Flow of cerebrospinal fluid (CSF) through perivascular spaces (PVSs) in the brain is important for clearance of metabolic waste. Arterial pulsations are thought to drive flow, but this has never been quantitatively shown. We used particle tracking to quantify CSF flow velocities in PVSs of live mice. CSF flow is pulsatile and driven primarily by the cardiac cycle. The speed of the arterial wall matches that of the CSF, suggesting arterial wall motion is the principal driving mechanism, via a process known as perivascular pumping. Increasing blood pressure leaves the artery diameter unchanged but changes the pulsations of the arterial wall, increasing backflow and thereby reducing net flow in the PVS. Perfusion-fixation alters the normal flow direction and causes a 10-fold reduction in PVS size. We conclude that particle tracking velocimetry enables the study of CSF flow in unprecedented detail and that studying the PVS in vivo avoids fixation artifacts.

Stroke affects millions each year. Poststroke brain edema predicts the severity of eventual stroke damage, yet our concept of how edema develops is incomplete and treatment options remain limited. In early stages, fluid accumulation occurs owing to a net gain of ions, widely thought to enter from the vascular compartment. Here, we used magnetic resonance imaging, radiolabeled tracers, and multiphoton imaging in rodents to show instead that cerebrospinal fluid surrounding the brain enters the tissue within minutes of an ischemic insult along perivascular flow channels. This process was initiated by ischemic spreading depolarizations along with subsequent vasoconstriction, which in turn enlarged the perivascular spaces and doubled glymphatic inflow speeds. Thus, our understanding of poststroke edema needs to be revised, and these findings could provide a conceptual basis for development of alternative treatment strategies.

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SUMMARY The emerging interest in brain fluid transport has prompted a need for techniques that provide an understanding of what factors regulate cerebrospinal fluid (CSF) production. Here, we describe a methodology for direct quantification of CSF production in awake mice. We measure CSF production by placing a catheter in a lateral ventricle, while physically blocking outflow from the 4 th ventricle. Using this methodology, we show that CSF production increases during isoflurane anesthesia, and to a lesser extent with ketamine/xylazine anesthesia, relative to the awake state. Aged mice have reduced CSF production, which is even lower in aged mice overexpressing amyloid-β. Unexpectedly, CSF production in young female mice is 30% higher than in age-matched males. Altogether, the present observations imply that a reduction in CSF production might contribute to the age-related risk of proteinopathies but that the rate of CSF production and glymphatic fluid transport are not directly linked.

The G-protein coupled, protease-activated receptor 1 (PAR1) is a membrane protein expressed in astrocytes. Fine astrocytic processes are in tight contact with neurons and blood vessels and shape excitatory synaptic transmission due to their abundant expression of glutamate transporters. PAR1 is proteolytically-activated by bloodstream serine proteases also involved in the formation of blood clots. PAR1 activation has been suggested to play a key role in pathological states like thrombosis, hemostasis and inflammation. What remains unclear is whether PAR1 activation also regulates glutamate uptake in astrocytes and how this shapes excitatory synaptic transmission among neurons. Here we show that, in the mouse hippocampus, PAR1 activation induces a rapid structural re-organization of the neuropil surrounding glutamatergic synapses, which is associated with faster clearance of synaptically-released glutamate from the extracellular space. This effect can be recapitulated using realistic 3D Monte Carlo reaction-diffusion simulations, based on axial scanning transmission electron microscopy (STEM) tomography reconstructions of excitatory synapses. The faster glutamate clearance induced by PAR1 activation leads to short- and long-term changes in excitatory synaptic transmission. Together, these findings identify PAR1 as an important regulator of glutamatergic signaling in the hippocampus and a possible target molecule to limit brain damage during hemorrhagic stroke.

Cerebral edema develops after anoxic brain injury. In two models of asphyxial and asystolic cardiac arrest without resuscitation, we found that edema develops shortly after anoxia secondary to terminal depolarizations and the abnormal entry of cerebrospinal fluid (CSF). Edema severity correlated with the availability of CSF with the age-dependent increase in CSF volume worsening the severity of edema. Edema was identified primarily in brain regions bordering CSF compartments in mice and humans. The degree of ex vivo tissue swelling was predicted by an osmotic model suggesting that anoxic brain tissue possesses a high intrinsic osmotic potential. This osmotic process was temperature-dependent, proposing an additional mechanism for the beneficial effect of therapeutic hypothermia. These observations show that CSF is a primary source of edema fluid in anoxic brain. This novel insight offers a mechanistic basis for the future development of alternative strategies to prevent cerebral edema formation after cardiac arrest.

Perivascular spaces (PVS) drain brain waste metabolites, but their specific flow paths are debated. Meningeal pia mater reportedly forms the outermost boundary that confines flow around blood vessels. Yet, we show that pia is perforated and permissive to PVS fluid flow. Furthermore, we demonstrate that pia is comprised of vascular and cerebral layers that coalesce in variable patterns along leptomeningeal arteries, often merging around penetrating arterioles. Heterogeneous pial architectures form variable sieve-like structures that differentially influence cerebrospinal fluid (CSF) transport along PVS. The degree of pial coverage correlates with macrophage density and phagocytosis of CSF tracer. In vivo imaging confirms transpial influx of CSF tracer, suggesting a role of pia in CSF filtration, but not flow restriction. Additionally, pial layers atrophy with age. Old mice also exhibit areas of pial denudation that are not observed in young animals, but pia is unexpectedly hypertrophied in a mouse model of Alzheimer’s disease. Moreover, pial thickness correlates with improved CSF flow and reduced β-amyloid deposits in PVS of old mice. We show that PVS morphology in mice is variable and that the structure and function of pia suggests a previously unrecognized role in regulating CSF transport and amyloid clearance in aging and disease.

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