Division of Cell Biology, Faculty of Health Sciences, Department of Human Biology, University of Cape Town, Cape Town, South Africa.
Faculty of Health Sciences, Neuroscience Institute, University of Cape Town, Groote Schuur Hospital, Cape Town, South Africa.
Methods Mol Biol. 2023;2667:31-45. doi: 10.1007/978-1-0716-3199-7_3.
Cryptococcal meningitis affects millions of people worldwide and is especially prevalent in regions with a high burden of HIV/AIDS. The study of the pathophysiology of this often fatal disease has been significantly hindered by the lack of reliable experimental models, especially at the level of the brain, which is the main organ of injury. Here we outline our novel protocol for the use of hippocampal organotypic brain slice cultures (HOCs) to study the host-fungal interactions during cryptococcal infections of the brain. HOCs are a powerful platform for investigating neuroimmune interactions as they allow for the preservation of all innate neuroglial cells including microglia, astrocytes, and neurons, all of which maintain their three-dimensional architecture and functional connectivity. We made HOCs from neonatal mice and infected these with a fluorescent strain of Cryptococcus neoformans for 24 h. Using immunofluorescent staining, we confirmed the presence and morphology of microglia, astrocytes, and neurons in HOCs prior to infection. Using fluorescent and light microscopy, we also confirmed that Cryptococcus neoformans encapsulates and buds in vitro, as it would in a host. Finally, we demonstrate that infection of HOCs with Cryptococcus neoformans results in close association of the fungal cells with host microglial cells. Our results demonstrate the utility of HOCs as a model to study the pathophysiology and host neuroimmune responses in neurocryptococcosis, which may assist in improving our collective understanding of the pathogenesis of this disease.
隐球菌性脑膜炎影响着全球数百万人，在艾滋病毒/艾滋病负担沉重的地区尤为普遍。由于缺乏可靠的实验模型，特别是在大脑这一主要损伤器官的水平上，这种经常致命疾病的病理生理学研究受到了极大的阻碍。在这里，我们概述了我们使用海马器官型脑片培养物（HOC）研究大脑隐球菌感染过程中宿主-真菌相互作用的新方案。HOC 是研究神经免疫相互作用的有力平台，因为它允许保留所有先天神经胶质细胞，包括小胶质细胞、星形胶质细胞和神经元，所有这些细胞都保持其三维结构和功能连接。我们从小鼠新生儿中制备 HOC，并将其感染荧光标记的新型隐球菌 24 小时。通过免疫荧光染色，我们在感染前确认了 HOC 中小胶质细胞、星形胶质细胞和神经元的存在和形态。通过荧光和光学显微镜，我们还证实了新型隐球菌在体外包裹和出芽，就像在宿主中一样。最后，我们证明了 HOC 被新型隐球菌感染会导致真菌细胞与宿主小胶质细胞密切关联。我们的结果表明，HOC 可用作研究神经隐球菌病病理生理学和宿主神经免疫反应的模型，这可能有助于提高我们对这种疾病发病机制的集体理解。
