New players in the neurovascular unit: Insights from experimental and clinical epilepsy

The conventional notion that neurons are exclusively responsible for brain signaling is increasingly challenged by the idea that brain function in fact depends on a complex interplay between neurons, glial cells, vascular endothelium, and immune-related blood cells. Recent data demonstrates that neuronal activity is profoundly affected by an entire cellular and extracellular ‘orchestra’, the so-called neurovascular unit (NVU). Among the ‘musical instruments’ of this orchestra, there may be molecules long-known in biomedicine as important mediators of inflammatory and immune responses in the organism, as well as non-neuronal cells, e.g., leukocytes. We here review recent evidence on the structure and function of the NVU, both in the healthy brain and in pathological conditions, such as the abnormal NVU activation observed in epilepsy. We will argue that a better understanding of NVU function will require the addition of new players to the ‘orchestra’.     

Neurovascular pathways to neurodegeneration in Alzheimer's disease and other disorders

The neurovascular unit (NVU) comprises brain endothelial cells, pericytes or vascular smooth muscle cells, glia and neurons. The NVU controls blood-brain barrier (BBB) permeability and cerebral blood flow, and maintains the chemical composition of the neuronal 'milieu', which is required for proper functioning of neuronal circuits. Recent evidence indicates that BBB dysfunction is associated with the accumulation of several vasculotoxic and neurotoxic molecules within brain parenchyma, a reduction in cerebral blood flow, and hypoxia. Together, these vascular-derived insults might initiate and/or contribute to neuronal degeneration. This article examines mechanisms of BBB dysfunction in neurodegenerative disorders, notably Alzheimer's disease, and highlights therapeutic opportunities relating to these neurovascular deficits.     

神经血管单元和Notch信号通路在缺血性卒中后的作用

缺血性卒中是临床常见疾病,且致死致残率高,幸存的患者预后多不同程度的患有偏瘫等后遗症,但目前还没有好的治疗方法。很长一段时间以来,卒中后的治疗关注点在于神经元的保护,割裂了神经元和周围细胞的联系。2001年,"神经血管单元"概念的提出为缺血性卒中的临床治疗提供了新的角度。此外,有研究表明Notch信号通路参与了神经、血管再生过程,对于卒中后神经血管单元的修复有调节作用。因此,本文从神经血管单元和Notch信号通路两个切入点综述了二者在缺血性卒中发生后的作用。     

How can microbial interactions with the blood–brain barrier modulate astroglial and neuronal function?

The vascular endothelium of the blood-brain barrier (BBB) is regarded as a part of the neurovascular unit (NVU). This emerging NVU concept emphasizes the need for homeostatic signalling among the neuronal, glial and vascular endothelial cellular compartments in maintaining normal brain function. Conversely, dysfunction in any component of the NVU affects another, thus contributing to disease. Brain endothelial activation and dysfunction is observed in various neurological diseases, such as (ischemic) stroke, seizure, brain inflammation and infectious diseases and likely contributes to or exacerbates neurological conditions. The role and impact of brain endothelial factors on astroglial and neuronal activation is unclear. Similarly, it is not clear which stages of BBB endothelial activation can be considered beneficial versus detrimental. Although the BBB plays an important role in context of encephalopathies caused by neurotropic microbes that must first penetrate into the brain, a crucial role of the BBB in contributing to neurological dysfunction may be seen in cerebral malaria (CM), where the Plasmodium parasite remains sequestered in the brain vasculature, does not enter the brain parenchyma, and yet causes coma and seizures. In this minireview some of the scenarios and factors that may play a role in BBB as a relay station to modulate astroneuronal functioning are discussed.     

