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Ischemic stroke is a predominant cause of disability worldwide, with thrombolytic or mechanical removal of the occlusion being the only therapeutic option. Reperfusion bears the risk of an acute deleterious calcium-dependent breakdown of the blood-brain barrier. Its mechanism, however, is unknown. Here, we identified type 5 NADPH oxidase (NOX5), a calcium-activated, ROS-forming enzyme, as the missing link. Using a humanized knockin (KI) mouse model and in vitro organotypic cultures, we found that reoxygenation or calcium overload increased brain ROS levels in a NOX5-dependent manner. In vivo, postischemic ROS formation, infarct volume, and functional outcomes were worsened in NOX5-KI mice. Of clinical and therapeutic relevance, in a human blood-barrier model, pharmacological NOX inhibition also prevented acute reoxygenation-induced leakage. Our data support further evaluation of poststroke recanalization in the presence of NOX inhibition for limiting stroke-induced damage., Introduction Ischemic stroke represents one of the most frequent causes of death and leading causes of disability worldwide (1). Thrombolytic or mechanical removal of the occlusion is the only therapeutic [...]

To maintain homeostasis autonomic control centers in the hypothalamus and medulla must respond appropriately to both external and internal stimuli. Although protected behind the blood-brain barrier, neurons in these autonomic control centers are known to be influenced by changing levels of important signaling molecules in the systemic circulation (e.g., osmolarity, glucose concentrations, and regulatory peptides). The subfornical organ belongs to a group of specialized central nervous system structures, the circumventricular organs, which are characterized by the lack of the normal blood-brain barrier, such that circulating lipophobic substances may act on neurons within this region and via well-documented efferent neural projections to hypothalamic autonomic control centers, influence autonomic function. This review focuses on the role of the subfornical organ in sensing peripheral signals and transmitting this information to autonomic control centers in the hypothalamus. autonomic; cardiovascular; satiety signals; immune regulation; blood-brain barrier doi: 10.1152/ajpregu.00103.2010.

Administration of synthetic or purified peptides directly into the brain ventricles is a method commonly used by neuroscientists for exploring physiological and behavioral functions of gene products. i.v. administration is controlled by the blood-brain barrier, which limits its effectiveness, and current approaches for acute or chronic intracerebroventricular delivery have significant technical drawbacks resulting from both the chemical properties of the delivered substance and the experimental procedures. Here we describe a genetic approach for the delivery of secreted peptides or proteins into the cerebrospinal fluid (CSF). Using a choroid plexus-specific promoter, we established a lentiviral-based system, which offers inducible and reversible delivery of a gene product into the CSF. The functionality of this system was demonstrated by using the overexpression of the two established neuropeptides, corticotropin-releasing factor and gonadotropin-releasing hormone, modulating anxiety-like behavior and estrus cycle, respectively. We show that this choroid plexus-specific lentiviral-based system is a reliable, effective, and adaptable research tool for intracerebroventricular delivery. blood--brain barrier | choroid plexus | Lentiviruses | secreted peptides | cerebrospinal fluid doi/10.1073/pnas.0907059107

Am J Physiol Heart Circ Physiol 298: H179-H188, 2010. First published October 23, 2009; doi: 10.1152/ajpheart.00828.2009.--We examined the expression of tight junction (TJ) proteins in the cerebral cortex, cerebellum, and spinal cord of fetuses after maternal treatment with single and multiple courses of dexamethasone. Ewes received either single courses of four 6-mg dexamethasone or placebo injections every 12 h for 48 h between 104 and 107 days or the same treatment once a week between 76-78 and 104-107 days of gestation. TJ protein expression was determined by Western immunoblot analysis on tissue harvested at 105-108 days of gestation. Blood-brain barrier permeability has been previously quantified with the blood-to-brain transfer constant ([K.sub.i]) with [alpha]-aminoisobutyric acid (39). After a single course of dexamethasone, claudin-5 increased (P < 0.05) in the cerebral cortex, occludin and claudin-1 increased in the cerebellum, and occludin increased in the spinal cord. After multiple dexamethasone courses, occludin and zonula occludens (ZO)-1 increased in the cerebral cortex, and occludin and claudin-1 increased in the cerebellum. Junctional adhesion molecule-A and ZO-2 expressions did not change. Linear regression comparing [K.sub.i] to TJ proteins showed inverse correlations with claudin-1 and claudin-5 in the cerebral cortex after a single course and ZO-2 in the spinal cord after multiple courses and direct correlations with ZO-1 in the cerebellum and spinal cord after multiple courses. We conclude that maternal glucocorticoid treatment increases the expression of specific TJ proteins in vivo, patterns of TJ protein expression vary after exposure to single and multiple glucocorticoid courses, and decreases in blood-brain barrier permeability are associated with increases in claudin-l, claudin-5, and ZO-2 expression and decreases in ZO-1 expression. In utero glucocorticoid exposure alters the molecular composition of the barrier and affects fetal blood-brain barrier function. blood-brain barrier; dexamethasone; steroids

