Bioluminesens og nær-infrarøde Imaging av optikusnevritt og hjernebetennelse i EAE Modell av multippel sklerose i Mus
Summary
Vi viser en teknikk for in vivo levende bioluminescens og nær-infrarød avbildning av optisk nevritt og hjernebetennelse i eksperimentell autoimmun encefalomyelitt (EAE) modell for multippel sklerose i SJL / J-mus.
Abstract
Eksperimentell autoimmun encefalomyelitt (EAE) i SJL / J mus er en modell for relapsing-remitting multippel sklerose (RRMS). Kliniske EAE score beskriver motoriske funksjons underskudd er grunnleggende avlesninger av immunmediert betennelse i ryggmargen. Men, score og kroppsvekt ikke tillate for en in vivo vurdering av hjernebetennelse og optikusnevritt. Det sistnevnte er et tidlig og hyppig manifestasjon i omtrent 2/3 av MS-pasienter. Her viser vi metoder for bioluminescens og nær-infrarøde levende avbildning for å vurdere EAE fremkalt optisk nevritt, hjernebetennelse, og blod-hjerne-barrieren (BBB) avbrudd i levende mus ved bruk av en in vivo bildedannende system. En selvlysende substrat aktiveres ved oksidase viste primært optikusnevritt. Signalet var bestemt og tillot visualisering av medisiner effekter og sykdoms tid kurs, som parallelt de kliniske score. Pegylerte fluorescerende nanopartikler som forble i vasculature over lengre tid ble brukt for å vurdere BBB integritet. Nær-infrarød avbildning avslørte en BBB lekkasje ved toppen av sykdommen. Signalet var den sterkeste rundt øynene. En nær-infrarødt substrat for matriksmetalloproteinaser ble brukt for å vurdere EAE-fremkalt betennelse. Auto-fluorescens forstyrret signalet, som krever spektral unmixing for kvantifisering. Totalt sett, bioluminesens bilde var en pålitelig metode for å vurdere EAE-forbundet optikusnevritt og medisiner effekter og var overlegne i forhold til nær-infrarøde teknikker i form av signal spesifisitet, robusthet, enkel kvantifisering og kostnad.
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
Den foreliggende video viser teknikker for bioluminescens og nær-infrarøde fluorescens in vivo avbildning av EAE i SJL / J-mus. Vi viser at bioluminescens avbildning ved hjelp av en betennelse følsom sonde viser hovedsakelig optisk nevritt, og kvantifiseringen stemmer overens med klinisk evaluering av EAE alvorlighetsgrad og virkningene av medikamenter. Imidlertid Bioluminescens avbildningsmetoden var ikke i stand til å detektere betennelse av den lumbale ryggmargen, som er en primær område av EAE manifes…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Denne forskningen ble støttet av Deutsche Forschungsgemeinschaft (CRC1039 A3) og forskningsmidler programmet "Landesoffensive zur Entwicklung wissenschaftlich-ökonomischer Exzellenz" (LOEWE) i delstaten Hessen, Research Center for translasjonell medisin og farmakologi TMP og Else Kröner-Fresenius Foundation (EKFS), forskerutdanning Gruppe translasjonell forskning innovasjon – 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 |
References
- Compston, A., Coles, A. Multiple sclerosis. Lancet. 372 (9648), 1502-1517 (2008).
- Dunn, J. Impact of mobility impairment on the burden of caregiving in individuals with multiple sclerosis. Expert Rev Pharmacoecon Outcomes Res. 10 (4), 433-440 (2010).
- Dutta, R., Trapp, B. D. Mechanisms of neuronal dysfunction and degeneration in multiple sclerosis. Prog Neurobiol. 93 (1), 1-12 (2011).
- Sawcer, S., et al. Genetic risk and a primary role for cell-mediated immune mechanisms in multiple sclerosis. Nature. 476 (7359), 214-219 (2011).
- Schmitz, K., et al. R-flurbiprofen attenuates experimental autoimmune encephalomyelitis in mice. EMBO Mol Med. 6 (11), 1398-1422 (2014).
- Balls, M. The origins and early days of the Three Rs concept. Altern Lab Anim. 37 (3), 255-265 (2009).
