Adult human brain tissue cultures to study neuroHIV

成人脑组织培养研究神经艾滋病毒

• 批准号: 10619170
• 负责人: Joshua Goodyear Jackson
• 金额: $ 22.73万
• 依托单位: DREXEL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10619170
• 关键词: Adjuvant Therapy; Adult; Affect; Animal Model; Animals; Anti-Retroviral Agents; Architecture; Astrocytes; Automobile Driving; Behavioral Symptoms; Biological Models; Blood donor; Blood specimen; Brain; Calcium; Calcium Signaling; Cell Survival; Cell model; Cells; Central Nervous System; Central Nervous System Infections; Cerebral cortex; Cerebrospinal Fluid; Characteristics; Clinical Research; Clinical Trials; Collaborations; Communication; Computer software; Confocal Microscopy; Culture Media; Dendritic Spines; Disease; Dissociation; Drug toxicity; Encephalitis; Environment; Evaluation; Excision; Experimental Models; Exposure to; Flow Cytometry; Fostering; Foundations; Future; Genetic; Glutamates; HIV; HIV Infections; HIV Seronegativity; HIV-associated neurocognitive disorder; Heterogeneity; Human; Image; Image Analysis; Impaired cognition; Impairment; Individual; Infection; Inflammation; Inflammation Mediators; Inflammatory; Label; Life; Longevity; Longitudinal Studies; Macrophage; Measures; Mediating; Modeling; Molecular; Monitor; Morphology; Neurobehavioral Manifestations; Neuroimmune; Neurons; Neuropathogenesis; Neurosurgeon; Operative Surgical Procedures; Pathologic; Pathology; Pathway interactions; Patients; Persons; Pharmaceutical Preparations; Pre-Clinical Model; Preclinical Testing; Property; Regimen; Research; Research Personnel; Scientist; Signal Transduction; Sister; Slice; Stains; Structure; Substance Use Disorder; System; Techniques; Thick; Tissue Donors; Tissues; Vertebral column; Viral; Virus Replication; Vulnerable Populations; antiretroviral therapy; automated analysis; base; brain surgery; brain tissue; cell type; clinical investigation; comorbidity; confocal imaging; density; drug action; effective therapy; fluorescence imaging; glutamatergic signaling; human model; human tissue; inflammatory milieu; insight; monocyte; motor symptom; multi-electrode arrays; neuroAIDS; neuroinflammation; neuron loss; neuronal circuitry; neuropathology; neuroprotection; neurotransmission; novel therapeutic intervention; older patient; pandemic disease; peripheral blood; preclinical study; preservation; standard of care; tissue culture

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.

项目摘要：科学家和临床医生研究了与HIV相关的基本机制 神经认知障碍（手）数十年，在动物模型中跨越临床试验和临床前研究 和蜂窝系统。尽管这项研究无疑已将现场推动，但仍然没有 批准的调整疗法以缓解影响大部分艺术治疗的认知障碍 患者。这可能部分是由于临床前模型本身，这可能不完全模型 人脑的细胞异质性，组织和特性。我们建议通过 开发人脑切片培养模型以研究HIV神经病的发生。提供人体组织 通过与神经外科医生的合作，从脑外科手术。这些包括成人正常人 脑组织，脑脊液和外周血。我们将分离单核细胞衍生的巨噬细胞（MDM） 从供体的血液样本中，用荧光HIV-1感染它们，并将感染的MDM暴露于患者 - 匹配的HIV阴性脑培养物。建立大脑切片感染后，HIV复制将是 由相关浓度的护理标准艺术控制。这个人类模型可以更好地概括 被认为是在治疗患者中出现的手的病理特征，包括感染适当的CNS 细胞类型，微妙的脑感染和神经传递受损，并充当高度相关的模型 对神经保护策略的神经hiv和临床前测试的未来研究系统。 AIM 1将优化 脑切片培养条件和检查神经元结构和功能（以及神经炎症） 文化的生活。细胞活力将在具有不同培养基和切片厚度的切片培养物中进行评估 通过纵向重要染色方法和流式细胞仪。神经元活动也将在纵向上 用多电极阵列或钙成像测量，树突分支和脊柱变化将是 使用DII染色和Neurolucida 360软件分析在多个时间点监测。神经胶质功能将 还在使用钙信号传导和谷氨酸问题进行研究。 AIM 2将检查优化的切片文化 感染患者匹配的HIV+ MDM并用ART治疗。感染进度将通过共焦点跟踪 荧光HIV，流式细胞术和HIV P24α的成像。艾滋病毒的炎症环境 将使用流式细胞术和alphalisa评估被感染/未感染的切片前和图片后进行选择 炎症介体和营养因素。艺术治疗的HIV+脑切片中的神经元状态将是 用AIM 1中列出的相同技术确定。项目的完成将为A奠定基础 基于人体组织的模型系统，在很大程度上保持成人的组成和局部连接 大脑，反映了当今患者中几乎被抑制的病理，并可以帮助验证重要的研究 动物模型。该模型可以促进对中枢神经系统中病毒持久性的理解，神经病发生和 神经保护机制，药物作用或毒性机制以及合并症研究。