Pia-FLOW: Deciphering hemodynamic maps of the pial vascular connectome and its response to arterial occlusion.

The pial vasculature is the sole source of blood supply to the neocortex. The brain is contained within the skull, a vascularized bone marrow with a unique anatomical connection to the brain meninges. Recent developments in tissue clearing have enabled detailed mapping of the entire pial and calvarial vasculature. However, what are the absolute flow rate values of those vascular networks? This information cannot accurately be retrieved with the commonly used bioimaging methods. Here, we introduce Pia-FLOW, a unique approach based on large-scale transcranial fluorescence localization microscopy, to attain hemodynamic imaging of the whole murine pial and calvarial vasculature at frame rates up to 1,000 Hz and spatial resolution reaching 5.4 µm. Using Pia-FLOW, we provide detailed maps of flow velocity, direction, and vascular diameters which can serve as ground-truth data for further studies, advancing our understanding of brain fluid dynamics. Furthermore, Pia-FLOW revealed that the pial vascular network functions as one unit for robust allocation of blood after stroke.

The role of leptomeningeal collaterals in redistributing blood flow during stroke.

Leptomeningeal collaterals (LMCs) connect the main cerebral arteries and provide alternative pathways for blood flow during ischaemic stroke. This is beneficial for reducing infarct size and reperfusion success after treatment. However, a better understanding of how LMCs affect blood flow distribution is indispensable to improve therapeutic strategies. Here, we present a novel in silico approach that incorporates case-specific in vivo data into a computational model to simulate blood flow in large semi-realistic microvascular networks from two different mouse strains, characterised by having many and almost no LMCs between middle and anterior cerebral artery (MCA, ACA) territories. This framework is unique because our simulations are directly aligned with in vivo data. Moreover, it allows us to analyse perfusion characteristics quantitatively across all vessel types and for networks with no, few and many LMCs. We show that the occlusion of the MCA directly caused a redistribution of blood that was characterised by increased flow in LMCs. Interestingly, the improved perfusion of MCA-sided microvessels after dilating LMCs came at the cost of a reduced blood supply in other brain areas. This effect was enhanced in regions close to the watershed line and when the number of LMCs was increased. Additional dilations of surface and penetrating arteries after stroke improved perfusion across the entire vasculature and partially recovered flow in the obstructed region, especially in networks with many LMCs, which further underlines the role of LMCs during stroke.

Vascular Response to Spreading Depolarization Predicts Stroke Outcome.

BACKGROUND: Cortical spreading depolarization (CSD) is a massive neuro-glial depolarization wave, which propagates across the cerebral cortex. In stroke, CSD is a necessary and ubiquitous mechanism for the development of neuronal lesions that initiates in the ischemic core and propagates through the penumbra extending the tissue injury. Although CSD propagation induces dramatic changes in cerebral blood flow, the vascular responses in different ischemic regions and their consequences on reperfusion and recovery remain to be defined. METHODS: Ischemia was performed using the thrombin model of stroke and reperfusion was induced by r-tPA (recombinant tissue-type plasminogen activator) administration in mice. We used in vivo electrophysiology and laser speckle contrast imaging simultaneously to assess both electrophysiological and hemodynamic characteristics of CSD after ischemia onset. Neurological deficits were assessed on day 1, 3, and 7. Furthermore, infarct sizes were quantified using 2,3,5-triphenyltetrazolium chloride on day 7. RESULTS: After ischemia, CSDs were evidenced by the characteristic propagating DC shift extending far beyond the ischemic area. On the vascular level, we observed 2 types of responses: some mice showed spreading hyperemia confined to the penumbra area (penumbral spreading hyperemia) while other showed spreading hyperemia propagating in the full hemisphere (full hemisphere spreading hyperemia). Penumbral spreading hyperemia was associated with severe stroke-induced damage, while full hemisphere spreading hyperemia indicated beneficial infarct outcome and potential viability of the infarct core. In all animals, thrombolysis with r-tPA modified the shape of the vascular response to CSD and reduced lesion volume. CONCLUSIONS: Our results show that different types of spreading hyperemia occur spontaneously after the onset of ischemia. Depending on their shape and distribution, they predict severity of injury and outcome. Furthermore, our data show that modulating the hemodynamic response to CSD may be a promising therapeutic strategy to attenuate stroke outcome.

