Neuroinflammation During RNA Viral Infections.

Neurotropic RNA viruses continue to emerge and are increasingly linked to diseases of the central nervous system (CNS) despite viral clearance. Indeed, the overall mortality of viral encephalitis in immunocompetent individuals is low, suggesting efficient mechanisms of virologic control within the CNS. Both immune and neural cells participate in this process, which requires extensive innate immune signaling between resident and infiltrating cells, including microglia and monocytes, that regulate the effector functions of antiviral T and B cells as they gain access to CNS compartments. While these interactions promote viral clearance via mainly neuroprotective mechanisms, they may also promote neuropathology and, in some cases, induce persistent alterations in CNS physiology and function that manifest as neurologic and psychiatric diseases. This review discusses mechanisms of RNA virus clearance and neurotoxicity during viral encephalitis with a focus on the cytokines essential for immune and neural cell inflammatory responses and interactions. Understanding neuroimmune communications in the setting of viral infections is essential for the development of treatments that augment neuroprotective processes while limiting ongoing immunopathological processes that cause ongoing CNS disease.

Neurovascular Interactions in the Nervous System.

Molecular cross talk between the nervous and vascular systems is necessary to maintain the correct coupling of organ structure and function. Molecular pathways shared by both systems are emerging as major players in the communication of the neuronal compartment with the endothelium. Here we review different aspects of this cross talk and how vessels influence the development and homeostasis of the nervous system. Beyond the classical role of the vasculature as a conduit to deliver oxygen and metabolites needed for the energy-demanding neuronal compartment, vessels emerge as powerful signaling systems that control and instruct a variety of cellular processes during the development of neurons and glia, such as migration, differentiation, and structural connectivity. Moreover, a broad spectrum of mild to severe vascular dysfunctions occur in various pathologies of the nervous system, suggesting that mild structural and functional changes at the neurovascular interface may underlie cognitive decline in many of these pathological conditions.

Development and Cell Biology of the Blood-Brain Barrier.

The vertebrate vasculature displays high organotypic specialization, with the structure and function of blood vessels catering to the specific needs of each tissue. A unique feature of the central nervous system (CNS) vasculature is the blood-brain barrier (BBB). The BBB regulates substance influx and efflux to maintain a homeostatic environment for proper brain function. Here, we review the development and cell biology of the BBB, focusing on the cellular and molecular regulation of barrier formation and the maintenance of the BBB through adulthood. We summarize unique features of CNS endothelial cells and highlight recent progress in and general principles of barrier regulation. Finally, we illustrate why a mechanistic understanding of the development and maintenance of the BBB could provide novel therapeutic opportunities for CNS drug delivery.

Astrocyte Endfeet in Brain Function and Pathology: Open Questions.

Astrocyte endfeet enwrap the entire vascular tree within the central nervous system, where they perform important functions in regulating the blood-brain barrier (BBB), cerebral blood flow, nutrient uptake, and waste clearance. Accordingly, astrocyte endfeet contain specialized organelles and proteins, including local protein translation machinery and highly organized scaffold proteins, which anchor channels, transporters, receptors, and enzymes critical for astrocyte-vascular interactions. Many neurological diseases are characterized by the loss of polarization of specific endfoot proteins, vascular dysregulation, BBB disruption, altered waste clearance, or, in extreme cases, loss of endfoot coverage. A role for astrocyte endfeet has been demonstrated or postulated in many of these conditions. This review provides an overview of the development, composition, function, and pathological changes of astrocyte endfeet and highlights the gaps in our knowledge that future research should address.

Functional Ultrasound Neuroimaging.

