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.

Venous-plexus-associated lymphoid hubs support meningeal humoral immunity.

There is increasing interest in how immune cells in the meninges-the membranes that surround the brain and spinal cord-contribute to homeostasis and disease in the central nervous system1,2. The outer layer of the meninges, the dura mater, has recently been described to contain both innate and adaptive immune cells, and functions as a site for B cell development3-6. Here we identify organized lymphoid structures that protect fenestrated vasculature in the dura mater. The most elaborate of these dural-associated lymphoid tissues (DALT) surrounded the rostral-rhinal confluence of the sinuses and included lymphatic vessels. We termed this structure, which interfaces with the skull bone marrow and a comparable venous plexus at the skull base, the rostral-rhinal venolymphatic hub. Immune aggregates were present in DALT during homeostasis and expanded with age or after challenge with systemic or nasal antigens. DALT contain germinal centre B cells and support the generation of somatically mutated, antibody-producing cells in response to a nasal pathogen challenge. Inhibition of lymphocyte entry into the rostral-rhinal hub at the time of nasal viral challenge abrogated the generation of germinal centre B cells and class-switched plasma cells, as did perturbation of B-T cell interactions. These data demonstrate a lymphoid structure around vasculature in the dura mater that can sample antigens and rapidly support humoral immune responses after local pathogen challenge.

Piezo1 Is a Mechanosensor Channel in Central Nervous System Capillaries.

Capillaries are equipped to sense neurovascular coupling agents released onto the outer wall of a capillary, translating these external signals into electrical/Ca2+ changes that play a crucial role in blood flow regulation and ensuring that neuronal demands are met. However, control mechanisms attributable to forces imposed onto the lumen are less clear. Here, we show that Piezo1 channels act as mechanosensors in central nervous system capillaries. Electrophysiological analyses confirmed expression and function of Piezo1 channels in brain cortical and retinal capillaries. Activation of Piezo1 channels evoked currents that were sensitive to endothelial cell-specific Piezo1 deletion. Using genetically encoded Ca2+ indicator mice and an ex vivo pressurized retina preparation, we found that activation of Piezo1 channels by mechanical forces triggered Ca2+ signals in capillary endothelial cells. Collectively, these findings indicate that Piezo1 channels are capillary mechanosensors that initiate crucial Ca2+ signals and could, therefore, have a profound impact on central nervous system blood flow control.

Leukaemia hijacks a neural mechanism to invade the central nervous system.

Acute lymphoblastic leukaemia (ALL) has a marked propensity to metastasize to the central nervous system (CNS). In contrast to brain metastases from solid tumours, metastases of ALL seldom involve the parenchyma but are isolated to the leptomeninges, which is an infrequent site for carcinomatous invasion. Although metastasis to the CNS occurs across all subtypes of ALL, a unifying mechanism for invasion has not yet been determined. Here we show that ALL cells in the circulation are unable to breach the blood-brain barrier in mice; instead, they migrate into the CNS along vessels that pass directly between vertebral or calvarial bone marrow and the subarachnoid space. The basement membrane of these bridging vessels is enriched in laminin, which is known to coordinate pathfinding of neuronal progenitor cells in the CNS. The laminin receptor α6 integrin is expressed in most cases of ALL. We found that α6 integrin-laminin interactions mediated the migration of ALL cells towards the cerebrospinal fluid in vitro. Mice with ALL xenografts were treated with either a PI3Kδ inhibitor, which decreased α6 integrin expression on ALL cells, or specific α6 integrin-neutralizing antibodies and showed significant reductions in ALL transit along bridging vessels, blast counts in the cerebrospinal fluid and CNS disease symptoms despite minimally decreased bone marrow disease burden. Our data suggest that α6 integrin expression, which is common in ALL, allows cells to use neural migratory pathways to invade the CNS.

A human cell type similar to murine central nervous system perivascular fibroblasts.

