Impaired dynamics of precapillary sphincters and pericytes at first-order capillaries predict reduced neurovascular function in the aging mouse brain.

The microvascular inflow tract, comprising the penetrating arterioles, precapillary sphincters and first-order capillaries, is the bottleneck for brain blood flow and energy supply. Exactly how aging alters the structure and function of the microvascular inflow tract remains unclear. By in vivo four-dimensional two-photon imaging, we reveal an age-dependent decrease in vaso-responsivity accompanied by a decrease in vessel density close to the arterioles and loss of vascular mural cell processes, although the number of mural cell somas and their alpha smooth muscle actin density were preserved. The age-related reduction in vascular reactivity was mostly pronounced at precapillary sphincters, highlighting their crucial role in capillary blood flow regulation. Mathematical modeling revealed impaired pressure and flow control in aged mice during vasoconstriction. Interventions that preserve dynamics of cerebral blood vessels may ameliorate age-related decreases in blood flow and prevent brain frailty.

Post-capillary venules are the key locus for transcytosis-mediated brain delivery of therapeutic nanoparticles.

Effective treatments of neurodegenerative diseases require drugs to be actively transported across the blood-brain barrier (BBB). However, nanoparticle drug carriers explored for this purpose show negligible brain uptake, and the lack of basic understanding of nanoparticle-BBB interactions underlies many translational failures. Here, using two-photon microscopy in mice, we characterize the receptor-mediated transcytosis of nanoparticles at all steps of delivery to the brain in vivo. We show that transferrin receptor-targeted liposome nanoparticles are sequestered by the endothelium at capillaries and venules, but not at arterioles. The nanoparticles move unobstructed within endothelium, but transcytosis-mediated brain entry occurs mainly at post-capillary venules, and is negligible in capillaries. The vascular location of nanoparticle brain entry corresponds to the presence of perivascular space, which facilitates nanoparticle movement after transcytosis. Thus, post-capillary venules are the point-of-least resistance at the BBB, and compared to capillaries, provide a more feasible route for nanoparticle drug carriers into the brain.

Precapillary sphincters and pericytes at first-order capillaries as key regulators for brain capillary perfusion.

Rises in local neural activity trigger local increases of cerebral blood flow, which is essential to match local energy demands. However, the specific location of microvascular flow control is incompletely understood. Here, we used two-photon microscopy to observe brain microvasculature in vivo. Small spatial movement of a three-dimensional (3D) vasculature makes it challenging to precisely measure vessel diameter at a single x-y plane. To overcome this problem, we carried out four-dimensional (x-y-z-t) imaging of brain microvessels during exposure to vasoactive molecules in order to constrain the impact of brain movements on the recordings. We demonstrate that rises in synaptic activity, acetylcholine, nitric oxide, cyclic guanosine monophosphate, ATP-sensitive potassium channels, and endothelin-1 exert far greater effects on brain precapillary sphincters and first-order capillaries than on penetrating arterioles or downstream capillaries, but with similar kinetics. The high level of responsiveness at precapillary sphincters and first-order capillaries was matched by a higher level of α-smooth muscle actin in pericytes as compared to penetrating arterioles and downstream capillaries. Mathematical modeling based on 3D vasculature reconstruction showed that precapillary sphincters predominantly regulate capillary blood flow and pressure as compared to penetrating arterioles and downstream capillaries. Our results confirm a key role for precapillary sphincters and pericytes on first-order capillaries as sensors and effectors of endothelium- or brain-derived vascular signals.

In Vivo Three-Dimensional Two-Photon Microscopy to Study Conducted Vascular Responses by Local ATP Ejection Using a Glass Micro-Pipette.

