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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. Utilizing a thrombin-based mouse model of stroke and the gold standard fibrinolytic treatment rt-PA, we here show that LMCs play a critical role in preserving vascular function in ischemic territories. We applied laser speckle contrast imaging, ultrafast ultrasound, and two-photon microscopy, to show that after thrombolysis, LMCs allow for gradual reperfusion resulting in small infarcts. On the contrary, in mice with poor LMCs, distal segments of recanalized arteries collapse and deleterious hyperemia causes hemorrhage and mortality. 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 after stroke. Future therapeutic interventions should aim to enhance collateral function, allowing for gradual reperfusion of ischemic tissues after 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.

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.

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 Pdgfb$^{ret/ret}$ 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 Pdgfb$^{ret/ret}$ mice throughout the cerebrovascular network and in different cortical layers by in vivo two-photon microscopy. We confirmed that Pdgfb$^{ret/ret}$ 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 Pdgfb$^{ret/ret}$ mice, which may impair oxygen delivery to the tissue. We also measured intravascular PO$_{2}$ and found that concentrations were higher in cortical Layer 2/3 in Pdgfb$^{ret/ret}$ mice, indicative of reduced blood oxygen extraction. Finally, we found that Pdgfb$^{ret/ret}$ 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.

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.

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 s

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. Keywords: Mural cells; calcium signaling; mouse; neuroscience; pericyte; vasculature.

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.