Blood-brain barrier (BBB) Na transporters are essential for brain water and electrolyte homeostasis. However, they also contribute to edema formation during the early hours of ischemic stroke by increased transport of Na from blood into brain across an intact BBB. We previously showed that a luminal BBB Na-K-Cl cotransporter is stimulated by hypoxia, aglycemia, and AVP and that inhibition of the cotransporter by intravenous bumetanide significantly reduces edema and infarct in the rat middle cerebral artery occlusion (MCAO) model of stroke. More recently, we found evidence that intravenous cariporide (HOE-642), a highly potent Na/H exchange inhibitor, also reduces brain edema after MCAO. The present study was conducted to investigate which Na/H exchange protein isoforms are present in BBB endothelial cells and to evaluate the effects of ischemic factors on BBB Na/H exchange activity. Western blot analysis of bovine cerebral microvascular endothelial cells (CMEC) and immunoelectron microscopy of perfusion-fixed rat brain revealed that Na/H exchanger isoforms 1 and 2 (NHE1 and NHE2) are present in BBB endothelial cells. Using microspectrofluorometry and the pH-sensitive dye BCECF, we found that hypoxia (2% 02, 30 min), aglycemia (30 min), and AVP (1-200 nM, 5 min) significantly increased CMEC Na/H exchange activity, assessed as Na-dependent, HOE-642-sensitive [H.sup.+] flux. We found that AVP stimulation of CMEC Na/H exchange activity is dependent on intracellular Ca concentration and is blocked by [V.sub.1], but not [V.sub.2], vasopressin receptor antagonists. Our findings support the hypothesis that a BBB Na/H exchanger, possibly NHE1 and/or NHE2, is stimulated during ischemia to participate in cerebral edema formation. blood-brain barrier; stroke; cerebral ischemia; brain edema; cariporide

A diverse array of infectious agents, including prions and certain neurotropic viruses, bind to the laminin receptor (LR), and this determines tropism to the CNS. Bacterial meningitis in childhood is almost exclusively caused by the respiratory tract pathogens Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae, but the mechanism by which they initiate contact with the vascular endothelium of the blood brain barrier (BBB) is unknown. We hypothesized that an interaction with LR might underlie their CNS tropism. Using affinity chromatography, coimmunoprecipitation, retagging, and in vivo imaging approaches, we identified 37/67-kDa LR as a common receptor for all 3 bacteria on the surface of rodent and human brain microvascular endothelial cells. Mutagenesis studies indicated that the corresponding bacterial LR-binding adhesins were pneumococcal CbpA, meningococcal PilQ and PorA, and OmpP2 of H. influenzae. The results of competitive binding experiments suggest that a common adhesin recognition site is present in the carboxyl terminus of LR. Together, these findings suggest that disruption or modulation of the interaction of bacterial adhesins with LR might engender unexpectedly broad protection against bacterial meningitis and may provide a therapeutic target for the prevention and treatment of disease., Introduction The 37-kDa laminin receptor (LR) precursor protein is highly conserved in eukaryotes. The precursor form is thought to mature into one or more homo- or heterodimeric 67-kDa dimeric forms [...]

Drug abuse is a worldwide health concern in which addiction involves activation of the dopaminergic signaling pathway in the brain. Here, we introduce a nanotechnology approach that utilizes gold nanorod-DARPP-32 siRNA complexes (nanoplexes) that target this dopaminergic signaling pathway in the brain. The shift in the localized longitudinal plasmon resonance peak of gold nanorods (GNRs) was used to show their interaction with siRNA. Plasmonic enhanced dark field imaging was used to visualize the uptake of these nanoplexes in dopaminergic neurons in vitro. Gene silencing of the nanoplexes in these cells was evidenced by the reduction in the expression of key proteins (DARPP-32, ERK, and PP-1) belonging to this pathway, with no observed cytotoxicity. Moreover, these nanoplexes were shown to transmigrate across an in vitro model of the blood-brain barrier (BBB). Therefore, these nanoplexes appear to be suited for brain-specific delivery of appropriate siRNA for therapy of drug addiction and other brain diseases. DARPP-32 | dark field imaging | surface plasmon resonance | nanoplexes | blood-brain barrier

The blood-brain barrier (BBB) is confined to the endothelium of brain capillaries and is indispensable for fluid homeostasis and neuronal function. In this study, we show that endothelial Wnt/[beta]-catenin ([beta]-cat) signaling regulates induction and maintenance of BBB characteristics during embryonic and postnatal development. Endothelial specific stabilization of [beta]-cat in vivo enhances barrier maturation, whereas inactivation of [beta]-cat causes significant down-regulation of claudin3 (Cldn3), up-regulation of plamalemma vesicle-associated protein, and BBB breakdown. Stabilization of [beta]-cat in primary brain endothelial cells (ECs) in vitro by N-terminal truncation or Wnt3a treatment increases Cldn3 expression, BBB-type tight junction formation, and a BBB characteristic gene signature. Loss of [beta]-cat or inhibition of its signaling abrogates this effect. Furthermore, stabilization of [beta]-cat also increased Cldn3 and barrier properties in nonbrain-derived ECs. These findings may open new therapeutic avenues to modulate endothelial barrier function and to limit the devastating effects of BBB breakdown.

We have demonstrated that after intraperitoneal lipopolysaccharide (LPS) injection, old rats mount fevers similar to those of young rats at an ambient temperature (Ta) of 31[degrees]C, but not at 21[degrees]C. The same is true for intraperitoneal or intravenous IL-113 administration. The underlying mechanism responsible for blunted fever in old rats may be a deficiency in communication between the periphery and the brain. Possibly, peripheral cytokine actions are altered in old rats, such that the signal that reaches the brain is diminished. Here, we hypothesized that at standard laboratory temperatures, not enough 1L-1[beta] is reaching the brain for fever to occur and that a warmer Ta would increase the influx of IL-1[beta] into the brain, enabling old rats to generate fever. Young (3-5 mo) and old (23-29 mo) Long-Evans rats were maintained for 3 days at either Ta 21 or 31[degrees]C prior to intravenous injection with radiolabeled IL-1[beta] to measure passage across the blood-brain barrier. Young rats showed similar influx of IL-1[beta] into the brain at the two Tas, but old rats showed significant influx only at the warmer Ta. These data suggest that the lack of fever at a cool Ta may be due to a reduced influx of IL-1[beta] into the brain. aging; blood-brain-barrier; cytokine; ambient temperature

