The transport of manganese across the blood-brain barrier

The mammalian central nervous system (CNS) possesses a unique and specialized capillary adaptation, referred to as the blood-brain barrier (BBB). The BBB maintains an optimal neuronal microenvironment, regulating blood-tissue exchange of macromolecules and nutrients. The BBB is characterized by individual endothelial cells that are continuously linked by tight junctions, inhibiting the diffusion of macromolecules and solutes between adjacent endothelial cells. This review will focus on pertinent issues to BBB maintenance, and survey recent dogmas on the transport mechanisms for the essential metal, manganese, across this barrier. Specifically, putative carriers for manganese into and out of the brain will be discussed.     

Alteration of iron homeostasis following chronic exposure to manganese in rats

Recent studies suggest that manganese-induced neurodegenerative toxicity may be partly due to its action on aconitase, which participates in cellular iron regulation and mitochondrial energy production. This study was performed to investigate whether chronic manganese exposure in rats influenced the homeostasis of iron in blood and cerebrospinal fluid (CSF). Groups of 8–10 rats received intraperitoneal injections of MnCl₂ at the dose of 6 mg Mn/kg/day or equal volume of saline for 30 days. Concentrations of manganese and iron in plasma and CSF were determined by atomic absorption spectrophotometry. Rats exposed to manganese showed a greatly elevated manganese concentration in both plasma and CSF. The magnitude of increase in CSF manganese (11-fold) was equivalent to that of plasma (10-fold). Chronic manganese exposure resulted in a 32% decrease in plasma iron (p<0.01) and no changes in plasma total iron binding capacity (TIBC). However, it increased CSF iron by 3-fold as compared to the controls (p<0.01). Northern blot analyses of whole brain homogenates revealed a 34% increase in the expression of glutamine synthetase (p<0.05) with unchanged metallothionein-I in manganese-intoxicated rats. When the cultured choroidal epithelial cells derived from rat choroid plexus were incubated with MnCl₂ (100 μM) for four days, the expression of transferrin receptor mRNA appeared to exceed by 50% that of control (p<0.002). The results indicate that chronic manganese exposure alters iron homeostasis possibly by expediting unidirectional influx of iron from the systemic circulation to cerebral compartment. The action appears likely to be mediated by manganese-facilitated iron transport at brain barrier systems.     

Mechanism and developmental changes in iron transport across the blood-brain barrier

Transferrin and iron uptake by the brain were measured using [(59)Fe-(125)I]transferrin injected intravenously in rats aged from 15 days to 22 weeks. The values for both decreased with age. In rats aged 18 and 70 days the uptake was measured at short time intervals after the injection. When expressed as the volume of distribution (Vd), which represents the volume of plasma from which the transferrin and iron were derived, the results for iron were greater than those of transferrin as early as 7 min after injection and the difference increased rapidly with time, especially in the younger animals. A very similar time course was found for uptake by bone marrow (femurs) where iron uptake involves receptor-mediated endocytosis of Fe-transferrin, release of iron in the cell and recycling of apo-transferrin to the blood. It is concluded that, during transport of transferrin-bound plasma iron into the brain, a similar process occurs in brain capillary endothelial cells (BCECs) and that transcytosis of transferrin into the brain interstitium is only a minor pathway. Also, the high rate of iron transport into the brain in young animals, when iron requirements are high due to rapid growth of the brain, is a consequence of the level of expression and rate of recycling of transferrin receptors on BCECs. As the animal and brain mature both decrease.     

