Innovative In-Situ Imaging Techniques for the Visualization of CNS associated HIV reservoirs in the Context of Substance Abuse - Despite advances in our understanding of HIV pathogenesis, a knowledge gap remains concerning HIV viral reservoirs. Unknowns include, their various locations, size of the reservoir and the spatial/temporal kinetics for reservoir establishment. The difficulty in quantifying and mapping HIV using common histological techniques has limited progress towards answering these questions. These challenges are recognized under RFA-DA-23- 001 in a call to action to “develop or exploit in situ imaging technologies to investigate HIV infection, or latent HIV/SIV reservoirs” whilst also in the context of substance abuse. Here we address the above by leveraging the latest in optical tissue clearing and volumetric imaging to resolve single cell information while maintaining the 3D structural arrangement in whole tissue. These techniques provide high spatial resolution that in-vivo imaging cannot match, affecting the ability to identify reservoirs that are sparse throughout the entire tissue. Importantly, this application will focus on novel CNS associated reservoirs including the calvarial bone marrow environment and CSF draining superior lymph nodes. Comparisons will be made to brain viral reservoirs within the basal ganglia and hypothalamus. The overall hypothesis is: Volumetric deep tissue microscopy and machine learning image analysis reveals the level of HIV present, spatial distribution and cells infected within novel CNS-associated sanctuary/reservoir sites. In the R61 development phase, Clarity will be performed to eliminate cellular lipids and generate a physical structural support via transparent acrylamide hydrogels that allows light to penetrate deeply for 3D reconstruction. Due to the inherent variability of organs and species-specific tissue composition, no universal protocol is appropriate. Thus, hydrogel formulations, mechanisms for lipid removal, and analytical determinations for optical clearing will be evaluated. The goal is to analytically determine parameters ideal for excellent structural retention and minimal tissue damage/protein loss in non-human primate whole tissue. Additionally, these clearing methods will be optimized for compatibility with antibody immunostaining and in-situ hybridization of HIV RNA/DNA probes in CNS associated HIV reservoirs. Our evaluations will include evidence for a novel HIV reservoir, the calvarial bone marrow (Aim 1), the superior cervical lymph node (the end site of CSF drainage) (Aim 2) and key areas within the brain (Aim 3). The significance of these studies is reflected on the fact that to date, no published work exists in whole tissue optical clearing of the aforementioned viral reservoirs in non-human primates. In the R33 application phase, protocols developed in the R61 phase will be utilized to determine standing questions about HIV reservoirs such as how soon the reservoir is established, which cell types are infected and to what degree do these sites harbor HIV. The above questions will be answered as a function of chronic opioid exposure (Aim 4) or presence of ART (Aim 5). Overall, these studies will provide critical information on CNS associated HIV reservoirs using novel in-situ imaging methodologies, fulfilling the main objective of RFA-DA-23-001.
