Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation

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

Human Immunodeficiency Virus 1 (HIV-1) invades the brain during the acute phase of virus infection, and productively infects both microglia and brain resident macrophages, leading to their activation - and the release of both host-derived inflammatory mediators and soluble HIV-1 virotoxins such as Tat and gp120 (reviewed in ^1,2). As a consequence, a chronic neuroinflammatory state becomes established in the CNS, which is thought to contribute to the pathogenesis of HIV-1 Associated Neurocognitive Disorders (HAND)^3-5.

Chronic overexpression of HIV-1 Tat or interleukin (IL)-17A within the CNS of mice has been shown to re....

Protocol

The University of Rochester's University Committee on Animal Resources approved all procedures performed in this paper.

1. Pre-surgical Preparation (and Mice)

1. Prepare the surgical area with all required equipment. Sterilize all tools used during the procedure beforehand using 70% ethanol. Optionally, use a glass-bead sterilizer or autoclave to sterilize the tools.
2. Place the mouse in an isoflurane induction chamber connected to an isoflurane vaporizer. Set isoflurane levels to 4% at a rate of 1 L/min.
   Note: for the experiments reported here, mice with a doxycycline (DOX) inducible HIV-1 Tat transgene driven b....

Results

The thin-skull cortical window allows for in vivo two-photon imaging of cortical capillaries (Figure 1). A suitable area to image shows numerous, distinct capillaries (Figure 1A). In the same field of view, there is no arterial cell wall autofluorescence, and there may be other fluorescent signals, such as collagen fluorescence, induced by second harmonic generation¹¹ (Figure 1B).

Discussion

The method described here can be applied to analyze brain microvascular structures in a wide range of experimental models/settings. For the success of this method, three critical steps must be mastered. First, the thin-skull window must not damage the skull or underlying brain. It is easy to puncture the skull during thinning, or cause heat induced vascular leakage. This can interfere with imaging as the fluorescent dye will leak into the plane of focus and obscure the capillaries. If the skull frequently breaks during t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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