697 Rapid Autopsy Isolation of Human Microglia from a Patient with HIV-1 Encephalitis: Recovery of Virus and Studies of Glial Function S. Swindells*, A. Ghorpade, Y. Persidsky, K. Borgmann, R. Persidsky, S. Holter, R. Cotter, K. Carlson, R. McComb, L. Bruch, H. Gendelman Univ of Nebraska Med Ctr, Omaha
Background: Mononuclear phagocyte (MP) infection and immune activation underlies the neurodegeneration of HIV-1-associated dementia (HAD). The dynamics of these processes are poorly understood. To address this issue, we established a rapid autopsy program for isolation, purification, and study of primary human neural cells from brain tissue.
Methods: An autopsy was performed within 4 hrs of death on a 44 yr old male with AIDS, a CD4+ T-cell count of 25 cells/mm3 and a viral load of 270,000 copies/ml. Pre-mortem neuro-imaging showed brain atrophy and neuropsychological testing showed mild cognitive impairment with an AIDS Dementia Complex (ADC) score of 1. Brain tissue was harvested for the isolation of microglia from diverse brain regions including basal ganglia, white matter, cortex, cerebellum, and anterior frontal cortex. Plasma, serum, and cerebrospinal fluid were also obtained.
Results: Highly pure preparations of microglial cells were obtained as demonstrated by CD68 antigen expression. Multi-nucleated giant cells were observed in cultures starting at 8-10 days after cell cultivation. Within two wks basal ganglia, cortex, and white matter microglia demonstrated significant cytopathic effects. Viral replication was confirmed by RT activity. Peripheral nerve and CD40 ligand were used to study microglial responses to immune stimuli. Tumor necrosis factor alpha (TNF-alpha) served as an indicator of immune activation. Microglial cells produced high levels of TNF-alpha after activation and responded readily to injury stimuli. Cellular extracts of microglial cells were analyzed on ProteinChip to obtain bio-marker profiles.
Conclusions: This is a first report of a rapid autopsy on a patient with HIV-1 encephalitis with successful isolation of microglia and infectious virus. These studies on primary, ex vivo neural cells provide novel tools to study neuropathogenesis of HIV-1-associated dementia.
