THREE-DIMENSIONAL BRAIN MAPPING REVEALS PERIVASCULAR AGGREGATION OF CRYPTOCOCCAL CELLS AND GLYMPHATIC DYSFUNCTION IN MURINE CRYPTOCOCCAL MENINGITIS

Cryptococcal meningitis (CM) is an invasive fungal infection of the brain associated with severe neurological complications including disruption of intracranial fluid dynamics. Clinical imaging commonly reveals dilated perivascular spaces (PVS), but the anatomical and functional basis of these abnormalities remains unclear. Here, we examined the spatial dissemination of cryptococcal cells during infection, its relationship to neurovascular architecture and fluid transport pathways. Twenty-four C57BL/6 mice were intravenously injected with either 5x10⁴ Cryptococcus neoformans, Cryptococcus gattii, or saline. Optically cleared whole brains were imaged using light-sheet fluorescent microscopy to enable three-dimensional reconstruction of fungal distribution relative to the cerebral vasculature. Three-dimensional segmentations of the vascular network were generated, and quantitative distance mapping was applied to measure the proximity of individual fungal cells to the nearest vessel. Glymphatic transport was assessed by intracisternal infusion of a fluorescent ovalbumin tracer, with brain-wide tracer penetration quantified across serial coronal sections. We observed a multi-compartmental dissemination across the vascular, perivascular and parenchymal compartments, with significant aggregation in close proximity to the vasculature. Fungal dissemination was associated with impaired brain-wide tracer influx, reflected by reduced tracer penetration and increased resistance to intracisternal infusion in infected animals. Our findings provide the first experimental evidence of glymphatic disruption during CM, potentially arising from occlusion of the PVS by aggregated cryptococcal cells. This mechanism provides insights into how fungal neuroinvasion may drive downstream neurofluid complications such as hydrocephalus and elevated intracranial pressure.