Se demuestra que la formación de imágenes de bioluminiscencia utilizando una sonda inflamación sensible muestra principalmente neuritis óptica, y la cuantificación de acuerdo con la evaluación clínica de la gravedad de EAE y los efectos de la medicación. Sin embargo, el método de formación de imágenes de bioluminiscencia no fue capaz de dete…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Esta investigación fue apoyada por la Deutsche Forschungsgemeinschaft (CRC1039 A3) y el programa de financiación de la investigación "Landesoffensive zur Entwicklung wissenschaftlich-ökonomischer Exzellenz" (LOEWE) del Estado de Hessen, Centro de Investigación de Medicina Traslacional y Farmacología TMP y el Else Fundación Kröner-Fresenius (EKFS), Grupo de Investigación de Formación de Investigación traslacional Innovación – 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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