Phe(13),Tyr(19)-melanin-concentration hormone and the blood-brain barrier: role of protein binding

Melanin-concentrating hormone (MCH), found both peripherally and centrally, is involved in food ingestion. Although its expression in brain is increased by fasting, it is not known whether it crosses the blood-brain barrier (BBB). Use of the sensitive method of multiple-time regression analysis has shown that almost all of the peptides and polypeptides tested cross the BBB at a rate faster than the vascular marker albumin. With this same method, however, we found that the 19-amino acid 125I-Phe13,Tyr19-MCH did not cross faster than 99mTc-albumin. Several mechanisms were excluded as possible explanations for the slow rate of influx. These included degradation, association with capillary endothelial cells, and transport from brain to blood. When Phe13,Tyr19-MCH was perfused in blood-free buffer, however, it entered the brain significantly faster than albumin. This suggested protein binding as an explanation for the slow rate of influx when the MCH was administered in blood. Protein binding was confirmed by capillary zone electrophoresis, which showed that almost all of the Phe13,Tyr19-MCH added to blood migrated with a large-molecular-weight substance. Sodium dodecyl sulfate-capillary gel electrophoresis of Phe13,Tyr19-MCH in buffer additionally showed that the MCH aggregated as a trimer, a factor not preventing its influx by blood-free perfusion. Thus, the results show that blood-borne Phe13,Tyr19-MCH does not significantly cross the BBB, probably because of its binding to serum proteins.     

The blood-brain barrier: connecting the gut and the brain

The BBB prevents the unrestricted exchange of substances between the central nervous system (CNS) and the blood. The blood-brain barrier (BBB) also conveys information between the CNS and the gastrointestinal (GI) tract through several mechanisms. Here, we review three of those mechanisms. First, the BBB selectively transports some peptides and regulatory proteins in the blood-to-brain or the brain-to-blood direction. The ability of GI hormones to affect functions of the BBB, as illustrated by the ability of insulin to alter the BBB transport of amino acids and drugs, represents a second mechanism. A third mechanism is the ability of GI hormones to affect the secretion by the BBB of substances that themselves affect feeding and appetite, such as nitric oxide and cytokines. By these and other mechanisms, the BBB regulates communications between the CNS and GI tract.     

Accumulation of Eu chelates in cells expressing or not P-glycoprotein: Implications for blood-brain barrier crossing

Alzheimer’s disease (AD) is the most commonly form of dementia in the elderly. The development of molecules able to detect biomarkers characteristic of AD is critical to its understanding and treatment. However, such molecules must be able to pass blood-brain barrier (BBB) which is a major impediment to the entry of many therapeutic drugs into the brain. Such a limitation applies to the development of magnetic resonance imaging molecular neuroimaging agents using biomarkers of AD-like β-amyloid deposits, as the common extracellular contrast agents (CAs) are not able to cross an intact BBB. In this work, we have studied the ability of a series of simple Eu³⁺ complexes to enter cells overexpressing or not the ABCB1 (P-gp or P-glycoprotein) protein, which is expressed at the BBB and in human embryonic astrocytes. The intracellular uptake of the Eu³⁺ complexes of linear and macrocyclic polyaminocarboxylate ligands with different charges and lipophilicities was followed by atomic absorption spectrometry. Based on biochemical argument, we propose that lipophilic contrast agents can be efficiently taken up by cells and accumulate inside mitochondria when they are positively charged. The important point is that they are not P-gp substrates, which is one of the major obstacles for them to cross the BBB.     

Development of the blood-brain barrier

The endothelial cells forming the blood-brain barrier (BBB) are highly specialized to allow precise control over the substances that leave or enter the brain. An elaborate network of complex tight junctions (TJ) between the endothelial cells forms the structural basis of the BBB and restricts the paracellular diffusion of hydrophilic molecules. Additonally, the lack of fenestrae and the extremely low pinocytotic activity of endothelial cells of the BBB inhibit the transcellular passage of molecules across the barrier. On the other hand, in order to meet the high metabolic needs of the tissue of the central nervous system (CNS), specific transport systems selectively expressed in the membranes of brain endothelial cells in capillaries mediate the directed transport of nutrients into the CNS or of toxic metabolites out of the CNS. Whereas the characteristics of the mature BBB endothelium are well described, the cellular and molecular mechanisms that control the development, differentiation and maintenance of the highly specialized endothelial cells of the BBB remain unknown to date, despite the recent explosion in our knowledge of the growth factors and their receptors specifically acting on vascular endothelium during development. This review summarizes our current knowledge of the cellular and molecular mechanisms involved in the development and maintenance of the BBB.     

