The Blood-Brain Barrier: Composition, Properties, and Roles in Brain Health.

Blood vessels are critical to deliver oxygen and nutrients to tissues and organs throughout the body. The blood vessels that vascularize the central nervous system (CNS) possess unique properties, termed the blood-brain barrier (BBB), which allow these vessels to tightly regulate the movement of ions, molecules, and cells between the blood and the brain. This precise control of CNS homeostasis allows for proper neuronal function and protects the neural tissue from toxins and pathogens, and alterations of this barrier are important components of the pathogenesis and progression of various neurological diseases. The physiological barrier is coordinated by a series of physical, transport, and metabolic properties possessed by the brain endothelial cells (ECs) that form the walls of the blood vessels. These properties are regulated by interactions between different vascular, perivascular, immune, and neural cells. Understanding how these cell populations interact to regulate barrier properties is essential for understanding how the brain functions in both health and disease contexts.

Astroglial Hmgb1 regulates postnatal astrocyte morphogenesis and cerebrovascular maturation.

Astrocytes are intimately linked with brain blood vessels, an essential relationship for neuronal function. However, astroglial factors driving these physical and functional associations during postnatal brain development have yet to be identified. By characterizing structural and transcriptional changes in mouse cortical astrocytes during the first two postnatal weeks, we find that high-mobility group box 1 (Hmgb1), normally upregulated with injury and involved in adult cerebrovascular repair, is highly expressed in astrocytes at birth and then decreases rapidly. Astrocyte-selective ablation of Hmgb1 at birth affects astrocyte morphology and endfoot placement, alters distribution of endfoot proteins connexin43 and aquaporin-4, induces transcriptional changes in astrocytes related to cytoskeleton remodeling, and profoundly disrupts endothelial ultrastructure. While lack of astroglial Hmgb1 does not affect the blood-brain barrier or angiogenesis postnatally, it impairs neurovascular coupling and behavior in adult mice. These findings identify astroglial Hmgb1 as an important player in postnatal gliovascular maturation.

Neonatal hyperoxia in mice triggers long-term cognitive deficits via impairments in cerebrovascular function and neurogenesis.

Preterm birth is the leading cause of death in children under 5 years of age. Premature infants who receive life-saving oxygen therapy often develop bronchopulmonary dysplasia (BPD), a chronic lung disease. Infants with BPD are at a high risk of abnormal neurodevelopment, including motor and cognitive difficulties. While neural progenitor cells (NPCs) are crucial for proper brain development, it is unclear whether they play a role in BPD-associated neurodevelopmental deficits. Here, we show that hyperoxia-induced experimental BPD in newborn mice led to lifelong impairments in cerebrovascular structure and function as well as impairments in NPC self-renewal and neurogenesis. A neurosphere assay utilizing nonhuman primate preterm baboon NPCs confirmed impairment in NPC function. Moreover, gene expression profiling revealed that genes involved in cell proliferation, angiogenesis, vascular autoregulation, neuronal formation, and neurotransmission were dysregulated following neonatal hyperoxia. These impairments were associated with motor and cognitive decline in aging hyperoxia-exposed mice, reminiscent of deficits observed in patients with BPD. Together, our findings establish a relationship between BPD and abnormal neurodevelopmental outcomes and identify molecular and cellular players of neonatal brain injury that persist throughout adulthood that may be targeted for early intervention to aid this vulnerable patient population.

Unbiased analysis of mouse brain endothelial networks from two- or three-dimensional fluorescence images.