The brain vasculature has several specific features, one of them being the blood-brain barrier (BBB), which supports and protects the brain by allowing for the passage of oxygen and nutrients, while at the same time preventing passage of pathogens and toxins. The BBB also prevents efficient delivery of drugs to the brain, e.g. for treatment of brain tumors. In the murine brain, perivascular fibroblasts were recently identified as a novel potential constituent of the BBB. Here we present the existence of human cells that could be the equivalent to the murine brain perivascular fibroblasts. Using RNA sequencing, we show a similar transcriptomic profile of cultured human brain cells and murine perivascular fibroblasts. These data open up a window for new hypotheses on cell types involved in human CNS diseases.

A critical role for microglia in maintaining vascular integrity in the hypoxic spinal cord.

Hypoxic preconditioning reduces disease severity in a mouse model of multiple sclerosis (MS), in part by enhancing the barrier properties of spinal cord blood vessels. Because other studies have shown that similar levels of hypoxia transiently increase permeability of central nervous system (CNS) blood vessels, the goal of this study was to define the impact of chronic mild hypoxia (CMH, 8% O2) on the integrity of spinal cord blood vessels and the responses of neighboring glial cells. Using extravascular fibrinogen as a marker of vascular disruption, we found that CMH triggered transient vascular leak in spinal cord blood vessels, particularly in white matter, which was associated with clustering and activation of Mac-1-positive microglia around disrupted vessels. Microglial depletion with the colony stimulating factor-1 receptor (CSF-1R) inhibitor PLX5622, while having no effect under normoxic conditions, profoundly increased vascular leak in both white and gray matter during CMH, and this was associated with disruption of astrocyte-vascular coupling and enhanced loss of tight junction proteins. Microglial repair of leaky blood vessels was blocked by a peptide that inhibits the interaction between fibrinogen and its Mac-1 integrin receptor. These findings highlight an important role for microglia in maintaining vascular integrity in the hypoxic spinal cord and suggest that a fibrinogen-Mac-1 interaction underpins this response. As relative hypoxia is experienced in many situations including high altitude, lung disease, obstructive sleep apnea, and age-related CNS ischemia/hypoxia, our findings have important implications regarding the critical role of microglia in maintaining vascular integrity in the CNS.

Endothelial-Tumor Cell Interaction in Brain and CNS Malignancies.

Glioblastoma and other brain or CNS malignancies (like neuroblastoma and medulloblastoma) are difficult to treat and are characterized by excessive vascularization that favors further tumor growth. Since the mean overall survival of these types of diseases is low, the finding of new therapeutic approaches is imperative. In this review, we discuss the importance of the interaction between the endothelium and the tumor cells in brain and CNS malignancies. The different mechanisms of formation of new vessels that supply the tumor with nutrients are discussed. We also describe how the tumor cells (TC) alter the endothelial cell (EC) physiology in a way that favors tumorigenesis. In particular, mechanisms of EC-TC interaction are described such as (a) communication using secreted growth factors (i.e., VEGF, TGF-ß), (b) intercellular communication through gap junctions (i.e., Cx43), and (c) indirect interaction via intermediate cell types (pericytes, astrocytes, neurons, and immune cells). At the signaling level, we outline the role of important mediators, like the gasotransmitter nitric oxide and different types of reactive oxygen species and the systems producing them. Finally, we briefly discuss the current antiangiogenic therapies used against brain and CNS tumors and the potential of new pharmacological interventions that target the EC-TC interaction.

Shrinkage-mediated imaging of entire organs and organisms using uDISCO.

Recent tissue-clearing approaches have become important alternatives to standard histology approaches. However, light scattering in thick tissues and the size restrictions on samples that can be imaged with standard light-sheet microscopy pose limitations for analyzing large samples such as an entire rodent body. We developed 'ultimate DISCO' (uDISCO) clearing to overcome these limitations in volumetric imaging. uDISCO preserves fluorescent proteins over months and renders intact organs and rodent bodies transparent while reducing their size up to 65%. We used uDISCO to image neuronal connections and vasculature from head to toe over 7 cm and to perform unbiased screening of transplanted stem cells within the entire body of adult mice. uDISCO is compatible with diverse labeling methods and archival human tissue, and it can readily be used in various biomedical applications to study organization of large organ systems throughout entire organisms.