Functional ultrasound (fUS) is a neuroimaging method that uses ultrasound to track changes in cerebral blood volume as an indirect readout of neuronal activity at high spatiotemporal resolution. fUS is capable of imaging head-fixed or freely behaving rodents and of producing volumetric images of the entire mouse brain. It has been applied to many species, including primates and humans. Now that fUS is reaching maturity, it is being adopted by the neuroscience community. However, the nature of the fUS signal and the different implementations of fUS are not necessarily accessible to nonspecialists. This review aims to introduce these ultrasound concepts to all neuroscientists. We explain the physical basis of the fUS signal and the principles of the method, present the state of the art of its hardware implementation, and give concrete examples of current applications in neuroscience. Finally, we suggest areas for improvement during the next few years.

Regulation of cerebral blood flow in humans: physiology and clinical implications of autoregulation.

Brain function critically depends on a close matching between metabolic demands, appropriate delivery of oxygen and nutrients, and removal of cellular waste. This matching requires continuous regulation of cerebral blood flow (CBF), which can be categorized into four broad topics: 1) autoregulation, which describes the response of the cerebrovasculature to changes in perfusion pressure; 2) vascular reactivity to vasoactive stimuli [including carbon dioxide (CO2)]; 3) neurovascular coupling (NVC), i.e., the CBF response to local changes in neural activity (often standardized cognitive stimuli in humans); and 4) endothelium-dependent responses. This review focuses primarily on autoregulation and its clinical implications. To place autoregulation in a more precise context, and to better understand integrated approaches in the cerebral circulation, we also briefly address reactivity to CO2 and NVC. In addition to our focus on effects of perfusion pressure (or blood pressure), we describe the impact of select stimuli on regulation of CBF (i.e., arterial blood gases, cerebral metabolism, neural mechanisms, and specific vascular cells), the interrelationships between these stimuli, and implications for regulation of CBF at the level of large arteries and the microcirculation. We review clinical implications of autoregulation in aging, hypertension, stroke, mild cognitive impairment, anesthesia, and dementias. Finally, we discuss autoregulation in the context of common daily physiological challenges, including changes in posture (e.g., orthostatic hypotension, syncope) and physical activity.

Sympathetic and Sensory-Motor Nerves in Peripheral Small Arteries.

Small arteries, which play important roles in controlling blood flow, blood pressure, and capillary pressure, are under nervous influence. Their innervation is predominantly sympathetic and sensory motor in nature, and while some arteries are densely innervated, others are only sparsely so. Innervation of small arteries is a key mechanism in regulating vascular resistance. In the second half of the previous century, the physiology and pharmacology of this innervation were very actively investigated. In the past 10-20 yr, the activity in this field was more limited. With this review we highlight what has been learned during recent years with respect to development of small arteries and their innervation, some aspects of excitation-release coupling, interaction between sympathetic and sensory-motor nerves, cross talk between endothelium and vascular nerves, and some aspects of their role in vascular inflammation and hypertension. We also highlight what remains to be investigated to further increase our understanding of this fundamental aspect of vascular physiology.

Pericytes and the Control of Blood Flow in Brain and Heart.

Pericytes, attached to the surface of capillaries, play an important role in regulating local blood flow. Using optogenetic tools and genetically encoded reporters in conjunction with confocal and multiphoton imaging techniques, the 3D structure, anatomical organization, and physiology of pericytes have recently been the subject of detailed examination. This work has revealed novel functions of pericytes and morphological features such as tunneling nanotubes in brain and tunneling microtubes in heart. Here, we discuss the state of our current understanding of the roles of pericytes in blood flow control in brain and heart, where functions may differ due to the distinct spatiotemporal metabolic requirements of these tissues. We also outline the novel concept of electro-metabolic signaling, a universal mechanistic framework that links tissue metabolic state with blood flow regulation by pericytes and vascular smooth muscle cells, with capillary KATP and Kir2.1 channels as primary sensors. Finally, we present major unresolved questions and outline how they can be addressed.

High-resolution omics of vascular ageing and inflammatory pathways in neurodegeneration.

