A Vascular-Centric Approach to Autism Spectrum Disorders.

Brain development and function are highly reliant on adequate establishment and maintenance of vascular networks. Early impairments in vascular health can impact brain maturation and energy metabolism, which may lead to neurodevelopmental anomalies. Our recent work not only provides novel insights into the development of cerebrovascular networks but also emphasizes the importance of their well-being for proper brain maturation. In particular, we have demonstrated that endothelial dysfunction in autism spectrum disorders (ASD) mouse models is causally related to altered behavior and brain metabolism. In the prenatal human brain, vascular cells change metabolic states in the second trimester. Such findings highlight the need to identify new cellular and molecular players in neurodevelopmental disorders, raising awareness about the importance of a healthy vasculature for brain development. It is thus essential to shift the mostly neuronal point of view in research on ASD and other neurodevelopmental disorders to also include vascular and metabolic features.

Sex, hormones and cerebrovascular function: from development to disorder.

Proper cerebrovascular development and neurogliovascular unit assembly are essential for brain growth and function throughout life, ensuring the continuous supply of nutrients and oxygen. This involves crucial events during pre- and postnatal stages through key pathways, including vascular endothelial growth factor (VEGF) and Wnt signaling. These pathways are pivotal for brain vascular growth, expansion, and blood-brain barrier (BBB) maturation. Interestingly, during fetal and neonatal life, cerebrovascular formation coincides with the early peak activity of the hypothalamic-pituitary-gonadal axis, supporting the idea of sex hormonal influence on cerebrovascular development and barriergenesis.Sex hormonal dysregulation in early development has been implicated in neurodevelopmental disorders with highly sexually dimorphic features, such as autism spectrum disorder (ASD) and attention-deficit/hyperactivity disorder (ADHD). Both disorders show higher prevalence in men, with varying symptoms between sexes, with boys exhibiting more externalizing behaviors, such as aggressivity or hyperactivity, and girls displaying higher internalizing behaviors, including anxiety, depression, or attention disorders. Indeed, ASD and ADHD are linked to high prenatal testosterone exposure and reduced aromatase expression, potentially explaining sex differences in prevalence and symptomatology. In line with this, high estrogen levels seem to attenuate ADHD symptoms. At the cerebrovascular level, sex- and region-specific variations of cerebral blood flow perfusion have been reported in both conditions, indicating an impact of gonadal hormones on the brain vascular system, disrupting its ability to respond to neuronal demands.This review aims to provide an overview of the existing knowledge concerning the impact of sex hormones on cerebrovascular formation and maturation, as well as the onset of neurodevelopmental disorders. Here, we explore the concept of gonadal hormone interactions with brain vascular and BBB development to function, with a particular focus on the modulation of VEGF and Wnt signaling. We outline how these pathways may be involved in the underpinnings of ASD and ADHD. Outstanding questions and potential avenues for future research are highlighted, as uncovering sex-specific physiological and pathological aspects of brain vascular development might lead to innovative therapeutic approaches in the context of ASD, ADHD and beyond.

Does Perinatal Intermittent Hypoxia Affect Cerebrovascular Network Development?

Perinatal hypoxia is an inadequate delivery of oxygen to the fetus in the period immediately before, during, or after the birth process. The most frequent form of hypoxia occurring in human development is chronic intermittent hypoxia (CIH) due to sleep-disordered breathing (apnea) or bradycardia events. CIH incidence is particularly high with premature infants. During CIH, repetitive cycles of hypoxia and reoxygenation initiate oxidative stress and inflammatory cascades in the brain. A dense microvascular network of arterioles, capillaries, and venules is required to support the constant metabolic demands of the adult brain. The development and refinement of this microvasculature is orchestrated throughout gestation and in the initial weeks after birth, at a critical juncture when CIH can occur. There is little knowledge on how CIH affects the development of the cerebrovasculature. However, since CIH (and its treatments) can cause profound abnormalities in tissue oxygen content and neural activity, there is reason to believe that it can induce lasting abnormalities in vascular structure and function at the microvascular level contributing to neurodevelopmental disorders. This mini-review discusses the hypothesis that CIH induces a positive feedback loop to perpetuate metabolic insufficiency through derailment of normal cerebrovascular development, leading to long-term deficiencies in cerebrovascular function.

