Differential long-term neurotoxicity of HIV-1 proteins in the rat hippocampal formation: a design-based stereological study

Abstract

The human immunodeficiency virus type 1 (HIV-1) proteins, gp120 and Tat, are believed to play a role in mediating central nervous system (CNS) pathology in HIV-1 infected patients. Using design-based stereology, we examined the role of neonatal intrahippocampal injections of gp120 and Tat on the adult hippocampus ( approximately 7(1/2) month). Postnatal day (P)1-treated Sprague-Dawley rats were bilaterally injected with vehicle (VEH, 0.5 microl sterile buffer), gp120 (100 ng), Tat (25 microg) or combined gp120 + Tat (100 ng + 25 microg). Using Nissl-stained tissue sections, we quantified total neurons in five subregions of the rat hippocampus [granual layer (GL), hilus of the dentate gyrus (DGH), cornu ammonis fields (CA)2/3, CA1, and subiculum (SUB)], and total glial cells (astrocytes and oligodendrocytes) in two subregions (DGH and SUB). Estimates of cell area and cell volume were taken in the DGH. There was a significant reduction of neuron number in the CA2/3 subfield by Tat and gp120, and a significant reduction in the DGH by Tat only. For glial cells, numbers of astrocytes in the DGH and SUB were increased by the Tat protein, whereas no effects were noted for gp120. Finally, for oligodendrocytes Tat increased cell number in the DGH but not in any other region; gp120 had no detectable effect in any brain region. Estimates of cell area and cell volume of the three different cell types revealed no significant differences between treatments. Collectively, these results suggest differential effects of gp120 and Tat on the estimated total number of neurons, as well as on the number of glial cells.

FIGURE 1

A: Calibration bar; 250 μm, × 4 magnification. Left Panel: The cytoarchitectonic delineation of hippocampal subfields cut in the horizontal plane. Middle Panel: Arrows indicate the hippocampal subfields for analysis. Arrowheads mark the border between the different hippocampal subfields. Right Panel: Boundaries of the cellular layers as outlined on the StereoInvestigator 7.0 system, i.e., the defined regions of interest within cells were counted and cell area and cell volume were taken; B: A more detailed illustration of the different hippocampal subregions with close ups by using × 60 and × 100 magnifications.

FIGURE 2

A photomicrograph of the hilus of the dentate gyrus at × 100 magnification, illustrating typical cell types based on morphology and Nissl-Staining. Neurons, indicated by the solid gray arrowheads, are heavily stained with a large nucleus and single nucleolus. Astrocytes, indicated by the open arrowheads, display pale staining of the nucleus and a thin rim of Nissl stained cytoplasm immediately adjacent to the nucleus. Oligodendrocytes, indicated by the filled black arrowheads, are identified by dark nuclei eccentrically located on one end of a polar cell body. Microglia, that are not shown here, are characterized by irregular shape, dark stained nuclei and patched of condensed chromatin with little cytoplasmic staining.; Calibration Bar: 25 μm.

FIGURE 3

Estimated total number of neurons across the five subregions of the rat hippocampus. Region specific illustration of the total neuron number in the (A) granual layer (GL), (B) hilus of the dentate gyrus (DGH), (C) cornu ammonis fields (CA)2/3, (D) CA1, and (E) subiculum (SUB)., P ≤ 0.05; P ≤ 0.01.

FIGURE 4

Estimated total number of neurons, astrocytes and oligodendrocytes in the (A) hilus of the dentate gyrus (DGH) and (B) subiculum (SUB), P ≤ 0.05; P ≤ 0.01, P ≤ 0.001.

FIGURE 5

Cell morphology data in the hilus of the dentate gyrus (DGH) illustrate (A) cell volume with a significant cell type effect and (B) cell area with a significant cell type effect.