By The Blood-brain Barrier Research Articles

Noninvasive and targeted gene delivery into the brain using microbubble-facilitated focused ultrasound.

Recombinant adeno-associated viral (rAAV) vectors are potentially powerful tools for gene therapy of CNS diseases, but their penetration into brain parenchyma is severely limited by the blood-brain barrier (BBB) and current delivery relies on invasive stereotactic injection. Here we evaluate the local, targeted delivery of rAAV vectors into the brains of mice by noninvasive, reversible, microbubble-facilitated focused ultrasound (FUS), resulting in BBB opening that can be monitored and controlled by magnetic resonance imaging (MRI). Using this method, we found that IV-administered AAV2-GFP (green fluorescence protein) with a low viral vector titer (1×109 vg/g) can successfully penetrate the BBB-opened brain regions to express GFP. We show that MRI monitoring of BBB-opening could serve as an indicator of the scale and distribution of AAV transduction. Transduction peaked at 3 weeks and neurons and astrocytes were affected. This novel, noninvasive delivery approach could significantly broaden the application of AAV-viral-vector-based genes for treatment of CNS diseases.

Design, Synthesis and Brain Uptake of LAT1-Targeted Amino Acid Prodrugs of Dopamine

Drug delivery to the brain is impeded by the blood-brain barrier (BBB). Here, we attempted to enhance the brain uptake of cationic dopamine by utilizing the large amino acid transporter 1 (LAT1) at the BBB by prodrug approach. Three amino acid prodrugs of dopamine were synthesized and their prodrug properties were examined in vitro. Their LAT1-binding and BBB-permeation were studied using the in situ rat brain perfusion technique. The brain uptake after intravenous administration and the dopamine-releasing ability in the rat striatum after intraperitoneal administration were also determined for the most promising prodrug. All prodrugs underwent adequate cleavage in rat tissue homogenates. The prodrug with phenylalanine derivative as the promoiety had both higher affinity for LAT1 and better brain uptake properties than those with an alkyl amino acid - mimicking promoiety. The phenylalanine prodrug was taken up into the brain after intravenous injection but after intraperitoneal injection the prodrug did not elevate striatal dopamine concentrations above those achieved by corresponding L-dopa treatment. These results indicate that attachment of phenylalanine to a cationic drug via an amide bond from the meta-position of its aromatic ring could be highly applicable in prodrug design for LAT1-mediated CNS-delivery of not only anionic but also cationic polar drugs.

Regulation of Immune Cell Infiltration into the CNS by Regional Neural Inputs Explained by the Gate Theory

The central nervous system (CNS) is an immune-privileged environment protected by the blood-brain barrier (BBB), which consists of specific endothelial cells that are brought together by tight junctions and tight liner sheets formed by pericytes and astrocytic end-feet. Despite the BBB, various immune and tumor cells can infiltrate the CNS parenchyma, as seen in several autoimmune diseases like multiple sclerosis (MS), cancer metastasis, and virus infections. Aside from a mechanical disruption of the BBB like trauma, how and where these cells enter and accumulate in the CNS from the blood is a matter of debate. Recently, using experimental autoimmune encephalomyelitis (EAE), an animal model of MS, we found a “gateway” at the fifth lumber cord where pathogenic autoreactive CD4+ T cells can cross the BBB. Interestingly, this gateway is regulated by regional neural stimulations that can be mechanistically explained by the gate theory. In this review, we also discuss this theory and its potential for treating human diseases.

Focal Delivery of AAV2/1-transgenes Into the Rat Brain by Localized Ultrasound-induced BBB Opening

Delivery of drugs and macromolecules to the central nervous system (CNS) is hindered by the blood–brain barrier (BBB). Several approaches have been used to overcome this hindrance to facilitate the treatment of various CNS diseases. We now present results showing that chimeric adeno-associated virus 2/1 (AAV2/1) particles containing the coding region for the LacZ gene are efficiently delivered into the rat brain upon intravenous (IV) administration after BBB opening by focused ultrasound in the presence of vascular acoustic resonators. We show that the transgene is correctly and efficiently expressed in cells located in the neighborhood of the insonated focus, especially in the vicinity of small vessels and capillaries. Histochemical LacZ staining allows the identification of large amounts of cells expressing the enzymatically active protein. Using double immunofluorescence (IF) with antibodies against tubulinIII and bacterial LacZ, we identified these cells to be mostly neurons. A small proportion of the transduced cells was recognized as glial cells, reacting positive in the IF with antibodies against astrocytic markers. These results demonstrate that our approach allows a very specific, localized, and efficient expression of intravenously administered transgenes in the brain of rats upon ultrasound-induced BBB opening.