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.

Endothelial structure contributes to heterogeneity in brain capillary diameter.

The high metabolic demand of brain tissue is supported by a constant supply of blood flow through dense microvascular networks. Capillaries are the smallest class of vessels in the brain and their lumens vary in diameter between ~2 and 5 µm. This diameter range plays a significant role in optimizing blood flow resistance, blood cell distribution, and oxygen extraction. The control of capillary diameter has largely been ascribed to pericyte contractility, but it remains unclear if the architecture of the endothelial wall also contributes to capillary diameter. Here, we use public, large-scale volume electron microscopy data from mouse cortex (MICrONS Explorer, Cortical mm3) to examine how endothelial cell number, endothelial cell thickness, and pericyte coverage relates to microvascular lumen size. We find that transitional vessels near the penetrating arteriole and ascending venule are composed of two to six interlocked endothelial cells, while the capillaries intervening these zones are composed of either one or two endothelial cells, with roughly equal proportions. The luminal area and diameter are on average slightly larger with capillary segments composed of two interlocked endothelial cells vs one endothelial cell. However, this difference is insufficient to explain the full range of capillary diameters seen in vivo. This suggests that both endothelial structure and other influences, including pericyte tone, contribute to the basal diameter and optimized perfusion of brain capillaries.

Endothelial structure contributes to heterogeneity in brain capillary diameter.

The high metabolic demand of brain tissue is supported by a constant supply of blood through dense microvascular networks. Capillaries are the smallest class of vessels and vary in diameter between ∼2 to 5 µm in the brain. This diameter range plays a significant role in the optimization of blood flow resistance, blood cell distribution, and oxygen extraction. The control of capillary diameter has largely been ascribed to pericyte contractility, but it remains unclear if endothelial wall architecture also contributes to capillary diameter heterogeneity. Here, we use public, large-scale volume electron microscopy data from mouse cortex (MICrONS Explorer, Cortical MM^3) to examine how endothelial cell number, endothelial cell thickness, and pericyte coverage relates to microvascular lumen size. We find that transitional vessels near the penetrating arteriole and ascending venule are composed of 2 to 5 interlocked endothelial cells, while the numerous capillary segments intervening these zones are composed of either 1 or 2 endothelial cells, with roughly equal proportions. The luminal area and diameter is on average slightly larger with capillary segments composed of 2 interlocked endothelial cells versus 1 endothelial cell. However, this difference is insufficient to explain the full range of capillary diameters seen in vivo. This suggests that both endothelial structure and other influences, such as pericyte tone, contribute to the basal diameter and optimized perfusion of brain capillaries.

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 drive fibrosis following injury but their homeostatic functions are not well detailed. In mice, 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-CreERT transgenic mice to track PVF developmental timing and progression in postnatal mice. Using a combination of 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 rapidly expands via mechanisms of local cell proliferation and migration from the meninges, reaching adult levels at P14. Finally, we show that PVFs and perivascular macrophages (PVMs) develop concurrently along postnatal cerebral blood vessels, where the location and depth of PVMs and PVFs highly correlate. 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. Summary: Brain perivascular fibroblasts migrate from their origin in the meninges and proliferate locally to fully cover penetrating vessels during postnatal mouse development.

Pericyte remodeling is deficient in the aged brain and contributes to impaired capillary flow and structure.

Deterioration of brain capillary flow and architecture is a hallmark of aging and dementia. It remains unclear how loss of brain pericytes in these conditions contributes to capillary dysfunction. Here, we conduct cause-and-effect studies by optically ablating pericytes in adult and aged mice in vivo. Focal pericyte loss induces capillary dilation without blood-brain barrier disruption. These abnormal dilations are exacerbated in the aged brain, and result in increased flow heterogeneity in capillary networks. A subset of affected capillaries experience reduced perfusion due to flow steal. Some capillaries stall in flow and regress, leading to loss of capillary connectivity. Remodeling of neighboring pericytes restores endothelial coverage and vascular tone within days. Pericyte remodeling is slower in the aged brain, resulting in regions of persistent capillary dilation. These findings link pericyte loss to disruption of capillary flow and structure. They also identify pericyte remodeling as a therapeutic target to preserve capillary flow dynamics.

