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The endothelial cells that form the blood–brain barrier (BBB) are coated with glycocalyx, on the luminal side, and with the basement membrane and astrocyte endfeet, on the abluminal side. However, it is unclear how exactly the glycocalyx and extravascular structures contribute to BBB properties. We used two-photon microscopy in anesthetized mice to record passive transport of four different-sized molecules—sodium fluorescein (376 Da), Alexa Fluor (643 Da), 40-kDa dextran, and 150-kDa dextran—from blood to brain, at the level of single cortical capillaries. Both fluorescein and Alexa penetrated nearly the entire glycocalyx volume, but the dextrans penetrated less than 60% of the volume. This suggested that the glycocalyx was a barrier for large but not small molecules. The estimated permeability of the endothelium was the same for fluorescein and Alexa but several-fold lower for the larger dextrans. In the extravascular compartment, co-localized with astrocyte endfeet, diffusion coefficients of the dyes were an order of magnitude lower than in the brain parenchyma. This suggested that the astrocyte endfeet and basement membrane also contributed to BBB properties. In conclusion, the passive transport of small and large hydrophilic molecules through the BBB was determined by three separate barriers: the glycocalyx, the endothelium, and the extravascular compartment. All three barriers must be taken into account in drug delivery studies and when considering BBB dysfunction in disease states.

The blood–brain barrier (BBB), built by brain endothelial cells (BECs), is impermeable to biologics. Liposomes and other nanoparticles are good candidates for the delivery of biologics across the BECs, as they can encapsulate numerous molecules of interest in an omnipotent manner. The liposomes need attachment of a targeting molecule, as BECs unfortunately are virtually incapable of uptake of non-targeted liposomes from the circulation. Experiments of independent research groups have qualified antibodies targeting the transferrin receptor as superior for targeted delivery of nanoparticles to BECs. Functionalization of nanoparticles via conjugation with anti-transferrin receptor antibodies leads to nanoparticle uptake by endothelial cells of both brain capillaries and post-capillary venules. Reducing the density of transferrin receptor-targeted antibodies conjugated to liposomes limits uptake in BECs. Opposing the transport of nanoparticles conjugated to high-affine anti-transferrin receptor antibodies, lowering the affinity of the targeting antibodies or implementing monovalent antibodies increase uptake by BECs and allows for further transport across the BBB. The novel demonstration of transport of targeted liposomes in post-capillary venules from blood to the brain is interesting and clearly warrants further mechanistic pursuit. The recent evidence for passing targeted nanoparticles through the BBB shows great promise for future drug delivery of biologics to the brain.

Functional neuroimaging, such as fMRI, is based on coupling neuronal activity and accompanying changes in cerebral blood flow (CBF) and metabolism. However, the relationship between CBF and events at the level of the penetrating arterioles and capillaries is not well established. Recent findings suggest an active role of capillaries in CBF control, and pericytes on capillaries may be major regulators of CBF and initiators of functional imaging signals. Here, using two-photon microscopy of brains in living mice, we demonstrate that stimulation-evoked increases in synaptic activity in the mouse somatosensory cortex evokes capillary dilation starting mostly at the first- or second-order capillary, propagating upstream and downstream at 5–20 μm/s. Therefore, our data support an active role of pericytes in cerebrovascular control. The gliotransmitter ATP applied to first- and second-order capillaries by micropipette puffing induced dilation, followed by constriction, which also propagated at 5–20 μm/s. ATP-induced capillary constriction was blocked by purinergic P2 receptors. Thus, conducted vascular responses in capillaries may be a previously unidentified modulator of cerebrovascular function and functional neuroimaging signals.

Cortical spreading depression (CSD) is associated with a dramatic failure of brain ion homeostasis and increased energy metabolism. There is strong clinical and experimental evidence to suggest that CSD is the mechanism of migraine, and involved in progressive neuronal injury in stroke and head trauma. Here we tested the hypothesis that single episodes of CSD induced acute hypoxia, and prolonged impairment of neurovascular and neurometabolic coupling. Cortical spreading depression was induced in rat frontal cortex, whereas cortical electrical activity and local field potentials (LFPs) were recorded by glass microelectrodes, cerebral blood flow (CBF) by laser—Doppler flowmetry, and tissue oxygen tension (tpO2) with Polarographic microelectrodes. Cortical spreading depression increased cerebral metabolic rate of oxygen (CMRO2) by 71% ± 6.7% and CBF by 238% ± 48.1% for 1 to 2 mins. For the following 2 h, basal tpO2 and CBF were reduced whereas basal CMRO2 was persistently elevated by 8.1% ± 2.9%. In addition, within first hour after CSD we found impaired neurovascular coupling (LFP versus CBF), whereas neurometabolic coupling (LFP versus CMRO2) remained unaffected. Impaired neurovascular coupling was explained by both reduced vascular reactivity and suppressed function of cortical inhibitory interneurons. The protracted effects of CSD on basal CMRO2 and neurovascular coupling may contribute to cellular dysfunction in patients with migraine and acutely injured cerebral cortex.