Effects of Thrombin on the Neurovascular Unit in Cerebral Ischemia

Cerebral ischemia is a cerebrovascular disease with high morbidity and mortality that poses a significant burden on society and the economy. About 60% of cerebral ischemia is caused by thrombus, and the formation of thrombus proceeds from insoluble fibrin, following its transformation from liquid fibrinogen. In thrombus-induced ischemia, increased permeability of the blood–brain barrier (BBB), followed by the extravasation of blood components into the brain results in an altered brain microenvironment. Changes in the brain microenvironment affect brain function and the neurovascular unit (NVU), the working unit of the brain. Recent studies have reported that coagulation factors interact with the NVU and its components, but the specific function of this interaction is highly speculative and warrants further investigations. In this article, we reviewed the role of coagulation factors in cerebral ischemia and the role of coagulation factors in thrombosis. Additionally, the influence of thrombin on the NVU is introduced, as well as in the function of NVU, which may help to explore part of brain injury mechanism during ischemia. Lastly, we propose some novel therapeutic approaches on ischemic stroke by reducing the risk of coagulation.

     

Purinergic mechanisms in gliovascular coupling

Regional elevations in cerebral blood flow (CBF) often occur in response to localized increases in cerebral neuronal activity. An ever expanding literature has linked this neurovascular coupling process to specific signaling pathways involving neuronal synapses, astrocytes and cerebral arteries and arterioles. Collectively, these structures are termed the "neurovascular unit" (NVU). Astrocytes are thought to be the cornerstone of the NVU. Thus, not only do astrocytes "detect" increased synaptic activity, they can transmit that information to proximal and remote astrocytic sites often through a Ca(2+)- and ATP-related signaling process. At the vascular end of the NVU, a Ca(2+)-dependent formation and release of vasodilators, or substances linked to vasodilation, can occur. The latter category includes ATP, which upon its appearance in the extracellular compartment, can be rapidly converted to the potent vasodilator, adenosine, via the action of ecto-nucleotidases. In the present review, we give consideration to experimental model-specific variations in purinergic influences on gliovascular signaling mechanisms, focusing on the cerebral cortex. In that discussion, we compare findings obtained using in vitro (rodent brain slice) models and multiple in vivo models (2-photon imaging; somatosensory stimulation-evoked cortical hyperemia; and sciatic nerve stimulation-evoked pial arteriolar dilation). Additional attention is given to the importance of upstream (remote) vasodilation; the key role played by extracellular ATP hydrolysis (via ecto-nucleotidases) in gliovascular coupling; and interactions among multiple signaling pathways.     

Automated image analysis methods for 3-D quantification of the neurovascular unit from multichannel confocal microscope images.

BACKGROUND: There is a need for integrative and quantitative methods to investigate the structural and functional relations among elements of complex systems, such as the neurovascular unit (NVU), that involve multiple cell types, microvasculatures, and various genomic/proteomic/ionic functional entities. METHODS: Vascular casting and selective labeling enabled simultaneous three-dimensional imaging of the microvasculature, cell nuclei, and cytoplasmic stains. Multidimensional segmentation was achieved by (i) bleed-through removal and attenuation correction; (ii) independent segmentation and morphometry for each corrected channel; and (iii) spatially associative feature computation across channels. The combined measurements enabled cell classification based on nuclear morphometry, cytoplasmic signals, and distance from vascular elements. Specific spatial relations among the NVU elements could be quantified. RESULTS: A software system combining nuclear and vessel segmentation codes and associative features was constructed and validated. Biological variability contributed to misidentified nuclei (9.3%), undersegmentation of nuclei (3.7%), hypersegmentation of nuclei (14%), and missed nuclei (4.7%). Microvessel segmentation errors occurred rarely, mainly due to nonuniform lumen staining. CONCLUSIONS: Associative features across fluorescence channels, in combination with standard features, enable integrative structural and functional analysis of the NVU. By labeling additional structural and functional entities, this method can be scaled up to larger-scale systems biology studies that integrate spatial and molecular information.     

Blood–Brain Barrier Breakdown in Stress and Neurodegeneration: Biochemical Mechanisms and New Models for Translational Research

Biochemistry (Moscow) - Blood-brain barrier (BBB) is a structural and functional element of the neurovascular unit (NVU), which includes cells of neuronal, glial, and endothelial nature. The main...     

