Phase Aberration Correction for in vivo Ultrasound Localization Microscopy Using a Spatiotemporal Complex-Valued Neural Network

Ultrasound Localization Microscopy (ULM) can map microvessels at a resolution of a few micrometers ({\mu}m). Transcranial ULM remains challenging in presence of aberrations caused by the skull, which lead to localization errors. Herein, we propose a deep learning approach based on recently introduced complex-valued convolutional neural networks (CV-CNNs) to retrieve the aberration function, which can then be used to form enhanced images using standard delay-and-sum beamforming. Complex-valued convolutional networks were selected as they can apply time delays through multiplication with in-phase quadrature input data. Predicting the aberration function rather than corrected images also confers enhanced explainability to the network. In addition, 3D spatiotemporal convolutions were used for the network to leverage entire microbubble tracks. For training and validation, we used an anatomically and hemodynamically realistic mouse brain microvascular network model to simulate the flow of microbubbles in presence of aberration. We then confirmed the capability of our network to generalize to transcranial in vivo data in the mouse brain (n=2). Qualitatively, vascular reconstructions using a pixel-wise predicted aberration function included additional and sharper vessels. The spatial resolution was evaluated by using the Fourier ring correlation (FRC). After correction, we measured a resolution of 16.7 {\mu}m in vivo, representing an improvement of up to 27.5 %. This work leads to different applications for complex-valued convolutions in biomedical imaging and strategies to perform transcranial ULM

Perivascular spaces in the brain:anatomy, physiology and pathology

Perivascular spaces include a variety of passageways around arterioles, capillaries and venules in the brain, along which a range of substances can move. Although perivascular spaces were first identified over 150 years ago, they have come to prominence recently owing to advances in knowledge of their roles in clearance of interstitial fluid and waste from the brain, particularly during sleep, and in the pathogenesis of small vessel disease, Alzheimer disease and other neurodegenerative and inflammatory disorders. Experimental advances have facilitated in vivo studies of perivascular space function in intact rodent models during wakefulness and sleep, and MRI in humans has enabled perivascular space morphology to be related to cognitive function, vascular risk factors, vascular and neurodegenerative brain lesions, sleep patterns and cerebral haemodynamics. Many questions about perivascular spaces remain, but what is now clear is that normal perivascular space function is important for maintaining brain health. Here, we review perivascular space anatomy, physiology and pathology, particularly as seen with MRI in humans, and consider translation from models to humans to highlight knowns, unknowns, controversies and clinical relevance

In vivo imaging with a water immersion objective affects brain temperature, blood flow and oxygenation

Previously, we reported the first oxygen partial pressure (Po2) measurements in the brain of awake mice, by performing two-photon phosphorescence lifetime microscopy at micrometer resolution (Lyons et al., 2016). However, this study disregarded that imaging through a cranial window lowers brain temperature, an effect capable of affecting cerebral blood flow, the properties of the oxygen sensors and thus Po2 measurements. Here, we show that in awake mice chronically implanted with a glass window over a craniotomy or a thinned-skull surface, the postsurgical decrease of brain temperature recovers within a few days. However, upon imaging with a water immersion objective at room temperature, brain temperature decreases by ~2–3°C, causing drops in resting capillary blood flow, capillary Po2, hemoglobin saturation, and tissue Po2. These adverse effects are corrected by heating the immersion objective or avoided by imaging through a dry air objective, thereby revealing the physiological values of brain oxygenation

Orai, RyR, and IP3R channels cooperatively regulate calcium signaling in brain mid-capillary pericytes

Abstract Pericytes are multifunctional cells of the vasculature that are vital to brain homeostasis, yet many of their fundamental physiological properties, such as Ca2+ signaling pathways, remain unexplored. We performed pharmacological and ion substitution experiments to investigate the mechanisms underlying pericyte Ca2+ signaling in acute cortical brain slices of PDGFRβ-Cre::GCaMP6f mice. We report that mid-capillary pericyte Ca2+ signalling differs from ensheathing type pericytes in that it is largely independent of L- and T-type voltage-gated calcium channels. Instead, Ca2+ signals in mid-capillary pericytes were inhibited by multiple Orai channel blockers, which also inhibited Ca2+ entry triggered by endoplasmic reticulum (ER) store depletion. An investigation into store release pathways indicated that Ca2+ transients in mid-capillary pericytes occur through a combination of IP3R and RyR activation, and that Orai store-operated calcium entry (SOCE) is required to sustain and amplify intracellular Ca2+ increases evoked by the GqGPCR agonist endothelin-1. These results suggest that Ca2+ influx via Orai channels reciprocally regulates IP3R and RyR release pathways in the ER, which together generate spontaneous Ca2+ transients and amplify Gq-coupled Ca2+ elevations in mid-capillary pericytes. Thus, SOCE is a major regulator of pericyte Ca2+ and a target for manipulating their function in health and disease

