Adult human brain tissue cultures to study neuroHIV

项目摘要：科学家和临床医生已经研究了艾滋病毒相关的潜在机制 神经认知障碍(HAND)数十年，跨越动物模型的临床试验和临床前研究 和蜂窝系统。虽然这项研究无疑推动了该领域的发展，但仍没有 批准的辅助治疗，以缓解影响ART治疗的相当大一部分的认知障碍 病人。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。我们建议通过以下方式弥合这一差距 建立人脑切片培养模型以研究HIV神经发病机制。提供人体组织 通过与神经外科医生同事的合作从脑外科手术中脱颖而出。这些人包括成年正常人 脑组织、脑脊液和外周血液。我们将分离单核细胞来源的巨噬细胞(MDM) 从捐赠者的血液样本中，用荧光HIV-1感染他们，并将感染的MDM暴露给患者- 与HIV阴性的大脑培养相匹配。在确定脑片感染后，艾滋病毒的复制将 以相应浓度的标准护理技术进行控制。这个人类模型可能会更好地概括 被认为存在于接受治疗的患者的手的病理特征，包括适当的中枢神经系统感染 细胞类型、轻微的脑部炎症和神经传递受损，并作为一个高度相关的模型 未来神经艾滋病毒研究和神经保护策略的临床前测试的系统。目标1将优化 脑片培养条件，并检查神经元结构和功能(和神经炎症)。 文化的生命。在不同的培养介质和切片厚度的切片培养中，将评估细胞的活性。 采用纵向活体染色方法和流式细胞术。神经元的活动也将是纵向的 用多电极阵列或钙成像测量，树突分支和棘突改变 使用DiI染色和NeuroLucida 360软件分析在多个时间点进行监测。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 感染了与患者匹配的艾滋病毒MDM，并接受抗逆转录病毒治疗。将通过共焦跟踪感染进展 HIV荧光成像、流式细胞术和HIV p24 Alphalisa。艾滋病毒的炎性环境- ART前后感染/未感染的切片将使用流式细胞仪和Alphalisa进行评估以选择 炎性介质和营养因子。接受抗逆转录病毒治疗的HIV脑片中的神经元状态将是 用目标1中列出的相同技术确定。该项目的完成将为 以人体组织为基础的模型系统，在很大程度上维持成人的组成和局部连接性 大脑，反映了当今患者中病毒抑制的病理，并有助于验证来自 动物模型。该模型可以促进对病毒在中枢神经系统中持续存在、神经发病机制和 神经保护机制，药物作用或毒性的机制，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.