- Barthelmes, J., et al. Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues. J Vis Exp. (111), (2016).
- Leahy, A. A., et al. Analysis of the trajectory of osteoarthritis development in a mouse model by serial near-infrared fluorescence imaging of matrix metalloproteinase activities. Arthritis Rheumatol. 67 (2), 442-453 (2015).
- Scales, H. E., et al. Assessment of murine collagen-induced arthritis by longitudinal non-invasive duplexed molecular optical imaging. Rheumatology (Oxford). 55 (3), 564-572 (2016).
- Nahrendorf, M., et al. Dual channel optical tomographic imaging of leukocyte recruitment and protease activity in the healing myocardial infarct. Circ Res. 100 (8), 1218-1225 (2007).
- Eaton, V. L., et al. Optical tomographic imaging of near infrared imaging agents quantifies disease severity and immunomodulation of experimental autoimmune encephalomyelitis in vivo. J Neuroinflammation. 10, (2013).
- Kandagaddala, L. D., Kang, M. J., Chung, B. C., Patterson, T. A., Kwon, O. S. Expression and activation of matrix metalloproteinase-9 and NADPH oxidase in tissues and plasma of experimental autoimmune encephalomyelitis in mice. Exp Toxicol Pathol. 64 (1-2), 109-114 (2012).
- Wang, C., et al. In situ fluorescence imaging of myelination. J Histochem Cytochem. 58 (7), 611-621 (2010).
- Wang, C., et al. Longitudinal near-infrared imaging of myelination. J Neurosci. 31 (7), 2382-2390 (2011).
- Engelhardt, B. Molecular mechanisms involved in T cell migration across the blood-brain barrier. J Neural Transm. 113 (4), 477-485 (2006).
- Badawi, A. H., et al. Suppression of EAE and prevention of blood-brain barrier breakdown after vaccination with novel bifunctional peptide inhibitor. Neuropharmacology. 62 (4), 1874-1881 (2012).
- Simmons, S. B., Pierson, E. R., Lee, S. Y., Goverman, J. M. Modeling the heterogeneity of multiple sclerosis in animals. Trends in immunology. 34 (8), 410-422 (2013).
- Lin, T. H., et al. Diffusion fMRI detects white-matter dysfunction in mice with acute optic neuritis. Neurobiol Dis. 67, 1-8 (2014).
- Knier, B., et al. Neutralizing IL-17 protects the optic nerve from autoimmune pathology and prevents retinal nerve fiber layer atrophy during experimental autoimmune encephalomyelitis. J Autoimmun. 56, 34-44 (2015).
- Schellenberg, A. E., Buist, R., Yong, V. W., Del Bigio, M. R., Peeling, J. Magnetic resonance imaging of blood-spinal cord barrier disruption in mice with experimental autoimmune encephalomyelitis. Magn Reson Med. 58 (2), 298-305 (2007).
- Mori, Y., et al. Early pathological alterations of lower lumbar cords detected by ultrahigh-field MRI in a mouse multiple sclerosis model. Int Immunol. 26 (2), 93-101 (2014).
- Bittner, S., et al. Endothelial TWIK-related potassium channel-1 (TREK1) regulates immune-cell trafficking into the CNS. Nat Med. 19 (9), 1161-1165 (2013).
- Theien, B. E., et al. Differential effects of treatment with a small-molecule VLA-4 antagonist before and after onset of relapsing EAE. Blood. 102 (13), 4464-4471 (2003).
- Hawkins, B. T., Davis, T. P. The blood-brain barrier/neurovascular unit in health and disease. Pharmacol Rev. 57 (2), 173-185 (2005).
- Coisne, C., Mao, W., Engelhardt, B. Cutting edge: Natalizumab blocks adhesion but not initial contact of human T cells to the blood-brain barrier in vivo in an animal model of multiple sclerosis. J Immunol. 182 (10), 5909-5913 (2009).
- Andresen, V., et al. High-resolution intravital microscopy. PLoS One. 7 (12), e50915 (2012).
- Bukilica, M., et al. Stress-induced suppression of experimental allergic encephalomyelitis in the rat. Int J Neurosci. 59 (1-3), 167-175 (1991).
