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

创新的原位成像技术，用于在药物滥用情况下可视化中枢神经系统相关的艾滋病毒储存库

• 批准号: 10682957
• 负责人: Allison Michelle Andrews
• 金额: $ 58.69万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10682957
• 关键词: 3-Dimensional; Acrylamides; Address; Affect; Anatomy; Antibodies; Antibody Specificity; Area; Basal Ganglia; Biology; Bone Density; Bone Marrow; Brain; Brain imaging; Brain region; Calvaria; Cells; Cellular Structures; Cervical lymph node group; Chronic; Computer software; DNA; DNA Probes; Decalcification; Detection; Development; Drainage procedure; Environment; Evaluation; Excision; Formulation; Goals; HIV; HIV Infections; Histologic; Histological Techniques; Home; Hydrogels; Hypothalamic structure; Image; Image Analysis; Imaging Techniques; Imaging technology; Immune; In Situ Hybridization; Infection; Intercellular Fluid; Kinetics; Knowledge; Light; Lipids; Location; Lymphatic System; Machine Learning; Maps; Methodology; Methods; Microscopy; Morphine; Morphology; Mus; Myelogenous; Neuroanatomy; Opiate Addiction; Opioid; Optics; Organ; Pathogenesis; Penetration; Pharmaceutical Preparations; Phase; Predisposition; Procedures; Proteins; Protocols documentation; Publishing; RNA; RNA Probes; Resolution; Route; SIV; Site; Slice; Spatial Distribution; Structure; Substance abuse problem; Techniques; Testing; Thinness; Time; Tissues; Vascular System; Viral; Viral reservoir; Virus; Virus Latency; Virus Replication; Visualization; Work; bone reconstruction; brain parenchyma; cell type; cohort; design; draining lymph node; humanized mouse; imaging capabilities; in situ imaging; in vivo; in vivo imaging; innovation; lateral ventricle; long bone; lymph nodes; neuropathology; neurovascular unit; nonhuman primate; nonhuman tissue; novel; novel therapeutics; opioid exposure; particle; reconstruction; scale up; simian human immunodeficiency virus

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.

尽管我们对艾滋病毒发病机制的了解取得了进展，但关于艾滋病毒病毒的知识差距仍然存在 未知的因素包括它们的不同位置、储层的大小以及空间/时间动力学。 使用常见的组织学技术量化和绘制 HIV 病毒库的建立存在困难。 RFA-DA-23- 承认这些挑战的进展有限。 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在感染 HIV/SIV 宿主”，同时也在药物滥用的背景下解决上述问题。 最新的光学组织透明和体积成像技术可解析单细胞信息，同时保持 这些技术提供了体内的高空间分辨率。 成像无法匹配，影响识别整个组织中稀疏的储库的能力。 重要的是，该应用将重点关注新型中枢神经系统相关储库，包括颅骨骨髓 环境和脑脊液引流上淋巴结将与脑内病毒库进行比较。 基底神经节和下丘脑的总体假设是：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、空间分布和感染细胞 在 R61 开发阶段，Clarity 将在新的 CNS 相关保护区/水库地点内进行。 消除细胞脂质并通过透明丙烯酰胺产生物理结构支撑 由于器官固有的可变性，水凝胶可以让光深入穿透以进行 3D 重建。 和物种特异性的组织成分，没有通用的方案是合适的。 将评估脂质去除机制和光学透明化的分析测定。 通过分析确定实现出色结构保留和最小组织损伤/蛋白质的理想参数 此外，这些清除方法将针对兼容性进行优化。 中枢神经系统相关 HIV 中的抗体免疫染色和 HIV RNA/DNA 探针原位杂交 我们的评估将包括新的 HIV 储存库——颅骨骨髓（目标 1）的证据， 颈上淋巴结（脑脊液引流的终点）（目标 2）和大脑内的关键区域（目标 3）。 这些研究的重要性体现在这样一个事实：迄今为止，还没有发表过完整组织的作品。 在 R33 应用阶段，对非人类灵长类动物中提到的病毒库进行光学清除。 R61 阶段制定的方案将用于确定有关 HIV 病毒库的长期问题 例如储存库多久建立、哪些细胞类型被感染以及这些位点受到感染的程度如何 上述问题将根据长期阿片类药物暴露（目标 4）或 总体而言，这些研究将提供有关中枢神经系统相关艾滋病毒的重要信息。 使用新颖的原位成像方法来检测储层，实现了 RFA-DA-23-001 的主要目标。