Synchrotron radiation micro-tomography for high-resolution neurovascular network morphology investigation.

There has been increasing interest in using high-resolution micro-tomography to investigate the morphology of neurovascular networks in the central nervous system, which remain difficult to characterize due to their microscopic size as well as their delicate and complex 3D structure. Synchrotron radiation X-ray imaging, which has emerged as a cutting-edge imaging technology with a high spatial resolution, provides a novel platform for the non-destructive imaging of microvasculature networks at a sub-micrometre scale. When coupled with computed tomography, this technique allows the characterization of the 3D morphology of vasculature. The current review focuses on recent progress in developing synchrotron radiation methodology and its application in probing neurovascular networks, especially the pathological changes associated with vascular abnormalities in various model systems. Furthermore, this tool represents a powerful imaging modality that improves our understanding of the complex biological interactions between vascular function and neuronal activity in both physiological and pathological states.

Astrocyte Signaling in the Neurovascular Unit After Central Nervous System Injury.

Astrocytes comprise the major non-neuronal cell population in the mammalian neurovascular unit. Traditionally, astrocytes are known to play broad roles in central nervous system (CNS) homeostasis, including the management of extracellular ion balance and pH, regulation of neurotransmission, and control of cerebral blood flow and metabolism. After CNS injury, cell⁻cell signaling between neuronal, glial, and vascular cells contribute to repair and recovery in the neurovascular unit. In this mini-review, we propose the idea that astrocytes play a central role in organizing these signals. During CNS recovery, reactive astrocytes communicate with almost all CNS cells and peripheral progenitors, resulting in the promotion of neurogenesis and angiogenesis, regulation of inflammatory response, and modulation of stem/progenitor response. Reciprocally, changes in neurons and vascular components of the remodeling brain should also influence astrocyte signaling. Therefore, understanding the complex and interdependent signaling pathways of reactive astrocytes after CNS injury may reveal fundamental mechanisms and targets for re-integrating the neurovascular unit and augmenting brain recovery.

[The application of hybrid operation suite in the management of cerebral and spinal vascular diseases and intracranial hypervascular tumors].

Objective: To evaluate the effect of hybrid operation suite in the treatment of cerebral and spinal vascular diseases and intracranial hypervascular tumors. Methods: A retrospective study was conducted on 132 patients with various cerebral and spinal vascular diseases and intracranial hypervascular tumors who were treated by hybrid surgery at Department of Neurosurgery, Huashan Hospital from October 2016 to December 2017.There were 70 male and 62 female patients with a mean age of 48.33 years (range: 14-78 years), including 64 cases of intracranial aneurysm (41 complicated aneurysm cases), 28 cases of brain arteriovenous malformation (BAVM), 12 cases of hypervascular tumor, 12 cases of dural arteriovenous fistula (DAVF), 6 cases of carotid artery stenosis, 5 cases of Moyamoya disease, 3 cases of intracranial aneurysm or BAVM combined with tumor, 1 case of scalp arteriovenous fistula and 1 case of critical brain trauma in which a foreign metal stick approached the basal vascular circuit.Abnormalities were found in 16 cases in intraoperative angiography. The clinical data of all patients was collected as a perspective cohort. The success rate of hybrid surgery, intra-operative and post-operative complications, morbidity, mortality, rate of infection, the length of hospital stay were all analyzed to illustrate the effect of hybrid operation mode to traditional surgical pattern. Results: For 64 cases with intracranial aneurysms, the immediate complete occlusion rate was 90.5%, with a mortality of 4.7% and a morbidity of 14.0%. For 28 cases of BAVM and 12 cases of DAVF, all patients achieved total obliteration and favorable social independent outcomes after hybrid surgery, with no complication.For 6 cases of carotid artery stenosis and 5 cases of Moyamoya, intra-operative confirmed good cerebral reperfusion without any new post-operative neurologic deficits. After tumor vessels embolization, 4 out of 12 cases of hypervascular tumor needed intra-operative blood transfusion, and all patients achieved total tumor resection in a single stage. Only one patient with medulla oblongata hemangioblastoma died 6 months after operation due to respiratory deficit related pneumonia. Compared to traditional surgeries, the hybrid operation pattern did not significantly increase the total infection rate, central nervous system infection rate, hospital stay days and post-operative hospital stay days (all P>0.05) while the in-patient cost increased mildly (119 332 yuan vs.98 215 yuan, t=2.38, P=0.02). Conclusions: The operations of complex cerebral and spinal vascular diseases and intracranial hypervascular tumors can be performed in hybrid operation suite safely.This surgical mode can ensure the quality of operation and promote the development of innovative and complicated surgical procedures.

