"Study of the Neurovascular Unit (NVU) of the mouse brain after LPS-induced systemic inflammation"

Abstract

The blood-brain barrier (BBB) is a highly selective barrier for the transport of substances between blood circulation and the central nervous system. Three cellular components of the brain microvasculature compose the BBB – the endothelial cells, the astrocyte end-feet and the pericytes (PCs). Neuroinflammation is a common pathological event observed in many different brain diseases, frequently associated with BBB dysfunction and also, characterized by microglia activation and extensive astrogliosis. Astrocytes express aquaporin 4 (AQP4), the water channel protein, involved in water homeostasis. Recent studies have started correlating neuroinflammation and BBB leakage with AQP4 channels dysregulation.Introduction: The blood-brain barrier (BBB) is a highly selective barrier for the transport of substances between blood circulation and the central nervous system. Three cellular components of the brain microvasculature compose the BBB – the endothelial cells, the astrocyte end-feet and the pericytes (PCs). Neuroinflammation is a common pathological event observed in many different brain diseases, frequently associated with BBB dysfunction and also, characterized by microglia activation and extensive astrogliosis. Astrocytes express aquaporin 4 (AQP4), the water channel protein, involved in water homeostasis. Recent studies have started correlating neuroinflammation and BBB leakage with AQP4 channels dysregulation.

Aim of this study is to visualize in real time the interactions between, blood vessels, microglia and perivascular astrocytes, under physiological conditions and following LPS-systemic inflammation, using both confocal microscopy to observe the localization of AQP4 participating in BBB formation and 2-photon laser scanning microscopy (2P-LSM) intravital imaging, study in real time in whole anaesthetised mice BBB leakage and dynamic interactions of microglia with blood vessels under physiological and neuroinflammatory conditions at certain time periods upon LPS administration.

Systemic administration of two doses of 5 mg/kg LPS is performed in transgenic CX3CR1-EGFP mice expressing fluorescent protein specifically in microglia in order to follow the attachment/detachment of microglial cells to brain vasculature following LPS administration. In parallel to intravital imaging C57/BL6 mice are injected with the same doses of LPS, in order to study the localization of AQP4 in perivascular astrocytes and BBB-endothelial cells respectively in fixed brain tissue. Finally, oral administration of a Chemical Factor PLX-X is performed in C57BL6 mice, in order to eliminate microglial cells and to study the topology, morphology and activation of astrocytes upon PLX-X and LPS injection.

Our first data demonstrates a translocation of AQP4 from astrocytic end-feet attached to the blood vessels to the astrocytic processes, following LPS administration. Also, our first data from in vivo imaging shows, that the contact of microglia with the blood vessels exhibits a 2-fold increase upon LPS administration. Finally, our first data indicates that Chemical Factor PLX-X administration induces a 90% elimination of microglia cells and also, these data shows microglia proliferation and astrocytes’ delayed activation upon PLX-X/LPS administration. The microglia proliferation and the level of astrocytic activation upon PLX-X/LPS treatment are lower than LPS administration on its own.

This study will add new information on the spatiotemporal sequence of events occurring in the neurovascular niche following brain inflammation, which is a response occurring in every neurological disease or trauma of the CNS affecting immune response, neuronal activity and survival mechanisms.

TSIOTI Ioanna