Dysfunctional glymphatic system with disrupted aquaporin-4 expression pattern on astrocytes causes bacterial product accumulation in the CSF during pneumococcal meningitis

Abstract

BACKGROUND: Pneumococcal meningitis, inflammation of the meninges due to an infection of the Central Nervous System caused by Streptococcus pneumoniae (the pneumococcus), is the most common form of community-acquired bacterial meningitis globally. The brain is separated from the systemic circulation by the blood-brain barrier (BBB), and meningitis triggers the host immune response increasing the BBB permeability, allowing peripheral immune cells to reach the cerebrospinal fluid (CSF), and increasing debris production. The glymphatic system is a glial-dependent clearance pathway that drives the exchange of compounds between the brain parenchyma and the CSF regulating the waste clearance away from the brain. Aquaporin-4 (AQP4)-water channels on astrocytic end feet regulate the solute transport of the glymphatic system. METHODS AND RESULTS: Wistar rats, either subjected to pneumococcal meningitis or to artificial-CSF (sham), received Evans blue albumin (EBA) intracisternal. Overall, the meningitis group presented a significant impairment of the glymphatic system by retaining the EBA in the brain without consistently releasing the EBA into the bloodstream compared to the sham non-infected group. Through western blot and immunofluorescence microscopy analysis using rat CSF and brain tissue sections, an increased accumulation of pneumococci was detected over time in the CSF, and because of a loss of drainage between CSF and brain interstitial space, such bacterial accumulation was not observed in the brain parenchyma. Western blot analysis for Iba1, TMEM119 and IFN-Ɣ in rat brain homogenates and NSE in serum showed increased neuroinflammation and neuronal damage in the brain over time during pneumococcal infection. Neurological impairment upon neuronal cell damage caused by meningitis with a malfunctioning glymphatic system was also demonstrated through open-field behavioral tests comparing rats from sham and meningitis groups. Lastly, protein expression analysis of AQP4 revealed no differences in AQP4 between the brains of the rats from the meningitis group and those from the sham non-infected rats. Importantly, confocal microscopy analysis showed a detachment of the astrocytic end feet from the BBB vascular endothelium with consequent misplacement of AQP4-water channels. CONCLUSIONS: These findings clearly indicate that pneumococcal meningitis decreases the glymphatic system's functionality, increasing the neurotoxic waste debris in the brain ultimately leading to brain-wide neuroinflammation and neuronal damage. Finally, our results clearly showed that during pneumococcal meningitis, the glymphatic system does not function because of a detachment of the astrocytic end feet from the BBB vascular endothelium, which leads to a misplacement of AQP4 with consequent the loss of the AQP4-water channel's functionality.