[Molecular mechanism for the establishment of blood-vessel gateway for immune cells in the CNS.]

We have been studying about the molecular mechanism responsible for the establishment of the blood-vessel gateway through which immune cells enter the CNS. We have discovered three kinds of gateways in a multiple sclerosis model, EAE, based on the neural stimulations and named them the gravity-gateway reflex, electric-gateway reflex, and pain-gateway reflex, respectively. All gateway reflexes are involved in specific crosstalk between sensory-sympathetic pathways. For example, in the gravity-gateway reflex, gravity-mediated sensory stimulation via the soleus muscles activates fifth lumber(L5)dorsal loot ganglions to activate L5 sympathetic ganglions, which express norepinephrine at specific vessels of the L5 cord. We explain these three types of gateway reflexes in this chapter.

Sleep Deprivation Impairs the Human Central and Peripheral Nervous System Discrimination of Social Threat.

Facial expressions represent one of the most salient cues in our environment. They communicate the affective state and intent of an individual and, if interpreted correctly, adaptively influence the behavior of others in return. Processing of such affective stimuli is known to require reciprocal signaling between central viscerosensory brain regions and peripheral-autonomic body systems, culminating in accurate emotion discrimination. Despite emerging links between sleep and affective regulation, the impact of sleep loss on the discrimination of complex social emotions within and between the CNS and PNS remains unknown. Here, we demonstrate in humans that sleep deprivation impairs both viscerosensory brain (anterior insula, anterior cingulate cortex, amygdala) and autonomic-cardiac discrimination of threatening from affiliative facial cues. Moreover, sleep deprivation significantly degrades the normally reciprocal associations between these central and peripheral emotion-signaling systems, most prominent at the level of cardiac-amygdala coupling. In addition, REM sleep physiology across the sleep-rested night significantly predicts the next-day success of emotional discrimination within this viscerosensory network across individuals, suggesting a role for REM sleep in affective brain recalibration. Together, these findings establish that sleep deprivation compromises the faithful signaling of, and the "embodied" reciprocity between, viscerosensory brain and peripheral autonomic body processing of complex social signals. Such impairments hold ecological relevance in professional contexts in which the need for accurate interpretation of social cues is paramount yet insufficient sleep is pervasive.

Incidence and risk factors for Central Nervous System thrombosis in paediatric acute lymphoblastic leukaemia during intensive asparaginase treatment: a single-centre cohort study.

Central Nervous System (CNS) thrombosis is a complication of acute lymphoblastic leukaemia (ALL) treatment that is potentially associated with significant morbidity and neurological sequelae. Its presumably multifactorial aetiology is poorly characterized. We conducted a single-centre, retrospective cohort study on 346 ALL paediatric patients (1-16 years old) treated with asparaginase intensive Dana Farber Cancer Institute (DFCI) protocols from 1998 to 2011. The incidence, risk factors and outcome of CNS thrombosis were evaluated. CNS thrombosis occurred in 3·8% (13/346) of the patients (95% confidence interval 2·0-6·3%). Twelve events were diagnosed during intensification, all of which resolved within 2 weeks without neurological sequelae or significant impact in survival. Obesity (body mass index above 95th percentile) and asparaginase formulation were the only factors associated with CNS thrombosis, with an increase in the odds of event in obese patients [odds ratio (OR) = 3·37; P = 0·064] and a reduction in patients receiving Erwinia asparaginase (OR = 0·12; P = 0·018). No association could be demonstrated for age, gender, DFCI risk-group, ALL phenotype, steroid or doxorubicin use, central venous line use or CNS radiotherapy. CNS thrombosis is a rare but manageable adverse event without significant sequelae or detrimental effects in survival. Increased awareness is recommended in obese patients particularly during intensive asparaginase use.

