Bioluminescência e Near-infrared imagem de neurite óptica e Cérebro Inflamação no modelo de EAE de esclerose múltipla em ratinhos
Summary
Mostramos uma técnica para in vivo bioluminescência vivo e no infravermelho próximo de imagem de neurite óptica e encefalite no modelo de encefalomielite autoimune experimental (EAE) para a esclerose múltipla em ratinhos SJL / J.
Abstract
Encefalomielite auto-imune experimental (EAE) em murganhos SJL / J é um modelo para a esclerose múltipla recorrente-remitente (EMRR). pontuações de EAE clínica que descrevem os défices de função de motor são leituras básicas da inflamação mediada por imune da medula espinhal. No entanto, a pontuação e peso corporal não permitem uma avaliação in vivo de inflamação do cérebro e neurite óptica. O último é uma manifestação precoce e frequente em cerca de 2/3 dos pacientes com EM. Aqui, mostramos métodos para a bioluminescência e imagens ao vivo do infravermelho próximo para avaliar EAE evocado neurite óptica, inflamação do cérebro, e barreira sangue-cérebro interrupção (BBB) em ratos vivos utilizando um sistema de imagem in vivo. Um substrato bioluminescente activado por oxidases mostrou principalmente neurite óptica. O sinal foi específico e permitiu a visualização de efeitos da medicação e cursos tempo de doença, que em paralelo os escores clínicos. nanopartículas fluorescentes peguilados que permaneceram dentro da vasculature por períodos de tempo prolongados foram usadas para avaliar a integridade da BHE. No infravermelho próximo de imagem revelou uma fuga de certificação no pico da doença. O sinal foi mais forte em torno dos olhos. Um substrato de infravermelho próximo para metaloproteinases de matriz foi usada para avaliar a inflamação evocados-EAE. Auto-fluorescência interferiu com o sinal, o que requer a separação espectral para efeitos de quantificação. Em geral, imagem de bioluminescência foi um método fiável para avaliar a neurite óptica e efeitos de medicação associada a EAE e foi superior ao das técnicas de infravermelho próximo, em termos de especificidade do sinal, robustez, facilidade de quantificação, e custo.
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
A presente vídeo mostra técnicas de bioluminescência e fluorescência no infravermelho próximo imagiologia in vivo de EAE em ratinhos SJL / J. Mostramos que imagem de bioluminescência utilizando uma sonda sensível à inflamação mostra principalmente a neurite óptica, e a quantificação está de acordo com a avaliação clínica da gravidade da EAE e os efeitos do medicamento. No entanto, o método de imagem de bioluminescência não foi capaz de detectar a inflamação da medula espinal lombar, que é…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Esta pesquisa foi apoiada pela Deutsche Forschungsgemeinschaft (CRC1039 A3) e do programa de financiamento da investigação "Landesoffensive zur Entwicklung wissenschaftlich-ökonomischer Exzellenz" (LOEWE) do Estado de Hessen, Centro de Pesquisa para Translational Medicine and Pharmacology TMP ea Else Kröner-Fresenius Foundation (EKFS), Research Training Group Translational Research Innovation – 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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