High-resolution omics, particularly single-cell and spatial transcriptomic profiling, are rapidly enhancing our comprehension of the normal molecular diversity of gliovascular cells, as well as their age-related changes that contribute to neurodegeneration. With more omic profiling studies being conducted, it is becoming increasingly essential to synthesise valuable information from the rapidly accumulating findings. In this review, we present an overview of the molecular features of neurovascular and glial cells that have been recently discovered through omic profiling, with a focus on those that have potentially significant functional implications and/or show cross-species differences between human and mouse, and that are linked to vascular deficits and inflammatory pathways in ageing and neurodegenerative disorders. Additionally, we highlight the translational applications of omic profiling, and discuss omic-based strategies to accelerate biomarker discovery and facilitate disease course-modifying therapeutics development for neurodegenerative conditions.

Meningeal origins and dynamics of perivascular fibroblast development on the mouse cerebral vasculature.

Perivascular fibroblasts (PVFs) are a fibroblast-like cell type that reside on large-diameter blood vessels in the adult meninges and central nervous system (CNS). PVFs contribute to fibrosis following injury but their homeostatic functions are not defined. PVFs were previously shown to be absent from most brain regions at birth and are only detected postnatally within the cerebral cortex. However, the origin, timing and cellular mechanisms of PVF development are not known. We used Col1a1-GFP and Col1a2-CreERT2 transgenic mice to track PVF development postnatally. Using lineage tracing and in vivo imaging we show that brain PVFs originate from the meninges and are first seen on parenchymal cerebrovasculature at postnatal day (P) 5. After P5, PVF coverage of the cerebrovasculature expands via local cell proliferation and migration from the meninges. Finally, we show that PVFs and perivascular macrophages develop concurrently. These findings provide the first complete timeline for PVF development in the brain, enabling future work into how PVF development is coordinated with cell types and structures in and around the perivascular spaces to support normal CNS vascular function.

Pericyte-Specific Secretome Profiling in Hypoxia Using TurboID in a Multicellular in Vitro Spheroid Model.

Cellular communication within the brain is imperative for maintaining homeostasis and mounting effective responses to pathological triggers like hypoxia. However, a comprehensive understanding of the precise composition and dynamic release of secreted molecules has remained elusive, confined primarily to investigations using isolated monocultures. To overcome these limitations, we utilized the potential of TurboID, a non-toxic biotin ligation enzyme, to capture and enrich secreted proteins specifically originating from human brain pericytes in spheroid cocultures with human endothelial cells and astrocytes. This approach allowed us to characterize the pericyte secretome within a more physiologically relevant multicellular setting encompassing the constituents of the blood-brain barrier. Through a combination of mass spectrometry and multiplex immunoassays, we identified a wide spectrum of different secreted proteins by pericytes. Our findings demonstrate that the pericytes secretome is profoundly shaped by their intercellular communication with other blood-brain barrier-residing cells. Moreover, we identified substantial differences in the secretory profiles between hypoxic and normoxic pericytes. Mass spectrometry analysis showed that hypoxic pericytes in coculture increase their release of signals related to protein secretion, mTOR signaling, and the complement system, while hypoxic pericytes in monocultures showed an upregulation in proliferative pathways including G2M checkpoints, E2F-, and Myc-targets. In addition, hypoxic pericytes show an upregulation of proangiogenic proteins such as VEGFA but display downregulation of canonical proinflammatory cytokines such as CXCL1, MCP-1, and CXCL6. Understanding the specific composition of secreted proteins in the multicellular brain microvasculature is crucial for advancing our knowledge of brain homeostasis and the mechanisms underlying pathology. This study has implications for the identification of targeted therapeutic strategies aimed at modulating microvascular signaling in brain pathologies associated with hypoxia.

Parvalbumin interneuron activity drives fast inhibition-induced vasoconstriction followed by slow substance P-mediated vasodilation.

