Bioluminescenza e vicino infrarosso Imaging di neurite ottica e cervello infiammazione nel modello EAE della sclerosi multipla nei topi
Summary
Mostriamo una tecnica per bioluminescenza vivo in vivo e vicino infrarosso per immagini di neurite ottica ed encefalite nel modello di encefalomielite autoimmune sperimentale (EAE) per la sclerosi multipla in topi SJL / J.
Abstract
Encefalomielite autoimmune sperimentale (EAE) in SJL / J topi è un modello per la sclerosi multipla recidivante-remittente (RRMS). punteggi EAE clinici che descrivono i deficit delle funzioni motorie sono letture di base della infiammazione immuno-mediata del midollo spinale. Tuttavia, i punteggi e il peso corporeo non permettono una valutazione in vivo di infiammazione del cervello e neurite ottica. Quest'ultima è una manifestazione precoce e frequente in circa 2/3 dei pazienti con SM. Qui vi mostriamo i metodi per bioluminescenza e vicino infrarosso immagini dal vivo per valutare EAE evocato neurite ottica, infiammazione del cervello, e la barriera emato-encefalica interruzione (BBB) nei topi che vivono con un sistema di imaging in vivo in. Un substrato bioluminescente attivato da ossidasi soprattutto ha mostrato neurite ottica. Il segnale era specifico e ha permesso la visualizzazione degli effetti di farmaci e gli andamenti temporali di malattia, che in parallelo i punteggi clinici. nanoparticelle fluorescenti pegilato che sono rimasti all'interno del vasculature per lunghi periodi di tempo sono stati utilizzati per valutare l'integrità BBB. Vicino infrarosso di imaging rivelato una perdita BBB al picco della malattia. Il segnale era più forte intorno agli occhi. Un substrato vicino infrarosso per metalloproteinasi della matrice è stato utilizzato per valutare l'infiammazione EAE-evocato. Auto-fluorescenza interferiva con il segnale, che richiede unmixing spettrale per la quantificazione. Nel complesso, imaging bioluminescenza è un metodo affidabile per valutare neurite ottica e farmaci effetti EAE-associata e era superiore alle tecniche vicino infrarosso in termini di specificità del segnale, robustezza, facilità di quantificazione, e 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
Il presente video mostra le tecniche di bioluminescenza e fluorescenza nel vicino infrarosso imaging in vivo di EAE in topi SJL / J. Abbiamo dimostrato che l'imaging bioluminescenza utilizzando una sonda infiammazione sensibile mostra principalmente neurite ottica, e la quantificazione concorda con la valutazione clinica di EAE gravità e gli effetti del farmaco. Tuttavia, il metodo di imaging bioluminescenza non era in grado di rilevare l'infiammazione del midollo spinale lombare, che è un sito primar…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Questa ricerca è stata sostenuta dalla Deutsche Forschungsgemeinschaft (CRC1039 A3) e il programma di finanziamento della ricerca "Landesoffensive zur Entwicklung Wissenschaftlich-ökonomischer Exzellenz" (LOEWE) dello Stato di Hessen, Centro di Ricerca per traslazionale Medicina e Farmacologia TMP e la Else Kröner-Fresenius Fondazione (EKFS), Gruppo formazione mediante la ricerca traslazionale 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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