Hypertensive encephalopathy is a life-threatening condition due to elevation of cerebral perfusion pressure beyond the limits of autoregulation. Breakdown of the blood-brain barrier (BBB) leads to cerebral edema and reduced blood flow. In this issue of the JCI, Mochly-Rosen and colleagues demonstrate a novel molecular strategy for preserving the BBB in a model of hypertension- induced encephalopathy (see the related article beginning on page 173). Using a rationally designed peptide inhibitor of [delta]PKC, they stabilized the BBB and improved mortality in hypertensive rats. This study highlights the therapeutic potential of [delta]PKC inhibitors in hypertensive encephalopathy and provides incentive to elucidate [delta]PKC signaling pathways that mediate BBB dysfunction in other disease states., The blood-brain barrier The blood-brain barrier (BBB) is a gliovascular unit composed of capillary endothelial cells and pericytes surrounded by basal lamina, astrocytic end-feet, and perivascular interneurons (Figure 1A) (1). [...]

Two libraries of mono-N-methylated and di-N-methylated diketopiperazines (DKPs) are prepared and evaluated in order to understand the features governing the passage of peptide molecules across the blood-brain barrier (BBB) by passive diffusion. The DKPs or cyclic dipeptide scaffolds belong to a novel family of brain delivery systems to transport to the brain drugs and other cargos that cannot cross the BBB without help.

It is recognized that ketone bodies, such as R-[beta]-hydroxybutyrate ([beta]-HB) and acetoacetate, are energy sources for the brain. As with glucose metabolism, monocarboxylate uptake by the brain is dependent on the function and regulation of its own transporter system. We concurrently investigated ketone body influx, blood flow, and regulation of monocarboxylate transporter (MCT-1) and glucose transporter (GLUT-1) in diet-induced ketotic (KG) rat brain. Regional blood-to-brain [beta]-HB influx ([micro]mol x [g.sup.-1] x [min.sup.-1]) increased 40-fold with ketosis (4.8 [+ or -] 1.8 plasma[beta]-HB; mM) in all regions compared with the nonketotic groups (standard and no-fat diets); there were no changes in regional blood flow. Immunohistochemical staining revealed that GLUT-1 density (number/[mm.sup.2]) in the cortex was significantly elevated (40%) in the ketotic group compared with the standard and no-fat diet groups. MCT-1 was also markedly (3-fold) upregulated in the ketotic group compared with the standard diet group. In the standard diet group, 40% of the brain capillaries stained positive for MCT-1; this amount doubled with the ketotic diet. Western blot analysis of isolated microvessels from ketotic rat brain showed an eightfold increase in GLUT-1 and a threefold increase in MCT-1 compared with the standard diet group. These data suggest that diet-induced ketosis results in increased vascular density at the blood-brain barrier without changes in blood flow. The increase in extraction fraction and capillary density with increased plasma ketone bodies indicates a significant flux of substrates available for brain energy metabolism. ketone bodies; monocarboxylate transporter-1; GLUT-1; [beta]-hydroxybutyrate; brain metabolism; blood-brain barrier doi: 10.1152/ajpendo.00512.2006

Blood-brain barrier (BBB) controls paracellular solute diffusion into the brain microenvironment and is maintained primarily by tight junctions between adjacent microvascular endothelial cells. Studies implicate blood flow-associated shear stress as a pathophysiological mediator of BBB function, although detailed biochemical data are scarce. We hypothesize that shear stress upregulates BBB function via direct modulation of expression and properties of pivotal tight-junction proteins occludin and zonula occludens-1 (ZO-1). Bovine brain microvascular endothelial cells (BBMvECs) were exposed to either steady or pulsatile shear stress (10 and 14 dyn/[cm.sup.2], respectively) for 24 h. Sheared BBMvECs were monitored for occludin-ZO-1 expression, association, and subcellular localization, and transendothelial permeability of BBMvECs to FITC-dextran and [sup.14][C]sucrose was assessed. Actin reorganization and BBMvEC realignment were observed following steady shear stress for 24 h. Substantial increases in occludin mRNA and protein expression (2.73 [+ or -] 0.26- and 1.83 [+ or -] 0.03-fold) and in occludin-ZO-1 association (2.12 [+ or -] 0.15-fold) were also observed. Steady shear stress also induced clear relocalization of both proteins to the cell-cell border in parallel with reduced transendothelial permeability to FITC-dextran (but not sucrose). Following pulsatile shear stress, increased protein levels for both occludin and ZO-1 (2.15 [+ or -] 0.02- and 1.67 [+ or -] 0.21-fold) and increased occludin-ZO-1 association (2.91 [+ or -] 0.14-fold) were observed in parallel with a reduction in transendothelial permeability to [sup.14][C]sucrose. Shear stress upregulates BBMvEC barrier function at the molecular level via modulation of expression, association, and localization of occludin and ZO-1. The pulsatile shear model appeared to give the most profound biochemical responses. blood-brain barrier; occludin; zonula occludens-1 doi:10.1152/ajpheart.01177.2006

Treatment of many neuronal degenerative disorders will require delivery of a therapeutic protein to neurons or glial cells across the whole CNS. The presence of the blood--brain barrier hampers the delivery of these proteins from the blood, thus necessitating a new method for delivery. Receptors on the blood--brain barrier bind ligands to facilitate their transport to the CNS; therefore, we hypothesized that by targeting these receptors, we may be able to deliver proteins to the CNS for therapy. Here, we report the use of the lentivirus vector system to deliver the lysosomal enzyme glucocerebrosidase and a secreted form of GFP to the neurons and astrocytes in the CNS. We fused the low-density lipoprotein receptor-binding domain of the apolipoprotein B to the targeted protein. This approach proved to be feasible for delivery of the protein and could possibly be used as a general method for delivery of therapeutic proteins to the CNS. fusion protein | lentiviral vectors | low-density lipoprotein receptor