Selective effects of alpha-MSH and MIF-1 on the blood-brain barrier

The effects of intravenously-injected alpha-MSH and MIF-1 (Pro-Leu-Gly-NH2) on the permeability of the blood-brain barrier (BBB) to a large protein and a small anion were studied using radioiodinated serum albumin (RISA) and 99mTc-labeled sodium pertechnetate. The permeability of the BBB to RISA was unaltered by either peptide. Permeability to the inorganic pertechnetate anion, however, was significantly increased by alpha-MSH but not by MIF-1 at doses known to evoke EEG and behavioral responses. The peptides did not cause a change in the systemic blood pressure. It is possible, therefore, that at least some CNS effects of peripherally administered peptides are exerted by alteration of the permeability of the BBB to other substances.     

Erythropoietin prevents the increase in blood-brain barrier permeability during pentylentetrazol induced seizures

Recombinant human erythropoietin (r-Hu EPO) has been shown to exert neuroprotection in ischemic, excitotoxicity, trauma, convulsions and neurodegenerative disorders. Blood-brain barrier (BBB) leakage plays a role in the pathogenesis of many pathological states of the brain including neurodegenerative disorders. This study aimed to investigate the effects of r-Hu EPO on BBB integrity in pentylentetrazol (PTZ) induced seizures in rats. Seizures were observed and evaluated regard to latency and intensity for an hour. Macroscopical and spectrophotometrical measurement of Evans Blue (EB) leakage were observed for BBB integrity. r-Hu EPO was given intraperitoneally 24 h prior to seizure induction. Total seizure duration of 720+/-50 s after single PTZ administration (80 mg/kg i.p.) was declined to 190+/-40 s in r-Hu EPO pretreatment. A typical BBB breakdown pattern (i.e. staining in cerebellum, cerebral cortex, midbrain, hippocampus, thalamus and corpus striatum) was observed in rat brains with PTZ induced seizures; whereas, EPO pretreatment confined BBB leakage to cerebellum and cortical areas, and lessened the intensity of tonic-clonic seizures observed in PTZ seizures. The protective effect of r-Hu EPO on BBB permeability in seizures is a new and original finding. The protective action of r-Hu EPO in seizures and some of CNS pathologies warrant further investigations.     

Influence of surgical pain stress on the blood-brain barrier permeability in rats

Effect of surgical pain stress on the blood-brain barrier permeability was investigated in rats. The animals were divided into four groups: Group 1: control, Group 2: immobilization stress, Group 3: acute hypertension, Group 4: immobilization stress + surgical pain stress.Bilateral hid paw surgical wounds for cannulations were applied in animals' inguinal regions under diethyl-ether anesthesia, then the animals were awaken from anesthesia to produce surgical pain stress. Evans-blue was used as a blood-brain barrier tracer. There is no significantly blood-brain barrier breakdown after short-time immobilization stress, but after adrenalin hypertension blood-brain barrier permeability was increased especially on frontal and occipital cortices in 50% of the animals. Surgical pain stress increased blood-brain barrier permeabiliy in comparison to acute adrenalin-induced hypertension (p < 0.01). In surgical pain stress-induced animals distinct Evans-blue leakage was observed in the occipital, frontal and parieto-temporal cortices.     

Effects of chronic (dietary) choline availability on the transport of choline across the blood-brain barrier

The effects of dietary choline availability on the transport of choline across the blood-brain barrier (BBB) were investigated using the intracarotid injection technique. Maintenance of rats on choline-deficient, basal choline, or choline-supplemented diets for 28-32 days led to respective increases in blood levels of choline and correlative increases in the velocity of transport of choline measured using a buffer injectate. When serum from these rats was included in the injectate and transport determined in control animals, there was a marked inhibition of choline transport that was related to the concentration of choline in the diets. Results suggest that the activity of the choline carrier at the BBB is antagonized by an inhibitory substance in serum whose concentration or activity may be modified by chronic alterations in circulating levels of choline and whose presence may normally regulate the velocity of choline transport.     

The blood-brain barrier in brain homeostasis and neurological diseases

Brain endothelial cells are unique among endothelial cells in that they express apical junctional complexes, including tight junctions, which quite resemble epithelial tight junctions both structurally and functionally. They form the blood-brain-barrier (BBB) which strictly controls the exchanges between the blood and the brain compartments by limiting passive diffusion of blood-borne solutes while actively transporting nutrients to the brain. Accumulating experimental and clinical evidence indicate that BBB dysfunctions are associated with a number of serious CNS diseases with important social impacts, such as multiple sclerosis, stroke, brain tumors, epilepsy or Alzheimer's disease. This review will focus on the implication of brain endothelial tight junctions in BBB architecture and physiology, will discuss the consequences of BBB dysfunction in these CNS diseases and will present some therapeutic strategies for drug delivery to the brain across the BBB.     