The Blood-Brain Barrier Choline Transporter

Drug delivery to the brain is made difficult by the blood-brain barrier (BBB) which is selectively permeable to organic drug compounds. Several membrane solute and nutrient transporters are expressed in the BBB vasculature, which may be utilized as mechanism of delivery of drugs to the brain. Of interest to us, are the organic cation transporters which could be used to transport cationic compounds into the CNS. In this mini-review, we will review the current understanding of the structural requirements for designing compounds which could effectively use organic cation transporters (OCT). For the first time, structural requirements for both OCT1 and OCT2 versus the BBB choline transporter (BBBCHT) are discussed and compared. The information gained here could increase the success rate in successful CNS drug delivery and therapeutics.

Targeting metabolic inflammation in Parkinson’s disease: Implications for prospective therapeutic strategies

1. Parkinson's disease (PD) is one of the most common neurodegenerative disorders and is characterized by a progressive loss of dopaminergic neurons in the substantia nigra pars compacta. Although the aetiology of PD has not been clarified as yet, it is believed that ageing, diet, diabetes and adiposity are associated with PD. 2. Type 2 diabetes and lipid abnormalities share multiple common pathophysiological mechanisms with PD. In particular, inflammation plays a critical role in the destruction of both pancreatic islet β-cells and dopaminergic neurons in the substantia nigra. Emerging evidence indicates that dysfunctions of energy metabolism evoke metabolic inflammation, which differs to the narrow concept of inflammation, participating in systemic pathological processes such as neurodegenerative disease and diabetes. 3. The brain is considered an immunologically privileged organ, free from immune reactions, because it is protected by the blood-brain barrier (BBB). However, studies have shown that there is gradual impairment of neurovascular function with ageing and in neurodegenerative disorders, resulting in abnormal states, including increased BBB permeability. Consequently, harmful elements that would not normally be able to cross the BBB, such as pro-inflammatory factors, reactive oxygen species and neurotoxins, infiltrate into the brain, triggering neural injury. 4. Currently, the drugs available for the treatment of PD only ameliorate the symptoms of the disease. Therapeutic strategies aimed at stopping or modifying disease progression are still being sought. Most recent studies suggest that both central and peripheral inflammation may be dysregulated in PD. Therefore, therapeutic strategies aimed at modulating systemic inflammatory reactions or energy metabolism may represent a goal in neuroprotection in PD.

Nanotechnology Based Diagnostic and Therapeutic Strategies for Neuroscience with Special Emphasis on Ischemic Stroke

Ischemic stroke is the second leading cause of death and long-term disability worldwide, for which no effective therapies are available. The increasing prevalence of ischemic stroke and related health risks, combined with the lack of effective therapies, highlight the desperate need for continued research for exploring the safe and effective drugs, which favourably influence multiple pathways leading to neuroprotection and extend the benefit to a larger number of patients diagnosed with stroke. Numerous preclinical studies have reported very promising results using "neuroprotectants", all of which have failed at clinical trials because of either safety issues or lack of efficacy. The delivery of many potentially therapeutic neuroprotectants and diagnostic compounds to specific areas of the brain is restricted by the blood-brain barrier (BBB). Nanoparticles (NPs) have colossal applications that could revolutionize the treatment of ischemic stroke. NPs can readily transmigrate across the BBB without compromising its integrity. Recent striking developments in nanotechnology have produced a great deal of nano-devices, which could be used for the treatment and neuronal regeneration following ischemic stroke. This article attempts to convey the untapped potentials of nanopharmaceuticals for the treatment of ischemic stroke. Looking towards the future, this review focuses on the potential applications of nanoparticulate systems for the delivery of therapeutic cargo into the brain for imparting neuroprotection against ischemic stroke. This review also provides an overview of targeted NPs, which are being used for imaging, neuroprotection and regeneration of ischemic brain.

Central nervous system: A modified immune surveillance circuit?