Previously, we reported a defect in the blood-brain barrier (BBB) of apolipoprotein E-deficient (apoE-/-) mice (24). Here, we investigate BBB permeability in wild-type (WT) and apoE-/- mice as a function of age. Both WT and apoE-/- mice showed significantly increased cortical BBB leakage with age. However, in apoE-/- mice, the leakage increased at a 3.7x higher rate compared with WT mice. Surprisingly, the cerebellum showed significantly more leakage than other brain regions across age, while there was no difference between the two hemispheres. To determine the contribution of tissue- vs. blood-borne apoE to vascular permeability, we generated chimeric mice by bone marrow transplantation and measured their BBB leakage. These experiments suggest that both blood- and tissue-derived apoE are equally important for BBB function. In sum, we find an age-dependent defect in the BBB that is exacerbated in apoE-/- mice. Since vascular defects are found in patients with age-related neurodegenerative diseases, such as Alzheimer's, age-related BBB leakage could underlie these defects and may thus be an important contributor to the cumulative neuronal damage of these diseases. apolipoprotein E; aging; age-related neurodegeneration; inflammation

Experimental allergic encephalomyelitis (EAE) is an inflammatory demyelinating disease of the CNS that is used to model certain parameters of multiple sclerosis. To establish the relative contributions of T cell reactivity, the loss of blood-brain barrier (BBB) integrity, CNS inflammation, and lesion formation toward the pathogenesis of EAE, we assessed the incidence of EAE and these parameters in mice lacking NF-[kappa]B, TNF-[alpha], IFN-[alpha][beta] receptors, IFN-[gamma] receptors, and inducible nitric oxide synthase. Although increased myelin oligodendrocyte glycoprotein-specific T cell reactivity was generally associated with a more rapid onset or increased disease severity, the loss of BBB integrity and cell accumulation in spinal cord tissues was invariably associated with the development of neurological disease signs. Histological and real-time RT-PCR analyses revealed differences in the nature of immune/inflammatory cell accumulation in the spinal cord tissues of the different mouse strains. On the other hand, disease severity during the acute phase of EAE directly correlated with the extent of BBB permeability. Thus, the loss of BBB integrity seems to be a requisite event in the development of EAE and can occur in the absence of important inflammatory mediators. gene knockout mice | multiple sclerosis

This study investigated the effects of streptozotocin-induced diabetes on the functional integrity of the blood-brain barrier in the rat at 7, 28, 56, and 90 days, using vascular space markers ranging in size from 342 to 65,000 Da. We also examined the effect of insulin treatment of diabetes on the formation and progression of cerebral microvascular damage and determined whether observed functional changes occurred globally throughout the brain or within specific brain regions. Results demonstrate that streptozotocin-induced diabetes produced a progressive increase in blood-brain barrier permeability to small molecules from 28 to 90 days and these changes in blood-brain barrier permeability were region specific, with the midbrain most susceptible to diabetes-induced microvascular damage. In addition, results showed that insulin treatment of diabetes attenuated blood-brain barrier disruption, especially during the first few weeks: however, as diabetes progressed, it was evident that microvascular damage occurred even when hyperglycemia was controlled. Overall, results of this study suggest that diabetes-induced perturbations to cerebral microvessels may disrupt homeostasis and contribute to long-term cognitive and functional deficits of the central nervous system. in situ; endothelial; cerebral blood flow; neurovascular; dementia

The transport of [sup.125]I-labeled thyroxine ([T.sub.4]) from the cerebrospinal fluid (CSF) into brain and choroid plexus (CP) was measured in anesthetized rabbit [0.5 mg/kg medetomidine (Domitor) and 10 mg/kg pentobarbitonal sodium (Sagatal) iv] using the ventriculocisternal (V-C) perfusion technique. [sup.125]I-labeled [T.sub.4] contained in artificial CSF was continually perfused into the lateral ventricles for up to 4 h and recovered from the cisterna magna. The %recovery of [sup.125]I-labeled T4 from the aCSF was 47.2 [+ or -] 5.6% (n = 10), indicating removal of [sup.125]I-labeled [T.sub.4] from the CSF. The recovery increased to 53.2 [+ or -] 6.3% (n = 4) and 57.8 [+ or -] 14.8% (n = 3), in the presence of 100 and 200 [micro]M unlabeled-[T.sub.4], respectively (P < 0.05), indicating a saturable component to [T.sub.4] removal from CSF. There was a large accumulation of [sup.125]I-labeled [T.sub.4] in the CP, and this was reduced by 80% in the presence of 200 [micro]M unlabeled [T.sub.4], showing saturation. In the presence of the thyroid-binding protein transthyretin (TTR), more [sup.125]I-labeled T4 was recovered from CSF, indicating that the binding protein acted to retain [T.sub.4] in CSF. However, [sup.125]I-labeled [T.sub.4] uptake into the ependymal region (ER) of the frontal cortex also increased by 13 times compared with control conditions. Elevation was also seen in the hippocampus (HC) and brain stem. Uptake was significantly inhibited by the presence of endocytosis inhibitors nocodazole and monensin by > 50%. These data suggest that the distribution of [T.sub.4] from CSF into brain and CP is carrier mediated, TTR dependent, and via RME. These results support a role for TTR in the distribution of [T.sub.4] from CSF into brain sites around the ventricular system, indicating those areas involved in neurogenesis (ER and HC). hippocampus; ependymal region; blood-brain barrier

