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Mouse Organotypic Brain Slice Cultures: A Novel Model for Studying Neuroimmune Responses to Cryptococcal Brain
Amalia N Awala1,2,3,4, Maahir Kauchali2,3,4, Anja de Lange1,2,3,4
1Division of Cell Biology, Faculty of Health Sciences, Department of Human Biology, University of Cape Town, Cape Town, South Africa.
Abstract:
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.
Insights
Hippocampal organotypic brain slice cultures (HOCs) offer a novel model for studying cryptococcal meningitis. This research demonstrates HOCs effectively model host-fungal interactions in the brain, aiding neurocryptococcosis research.
Area of Science:
- Neuroscience
- Infectious Diseases
- Immunology
Background:
- Cryptococcal meningitis is a major global health threat, particularly in HIV/AIDS populations.
- Studying brain pathophysiology is crucial but limited by inadequate experimental models.
- Neuroimmune interactions are central to cryptococcal meningitis pathogenesis.
Purpose of the Study:
- To introduce and validate hippocampal organotypic brain slice cultures (HOCs) as a model for neurocryptococcosis.
- To investigate host-fungal interactions within a preserved brain microenvironment.
- To facilitate the study of neuroimmune responses in cryptococcal brain infections.
Main Methods:
- Preparation of hippocampal organotypic brain slice cultures (HOCs) from neonatal mice.
- Infection of HOCs with a fluorescent strain of Cryptococcus neoformans.
- Analysis using immunofluorescent staining, fluorescent microscopy, and light microscopy.
Main Results:
- HOCs preserve key neuroglial cells (microglia, astrocytes, neurons) with intact architecture and connectivity.
- Cryptococcus neoformans demonstrated characteristic encapsulation and budding in vitro within HOCs.
- Infection led to close association between fungal cells and host microglial cells in HOCs.
Conclusions:
- HOCs represent a valuable and viable model for studying neurocryptococcosis.
- This model system aids in understanding the pathophysiology and host neuroimmune responses to brain cryptococcal infections.
- The HOC model can advance research into the pathogenesis of cryptococcal meningitis.
