تلألؤ بيولوجي والقريبة من تحت الحمراء التصوير من التهاب العصب البصري والمخ التهاب في نموذج EAE من مرض التصلب العصبي المتعدد في الفئران
Summary
وتبين لنا تقنية لفي الجسم الحي تلألؤ بيولوجي الحية والأشعة تحت الحمراء القريبة التصوير من التهاب العصب البصري والتهاب الدماغ في النموذج التجريبي التهاب الدماغ والنخاع المناعي الذاتي (بنت) لمرض التصلب المتعدد في الفئران SJL / J.
Abstract
التجريبية التهاب الدماغ والنخاع المناعي الذاتي (بنت) في SJL / J الفئران هو نموذج للتحويل بين التصلب المتعدد الانتكاس (RRMS). عشرات EAE السريرية واصفا العجز وظيفة الحركة هي قراءات الأساسية للالتهاب بوساطة في مأمن من الحبل الشوكي. ومع ذلك، عشرات ووزن الجسم لا تسمح لإجراء تقييم في الجسم الحي من التهاب الدماغ والتهاب العصب البصري. وهذا الأخير هو مظهر باكر ومتكررة في حوالي 2/3 من مرضى التصلب المتعدد. هنا، وتبين لنا طرق تلألؤ بيولوجي والتصوير الحي القريب من الأشعة تحت الحمراء لتقييم EAE أثار التهاب العصب البصري، التهاب الدماغ، وحاجز الدم في الدماغ (BBB) اضطراب في الفئران الحية باستخدام المجراة نظام التصوير. والركيزة إضاءة الحيوية تفعيلها من خلال oxidases أظهر في المقام الأول التهاب العصب البصري. كانت إشارة محددة وسمحت التصور من آثار الدواء والمرض مرة الدورات، التي توازي عشرات من السريرية. النانوية الفلورسنت مضاد للفيروسات التي بقيت داخل vasculaturاستخدمت البريد لفترات طويلة من الوقت لتقييم سلامة BBB. بالقرب من الأشعة تحت الحمراء وكشف التصوير تسرب BBB في ذروة المرض. كانت إشارة أقوى حول العينين. تم استخدام الركيزة القريب من الأشعة تحت الحمراء للمصفوفة metalloproteinases لتقييم التهاب أثار EAE. تدخلت لصناعة السيارات في مضان مع إشارة، تتطلب unmixing الطيفي للالكمي. وعموما، كان التصوير تلألؤ بيولوجي طريقة موثوق بها لتقييم التهاب العصب البصري والدواء الآثار المرتبطة EAE وكان متفوقة على التقنيات القريب من الأشعة تحت الحمراء من حيث خصوصية إشارة، متانة، وسهولة القياس الكمي، والتكلفة.
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
ويظهر شريط الفيديو الحالي تقنيات تلألؤ بيولوجي ومضان الأشعة تحت الحمراء القريبة في التصوير المجراة من EAE في الفئران SJL / J. وتبين لنا أن تلألؤ بيولوجي التصوير باستخدام مسبار الحساسة للالتهاب يظهر أساسا التهاب العصب البصري، ويوافق على القياس الكمي مع التقييم…
Disclosures
The authors have nothing to disclose.
Acknowledgements
وأيد هذا البحث من قبل جمعية الألمانية للبحوث (CRC1039 A3) وبرنامج تمويل البحوث "Landesoffensive زور ENTWICKLUNG wissenschaftlich-ökonomischer Exzellenz" (LOEWE) في ولاية هيسن، مركز بحوث الطب بالحركة والصيدلة TMP وعدا كورون سهم Fresenius مؤسسة (EKFS)، مجموعة بحوث التدريب بالحركة البحوث الاكتشافات – فارما (رحلة).
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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