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 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.

Is CAA a perivascular brain clearance disease? A discussion of the evidence to date and outlook for future studies.

The brain's network of perivascular channels for clearance of excess fluids and waste plays a critical role in the pathogenesis of several neurodegenerative diseases including cerebral amyloid angiopathy (CAA). CAA is the main cause of hemorrhagic stroke in the elderly, the most common vascular comorbidity in Alzheimer's disease and also implicated in adverse events related to anti-amyloid immunotherapy. Remarkably, the mechanisms governing perivascular clearance of soluble amyloid ß-a key culprit in CAA-from the brain to draining lymphatics and systemic circulation remains poorly understood. This knowledge gap is critically important to bridge for understanding the pathophysiology of CAA and accelerate development of targeted therapeutics. The authors of this review recently converged their diverse expertise in the field of perivascular physiology to specifically address this problem within the framework of a Leducq Foundation Transatlantic Network of Excellence on Brain Clearance. This review discusses the overarching goal of the consortium and explores the evidence supporting or refuting the role of impaired perivascular clearance in the pathophysiology of CAA with a focus on translating observations from rodents to humans. We also discuss the anatomical features of perivascular channels as well as the biophysical characteristics of fluid and solute transport.

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.

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.

The Neurovasculome: Key Roles in Brain Health and Cognitive Impairment: A Scientific Statement From the American Heart Association/American Stroke Association.

BACKGROUND: Preservation of brain health has emerged as a leading public health priority for the aging world population. Advances in neurovascular biology have revealed an intricate relationship among brain cells, meninges, and the hematic and lymphatic vasculature (the neurovasculome) that is highly relevant to the maintenance of cognitive function. In this scientific statement, a multidisciplinary team of experts examines these advances, assesses their relevance to brain health and disease, identifies knowledge gaps, and provides future directions. METHODS: Authors with relevant expertise were selected in accordance with the American Heart Association conflict-of-interest management policy. They were assigned topics pertaining to their areas of expertise, reviewed the literature, and summarized the available data. RESULTS: The neurovasculome, composed of extracranial, intracranial, and meningeal vessels, as well as lymphatics and associated cells, subserves critical homeostatic functions vital for brain health. These include delivering O2 and nutrients through blood flow and regulating immune trafficking, as well as clearing pathogenic proteins through perivascular spaces and dural lymphatics. Single-cell omics technologies have unveiled an unprecedented molecular heterogeneity in the cellular components of the neurovasculome and have identified novel reciprocal interactions with brain cells. The evidence suggests a previously unappreciated diversity of the pathogenic mechanisms by which disruption of the neurovasculome contributes to cognitive dysfunction in neurovascular and neurodegenerative diseases, providing new opportunities for the prevention, recognition, and treatment of these conditions. CONCLUSIONS: These advances shed new light on the symbiotic relationship between the brain and its vessels and promise to provide new diagnostic and therapeutic approaches for brain disorders associated with cognitive dysfunction.

Endothelial Nitric Oxide Synthase-Deficient Mice: A Model of Spontaneous Cerebral Small-Vessel Disease.

Age-related cerebral small-vessel disease (CSVD) is a major cause of stroke and dementia. Despite a widespread acceptance of small-vessel arteriopathy, lacunar infarction, diffuse white matter injury, and cognitive impairment as four cardinal features of CSVD, a unifying pathologic mechanism of CSVD remains elusive. Herein, we introduce partial endothelial nitric oxide synthase (eNOS)-deficient mice as a model of age-dependent, spontaneous CSVD. These mice developed cerebral hypoperfusion and blood-brain barrier leakage at a young age, which progressively worsened with advanced age. Their brains exhibited elevated oxidative stress, astrogliosis, cerebral amyloid angiopathy, microbleeds, microinfarction, and white matter pathology. Partial eNOS-deficient mice developed gait disturbances at middle age, and hippocampus-dependent memory deficits at older ages. These mice also showed enhanced expression of bone morphogenetic protein 4 (BMP4) in brain pericytes before myelin loss and white matter pathology. Because BMP4 signaling not only promotes astrogliogenesis but also blocks oligodendrocyte differentiation, we posit that paracrine actions of BMP4, localized within the neurovascular unit, promote white matter disorganization and neurodegeneration. These observations point to BMP4 signaling pathway in the aging brain vasculature as a potential therapeutic target. Finally, because studies in partial eNOS-deficient mice corroborated recent clinical evidence that blood-brain barrier disruption is a primary cause of white matter pathology, the mechanism of impaired nitric oxide signaling-mediated CSVD warrants further investigation.

