The blood-brain barrier significantly limits eflornithine entry into Trypanosoma brucei brucei infected mouse brain

Drugs to treat African trypanosomiasis are toxic, expensive and subject to parasite resistance. New drugs are urgently being sought. Although the existing drug, eflornithine, is assumed to reach the brain in high concentrations, little is known about how it crosses the healthy and infected blood-brain barrier. This information is essential for the design of drug combinations and new drugs. This study used novel combinations of animal models to address these omissions. Eflornithine crossed the healthy blood-CNS interfaces poorly, but this could be improved by co-administering suramin, but not nifurtimox, pentamidine or melarsoprol. Work using a murine model of sleeping sickness demonstrated that Trypanosoma brucei brucei crossed the blood-CNS interfaces, which remained functional, early in the course of infection. Concentrations of brain parasites increased during the infection and this resulted in detectable blood-brain barrier, but not choroid plexus, dysfunction at day 28 post-infection with resultant increases in eflornithine brain delivery. Barrier integrity was never restored and the animals died at day 37.9 +/- 1.2. This study indicates why an intensive treatment regimen of eflornithine is required (poor blood-brain barrier penetration) and suggests a possible remedy (combining eflornithine with suramin). The blood-brain barrier retains functionality until a late, possibly terminal stage, of trypanosoma infection.     

Claudin‐5a in developing zebrafish brain barriers: another brick in the wall

Claudins serve essential roles in regulating paracellular permeability properties within occluding junctions. Recent studies have begun to elucidate developmental roles of claudins within immature tissues. This work has uncovered an involvement of several claudins in determining tight junction properties that have an effect on embryonic morphogenesis and physiology. During zebrafish brain morphogenesis, Claudin‐5a determines the paracellular permeability of tight junctions within a transient neuroepithelial‐ventricular barrier that maintains the hydrostatic fluid pressure required for brain ventricular lumen expansion. However, the roles of Claudins in development may well extend beyond being mere junctional components. Several post‐translational modifications of Claudins have been characterized that indicate a direct regulation by developmental signals. This review focuses on the involvement of Claudin‐5a in cerebral barrier formation in the zebrafish embryo and includes some speculations about possible modes of regulation.     

Expression and possible role of creatine transporter in the brain and at the blood-cerebrospinal fluid barrier as a transporting protein of guanidinoacetate, an endogenous convulsant

Little is known about the cerebral distribution and clearance of guanidinoacetate (GAA), the accumulation of which induces convulsions. The purpose of the present study was to identify creatine transporter (CRT)-mediated GAA transport and to clarify its cerebral expression and role in GAA efflux transport at the blood-cerebrospinal fluid barrier (BCSFB). CRT mediated GAA transport with a K(m) value of 269 microM/412 microM which was approximately 10-fold greater than that of CRT for creatine. There was wide and distinct cerebral expression of CRT and localization of CRT on the brush-border membrane of choroid plexus epithelial cells. The in vivo elimination clearance of GAA from the CSF was 13-fold greater than that of d-mannitol reflecting bulk flow of the CSF. This process was partially inhibited by creatine. The characteristics of GAA uptake by isolated choroid plexus and an immortalized rat choroid plexus epithelial cell line (TR-CSFB cells) used as an in vitro model of BCSFB are partially consistent with those of CRT. These results suggest that CRT plays a role in the cerebral distribution of GAA and GAA uptake by the choroid plexus. However, in the presence of endogenous creatine in the CSF, CRT may make only a limited contribution to the GAA efflux transport at the BCSFB.     

Epithelial V-like antigen regulates permeability of the blood-CSF barrier

Epithelial V-like antigen (EVA), a CD3-binding immunoglobulin-like protein, regulates embryonic thymic development. Here we demonstrate that EVA is expressed in choroid plexus from mature immune competent and lymphocyte-deficient (RAG−/−) mice. Choroid plexus epithelial cells from RAG−/− mice demonstrated reduced junctional integrity and enhanced permeability that was associated with decreased expression of E-cadherin and EVA mRNA as compared to wild-type mice. Following iv infusion of an anti-CD3 antibody (145-2C11) that also binds EVA, expression of E-cadherin and EVA mRNA approached levels seen in wild-type mice. Immuno-fluorescent staining for cadherin also revealed decreased expression in untreated RAG−/− mice that could be increased by 145-2C11 treatment. Expression of mouse EVA in HEK-293 cells followed by challenge with 145-2C11 resulted in increased cytosolic calcium that was not seen in control cells. These results suggest that EVA expressed in choroid plexus cells may regulate the permeability of the blood-CSF barrier.     

