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There is renewed interest in how central nervous system (CNS) fluids and barriers contribute to neurological disorders, including cerebral amyloid angiopathy (CAA). Evidence suggests that impaired clearance of amyloid-β (Aβ) plays a role in CAA formation, highlighting the need to better understand brain fluid clearance mechanisms. Nonetheless, the mechanisms for fluid circulation and solute exchange in the brain remain debated, and many studies overlook the brain’s compartmentalizing barriers. To address this gap, we developed new transgenic mouse models to visualize key CNS barriers.

We have developed two dual-reporter mouse strains: a Vascular reporter (Claudin5-GFP for cerebral vessels, Prox1-tdTom for lymphatics) and a Border reporter (Aqp4-mRuby3 for astrocytic glia limitans, VE-cadherin-GFP for leptomeninges and blood vessels). These two strains were crossed with the ArcAβ transgenic line, yielding two triple-transgenic models of cerebral amyloidosis. We analyzed Aβ deposition at different disease stages and examined solute distribution after parenchymal tracer infusion using ex vivo imaging of decalcified skulls and fixed brains.

Aβ deposition patterns and tracer studies revealed multiple drainage pathways that are dependant on the region of the brain and the size of the solute. These include transport along white matter tracts and across the glia limitans—findings that diverge from the popular “glymphatic” model of perivascular bulk flow. Aβ was found to accumulate at the leptomeninges in a pattern that was not always associated with pial blood vessels. The potential links between Aβ accumulation and changes in barrier integrity are under further investigation.

Our novel reporter models provide powerful tools to study CNS barriers and solute clearance in CAA. These findings offer new insights into alternative drainage mechanisms besides glymphatic flow. The involvement of each pathway upon the onset of Aβ deposition remains to be elucidated and we speculate that certain types of deposition result from the rerouting of clearance due to the failure of other pathways. Future validation with in vivo imaging techniques, such as two-photon microscopy and synchrotron X-ray imaging, will further elucidate the spatial and functional dynamics of fluid movement in the diseased brain.

Brain barriers establish distinct compartments that regulate the accessibility of immune cells and mediators to the brain parenchyma. Under pathological conditions, when barrier integrity is compromised, immune cells and mediators gain access to the CNS parenchyma, contributing to disease progression. For example, in stroke, neutrophils might access the parenchyma and influence the course of the disease.
Two-photon intravital microscopy (2P-IVM) enables the investigation of immune cell dynamics and barrier functions of the leptomeninges and the blood-brain barrier in vivo, offering critical insights into the functions of different immune cell populations. This study aims to unravel the inflammatory response induced by the surgical procedures used for IVM of the brain by studying neutrophil accumulation and the barrier properties of the meninges and the CNS vasculature.

We made use of a newly established border reporter mouse obtained by crossing VE-cadherin-GFP and aquaporin 4 (Aqp4)-mRuby3 knock-in mice. The border reporter mouse allows for visualization of the vascular and leptomeningeal adherens junctions and the glia limitans, respectively. Crossing the border reporter mice with the Catchup mouse (Ly6G-cre, -tdTomato; Ai14), allowed for in vivo neutrophil imaging in the different CNS compartments.

Performing 2P-IVM in the Catchup-border reporter mouse, we found that both skull thinning and cranial window preparations induced a sustained inflammatory response characterized by neutrophil recruitment specifically into the dura mater but not into the leptomeninges or CNS parenchyma. Notably, these interventions seem to also result in the formation of an artificial subdural space—a space that, without visualization of the respective landmarks, may be misinterpreted as subarachnoid space.

Our findings underscore the importance of recognizing the inflammatory and structural changes induced by surgical preparations, allowing for brain in vivo imaging and highlighting the need for optimized surgical protocols to minimize sterile inflammation, which is essential for ensuring accurate interpretations in intravital imaging studies of both physiological and pathological states, such as stroke. Moreover, we show that the Catchup-border reporter mouse is a valuable model for investigating neutrophil behavior in homeostasis and pathological and inflammatory conditions.

Blood vessels are remarkably specialized structures that present morphological, functional, and transcriptional heterogeneity. Hence, proper specification into arteries, veins, capillaries and lymphatic vasculature is crucial and under intense investigation. Endothelial cell (EC) metabolism is an important regulator of vessel formation in physiological and pathological conditions. ECs are highly glycolytic and metabolism crucially controls the angiogenic potential of ECs. However, recent single cell studies have also suggested that arterial and venous ECs have different metabolic properties. Even though it has already been suggested that a metabolic arrest precedes arterialization, functional evidence that endothelial metabolism can (co)define arteriovenous specification is so far missing.

We use the postnatal mouse retina as a model system of angiogenesis. Previous studies have shown that the pool of proliferating ECs that expands the growing vascular network derives from venous endothelium. ECs that get exposed to the highest levels of VEGF are subsequently selected to become tip cells, which are specialized, migratory ECs at the growing vessel front. Arterial ECs are specified at the tip cell position from where they migrate into the artery or capillary system, but rarely back into the vein. Using a tip-cell specific Cre line (Esm1-CreERT2) combined with different fate tracing strategies, we are studying the sorting of tip ECs into arteries after PFKFB3 deletion (PFKFB3ΔtipEC) or overexpression (PFKFB3-tip-OE).

PFKFB3 deletion promotes the migration of tip ECs into the arteries in PFKFB3ΔtipEC mice. However, arteries were overall less complex than in WT littermates. Interestingly, when tracing PFKFB3-tip-OE ECs in which one extra copy of HA-tagged PFKFB3 is expressed under the Rosa26 promoter, we found a very low number of HA-tagged ECs, suggesting that PFKFB3 is also downregulated on post-transcriptional level. Indeed, when PFKFB3-tip-OE pups were injected with a proteasome inhibitor (MG132), a significant number of HA-tagged ECs was found in arteries, capillaries and, surprisingly, veins.

Our data suggests that PFKFB3 expression is tightly regulated both on a transcriptional and translational level. This is required for the proper formation of arteries, and sorting of tip-cell derived ECs specifically into the arterial and not venous compartment.