Multiple hurdle mechanism and blood-brain barrier in epilepsy: glucocorticoid receptor-heat shock proteins on drug regulation

正Epilepsy is a complex neurologic condition which affects over 50 million people worldwide.Pharmacotherapy,primarily involving the use of anti-seizure drugs (ASDs),is an essential part of controlling seizures.However,nearly 30%of patients develop drug-resistant epilepsy,clinically defined as the persistence of seizure following trials of two ASDs (Kwan et al.,     

Expression of the iron transporter ferroportin in synaptic vesicles and the blood-brain barrier

Iron homeostasis in the mammalian brain is an important and poorly understood subject. Transferrin-bound iron enters the endothelial cells of the blood-brain barrier from the systemic circulation, and iron subsequently dissociates from transferrin to enter brain parenchyma by an unknown mechanism. In recent years, several iron transporters, including the iron importer DMT1 (Ireg1, MTP, DCT1) and the iron exporter ferroportin (SLC11A3, Ireg, MTP1) have been cloned and characterized. To better understand brain iron homeostasis, we have characterized the distribution of ferroportin, the presumed intestinal iron exporter, and have evaluated its potential role in regulation of iron homeostasis in the central nervous system. We discovered using in situ hybridization and immunohistochemistry that ferroportin is expressed in the endothelial cells of the blood-brain barrier, in neurons, oligodendrocytes, astrocytes, and the choroid plexus and ependymal cells. In addition, we discovered using techniques of immunoelectron microscopy and biochemical purification of synaptic vesicles that ferroportin is associated with synaptic vesicles. In the blood-brain barrier, it is likely that ferroportin serves as a molecular transporter of iron on the abluminal membrane of polarized endothelial cells. The role of ferroportin in synaptic vesicles is unknown, but its presence at that site may prove to be of great importance in neuronal iron toxicity. The widespread representation of ferroportin at sites such as the blood-brain barrier and synaptic vesicles raises the possibility that trafficking of elemental iron may be instrumental in the distribution of iron in the central nervous system.     

Cyclosporin-A treatment of experimental allergic encephalomyelitis: Changes in immunological regulation and blood-CSF barrier function

Strain 13 guinea pigs at 18-21 days of age were sensitised with spinal cord and Freund's complete adjuvant to induce experimental allergic encephalomyelitis (EAE). Treatment with cyclosporin-A (CS-A) from one day before sensitisation until from 12 to 39 days after sensitisation resulted in a suppression of the disease. Suppression was indicated by an absence or reduction in severity of clinical signs together with a lack of increase in blood-cerebrospinal fluid (CSF) barrier permeability to proteins and a lower white cell count in the CSF from treated animals. When CS-A treatment was withdrawn, clinical disease reappeared but changes in IgG concentration in the CSF indicated that intrathecal synthesis of IgG had occurred, which was not the case in the untreated disease. Modification by CS-A of the immunoregulatory processes involved in the development of EAE, provides a model to study the special conditions which operate in neuroimmunological disorders.     

Examination of the blood-to-brain transfer of alpha-aminoisobutyric acid and horseradish peroxidase: regional alterations in blood-brain barrier function following acute hypertension

Blood-brain barrier (BBB) alterations following acute hypertension were studied in rats, employing as tracers in each animal both horseradish peroxidase (HRP) (MW 40,000) and [14C]alpha-aminoisobutyric acid ([14C]AIB) (MW 104). Eighteen animals were subjected to acute hypertension induced by the intravenous infusion of norepinephrine bitartrate (NE) (Levophed). Five animals injected with both tracers but not infused with NE served as controls. The brain of each animal was serially sectioned with adjacent sections processed either for macroautoradiography or for light microscopic visualization of HRP reaction product via histochemical reaction with tetramethylbenzidine. Quantitative blood-to-brain transfer constants for AIB were determined in each of 14 brain regions. Qualitative comparisons were also made between the AIB and HRP blood-to-brain extravasation patterns in each group. Acute hypertension increased cerebrovascular permeability to both AIB and HRP in most animals. Topographically, the sites of the most highly elevated AIB transfer corresponded with sites of HRP extravasation. Conversely, all sites of protein passage corresponded spatially to sites of elevated AIB transfer. Brain regions commonly showing increased permeability to both tracers included the cerebral cortices, corpus callosum, and thalamus. Importantly, some brain regions showed elevated AIB transfer constants where protein extravasation was absent. These regions included the caudate-putamen, hippocampus, basal forebrain, and cerebellum. These observations suggest that following acute hypertension, alterations in BBB permeability are not limited to vascular segments allowing protein extravasation.     