Significance: A growing body of research supports the significant role of cerebrovascular abnormalities in neurological disorders. As these insights develop, standardized tools for unbiased and high-throughput quantification of cerebrovascular structure are needed. Aim: We provide a detailed protocol for performing immunofluorescent labeling of mouse brain vessels, using thin ( 25 µ m ) or thick (50 to 150 µ m ) tissue sections, followed respectively by two- or three-dimensional (2D or 3D) unbiased quantification of vessel density, branching, and tortuosity using digital image processing algorithms. Approach: Mouse brain sections were immunofluorescently labeled using a highly selective antibody raised against mouse Cluster of Differentiation-31 (CD31), and 2D or 3D microscopy images of the mouse brain vasculature were obtained using optical sectioning. An open-source toolbox, called Pyvane, was developed for analyzing the imaged vascular networks. The toolbox can be used to identify the vasculature, generate the medial axes of blood vessels, represent the vascular network as a graph, and calculate relevant measurements regarding vascular morphology. Results: Using Pyvane, vascular parameters such as endothelial network density, number of branching points, and tortuosity are quantified from 2D and 3D immunofluorescence micrographs. Conclusions: The steps described in this protocol are simple to follow and allow for reproducible and unbiased analysis of mouse brain vascular structure. Such a procedure can be applied to the broader field of vascular biology.

Sex differences in developmental patterns of neocortical astroglia: A mouse translatome database.

Astroglial cells are key players in the development and maintenance of neurons and neuronal networks. Astroglia express steroid hormone receptors and show rapid responses to hormonal manipulations. However, despite important sex differences in the cortex and hippocampus, few studies have examined sex differences in astroglial cells in telencephalic development. To characterize the cortical astroglial translatome in male and female mice across postnatal development, we use translating ribosome affinity purification together with RNA sequencing and immunohistochemistry to phenotype astroglia at six developmental time points. Overall, we find two distinct astroglial phenotypes between early (P1-P7) and late development (P14-adult), independent of sex. We also find sex differences in gene expression patterns across development that peak at P7 and appear to result from males reaching a mature astroglial phenotype earlier than females. These developmental sex differences could have an impact on the construction of neuronal networks and windows of vulnerability to perturbations and disease.

Maternal high-fat diet in mice induces cerebrovascular, microglial and long-term behavioural alterations in offspring.

Various environmental exposures during pregnancy, like maternal diet, can compromise, at critical periods of development, the neurovascular maturation of the offspring. Foetal exposure to maternal high-fat diet (mHFD), common to Western societies, has been shown to disturb neurovascular development in neonates and long-term permeability of the neurovasculature. Nevertheless, the effects of mHFD on the offspring's cerebrovascular health remains largely elusive. Here, we sought to address this knowledge gap by using a translational mouse model of mHFD exposure. Three-dimensional and ultrastructure analysis of the neurovascular unit (vasculature and parenchymal cells) in mHFD-exposed offspring revealed major alterations of the neurovascular organization and metabolism. These alterations were accompanied by changes in the expression of genes involved in metabolism and immunity, indicating that neurovascular changes may result from abnormal brain metabolism and immune regulation. In addition, mHFD-exposed offspring showed persisting behavioural alterations reminiscent of neurodevelopmental disorders, specifically an increase in stereotyped and repetitive behaviours into adulthood.

Structural and Functional Remodeling of the Brain Vasculature Following Stroke.

Maintenance of cerebral blood vessel integrity and regulation of cerebral blood flow ensure proper brain function. The adult human brain represents only a small portion of the body mass, yet about a quarter of the cardiac output is dedicated to energy consumption by brain cells at rest. Due to a low capacity to store energy, brain health is heavily reliant on a steady supply of oxygen and nutrients from the bloodstream, and is thus particularly vulnerable to stroke. Stroke is a leading cause of disability and mortality worldwide. By transiently or permanently limiting tissue perfusion, stroke alters vascular integrity and function, compromising brain homeostasis and leading to widespread consequences from early-onset motor deficits to long-term cognitive decline. While numerous lines of investigation have been undertaken to develop new pharmacological therapies for stroke, only few advances have been made and most clinical trials have failed. Overall, our understanding of the acute and chronic vascular responses to stroke is insufficient, yet a better comprehension of cerebrovascular remodeling following stroke is an essential prerequisite for developing novel therapeutic options. In this review, we present a comprehensive update on post-stroke cerebrovascular remodeling, an important and growing field in neuroscience, by discussing cellular and molecular mechanisms involved, sex differences, limitations of preclinical research design and future directions.