Special Section Guest Editorial: Imaging Neuroimmune, Neuroglial, and Neurovascular Interfaces.

The guest editorial provides an introduction to Parts 1 and 2 of the Neurophotonics Special Section on Imaging Neuroimmune, Neuroglial, and Neurovascular Interfaces.

Reinforced thinned-skull window for repeated imaging of the neonatal mouse brain.

Significance: Two-photon microscopy is a powerful tool for in vivo imaging of the mammalian brain at cellular to subcellular resolution. However, resources that describe methods for imaging live newborn mice have remained sparse. Aim: We describe a non-invasive cranial window procedure for longitudinal imaging of neonatal mice. Approach: We demonstrate construction of the cranial window by iterative shaving of the calvarium of P0 to P12 mouse pups. We use the edge of a syringe needle and scalpel blades to thin the bone to ∼ 15 - µ m thickness. The window is then reinforced with cyanoacrylate glue and a coverslip to promote stability and optical access for at least a week. The head cap also includes a light-weight aluminum flange for head-fixation during imaging. Results: The resulting chronic thinned-skull window enables in vivo imaging to a typical cortical depth of ∼ 200 µ m without disruption of the intracranial environment. We highlight techniques to measure vascular structure and blood flow during development, including use of intravenous tracers and transgenic mice to label the blood plasma and vascular cell types, respectively. Conclusions: This protocol enables direct visualization of the developing neurogliovascular unit in the live neonatal brain during both normal and pathological states.

Public Volume Electron Microscopy Data: An Essential Resource to Study the Brain Microvasculature.

Electron microscopy is the primary approach to study ultrastructural features of the cerebrovasculature. However, 2D snapshots of a vascular bed capture only a small fraction of its complexity. Recent efforts to synaptically map neuronal circuitry using volume electron microscopy have also sampled the brain microvasculature in 3D. Here, we perform a meta-analysis of 7 data sets spanning different species and brain regions, including two data sets from the MICrONS consortium that have made efforts to segment vasculature in addition to all parenchymal cell types in mouse visual cortex. Exploration of these data have revealed rich information for detailed investigation of the cerebrovasculature. Neurovascular unit cell types (including, but not limited to, endothelial cells, mural cells, perivascular fibroblasts, microglia, and astrocytes) could be discerned across broad microvascular zones. Image contrast was sufficient to identify subcellular details, including endothelial junctions, caveolae, peg-and-socket interactions, mitochondria, Golgi cisternae, microvilli and other cellular protrusions of potential significance to vascular signaling. Additionally, non-cellular structures including the basement membrane and perivascular spaces were visible and could be traced between arterio-venous zones along the vascular wall. These explorations revealed structural features that may be important for vascular functions, such as blood-brain barrier integrity, blood flow control, brain clearance, and bioenergetics. They also identified limitations where accuracy and consistency of segmentation could be further honed by future efforts. The purpose of this article is to introduce these valuable community resources within the framework of cerebrovascular research. We do so by providing an assessment of their vascular contents, identifying features of significance for further study, and discussing next step ideas for refining vascular segmentation and analysis.

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.

Imaging the construction of capillary networks in the neonatal mouse brain.

Capillary networks are essential for distribution of blood flow through the brain, and numerous other homeostatic functions, including neurovascular signal conduction and blood-brain barrier integrity. Accordingly, the impairment of capillary architecture and function lies at the root of many brain diseases. Visualizing how brain capillary networks develop in vivo can reveal innate programs for cerebrovascular growth and repair. Here, we use longitudinal two-photon imaging through noninvasive thinned skull windows to study a burst of angiogenic activity during cerebrovascular development in mouse neonates. We find that angiogenesis leading to the formation of capillary networks originated exclusively from cortical ascending venules. Two angiogenic sprouting activities were observed: 1) early, long-range sprouts that directly connected venules to upstream arteriolar input, establishing the backbone of the capillary bed, and 2) short-range sprouts that contributed to expansion of anastomotic connectivity within the capillary bed. All nascent sprouts were prefabricated with an intact endothelial lumen and pericyte coverage, ensuring their immediate perfusion and stability upon connection to their target vessels. The bulk of this capillary expansion spanned only 2 to 3 d and contributed to an increase of blood flow during a critical period in cortical development.