We examined the effects of hyperosmolality on blood-brain barrier (BBB) permeability during development to test the vulnerability of the immature barrier to stress. The BBB response to hyperosmolality was quantified using the blood-to-brain transfer constant ([K.sub.i]) with [alpha]-aminoisobutyric acid in fetuses at 60% and 90% gestation, premature, newborn, and older lambs. [K.sub.i] plotted against osmolality increased as a function of increases in osmolality in all groups and brain regions. The relationship was described (P < 0.05) by a segmented regression model. At lower osmolalities, changes in [K.sub.i] were minimal, but after a break point (threshold) was reached, the increase (P < 0.05) was linear. We examined the responses of [K.sub.i] to hyperosmolality within each brain region by comparing the thresholds and slopes of the second regression segment. Lower thresholds and higher slopes imply greater vulnerability to hyperosmolality in the younger groups. Thresholds increased (P < 0.05) with development in the thalamus, superior colliculus, pons, and spinal cord, and slopes of the second regression segment decreased (P < 0.05) in the cerebellum, hippocampus, inferior colliculus, medulla, and spinal cord. BBB resistance to hyperosmolality increased (P < 0.05) with development in most brain regions. The pattern of the Ki plotted against osmolality was (P < 0.05) heterogenous among brain regions in fetuses and premature and newborn lambs, but not in older lambs. We conclude that 1) BBB permeability increased as a function of changes in osmolality, 2) the barrier becomes more resistant to hyperosmolality during development, and 3) the permeability response to hyperosmolality is heterogenous among brain regions in fetuses and premature and newborn lambs. [alpha]-aminoisobutyric acid; brain; barrier; development; immature; lambs; mannitol; sheep

There is substantial evidence linking blood-brain barrier (BBB) failure during cerebral ischemia to matrix metalloproteinases (MMP). BBB function may be affected by loss of shear stress under normoxia/normoglycemia, as during cardiopulmonary bypass procedures. The present study used an in vitro flow-perfused BBB model to analyze the individual contributions of flow, cytokine levels, and circulating blood leukocytes on the release/ activity of MMP-9, MMP-2, and their endogenous inhibitors, the tissue inhibitors of MMPs (TIMPs), TIMP-1, and TIMP-2. The presence of circulating blood leukocytes under normoxic/normoglycemic flow cessation/reperfusion significantly increased the luminal levels of MMP-9 and activity of MMP-2, accompanied by partial reduction of TIMP-1, complete reduction of TIMP-2 and increased BBB permeability. These changes were not observed during constant flow with circulating blood leukocytes, or after normoxic/normoglycemic or hypoxic/hypoglycemic flow cessation/reperfusion without circulating blood leukocytes. The addition of anti-1L-6 or anti-TNF-[alpha] antibody in the lumen before reperfusion suppressed the levels of MMP-9 and activity of MMP-2, had no effect on TIMP-1, and completely restored TIMP-2 and BBB integrity. Injection of TIMP-2 in the lumen before reperfusion prevented the activation of MMP-2 and BBB permeability. These data indicate that blood leukocytes and loss of flow are major factors in the activation of MMP-2, and that cytokine-mediated differential regulation of TIMP-1 and TIMP-2 may contribute significantly to BBB failure. shear stress; inflammation; matrix metalloproteinases

Cationic amino acid (CAA) transport is brought about by two families of proteins that are found in various tissues: Cat (CAA transporter), referred to as system [y.sup.+], and Bat [broad-scope amino acid (AA) transporter], which comprises systems [b.sup.0,+], [B.sup.0,+], and [y.sup.+]L. CAA traverse the blood-brain barrier (BBB), but experiments done in vivo have only been able to examine the BBB from the luminal (blood-facing) side. In the present study, plasma membranes isolated from bovine brain microvessels were used to identify and characterize the CAA transporter(s) on both sides of the BBB. From these studies, it was concluded that system [y.sup.+] was the only transporter present, with a prevalence of activity on the abluminal membrane. System [y.sup.+] was voltage dependent and had a [K.sub.m] of 470 [+ or -] 106 [micro]M (SE) for lysine, a [K.sub.i] of 34 [micro]M for arginine, and a [K.sub.i] of 290 [micro]M for omithine. In the presence of [Na.sup.+], system y + was inhibited by several essential neutral AAs. The [K.sub.i] values were 3-10 times the plasma concentrations, suggesting that system [y.sup.+] was not as important a point of access for these AAs as system L1. Several small nonessential AAs (serine, glutamine, alanine,and glycine) inhibited system [y.sup.+] with [K.sub.i] values similar to their plasma concentrations, suggesting that system [y.sup.+] may account for the permeability of the BBB to these AAs. System [y.sup.+] may be important in the provision of arginine for NO synthesis. Real-time PCR and Western blotting techniques established the presence of the three known nitric oxide synthases in cerebral endothelial cells: NOS-1 (neuronal), NOS-2 (inducible), and NOS-3 (endothelial). These results confirm that system [y.sup.+] is the only CAA transporter in the BBB and suggest that NO can be produced in brain endothelial cells. amino acid active transport; brain capillaries; endothelial cells; essential amino acids; nonessential amino acids; polarity doi:10.1152/ajpendo.00007.2006