Brain capillary endothelium and choroid plexus epithelium regulate transport of transferrin-bound and free iron into the rat brain

Iron transport into the CNS is still not completely understood. Using a brain perfusion technique in rats, we have shown a significant brain capillary uptake of circulating transferrin (Tf)-bound and free 59Fe (1 nm) at rates of 136 +/- 26 and 182 +/- 23 microL/g/min, respectively, while their respective transport rates into brain parenchyma were 1.68 +/- 0.56 and 1.52 +/- 0.48 microL/g/min. Regional Tf receptor density (Bmax) in brain endothelium determined with 125I-holo-Tf correlated well with 59Fe-Tf regional brain uptake rates reflecting significant vascular association of iron. Tf-bound and free circulating 59Fe were sequestered by the choroid plexus and transported into the CSF at low rates of 0.17 +/- 0.01 and 0.09 +/- 0.02 microL/min/g, respectively, consistent with a 10-fold brain-CSF concentration gradient for 59Fe, Tf-bound or free. We conclude that transport of circulating Tf-bound and free iron could be equally important for its delivery to the CNS. Moreover, data suggest that entry of Tf-bound and free iron into the CNS is determined by (i) its initial sequestration by brain capillaries and choroid plexus, and (ii) subsequent controlled and slow release from vascular structures into brain interstitial fluid and CSF.     

RNAi-mediated reversible opening of the blood-brain barrier

Background

The blood‐brain barrier (BBB) contains tight junctions (TJs) which reduce the space between adjacent endothelial cells lining the fine capillaries of the microvasculature of the brain to form a selective and regulatable barrier.

Methods

Using a hydrodynamic approach, we delivered siRNA targeting the TJ protein claudin‐5 to the endothelial cells of the BBB in mice.

Results

We have shown a significant decrease in claudin‐5 mRNA levels 24 and 48 hours post‐delivery of siRNA, with levels of protein expression decreasing up to 48 hours post‐injection compared to uninjected, phosphate‐buffered saline (PBS)‐injected and non‐targeting siRNA‐injected mice. We observed increased permeability at the BBB to molecules up to 742 Da, but not 4400 Da, using tracer molecule perfusion and MRI analysis. To illustrate the functional efficacy of size‐selective and transient barrier opening, we have shown that enhanced delivery of the small neuropeptide thyrotropin‐releasing hormone (TRH) (MW 360 Da) to the brains of mice 48 hours post‐injection of siRNA targeting claudin‐5 significantly modifies behavioural output.

Conclusions

These data demonstrate that it is now possible to transiently and size‐selectively open the BBB in mice, allowing in principle the delivery of a wide range of agents for the establishment and treatment of experimental mouse models of neurodegenerative, neuropsychiatric and malignant diseases. Copyright © 2008 John Wiley & Sons, Ltd.     

The significance of the mutated divalent metal transporter (DMT1) on iron transport into the Belgrade rat brain

Brain iron transport and distributional pattern of divalent metal transporter I (DMT1) were studied in homozygous Belgrade rats (b/b) which suffer from a mutation in the DMT1 gene. In adult rats, brain uptake of transferrin-bound iron injected intravenously (i.v.) was significantly lower compared with that in heterozygous Belgrade (+/b) and Wistar rats, whereas transferrin uptake was identical. The difference in iron uptake was not apparent until 30 min after injection. The brain iron concentration was lower, and neuronal transferrin receptor-immunoreactivity higher, in adult b/b rats, thus confirming their iron-deficient stage. Antibodies targeting different sites on the DMT1 molecule consistently detected DMT1 in neurones and choroid plexus at the same level irrespective of strain, but failed to detect DMT1 in brain capillary endothelial cells (BCECs), or macro- or microglial cells. The absence of DMT1 in BCECs was confirmed in immunoblots of purified BCECs. DMT1 was virtually undetectable in neurones of rats aged 18 post-natal days irrespective of strain. Neuronal expression of transferrin receptors and DMT1 in adult rats implies that neurones at this age acquire iron by receptor-mediated endocytosis of transferrin followed by iron transport out of endosomes mediated by DMT1. The existence of the mutated DMT1 molecule in neurones suggests that the low cerebral iron uptake in b/b rats derives from a reduced neuronal uptake rather than an impaired iron transport through the blood-brain barrier.     