Understanding How Exercise Promotes Cognitive Integrity in the Aging Brain

Alterations in the structure and organization of the aging central nervous system (CNS), and associated functional deficits, result in cognitive decline and increase susceptibility to neurodegeneration. Age-related changes to the neurovascular unit (NVU), and their consequences for cerebrovascular function, are implicated as driving cognitive impairment during aging as well as in neurodegenerative disease. The molecular events underlying these effects are incompletely characterized. Similarly, the mechanisms underlying effects of factors that reduce the impact of aging on the brain, such as physical exercise, are also opaque. A study in this issue of PLOS Biology links the NVU to cognitive decline in the aging brain and suggests a potential underlying molecular mechanism. Notably, the study further links the protective effects of chronic exercise on cognition to neurovascular integrity during aging.     

In Vitro Modeling of the Neurovascular Environment by Coculturing Adult Human Brain Endothelial Cells with Human Neural Stem Cells

Brain and vascular cells form a functionally integrated signalling network that is known as the neurovascular unit (NVU). The signalling (autocrine, paracrine and juxtacrine) between different elements of this unit, especially in humans, is difficult to disentangle in vivo. Developing representative in vitro models is therefore essential to better understand the cellular interactions that govern the neurovascular environment. We here describe a novel approach to assay these cellular interactions by combining a human adult cerebral microvascular endothelial cell line (hCMEC/D3) with a fetal ganglionic eminence-derived neural stem cell (hNSC) line. These cell lines provide abundant homogeneous populations of cells to produce a consistently reproducible in vitro model of endothelial morphogenesis and the ensuing NVU. Vasculature-like structures (VLS) interspersed with patches of differentiating neural cells only occurred when hNSCs were seeded onto a differentiated endothelium. These VLS emerged within 3 days of coculture and by day 6 were stabilizing. After 7 days of coculture, neuronal differentiation of hNSCs was increased 3-fold, but had no significant effect on astrocyte or oligodendrocyte differentiation. ZO1, a marker of adherens and tight junctions, was highly expressed in both undifferentiated and differentiated endothelial cells, but the adherens junction markers CD31 and VE-cadherin were significantly reduced in coculture by approximately 20%. A basement membrane, consisting of laminin, vitronectin, and collagen I and IV, separated the VLS from neural patches. This simple assay can assist in elucidating the cellular and molecular signaling involved in the formation of VLS, as well as the enhancement of neuronal differentiation through endothelial signaling.     

Combined treatment of sodium ferulate,n‐butylidenephthalide,and ADSCs rehabilitates neurovascular unit in rats after photothrombotic stroke

The remodelling of structural and functional neurovascular unit (NVU) becomes a central therapeutic strategy after cerebral ischaemic stroke. In the present study, we investigated the effect of combined therapy of sodium ferulate (SF), n‐butylidenephthalide (BP) and adipose‐derived stromal cells (ADSCs) to ameliorate the injured NVU in the photochemically induced thrombotic stroke in rats. After solely or combined treatment, the neovascularization, activation of astrocytes, neurogenesis, expressions of vascular endothelial growth factor (VEGF) and claudin‐5 were assessed by immunohistochemical or immunofluorescence staining. In order to uncover the underlying mechanism of therapeutic effect, signalling of protein kinase B/mammalian target of rapamycin (AKT/mTOR), extracellular signal‐regulated kinase 1/2 (ERK1/2), and Notch1 in infarct zone were analysed by western blot. ¹⁸F‐2‐deoxy‐glucose/positron emission tomography, magnetic resonance imaging, Evans blue staining were employed to evaluate the glucose metabolism, cerebral blood flow (CBF), and brain‐blood barrier (BBB) permeability, respectively. The results showed that combined treatment increased the neovascularization, neurogenesis, and VEGF secretion, modulated the astrocyte activation, enhanced the regional CBF, and glucose metabolism, as well as reduced BBB permeability and promoted claudin‐5 expression, indicating the restoration of structure and function of NVU. The activation of ERK1/2 and Notch1 pathways and inhibition of AKT/mTOR pathway might be involved in the therapeutic mechanism. In summary, we have demonstrated that combined ADSCs with SF and BP, targeting the NVU remodelling, is a potential treatment for ischaemic stroke. These results may provide valuable information for developing future combined cellular and pharmacological therapeutic strategy for ischaemic stroke.     

