Kvantificering af cerebral vaskulær arkitektur ved hjælp af to-foton mikroskopi i en musemodel af HIV-induceret 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) invaderer hjernen under den akutte fase af virusinfektion og produktivt inficerer både microglia og hjerne residente makrofager, hvilket fører til deres aktivering – og frigivelse af begge værtsafledte inflammatoriske mediatorer og opløseligt HIV-1 virotoxins som Tat og gp120 (revideret i 1,2). Som følge heraf bliver en kronisk neuroinflammatoriske, der er etableret i CNS, hvilket menes at bidrage til patogenesen af HIV-1 associeret neurokognitive Disorders (hånd) 3-5.
Kronisk overekspression af HIV-1 Tat eller interleukin (IL) -17a i CNS hos mus har vist sig at resultere i mikrovaskulære fortynding 6,7. Dette giver den mulighed, at kronisk neuroinflammation kan bidrage til patogenesen af hånden gennem virkninger på de cerebrale kar. For yderligere at undersøge dette spørgsmål, har vi udviklet metoder til at kvantificere cerebrale vaskulære Structurer.
Dette papir beskriver en fremgangsmåde til kvantificering af antallet af kapillære knudepunkter, kapillære segmenter, betyder segmentlængde, total segmentlængde, betyder kapillær diameter, og den samlede kapillarvolumen hjælp in vivo billeddannelse af kapillære netværk gennem et tyndt kranium cortical vindue (modificeret fra den tidligere beskrevne protokoller) 8,9, samt ex vivo billeddannelse af hjernen sektioner, ved anvendelse af to-foton mikroskopi. Dette kombineret tilgang giver en holistisk kvantificering af cerebrale vaskulære parametre, da di vivo tynde kranium kortikal vindue giver mulighed for bevarelse af den cerebrale miljø, mens ex vivo billeddannelse af kapillarnetværk i hjernen skiver muliggør genopbygning af komplette, tre-dimensionelle kapillære netværk – som derefter kan kvantificeres ved anvendelse af kommercielt tilgængeligt software.
Protocol
Representative Results
Discussion
Den her beskrevne fremgangsmåde kan anvendes til at analysere hjerne mikrovaskulære strukturer i en lang række forsøgsmodeller / indstillinger. For succes i denne metode, skal tre kritiske trin skal beherskes. For det første skal det tynde kranium vinduet ikke skade kraniet eller underliggende hjerne. Det er let at punktere kraniet under udtynding eller forårsage varmeinduceret vaskulær lækage. Dette kan forstyrre billeddannelse som det fluorescerende farvestof vil sive ind i fokusplanet og skjule kapillærerne….
Divulgazioni
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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