Kwantificering van cerebrale vasculaire architectuur met behulp van twee-foton microscopie in een muismodel van de HIV-geïnduceerde Neuroinflammation
Summary
This paper describes a method by which the vascular architecture in the brain can be quantified using in vivo and ex vivo two-photon microscopy.
Abstract
Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic neuroinflammatory state within the central nervous system (CNS), thought to be driven principally by virally-mediated activation of microglia and brain resident macrophages. HIV-1 infection is also accompanied by changes in cerebrovascular blood flow (CBF), raising the possibility that HIV-associated chronic neuroinflammation may lead to changes in CBF and/or in cerebral vascular architecture. To address this question, we have used a mouse model for HIV-induced neuroinflammation, and we have tested whether long-term exposure to this inflammatory environment may damage brain vasculature and result in rarefaction of capillary networks. In this paper we describe a method to quantify changes in cortical capillary density in a mouse model of neuroinflammatory disease (HIV-1 Tat transgenic mice). This generalizable approach employs in vivo two-photon imaging of cortical capillaries through a thin-skull cortical window, as well as ex vivo two-photon imaging of cortical capillaries in mouse brain sections. These procedures produce images and z-stack files of capillary networks, respectively, which can be then subjected to quantitative analysis in order to assess changes in cerebral vascular architecture.
Introduction
Human Immunodeficiency Virus 1 (HIV-1) binnendringt de hersenen tijdens de acute fase van virusinfectie en productief infecteert zowel microglia en brein macrofagen, waardoor hun activering – de afgifte van zowel gastheer afkomstig ontstekingsmediatoren en oplosbare HIV-1 virotoxins zoals Tat en gp120 (besproken in 1,2). Bijgevolg wordt een chronische neuro-inflammatoire toestand ingesteld in het CZS, waarvan wordt gedacht dat bijdraagt aan de pathogenese van HIV-1 geassocieerde Neurocognitieve Disorders (HAND) 3-5.
Chronische overexpressie van HIV-1 Tat en interleukine (IL) -17A in het CZS van muizen is aangetoond dat het resulteert in microvasculaire verdunning 6,7. Dit verhoogt de mogelijkheid dat chronische inflammatoire processen kan bijdragen tot de pathogenese van de hand door effecten op de cerebrale vasculatuur. Om dit vraagstuk nader te bestuderen, hebben we methoden om cerebrovasculaire struc kwantificeren ontwikkeldturen.
Dit document beschrijft een werkwijze voor het kwantificeren van het aantal capillaire knooppunten capillaire segmenten gemiddelde segmentlengte, totale segmentlengte, gemiddelde capillair diameter en totale capillaire volume met in vivo beeldvorming van capillaire netwerken via een dunne schedel corticale venster (gemodificeerd van eerder beschreven protocols) 8,9, en ex vivo beeldvorming van hersencoupes, met behulp van twee-foton microscopie. Deze gecombineerde aanpak voorziet in een holistische kwantificering van cerebrale vasculaire parameters, omdat de in vivo dunne schedel corticale venster zorgt voor het behoud van de cerebrale milieu, maar ex vivo beeldvorming van capillaire netwerken in hersencoupes maakt de reconstructie van de volledige driedimensionale capillaire netwerken – dat vervolgens kan worden gekwantificeerd met behulp van commercieel verkrijgbare software.
Protocol
Representative Results
Discussion
De hier beschreven methode kan worden toegepast op de hersenen microvasculaire te analyseren in een groot aantal experimentele modellen / instellingen. Voor het succes van deze werkwijze moet drie cruciale stappen worden beheerst. Ten eerste moet de dunne schedel raam schedel of onderliggende hersenen niet beschadigen. Het is gemakkelijk om de schedel te doorboren tijdens dunner, of ervoor zorgen dat de warmte-geïnduceerde vasculaire lekkage. Dit kan interfereren met beeldvorming als de fluorescerende kleurstof zal lek…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Maria Jepson, Dr. Paivi Jordan, and Dr. Linda Callahan at the University of Rochester Multiphoton Core for technical advice throughout the completion of this protocol. We also thank Dr. Changyong Feng for expert statistical advice, and Dr. Maiken Nedergaard at the University of Rochester Medical Center for the headplate design used in this paper. This work was supported in part by grants T32GM007356 and R01DA026325 from the National Institutes of Health (NIH); and by the University of Rochester Center for AIDS Research grant P30AI078498 (NIH).
Materials
| Leica Microscope | Leica Inc. | MZ8 | |
| High Intensity Illuminator | Dolan-Jenner | 180 | |
| Heating Pad | Stryker | TP3E | |
| T/PUMP | Gaymar Industries, Inc. | TP-500 | |
| TEC-4 Isoflurane Vaporizer | Datex Ohmeda | 447 | |
| Artificial Tear Gel | Butler AHS | 7312 | |
| Povidone-Iodine solution | Aplicare | 52380-1855-9 | |
| Extra Fine Bonn Scissors | Fine Science Tools | 14084-08 | |
| Dumot #5 Forceps | Fine Science Tools | 11295-10 | |
| Dumont #5/45 Forceps | Fine Science Tools | 11251-35 | |
| Ferric Chloride Solution | Ricca Chemical Company | 3120-16 | |
| Loctite 454 Prism Instant Adhesive Gel | Henkel | 45404 | |
| Dental Cement | Stoelting | 51459 | |
| Microtoruqe II Handpiece Kit | Pearson Dental | R14-0002 | |
| 005 Burr for Micro Drill | Fine Science Tools | 19007-05 | |
| Norland Blade (Dental Microblade) | Salvin Dental | 6900 | |
| Urethane | Sigma-Aldrich | U2500 | Group 2B Carcinogen |
| Braided Suture | Ethicon | 735G | |
| Vannas Spring Scissors | Fine Science Tools | 15000-03 | |
| Arterial Catheter | SAI Infusion Technologies | MAC-01 | The end of the catheter was manually stretched out in order to decrease its diameter. |
| Blood Pressure Moniter | World Precision Intruments | SYS-BP1 | |
| Blood Pressure Transducer and Cable | World Precision Intruments | BLPR2 | |
| RAPIDLab Blood Gas Analyzer | Siemens | 248 | |
| 40 μl Capillary Tube | VWR | 15401-413 | |
| Texas Red-dextran (70,000 MW, 10 mg/kg dissolved in saline) | Invitrogen | D-1830 | |
| Adult Mouse Brain Slicer Matrix | Zivic Instruments | BSMAS001-1 | |
| Olympus Fluoview 1000 AOM-MPM Multiphoton Microscope | Olypmus | FV-1000 MPE | |
| MaiTai HP DeepSee Ti:Sa laser | Spectra-Physics | ||
| ImageJ Software | National Institutes of Health (NIH) | Available at http://rsb.info.nih.gov/ij/download.html | |
| Amira Software | Visage Imaging |
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