Three-dimensional ultrastructure of the brain pericyte-endothelial interface.

Pericytes and endothelial cells share membranous interdigitations called "peg-and-socket" interactions that facilitate their adhesion and biochemical crosstalk during vascular homeostasis. However, the morphology and distribution of these ultrastructures have remained elusive. Using a combination of 3D electron microscopy techniques, we examined peg-and-socket interactions in mouse brain capillaries. We found that pegs extending from pericytes to endothelial cells were morphologically diverse, exhibiting claw-like morphologies at the edge of the cell and bouton-shaped swellings away from the edge. Reciprocal endothelial pegs projecting into pericytes were less abundant and appeared as larger columnar protuberances. A large-scale 3D EM data set revealed enrichment of both pericyte and endothelial pegs around pericyte somata. The ratio of pericyte versus endothelial pegs was conserved among the pericytes examined, but total peg abundance was heterogeneous across cells. These data show considerable investment between pericytes and endothelial cells, and provide morphological evidence for pericyte somata as sites of enriched physical and biochemical interaction.

Protective effect of neuropeptide Y2 receptor activation against methamphetamine-induced brain endothelial cell alterations.

Methamphetamine (METH) consumption is a health problem that leads to neurological and psychiatric disturbances. The cellular alterations behind these conditions have been extensively investigated and it is now well-established that METH causes cerebrovascular alterations being a key feature in drug-induced neuropathology. Although promising advances in understanding the blood-brain barrier (BBB) alterations induced by METH, there is still no available approach to counteract or diminish such effects. Interestingly, several studies show that neuropeptide Y (NPY) has an important protective role against METH-induced neuronal and glial toxicity, as well as behavioral deficits. Despite these beneficial effects of the NPY system, nothing is known about its role in brain endothelial cells under conditions of METH exposure. Thus, our aim was to unravel the effect of NPY and its receptors against METH-induced endothelial cell dysfunction. For that, we used a human brain microvascular endothelial cell line (hCMEC/D3) and our results demonstrate that endothelial cells express both NPY Y1 (Y1R) and Y2 (Y2R) receptors, but only Y2R is upregulated after METH exposure. Moreover, this drug of abuse induced endothelial cell death and elicited the production of reactive oxygen species (ROS) by these cells, which were prevented by the activation of Y2R. Additional, cell death and oxidative stress triggered by METH were dependent on the concentration of the drug. In sum, with the present study we identified for the first time the NPY system, and particularly the Y2R subtype, as a promising target to protect against METH-induced neurovascular dysfunction.

Microglia Dysfunction Caused by the Loss of Rhoa Disrupts Neuronal Physiology and Leads to Neurodegeneration.

Nervous tissue homeostasis requires the regulation of microglia activity. Using conditional gene targeting in mice, we demonstrate that genetic ablation of the small GTPase Rhoa in adult microglia is sufficient to trigger spontaneous microglia activation, producing a neurological phenotype (including synapse and neuron loss, impairment of long-term potentiation [LTP], formation of ß-amyloid plaques, and memory deficits). Mechanistically, loss of Rhoa in microglia triggers Src activation and Src-mediated tumor necrosis factor (TNF) production, leading to excitotoxic glutamate secretion. Inhibiting Src in microglia Rhoa-deficient mice attenuates microglia dysregulation and the ensuing neurological phenotype. We also find that the Rhoa/Src signaling pathway is disrupted in microglia of the APP/PS1 mouse model of Alzheimer disease and that low doses of Aß oligomers trigger microglia neurotoxic polarization through the disruption of Rhoa-to-Src signaling. Overall, our results indicate that disturbing Rho GTPase signaling in microglia can directly cause neurodegeneration.

Postnatal development of cerebrovascular structure and the neurogliovascular unit.