Immune surveillance in the central nervous system (CNS) was considered impossible because: (i) the brain parenchyma is separated from the blood circulation by the blood-brain barrier (BBB); (ii) the brain lacks lymphatic drainage and (iii) the brain displays low major histocompatibility complex class II (MHCII) expression. In this context, the BBB prevents entry of immune molecules and effector cells to the CNS. The absence of lymphatic vessels avoids CNS antigens from reaching the lymph nodes for lymphocyte presentation and activation. Finally, the low MHCII expression hinders effective antigen presentation and re-activation of T cells for a competent immune response. All these factors limit the effectiveness of the afferent and efferent arms necessary to carry out immune surveillance. Nevertheless, recent evidence supports that CNS is monitored by the immune system through a modified surveillance circuit; this work reviews these findings.

A Carrier for Non-Covalent Delivery of Functional Beta-Galactosidase and Antibodies against Amyloid Plaques and IgM to the Brain

BackgroundTherapeutic intervention of numerous brain-associated disorders currently remains unrealized due to serious limitations imposed by the blood-brain-barrier (BBB). The BBB generally allows transport of small molecules, typically <600 daltons with high octanol/water partition coefficients, but denies passage to most larger molecules. However, some receptors present on the BBB allow passage of cognate proteins to the brain. Utilizing such receptor-ligand systems, several investigators have developed methods for delivering proteins to the brain, a critical requirement of which involves covalent linking of the target protein to a carrier entity. Such covalent modifications involve extensive preparative and post-preparative chemistry that poses daunting limitations in the context of delivery to any organ. Here, we report creation of a 36-amino acid peptide transporter, which can transport a protein to the brain after routine intravenous injection of the transporter-protein mixture. No covalent linkage of the protein with the transporter is necessary.ApproachA peptide transporter comprising sixteen lysine residues and 20 amino acids corresponding to the LDLR-binding domain of apolipoprotein E (ApoE) was synthesized. Transport of beta-galactosidase, IgG, IgM, and antibodies against amyloid plques to the brain upon iv injection of the protein-transporter mixture was evaluated through staining for enzyme activity or micro single photon emission tomography (micro-SPECT) or immunostaining. Effect of the transporter on the integrity of the BBB was also investigated.Principal FindingsThe transporter enabled delivery to the mouse brain of functional beta-galactosidase, human IgG and IgM, and two antibodies that labeled brain-associated amyloid beta plaques in a mouse model of Alzheimer's disease.SignificanceThe results suggest the transporter is able to transport most or all proteins to the brain without the need for chemically linking the transporter to a protein. Thus, the approach offers an avenue for rapid clinical evaluation of numerous candidate drugs against neurological diseases including cancer. (299 words).

Blood-brain barrier P450 enzymes and multidrug transporters in drug resistance: a synergistic role in neurological diseases.

Drug penetration into the central nervous system (CNS) is controlled by the blood-brain barrier (BBB). Even though a number of strategies to circumvent the BBB and to improve drug access have been developed, drug resistance in CNS diseases remains an unmet clinical problem. We here review the mechanisms by which a healthy or pathological BBB influences drug distribution in the brain, with emphasis on the role of P450 metabolic enzymes and multi-drug transporter (MDT) proteins. In addition to the classic hepatic and gut biotransformation pathways, CNS expression of P450 enzymes may bear pharmacokinetic and pharmacodynamic significance exerting a metabolic activity and transforming parent drugs into specific products. We propose these mechanisms to play a major role in CNS drug resistant pathologies including refractory forms of epilepsy. Changes in the cerebrovascular hemodynamic conditions can affect expression of P450 enzymes and MDT proteins. This should be taken into account when developing in vitro experimental approaches to reproduce the physiological or pathological properties of the BBB. Finally, a link between P450 and MDT expression in the diseased brain and cell survival is discussed.