Maintenance of normal brain function requires a sufficient and efficient supply of oxygen and nutrition by a complex network of vessels. However, the regulation of cerebral blood flow (CBF) is incompletely understood, especially at the capillary level. Two-photon microscopy is a powerful tool widely used to study CBF and its regulation. Currently, this field is limited by the lack of in vivo two-photon microscopy studies examining (1) CBF responses in three-dimensions, (2) conducted vascular responses, and (3) localized interventions within the vascular network. Here, we describe a 3D in vivo method using two-photon microscopy to study conducted vascular responses elicited by local ejection of ATP with a glass micro-pipette. Our method uses fast and repetitive hyperstack two-photon imaging providing precise diameter measurements by maximal intensity projection of the obtained images. Furthermore, we show that this method can also be used to study 3D astrocytic calcium responses. We also discuss the advantages and limitations of glass micro-pipette insertion and two-photon hyperstack imaging.

Spontaneous astrocytic Ca2+ activity abounds in electrically suppressed ischemic penumbra of aged mice.

Experimental focal cortical ischemic lesions consist of an ischemic core and a potentially salvageable peri-ischemic region, the ischemic penumbra. The activity of neurons and astrocytes is assumed to be suppressed in the penumbra because the electrical function is interrupted, but this is incompletely elucidated. Most experimental stroke studies used young adult animals, whereas stroke is prevalent in the elderly population. Using two-photon imaging in vivo, we here demonstrate extensive but electrically silent, spontaneous Ca2+ activity in neurons and astrocytes in the ischemic penumbra of 18- to 24-month-old mice 2-4 hr after middle cerebral artery occlusion. In comparison, stroke reduced spontaneous Ca2+ activity in neurons and astrocytes in adult mice (3-4 months of age). In aged mice, stroke increased astrocytic spontaneous Ca2+ activity considerably while neuronal spontaneous Ca2+ activity was unchanged. Blockade of action potentials and of purinergic receptors strongly reduced spontaneous Ca2+ activity in both neurons and astrocytes in the penumbra of old stroke mice. This indicates that stroke had a direct influence on mechanisms in presynaptic terminals and on purinergic signaling. Thus, highly dynamic variations in spontaneous Ca2+ activity characterize the electrically compromised penumbra, with remarkable differences between adult and old mice. The data are consistent with the notion that aged neurons and astrocytes take on a different phenotype than young mice. The increased activity of the aged astrocyte phenotype may be harmful to neurons. We suggest that the abundant spontaneous Ca2+ activity in astrocytes in the ischemic penumbra of old mice may be a novel target for neuroprotection strategies. A video abstract of this article can be found at https://youtu.be/AKlwKFsz1qE.

Stimulation-induced increases in cerebral blood flow and local capillary vasoconstriction depend on conducted vascular responses.

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.

Monitoring of blood oxygenation in brain by resonance Raman spectroscopy.

Blood oxygenation in cerebral vessels is an essential parameter to evaluate brain function and to investigate the coupling between local blood flow and neuronal activity. We apply resonance Raman spectroscopy in vivo to study hemoglobin oxygenation in cortex vessels of anesthetized ventilated mice. We demonstrate that the pairs of Raman peaks at 1355 and1375 cm-1 (symmetric vibrations of pyrrol half-rings in the heme molecule), 1552 and 1585 cm-1 and 1602 and 1638 cm-1 (vibrations of methine bridges in heme molecule) are reliable markers for quantitative estimation of the relative amount of oxyhemoglobin in venules, arterioles, and capillaries. in vivo measurements of blood oxygenation in the cortex of mice ventilated with inspiratory gas mixtures containing different amounts of oxygen-normoxia, hyperoxia and hypoxia-validate the proposed approach. Our method allows to visualize blood saturation with O2 in different microvascular networks.

Fast Ca2+ responses in astrocyte end-feet and neurovascular coupling in mice.