Vascular contributions to 16p11.2 deletion autism syndrome modeled in mice.

While the neuronal underpinnings of autism spectrum disorder (ASD) are being unraveled, vascular contributions to ASD remain elusive. Here, we investigated postnatal cerebrovascular development in the 16p11.2df/+ mouse model of 16p11.2 deletion ASD syndrome. We discover that 16p11.2 hemizygosity leads to male-specific, endothelium-dependent structural and functional neurovascular abnormalities. In 16p11.2df/+ mice, endothelial dysfunction results in impaired cerebral angiogenesis at postnatal day 14, and in altered neurovascular coupling and cerebrovascular reactivity at postnatal day 50. Moreover, we show that there is defective angiogenesis in primary 16p11.2df/+ mouse brain endothelial cells and in induced-pluripotent-stem-cell-derived endothelial cells from human carriers of the 16p11.2 deletion. Finally, we find that mice with an endothelium-specific 16p11.2 deletion (16p11.2ΔEC) partially recapitulate some of the behavioral changes seen in 16p11.2 syndrome, specifically hyperactivity and impaired motor learning. By showing that developmental 16p11.2 haploinsufficiency from endothelial cells results in neurovascular and behavioral changes in adults, our results point to a potential role for endothelial impairment in ASD.

Danegaptide Enhances Astrocyte Gap Junctional Coupling and Reduces Ischemic Reperfusion Brain Injury in Mice.

Ischemic stroke is a complex and devastating event characterized by cell death resulting from a transient or permanent arterial occlusion. Astrocytic connexin43 (Cx43) gap junction (GJ) proteins have been reported to impact neuronal survival in ischemic conditions. Consequently, Cx43 could be a potential target for therapeutic approaches to stroke. We examined the effect of danegaptide (ZP1609), an antiarrhythmic dipeptide that specifically enhances GJ conductance, in two different rodent stroke models. In this study, danegaptide increased astrocytic Cx43 coupling with no significant effects on Cx43 hemichannel activity, in vitro. Using matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI IMS) the presence of danegaptide within brain tissue sections were detected one hour after reperfusion indicating successful transport of the dipeptide across the blood brain barrier. Furthermore, administration of danegaptide in a novel mouse brain ischemia/reperfusion model showed significant decrease in infarct volume. Taken together, this study provides evidence for the therapeutic potential of danegaptide in ischemia/reperfusion stroke.

Targeting MAPK phosphorylation of Connexin43 provides neuroprotection in stroke.

Connexin43 (Cx43) function is influenced by kinases that phosphorylate specific serine sites located near its C-terminus. Stroke is a powerful inducer of kinase activity, but its effect on Cx43 is unknown. We investigated the impact of wild-type (WT) and knock-in Cx43 with serine to alanine mutations at the protein kinase C (PKC) site Cx43S368A, the casein kinase 1 (CK1) sites Cx43S325A/328Y/330A, and the mitogen-activated protein kinase (MAPK) sites Cx43S255/262/279/282A (MK4) on a permanent middle cerebral artery occlusion (pMCAO) stroke model. We demonstrate that MK4 transgenic animals exhibit a significant decrease in infarct volume that was associated with improvement in behavioral performance. An increase in astrocyte reactivity with a concomitant decrease in microglial reactivity was observed in MK4 mice. In contrast to WT, MK4 astrocytes displayed reduced Cx43 hemichannel activity. Pharmacological blockade of Cx43 hemichannels with TAT-Gap19 also significantly decreased infarct volume in WT animals. This study provides novel molecular insights and charts new avenues for therapeutic intervention associated with Cx43 function.

Matrix-assisted laser desorption/ionization imaging mass spectrometry of intraperitoneally injected danegaptide (ZP1609) for treatment of stroke-reperfusion injury in mice.

