Quantificação de Vascular Cerebral Arquitetura usando dois fótons Microscopia em um modelo de mouse Neuroinflammation induzida por HIV
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
Vírus da Imunodeficiência Humana tipo 1 (VIH-1) invade o cérebro durante a fase aguda da infecção pelo vírus, e produtivamente infecta ambas as microglia e macrófagos residentes cérebro, que conduz à sua activação – e a libertação de ambos os mediadores inflamatórios derivados do hospedeiro e solúvel HIV-1 virotoxins como Tat e gp120 (revisto em 1,2). Como consequência, um estado neuroinflamatória crônica torna-se estabelecido no CNS, que é pensado para contribuir para a patogênese do HIV-1 Distúrbios Associated neurocognitivos (a mão) 3-5.
Superexpressão crónica do HIV-1 Tat ou interleucina (IL) -17 no SNC de ratinhos foi mostrado para resultar em 6,7 rarefacção microvascular. Isto levanta a possibilidade de que neuroinflama�o crónica pode contribuir para a patogénese da mão através de efeitos sobre a vasculatura cerebral. A fim de analisar melhor esta questão, temos desenvolvido métodos para quantificar struc vasculares cerebraisturas.
Este artigo descreve um método para a quantificação do número de nodos capilares, segmentos capilares, comprimento do segmento, o comprimento total do segmento dizer, o diâmetro médio do capilar, e o volume total do capilar utilizando imagiologia in vivo de redes capilares através de uma janela cortical crânio fina (modificado a partir do descrito anteriormente protocolos de 8,9), assim como ex vivo de imagens de secções do cérebro, utilizando microscopia de dois fotões. Esta abordagem combinada fornece para uma quantificação global de parâmetros vasculares cerebrais, desde fina-crânio janela cortical in vivo permite a preservação do meio ambiente cerebral, enquanto ex vivo de imagens de redes capilares em fatias de cérebro permite a reconstrução completa, tridimensional – redes capilares que pode, então, ser quantificada utilizando software disponível comercialmente.
Protocol
Representative Results
Discussion
O método aqui descrito pode ser aplicado para analisar as estruturas microvasculares cerebrais em uma ampla gama de modelos / configurações experimentais. Para o sucesso deste método, três passos críticos deve ser dominado. Em primeiro lugar, a janela crânio-fina não deve danificar o crânio ou cérebro subjacente. É fácil de perfurar o crânio durante o desbaste, ou causar perda vascular induzida pelo calor. Isto pode interferir com a formação de imagens conforme o corante fluorescente vai infiltrar-se no p…
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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