The unceasing metabolic demands of brain function are supported by an intricate three-dimensional network of arterioles, capillaries, and venules, designed to effectively distribute blood to all neurons and to provide shelter from harmful molecules in the blood. The development and maturation of this microvasculature involves a complex interplay between endothelial cells with nearly all other brain cell types (pericytes, astrocytes, microglia, and neurons), orchestrated throughout embryogenesis and the first few weeks after birth in mice. Both the expansion and regression of vascular networks occur during the postnatal period of cerebrovascular remodeling. Pial vascular networks on the brain surface are dense at birth and are then selectively pruned during the postnatal period, with the most dramatic changes occurring in the pial venular network. This is contrasted to an expansion of subsurface capillary networks through the induction of angiogenesis. Concurrent with changes in vascular structure, the integration and cross talk of neurovascular cells lead to establishment of blood-brain barrier integrity and neurovascular coupling to ensure precise control of macromolecular passage and metabolic supply. While we still possess a limited understanding of the rules that control cerebrovascular development, we can begin to assemble a view of how this complex process evolves, as well as identify gaps in knowledge for the next steps of research. This article is categorized under: Nervous System Development > Vertebrates: Regional Development Vertebrate Organogenesis > Musculoskeletal and Vascular Nervous System Development > Vertebrates: General Principles.

The interplay between glioblastoma and microglia cells leads to endothelial cell monolayer dysfunction via the interleukin-6-induced JAK2/STAT3 pathway.

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor, with an average life expectancy of 12-15 months. GBM is highly infiltrated by microglial cells (MG) promoting tumor growth and invasiveness. Moreover, microglia activation and subsequent neuroinflammation seem to be involved in blood-brain barrier (BBB) dysfunction commonly observed in several central nervous system diseases, including brain tumors. Nevertheless, how the crosstalk between microglia and tumor cells interferes with BBB function is far from being clarified. Herein, we evaluated the effects of reciprocal interactions between MG and GBM cells in the barrier properties of brain endothelial cells (ECs), using an in vitro approach. The exposure of ECs to the inflammatory microenvironment mediated by MG-GBM crosstalk induced a decrease in the transendothelial electric resistance and an increase in permeability across the ECs (macromolecular flux of 4 kDa-fluorescein isothiocyanate and 70 kDa-Rhodamine B isothiocyanate-Dextran). These effects were accompanied by a downregulation of the intercellular junction proteins, ß-catenin and zonula occludens. Moreover, the dynamic interaction between microglia and tumor cells triggered the release of interleukin-6 (IL-6) by microglia and subsequent activation of the downstream Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) pathway. Interestingly, the depletion of IL-6 or the blockade of the JAK/STAT3 signaling with AG490 were able to prevent the EC hyperpermeability. Overall, we demonstrated that IL-6 released during MG-GBM crosstalk leads to barrier dysfunction through the activation of the JAK/STAT3 pathway in ECs and downregulation of intercellular junction proteins. These results provide new insights into the mechanisms underlying the disruption of BBB permeability in GBM.

Effect of chronic methylphenidate treatment on hippocampal neurovascular unit and memory performance in late adolescent rats.

Methylphenidate (MPH) is the classic treatment for attention deficit hyperactivity disorder (ADHD) among children and adults. Despite its beneficial effects, non-medical use of MPH is nowadays a problem with high impact on society. Thus, our goal was to uncover the neurovascular and cognitive effects of MPH chronic use during a critical period of development in control conditions. For that, male Wistar Kyoto rats were treated with MPH (1.5 or 5 mg/kg/day at weekdays, per os) from P28 to P55. We concluded that the higher dose of MPH caused hippocampal blood-brain barrier (BBB) hyperpermeability by vesicular transport (transcytosis) concomitantly with the presence of peripheral immune cells in the brain parenchyma. These observations were confirmed by in vitro studies, in which the knockdown of caveolin-1 in human brain endothelial cells prevented the increased permeability and leukocytes transmigration triggered by MPH (100 µM, 24 h). Furthermore, MPH led to astrocytic atrophy and to a decrease in the levels of several synaptic proteins and impairment of AKT/CREB signaling, together with working memory deficit assessed in the Y-maze test. On the contrary, we verified that the lower dose of MPH (1.5 mg/kg/day) increased astrocytic processes and upregulated several neuronal proteins as well as signaling pathways involved in synaptic plasticity culminating in working memory improvement. In conclusion, the present study reveals that a lower dose of MPH in normal rats improves memory performance being associated with the modulation of astrocytic morphology and synaptic machinery. However, a higher dose of MPH leads to BBB dysfunction and memory impairment.