Project summary: Scientists and clinicians have studied the underlying mechanisms of HIV-associated
neurocognitive disorder (HAND) for decades, spanning clinical trials and preclinical studies in animal models
and cellular systems. Although this research has undoubtedly pushed the field forward, there is still no
approved adjuvant therapy to relieve the cognitive impairment affecting a significant portion of ART-treated
patients. This could be partially due to the preclinical models themselves, which may incompletely model
human brain’s cellular heterogeneity, organization, and properties. We propose to bridge this gap by
developing a human brain slice culture model to study HIV neuropathogenesis. Human tissues are provided
from brain surgeries via a collaboration with a neurosurgeon colleague. These include adult normal human
brain tissues, cerebrospinal fluid, and peripheral blood. We will isolate monocyte-derived macrophages (MDM)
from a donor’s blood sample, infect them with a fluorescent HIV-1, and expose the infected MDMs to patient-
matched HIV-negative brain cultures. After the brain slice infection is established, HIV replication will be
controlled with relevant concentrations of standard-of-care ART. This human model may better recapitulate
pathological features of HAND thought to be present in treated patients, including infection of appropriate CNS
cell types, subtle brain inflammation, and impaired neurotransmission, and serve as a highly relevant model
system for future studies on neuroHIV and preclinical testing of neuroprotective strategies. Aim 1 will optimize
brain slice culture conditions and examine neuronal structure and function (and neuroinflammation) over the
life of the culture. Cell viability will be assessed in slice cultures with different culture media and slice thickness
by longitudinal vital staining approaches and flow cytometry. Neuronal activity will also be longitudinally
measured with multielectrode arrays or calcium imaging, and dendritic branching and spine changes will be
monitored at multiple timepoints using DiI staining and Neurolucida 360 software analysis. Glial function will
also be studied using calcium signaling and glutamate probes. Aim 2 will examine optimized slice cultures
infected with patient-matched HIV+ MDMs and treated with ART. Infection progress will be tracked by confocal
imaging of the fluorescent HIV, flow cytometry, and HIV p24 Alphalisa. The inflammatory environment of HIV-
infected/uninfected slices pre- and post-ART will be assessed using flow cytometry and Alphalisa for select
inflammatory mediators and trophic factors. Neuronal status in ART-treated HIV+ brain slices will be
determined with the same techniques listed in Aim 1. Completion of the project will lay the foundation for a
human tissue-based model system that largely maintains the composition and local connectivity of the adult
brain, reflects the virally suppressed pathology in today’s patients, and can help validate important studies from
animal models. The model could foster understanding of viral persistence in the CNS, neuropathogenesis and
neuroprotective mechanisms, mechanism of drug action or toxicity, and comorbidities studies.