RATIONALE: This work focuses on direct matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) detection of intraperitoneally (IP)-injected dipeptide ZP1609 in mouse brain tissue. Direct analysis of drug detection in intact tissue sections provides distribution information that can impact drug development. MALDI-IMS capabilities of uncovering drug transport across the blood-brain barrier are demonstrated. METHODS: Successful peptide detection using MALDI-IMS was achieved using a MALDI TOF/TOF system. Upon optimization of sample preparation procedures for dipeptide ZP1609, an additional tissue acidification procedure was found to greatly enhance signal detection. The imaging data acquired was able to determine successful transport of ZP1609 across the blood-brain barrier. Data obtained from MALDI-IMS can help shape our understanding of biological functions, disease progression, and effects of drug delivery. RESULTS: Direct detection of ZP1609 throughout the brain tissue sections was observed from MALDI-MS images. However, in cases where there was induction of stroke, a peak of lower signal intensity was also detected in the target m/z region. Although distinct differences in signal intensity can be seen between control and experimental groups, fragments and adducts of ZP1609 were investigated using MALDI-IMS to verify detection of the target analyte. CONCLUSIONS: Overall, the data reveals successful penetration of ZP1609 across the blood-brain barrier. The benefits of tissue acidification in the enhancement of detection sensitivity for low-abundance peptides were demonstrated. MALDI-IMS has been shown to be a useful technique in the direct detection of drugs within intact brain tissue sections.

Acute connexin43 temporal and spatial expression in response to ischemic stroke.

Connexin43 (Cx43) gap junctions expressed in astrocytes can significantly impact neuronal survival in stroke. However, little is known regarding Cx43 spatial and temporal expression during the initial stages of brain ischemia. Using immunohistochemistry and Western blot analysis, we examined Cx43 spatial and temporal expression as a function of neuronal injury within the first 24 h after permanent middle cerebral artery occlusion (pMCAO). Western blot analysis showed a significant increase in Cx43 protein expression in the core ischemic area at 2 and 3 h after pMCAO. However, after 6 h of pMCAO Cx43 levels were significantly reduced. This reduction was due to cell death and concomitant Cx43 degradation in the expanding focal ischemic region, while the peri-infarct zone revealed intense Cx43 staining. The neuronal cell-death marker Fluoro-Jade C labeled injured neurons faintly at 1 h post-pMCAO with a time-dependent increase in both intensity and size of punctate staining. In addition, decreased microtubule-associated protein 2 (MAP2) immunoreactivity and thionin staining similarly indicated cell damage beginning at 1 h after pMCAO. Taken together, Cx43 expression is sensitive to neuronal injury and can be detected as early as 2 h post-pMCAO. These findings underscore Cx43 gap junction as a potential early target for therapeutic intervention in ischemic stroke.

Pannexin1 knockout and blockade reduces ischemic stroke injury in female, but not in male mice.

The membrane channel Pannexin 1 (Panx1) mediates apoptotic and inflammatory signaling cascades in injured neurons, responses previously shown to be sexually dimorphic under ischemic conditions. We tested the hypothesis that Panx1 plays an underlying role in mediating sex differences in stroke outcome responses. Middle-aged, 8-9 month old male and female wild type and Panx1 KO mice were subjected to permanent middle cerebral artery (MCA) occlusion, and infarct size and astrocyte and microglia activation were assessed 4 days later. The sexually dimorphic nature of Panx1 deletion was also explored by testing the effect of probenecid a known Panx1 blocker to alter stroke volume. Panx1 KO females displayed significantly smaller infarct volumes (~ 50 % reduction) compared to their wild-type counterparts, whereas no such KO effect occurred in males. This sex-specific effect of Panx1 KO was recapitulated by significant reductions in peri-infarct inflammation and astrocyte reactivity, as well as smaller infarct volumes in probenecid treated females, but not males. Finally, females showed overall, higher Panx1 protein levels than males under ischemic conditions. These findings unmask a deleterious role for Panx1 in response to permanent MCA occlusion, that is unique to females, and provide several new frameworks for understanding sex differences in stroke outcome.

