Hyperammonemia enhances the function and expression of P‐glycoprotein and Mrp2 at the blood–brain barrier through NF‐κB

Ammonia is considered to be the main neurotoxin responsible for hepatic encephalopathy resulting from liver failure. Liver failure has been reported to alter expression and activity of P‐glycoprotein (P‐gp) and multidrug resistance‐associated protein 2 (Mrp2) at the blood–brain barrier (BBB). The aim of this study was to investigate whether ammonia is involved in abnormalities of expression and activity of P‐gp and Mrp2 at the BBB. Hyperammonemic rats were developed by an intraperitoneal injection of ammonium acetate (NH₄Ac, 4.5 mmol/kg). Results showed that Mrp2 function markedly increased in cortex and hippocampus of rats at 6 h following NH₄Ac administration. Significant increase in function of P‐gp was observed in hippocampus of rats. Meanwhile, such alterations were in line with the increase in mRNA and protein levels of P‐gp and Mrp2. Significant increase in levels of nuclear amount of nuclear factor‐κB (NF‐κB) p65 was also observed. Primarily cultured rat brain microvessel endothelial cells (rBMECs) were used for in vitro study. Data indicated that 24 h exposure to ammonia significantly increased function and expression of P‐gp and Mrp2 in rBMECs, accompanied with activation of NF‐κB. Furthermore, such alterations induced by ammonia were reversed by NF‐κB inhibitor. In conclusion, this study demonstrates that hyperammonemia increases the function and expression of P‐gp and Mrp2 at the BBB via activating NF‐κB pathway.

     

Type 1 diabetes exacerbates blood–brain barrier alterations during experimental epileptic seizures in an animal model

The aim of this study was to perform the effects of diabetes on the permeability of the blood–brain barrier (BBB) during pentylenetetrazole (PTZ)‐induced epileptic attacks. For this propose, the animals were divided into four groups. These groups contained were intact, PTZ‐treated, diabetic and PTZ‐treated diabetic individuals, respectively. To evaluate the functioning of the BBB, Evans blue was used as a BBB permeability indicator, and the expressions of zonula occludens‐1 and glial fibrillary acidic protein involving the functioning of the BBB were determined immunohistochemically. Also, the changes in the release of serum tumour necrosis factor‐alpha and interleukin‐10 and interleukin‐12 were studied by using enzyme‐linked immunosorbent assay method. BBB permeability in the seizures under diabetic conditions showed a considerable increase (p < 0·01) in all of the brain we studied. The immunoreactive staining intensity of zonula occludens‐1 and glial fibrillary acidic protein was found reduced in the brain regions of diabetic rats (p < 0·01). However, the serum level of tumour necrosis factor‐alpha increased in diabetes and diabetes + PTZ groups, and the serum level of interleukin‐12 increased significantly in all experimental groups (p < 0·05). In conclusion, diabetes dramatically increases BBB damage during epileptic seizures, and it may be derived from an elevation of paracellular passage. Copyright © 2015 John Wiley & Sons, Ltd.     

Dl‐3n‐butylphthalide improves traumatic brain injury recovery via inhibiting autophagy‐induced blood‐brain barrier disruption and cell apoptosis

Blood‐brain barrier (BBB) disruption and neuronal apoptosis are important pathophysiological processes after traumatic brain injury (TBI). In clinical stroke, Dl‐3n‐butylphthalide (Dl‐NBP) has a neuroprotective effect with anti‐inflammatory, anti‐oxidative, anti‐apoptotic and mitochondrion‐protective functions. However, the effect and molecular mechanism of Dl‐NBP for TBI need to be further investigated. Here, we had used an animal model of TBI and SH‐SY5Y/human brain microvascular endothelial cells to explore it. We found that Dl‐NBP administration exerts a neuroprotective effect in TBI/OGD and BBB disorder, which up‐regulates the expression of tight junction proteins and promotes neuronal survival via inhibiting mitochondrial apoptosis. The expressions of autophagy‐related proteins, including ATG7, Beclin1 and LC3II, were significantly increased after TBI/OGD, and which were reversed by Dl‐NBP treatment both in vivo and in vitro. Moreover, rapamycin treatment had abolished the effect of Dl‐NBP for TBI recovery. Collectively, our current studies indicate that Dl‐NBP treatment improved locomotor functional recovery after TBI by inhibiting the activation of autophagy and consequently blocking the junction protein loss and neuronal apoptosis. Dl‐NBP, as an anti‐inflammatory and anti‐oxidative drug, may act as an effective strategy for TBI recovery.     