Brain capillary endothelial cells form the blood-brain barrier (BBB). They are connected by extensive tight junctions, and are polarized into luminal (blood-facing) and abluminal (brain-facing) plasma membrane domains. The polar distribution of transport proteins mediates amino acid (AA) homeostasis in the brain. The existence of two facilitative transporters for neutral amino acids (NAAs) on both membranes provides the brain access to essential AAs. Four [Na.sup.+]-dependent transporters of NAA exist in the abluminal membranes of the BBB. Together these systems have the capability to actively transfer every naturally occurring NAA from the extracellular fluid (ECF) to endothelial cells and from there into circulation. The presence of [Na.sup.+]-dependent carriers on the abluminal membrane provides a mechanism by which NAA concentrations in the ECF of brain are maintained at ~10% those of the plasma. Also present on the abluminal membrane are at least three [Na.sup.+]-dependent systems transporting acidic AAs (EAAT) and a [Na.sup.+]-dependent system transporting glutamine (N). Facilitative carriers for glutamine and glutamate are found only in the luminal membrane of the BBB. This organization promotes the net removal of acidic- and nitrogen-rich AAs from the brain and accounts for the low level of glutamate penetration into the central nervous system. The presence of a [gamma]-glutamyl cycle at the luminal membrane and [Na.sup.+]-dependent AA transporters at the abluminal membrane may serve to modulate movement of AAs from blood to the brain. The [gamma]-glutamyl cycle is expected to generate pyroglutamate (synonymous with oxyproline) within the endothelial cells. Pyroglutamate stimulates secondary active AA transporters at the abluminal membrane, thereby reducing the net influx of AAs to the brain. It is now clear that BBB participates in the active regulation of the AA content of the brain. KEY WORDS: * brain capillaries * endothelial cells * polarity * sodium dependent transport * facilitative transport

Brain edema that forms during the early stages of stroke involves increased transport of [Na.sup.+] and C1- across an intact blood-brain barrier (BBB). Our previous studies have shown that a luminal BBB [Na.sup.+]-[K.sup.+]-C[1.sup.-] cotransporter is stimulated by conditions present during ischemia and that inhibition of the cotransporter by intravenous bumetanide greatly reduces edema formation in the rat middle cerebral artery occlusion model of stroke. The present study focused on investigating the effects of hypoxia, which develops rapidly in the brain during ischemia, on the activity and expression of the BBB [Na.sup.+]-[K.sup.+]-C[1.sup.-] cotransporter, as well as on [Na.sup.+]-[K.sup.+]-ATPase activity, cell ATP content, and intracellular volume. Cerebral microvascular endothelial cells (CMECs) were assessed for [Na.sup.+]-[K.sup.+]-C[1.sup.-] cotransporter and [Na.sup.+]-[K.sup.+]-ATPase activities as bumetanide-sensitive and ouabain-sensitive [sup.86]Rb influxes, respectively. ATP content was assessed by luciferase assay and intracellular volume by [[sup.3]H]-3-O-methyl-D-glucose and [[sup.14]C]-sucrose equilibration. We found that 30-min exposure of CMECs to hypoxia ranging from 7.5% to 0.5% 02 (vs. 19% normoxic [O.sub.2]) significantly increased cotransporter activity as did 7.5% or 2% [O.sub.2] for up to 2 h. This was not associated with reduction in [Na.sup.+]-[K.sup.+]-ATPase activity or ATP content. CMEC intracellular volume increased only after 4 to 5 h of hypoxia. Furthermore, glucose and pyruvate deprivation increased cotransporter activity under both normoxic and hypoxic conditions. Finally, we found that hypoxia increased phosphorylation but not abundance of the cotransporter protein. These findings support the hypothesis that hypoxia stimulation of the BBB [Na.sup.+]-[K.sup.+]-C[1.sup.-] cotransporter contributes to ischemia-induced brain edema formation. edema; blood-brain barrier; bumetanide; cell volume

The blood-brain barrier (BBB) is a metabolic and physiological barrier important for maintaining brain homeostasis. The aim of this study was to determine the role of PKC activation in BBB paracellular permeability changes induced by hypoxia and posthypoxic reoxygenation using in vitro and in vivo BBB models. In rat brain microvessel endothelial cells (RMECs) exposed to hypoxia (1% [O.sub.2]-99% [N.sub.2]; 24 h), a significant increase in total PKC activity was observed, and this was reduced by posthypoxic reoxygenation (95% room air-5% C[O.sub.2]) for 2 h. The expression of PKC-[beta]II, PKC-[gamma] PKC-[eta], PKC-[mu], and PKC-[lambda] also increased following hypoxia (1% [O.sub.2]-99% [N.sub.2]; 24 h), and these protein levels remained elevated following posthypoxic reoxygenation (95% room air-5% C[O.sub.2]; 2 h). Increases in the expression of PKC-[epsilon] and PKC-[zeta] were also observed following posthypoxic reoxygenation (95% room air-5% C[O.sub.2]; 2 h). Moreover, inhibition of PKC with chelerythrine chloride (10 [micro]M) attenuated the hypoxia-induced increases in [[sup.14]C]sucrose permeability. Similar to what was observed in RMECs, total PKC activity was also stimulated in cerebral microvessels isolated from rats exposed to hypoxia (6% [O.sub.2]-94% [N.sub.2]; 1 h) and posthypoxic reoxygenation (room air; 10 min). In contrast, hypoxia (6% [O.sub.2]-94% [N.sub.2]; 1 h) and posthypoxic reoxygenation (room air; 10 min) significantly increased the expression levels of only PKC-[gamma] and PKC-[theta] in the in vivo hypoxia model. These data demonstrate that hypoxia-induced BBB paracellular permeability changes occur via a PKC-dependent mechanism, possibly by differentially regulating the protein expression of the 11 PKC isozymes. protein kinase C; paracellular; neurovascular unit; rat

The Gamma-Hydroxybutyric Acid (GHB) was synthesized in 1960 in an attempt to create an analogue of the ubiquitous inhibitory brain neurotransmitter gamma-aminobutyric acid (GABA) that would cross the blood-brain barrier. The findings indicate that GHB is expected to have sedative properties similar to those that were reported for beta-butyrolactone in 1992.

