Vi visar en teknik för in vivo levande bioluminiscens och nära infraröda avbildning av optikusneurit och encefalit i experimentell autoimmun encefalomyelit (EAE) modell för multipel skleros i SJL / J-möss.
Experimentell autoimmun encefalomyelit (EAE) i SJL / J-möss är en modell för relapserande remitterande multipel skleros (RRMS). Kliniska EAE-poäng som beskriver motorfunktions underskott är basiska utläsningar av immunmedierad inflammation av ryggmärgen. Dock inte poäng och kroppsvikt inte möjliggöra en in vivo bedömning av hjärninflammation och optikusneurit. Det senare är en tidig och frekvent manifestation i omkring 2/3 av MS-patienter. Här visar vi metoder för mareld och nära infraröda levande avbildning att bedöma EAE framkallade optikusneurit, hjärninflammation, och blod-hjärnbarriären (BBB) störningar i levande möss med användning av ett in vivo imaging systemet. En självlysande substrat aktiveras av oxidaser visade främst optikusneurit. Signalen var specifik och tillät visualisering av läkemedelseffekter och sjukdomar tidsförlopp, som parallellt de kliniska poängen. Pegylerade fluorescerande nanopartiklar som återstod i vasculature under längre tidsperioder har använts för att bedöma BBB integritet. Nära infraröda imaging visade en BBB läcka på toppen av sjukdomen. Signalen var starkast runt ögonen. En nära-infraröd substrat för matrismetalloproteinaser användes för att bedöma EAE-framkallade inflammation. Auto-fluorescens störde signalen kräver spektral unmixing för kvantifiering. Sammantaget mareld avbildning var en tillförlitlig metod för att bedöma EAE-associerad optikusneurit och medicinering effekter och var överlägsen de nära infraröda tekniker när det gäller signal specificitet, robusthet, enkel kvantifiering och kostnad.
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.
Föreliggande video visar tekniker för mareld och nära infraröda fluorescens in vivo avbildning av EAE i SJL / J-möss. Vi visar att mareld avbildning med en inflammation känslig sond visar huvudsakligen optikusneurit och kvantifiering instämmer med den kliniska utvärderingen av EAE svårighetsgrad och effekterna av medicinering. Emellertid bioluminescens avbildningsmetod kunde inte detektera inflammation i ryggradens ryggmärgen, som är ett primärt ställe av EAE manifestation 17,</su…
The authors have nothing to disclose.
Denna forskning stöddes av Deutsche Forschungsgemeinschaft (CRC1039 A3) och forskningsmedel programmet "Landesoffensive Zur Entwicklung Wissenschaftlich-ökonomischer Exzellenz" (LOEWE) i delstaten Hessen, Research Center för Translational Medicine och farmakologi TMP och Else Kröner-Fresenius Foundation (EKFS), Research Training Group Translational Research Innovation – Pharma (TRIP).
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 |