Structural insights into the binding and catalytic mechanisms of the Listeria monocytogenes bacteriophage glycosyl hydrolase PlyP40.

Endolysins are bacteriophage-encoded peptidoglycan hydrolases that specifically degrade the bacterial cell wall at the end of the phage lytic cycle. They feature a distinct modular architecture, consisting of enzymatically active domains (EADs) and cell wall-binding domains (CBDs). Structural analysis of the complete enzymes or individual domains is required for better understanding the mechanisms of peptidoglycan degradation and provides guidelines for the rational design of chimeric enzymes. We here report the crystal structure of the EAD of PlyP40, a member of the GH-25 family of glycosyl hydrolases, and the first muramidase reported for Listeria phages. Site-directed mutagenesis confirmed key amino acids (Glu98 and Trp10) involved in catalysis and substrate stabilization. In addition, we found that PlyP40 contains two heterogeneous CBD modules with homology to SH3 and LysM domains. Truncation analysis revealed that both domains are required for full activity but contribute to cell wall recognition and lysis differently. Replacement of CBDP40 with a corresponding domain from a different Listeria phage endolysin yielded an enzyme with a significant shift in pH optimum. Finally, domain swapping between PlyP40 and the streptococcal endolysin Cpl-1 produced an intergeneric chimera with activity against Listeria cells, indicating that structural similarity of individual domains determines enzyme function.

Cortex-wide transcranial localization microscopy with fluorescently labeled red blood cells.

Large-scale imaging of brain activity with high spatio-temporal resolution is crucial for advancing our understanding of brain function. The existing neuroimaging techniques are largely limited by restricted field of view, slow imaging speed, or otherwise do not have the adequate spatial resolution to capture brain activities on a capillary and cellular level. To address these limitations, we introduce fluorescence localization microscopy aided with sparsely-labeled red blood cells for cortex-wide morphological and functional cerebral angiography with 4.9 µm spatial resolution and 1 s temporal resolution. When combined with fluorescence calcium imaging, the proposed method enables extended recordings of stimulus-evoked neuro-vascular changes in the murine brain while providing simultaneous multiparametric readings of intracellular neuronal activity, blood flow velocity/direction/volume, and vessel diameter. Owing to its simplicity and versatility, the proposed approach will become an invaluable tool for deciphering the regulation of cortical microcirculation and neurovascular coupling in health and disease.

A chemogenetic approach for dopamine imaging with tunable sensitivity.

Genetically-encoded dopamine (DA) sensors enable high-resolution imaging of DA release, but their ability to detect a wide range of extracellular DA levels, especially tonic versus phasic DA release, is limited by their intrinsic affinity. Here we show that a human-selective dopamine receptor positive allosteric modulator (PAM) can be used to boost sensor affinity on-demand. The PAM enhances DA detection sensitivity across experimental preparations (in vitro, ex vivo and in vivo) via one-photon or two-photon imaging. In vivo photometry-based detection of optogenetically-evoked DA release revealed that DETQ administration produces a stable 31 minutes window of potentiation without effects on animal behavior. The use of the PAM revealed region-specific and metabolic state-dependent differences in tonic DA levels and enhanced single-trial detection of behavior-evoked phasic DA release in cortex and striatum. Our chemogenetic strategy can potently and flexibly tune DA imaging sensitivity and reveal multi-modal (tonic/phasic) DA signaling across preparations and imaging approaches.

Leptomeningeal collaterals regulate reperfusion in ischemic stroke and rescue the brain from futile recanalization.

