HIV誘導神経炎症のマウスモデルでは二光子顕微鏡を用いた脳血管のアーキテクチャの定量化
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
ヒト免疫不全ウイルス1（HIV-1）ウイルス感染の急性期の間に脳に侵入し、かつ生産両方ミクログリア及び脳の常在マクロファージ、それらの活性化につながる感染 – 及び両方宿主由来の炎症性メディエーターおよび可溶性HIV-1の放出をそのような（1,2に見直さ）Tatおよびgp120のようvirotoxins。その結果、慢性神経炎症状態は、HIV-1関連神経認知障害（手）3-5の病因に寄与していると考えられるCNS、に設立されてしまいます。
HIV-1のTatまたはマウスのCNS 内のインターロイキン（IL）-17Aの慢性的過剰発現は、微小血管希薄6,7をもたらすことが示されています。これには、慢性神経炎症が脳血管系への影響を介して手の病因に貢献するかもしれないという可能性を提起します。さらに、この問題を調べるために、我々は、脳血管STRUCを定量化する方法を開発しましたトゥーレス。
この論文では、セグメント長、総セグメントの長さを意味する毛細管の直径、及び薄い頭骨皮質窓を通して毛細管ネットワークのインビボイメージングに用いて全毛細管容積平均、毛細管ノード、キャピラリーセグメントの数を定量化するための方法を記載して（前述の修飾されましたプロトコル）8,9、ならびに二光子顕微鏡を用いて脳切片の ex vivoイメージング。脳スライス内の毛細血管網の ex vivoイメージングは、三次元、完全な再構築を可能にしながら、in vivoでの薄頭蓋骨皮質ウィンドウは、脳環境の保全を可能にするので、この組み合わせアプローチは、脳血管のパラメータの全体的な定量を提供します毛細血管ネットワーク – 次に、市販のソフトウェアを用いて定量することができます。
Protocol
Representative Results
Discussion
ここに記載された方法は、実験モデル/設定の広範囲の脳微小血管構造を分析するために適用することができます。この方法の成功のために、3つの重要なステップを習得する必要があります。まず、薄頭蓋骨窓は、頭蓋骨または基礎脳に損傷を与えてはなりません。それは間伐時に頭蓋骨に穴を開け、または熱誘導性の血管漏出を引き起こすことは容易です。蛍光色素は、焦点面に漏出し、?…
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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