Naphthoquine-induced Central Nervous System and Hepatic Vasculocentric Toxicity in the Beagle Dog.

Naphthoquine phosphate (NP) was considered as a partner drug with a promising antimalarial drug candidate. Here we report unexpected adverse clinical signs and microscopic findings in a canine pilot toxicology study with NP. Male and female dogs were dosed daily by oral gavage with NP at 2, 10, or 50 mg/kg/day for a maximum of 14 days. NP was not tolerated at ≥10 mg/kg/day; several animals were sacrificed in moribund condition and marked neurological clinical signs were noted at 50 mg/kg/day. The main microscopic observation was central nervous system vasculocentric inflammation (mainly lymphocytes and macrophages) in the white and gray matter of various regions of the brain at ≥2 mg/kg/day and at lower incidence in the spinal cord at ≥10 mg/kg/day. Vasculocentric microscopic changes predominantly centered on the centrilobular vein were also observed in the liver at ≥2 mg/kg/day. Females were more sensitive than males with comparable NP plasma exposure. In conclusion, under the conditions of this study, the administration of NP to dogs via daily oral gavage for up to 2 weeks was not tolerated causing moribundity, marked neurological clinical signs, and vasculocentric microscopic changes in the central nervous system and the liver.

Pericytes are required for blood-brain barrier integrity during embryogenesis.

Vascular endothelial cells in the central nervous system (CNS) form a barrier that restricts the movement of molecules and ions between the blood and the brain. This blood-brain barrier (BBB) is crucial to ensure proper neuronal function and protect the CNS from injury and disease. Transplantation studies have demonstrated that the BBB is not intrinsic to the endothelial cells, but is induced by interactions with the neural cells. Owing to the close spatial relationship between astrocytes and endothelial cells, it has been hypothesized that astrocytes induce this critical barrier postnatally, but the timing of BBB formation has been controversial. Here we demonstrate that the barrier is formed during embryogenesis as endothelial cells invade the CNS and pericytes are recruited to the nascent vessels, over a week before astrocyte generation. Analysing mice with null and hypomorphic alleles of Pdgfrb, which have defects in pericyte generation, we demonstrate that pericytes are necessary for the formation of the BBB, and that absolute pericyte coverage determines relative vascular permeability. We demonstrate that pericytes regulate functional aspects of the BBB, including the formation of tight junctions and vesicle trafficking in CNS endothelial cells. Pericytes do not induce BBB-specific gene expression in CNS endothelial cells, but inhibit the expression of molecules that increase vascular permeability and CNS immune cell infiltration. These data indicate that pericyte-endothelial cell interactions are critical to regulate the BBB during development, and disruption of these interactions may lead to BBB dysfunction and neuroinflammation during CNS injury and disease.

Pericytes regulate the blood-brain barrier.