激光间质热治疗开放血脑屏障的研究

目的：激光间质热治疗（Ｉｎｔｅｒｓｔｉｔｉａｌ Ｌａｓｅｒ Ｔｈｅｒｍｏｔｈｅｒａｐｙ,ＩＬＴＴ）是一种侵袭性小的脑肿瘤治疗新方法,为查明激光间质热治疗对血脑屏障的影响。方法：本实验利用伊文思蓝作为示踪剂,观察激光间质热治疗后血脑屏障开放范围和程度。结果：激光间质热治疗后效应灶脑组织伊文思蓝含量立即增工随ＩＬＴＴ后时间的延长而增加。至４８小时达到高峰,其后下降,可维持一周,结论：激光间质热治疗开放     

Alterations in cellular IRP-dependent iron regulation by in vitro manganese exposure in undifferentiated PC12 cells

Manganese (Mn) may interfere with iron regulation by altering the binding of iron regulatory proteins (IRPs) to their response elements found on the mRNA encoding proteins critical to iron homeostasis. To explore this, the effects of 24-h in vitro manganese exposure (1, 10, 50, and 200 microM Mn) on: (i) total intracellular and labile iron concentrations; (ii) the cellular abundance of transferrin receptor (TfR), H- and L-ferritin, and mitochondrial aconitase proteins; and (iii) IRP binding to a [32P](-) labeled mRNA sequence of L-ferritin were evaluated in undifferentiated PC12 cells. In vitro manganese exposure altered the cellular abundance of TfR, H-/L-ferritin, and m-aconitase, resulting in an increase in labile iron. This latter effect led to a decrease in IRP binding activity at the lower (10 and 50 microM) manganese exposures. In contrast, 200 microM manganese exposure increased IRP binding, in spite of the significant increase in labile iron. These data indicate that at lower exposures, manganese directly interfered with IRP-dependent translational events, producing an increase in labile iron, which in turn signaled a decrease in IRP binding at 24 h. At higher exposures, the intracellular burden of manganese resulted in overt cytotoxicity and appeared to compromise the normal compensatory response to increased labile iron, producing increased IRP binding. We conclude that low to moderate manganese exposure interferes with cellular iron regulation, and thus may serve as a contributory mechanism underlying manganese neurotoxicity.     

Comparative in vivo and pathological analysis of the blood-brain barrier in mouse telencephalic transplants

The post-transplantation status of the blood-brain barrier (BBB) is still a matter of debate. In an attempt to define BBB properties after neural transplantation in mice of a defined genetic background, we have used two exogenous markers (horseradish peroxidase and Evans blue), one endogenous marker (immunoglobulins), and in vivo contrast enhanced magnetic resonance imaging (MRI) and compared the results obtained with the different methods. With all four techniques employed, we found the BBB to be reconstituted in 67% of the grafts 3 weeks after grafting, and in more than 90% of all grafts 50 days after grafting. Horseradish peroxidase and contrast enhanced MRI were the most sensitive techniques, the latter offering the unique advantage of repetitive scanning of individual grafts. Our findings provide important information for transplantation studies in mouse models for neurodegenerative diseases.     

Studies of the slow bidirectional transport of iron and transferrin across the blood-brain barrier

Although iron is involved in brain function, very little is known about the regulation of its concentrations in the central nervous system. We quantitatively measured the entry and exit rates of iron, transferrin (its major transport protein), and albumin in mice. The blood to brain transport of iron greater than transferrin greater than albumin and the brain to blood transport of transferrin greater than albumin greater than iron. The results suggest that iron and transferrin have slow, bidirectional, probably saturable, and to some degree independent transport systems, although iron introduced directly into the brain is not readily available for brain to blood transport.     