Effects of radiofrequency radiation exposure on blood-brain barrier permeability in male and female rats

During the last several decades, numerous studies have been performed aiming at the question of whether or not exposure to radiofrequency radiation (RFR) influences the permeability of the blood-brain barrier (BBB). The objective of this study was to investigate the effect of RFR on the permeability of BBB in male and female Wistar albino rats. Right brain, left brain, cerebellum, and total brain were analyzed separately in the study. Rats were exposed to 0.9 and 1.8 GHz continuous-wave (CW) RFR for 20 min (at SARs of 4.26 mW/kg and 1.46 mW/kg, respectively) while under anesthesia. Control rats were sham-exposed. Disruption of BBB integrity was detected spectrophotometrically using the Evans-blue dye, which has been used as a BBB tracer and is known to be bound to serum albumin. Right brain, left brain, cerebellum, and total brain were evaluated for BBB permeability. In female rats, no albumin extravasation was found in in the brain after RFR exposure. A significant increase in albumin was found in the brains of the RF-exposed male rats when compared to sham-exposed male brains. These results suggest that exposure to 0.9 and 1.8 GHz CW RFR at levels below the international limits can affect the vascular permeability in the brain of male rats. The possible risk of RFR exposure in humans is a major concern for the society. Thus, this topic should be investigated more thoroughly in the future.     

Linoleic acid passage through the blood-brain barrier and a possible effect of age

It has already been established that the blood-brain barrier is readily crossed by unsaturated fatty acids, while saturated fatty acid transport appears to be protein mediated. When the passage of the fatty acids is tested in vivo by using perfusion buffers containing both linoleate and palmitate in different concentrations, linoleate is able to decrease the palmitate passage, while palmitate increases the linoleate passage. These results could be related to the effect of two fatty acids on the ratio between the fatty acids bound to the serum albumin and the free fatty acid pool, which is only available for transport through membranes. However, on the basis of some results obtained with aged rats, the possibility that a relationship may exist between palmitate and linoleate during their passage through the BBB is discussed. Moreover, it seems likely that in aged rats a moderate modification for fatty acids takes place in the BBB.     

TIMP-3: A novel target for glucocorticoid signaling at the blood-brain barrier

Glucocorticoids (GCs) are used in the treatment of neuroinflammatory diseases such as multiple sclerosis. Several studies have demonstrated the beneficial effect of GCs on the balance between matrix metalloproteinases (MMPs) and their endogenous inhibitors, the TIMPs (tissue inhibitors of metalloproteinases). We could demonstrate that all four known TIMPs are present at the blood-brain barrier (BBB) endothelium. Hydrocortisone (HC) selectively upregulates TIMP-3 while TIMP-1, TIMP-2 and TIMP-4 were downregulated on the mRNA-level. This effect could be completely reversed by the glucocorticoid receptor inhibitor mifepristone (Mife). On the protein-level all TIMPs could be detected in the apical supernatants whereas in the isolated extracellular matrix (ECM) only TIMP-3 was found. The application of HC led to a strong enrichment of TIMP-3 in the ECM. Our findings demonstrate that HC directly targets TIMP-3 at the BBB assuming a protective role against matrix disruption and thus to guarantee the barrier integrity.     

Microbial translocation of the blood-brain barrier

A major contributing factor to high mortality and morbidity associated with CNS infection is the incomplete understanding of the pathogenesis of this disease. Relatively small numbers of pathogens account for most cases of CNS infections in humans, but it is unclear how such pathogens cross the blood-brain barrier (BBB) and cause infections. The development of the in vitro BBB model using human brain microvascular endothelial cells has facilitated our understanding of the microbial translocation of the BBB, a key step for the acquisition of CNS infections. Recent studies have revealed that microbial translocation of the BBB involves host cell actin cytoskeletal rearrangements, most likely as the result of specific microbial-host interactions. A better understanding of microbial-host interactions that are involved in microbial translocation of the BBB should help in developing new strategies to prevent CNS infections. This review summarises our current understanding of the pathogenic mechanisms involved in translocation of the BBB by meningitis-causing bacteria, fungi and parasites.     