Oxygen-glucose deprivation and interleukin-1α trigger the release of perlecan LG3 by cells of neurovascular unit

Two of the main stresses faced by cells at the neurovascular unit (NVU) as an immediate result of cerebral ischemia are oxygen-glucose deprivation (OGD)/reperfusion and inflammatory stress caused by up regulation of IL-1. As a result of these stresses, perlecan, an important component of the NVU extracellular matrix, is highly proteolyzed. In this study, we describe that focal cerebral ischemia in rats results in increased generation of laminin globular domain 3 (LG3), the c-terminal bioactive fragment of perlecan. Further, in vitro study of the cells of the NVU was performed to locate the source of this increased perlecan-LG3. Neurons, astrocytes, brain endothelial cells and pericytes were exposed to OGD/reperfusion and IL-1α/β. It was observed that neurons and pericytes showed increased levels of LG3 during OGD in their culture media. During in vitro reperfusion, neurons, astrocytes and pericytes showed elevated levels of LG3, but only after exposure to brief durations of OGD. IL-1α and IL-1β treatment tended to have opposite effects on NVU cells. While IL-1α increased or had minimal to no effect on LG3 generation, high concentrations of IL-1β decreased it in most cells studied. Finally, LG3 was determined to be neuroprotective and anti-proliferative in brain endothelial cells, suggesting a possible role for the generation of LG3 in the ischemic brain.     

Integrin-mediated regulation of neurovascular development,physiology and disease

The mammalian central nervous system (CNS) is comprised of billions of neurons and glia that are intertwined with an elaborate network of blood vessels. These various neural and vascular cell types actively converse with one another to form integrated, multifunctional complexes, termed neurovascular units. Cell-cell communication within neurovascular units promotes normal CNS development and homeostasis, and abnormal regulation of these events leads to a variety of debilitating CNS diseases. This review will summarize (i) cellular and molecular mechanisms that regulate physiological assembly and maintenance of neurovascular units; and (ii) signaling events that induce pathological alterations in neurovascular unit formation and function. An emphasis will be placed on neural-vascular cell adhesion events mediated by integrins and their extracellular matrix (ECM) ligands. I will highlight the role of a specific adhesion and signaling axis involving αvβ8 integrin, latent transforming growth factor β’s (TGFβ’s), and canonical TGFβ receptors. Possible functional links between components of this axis and other signal transduction cascades implicated in neurovascular development and disease will be discussed. In summary, comprehensively understanding the pathways that regulate bidirectional neural-vascular cell contact and communication will provide new insights into the mechanisms of neurovascular unit development, physiology and disease.     

A Working Module for the Neurovascular Unit in the Sexually Dimorphic Nucleus of the Preoptic Area

The neurovascular unit (NVU) can be conceptualized as a functional entity consisting of neurons, astrocytes, pericytes, and endothelial and smooth muscle cells that operate in concert to affect blood flow to a very circumscribed area. Although we are currently in a “golden era” of bioengineering, there are, as yet, no living NVUs-on-a-chip modules available and the development of a neural chip that would mimic NVUs is a seemingly lofty goal. The sexually dimorphic nucleus of the preoptic area (SDN-POA) is a tiny brain structure (between 0.001~0.007 mm³ in rats) with an assessable biological function (i.e., male sexual behavior). The present effort was undertaken to determine whether there are identifiable NVUs in the SDN-POA by assessing its vasculature relative to its known neural components. First, a thorough and systematic review of thousands of histologic and immunofluorescent images from 201 weanling and adult rats was undertaken to define the characteristics of the vessels supplying the SDN-POA: its primary supply artery/arteriole and capillaries are physically inseparable from their neural elements. A subsequent immunofluorescent study targeting α-smooth muscle actin confirmed the identity of an artery/arteriole supplying the SDN-POA. In reality, the predominant components of the SDN-POA are calbindin D28k-positive neurons that are comingled with tyrosine hydroxylase-positive projections. Finally, a schematic of an SDN-POA NVU is proposed as a working model of the basic building block of the CNS. Such modules could serve the study of neurovascular mechanisms and potentially inform the development of next generation bioengineered neural transplants, i.e., the construct of an NVU neural chip.     