Delayed astrocytic contact with cerebral blood vessels in FGF‐2 deficient mice does not compromise permeability properties at the developing blood‐brain barrier

The brain functions within a specialized environment tightly controlled by brain barrier mechanisms. Understanding the regulation of barrier formation is important for understanding brain development and may also lead to finding new ways to deliver pharmacotherapies to the brain; access of many potentially promising drugs is severely hindered by these barrier mechanisms. The cellular composition of the neurovascular unit of the blood‐brain barrier proper and their effects on regulation of its function are beginning to be understood. One hallmark of the neurovascular unit in the adult is the astroglial foot processes that tightly surround cerebral blood vessels. However their role in barrier formation is still unclear. In this study we examined barrier function in newborn, juvenile and adult mice lacking fibroblast growth factor‐2 (FGF‐2), which has been shown to result in altered astroglial differentiation during development. We show that during development of FGF‐2 deficient mice the astroglial contacts with cerebral blood vessels are delayed compared with wild‐type animals. However, this delay did not result in changes to the permeability properties of the blood brain barrier as assessed by exclusion of either small or larger sized molecules at this interface. In addition cerebral vessels were positive for tight‐junction proteins and we observed no difference in the ultrastructure of the tight‐junctions. The results indicate that the direct contact of astroglia processes to cerebral blood vessels is not necessary for either the formation of the tight‐junctions or for basic permeability properties and function of the blood‐brain barrier. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1201–1212, 2016     

Inhibition of the rat blood–brain barrier choline transporter by manganese chloride

Choline transport has been characterized by multiple mechanisms including the blood-brain barrier (BBB), and high- and low-affinity systems. Each mechanism has unique locations and characteristics yet retain some similarities. Previous studies have demonstrated cationic competition by monovalent cations at the BBB and cation divalent manganese in the high-affinity system. To evaluate the effects of divalent manganese inhibition as well as other cationic metals at the BBB choline transporter, brain choline uptake was evaluated in the presence of certain metals of interest in Fischer-344 rats using the in situ brain perfusion technique. Brain choline uptake was inhibited in the presence of Cd(2+) (73 +/- 2%) and Mn(2+) (44 +/- 6%), whereas no inhibition was observed with Cu(2+) and Al(3+). Furthermore, it was found that manganese caused a reduction in brain choline uptake and significant regional choline uptake inhibition in the frontal and parietal cortex, the hippocampus and the caudate putamen (45 +/- 3%, 68 +/- 18%, 58 +/- 9% and 46 +/- 15%, respectively). These results suggest that choline uptake into the CNS can be inhibited by divalent cationic metals and monovalent cations. In addition, the choline transporter may be a means by which manganese enters the brain.     

D1 receptor‐mediated endogenous tPA upregulation contributes to blood‐brain barrier injury after acute ischaemic stroke

Blood‐brain barrier (BBB) integrity injury within the thrombolytic time window is becoming a critical target to reduce haemorrhage transformation (HT). We have previously reported that BBB damage was initially damaged in non‐infarcted striatum after acute ischaemia stroke. However, the underlying mechanism is not clear. Since acute ischaemic stroke could induce a significant increase of dopamine release in striatum, in current study, our aim is to investigate the role of dopamine receptor signal pathway in BBB integrity injury after acute ischaemia using rat middle cerebral artery occlusion model. Our data showed that 2‐h ischaemia induced a significant increase of endogenous tissue plasminogen activator (tPA) in BBB injury area and intra‐striatum infusion of tPA inhibitor neuroserpin, significantly alleviated 2‐h ischaemia‐induced BBB injury. In addition, intra‐striatum infusion of D1 receptor antagonist SCH23390 significantly decreased ischaemia‐induced upregulation of endogenous tPA, accompanied by decrease of BBB injury and occludin degradation. More important, inhibition of hypoxia‐inducible factor‐1 alpha with inhibitor YC‐1 significantly decreased 2‐h ischaemia‐induced endogenous tPA upregulation and BBB injury. Taken together, our data demonstrate that acute ischaemia disrupted BBB through activation of endogenous tPA via HIF‐1α upregulation, thus representing a new therapeutic target for protecting BBB after acute ischaemic stroke.     

Occludin oligomeric assembly at tight junctions of the blood-brain barrier is disrupted by peripheral inflammatory hyperalgesia

Tight junctions (TJs) at the blood-brain barrier (BBB) dynamically alter paracellular diffusion of blood-borne substances from the peripheral circulation to the CNS in response to external stressors, such as pain, inflammation, and hypoxia. In this study, we investigated the effect of lambda-carrageenan-induced peripheral inflammatory pain (i.e., hyperalgesia) on the oligomeric assembly of the key TJ transmembrane protein, occludin. Oligomerization of integral membrane proteins is a critical step in TJ complex assembly that enables the generation of tightly packed, large multiprotein complexes capable of physically obliterating the interendothelial space to inhibit paracellular diffusion. Intact microvessels isolated from rat brains were fractionated by detergent-free density gradient centrifugation, and gradient fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/ Western blot. Injection of lambda-carrageenan into the rat hind paw produced after 3 h a marked change in the relative amounts of oligomeric, dimeric, and monomeric occludin isoforms associated with different plasma membrane lipid raft domains and intracellular compartments in endothelial cells at the BBB. Our findings suggest that increased BBB permeability (i.e., leak) associated with lambda-carrageenan-induced peripheral inflammatory pain is promoted by the disruption of disulfide-bonded occludin oligomeric assemblies, which renders them incapable of forming an impermeant physical barrier to paracellular transport.     

