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.

Chronic central nervous system expression of HIV-1 Tat leads to accelerated rarefaction of neocortical capillaries and loss of red blood cell velocity heterogeneity.

OBJECTIVES: HIV-1 infection of the CNS is associated with impairment of CBF and neurocognitive function, and accelerated signs of aging. As normal aging is associated with rarefaction of the cerebral vasculature, we set out to examine chronic viral effects on the cerebral vasculature. METHODS: DOX-inducible HIV-1 Tat-tg and WT control mice were used. Animals were treated with DOX for three weeks or five to seven months. Cerebral vessel density and capillary segment length were determined from quantitative image analyses of sectioned cortical tissue. In addition, movement of red blood cells in individual capillaries was imaged in vivo using multiphoton microscopy, to determine RBCV and flux. RESULTS: Mean RBCV was not different between Tat-tg mice and age-matched WT controls. However, cortical capillaries from Tat-tg mice showed a significant loss of RBCV heterogeneity and increased RBCF that was attributed to a marked decrease in total cortical capillary length (35-40%) compared to WT mice. CONCLUSIONS: Cerebrovascular rarefaction is accelerated in HIV-1 Tat-transgenic mice, and this is associated with alterations in red cell blood velocity. These changes may have relevance to the pathogenesis of HIV-associated neurocognitive disorders in an aging HIV-positive population.

Semen-derived enhancer of viral infection (SEVI) binds bacteria, enhances bacterial phagocytosis by macrophages, and can protect against vaginal infection by a sexually transmitted bacterial pathogen.

The semen-derived enhancer of viral infection (SEVI) is a positively charged amyloid fibril that is derived from a self-assembling proteolytic cleavage fragment of prostatic acid phosphatase (PAP(248-286)). SEVI efficiently facilitates HIV-1 infection in vitro, but its normal physiologic function remains unknown. In light of the fact that other amyloidogenic peptides have been shown to possess direct antibacterial activity, we investigated whether SEVI could inhibit bacterial growth. Neither SEVI fibrils nor the unassembled PAP(248-286) peptide had significant direct antibacterial activity in vitro. However, SEVI fibrils bound to both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli and Neisseria gonorrhoeae) bacteria, in a charge-dependent fashion. Furthermore, SEVI fibrils but not the monomeric PAP(248-286) peptide promoted bacterial aggregation and enhanced the phagocytosis of bacteria by primary human macrophages. SEVI also enhanced binding of bacteria to macrophages and the subsequent release of bacterially induced proinflammatory cytokines (tumor necrosis factor alpha [TNF-α], interleukin-6 [IL-6], and IL-1ß). Finally, SEVI fibrils inhibited murine vaginal colonization with Neisseria gonorrhoeae. These findings demonstrate that SEVI has indirect antimicrobial activity and that this activity is dependent on both the cationic charge and the fibrillar nature of SEVI.

Detection of microregional hypoxia in mouse cerebral cortex by two-photon imaging of endogenous NADH fluorescence.

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg(1). We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 µm. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.