利用双光子显微镜的艾滋病毒引起的神经炎症的小鼠模型脑血管结构的量化
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
在病毒感染的急性期的人类免疫缺陷病毒-1（HIV-1）的侵入脑，和生产性感染都小胶质细胞和脑定居巨噬细胞，导致它们的活化 – 并且两个来自宿主的炎症介质和可溶性HIV-1的释放virotoxins如Tat和gp120的（ 在 1,2-综述）。因此，慢性神经炎状态成为中枢神经系统，这被认为是有助于HIV-1相关的神经认知障碍（手），3-5的发病机制建立。
HIV-1的Tat或小鼠的中枢神经系统内白细胞介素（IL）-17A的慢性过度表达已被证明导致微血管稀疏6,7。这就提出了慢性神经炎症可以通过对脑血管的影响造成的手发病的可能性。为了进一步探讨这个问题，我们已经制定的方法来量化脑血管STRUCTures的。
本文介绍的方法用于定量毛细管节点，毛细管段的数量，平均段长度，总段长度，平均毛细管直径，并通过一薄的颅骨皮质窗口使用毛细管网络的体内显像总毛细管体积（从先前描述的改性协议）8,9，以及离体成像的脑切片中，使用双光子显微镜。这种组合方法提供用于脑血管的参数的整体定量，由于体内薄颅骨皮质窗口允许大脑保护环境，同时毛细管网络在脑切片的离体成像能够重建完整，立体毛细管网络 – 然后可以使用市售软件定量。
Protocol
Representative Results
Discussion
此处所描述的方法可用于分析脑微血管结构在大范围的实验模型/设置。对于这种方法的成功，有三个关键步骤，必须要掌握。首先，将薄颅骨窗不能损伤颅骨或底层大脑。这是很容易变薄时穿破头骨，或引起热诱导的血管渗漏。这可以通过摄像干扰作为荧光染料将泄漏到焦点的平面中，并掩盖了毛细血管。如果颅骨薄头骨制备过程中频频打破，它很可能是过大的下行压力造成的。握住微扭矩钻尽…
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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