Quantificazione dei cerebrale vascolare Architettura con due fotoni Microscopia in un modello murino di Neuroinflammation HIV-indotta
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
Virus dell'immunodeficienza umana 1 (HIV-1) invade il cervello durante la fase acuta di infezione da virus, e infetta produttivo sia microglia e macrofagi residenti del cervello, portando a loro attivazione – e il rilascio di entrambi mediatori dell'infiammazione ospitanti di derivazione e l'HIV-1 solubile virotoxins come la Tat e gp120 (rivisto in 1,2). Di conseguenza, uno stato cronico neuroinflammatory si stabilisce nel SNC, che è pensato per contribuire alla patogenesi di HIV-1 disturbi associati neurocognitivi (mano) 3-5.
Sovraespressione cronica di HIV-1 Tat o interleuchina (IL) -17A nel SNC di topi ha mostrato di provocare microvascolare rarefazione 6,7. Ciò solleva la possibilità che neuroinfiammazione cronica può contribuire alla patogenesi di mano attraverso effetti sul sistema vascolare cerebrale. Al fine di esaminare ulteriormente la questione, abbiamo sviluppato metodi per quantificare strut vascolari cerebraliture.
Questo documento descrive un metodo per quantificare il numero di nodi capillari, segmenti capillari, significa la lunghezza del segmento, la lunghezza totale del segmento, diametro medio capillare, e il volume totale capillare usando immagini in vivo di reti capillari attraverso una finestra corticale sottile cranio (modificato da precedentemente descritta protocolli) 8,9, nonché ex vivo imaging sezioni cerebrali, utilizzando la microscopia a due fotoni. Questo approccio combinato prevede una quantificazione olistica parametri vascolari cerebrali, poiché il sottile cranio finestra corticale in vivo permette la preservazione dell'ambiente cerebrale, mentre ex vivo imaging reti capillari in fettine di cervello permette la ricostruzione di completo, tridimensionale reti capillari – che possono poi essere quantificati utilizzando il software disponibile in commercio.
Protocol
Representative Results
Discussion
Il metodo qui descritto può essere applicato per analizzare strutture microvascolari cerebrali in un'ampia gamma di modelli sperimentali / impostazioni. Per il successo di questo metodo, tre passaggi critici devono essere padroneggiato. In primo luogo, la finestra sottile cranio non deve danneggiare il cranio o cerebrale sottostante. E 'facile forare il cranio durante diradamento, o causare perdite di calore indotta vascolare. Questo può interferire con le immagini come il colorante fluorescente colerà nel pi…
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 |
Riferimenti
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