Pericytes as Inducers of Rapid, Matrix Metalloproteinase-9-Dependent Capillary Damage during Ischemia.

Blood-brain barrier disruption (BBB) and release of toxic blood molecules into the brain contributes to neuronal injury during stroke and other cerebrovascular diseases. While pericytes are builders and custodians of the BBB in the normal brain, their impact on BBB integrity during ischemia remains unclear. We imaged pericyte-labeled transgenic mice with in vivo two-photon microscopy to examine the relationship between pericytes and blood plasma leakage during photothrombotic occlusion of cortical capillaries. Upon cessation of capillary flow, we observed that plasma leakage occurred with three times greater frequency in regions where pericyte somata adjoined the endothelium. Pericyte somata covered only 7% of the total capillary length in cortex, indicating that a disproportionate amount of leakage occurred from a small fraction of the capillary bed. Plasma leakage was preceded by rapid activation of matrix metalloproteinase (MMP) at pericyte somata, which was visualized at high resolution in vivo using a fluorescent probe for matrix metalloproteinase-2/9 activity, fluorescein isothiocyanate (FITC)-gelatin. Coinjection of an MMP-9 inhibitor, but not an MMP-2 inhibitor, reduced pericyte-associated FITC-gelatin fluorescence and plasma leakage. These results suggest that pericytes contribute to rapid and localized proteolytic degradation of the BBB during cerebral ischemia. SIGNIFICANCE STATEMENT: Pericytes are a key component of the neurovascular unit and are essential for normal BBB function. However, during acute ischemia, we find that pericytes are involved in creating rapid and heterogeneous BBB disruption in the capillary bed. The mechanism by which pericytes contribute to BBB damage warrants further investigation, as it may yield new therapeutic targets for acute stroke injury and other neurological diseases involving capillary flow impairment.

Does pathology of small venules contribute to cerebral microinfarcts and dementia?

Microinfarcts are small, but strikingly common, ischemic brain lesions in the aging human brain. There is mounting evidence that microinfarcts contribute to vascular cognitive impairment and dementia, but the origins of microinfarcts are unclear. Understanding the vascular pathologies that cause microinfarcts may yield strategies to prevent their occurrence and reduce their deleterious effects on brain function. Current thinking suggests that cortical microinfarcts arise from the occlusion of penetrating arterioles, which are responsible for delivering oxygenated blood to small volumes of tissue. Unexpectedly, pre-clinical studies have shown that the occlusion of penetrating venules, which drain deoxygenated blood from cortex, lead to microinfarcts that appear identical to those resulting from arteriole occlusion. Here we discuss the idea that cerebral venule pathology could be an overlooked source for brain microinfarcts in humans. This article is part of the Special Issue "Vascular Dementia". Cover Image for this Issue: doi: 10.1111/jnc.14167.

Structural plasticity of the ventral stream and aphasia recovery.

Restrengthening of the residual language network is likely to be crucial for speech recovery in poststroke aphasia. Eight participants with chronic aphasia received intensive speech therapy for 3 weeks, with standardized naming tests and brain magnetic resonance imaging before and after therapy. Kurtosis-based diffusion tensor tractography was used to measure mean kurtosis (MK) along a segment of the inferior longitudinal fasciculus (ILF). Therapy-related reduction in the number of semantic but not phonemic errors was associated with strengthening (renormalization) of ILF MK (r = -0.90, p < 0.05 corrected), suggesting that speech recovery is related to structural plasticity of language-specific components of the residual language network. Ann Neurol 2017;82:147-151.

A murine toolbox for imaging the neurovascular unit.

