HIV kaynaklı Nöroinflamasyondan bir Fare Modeli İki foton Mikroskop kullanarak Serebral Vasküler Mimarlık Niceleme
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
İnsan Bağışıklık Yetmezliği Virüsü 1 (HIV-1) virüs enfeksiyonunun akut fazında beyni işgal ve verimli onların aktivasyon yol mikroglia ve beyin ikamet makrofajlar hem bozar – ve her iki konak kaynaklı inflamatuar mediatörlerin ve çözünür HIV-1 serbest bırakma Böyle Tat ve (1,2 gözden) gp120 olarak virotoxins. Sonuç olarak, kronik nöroenflamatuar devlet HIV-1 İlişkili Nörokognitif Bozuklukları (EL) 3-5 patogenezine katkıda düşünülmektedir MSS içinde kurulmuş olur.
HIV-1 Tat veya farelerin merkezi sinir sistemi içinde, interlökin (IL) -17A arasında kronik aşırı mikrovasküler seyrelmesi 6,7 ile sonuçlandığı gösterilmiştir. Bu kronik Nöroenflamasyon serebral damarlarda üzerindeki etkileri yoluyla EL patogenezine katkıda bulunabileceği ihtimalini yükseltir. Ayrıca bu soruyu incelemek için, biz serebral vasküler yapıda çalışılmıştır ölçmek için yöntemler geliştirmişlerdirtures.
Bu kağıt, kademeli bir uzunluğu, toplam kademeli bir uzunluk ve ortalama kılcal çap ve ince bir kafatası kortikal pencereden kılcal ağ şebekeleri in vivo görüntüleme kullanılarak total kılcal hacmi ve ortalama kılcal düğümleri, kılcal kesimlerinin sayısı ölçülmesi için bir yöntem tarif eder (daha önce tarif edilen modifiye protokolleri) 8,9, hem de iki foton mikroskopi kullanılarak beyin bölümlerinin ex vivo görüntüleme. In vivo ince kafatası kortikal pencere serebral çevrenin korunması için izin verir çünkü beyin dilimleri kılcal ağları ex vivo görüntüleme rekonstrüksiyonu sağlarken Bu kombine yaklaşım, serebral vasküler parametrelerin bütünsel bir kantitatif sağlar üç boyutlu, tam kılcal ağ şebekeleri, – daha sonra ticari olarak temin edilebilen yazılımı kullanılarak belirlenebilir.
Protocol
Representative Results
Discussion
Burada tarif edilen yöntem, deneysel modeller / ayarları geniş bir beyin mikrovasküler yapılarını analiz etmek için uygulanabilir. Bu yöntemin başarılı olması için, üç kritik adım hakim olması gerekmektedir. İlk olarak, ince kafatası penceresi kafatası ya da altta yatan beyin zarar vermemelidir. Bu inceltme sırasında kafatası delinme ya da ısı kaynaklı damar kaçağa neden kolaydır. Floresan boya odak düzlemi içine sızabilir ve kılcal belirsiz gibi bu görüntüleme engelleyebilir. Kafata…
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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