Name: quantification of cerebral vascular architecture using two-photon microscopy in a mouse model of hiv-induced neuroinflammation
Uploaded: 2016-01-12T18:00:00
Description: Watch this Scientific Journal Video about Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation at JoVE.com

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).

The University of Rochester&#39;s University Committee on Animal Resources approved all procedures performed in this paper.
1. Pre-surgical Preparation (and Mice)
<ol>
	<li>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.</li>
	<li>Place the mouse in an isoflurane induction chamber connected to an isoflurane vaporizer. Set isoflurane l...

<ol>
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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...

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 result in microvascular rarefaction6,7. This raises the possibility that chronic neuroinflammation may contribute to the pathogenesis of HAND through effects on the cerebral vasculature. In order to further examine this question, we have developed methods to quantify cerebral vascular structures.
This paper describes a method for quantifying the number of capillary nodes, capillary segments, mean segment length, total segment length, mean capillary diameter, and total capillary volume using in vivo imaging of capillary networks through a thin skull cortical window (modified from previously described protocols)8,9, as well as ex vivo imaging of brain sections, using two-photon microscopy. This combined approach provides for a holistic quantitation of cerebral vascular parameters, since the in vivo thin-skull cortical window allows for the preservation of the cerebral environment, while ex vivo imaging of capillary networks in brain slices enables the reconstruction of complete, three-dimensional capillary networks - which can then be quantified using commercially available software.

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quantification of cerebral vascular architecture using two-photon microscopy in a mouse model of hiv-induced neuroinflammation

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.

The thin-skull cortical window allows for in vivo&#160;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).
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Watch this Scientific Journal Video about Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation at JoVE.com

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

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.

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic ...

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