Cerebral malaria: role of microparticles and platelets in alterations of the blood-brain barrier

Brain lesions of cerebral malaria (CM) are characterised by a sequestration of Plasmodium falciparum-parasitised red blood cells (PRBC), leucocytes and platelets within brain microvessels, by an excessive release of pro-inflammatory cytokines as well as by disruption of the blood-brain barrier (BBB). We evaluated the possibility that PRBC and platelets interact and induce functional alterations in brain endothelium. Using an in vitro model of endothelial lesion, we showed that platelets can act as bridges between PRBC and endothelial cells (EC) allowing the binding of PRBC to endothelium devoid of cytoadherence receptors. Furthermore, platelets potentiated the cytotoxicity of PRBC for brain EC by inducing an alteration of the integrity of their monolayer and increasing their apoptosis. These findings provide insights into the mechanisms by which platelets can be deleterious to the brain endothelium during CM. Another aspect of inflammatory and infectious diseases is that they often lead to activation of vascular and blood cells. Such activation results in an enhanced vesiculation, i.e. the release of circulating microparticles (MP). We thus explored plasma levels of endothelial MP in Malawian children with malaria. Plasma MP numbers were markedly increased on admission only in patients with severe malaria complicated with coma. Using the experimental mouse model of CM, we evaluated the pathogenic implications of MP using genetically deficient mice in which the capacity to vesiculate is impaired. Such mice, lacking the ABCA-1 gene, upon infection by Plasmodium berghei ANKA, showed complete resistance to CM. When purified from infected susceptible animals, MP were able to reduce normal plasma clotting time and to significantly enhance tumour necrosis factor release from na?ve macrophages. Altogether these data provide a novel insight into the pathogenic mechanisms leading to the neurological syndrome. The finding that ABCA-1 gene deletion confers complete protection against cerebral pathology, linked to an impaired MP production, provides new potential targets for therapeutic amelioration of severe malaria.     

Effects of losartan on the blood-brain barrier permeability in long-term nitric oxide blockade-induced hypertensive rats

Hypertension is closely associated with vascular endothelial dysfunction. The aim of this study was to investigate the effects of Angiotensin II (ANG II) receptor antagonist losartan on the blood-brain barrier (BBB) permeability in L-NAME-induced hypertension and/or in ANG II-induced acute hypertension in normotensive and hypertensive rats. Systolic blood pressure was measured by tail cuff method before, during and following L-NAME treatment (1 g/L). Losartan (3 mg/kg) was given to the animal for five days. Acute hypertension was induced by ANG II (60 microg/kg). Arterial blood pressure was directly measured on the day of the experiment. BBB disruption was quantified according to the extravasation of the albumin-bound Evans blue dye. Losartan significantly reduced the mean arterial blood pressure from 169 +/- 3.9 mmHg to 82 +/- 2.9 mmHg in L-NAME and from 171 +/- 2.9 mmHg to 84 +/- 2.9 in L-NAME plus losartan plus ANG II groups (p < 0.05). The content of Evans blue dye in the cerebral cortex significantly increased in L-NAME (p < 0.01). Moreover, the content of Evans blue dye markedly increased in the cerebellum (p < 0.001) and slightly increased in diencephalon region (p < 0.05) in L-NAME plus ANG II. Losartan reduced the increased BBB permeability to Evans blue dye in L-NAME (p < 0.01) and L-NAME plus ANG II (p < 0.001). These results indicate that L-NAME and L-NAME plus ANG II both lead to an increase in microvascular Evans blue dye efflux to brain, and losartan treatment attenuates this protein-bound dye transport into brain tissue presumably due to its protective effect on endothelial cells of brain vessels.     

Targeted delivery of antibodies through the blood-brain barrier by MRI-guided focused ultrasound

The blood-brain barrier (BBB) is a persistent obstacle for the local delivery of macromolecular therapeutic agents to the central nervous system (CNS). Many drugs that show potential for treating CNS diseases cannot cross the BBB and there is a need for a non-invasive targeted drug delivery method that allows local therapy of the CNS using larger molecules. We developed a non-invasive technique that allows the image-guided delivery of antibody across the BBB into the murine CNS. Here, we demonstrate that subsequent to MRI-targeted focused ultrasound induced disruption of BBB, intravenously administered dopamine D(4) receptor-targeting antibody crossed the BBB and recognized its antigens. Using MRI, we were able to monitor the extent of BBB disruption. This novel technology should be useful in delivering macromolecular therapeutic or diagnostic agents to the CNS for the treatment of various CNS disorders.     