Recanalization is the mainstay of ischemic stroke treatment. However, even with timely clot removal, many stroke patients recover poorly. Leptomeningeal collaterals (LMCs) are pial anastomotic vessels with yet-unknown functions. We applied laser speckle imaging, ultrafast ultrasound, and two-photon microscopy in a thrombin-based mouse model of stroke and fibrinolytic treatment to show that LMCs maintain cerebral autoregulation and allow for gradual reperfusion, resulting in small infarcts. In mice with poor LMCs, distal arterial segments collapse, and deleterious hyperemia causes hemorrhage and mortality after recanalization. In silico analyses confirm the relevance of LMCs for preserving perfusion in the ischemic region. Accordingly, in stroke patients with poor collaterals undergoing thrombectomy, rapid reperfusion resulted in hemorrhagic transformation and unfavorable recovery. Thus, we identify LMCs as key components regulating reperfusion and preventing futile recanalization after stroke. Future therapeutic interventions should aim to enhance collateral function, allowing for beneficial reperfusion after stroke.

Ultrasound trapping and navigation of microrobots in the mouse brain vasculature.

The intricate and delicate anatomy of the brain poses significant challenges for the treatment of cerebrovascular and neurodegenerative diseases. Thus, precise local drug delivery in hard-to-reach brain regions remains an urgent medical need. Microrobots offer potential solutions; however, their functionality in the brain remains restricted by limited imaging capabilities and complications within blood vessels, such as high blood flows, osmotic pressures, and cellular responses. Here, we introduce ultrasound-activated microrobots for in vivo navigation in brain vasculature. Our microrobots consist of lipid-shelled microbubbles that autonomously aggregate and propel under ultrasound irradiation. We investigate their capacities in vitro within microfluidic-based vasculatures and in vivo within vessels of a living mouse brain. These microrobots self-assemble and execute upstream motion in brain vasculature, achieving velocities up to 1.5 µm/s and moving against blood flows of ~10 mm/s. This work represents a substantial advance towards the therapeutic application of microrobots within the complex brain vasculature.

Altered hemodynamics and vascular reactivity in a mouse model with severe pericyte deficiency.

Pericytes are the mural cells of the microvascular network that are in close contact with underlying endothelial cells. Endothelial-secreted PDGFB leads to recruitment of pericytes to the vessel wall, but this is disrupted in Pdgfbret/ret mice when the PDGFB retention motif is deleted. This results in severely reduced pericyte coverage on blood vessels. In this study, we investigated vascular abnormalities and hemodynamics in Pdgfbret/ret mice throughout the cerebrovascular network and in different cortical layers by in vivo two-photon microscopy. We confirmed that Pdgfbret/ret mice are severely deficient in pericytes throughout the vascular network, with enlarged brain blood vessels and a reduced number of vessel branches. Red blood cell velocity, linear density, and tube hematocrit were reduced in Pdgfbret/ret mice, which may impair oxygen delivery to the tissue. We also measured intravascular PO2 and found that concentrations were higher in cortical Layer 2/3 in Pdgfbret/ret mice, indicative of reduced blood oxygen extraction. Finally, we found that Pdgfbret/ret mice had a reduced capacity for vasodilation in response to an acetazolamide challenge during functional MRI imaging. Taken together, these results suggest that severe pericyte deficiency can lead to vascular abnormalities and altered cerebral blood flow, reminiscent of pathologies such as arteriovenous malformations.

Deep optoacoustic localization microangiography of ischemic stroke in mice.

Super-resolution optoacoustic imaging of microvascular structures deep in mammalian tissues has so far been impeded by strong absorption from densely-packed red blood cells. Here we devised 5 µm biocompatible dichloromethane-based microdroplets exhibiting several orders of magnitude higher optical absorption than red blood cells at near-infrared wavelengths, thus enabling single-particle detection in vivo. We demonstrate non-invasive three-dimensional microangiography of the mouse brain beyond the acoustic diffraction limit (<20 µm resolution). Blood flow velocity quantification in microvascular networks and light fluence mapping was also accomplished. In mice affected by acute ischemic stroke, the multi-parametric multi-scale observations enabled by super-resolution and spectroscopic optoacoustic imaging revealed significant differences in microvascular density, flow and oxygen saturation in ipsi- and contra-lateral brain hemispheres. Given the sensitivity of optoacoustics to functional, metabolic and molecular events in living tissues, the new approach paves the way for non-invasive microscopic observations with unrivaled resolution, contrast and speed.

