Development of an in vitro blood-brain barrier model to study molecular neuropathogenesis and neurovirologic disorders induced by human immunodeficiency virus type 1 infection.

Abstract

Objective: An in vitro blood-brain barrier (BBB) system was developed using primary cultures of human brain-derived microvascular endothelial cells (MVECs), macrophages, neuronal cells, and human fetal astrocytes. This BBB system simulates important morphologic and permeability characteristics of the BBB in vivo. This system could be used to study human neurologic/neurovirologic disorders.

Study design and methods: Microvascular endothelial cells were cultured to 100% confluency on the upper side of 0.45-microm polyethylene tetraphthalte (PET) membrane inserts coated with MVEC attachment factor. Expression of ZO-1 (Zona Occludens 1), a protein specifically associated with tight junctions and the intercellular sealing of adjacent MVECs, was analyzed by immunocytochemical methods. The integrity of the BBB formed on the insert membrane was also assessed by the measurement of electrical current passage through the membrane; and, after the formation of complete BBB, a two-compartment system was developed using the cell culture insert upper surface, essentially a confluent monolayer of brain-derived MVECs housed on a six-well chamber surface. In the six-well chamber surface, there are different central nervous system (CNS)-based cells, ie, human astrocytes, immature neurons, mature neurons, and MVEC. The cell culture inserts were in close juxtaposition to the surface of the chamber, making an intimate contact with the cells, separated by an insert membrane. The MVEC surface of the insert was exposed to human immunodeficiency virus type 1 (HIV-1) strains 89.6, NL4-3, and IIIB. After 72 hours, the cells were fixed and used for in situ polymerase chain reaction (IS-PCR), whereas the supernatant was subjected to HIV-1 p24 antigen determination.

Results: Primary human brain MVECs are capable of forming tight junctions, revealed by the expression of ZO-1, as well as elevated transendothelial electrical resistance. Based on these characteristics, these cells formed an in vitro BBB, which then was used to study the transfer of HIV-1 through this barrier. It was observed that HIV-1 can infect MVEC and can cross it in vitro and infect the cells growing on the opposite side of the membrane. Infection of various CNS-based cells was confirmed by IS-PCR, as well as by HIV-1 p24-antigen determination. It was observed that the dual-tropic strain, 89.6, had a greater potential to create a breach in the in vitro BBB, followed by NL4-3 and IIIB.

Conclusion: This model system is relevant for evaluating HIV-1 neuropathogenesis and therapeutics designed to alter HIV-1 expression in human CNS-based cells. As such, the effects of highly active antiretroviral therapy on HIV-1 infection of the human CNS, a possible drug sanctuary site, can be evaluated using this technology.