Maintaining brain fluid homeostasis is of critical importance for creating a stable environment conducive to optimal neuronal functioning, nutrient distribution, and waste product removal. In this study, we employed previously published data on brain oxygen and glucose consumption in awake rodents or humans to quantify the metabolic water production associated with distinct pathways of glucose metabolism. It is predicted that neuronal mitochondria are the primary source of metabolic water at rest, resulting in a continuous efflux into the cytosol, interstitial fluid, and cerebrospinal fluid. Net metabolic water production is predicted to be reduced by increases in activity due to a shift in metabolism from glucose oxidation to include glycolysis in neurons and ATP hydrolysis by the major cation pumps, which involves water consumption (ATP + H 2 O → ADP + Pi). In comparison, glycogenolysis, which occurs concurrently with the activation of astrocytes, potentially represents a major but previously unidentified contributor to metabolic water. Metabolic water production is dependent on the state of the brain, with a reduction of 30–40% occurring during deep sleep. Our estimates indicate that metabolic water functions as a conduit for interstitial fluid production within the brain, enabling flexible and efficient distribution of fluid that flows seamlessly from the parenchyma to the subarachnoid space and lymphatic vessels to facilitate the removal of brain waste, independent of the glymphatic system.

Cortical spreading depression (CSD) and depolarization waves are associated with dramatic failure of brain ion homeostasis, efflux of excitatory amino acids from nerve cells, increased energy metabolism and changes in cerebral blood flow (CBF). There is strong clinical and experimental evidence to suggest that CSD is involved in the mechanism of migraine, stroke, subarachnoid hemorrhage and traumatic brain injury. The implications of these findings are widespread and suggest that intrinsic brain mechanisms have the potential to worsen the outcome of cerebrovascular episodes or brain trauma. The consequences of these intrinsic mechanisms are intimately linked to the composition of the brain extracellular microenvironment and to the level of brain perfusion and in consequence brain energy supply. This paper summarizes the evidence provided by novel invasive techniques, which implicates CSD as a pathophysiological mechanism for this group of acute neurological disorders. The findings have implications for monitoring and treatment of patients with acute brain disorders in the intensive care unit. Drawing on the large body of experimental findings from animal studies of CSD obtained during decades we suggest treatment strategies, which may be used to prevent or attenuate secondary neuronal damage in acutely injured human brain cortex caused by depolarization waves.

A Comment to our recent paper that described a budget for brain metabolic water production claimed that all ATP produced by oxidation of glucose is consumed by hydrolysis, and that the net calculated production of metabolic water is equal to that obtained by combustion of glucose. However, ATP is synthesized and consumed by enzymatic reactions that do not involve water in the mechanism. Not all ATP consumed is hydrolyzed.

Increased neuron and astrocyte activity triggers increased brain blood flow, but controversy exists over whether stimulation-induced changes in astrocyte activity are rapid and widespread enough to contribute to brain blood flow control. Here, we provide evidence for stimulus-evoked Ca ²⁺ elevations with rapid onset and short duration in a large proportion of cortical astrocytes in the adult mouse somatosensory cortex. Our improved detection of the fast Ca ²⁺ signals is due to a signal-enhancing analysis of the Ca ²⁺ activity. The rapid stimulation-evoked Ca ²⁺ increases identified in astrocyte somas, processes, and end-feet preceded local vasodilatation. Fast Ca ²⁺ responses in both neurons and astrocytes correlated with synaptic activity, but only the astrocytic responses correlated with the hemodynamic shifts. These data establish that a large proportion of cortical astrocytes have brief Ca ²⁺ responses with a rapid onset in vivo, fast enough to initiate hemodynamic responses or influence synaptic activity.

Active nerve cells release vasodilators that increase their energy supply by dilating local blood vessels, a mechanism termed neurovascular coupling and the basis of BOLD functional neuroimaging signals. Here, we reveal a mechanism for cerebral blood flow control, a precapillary sphincter at the transition between the penetrating arteriole and first order capillary, linking blood flow in capillaries to the arteriolar inflow. The sphincters are encircled by contractile mural cells, which are capable of bidirectional control of the length and width of the enclosed vessel segment. The hemodynamic consequence is that precapillary sphincters can generate the largest changes in the cerebrovascular flow resistance of all brain vessel segments, thereby controlling capillary flow while protecting the downstream capillary bed and brain tissue from adverse pressure fluctuations. Cortical spreading depolarization constricts sphincters and causes vascular trapping of blood cells. Thus, precapillary sphincters are bottlenecks for brain capillary blood flow. Precapillary sphincters are mural cells encircling an indentation of blood vessels where capillaries branch off from penetrating arterioles (PAs), but their existence and role in the brain is not fully understood. Here authors describe these structures at PAs in the cortex and show that they constrict during cortical spreading depolarization in mice.

The blood-brain barrier (BBB) is formed by the endothelial cells lining cerebral microvessels, but how blood-borne signaling molecules influence permeability is incompletely understood. We here examined how the apolipoprotein M (apoM)-bound sphingosine 1–phosphate (S1P) signaling pathway affects the BBB in different categories of cerebral microvessels using ApoM deficient mice (Apom-/-). We used two-photon microscopy to monitor BBB permeability of sodium fluorescein (376 Da), Alexa Fluor (643 Da), and fluorescent albumin (45 kDA). We show that BBB permeability to small molecules increases in Apom-/- mice. Vesicle-mediated transfer of albumin in arterioles increased 3 to 10-fold in Apom-/- mice, whereas transcytosis in capillaries and venules remained unchanged. The S1P receptor 1 agonist SEW2871 rapidly normalized paracellular BBB permeability in Apom-/- mice, and inhibited transcytosis in penetrating arterioles, but not in pial arterioles. Thus, apoM-bound S1P maintains low paracellular BBB permeability in all cerebral microvessels and low levels of vesicle-mediated transport in penetrating arterioles.