A Novel Brain Neurovascular Unit Model with Neurons,Astrocytes and Microvascular Endothelial Cells of Rat

A novel triple cell neurovascular unit (NVU) model co-culturing with neurons, brain microvascular endothelial cells (BMECs) and astrocytes was established in this study for investigating the cerebral diseases and screening the candidates of therapeutic drug. We have first performed the cell identification and morphological characterization, analyzed the specific protein expression and determined the blood-brain barrier (BBB) function of the co-culture model under normal condition. Then, we further determined the BBB function, inflammation, cell injury and the variation of neuroprotective factor in this model after anoxia-reoxygenation. The results suggest that this model exhibited a better BBB function and significantly increased expression of P-glycoprotein (Pg-P) and ZO-1 compared with BMECs only or co-culture with astrocytes or neurons. After anoxia-reoxygenation, the pathological changes of this model were basically resemblance to the pathological changes of brain cells and BBB in vivo. And nimodipine, an antagonist of calcium, could reverse those changes as well. According to our observations, we deduce that this triple cell co-culture model exhibits the basic structure, function and cell-cell interaction of NVU, which may offer a more proper in vitro system of NVU for the further investigation of cerebral diseases and drug screening.     

A Novel In Vitro Model to Study Pericytes in the Neurovascular Unit of the Developing Cortex

Cortical function is impaired in various disorders of the central nervous system including Alzheimer’s disease, autism and schizophrenia. Some of these disorders are speculated to be associated with insults in early brain development. Pericytes have been shown to regulate neurovascular integrity in development, health and disease. Hence, precisely controlled mechanisms must have evolved in evolution to operate pericyte proliferation, repair and cell fate within the neurovascular unit (NVU). It is well established that pericyte deficiency leads to NVU injury resulting in cognitive decline and neuroinflammation in cortical layers. However, little is known about the role of pericytes in pathophysiological processes of the developing cortex. Here we introduce an in vitro model that enables to precisely study pericytes in the immature cortex and show that moderate inflammation and hypoxia result in caspase-3 mediated pericyte loss. Using heterozygous EYFP-NG2 mouse mutants we performed live imaging of pericytes for several days in vitro. In addition we show that pericytes maintain their capacity to proliferate which may allow cell-based therapies like reprogramming of pericytes into induced neuronal cells in the presented approach.     

Integrin-mediated regulation of neurovascular development,physiology and disease

The mammalian central nervous system (CNS) is comprised of billions of neurons and glia that are intertwined with an elaborate network of blood vessels. These various neural and vascular cell types actively converse with one another to form integrated, multifunctional complexes, termed neurovascular units. Cell-cell communication within neurovascular units promotes normal CNS development and homeostasis, and abnormal regulation of these events leads to a variety of debilitating CNS diseases. This review will summarize (1) cellular and molecular mechanisms that regulate physiological assembly and maintenance of neurovascular units; and (2) signaling events that induce pathological alterations in neurovascular unit formation and function. An emphasis will be placed on neural-vascular cell adhesion events mediated by integrins and their extracellular matrix (ECM) ligands. I will highlight the role of a specific adhesion and signaling axis involving αvβ8 integrin, latent transforming growth factor β''s (TGFβ''s), and canonical TGFβ receptors. Possible functional links between components of this axis and other signal transduction cascades implicated in neurovascular development and disease will be discussed. Comprehensively understanding the pathways that regulate bidirectional neural-vascular cell contact and communication will provide new insights into the mechanisms of neurovascular unit development, physiology and disease.Key words: αvβ8 integrin, latent TGFβ, neurovascular unit, brain angiogenesis, cerebral hemorrhage     