Temporal responses in the disruption of iron regulation by manganese

Manganese (Mn) is an essential trace element, though at elevated exposures it is also a neurotoxicant. Several mechanisms underlying manganese toxicity have been investigated, although a consistent mechanism(s) of action at low exposures has not been fully elucidated. Here we systematically evaluated the effects of in vitro manganese exposure on intracellular iron (Fe) homeostasis and iron-regulatory protein (IRP) binding activity in undifferentiated PC12 cells over a range of manganese exposure concentrations (1, 10, 50, and 200 microM MnCl(2)) and exposure durations (12, 24, 36, and 48 hr), to test the hypothesis that moderately elevated manganese exposure disrupts cellular iron regulation. Results demonstrate that manganese exposure produces a rapid and sustained dose-dependent dysregulation of cellular iron metabolism, with effects occurring as early as 12 hr exposure and at manganese doses as low as 1 microM. Manganese exposure altered the dynamics of IRP-1 binding and the intracellular abundance of IRP-2, and altered the cellular abundance of transferrin receptor, ferritin, and mitochondrial aconitase protein levels. Cellular levels of labile iron were significantly increased with manganese exposure, although total cellular iron levels were not. The overall pattern of effects shows that manganese produced an inappropriate cellular response akin to iron deficiency, to which the cells were able to mount a compensatory response. Consistent with our previous studies, these data indicate that even low to moderate exposures to Manganese in vitro significantly disrupt cellular iron metabolism, which may be an important contributory mechanism of manganese neurotoxicity.     

Maternal-fetal distribution of manganese in the rat following inhalation exposure to manganese sulfate

Studies examining the pharmacokinetics of manganese during pregnancy have largely focused on the oral route of exposure and have shown that the amount of manganese that crosses the rodent placenta is low. However, limited information exists regarding the distribution of manganese in fetal tissues following inhalation. The objective of this study was to determine manganese body burden in CD rats and fetuses following inhalation of a MnSO4 aerosol during pregnancy. Animals were evaluated following pre-breeding (2 weeks), mating (up to 14 days) and gestational (from gestation day (GD) 0 though 20) exposure to air or MnSO4 (0.05, 0.5, or 1 mg Mn/m(3)) for 6h/day, 7 days/week. The following maternal samples were collected for manganese analysis: whole blood, lung, pancreas, liver, brain, femur, and placenta. Fetal tissues were examined on GD 20 and included whole blood, lung, liver, brain, and skull cap. Maternal lung manganese concentrations were increased following exposure to MnSO4 at >or=0.05 mg Mn/m(3). Maternal brain and placenta manganese concentrations were increased following exposure of pregnant rats to MnSO4 at >or=0.5 mg Mn/m(3). Increased fetal liver manganese concentrations were observed following in utero exposure to MnSO4 at >or=0.5 mg Mn/m(3). Manganese concentrations within all other fetal tissues were not different from air-exposed controls. The results of this study demonstrate that the placenta partially sequesters inhaled manganese, thereby limiting exposure to the fetus.     

Looking at the blood-brain barrier: Molecular anatomy and possible investigation approaches

The blood-brain barrier (BBB) is a dynamic and complex interface between blood and the central nervous system that strictly controls the exchanges between the blood and brain compartments, therefore playing a key role in brain homeostasis and providing protection against many toxic compounds and pathogens. In this review, the unique properties of brain microvascular endothelial cells and intercellular junctions are examined. The specific interactions between endothelial cells and basement membrane as well as neighboring perivascular pericytes, glial cells and neurons, which altogether constitute the neurovascular unit and play an essential role in both health and function of the central nervous system, are also explored. Some relevant pathways across the endothelium, as well as mechanisms involved in the regulation of BBB permeability, and the emerging role of the BBB as a signaling interface are addressed as well. Furthermore, we summarize some of the experimental approaches that can be used to monitor BBB properties and function in a variety of conditions and have allowed recent advances in BBB knowledge. Elucidation of the molecular anatomy and dynamics of the BBB is an essential step for the development of new strategies directed to maintain or restore BBB integrity and barrier function and ultimately preserve the delicate interstitial brain environment.     

