Innovative In-Situ Imaging Techniques for the Visualization of CNS associated HIV reservoirs in the Context of Substance Abuse

尽管我们对艾滋病毒发病机制的了解取得了进展，但对艾滋病毒病毒的了解仍然存在差距 水库。未知数包括，它们的不同位置，水库的大小，以及 水库建设。用普通组织学技术量化和绘制HIV的难度 在回答这些问题方面进展有限。这些挑战在RFA-DA-23中得到承认- 001呼吁采取行动，“开发或利用原位成像技术来调查艾滋病毒感染或潜伏 艾滋病毒/SIV储藏室“，同时也在药物滥用的背景下。在这里，我们通过利用 最新的光学组织清除和体积成像，在保持单细胞信息的同时解析单细胞信息 整个组织的三维结构排列。这些技术提供了高空间分辨率，在体内 成像无法匹配，影响了识别整个组织中稀疏的储集层的能力。 重要的是，这项应用将集中在新的中枢神经系统相关的储存库，包括颅骨骨髓 环境和脑脊液引流上淋巴结。将与脑部病毒库进行比较 基底节和下丘脑。总体假设是：体积深部组织显微镜和 机器学习图像分析揭示了HIV的存在水平、空间分布和感染的细胞 在新的与中枢神经系统相关的保护区/水库内。在R61开发阶段，清晰度将 通过透明丙烯酰胺消除细胞脂质并产生物理结构支撑 水凝胶，可以让光线深入穿透进行3D重建。由于器官的内在变异性 和物种特定的组织成分，没有通用的方案是合适的。因此，水凝胶制剂， 将评估去除脂肪的机制和光学清除的分析测定。我们的目标是 分析确定的参数非常适合出色的结构保持性和最小的组织损伤/蛋白质 在非人灵长类动物的整个组织中丢失。此外，这些清理方法将针对兼容性进行优化 中枢神经系统相关HIV中HIV RNA/DNA探针的抗体免疫染色和原位杂交 水库。我们的评估将包括一种新的艾滋病毒宿主--头盖骨骨髓的证据(目标1)， 颈上淋巴结(脑脊液引流的终点)(目标2)和脑内关键区域(目标3)。 这些研究的意义反映在这样一个事实上，即到目前为止，在整个组织中还没有发表过的工作 光学清除上述非人类灵长类动物的病毒库。在R33应用阶段， 在R61阶段制定的方案将被用来确定有关艾滋病毒宿主的长期问题 例如，多快就建立了水库，哪些细胞类型被感染，以及这些部位感染的程度 窝藏艾滋病病毒。上述问题将根据长期阿片类药物暴露(目标4)或 艺术的存在(目标5)。总体而言，这些研究将提供与中枢神经系统相关的艾滋病毒的关键信息 使用新的原位成像方法，实现了RFA-DA-23-001的主要目标。

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.