The neurovascular unit (NVU) coordinates many essential functions in the brain including blood flow control, nutrient delivery, and maintenance of BBB integrity. These functions are the result of a cellular and molecular interplay that we are just beginning to understand. Cells of the NVU can now be investigated in the intact brain through the combined use of high-resolution in vivo imaging and non-invasive molecular tools to observe and manipulate cell function. Mouse lines that target transgene expression to cells of the NVU will be of great value in future work. However, a detailed evaluation of target cell specificity and expression pattern within the brain is required for many existing lines. The purpose of this review was to catalog mouse lines available to cerebrovascular biologists and to discuss their utility and limitations in future imaging studies.

Robust and fragile aspects of cortical blood flow in relation to the underlying angioarchitecture.

We review the organizational principles of the cortical vasculature and the underlying patterns of blood flow under normal conditions and in response to occlusion of single vessels. The cortex is sourced by a two-dimensional network of pial arterioles that feeds a three-dimensional network of subsurface microvessels in close proximity to neurons and glia. Blood flow within the surface and subsurface networks is largely insensitive to occlusion of a single vessel within either network. However, the penetrating arterioles that connect the pial network to the subsurface network are bottlenecks to flow; occlusion of even a single penetrating arteriole results in the death of a 500 µm diameter cylinder of cortical tissue despite the potential for collateral flow through microvessels. This pattern of flow is consistent with that calculated from a full reconstruction of the angioarchitecture. Conceptually, collateral flow is insufficient to compensate for the occlusion of a penetrating arteriole because penetrating venules act as shunts of blood that flows through collaterals. Future directions that stem from the analysis of the angioarchitecture concern cellular-level issues, in particular the regulation of blood flow within the subsurface microvascular network, and system-level issues, in particular the role of penetrating arteriole occlusions in human cognitive impairment.

Fluctuating and sensory-induced vasodynamics in rodent cortex extend arteriole capacity.

Neural activity in the brain is followed by localized changes in blood flow and volume. We address the relative change in volume for arteriole vs. venous blood within primary vibrissa cortex of awake, head-fixed mice. Two-photon laser-scanning microscopy was used to measure spontaneous and sensory evoked changes in flow and volume at the level of single vessels. We find that arterioles exhibit slow (<1 Hz) spontaneous increases in their diameter, as well as pronounced dilation in response to both punctate and prolonged stimulation of the contralateral vibrissae. In contrast, venules dilate only in response to prolonged stimulation. We conclude that stimulation that occurs on the time scale of natural stimuli leads to a net increase in the reservoir of arteriole blood. Thus, a "bagpipe" model that highlights arteriole dilation should augment the current "balloon" model of venous distension in the interpretation of fMRI images.

Pericytes: Unsung heroes in myelin repair after neonatal brain hypoxia.

Preterm infants can face lasting neurodevelopmental challenges due to hypoxia-induced injury of the cerebral white matter. In this issue of Neuron, Ren et al.1 identify microvascular pericytes as unexpected targets for growth hormone signaling, which enhances angiogenesis and remyelination after hypoxic injury in the developing mouse brain.

Impaired drainage through capillary-venous networks contributes to age-related white matter loss.

The gradual loss of cerebral white matter contributes to cognitive decline during aging. However, microvascular networks that support the metabolic demands of white matter remain poorly defined. We used in vivo deep multi-photon imaging to characterize microvascular networks that perfuse cortical layer 6 and corpus callosum, a highly studied region of white matter in the mouse brain. We show that these deep tissues are exclusively drained by sparse and wide-reaching venules, termed principal cortical venules, which mirror vascular architecture at the human cortical-U fiber interface. During aging, capillary networks draining into deep branches of principal cortical venules are selectively constricted, reduced in density, and diminished in pericyte numbers. This causes hypo-perfusion in deep tissues, and correlates with gliosis and demyelination, whereas superficial tissues become relatively hyper-perfused. Thus, age-related impairment of capillary-venular drainage is a key vascular deficit that contributes to the unique vulnerability of cerebral white matter during brain aging.

Chronic optical access through a polished and reinforced thinned skull.

We present a method to form an optical window in the mouse skull that spans millimeters and is stable for months without causing brain inflammation. This enabled us to repeatedly image blood flow in cortical capillaries of awake mice and determine long-range correlations in speed. We also repeatedly imaged dendritic spines, microglia and angioarchitecture, as well as used illumination to drive motor output via optogenetics and induce microstrokes via photosensitizers.