Three-dimensional wide-field fluorescence microscopy for transcranial mapping of cortical microcirculation.

Wide-field fluorescence imaging is an indispensable tool for studying large-scale biodynamics. Limited space-bandwidth product and strong light diffusion make conventional implementations incapable of high-resolution mapping of fluorescence biodistribution in three dimensions. We introduce a volumetric wide-field fluorescence microscopy based on optical astigmatism combined with fluorescence source localization, covering 5.6×5.6×0.6 mm3 imaging volume. Two alternative configurations are proposed exploiting multifocal illumination or sparse localization of point emitters, which are herein seamlessly integrated in one system. We demonstrate real-time volumetric mapping of the murine cortical microcirculation at capillary resolution without employing cranial windows, thus simultaneously delivering quantitative perfusion information across both brain hemispheres. Morphological and functional changes of cerebral vascular networks are further investigated after an acute ischemic stroke, enabling cortex-wide observation of concurrent collateral recruitment events occurring on a sub-second scale. The reported technique thus offers a wealth of unmatched possibilities for non- or minimally invasive imaging of biodynamics across scales.

Distinct signatures of calcium activity in brain mural cells.

Pericytes have been implicated in various neuropathologies, yet little is known about their function and signaling pathways in health. Here, we characterized calcium dynamics of cortical mural cells in anesthetized or awake Pdgfrb-CreERT2;Rosa26< LSL-GCaMP6s > mice and in acute brain slices. Smooth muscle cells (SMCs) and ensheathing pericytes (EPs), also named as terminal vascular SMCs, revealed similar calcium dynamics in vivo. In contrast, calcium signals in capillary pericytes (CPs) were irregular, higher in frequency, and occurred in cellular microdomains. In the absence of the vessel constricting agent U46619 in acute slices, SMCs and EPs revealed only sparse calcium signals, whereas CPs retained their spontaneous calcium activity. Interestingly, chemogenetic activation of neurons in vivo and acute elevations of extracellular potassium in brain slices strongly decreased calcium activity in CPs. We propose that neuronal activation and an extracellular increase in potassium suppress calcium activity in CPs, likely mediated by Kir2.2 and KATP channels.

Neutrophils Obstructing Brain Capillaries Are a Major Cause of No-Reflow in Ischemic Stroke.

Despite successful clot retrieval in large vessel occlusion stroke, ∼50% of patients have an unfavorable clinical outcome. The mechanisms underlying this functional reperfusion failure remain unknown, and therapeutic options are lacking. In the thrombin-model of middle cerebral artery (MCA) stroke in mice, we show that, despite successful thrombolytic recanalization of the proximal MCA, cortical blood flow does not fully recover. Using in vivo two-photon imaging, we demonstrate that this is due to microvascular obstruction of ∼20%-30% of capillaries in the infarct core and penumbra by neutrophils adhering to distal capillary segments. Depletion of circulating neutrophils using an anti-Ly6G antibody restores microvascular perfusion without increasing the rate of hemorrhagic complications. Strikingly, infarct size and functional deficits are smaller in mice treated with anti-Ly6G. Thus, we propose neutrophil stalling of brain capillaries to contribute to reperfusion failure, which offers promising therapeutic avenues for ischemic stroke.

Cortical Circuit Activity Evokes Rapid Astrocyte Calcium Signals on a Similar Timescale to Neurons.

Sensory stimulation evokes intracellular calcium signals in astrocytes; however, the timing of these signals is disputed. Here, we used novel combinations of genetically encoded calcium indicators for concurrent two-photon imaging of cortical astrocytes and neurons in awake mice during whisker deflection. We identified calcium responses in both astrocyte processes and endfeet that rapidly followed neuronal events (∼120 ms after). These fast astrocyte responses were largely independent of IP3R2-mediated signaling and known neuromodulator activity (acetylcholine, serotonin, and norepinephrine), suggesting that they are evoked by local synaptic activity. The existence of such rapid signals implies that astrocytes are fast enough to play a role in synaptic modulation and neurovascular coupling. VIDEO ABSTRACT.