P-糖蛋白对卡马西平和苯妥英钠通过大鼠血脑屏障影响的研究

目的观察P糖蛋白(Pglycoprotein,PGP)拮抗剂维拉帕米对卡马西平和苯妥英钠通过大鼠血脑屏障的影响,探讨PGP在难治性癫癎多药耐药机制中的作用。方法在健康大鼠大脑皮质内安置微透析探针,腹腔注射卡马西平(20mg/kg)和苯妥英钠(50mg/kg),在给药后不同时间点收集透析液,并用高效液相技术检测其中的药物浓度,通过微透析探针局部给予维拉帕米,观察后者能否提高大鼠皮质脑细胞外液中抗癫药物的浓度。结果维拉帕米升高了脑细胞外液中卡马西平[60min:(1.74±0.28)μg/ml,90min:(1.87±0.31)μg/ml]和苯妥英钠[30min:(1.08±0.30)μg/ml;60min:(1.54±0.22)μg/ml;150min:(0.91±0.19)μg/ml]的药物浓度,前者在给药后60～90min显著增高(P<0.05),后者在给药后30～150min显著增高(P<0.05)。结论PGP限制卡马西平和苯妥英钠顺利通过血脑屏障,降低脑皮质细胞外液抗癫癎药物浓度,难治性癫时PGP表达增加可能是引起患者对抗癫癎药物产生多药耐药的原因。     

The adhesion molecule ICAM-1 and its regulation in relation with the blood-brain barrier

Mucosal administration of copolymer 1 (Cop 1, Copaxone(R), glatiramer acetate) suppresses experimental autoimmune encephalomyelitis (EAE), and is currently tested for its efficacy in the treatment of multiple sclerosis (MS). Here we demonstrate that oral treatment with Cop 1 induces, in mice, specific Th2 cells in the central nervous system (CNS), as manifested by their isolation from brains of actively sensitized Cop 1-fed mice, as well as, by the localization of orally induced Cop 1 specific suppressor cells in the brain, after their passive transfer to the periphery. Feeding with Cop 1 results in the accumulation in the CNS of cells that secrete Th2 cytokines in response to either Cop 1 or the autoantigen myelin basic protein (MBP), even in Th1 shifting environment, which consequently would lead to therapeutic effect in the MS diseased organ.     

Minocycline protects the blood-brain barrier and reduces edema following intracerebral hemorrhage in the rat

Intracerebral hemorrhage (ICH) results from rupture of a blood vessel in the brain. After ICH, the blood–brain barrier (BBB) surrounding the hematoma is disrupted, leading to cerebral edema. In both animals and humans, edema coincides with inflammation, which is characterized by production of pro-inflammatory cytokines, activation of resident brain microglia and migration of peripheral immune cells into the brain. Accordingly, inflammation is an attractive target for reducing edema following ICH. In the present study, BBB damage was assessed by quantifying intact microvessels surrounding the hematoma, monitoring extravasation of IgG and measuring brain water content 3 days after ICH induced by collagenase injection into the rat striatum. In the injured brain, the water content increased in both ipsilateral and contralateral hemispheres compared with the normal brain. Quantitative real-time RT-PCR revealed an up-regulation of inflammatory genes associated with BBB damage; IL1β, TNFα and most notably, MMP-12. Immunostaining showed MMP-12 in damaged microvessels and their subsequent loss from tissue surrounding the hematoma. MMP-12 was also observed for the first time in neurons. Dual-antibody labeling demonstrated that neutrophils were the predominant source of TNFα protein. Intraperitoneal injection of the tetracycline derivative, minocycline, beginning 6 h after ICH ameliorated the damage by reducing microvessel loss, extravasation of plasma proteins and edema; decreasing TNFα and MMP-12 expression; and reducing the numbers of TNFα-positive cells and neutrophils in the brain. Thus, minocycline, administered at a clinically relevant time, appears to target the inflammatory processes involved in edema development after ICH.     