Seven active men were recruited to examine changes in the serum concentration of S10013, a proposed peripheral marker of blood-brain barrier permeability, following prolonged exercise in temperate (T) and warm (W) conditions. Subjects were seated immersed to the neck in water at 35.0 (0.1)[degrees]C (T) or 39.0 (0.1)[degrees]C (W) for 30 min. Subjects then entered a room maintained at either 18.3 (1.8)[degrees]C (T) or 35.0 (0.3)[degrees]C (W) and completed 60 min of cycle exercise at 60% peak oxygen uptake. Serum S100[beta] concentration was elevated after exercise in the W trial (+0.12 (0.10) [micro]g/l; P = 0.02) but not after the T trial (P = 0.238). Water immersion and exercise elevated core temperature by 2.1 (0.5)[degrees]C to 39.5 (0.3)[degrees]C at the end of exercise in the W trial compared with a 0.9 (0.2)[degrees]C increase during the T trial (P < 0,001 ). Weighted mean skin temperature was higher throughout the W trial compared with the T trial (P < 0.001). Heart rate (P < 0.001) and blood glucose (P < 0.001) and lactate (P < 0.001) concentrations were elevated to a greater extent during exercise in the W trial than in the T trial. Ratings of perceived exertion (P < 0.001) and thermal comfort (P < 0.001) were markedly higher throughout the W trial than in the T trial. The results of this study demonstrate that serum S100[beta] was elevated after water immersion and prolonged exercise in a warm environment, suggesting that blood-brain barrier permeability may be altered. central fatigue; central nervous system; hyperthermia

Solubility and permeability are intimately linked in drug absorption processes. They have, however, been traditionally assayed separately. To support this linkage, a combined solubility/permeability assay was developed for determining absorption [...]

Mucopolysaccharidosis type VII is a lysosomal storage disorder resulting from inherited deficiency of [beta]-glucuronidase (GUS). Mucopolysaccharidosis type VII is characterized by glycosaminoglycan storage in most tissues, including brain. In these disorders, enzyme delivery across the blood-brain barrier (BBB) is the main obstacle to correction of lysosomal storage in the CNS. Prior studies suggested mouse brain is accessible to GUS in the first 2 weeks of life but not later. To explore a possible role for the mannose 6-phosphate/insulin-like growth factor II receptor in GUS transport across the BBB in neonatal mice, we compared brain uptake of phosphorylated GUS (P-GUS) and nonphosphorylated GUS (NP-GUS) in newborn and adult mice. [sup.131]I-P-GUS was transported across the BBB after i.v. injection in 2-day-old mice. The brain influx rate ([K.sub.in]) of [sup.131]I-P-GUS in 2-day-old mice was 0.21 [micro]l/g*min and decreased with age. By 7 weeks of age, transport of [sup.131]I-P-GUS was not significant. Capillary depletion revealed that 62% of the [sup.131]I-P-GUS in brain was in brain parenchyma in 2-day-old mice. In addition, uptake of [sup.131]I-P-GUS into brain was significantly reduced by coinjection of unlabeled P-GUS or M6P in a dose-dependent manner, in contrast, the [K.sub.in] of [sup.131]I-NP-GUS (0.04 [micro]l/g*min) was significantly lower than [sup.131]I-P-GUS in 2-day-old mice. Transcardiac brain perfusion confirmed that neither [sup.131]I-P-GUS nor [sup.131]I-NP-GUS crossed the BBB in adult mice. These results indicate that [sup.131]I-P-GUS transport into brain parenchyma in early postnatal life is mediated by the mannose 6-phosphate/insulin-like growth factor II receptor. This receptor-mediated transport is not observed in adult mice. beta-glucuronidase | mannose 6-phosphate/insulin-like growth factor II receptor | central nervous system | lysosomal storage disease | phosphorylated [beta]-glucuronidase

The blood--brain barrier (BBB) represents a significant impediment to a large variety of central nervous system-active agents. In the current study, we applied fluorescent polystyrene nanospheres (20 nm) to study the BBB permeability following cerebral ischemia and reperfusion. A microdialysis probe was implanted in the cerebral cortex of an anesthetized rat injected with fluorescent polystyrene nanospheres. The circulating nanospheres extravasating to the brain extracellular fluids were collected by the probe. Fluorescence intensity in the microdialysates throughout the course of cerebral ischemia/ reperfusion was measured. Cerebral ischemia and reperfusion induced transient accumulations of extracellular nanospheres in the brain. The accumulation of nanospheres may result from their extravasation from the blood vessels. The concurrent cerebral oxygen levels monitored using oxygen-dependent quenching of phosphorescence decreased following ischemia and returned to their original levels after reperfusion. In conclusion, we demonstrated that high temporal resolution measurements of BBB permeability in vivo can be obtained using fluorescence polystyrene nanospheres and that these data correlate with changes of cerebral oxygen concentration. This present investigation indicates that nanoparticles have potential clinical applications involving drug delivery and determination of therapeutic efficacy and on-site diagnosis.