Gap junctions and hemichannels: communicating cell death in neurodevelopment and disease.

Gap junctions are unique membrane channels that play a significant role in intercellular communication in the developing and mature central nervous system (CNS). These channels are composed of connexin proteins that oligomerize into hexamers to form connexons or hemichannels. Many different connexins are expressed in the CNS, with some specificity with regard to the cell types in which distinct connexins are found, as well as the timepoints when they are expressed in the developing and mature CNS. Both the main neuronal Cx36 and glial Cx43 play critical roles in neurodevelopment. These connexins also mediate distinct aspects of the CNS response to pathological conditions. An imbalance in the expression, translation, trafficking and turnover of connexins, as well as mutations of connexins, can impact their function in the context of cell death in neurodevelopment and disease. With the ever-increasing understanding of connexins in the brain, therapeutic strategies could be developed to target these membrane channels in various neurological disorders.

Podoplanin: a marker for reactive gliosis in gliomas and brain injury.

Reactive astrogliosis is associated with many pathologic processes in the central nervous system, including gliomas. The glycoprotein podoplanin (PDPN) is upregulated in malignant gliomas. Using a syngeneic intracranial glioma mouse model, we show that PDPN is highly expressed in a subset of glial fibrillary acidic protein-positive astrocytes within and adjacent to gliomas. The expression of PDPN in tumor-associated reactive astrocytes was confirmed by its colocalization with the astrocytic marker S100ß and with connexin43, a major astrocytic gap junction protein. To determine whether the increase in PDPN is a general feature of gliosis, we used 2 mouse models in which astrogliosis was induced either by a needle injury or ischemia and observed similar upregulation of PDPN in reactive astrocytes in both models. Astrocytic PDPN was also found to be coexpressed with nestin, an intermediate filament marker for neural stem/progenitor cells. Our findings confirm that expression of PDPN is part of the normal host response to brain injury and gliomas, and suggest that it may be a novel cell surface marker for a specific population of reactive astrocytes in the vicinity of gliomas and nonneoplastic brain lesions. The findings also highlight the heterogeneity of glial fibrillary acidic protein-positive astrocytes in reactive gliosis.

The connexin43 mimetic peptide Gap19 inhibits hemichannels without altering gap junctional communication in astrocytes.

In the brain, astrocytes represent the cellular population that expresses the highest amount of connexins (Cxs). This family of membrane proteins is the molecular constituent of gap junction channels and hemichannels that provide pathways for direct cytoplasm-to-cytoplasm and inside-out exchange, respectively. Both types of Cx channels are permeable to ions and small signaling molecules allowing astrocytes to establish dynamic interactions with neurons. So far, most pharmacological approaches currently available do not distinguish between these two channel functions, stressing the need to develop new specific molecular tools. In astrocytes two major Cxs are expressed, Cx43 and Cx30, and there is now evidence indicating that at least Cx43 operates as a gap junction channel as well as a hemichannel in these cells. Based on studies in primary cultures as well as in acute hippocampal slices, we report here that Gap19, a nonapeptide derived from the cytoplasmic loop of Cx43, inhibits astroglial Cx43 hemichannels in a dose-dependent manner, without affecting gap junction channels. This peptide, which not only selectively inhibits hemichannels but is also specific for Cx43, can be delivered in vivo in mice as TAT-Gap19, and displays penetration into the brain parenchyma. As a result, Gap19 combined with other tools opens up new avenues to decipher the role of Cx43 hemichannels in interactions between astrocytes and neurons in physiological as well as pathological situations.

Histological assessment of angiogenesis in the hypoxic central nervous system.