Several rAAV Vectors Efficiently Cross the Blood–brain Barrier and Transduce Neurons and Astrocytes in the Neonatal Mouse Central Nervous System

Noninvasive systemic gene delivery to the central nervous system (CNS) has largely been impeded by the blood-brain barrier (BBB). Recent studies documented widespread CNS gene transfer after intravascular delivery of recombinant adeno-associated virus 9 (rAAV9). To investigate alternative and possibly more potent rAAV vectors for systemic gene delivery across the BBB, we systematically evaluated the CNS gene transfer properties of nine different rAAVEGFP vectors after intravascular infusion in neonatal mice. Several rAAVs efficiently transduce neurons, motor neurons, astrocytes, and Purkinje cells; among them, rAAVrh.10 is at least as efficient as rAAV9 in many of the regions examined. Importantly, intravenously delivered rAAVs did not cause abnormal microgliosis in the CNS. The rAAVs that achieve stable widespread gene transfer in the CNS are exceptionally useful platforms for the development of therapeutic approaches for neurological disorders affecting large regions of the CNS as well as convenient biological tools for neuroscience research.

Side Chain-oxidized Oxysterols Regulate the Brain Renin-Angiotensin System through a Liver X Receptor-dependent Mechanism

Disturbances in cholesterol metabolism have been associated with hypertension and neurodegenerative disorders. Because cholesterol metabolism in the brain is efficiently separated from plasma cholesterol by the blood-brain barrier (BBB), it is an unsolved paradox how high blood cholesterol can cause an effect in the brain. Here, we discuss the possibility that cholesterol metabolites permeable to the BBB might account for these effects. We show that 27-hydroxycholesterol (27-OH) and 24S-hydroxycholesterol (24S-OH) up-regulate the renin-angiotensin system (RAS) in the brain. Brains of mice on a cholesterol-enriched diet showed up-regulated angiotensin converting enzyme (ACE), angiotensinogen (AGT), and increased JAK/STAT activity. These effects were confirmed in in vitro studies with primary neurons and astrocytes exposed to 27-OH or 24S-OH, and were partially mediated by liver X receptors. In contrast, brain RAS activity was decreased in Cyp27a1-deficient mice, a model exhibiting reduced 27-OH production from cholesterol. Moreover, in humans, normocholesterolemic patients with elevated 27-OH levels, due to a CYP7B1 mutation, had markers of activated RAS in their cerebrospinal fluid. Our results demonstrate that side chain-oxidized oxysterols are modulators of brain RAS. Considering that levels of cholesterol and 27-OH correlate in the circulation and 27-OH can pass the BBB into the brain, we suggest that this cholesterol metabolite could be a link between high plasma cholesterol levels, hypertension, and neurodegeneration.

Honokiol Crosses BBB and BCSFB, and Inhibits Brain Tumor Growth in Rat 9L Intracerebral Gliosarcoma Model and Human U251 Xenograft Glioma Model

BackgroundGliosarcoma is one of the most common malignant brain tumors, and anti-angiogenesis is a promising approach for the treatment of gliosarcoma. However, chemotherapy is obstructed by the physical obstacle formed by the blood-brain barrier (BBB) and blood-cerebrospinal fluid barrier (BCSFB). Honokiol has been known to possess potent activities in the central nervous system diseases, and anti-angiogenic and anti-tumor properties. Here, we hypothesized that honokiol could cross the BBB and BCSFB for the treatment of gliosarcoma.MethodologiesWe first evaluated the abilities of honokiol to cross the BBB and BCSFB by measuring the penetration of honokiol into brain and blood-cerebrospinal fluid, and compared the honokiol amount taken up by brain with that by other tissues. Then we investigated the effect of honokiol on the growth inhibition of rat 9L gliosarcoma cells and human U251 glioma cells in vitro. Finally we established rat 9L intracerebral gliosarcoma model in Fisher 344 rats and human U251 xenograft glioma model in nude mice to investigate the anti-tumor activity.Principal FindingsWe showed for the first time that honokiol could effectively cross BBB and BCSFB. The ratios of brain/plasma concentration were respectively 1.29, 2.54, 2.56 and 2.72 at 5, 30, 60 and 120 min. And about 10% of honokiol in plasma crossed BCSFB into cerebrospinal fluid (CSF). In vitro, honokiol produced dose-dependent inhibition of the growth of rat 9L gliosarcoma cells and human U251 glioma cells with IC50 of 15.61 µg/mL and 16.38 µg/mL, respectively. In vivo, treatment with 20 mg/kg body weight of honokiol (honokiol was given twice per week for 3 weeks by intravenous injection) resulted in significant reduction of tumor volume (112.70±10.16 mm3) compared with vehicle group (238.63±19.69 mm3, P = 0.000), with 52.77% inhibiting rate in rat 9L intracerebral gliosarcoma model, and (1450.83±348.36 mm3) compared with vehicle group (2914.17±780.52 mm3, P = 0.002), with 50.21% inhibiting rate in human U251 xenograft glioma model. Honokiol also significantly improved the survival over vehicle group in the two models (P<0.05).Conclusions/SignificanceThis study provided the first evidence that honokiol could effectively cross BBB and BCSFB and inhibit brain tumor growth in rat 9L intracerebral gliosarcoma model and human U251 xenograft glioma model. It suggested a significant strategy for offering a potential new therapy for the treatment of gliosarcoma.