Aquaporin 4 expression and ultrastructure of the blood-brain barrier following cerebral contusion injury

This study aimed to investigate aquaporin 4 expression and the ultrastructure of the blood-brain barrier at 2–72 hours following cerebral contusion injury, and correlate these changes to the formation of brain edema. Results revealed that at 2 hours after cerebral contusion and laceration injury, aquaporin 4 expression significantly increased, brain water content and blood-brain barrier permeability increased, and the number of pinocytotic vesicles in cerebral microvascular endothelial cells increased. In addition, the mitochondrial accumulation was observed. As contusion and laceration injury became aggravated, aquaporin 4 expression continued to increase, brain water content and blood-brain barrier permeability gradually increased, brain capillary endothelial cells and astrocytes swelled, and capillary basement membrane injury gradually increased. The above changes were most apparent at 12 hours after injury, after which they gradually attenuated. Aquaporin 4 expression positively correlated with brain water content and the blood-brain barrier index. Our experimental findings indicate that increasing aquaporin 4 expression and blood-brain barrier permeability after cerebral contusion and laceration injury in humans is involved in the formation of brain edema.     

Taurine transport at the blood-brain barrier: an in vivo brain perfusion study

Taurine transport into six brain regions of equithesin-anesthetized rats was studied by the in situ brain perfusion technique. This technique gives both accurate measurements of cerebrovascular amino acid transport and allows complete control of the perfusate amino acid composition. Final wash procedure showed that taurine efflux occurred rapidly from endothelial cells. The taurine influx into endothelial cells was sodium and chloride dependent suggesting that the sodium and chloride gradients are the principal source of energy for taurine transport into endothelial cells. Taurine transport could be fitted by a model with saturable components. The kinetic constants in the parietal cortex were 1.4 × 10⁻⁴ μmol/s/g for the apparentV_max and 0.078 mM for the apparentK_m. Competition experiments in the presence of sodium ions showed that [¹⁴C]taurine uptake was strongly inhibited by the structural analogs of taurine, hypotaurine and β-alanine.     

Manganese distribution across the blood-brain barrier III. The divalent metal transporter-1 is not the major mechanism mediating brain manganese uptake

Manganese (Mn) is essential for and toxic to the brain. Brain Mn uptake utilizes both diffusion and transporter-mediated pathways. The divalent metal transporter-1 (DMT-1) has been suggested to mediate brain Mn uptake. The b/b Belgrade rat does not express significant amounts of functional DMT-1. In the present work, brain influx transfer coefficients of (54) Mn ion and (54) Mn transferrin (Mn Tf) were determined in b/b and +/b Belgrade and Wistar rats using the in situ brain perfusion technique. Brain Mn uptake was not significantly different among the three rat strains for either Mn species. We hypothesized that Mn may enter brain endothelial cells by a DMT-1-independent process but not be able to distribute across those cells into brain tissue due to the absence of DMT-1 activity. To test this hypothesis the brain capillary endothelial cells were isolated from b/b and +/b Belgrade rats and Wistar rats after in situ brain perfusion. Some animals received cerebrovascular washout after in situ brain perfusion to ascertain any affect of genotype on (54) Mn adsorption to the endothelial cell luminal surface. Less than 30% of the brain (54) Mn after (54) Mn ion or (54) Mn Tf perfusion remained associated with endothelial cells, suggesting the majority had distributed into brain extracellular fluid (ECF) and/or brain cells. Mn appears to distribute across the rat blood-brain barrier (BBB) into the brain by one or more carrier-mediated processes other than the DMT-1.