Adult human brain tissue cultures to study neuroHIV

项目概述：科学家和临床医生已经研究了HIV相关的潜在机制， 神经认知障碍（HAND）几十年来，跨越临床试验和动物模型的临床前研究 和蜂窝系统。尽管这项研究无疑推动了该领域的发展，但目前还没有 批准的辅助治疗，以减轻认知障碍，影响了相当一部分ART治疗的患者 患者这可能部分是由于临床前模型本身，其可能不完全建模 人脑细胞的异质性、组织和特性。我们建议采取以下措施， 建立人脑切片培养模型，研究HIV神经发病机制。提供了人体组织， 通过与一位神经外科医生同事的合作，其中包括成年正常人 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） 从捐献者的血液样本中，用荧光HIV-1感染他们，并将感染的MDM暴露给患者- 与HIV阴性的脑组织培养结果相匹配在大脑切片感染建立后，艾滋病毒复制将被阻止。 用相关浓度的标准治疗ART进行控制。这种人类模型可以更好地概括 认为治疗患者中存在HAND的病理学特征，包括适当CNS感染 细胞类型，轻微的脑炎症和受损的神经传递，并作为高度相关的模型 该系统用于未来对neuroHIV的研究和神经保护策略的临床前测试。目标1将优化 脑切片培养条件，并检查神经元结构和功能（和神经炎症）， 文化的生命。将在具有不同培养基和切片厚度的切片培养物中评估细胞活力 通过纵向活体染色方法和流式细胞术。神经元活动也将纵向 用多电极阵列或钙成像测量，树突分支和棘的变化将被 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 也可以使用钙信号和谷氨酸探针进行研究。目标2将检查优化的切片培养物 感染患者匹配的HIV+ MDM并接受ART治疗。感染进展将通过共聚焦显微镜跟踪。 荧光HIV成像、流式细胞术和HIV p24 Alphalisa。HIV的炎症环境- 将使用流式细胞术和Alphalisa评估ART前和后感染/未感染的切片， 炎症介质和营养因子。ART治疗的HIV+脑切片中的神经元状态将被 用目标1中列出的相同技术测定。该项目的完成将奠定基础， 一种基于人体组织的模型系统，它在很大程度上保持了成年人的组成和局部连通性 大脑，反映了当今患者的病毒抑制病理学，并有助于验证来自 动物模型该模型可以促进对病毒在CNS中的持续存在、神经发病机制和 神经保护机制、药物作用或毒性机制以及合并症研究。

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.