In vivo imaging with a water immersion objective affects brain temperature, blood flow and oxygenation

Previously, we reported the first oxygen partial pressure (Po2) measurements in the brain of awake mice, by performing two-photon phosphorescence lifetime microscopy at micrometer resolution (Lyons et al., 2016). However, this study disregarded that imaging through a cranial window lowers brain temperature, an effect capable of affecting cerebral blood flow, the properties of the oxygen sensors and thus Po2 measurements. Here, we show that in awake mice chronically implanted with a glass window over a craniotomy or a thinned-skull surface, the postsurgical decrease of brain temperature recovers within a few days. However, upon imaging with a water immersion objective at room temperature, brain temperature decreases by ~2–3°C, causing drops in resting capillary blood flow, capillary Po2, hemoglobin saturation, and tissue Po2. These adverse effects are corrected by heating the immersion objective or avoided by imaging through a dry air objective, thereby revealing the physiological values of brain oxygenation.ISSN:2050-084

Transfer functions linking neural calcium to single voxel functional ultrasound signal

Neurovascular coupling refers to changes in cerebral blood flow in response to neuronal stimulation, but to what extent this change can report neuronal activation is not known. Here the authors develop transfer functions between neural calcium signals and functional ultrasound changes in blood volume in co-registered single voxel brain volumes

Ca_V3.2 drives sustained burst-firing, which is critical for absence seizure propagation in reticular thalamic neurons

OBJECTIVE: Genetic alterations have been identified in the CACNA1H gene, encoding the CaV 3.2 T-type calcium channel in patients with absence epilepsy, yet the precise mechanisms relating to seizure propagation and spike-wave-discharge (SWD) pacemaking remain unknown. Neurons of the thalamic reticular nucleus (TRN) express high levels of CaV 3.2 calcium channels, and we investigated whether a gain-of-function mutation in the Cacna1h gene in Genetic Absence Epilepsy Rats from Strasbourg (GAERS) contributes to seizure propagation and pacemaking in the TRN. METHODS: Pathophysiological contributions of CaV 3.2 calcium channels to burst firing and absence seizures were assessed in vitro using acute brain slice electrophysiology and quantitative real-time polymerase chain reaction (PCR) and in vivo using free-moving electrocorticography recordings. RESULTS: TRN neurons from GAERS display sustained oscillatory burst-firing that is both age- and frequency-dependent, occurring only in the frequencies overlapping with GAERS SWDs and correlating with the expression of a CaV 3.2 mutation-sensitive splice variant. In vivo knock-down of CaV 3.2 using direct thalamic injection of lipid nanoparticles containing CaV 3.2 dicer small interfering (Dsi) RNA normalized TRN burst-firing, and in free-moving GAERS significantly shortened seizures. SIGNIFICANCE: This supports a role for TRN CaV 3.2 T-type channels in propagating thalamocortical network seizures and setting the pacemaking frequency of SWDs

Pannexin-1 opening in neuronal edema causes cell death but also leads to protection via increased microglia contacts

Summary: Neuronal swelling during cytotoxic edema is triggered by Na+ and Cl− entry and is Ca2+ independent. However, the causes of neuronal death during swelling are unknown. Here, we investigate the role of large-conductance Pannexin-1 (Panx1) channels in neuronal death during cytotoxic edema. Panx1 channel inhibitors reduce and delay neuronal death in swelling triggered by voltage-gated Na+ entry with veratridine. Neuronal swelling causes downstream production of reactive oxygen species (ROS) that opens Panx1 channels. We confirm that ROS activates Panx1 currents with whole-cell electrophysiology and find scavenging ROS is neuroprotective. Panx1 opening and subsequent ATP release attract microglial processes to contact swelling neurons. Depleting microglia using the CSF1 receptor antagonist PLX3397 or blocking P2Y12 receptors exacerbates neuronal death, suggesting that the Panx1-ATP-dependent microglia contacts are neuroprotective. We conclude that cytotoxic edema triggers oxidative stress in neurons that opens Panx1 to trigger death but also initiates neuroprotective feedback mediated by microglia contacts