The blood-brain barrier (BBB) consists of specific physical barriers, enzymes and transporters, which together maintain the necessary extracellular environment of the central nervous system (CNS). The main physical barrier is found in the CNS endothelial cell, and depends on continuous complexes of tight junctions combined with reduced vesicular transport. Other possible constituents of the BBB include extracellular matrix, astrocytes and pericytes, but the relative contribution of these different components to the BBB remains largely unknown. Here we demonstrate a direct role of pericytes at the BBB in vivo. Using a set of adult viable pericyte-deficient mouse mutants we show that pericyte deficiency increases the permeability of the BBB to water and a range of low-molecular-mass and high-molecular-mass tracers. The increased permeability occurs by endothelial transcytosis, a process that is rapidly arrested by the drug imatinib. Furthermore, we show that pericytes function at the BBB in at least two ways: by regulating BBB-specific gene expression patterns in endothelial cells, and by inducing polarization of astrocyte end-feet surrounding CNS blood vessels. Our results indicate a novel and critical role for pericytes in the integration of endothelial and astrocyte functions at the neurovascular unit, and in the regulation of the BBB.

The role and mechanism-of-action of Sema3E and Plexin-D1 in vascular and neural development.

Class 3 secreted semaphorins (Sema3A-3G) participate in many aspects of axon guidance through holoreceptor complexes that include Neuropilin-1 (Npn-1) or Neuropilin-2 and one of the four class A plexin proteins. However, unlike other Sema3 family proteins, Sema3E directly binds to Plexin-D1 without neuropilins. Its biological function was first explored in intersomitic vessel formation and since its initial discovery, Sema3E-Plexin-D1 signaling has been found to participate in the many biological systems in addition to vascular development, via seemingly different mode of actions. For example, temporal and spatial control of ligand vs. receptor results in two different mechanisms governing vascular patterning. Interactions with other transmembrane proteins such as neuropilin and VEGFR2 result in different axonal behaviors. Ligand receptor localization on pre- vs. post-synaptic neurons is used to control different types of synapse formation. Perhaps different downstream effectors will also result in different functional outcomes. Given the limited number of ligands and receptors in the genome and their multifunctional nature, we expect that more modes of action will be discovered in the future. In this review, we highlight current advances on the mechanisms of how Sema3E-Plexin-D1 interaction shapes the networks of multiple biological systems, in particular the vascular and nervous systems.

Sulfur as a signaling nutrient through hydrogen sulfide.

Hydrogen sulfide (H2S) has emerged as an important signaling molecule with beneficial effects on various cellular processes affecting, for example, cardiovascular and neurological functions. The physiological importance of H2S is motivating efforts to develop strategies for modulating its levels. However, advancement in the field of H2S-based therapeutics is hampered by fundamental gaps in our knowledge of how H2S is regulated, its mechanism of action, and its molecular targets. This review provides an overview of sulfur metabolism; describes recent progress that has shed light on the mechanism of H2S as a signaling molecule; and examines nutritional regulation of sulfur metabolism, which pertains to health and disease.

Comparison of vascular growth factors in the murine brain reveals placenta growth factor as prime candidate for CNS revascularization.

Vascular bypass procedures in the central nervous system (CNS) remain technically challenging, hindered by complications and often failing to prevent adverse outcome such as stroke. Thus, there is an unmet clinical need for a safe and effective CNS revascularization. Vascular endothelial growth factors (VEGFs) are promising candidates for revascularization; however, their effects appear to be tissue-specific and their potential in the CNS has not been fully explored. To test growth factors for angiogenesis in the CNS, we characterized the effects of endothelium-specific growth factors on the brain vasculature and parenchyma. Recombinant adeno-associated virus (AAV) vectors encoding the growth factors were injected transcranially to the frontoparietal cerebrum of mice. Angiogenesis, mural cell investment, leukocyte recruitment, vascular permeability, reactive gliosis and neuronal patterning were evaluated by 3-dimensional immunofluorescence, electron microscopy, optical projection tomography, and magnetic resonance imaging. Placenta growth factor (PlGF) stimulated robust angiogenesis and arteriogenesis without significant side effects, whereas VEGF and VEGF-C incited growth of aberrant vessels, severe edema, and inflammation. VEGF-B, angiopoietin-1, angiopoietin-2, and a VEGF/angiopoietin-1 chimera had minimal effects on the brain vessels or parenchyma. Of the growth factors tested, PlGF emerged as the most efficient and safe angiogenic factor, hence making it a candidate for therapeutic CNS revascularization.