The relationship between erythropoietin pretreatment with blood-brain barrier and lipid peroxidation after ischemia/reperfusion in rats

Blood-brain barrier (BBB) leakage plays a role in the pathogenesis of many pathological states of the brain including ischemia and some neurodegenerative disorders. In recent years, erythropoietin (EPO) has been shown to exert neuroprotection in many pathological conditions including ischemia in the brain. This study aimed to investigate the effects of EPO on BBB integrity, infarct size and lipid peroxidation following global brain ischemia/reperfusion in rats. Wistar male rats were divided into four groups (each group n=8); Group I; control group (sham-operated), Group II; ischemia/reperfusion group, Group III; EPO treated group (24 h before decapitation--000 U/kg r-Hu EPO i.p.), Group IV; EPO+ ischemia/reperfusion group (24 h before ischemia/reperfusion--3000 U/kg r-Hu EPO i.p.). Global brain ischemia was produced by the combination of bilateral common carotid arteries occlusion and hemorrhagic hypotension. Macroscopical and spectrophotometrical measurement of Evans Blue (EB) leakage was observed for BBB integrity. Infarct size was calculated based on 2,3,5-triphenyltetrazolium chloride (TTC) staining. Lipid peroxidation in the brain tissue was determined as the concentration of thiobarbituric acid-reactive substances (TBARS) for each group. Ischemic insult caused bilateral and regional BBB breakdown (hippocampus, cortex, corpus striatum, midbrain, brain stem and thalamus). EPO pretreatment reduced BBB disruption, infarct size and lipid peroxide levels in brain tissue with 20 min ischemia and 20 min reperfusion. These results suggest that EPO plays an important role in protecting against brain ischemia/reperfusion through inhibiting lipid peroxidation and decreasing BBB disruption.     

Alteration of blood-brain barrier function by methamphetamine and cocaine

The integrity of the blood-brain barrier (BBB) plays an important role in maintaining a safe neural microenvironment in the brain. Loss of BBB integrity has been recognized as a major cause of profound brain alterations. Psychoactive drugs such as methamphetamine (METH) or cocaine are well-known drugs of abuse that can alter the permeability of the BBB via various mechanisms. In addition, the neurotoxicity of METH is well documented, and alterations in BBB function can contribute to this toxicity. A great deal of effort has been devoted to understanding the cellular and molecular mechanisms of the action of these drugs in the central nervous system. However, only a few investigations have focused on the effects of METH and cocaine on BBB function. The aim of this short review is to summarize our present knowledge of this subject.     

The blood-brain barrier penetration and distribution of PEGylated fluorescein-doped magnetic silica nanoparticles in rat brain

PEGylated PAMAM conjugated fluorescein-doped magnetic silica nanoparticles (PEGylated PFMSNs) have been synthesized for evaluating their ability across the blood-brain barrier (BBB) and distribution in rat brain. The obtained nanoparticles were characterized by transmission electron microscopy (TEM), thermal gravimetry analyses (TGA), zeta potential (ζ-potential) titration, and X-ray photoelectron spectroscopy (XPS). The BBB penetration and distribution of PEGylated PFMSNs and FMSNs in rat brain were investigated not only at the cellular level with Confocal laser scanning microscopy (CLSM), but also at the subcellular level with transmission electron microscopy (TEM). The results provide direct evidents that PEGylated PFMSNs could penetrate the BBB and spread into the brain parenchyma.     

Effects of lipopolysaccharide on blood-brain barrier permeability during pentylenetetrazole-induced epileptic seizures in rats

We investigated the effects of lipopolysachharide (LPS) on functional and structural properties of the blood-brain barrier (BBB) during pentylenetetrazole (PTZ)-induced epileptic seizures in rats. Arterial blood pressure was significantly elevated during epileptic seizures irrespective of LPS pretreatment. Plasma levels of interleukin (IL)-1, interleukin (IL)-6, nitric oxide (NO) and malondialdehyde (MDA) increased while catalase concentrations decreased in animals treated with LPS, PTZ and LPS plus PTZ. The significantly increased BBB permeability to Evans blue (EB) dye in the cerebral cortex, diencephalon and cerebellum regions of rats by PTZ-induced seizures was markedly reduced upon LPS pretreatment. Immunoreactivity for tight junction proteins, zonula occludens-1 and occludin, did not change in brain vessels of animals treated with PTZ and LPS plus PTZ. Glial fibrillary acidic protein immunoreactivity was increased in LPS, but not in PTZ and LPS plus PTZ. These results indicate that LPS pretreatment reduces the passage of EB dye bound to albumin into the brain, at least partly, by increasing plasma NO and IL-6 levels during PTZ-induced epileptic seizures. We suggest that LPS may provide protective effects on the BBB integrity during epileptic seizures through transcellular pathway, since the paracellular route remained unaffected by LPS and LPS plus PTZ.