Characterisation of the leptomeningeal cells in vitro under conditions that mimic cerebrospinal fluid flow in healthy and diseased states

Leptomeningeal cells form the arachnoid and pia mater layers of the meninges and play a crucial role in maintaining and protecting the brain. Traditionally viewed as passive structural components, leptomeningeal cells are now recognised for their active contributions to CNS homeostasis and involvement in neurodegenerative diseases. Leptomeningeal cells interact with cerebrospinal fluid within the subarachnoid space, collectively clearing waste and maintaining homeostasis. Significant knowledge gaps remain regarding how leptomeningeal cells interact with cerebrospinal fluid, especially in terms of mechanotransductive responses to the dynamic flow of cerebrospinal fluid in healthy and diseased states. However, established in vitro models to study these cells in isolation are lacking, particularly models that replicate the effects of flow and disease conditions on leptomeningeal cells. This thesis aims to advance the understanding of leptomeningeal cells' response to flow and refine in vitro models for the study of these cells.

First, commercially obtained human leptomeningeal cells were characterised. The leptomeningeal cells’ morphology, protein expression, and barrier properties were examined under in vitro culture conditions. The cells were found to express both pial and arachnoidal markers, suggesting that these cells possess characteristics of both layers in vitro and can therefore serve as a representative model for overall leptomeningeal cell behaviour in experimental studies. Furthermore, the results revealed that cultured leptomeningeal cells exhibit altered phenotypes compared to their in vivo counterparts and weak barrier formation in culture, indicating that more in vivo representative culture conditions, such as the application of flow, are necessary.

Oscillatory shear stresses of 8 mPa, 50 mPa, and 100 mPa, designed to mimic in vivo cerebrospinal fluid flow, were applied to the leptomeningeal cells. The results showed that while flow generally reduced cellular stress and inflammation, low shear stress of 8 mPa particularly stabilised cellular behaviour compared to static conditions. Additionally, at the 8 mPa shear stress, leptomeningeal cells exhibited enhanced barrier properties, with transendothelial electrical resistivity values of 119 Ω.cm² compared to 38 Ω.cm² in static conditions. These findings emphasise the importance of replicating physiological conditions to achieve more representative in vitro models of leptomeningeal cells in both healthy and diseased states.

Accordingly, leptomeningeal cells were examined in an Alzheimer's disease model under both static and oscillatory flow conditions. The cells exhibited distinct responses under each condition, with the flow condition producing a more physiologically representative response, including cytokine expressions (e.g., IL-15, IL-33) typically associated with amyloid-beta exposure. Moreover, conditioned media from amyloid-beta-treated leptomeningeal cells altered S100β expression in astrocytes, suggesting that leptomeningeal cells can influence the behaviour of surrounding cells in the central nervous system, particularly in neurodegenerative conditions like Alzheimer's disease. This interaction could be crucial in understanding how Alzheimer's disease progresses and may offer new therapeutic targets by disrupting harmful cell-to-cell communications, further underscoring the importance of developing representative models for future studies.

Overall, this research enhances our understanding of the interaction between leptomeningeal cells and cerebrospinal fluid and establishes that leptomeningeal cells adapt to flow-induced shear stress, which significantly influences their behaviour in both healthy and diseased states.