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.

尽管我们对艾滋病发病机制的理解有了进步，但对艾滋病病毒的认识仍然存在差距。 水库未知数包括它们的各种位置、储层的大小和储层的空间/时间动力学。 水库建设使用普通组织学技术定量和绘制HIV图谱的困难 在回答这些问题方面进展有限。这些挑战在RFA-DA-23- 001在呼吁采取行动，“开发或利用原位成像技术，以调查艾滋病毒感染，或潜伏 艾滋病毒/SIV储存库”，同时也涉及药物滥用。在这里，我们通过利用 最新的光学组织清除和体积成像技术，可解析单细胞信息， 整个组织中的三维结构排列。这些技术提供了高空间分辨率， 成像不能匹配，从而影响识别在整个组织中稀疏的储库的能力。 重要的是，该应用将集中于新的CNS相关储库，包括颅骨骨髓 环境和脑脊液引流上级淋巴结。将进行比较，以脑病毒水库内 基底神经节和下丘脑。总体假设是：体积深层组织显微镜和 机器学习图像分析揭示了艾滋病毒的存在水平、空间分布和感染细胞 在新的CNS相关的避难所/水库网站。在R61开发阶段，Clarity将 通过透明丙烯酰胺消除细胞脂质并产生物理结构支持 这种水凝胶可以让光线深入穿透，进行3D重建。由于器官固有的变异性 和物种特异性组织组成，没有通用的协议是适当的。因此，水凝胶制剂， 将评估脂质去除的机制和光学透明的分析测定。目标是 分析确定理想的参数，以实现出色的结构保持和最小的组织损伤/蛋白质 非人类灵长类动物整个组织的损失。此外，这些清算方法将针对兼容性进行优化 用抗体免疫染色和HIV RNA/DNA探针在CNS相关HIV中的原位杂交 水库我们的评估将包括一个新的艾滋病毒储存库，颅骨骨髓（目标1）， 上级颈淋巴结（CSF引流的终点）（目标2）和脑内的关键区域（目标3）。 这些研究的重要性反映在这样一个事实上，即到目前为止，没有发表的工作存在于整个组织中 光学清除非人灵长类动物中的上述病毒储库。在R33应用阶段， 在R61阶段制定的协议将用于确定有关艾滋病毒储存库的长期问题 例如储库建立的时间，哪些细胞类型被感染，以及这些位点在多大程度上 携带艾滋病毒。上述问题将作为慢性阿片类药物暴露的函数（目标4）或 抗逆转录病毒疗法（Aim 5）总之，这些研究将提供CNS相关HIV的关键信息 使用新型原位成像方法对储层进行成像，实现RFA-DA-23-001的主要目标。