चूहे में मल्टीपल स्केलेरोसिस की EAE मॉडल में ऑप्टिक न्युरैटिस और मस्तिष्क में सूजन के bioluminescence और लगभग अवरक्त इमेजिंग
Summary
हम इन विवो लाइव bioluminescence के लिए और लगभग अवरक्त ऑप्टिक न्युरैटिस और SJL / जम्मू चूहों में एकाधिक काठिन्य के लिए प्रयोगात्मक autoimmune encephalomyelitis (EAE) मॉडल में इन्सेफेलाइटिस की इमेजिंग के लिए एक तकनीक दिखा।
Abstract
प्रायोगिक स्व-प्रतिरक्षित encephalomyelitis (EAE) SJL / जम्मू चूहों में पुनरावर्तन-प्रेषण मल्टीपल स्केलेरोसिस (RR एम्एस) के लिए एक मॉडल है। नैदानिक EAE मोटर समारोह घाटे का वर्णन स्कोर रीढ़ की हड्डी की प्रतिरक्षा की मध्यस्थता सूजन के बुनियादी readouts हैं। हालांकि, स्कोर और शरीर के वजन को मस्तिष्क में सूजन और ऑप्टिक न्युरैटिस का इन विवो मूल्यांकन के लिए अनुमति नहीं है। बाद के बारे में 2/3 एमएस रोगियों के में एक प्रारंभिक और लगातार अभिव्यक्ति है। यहाँ, हम vivo इमेजिंग प्रणाली का उपयोग रहने वाले चूहों में bioluminescence और लगभग अवरक्त रहते इमेजिंग EAE ऑप्टिक न्युरैटिस पैदा आकलन करने के लिए, मस्तिष्क में सूजन, और रक्त मस्तिष्क बाधा (BBB) विघटन के लिए तरीकों को दिखाने के। एक bioluminescent सब्सट्रेट oxidases से सक्रिय मुख्य रूप से ऑप्टिक न्युरैटिस दिखाया। संकेत विशिष्ट था और दवा के प्रभाव और रोग समय पाठ्यक्रम के दृश्य है, जो नैदानिक स्कोर paralleled की अनुमति दी। PEGylated फ्लोरोसेंट नैनोकणों कि vasculatur के भीतर बनेसमय की विस्तारित अवधि के लिए ई बीबीबी अखंडता का आकलन करने के लिए इस्तेमाल किया गया। लगभग अवरक्त इमेजिंग रोग के चरम पर एक बीबीबी रिसाव का पता चला। संकेत आंखों के आसपास मजबूत था। मैट्रिक्स metalloproteinases के लिए एक के पास अवरक्त सब्सट्रेट EAE पैदा सूजन का आकलन करने के लिए इस्तेमाल किया गया था। ऑटो प्रतिदीप्ति संकेत के साथ दखल, मात्रा का ठहराव के लिए वर्णक्रमीय unmixing की जरूरत पड़ेगी। कुल मिलाकर, bioluminescence इमेजिंग EAE जुड़े ऑप्टिक न्युरैटिस और दवा प्रभाव का आकलन करने के लिए एक विश्वसनीय विधि थी और संकेत विशिष्टता, मजबूती, मात्रा का ठहराव में आसानी, और लागत के मामले में लगभग अवरक्त तकनीक से बेहतर था।
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
वर्तमान वीडियो bioluminescence और SJL / जम्मू चूहों में EAE के vivo इमेजिंग में लगभग अवरक्त प्रतिदीप्ति के लिए तकनीक का पता चलता है। हम जानते हैं कि bioluminescence एक सूजन के प्रति संवेदनशील जांच का उपयोग मुख्य रूप से इमेज…
Disclosures
The authors have nothing to disclose.
Acknowledgements
इस शोध ड्यूश Forschungsgemeinschaft (CRC1039 ए 3) और अनुसंधान के वित्तपोषण कार्यक्रम हेसन, अनुसंधान ट्रांसलेशनल मेडिसिन और औषध TMP के लिए केंद्र के राज्य के 'Landesoffensive सूर Entwicklung wissenschaftlich-ökonomischer Exzellenz "(LOEWE) और बाकी क्रोनर-फ्रेसेनियस फाउंडेशन द्वारा समर्थित किया गया (EKFS), अनुसंधान प्रशिक्षण समूह ट्रांसलेशनल अनुसंधान नवाचार – फार्मा (यात्रा)।
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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