Intracerebroventricular enzyme infusion corrects central nervous system pathology and dysfunction in a mouse model of metachromatic leukodystrophy

Arylsulfatase A (ASA) catalyzes the desulfation of sulfatide, a major lipid component of myelin. Inherited functional deficiencies of ASA cause the lysosomal storage disease (LSD) metachromatic leukodystrophy (MLD), which is characterized by intralysosomal accumulation of sulfatide, progressive neurological symptoms and early death. Enzyme replacement therapy (ERT) using intravenous injection of active enzyme is a treatment option for many LSDs as exogenous lysosomal enzymes are delivered to lysosomes of patient's cells via receptor-mediated endocytosis. Efficient treatment of MLD and other LSDs with central nervous system (CNS) involvement is, however, hampered by the blood-brain barrier (BBB), which limits transfer of therapeutic enzymes from the circulation to the brain parenchyma. To bypass the BBB, we infused recombinant human ASA (rhASA) by implanted miniature pumps into the cerebrospinal fluid (CSF) of a conventional and a novel, genetically aggravated ASA knockout mouse model of MLD. rhASA continuously delivered to the lateral ventricle for 4 weeks penetrated the brain parenchyma and was targeted to the lysosomes of brain cells. Histological analysis revealed complete reversal of lysosomal storage in the infused hemisphere. rhASA concentrations and sulfatide clearance declined with increasing distance from the infusion site. Correction of the ataxic gait indicated reversal of central nervous system dysfunctions. The profound histopathological and functional improvements, the requirement of low enzyme doses and the absence of immunological side effects suggest intracerebroventricular ERT to be a promising treatment option for MLD and other LSDs with prevailing CNS disease.

Desipramine treatment has minimal effects on the brain accumulation of glucocorticoids in P-gp-deficient and wild-type mice

SummaryHyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis in patients with depression can be reduced by antidepressants, which are thought to improve endogenous glucocorticoid-mediated negative feedback. A proportion of peripherally released glucocorticoids need to enter brain tissue, protected by the blood–brain barrier (BBB), in order to achieve this negative feedback effect at the level of the central nervous systems (CNS). The multidrug resistance transporter P-glycoprotein (P-gp) has been shown to actively transport glucocorticoid hormones and has been implicated in the regulation of glucocorticoid access to the CNS. Using an in situ brain/choroid plexus perfusion method, we tested the hypothesis that the antidepressant desipramine increases glucocorticoid accumulation in the mouse brain by inhibiting P-gp, following either chronic treatment (8 days, 20 mg/kg/day, IP) or acute administration (20 min brain perfusion in the presence of either 0.9 μM or 10 μM desipramine). Contrary to our hypothesis, chronic treatment with desipramine did not affect the accumulation of [3H]dexamethasone in any sample compared to saline-treated mice. Acute desipramine had limited and variable effects on glucocorticoid accumulation in the CNS, with accumulation of [3H]dexamethasone increased in the cerebellum, accumulation of [3H]cortisol reduced in the frontal cortex, hypothalamus, and cerebellum, and accumulation of [3H]corticosterone (the endogenous glucocorticoid in rodents) not affected. Overall, under the conditions tested, these results do not support the hypothesis that treatment with desipramine can inhibit P-gp at the BBB and subsequently increase the accumulation of glucocorticoids in the brain.