Cerebral blood flow (CBF) is regulated by the activity of neurons and astrocytes. Understanding how these cells control activity-dependent increases in CBF is crucial to interpreting functional neuroimaging signals. The relative importance of neurons and astrocytes is debated, as are the functional implications of fast Ca2+ changes in astrocytes versus neurons. Here, we used two-photon microscopy to assess Ca2+ changes in neuropil, astrocyte processes, and astrocyte end-feet in response to whisker pad stimulation in mice. We also developed a pixel-based analysis to improve the detection of rapid Ca2+ signals in the subcellular compartments of astrocytes. Fast Ca2+ responses were observed using both chemical and genetically encoded Ca2+ indicators in astrocyte end-feet prior to dilation of arterioles and capillaries. A low dose of the NMDA receptor antagonist (5R,10s)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine-hydrogen-maleate (MK801) attenuated fast Ca2+ responses in the neuropil and astrocyte processes, but not in astrocyte end-feet, and the evoked CBF response was preserved. In addition, a low dose of 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), an agonist for the extrasynaptic GABAA receptor (GABAA R), increased CBF responses and the fast Ca2+ response in astrocyte end-feet but did not affect Ca2+ responses in astrocyte processes and neuropil. These results suggest that fast Ca2+ increases in the neuropil and astrocyte processes are not necessary for an evoked CBF response. In contrast, as local fast Ca2+ responses in astrocyte end-feet are unaffected by MK801 but increase via GABAA R-dependent mechanisms that also increased CBF responses, we hypothesize that the fast Ca2+ increases in end-feet adjust CBF during synaptic activity.

Multi-modal assessment of neurovascular coupling during cerebral ischaemia and reperfusion using remote middle cerebral artery occlusion.

Hyperacute changes in cerebral blood flow during cerebral ischaemia and reperfusion are important determinants of injury. Cerebral blood flow is regulated by neurovascular coupling, and disruption of neurovascular coupling contributes to brain plasticity and repair problems. However, it is unknown how neurovascular coupling is affected hyperacutely during cerebral ischaemia and reperfusion. We have developed a remote middle cerebral artery occlusion model in the rat, which enables multi-modal assessment of neurovascular coupling immediately prior to, during and immediately following reperfusion. Male Wistar rats were subjected to remote middle cerebral artery occlusion, where a long filament was advanced intraluminally through a guide cannula in the common carotid artery. Transcallosal stimulation evoked increases in blood flow, tissue oxygenation and neuronal activity, which were diminished by middle cerebral artery occlusion and partially restored during reperfusion. These evoked responses were not affected by administration of the thrombolytic alteplase at clinically used doses. Evoked cerebral blood flow responses were fully restored at 24 h post-middle cerebral artery occlusion indicating that neurovascular dysfunction was not sustained. These data show for the first time that the rat remote middle cerebral artery occlusion model coupled with transcallosal stimulation provides a novel method for continuous assessment of hyperacute neurovascular coupling changes during ischaemia and reperfusion, and offers unique insight into hyperacute ischaemic pathophysiology.

Increased 20-HETE synthesis explains reduced cerebral blood flow but not impaired neurovascular coupling after cortical spreading depression in rat cerebral cortex.

Cortical spreading depression (CSD) is associated with release of arachidonic acid, impaired neurovascular coupling, and reduced cerebral blood flow (CBF), caused by cortical vasoconstriction. We tested the hypothesis that the released arachidonic acid is metabolized by the cytochrome P450 enzyme to produce the vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE), and that this mechanism explains cortical vasoconstriction and vascular dysfunction after CSD. CSD was induced in the frontal cortex of rats and the cortical electrical activity and local field potentials recorded by glass microelectrodes, CBF by laser Doppler flowmetry, and tissue oxygen tension (tpO(2)) using polarographic microelectrodes. 20-HETE synthesis was measured in parallel experiments in cortical brain slices exposed to CSD. We used the specific inhibitor HET0016 (N-hydroxy-N'-(4-n-butyl-2-methylphenyl)formamidine) to block 20-HETE synthesis. CSD increased 20-HETE synthesis in brain slices for 120 min, and the time course of the increase in 20-HETE paralleled the reduction in CBF after CSD in vivo. HET0016 blocked the CSD-induced increase in 20-HETE synthesis and ameliorated the persistent reduction in CBF, but not the impaired neurovascular coupling after CSD. These findings suggest that CSD-induced increments in 20-HETE cause the reduction in CBF after CSD and that the attenuation of stimulation-induced CBF responses after CSD has a different mechanism. We suggest that blockade of 20-HETE synthesis may be clinically relevant to ameliorate reduced CBF in patients with migraine and acute brain cortex injuries.