Angiogenesis, the sprouting of new capillaries from preexisting vessels, is an integral part of both normal development and numerous pathological conditions such as tumor growth, inflammation, and stroke. The development of angiogenesis assays has been critical in understanding this process in both the context of disease and normal physiology. With the growing availability of antibodies against angiogenic markers as well as advances in microscopy and imaging analysis software, a more comprehensive assessment of the angiogenesis process is beginning to take form (Milner et al., Stroke 39:191-197, 2008; Freitas-Andrade et al., J Cereb Blood Flow Metab 32:663-675, 2012; Li et al., Glia 58:1157-1167, 2010; Dore-Duffy and LaManna, Antioxid Redox Signal 9:1363-1371, 2007). This chapter describes an in vivo method of inducing brain angiogenesis in mice by chronic exposure to mild hypoxia. In addition, a detailed procedure of quantifying angiogenesis using multiple immunofluorescent labeling of mouse brain tissue sections is also presented.

PlGF knockout delays brain vessel growth and maturation upon systemic hypoxic challenge.

In this study, we have investigated the potential role of placental growth factor (PlGF) in hypoxia-induced brain angiogenesis. To this end, PlGF wild-type (PlGF(+/+)) and PlGF knockout (PlGF(-/-)) mice were exposed to whole body hypoxia (10% oxygen) for 7, 14, and 21 days. PlGF(+/+) animals exhibited a significant ~40% increase in angiogenesis after 7 days of hypoxia compared with controls, while in PlGF(-/-) this effect only occurred after 14 days of hypoxia. No differences in pericyte/smooth muscle cell (SMC) coverage between the two genotypes were observed. After 14 days of hypoxia, PlGF(-/-) microvessels had a significant increase in fibrinogen accumulation and extravasation compared with those of PlGF(+/+), which correlated with endothelial cell disruption of the tight junction protein claudin-5. These vessels displayed large lumens, were surrounded by reactive astrocytes, lacked both pericyte/SMC coverage and endothelial vascular endothelial growth factor expression, and regressed after 21 days of hypoxia. Vascular endothelial growth factor expression levels were found to be significantly lower in the frontal cortex of PlGF(-/-) compared with those in PlGF(+/+) animals during the first 5 days of hypoxia, which in combination with the lack of PlGF may have contributed to the delayed angiogenic response and the prothrombotic phenotype observed in the PlGF(-/-)animals.

VEGFR-2-mediated increased proliferation and survival in response to oxygen and glucose deprivation in PlGF knockout astrocytes.

In hypoxic/ischemic conditions, astrocytes are involved in neuroprotection and angiogenesis. Vascular endothelial growth factor (VEGF) induces angiogenesis and exhibits neuroprotective and neurotrophic properties. However, the role of placental growth factor (PlGF), a VEGF homolog, in these processes is unclear. Therefore, proliferation and survival studies were performed on PlGF knockout (PlGF-/-) and wild-type (PlGF+/+) mouse astrocytes. A significant increase in cell proliferation and survival to oxygen and glucose deprivation (OGD) was observed in PlGF-/- compared to PlGF+/+ astrocytes. Interestingly, no PlGF protein expression was detected in PlGF+/+ astrocytes and no changes in VEGF protein levels were observed between the two genotypes. Real-time PCR and immunocytochemistry showed over-expression of VEGF receptor-2 (VEGFR-2) in PlGF-/- compared with PlGF+/+ astrocytes. Confocal microscopy revealed nuclear, membrane, and cytoplasmic localization of VEGFR-2. In vivo over-expression of VEGFR-2 mRNA was also detected in PlGF-/- compared with PlGF+/+ astrocytes. Stimulation with VEGF165 resulted in increased proliferation in PlGF-/- compared with PlGF+/+ astrocytes. This effect was blocked by the VEGFR-2 antagonist, VEGF165b. The enhanced proliferation of PlGF-/- astrocytes correlated with increased phospho-extracellular-signal-regulated kinase-1/2 levels, while the resistance to OGD was independent of the phosphatidylinositol 3'-kinase/Akt pathway. These results suggest that VEGFR-2 mediates the enhanced proliferative/OGD resistant phenotype observed in PlGF-/- astrocytes.