The role of parvalbumin (PV) interneurons in vascular control is poorly understood. Here, we investigated the hemodynamic responses elicited by optogenetic stimulation of PV interneurons using electrophysiology, functional magnetic resonance imaging (fMRI), wide-field optical imaging (OIS), and pharmacological applications. As a control, forepaw stimulation was used. Stimulation of PV interneurons in the somatosensory cortex evoked a biphasic fMRI response in the photostimulation site and negative fMRI signals in projection regions. Activation of PV neurons engaged two separable neurovascular mechanisms in the stimulation site. First, an early vasoconstrictive response caused by the PV-driven inhibition is sensitive to the brain state affected by anesthesia or wakefulness. Second, a later ultraslow vasodilation lasting a minute is closely dependent on the sum of interneuron multiunit activities, but is not due to increased metabolism, neural or vascular rebound, or increased glial activity. The ultraslow response is mediated by neuropeptide substance P (SP) released from PV neurons under anesthesia, but disappears during wakefulness, suggesting that SP signaling is important for vascular regulation during sleep. Our findings provide a comprehensive perspective about the role of PV neurons in controlling the vascular response.

Pericyte Control of Blood Flow Across Microvascular Zones in the Central Nervous System.

The vast majority of the brain's vascular length is composed of capillaries, where our understanding of blood flow control remains incomplete. This review synthesizes current knowledge on the control of blood flow across microvascular zones by addressing issues with nomenclature and drawing on new developments from in vivo optical imaging and single-cell transcriptomics. Recent studies have highlighted important distinctions in mural cell morphology, gene expression, and contractile dynamics, which can explain observed differences in response to vasoactive mediators between arteriole, transitional, and capillary zones. Smooth muscle cells of arterioles and ensheathing pericytes of the arteriole-capillary transitional zone control large-scale, rapid changes in blood flow. In contrast, capillary pericytes downstream of the transitional zone act on slower and smaller scales and are involved in establishing resting capillary tone and flow heterogeneity. Many unresolved issues remain, including the vasoactive mediators that activate the different pericyte types in vivo, the role of pericyte-endothelial communication in conducting signals from capillaries to arterioles, and how neurological disease affects these mechanisms.

Differential Vulnerability and Response to Injury among Brain Cell Types Comprising the Neurovascular Unit.

The neurovascular unit (NVU) includes multiple different cell types, including neurons, astrocytes, endothelial cells, and pericytes, which respond to insults on very different time or dose scales. We defined differential vulnerability among these cell types, using response to two different insults: oxygen-glucose deprivation (OGD) and thrombin-mediated cytotoxicity. We found that neurons are most vulnerable, followed by endothelial cells and astrocytes. After temporary focal cerebral ischemia in male rats, we found significantly more injured neurons, compared with astrocytes in the ischemic area, consistent with differential vulnerability in vivo. We sought to illustrate different and shared mechanisms across all cell types during response to insult. We found that gene expression profiles in response to OGD differed among the cell types, with a paucity of gene responses shared by all types. All cell types activated genes relating to autophagy, apoptosis, and necroptosis, but the specific genes differed. Astrocytes and endothelial cells also activated pathways connected to DNA repair and antiapoptosis. Taken together, the data support the concept of differential vulnerability in the NVU and suggest that different elements of the unit will evolve from salvageable to irretrievable on different time scales while residing in the same brain region and receiving the same (ischemic) blood flow. Future work will focus on the mechanisms of these differences. These data suggest future stroke therapy development should target different elements of the NVU differently.

Osteoblasts pattern endothelium and somatosensory axons during zebrafish caudal fin organogenesis.

Skeletal elements frequently associate with vasculature and somatosensory nerves, which regulate bone development and homeostasis. However, the deep, internal location of bones in many vertebrates has limited in vivo exploration of the neurovascular-bone relationship. Here, we use the zebrafish caudal fin, an optically accessible organ formed of repeating bony ray skeletal units, to determine the cellular relationship between nerves, bones and endothelium. In adult zebrafish, we establish the presence of somatosensory axons running through the inside of the bony fin rays, juxtaposed with osteoblasts on the inner hemiray surface. During development we show that the caudal fin progresses through sequential stages of endothelial plexus formation, bony ray addition, ray innervation and endothelial remodeling. Surprisingly, the initial stages of fin morphogenesis proceed normally in animals lacking either fin endothelium or somatosensory nerves. Instead, we find that sp7+ osteoblasts are required for endothelial remodeling and somatosensory axon innervation in the developing fin. Overall, this study demonstrates that the proximal neurovascular-bone relationship in the adult caudal fin is established during fin organogenesis and suggests that ray-associated osteoblasts pattern axons and endothelium.

