Histopathology of brain subregions. Representative brain regions in control uninfected (HIV-1⁻) m975 and infected (HIV-1⁺) m983 animals. Paraffin sections (5μ) of brain were labeled with anti-HLA-DR to identify human activated cells, anti-Iba-1 to detect macrophages and microglia, and anti-GFAP antibodies for astrocytes (all positive labeling is brown). Sections were counterstained with hematoxylin. Images of cortex, cerebellum, hippocampus, brain stem and corpus collosum were captured with a 40× objective. Accumulation of human cells in meninges, perivascular spaces with activation of microglial cells (pons/medulla) and astrocytes (cortex, hippocampus and pons/medulla) in infected mouse were noted. Arrows indicate perivascular cuff of HLA-DR⁺ cells, arrowhead microglial nodules formation.

DTI tests. A, Fractional Anisotropy (FA), B, Mean Diffusivity (D_av), C, Longitudinal Diffusivity (λ_ll), and D - Transverse Diffusivity (λ_⊥). Notable and consistent changes include increased FA in the frontal cortex (FC) and hippocampus (Hip), as well as decreased diffusivity in the whisker barrels (WB), cerebral cortex (CCx), and hippocampus of humanized mice group with more severe brain pathology. Statistically significant changes were not observed in the corpus callosum (CC), substantia nigra (SN), Medulla (M) and cerebellum (C). E, Correlation analyses of endpoint viral load with endpoint DTI metrics in WB. F, Correlation analysis of magnitude of changes of DTI metrics (Δ) pre and post HIV infection with viral load in WB region.

Quantitative fluorescence microscopy of neuronal protein expressions in brains of CD34-NSG mice. A, Schematic representation of the strategy of analysis. Slide-mounted brain sections (5μ thick) that included the corresponding regions (mouse brain atlas) analyzed by DTI were analyzed by quantitative fluorescence microscopy with an automated fluorescence microscope (Leica), multispectral imaging system (Nuance EX) and ImagePro® Plus image analysis software. Image cubes consisting of a Z-stack of images over a wide range of wavelengths were captured from each microscope field with a 20× objective. Cubes were unmixed by spectra corresponding to each fluorescence label and to autofluorescence into quantitative gray scale images for analysis of intensity and area of fluorescence emission. B, Individual mouse data represent expression of the neuronal proteins MAP2 and SYN (fluorescence intensity) and NF and GFAP (area of positive fluorescence labeling). The reduction of MAP2 and SYN were detected in cortical regions, but not in pons/medulla. The significant drop of NF was found in WB in all analyzed mice and in frontal cortex of two mice with significant immune pathology (m983, m985). It was not clearly observed in pons/medulla region. Variable changes in GFAP-labeled areas were noted for different regions.

Time-frame of observation, immune and viral load status of experimental humanized mice. A, Human CD34⁺ cell reconstitution, MRSI/DTI analysis, HIV-1 infection, and splenic histological analysis of NSG mice are mapped to a study time line B and C, Analyses of CD4 and CD8 cell populations in peripheral blood of four HIV-1-infected mice and six control mice. D, The dynamics of VL in peripheral blood of infected animals are shown. Mice were bled once every 4 weeks starting 2^nd week post HIV-infection. E, Percent of human T and B lymphocytes and macrophages in spleen were determined at the end point of the study and mapped with end point viral load (VL). B, C and D, Mean and S.E.M are shown, and E, individual numbers for each mouse.

¹H-MRS analyses. A, Brain regions of interests. B, N-acetyl aspartate concentrations in HIV-1-infected humanized mice. The means ± SEM of [NAA] from selected regions of brain are shown. The values were normalized to water for quantitative assessment. The values were drawn at *p < 0.05 vs pre-infection and ⁺p < 0.1 vs pre-infection. It can be clearly seen that NAA is significantly reduced over the course of infection in the cerebral cortex but not in the cerebellum, hippocampus or brainstem/pons region.

Immunofluorescence labeling of astrocytes, neurofilaments and synapses in brain regions of both HIV-infected and noninfected CD34-NSG mice. Slide-mounted sections (5μ) of brain from a noninfected mouse with the least brain damage (control M975) and an infected mouse with a markedly elevated viral load and exhibiting the most brain damage (M983) were stained for GFAP (an astrocyte specific marker; red) and Neurofilament (green). Images of cerebral cortex, whisker barrel and brainstem were captured at 40× objective. Dendritic and synaptic morphology of cortical neurons are illustrated. Sections of mouse M975 and mouse M983 were labeled for MAP2 (red), Synaptophysin (green), and nuclei (DAPI; blue). Panels to the far left map tissue labeling to regions in the context of whole brain sections.