Glycolysis-mediated control of blood-brain barrier development and function

The blood-brain barrier (BBB) consists of differentiated cells integrating in one ensemble to control transport processes between the central nervous system (CNS) and peripheral blood. Molecular organization of BBB affects the extracellular content and cell metabolism in the CNS. Developmental aspects of BBB attract much attention in recent years, and barriergenesis is currently recognized as a very important and complex mechanism of CNS development and maturation. Metabolic control of angiogenesis/barriergenesis may be provided by glucose utilization within the neurovascular unit (NVU). The role of glycolysis in the brain has been reconsidered recently, and it is recognized now not only as a process active in hypoxic conditions, but also as a mechanism affecting signal transduction, synaptic activity, and brain development. There is growing evidence that glycolysis-derived metabolites, particularly, lactate, affect barriergenesis and functioning of BBB. In the brain, lactate produced in astrocytes or endothelial cells can be transported to the extracellular space via monocarboxylate transporters (MCTs), and may act on the adjoining cells via specific lactate receptors. Astrocytes are one of the major sources of lactate production in the brain and significantly contribute to the regulation of BBB development and functioning. Active glycolysis in astrocytes is required for effective support of neuronal activity and angiogenesis, while endothelial cells regulate bioavailability of lactate for brain cells adjusting its bidirectional transport through the BBB. In this article, we review the current knowledge with regard to energy production in endothelial and astroglial cells within the NVU. In addition, we describe lactate-driven mechanisms and action of alternative products of glucose metabolism affecting BBB structural and functional integrity in developing and mature brain.     

三七总皂苷对脑缺血再灌注大鼠神经血管单元超微结构的影响

为了观察脑缺血再灌注(cerebral ischemia reperfusion, CIR)大鼠缺血灶周边脑组织不同时间点神经血管单元(neurovascular unit, NVU)超微结构变化,研究三七总皂苷(Panax notoginseng saponins, PNS)对脑缺血再灌注大鼠脑组织NVU超微结构的影响,本研究采用改良Zea Longa法制作局灶性大脑中动脉闭塞(MCAO)模型,缺血2 h后再灌注;采用Longa法评分标准检测各组大鼠术后4 h神经功能评分,随之各组进行干预,分别在缺血再灌注后24 h、72 h、7 d、3周进行神经功能评分和透射电镜下观察各组大鼠缺血灶周边脑组织的NVU超微结构变化。研究结果表明,干预前即术后4 h治疗组和对照组神经功能评分比较无明显差异;PNS干预后治疗组大鼠神经功能评分逐渐改善,缺血再灌注后24 h与对照组比较,差异无统计学意义(p>0.05),再灌注72 h、7 d、3周的大鼠神经缺损评分与同时间点对照组相比差异具有统计学意义(p<0.05)。电镜观察发现再灌注24 h、72 h、7 d、3周治疗组大鼠脑组织NVU超微结构的病理形态损伤均较同时间点对照组明显减轻。本研究结论认为,PNS通过整合促进脑缺血后NVU的神经元、胶质细胞和微血管的修复,改善神经功能缺损症状,对脑缺血具有保护作用。     

Cell–cell Signaling in the Neurovascular Unit

Historically, the neuron has been the conceptual focus for almost all of neuroscience research. In recent years, however, the concept of the neurovascular unit has emerged as a new paradigm for investigating both physiology and pathology in the CNS. This concept proposes that a purely neurocentric focus is not sufficient, and emphasizes that all cell types in the brain including neuronal, glial and vascular components, must be examined in an integrated context. Cell–cell signaling and coupling between these different compartments form the basis for normal function. Disordered signaling and perturbed coupling form the basis for dysfunction and disease. In this mini-review, we will survey four examples of this phenomenon: hemodynamic neurovascular coupling linking blood flow to brain activity; cellular communications that evoke the blood–brain barrier phenotype; parallel systems that underlie both neurogenesis and angiogenesis in the CNS; and finally, the potential exchange of trophic factors that may link neuronal, glial and vascular homeostasis. Special issue in honor of Naren Banik.