Transcranial ultrasound stimulation selectively affects cortical neurovascular coupling across neuronal types and LFP frequency bands.

Previous studies have affirmed that transcranial ultrasound stimulation (TUS) can influence cortical neurovascular coupling across low-frequency (0-2 Hz)/high-frequency (160-200 Hz) neural oscillations and hemodynamics. Nevertheless, the selectivity of this coupling triggered by transcranial ultrasound stimulation for spike activity (> 300 Hz) and additional frequency bands (4-150 Hz) remains elusive. We applied transcranial ultrasound stimulation to mice visual cortex while simultaneously recording total hemoglobin concentration, spike activity, and local field potentials. Our findings include (1) a significant increase in coupling strength between spike firing rates of putative inhibitory neurons/putative excitatory neurons and total hemoglobin concentration post-transcranial ultrasound stimulation; (2) an ~ 2.1-fold higher Pearson correlation coefficient between putative inhibitory neurons and total hemoglobin concentration compared with putative excitatory neurons and total hemoglobin concentration (*P < 0.05); (3) a notably greater cross-correlation between putative inhibitory neurons and total hemoglobin concentration than that between putative excitatory neurons and total hemoglobin concentration (*P < 0.05); (4) an enhancement of Pearson correlation coefficient between the relative power of γ frequency band (30-80 Hz), hγ frequency band (80-150 Hz) and total hemoglobin concentration following transcranial ultrasound stimulation (*P < 0.05); and (5) strongest cross-correlation observed at negative delay for θ frequency band, and positive delay for α, ß, γ, hγ frequency bands. Collectively, these results demonstrate that cortical neurovascular coupling evoked by transcranial ultrasound stimulation exhibits selectivity concerning neuronal types and local field potential frequency bands.

The significance and limited influence of cerebrovascular reactivity on age and sex effects in task- and resting-state brain activity.

Functional MRI measures the blood-oxygen-level dependent signals, which provide an indirect measure of neural activity mediated by neurovascular responses. Cerebrovascular reactivity affects both task-induced and resting-state blood-oxygen-level dependent activity and may confound inter-individual effects, such as those related to aging and biological sex. We examined a large dataset containing breath-holding, checkerboard, and resting-state tasks. We used the breath-holding task to measure cerebrovascular reactivity, used the checkerboard task to obtain task-based activations, and quantified resting-state activity with amplitude of low-frequency fluctuations and regional homogeneity. We hypothesized that cerebrovascular reactivity would be correlated with blood-oxygen-level dependent measures and that accounting for these correlations would result in better estimates of age and sex effects. We found that cerebrovascular reactivity was correlated with checkerboard task activations in the visual cortex and with amplitude of low-frequency fluctuations and regional homogeneity in widespread fronto-parietal regions, as well as regions with large vessels. We also found significant age and sex effects in cerebrovascular reactivity, some of which overlapped with those observed in amplitude of low-frequency fluctuations and regional homogeneity. However, correcting for the effects of cerebrovascular reactivity had very limited influence on the estimates of age and sex. Our results highlight the limitations of accounting for cerebrovascular reactivity with the current breath-holding task.

Targeting lipid nanoparticles to the blood-brain barrier to ameliorate acute ischemic stroke.