Molecular biology of the blood-brain and the blood-cerebrospinal fluid barriers: similarities and differences

Efficient processing of information by the central nervous system (CNS) represents an important evolutionary advantage. Thus, homeostatic mechanisms have developed that provide appropriate circumstances for neuronal signaling, including a highly controlled and stable microenvironment. To provide such a milieu for neurons, extracellular fluids of the CNS are separated from the changeable environment of blood at three major interfaces: at the brain capillaries by the blood-brain barrier (BBB), which is localized at the level of the endothelial cells and separates brain interstitial fluid (ISF) from blood; at the epithelial layer of four choroid plexuses, the blood-cerebrospinal fluid (CSF) barrier (BCSFB), which separates CSF from the CP ISF, and at the arachnoid barrier. The two barriers that represent the largest interface between blood and brain extracellular fluids, the BBB and the BCSFB, prevent the free paracellular diffusion of polar molecules by complex morphological features, including tight junctions (TJs) that interconnect the endothelial and epithelial cells, respectively. The first part of this review focuses on the molecular biology of TJs and adherens junctions in the brain capillary endothelial cells and in the CP epithelial cells. However, normal function of the CNS depends on a constant supply of essential molecules, like glucose and amino acids from the blood, exchange of electrolytes between brain extracellular fluids and blood, as well as on efficient removal of metabolic waste products and excess neurotransmitters from the brain ISF. Therefore, a number of specific transport proteins are expressed in brain capillary endothelial cells and CP epithelial cells that provide transport of nutrients and ions into the CNS and removal of waste products and ions from the CSF. The second part of this review concentrates on the molecular biology of various solute carrier (SLC) transport proteins at those two barriers and underlines differences in their expression between the two barriers. Also, many blood-borne molecules and xenobiotics can diffuse into brain ISF and then into neuronal membranes due to their physicochemical properties. Entry of these compounds could be detrimental for neural transmission and signalling. Thus, BBB and BCSFB express transport proteins that actively restrict entry of lipophilic and amphipathic substances from blood and/or remove those molecules from the brain extracellular fluids. The third part of this review concentrates on the molecular biology of ATP-binding cassette (ABC)-transporters and those SLC transporters that are involved in efflux transport of xenobiotics, their expression at the BBB and BCSFB and differences in expression in the two major blood-brain interfaces. In addition, transport and diffusion of ions by the BBB and CP epithelium are involved in the formation of fluid, the ISF and CSF, respectively, so the last part of this review discusses molecular biology of ion transporters/exchangers and ion channels in the brain endothelial and CP epithelial cells.

Systemic combinatorial peptide selection yields a non-canonical iron-mimicry mechanism for targeting tumors in a mouse model of human glioblastoma

The management of CNS tumors is limited by the blood-brain barrier (BBB), a vascular interface that restricts the passage of most molecules from the blood into the brain. Here we show that phage particles targeted with certain ligand motifs selected in vivo from a combinatorial peptide library can cross the BBB under normal and pathological conditions. Specifically, we demonstrated that phage clones displaying an iron-mimic peptide were able to target a protein complex of transferrin and transferrin receptor (TfR) through a non-canonical allosteric binding mechanism and that this functional protein complex mediated transport of the corresponding viral particles into the normal mouse brain. We also showed that, in an orthotopic mouse model of human glioblastoma, a combination of TfR overexpression plus extended vascular permeability and ligand retention resulted in remarkable brain tumor targeting of chimeric adeno-associated virus/phage particles displaying the iron-mimic peptide and carrying a gene of interest. As a proof of concept, we delivered the HSV thymidine kinase gene for molecular-genetic imaging and targeted therapy of intracranial xenografted tumors. Finally, we established that these experimental findings might be clinically relevant by determining through human tissue microarrays that many primary astrocytic tumors strongly express TfR. Together, our combinatorial selection system and results may provide a translational avenue for the targeted detection and treatment of brain tumors.

Current concepts of blood-brain barrier development

Homeostasis of the central nervous system (CNS) microenvironment is essential for its normal function and is maintained by the blood-brain barrier (BBB). The BBB proper is made up of endothelial cells (ECs) interconnected by tight junctions (TJs) that reveal a unique morphology and biochemical composition of the body's vasculature. In this article, we focus on developmental aspects of the BBB and describe morphological as well as molecular special features of the neuro-vascular unit (NVU) involved in barrier induction. Recently, we and others identified the Wnt/b-catenin pathway as crucial for brain angiogenesis, TJ and BBB formation. Based on these findings we discuss other pathways and molecular interactions for BBB establishment and maintenance. At the morphological level, our concept favors a major role for polarized astrocytes (ACs) therein. Orthogonal arrays of particles (OAPs) that are the morphological correlate of the water channel protein aquaporin-4 (AQP4) are specifically formed in the membrane of the AC endfoot. The polarized AC endfoot and hence OAPs are dependent on agrin and dystroglycan, of which agrin is a developmentally regulated extracellular matrix (ECM) component. Understanding the mechanisms leading to BBB development will be key to the understanding of barrier maintenance that is crucial for, but frequently disturbed, in the diseased adult brain.