Pathogen translocation across the blood–brain barrier

Neurological manifestations caused by neuroinvading pathogens are typically attributed to penetration of the blood–brain barrier (BBB) and invasion of the central nervous system. However, the mechanisms used by many pathogens (such as Borrelia ) to traverse the BBB are still unclear. Recent studies revealed that microbial translocation across the BBB must involve a repertoire of microbial–host interactions (receptor–ligand interactions). However, the array of interacting molecules responsible for the borrelial translocation is not yet clearly known. Pathogens bind several host molecules (plasminogen, glycosaminoglycans, factor H, etc.) that might mediate endothelial interactions in vivo . This review summarizes our current understanding of the pathogenic mechanisms involved in the translocation of the BBB by neuroinvasive pathogens.     

Age‐related ultrastructural changes of the basement membrane in the mouse blood‐brain barrier

The blood‐brain barrier (BBB) is essential for a functional neurovascular unit. Most studies focused on the cells forming the BBB, but very few studied the basement membrane (BM) of brain capillaries in ageing. We used transmission electron microscopy and electron tomography to investigate the BM of the BBB in ageing C57BL/6J mice. The thickness of the BM of the BBB from 24‐month‐old mice was double as compared with that of 6‐month‐old mice (107 nm vs 56 nm). The aged BBB showed lipid droplets gathering within the BM which further increased its thickness (up to 572 nm) and altered its structure. The lipids appeared to accumulate toward the glial side of the BM. Electron tomography showed that the lipid‐rich BM regions are located in small pockets formed by the end‐feet of astrocytes. These findings suggest an imbalance of the lipid metabolism and that may precede the structural alteration of the BM. These alterations may favour the accretion of abnormal proteins that lead to neurodegeneration in ageing. These findings warrant further investigation of the BM of brain capillaries and of adjoining cells as potential targets for future therapies.     

Occludin oligomeric assemblies at tight junctions of the blood–brain barrier are altered by hypoxia and reoxygenation stress

Hypoxic (low oxygen) and reperfusion (post‐hypoxic reoxygenation) phases of stroke promote an increase in microvascular permeability at tight junctions (TJs) of the blood–brain barrier (BBB) that may lead to cerebral edema. To investigate the effect of hypoxia (Hx) and reoxygenation on oligomeric assemblies of the transmembrane TJ protein occludin, rats were subjected to either normoxia (Nx, 21% O₂, 60 min), Hx (6% O₂, 60 min), or hypoxia/reoxygenation (H/R, 6% O₂, 60 min followed by 21% O₂, 10 min). After treatment, cerebral microvessels were isolated, fractionated by detergent‐free density gradient centrifugation, and occludin oligomeric assemblies associated with plasma membrane lipid rafts were solubilized by perfluoro‐octanoic acid (PFO) exclusively as high molecular weight protein complexes. Analysis by non‐reducing and reducing sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis/western blot of PFO‐solubilized occludin revealed that occludin oligomeric assemblies co‐localizing with ‘TJ‐associated’ raft domains contained a high molecular weight ‘structural core’ that was resistant to disassembly by either SDS or a hydrophilic reducing agent ex vivo, and by Hx and H/R conditions in vivo. However, exposure of PFO‐solubilized occludin oligomeric assemblies to SDS ex vivo revealed the non‐covalent association of a significant amount of dimeric and monomeric occludin isoforms to the disulfide‐bonded inner core, and dispersal of these non‐covalently attached occludin subunits to lipid rafts of higher density in vivo was differentially promoted by Hx and H/R. Our data suggest a model of isoform interaction within occludin oligomeric assemblies at the BBB that enables occludin to simultaneously perform a structural role in inhibiting paracellular diffusion, and a signaling role involving interactions of dimeric and monomeric occludin isoforms with a variety of regulatory molecules within different plasma membrane lipid raft domains.     

Evidence for the effectiveness of the blood--CSF barrier in the fetal rat choroid plexus. A freeze-fracture and peroxidase diffusion study

The blood--cerebrospinal fluid (CSF) barrier in the choroid plexus is principally constituted of apical junctional complexes between epithelial cells. The effectiveness of this barrier was studied during the fetal development in the rat. Choroid plexuses from fetuses (14th and 18th embryonic day) and newborn (1 and 6 day old) rats were examined after intravascular administration of a proteic tracer (horseradish peroxidase) and investigated by freeze-fracture. From the 14th day of fetal life, apical junctions were seen to constitute a barrier that prevents the passage of peroxidase from blood to CSF; the tight junctions were morphologically similar to those of the mature animals; the junctional fibrils appeared continuous on complementary replicas. These data suggest that, from the 14th day of fetal development, the blood--CSF barrier is both morphologically and physiologically mature.