Effective delivery of mRNA or small molecule drugs to the brain is a significant challenge in developing treatment for acute ischemic stroke (AIS). To address the problem, we have developed targeted nanomedicine to increase drug concentrations in endothelial cells of the blood-brain barrier (BBB) of the injured brain. Inflammation during ischemic stroke causes continuous neuronal death and an increase in the infarct volume. To enable targeted delivery to the inflamed BBB, we conjugated lipid nanocarriers (NCs) with antibodies that bind cell adhesion molecules expressed at the BBB. In the transient middle cerebral artery occlusion mouse model, NCs targeted to vascular cellular adhesion molecule-1 (VCAM) achieved the highest level of brain delivery, nearly two orders of magnitude higher than untargeted ones. VCAM-targeted lipid nanoparticles with luciferase-encoding mRNA and Cre-recombinase showed selective expression in the ischemic brain. Anti-inflammatory drugs administered intravenously after ischemic stroke reduced cerebral infarct volume by 62% (interleukin-10 mRNA) or 35% (dexamethasone) only when they were encapsulated in VCAM-targeted NCs. Thus, VCAM-targeted lipid NCs represent a new platform for strongly concentrating drugs within the compromised BBB of penumbra, thereby ameliorating AIS.

Endothelial PDGF-D contributes to neurovascular protection after ischemic stroke by rescuing pericyte functions.

Ischemic stroke induces neovascularization of the injured tissue as an attempt to promote structural repair and neurological recovery. Angiogenesis is regulated by pericytes that potently react to ischemic stroke stressors, ranging from death to dysfunction. Platelet-derived growth factor (PDGF) receptor (PDGFR)ß controls pericyte survival, migration, and interaction with brain endothelial cells. PDGF-D a specific ligand of PDGFRß is expressed in the brain, yet its regulation and role in ischemic stroke pathobiology remains unexplored. Using experimental ischemic stroke mouse model, we found that PDGF-D is transiently induced in brain endothelial cells at the injury site in the subacute phase. To investigate the biological significance of PDGF-D post-ischemic stroke regulation, its subacute expression was either downregulated using siRNA or upregulated using an active recombinant form. Attenuation of PDGF-D subacute induction exacerbates neuronal loss, impairs microvascular density, alters vascular permeability, and increases microvascular stalling. Increasing PDGF-D subacute bioavailability rescues neuronal survival and improves neurological recovery. PDGF-D subacute enhanced bioavailability promotes stable neovascularization of the injured tissue and improves brain perfusion. Notably, PDGF-D enhanced bioavailability improves pericyte association with brain endothelial cells. Cell-based assays using human brain pericyte and brain endothelial cells exposed to ischemia-like conditions were applied to investigate the underlying mechanisms. PDGF-D stimulation attenuates pericyte loss and fibrotic transition, while increasing the secretion of pro-angiogenic and vascular protective factors. Moreover, PDGF-D stimulates pericyte migration required for optimal endothelial coverage and promotes angiogenesis. Our study unravels new insights into PDGF-D contribution to neurovascular protection after ischemic stroke by rescuing the functions of pericytes.

Insulin regulates neurovascular coupling through astrocytes.

Mice with insulin receptor (IR)-deficient astrocytes (GFAP-IR knockout [KO] mice) show blunted responses to insulin and reduced brain glucose uptake, whereas IR-deficient astrocytes show disturbed mitochondrial responses to glucose. While exploring the functional impact of disturbed mitochondrial function in astrocytes, we observed that GFAP-IR KO mice show uncoupling of brain blood flow with glucose uptake. Since IR-deficient astrocytes show higher levels of reactive oxidant species (ROS), this leads to stimulation of hypoxia-inducible factor-1α and, consequently, of the vascular endothelial growth factor angiogenic pathway. Indeed, GFAP-IR KO mice show disturbed brain vascularity and blood flow that is normalized by treatment with the antioxidant N-acetylcysteine (NAC). NAC ameliorated high ROS levels, normalized angiogenic signaling and mitochondrial function in IR-deficient astrocytes, and normalized neurovascular coupling in GFAP-IR KO mice. Our results indicate that by modulating glucose uptake and angiogenesis, insulin receptors in astrocytes participate in neurovascular coupling.