The Mysterious Link between Cholesterol and Alzheimer?s Disease: Is the Blood-Brain Barrier a Suspect?

It is now well accepted that the deposition and aggregation of amyloid β (Aβ) peptides are hallmarks of Alzheimer’s disease (AD) [1]. These peptides are constitutively secreted by neurons but (for reasons that remain to be established) Aβ metabolism is altered in AD patients and leads slowly to the neurodegenerative process. With these considerations in mind, the identification of genetic, biochemical, environmental and dietary factors influencing Aβ peptide synthesis, clearance and degradation became a high priority for researchers working on AD. The enzyme responsible for Aβ peptide synthesis was soon identified as a β-secretase called β-site amyloid precursor protein cleaving enzyme 1 (BACE1) [2]. The most important genetic risk factor for late-onset AD was identified as the APOe4 allele [ 3]. This gene encodes an apolipoprotein, a protein component of the low-density lipoprotein (LDL) particles involved in lipid transport throughout the body; this finding highlighted a probable relationship between AD and cholesterol metabolism. More recently, two other susceptibility loci linked to cholesterol metabolism have been identified in AD: CLU (coding for clusterin, another apolipoprotein) [4] and ABCA7 [5] (coding for the ATP-binding cassette sub-family A member 7 transporter involved in cholesterol homeostasis [6] ). This link between AD and cholesterol metabolism was reinforced by the observation that the sterol can modulate BACE1 activity and promote Aβ peptide biosynthesis [7]. Cholesterol was also identified as dietary risk factor in humans; since elevated serum cholesterol is a risk factor for developing AD and amount of Aβ peptide in the brain correlates with serum levels of LDL and total cholesterol [8,9]. Moreover, rabbits and mice on a cholesterol-enriched diet show elevated serum cholesterol levels, increased Aβ peptide deposition and cognitive impairment [10-14]. However, the reasons why serum cholesterol influences AD are difficult to determine, for two main reasons. Firstly, most of the accretion of cholesterol in the central nervous system (CNS) is due to de novo synthesis (reviewed in [15]). Secondly, the brain is isolated from the rest of body by the blood-brain barrier (BBB), a dynamic interface formed by brain capillary endothelial cells (BCECs) and sealed by complex tight junctions that restrict the paracellular pathway [16]. One of the BBB’s main roles is to supply the brain with essential nutrients and mediate the efflux of many waste products. To this end, BCECs express specific receptors or transporters in their luminal (plasma-side) and/ or abluminal (brain-side) membranes that force molecules to take the transcellular route.

Contrast‐enhanced in vivo magnetic resonance microscopy of the mouse brain enabled by noninvasive opening of the blood‐brain barrier with ultrasound

The use of contrast agents for neuroimaging is limited by the blood-brain barrier (BBB), which restricts entry into the brain. To administer imaging agents to the brain of rats, intracarotid infusions of hypertonic mannitol have been used to open the BBB. However, this technically challenging approach is invasive, opens only a limited region of the BBB, and is difficult to extend to mice. In this work, the BBB was opened in mice, using unfocused ultrasound combined with an injection of microbubbles. This technique has several notable features: it (a) can be performed transcranially in mice; (b) takes only 3 min and uses only commercially available components; (c) opens the BBB throughout the brain; (d) causes no observed histologic damage or changes in behavior (with peak-negative acoustic pressures of 0.36 MPa); and (e) allows recovery of the BBB within 4 h. Using this technique, Gadopentetate Dimeglumine (Gd-DTPA) was administered to the mouse brain parenchyma, thereby shortening T(1) and enabling the acquisition of high-resolution (52 × 52 × 100 micrometers(3)) images in 51 min in vivo. By enabling the administration of both existing anatomic contrast agents and the newer molecular/sensing contrast agents, this technique may be useful for the study of mouse models of neurologic function and pathology with MRI.