Byline: N.C. Silver (1), C.D. Good (1), M.P. Sormani (2), D.G. MacManus (1), A.J. Thompson (1), M. Filippi (2), D.H. Miller (1) Keywords: Key words Multiple sclerosis; Magnetic resonance imaging; Triple-dose gadolinium; Magnetic transfer; Doamed imaging Abstract: By detecting focal blood-brain barier (BBB) breakdown, gadolinium (GD-DTPA) contrast-enhanced T1-weighted magnetic resonance imaging (MRI) allows assessment of inflammatory activity in multiple sclerosis (MS) and provides a sensitive means of monitoring immunomodulatory therapies in exploratory trials. Serial monthly studies were performed in eight relapsing-remitting and eight secondary progressive patients to assess new and more sensitive techniques for enhanced MRI. Brain and spine imaging was carried out at 1.5-T on two occasions 24--72 h apart using a conventional imaging protocol with T1-weighted MRI at single-dose (0.1 mmol/kg) Gd-DTPA and a potentially more sensitive 'modified' protocol with T1-weighted MRI at triple-dose (0.3 mmol/kg) Gd-DTPA (with addition of delay and magnetisation transfer presaturation for brain imaging). For each MRI protocol the total numbers of enhancing lesions (97 paired studies) and new enhancing lesions (81 paired studies) were assessed. The total number of enhancing lesions seen was 347/75 on conventional brain/cord MRI respectively, and 754/123 on modified brain/cord MRI. The respective numbers of new enhancing lesions were 168/40 on conventional and 276/71 on modified scans. Smaller increases were seen in the proportion of active scans using the modified protocol. Sample size calculations showed no reduction in sample sizes required for a parallel group study but a reduced sample size for crossover studies using the modified protocol: the addition of cord to brain imaging did not improve power for either trial design. A combined modified brain and cord imaging protocol markedly improves the detection of areas of focal BBB leakage in MS and many be useful in selected natural history studies. The modified brain protocol reduces sample size requirements for crossover studies but not necessarily for parallel design trials.. Author Affiliation: (1) NMR Research Unit, Institute of Neurology, University College Londen, Queen Square, Londen WC1N 3BG, UK, Tel.: +44-171-8373611, Fax: +44-171-2785616, GB (2) Department of Neurology, Scientific Institute Ospedale San Raffaele, University of Milan, Milan, Italy, IT Article note: Received: 23 May 2000, Received in revised form: 25 September 2000, Accepted: 19 October 2000

The mechanism of hypoxia-induced hyperpermeability was evaluated using an in vitro model of the blood-brain barrier which consists of porcine brain-derived microvascular endothelial cells (BMEC). It was shown that hypoxia-induced permeability in BMEC was abolished by an antibody to vascular endothelial growth factor (VGEF). The results suggest that hypoxia-induced permeability in vitro is mediated by the VEGF/VEGF receptor system.

The blood-brain barrier and a blood - cerebrospinal-fluid (CSF) barrier function together to isolate the brain from circulating drugs, toxins, and xenobiotics. The blood - CSF drug-permeability barrier is localized to the epithelium of the choroid plexus (CP). However, the molecular mechanisms regulating drug permeability across the CP epithelium are defined poorly. Herein, we describe a drug-permeability barrier in human and rodent CP mediated by epithelial-specific expression of the MDR1 (multidrug resistance) P glycoprotein (Pgp) and the multidrug resistance-associated protein (MRP). Noninvasive single-photon.emission computed tomography with 99mTc-sestamibi, a membrane-permeant radiopharmaceutical whose transport is mediated by both Pgp and MRP, shows a large blood-to-CSF concentration gradient across intact CP epithelium in humans in vivo. In rats, pharmacokinetic analysis with 99mTc-sestamibi determined the concentration gradient to be greater than 100-fold. In membrane fractions of isolated native CP from rat, mouse, and human, the 170-kDa Pgp and 190-kDa MRP are identified readily. Furthermore, the murine proteins are absent in CP isolated from their respective mdr1a/1b(-/-) and mrp(-/-) gene knockout littermates. As determined by immunohistochemical and drug-transport analysis of native CP and polarized epithelial cell cultures derived from neonatal rat CP, Pgp localizes subapically, conferring an apical-to-basal transepithelial permeation barrier to radiolabeled drugs. Conversely, MRP localizes basolaterally, conferring an opposing basal-to-apical drug-permeation barrier. Together, these transporters may coordinate secretion and reabsorption of natural product substrates and therapeutic drugs, including chemotherapeuric agents, antipsychotics, and HIV protease inhibitors, into and out of the central nervous system.

A study was conducted to test the hypothesis that antenatal corticosteroids reduce blood-brain barrier permeability in the pre-term ovine fetus. The hypothesis was based on the fact that antenatal corticosteroid therapy decreases the incidence of intraventricular hemorrhage in premature human infants. By administering dexamethasone in ewes, the study found that the blood-brain barrier permeability decreased across brain regions. The results suggest that corticosteroids could accelerate cardiovascular maturation and affect blood-brain function in unborn humans.

Dorothy Sipkins of Duke University School of Medicine and Fanny Herisson of Harvard Medical School had independently begun asking similar questions during clinical medical work early in their careers. Sipkins, [...]

A study indicates that the microvascular network instead of the brain regions lacking a blood-brain barrier (BBB) may serve as a major pathway for the delivery of blood-borne interleukin-1alpha to the brain. The finding is based on experiments that involve intravenously injecting mice with with radioiodinated interleukin-1alpha and subjecting their brains to emulsion audioradiography. Results showed that the brain regions lacking a BBB contain a high degree of blood-borne interleukin-1alpha.

Introduction Endothelial cells line all of the vessels of the circulatory system, providing a non-thrombogenic conduit for blood flow; they regulate many complex functions in the vasculature, such as coagulation, [...]

Isolated plasma membrane vesicles from the luminal and abluminal side of bovine brain capillaries were examined to investigate the mechanism of nitrogen waste removal in the brain given the blood brain barrier. Results reveal that the capillaries in the brain are active participants in the removal of nitrogenous wastes from the brain in the form of glutamine, glutamate and ammonia.