Topological basis for the robust distribution of blood to rodent neocortex.

The maintenance of robust blood flow to the brain is crucial to the health of brain tissue. We examined the pial network of the middle cerebral artery, which distributes blood from the cerebral arteries to the penetrating arterioles that source neocortical microvasculature, to characterize how vascular topology may support such robustness. For both mice and rats, two features dominate the topology. First, interconnected loops span the entire territory sourced by the middle cerebral artery. Although the loops comprise <10% of all branches, they maintain the overall connectivity of the network after multiple breaks. Second, >80% of offshoots from the loops are stubs that end in a single penetrating arteriole, as opposed to trees with multiple penetrating arterioles. We hypothesize that the loops and stubs protect blood flow to the parenchyma from an occlusion in a surface vessel. To test this, we assayed the viability of tissue that was sourced by an individual penetrating arteriole following occlusion of a proximal branch in the surface loop. We observed that neurons remained healthy, even when occlusion led to a reduction in the local blood flow. In contrast, direct blockage of a single penetrating arteriole invariably led to neuronal death and formation of a cyst. Our results show that the surface vasculature functions as a grid for the robust allocation of blood in the event of vascular dysfunction. The combined results of the present and prior studies imply that the pial network reallocates blood in response to changing metabolic needs.

The elusive brain perivascular fibroblast: a potential role in vascular stability and homeostasis.

In the brain, perivascular fibroblasts (PVFs) reside within the perivascular spaces (PVSs) of arterioles and large venules, however their physiological and pathophysiological roles remain largely unknown. PVFs express numerous extracellular matrix proteins that are found in the basement membrane and PVS surrounding large diameter vessels. PVFs are sandwiched between the mural cell layer and astrocytic endfeet, where they are poised to interact with mural cells, perivascular macrophages, and astrocytes. We draw connections between the more well-studied PVF pro-fibrotic response in ischemic injury and the less understood thickening of the vascular wall and enlargement of the PVS described in dementia and neurodegenerative diseases. We postulate that PVFs may be responsible for stability and homeostasis of the brain vasculature, and may also contribute to changes within the PVS during disease.

Capillary regression leads to sustained local hypoperfusion by inducing constriction of upstream transitional vessels.

In the brain, a microvascular sensory web coordinates oxygen delivery to regions of neuronal activity. This involves a dense network of capillaries that send conductive signals upstream to feeding arterioles to promote vasodilation and blood flow. Although this process is critical to the metabolic supply of healthy brain tissue, it may also be a point of vulnerability in disease. Deterioration of capillary networks is a hallmark of many neurological disorders and how this web is engaged during vascular damage remains unknown. We performed in vivo two-photon microscopy on young adult mural cell reporter mice and induced focal capillary injuries using precise two-photon laser irradiation of single capillaries. We found that ∼63% of the injuries resulted in regression of the capillary segment 7-14 days following injury, and the remaining repaired to re-establish blood flow within 7 days. Injuries that resulted in capillary regression induced sustained vasoconstriction in the upstream arteriole-capillary transition (ACT) zone at least 21 days post-injury in both awake and anesthetized mice. This abnormal vasoconstriction involved attenuation of vasomotor dynamics and uncoupling from mural cell calcium signaling following capillary regression. Consequently, blood flow was reduced in the ACT zone and in secondary, uninjured downstream capillaries. These findings demonstrate how capillary injury and regression, as often seen in age-related neurological disease, can impair the microvascular sensory web and contribute to cerebral hypoperfusion. SIGNIFICANCE: Deterioration of the capillary network is a characteristic of many neurological diseases and can exacerbate neuronal dysfunction and degeneration due to poor blood perfusion. Here we show that focal capillary injuries can induce vessel regression and elicit sustained vasoconstriction in upstream transitional vessels that branch from cortical penetrating arterioles. This reduces blood flow to broader, uninjured regions of the same microvascular network. These findings suggest that widespread and cumulative damage to brain capillaries in neurological disease may broadly affect blood supply and contribute to hypoperfusion through their remote actions.