Annexin A1 restores Aβ1‐42‐induced blood–brain barrier disruption through the inhibition of RhoA‐ROCK signaling pathway

The blood–brain barrier (BBB) is composed of brain capillary endothelial cells and has an important role in maintaining homeostasis of the brain separating the blood from the parenchyma of the central nervous system (CNS). It is widely known that disruption of the BBB occurs in various neurodegenerative diseases, including Alzheimer's disease (AD). Annexin A1 (ANXA1), an anti‐inflammatory messenger, is expressed in brain endothelial cells and regulates the BBB integrity. However, its role and mechanism for protecting BBB in AD have not been identified. We found that β‐Amyloid 1‐42 (Aβ42)‐induced BBB disruption was rescued by human recombinant ANXA1 (hrANXA1) in the murine brain endothelial cell line bEnd.3. Also, ANXA1 was decreased in the bEnd.3 cells, the capillaries of 5XFAD mice, and the human serum of patients with AD. To find out the mechanism by which ANXA1 recovers the BBB integrity in AD, the RhoA‐ROCK signaling pathway was examined in both Aβ42‐treated bEnd.3 cells and the capillaries of 5XFAD mice as RhoA was activated in both cases. RhoA inhibitors alleviated Aβ42‐induced BBB disruption and constitutively overexpressed RhoA‐GTP (active form of RhoA) attenuated the protective effect of ANXA1. When pericytes were cocultured with bEnd.3 cells, Aβ42‐induced RhoA activation of bEnd.3 cells was inhibited by the secretion of ANXA1 from pericytes. Taken together, our results suggest that ANXA1 restores Aβ42‐induced BBB disruption through inhibition of RhoA‐ROCK signaling pathway and we propose ANXA1 as a therapeutic reagent, protecting against the breakdown of the BBB in AD.     

Strategies used by bacterial pathogens to cross the blood–brain barrier

The skull, spine, meninges, and cellular barriers at the blood–brain and the blood–cerebrospinal fluid interfaces well protect the brain and meningeal spaces against microbial invasion. However, once in the bloodstream, a range of pathogenic bacteria is able to reach the brain and cause meningitis. Despite advances in antibacterial therapy, bacterial meningitis remains one of the most important infectious diseases worldwide. The most common causative bacteria in children and adults are Streptococcus pneumoniae and Neisseria meningitidis associated with high morbidity and mortality, while among neonates, most cases of bacterial meningitis are due to group B Streptococcus and Escherichia coli. Here we summarise our current knowledge on the strategies used by these bacterial pathogens to survive in the bloodstream, to colonise the brain vasculature and to cross the blood–brain barrier.     

S‐nitrosoglutathione prevents blood–brain barrier disruption associated with increased matrix metalloproteinase‐9 activity in experimental diabetes

Hyperglycemia is known to induce microvascular complications, thereby altering blood–brain barrier (BBB) permeability. This study investigated the role of matrix metalloproteinases (MMPs) and their endogenous inhibitors in increased BBB permeability and evaluated the protective effect of S‐nitrosoglutathione (GSNO) in diabetes. Diabetes was induced in mice by intraperitoneal injection of streptozotocin (40 mg/kg body weight) for 5 days and GSNO was administered orally (100 μg/kg body weight) daily for 8 weeks after the induction of diabetes. A significant decline in cognitive functions was observed in diabetic mice assessed by Morris water maze test. Increased permeability to different molecular size tracers accompanied by edema and ion imbalance was observed in cortex and hippocampus of diabetic mice. Furthermore, activity of both pro and active MMP‐9 was found to be significantly elevated in diabetic animals. Increased in situ gelatinase activity was observed in tissue sections and isolated microvessels from diabetic mice brain. The increase in activity of MMP‐9 was attributed to increased mRNA and protein expression in diabetic mice. In addition, a significant decrease in mRNA and protein expression of tissue inhibitor of matrix metalloproteinase‐1 was also observed in diabetic animals. However, GSNO supplementation to diabetic animals was able to abridge MMP‐9 activation as well as tissue inhibitor of matrix metalloproteinase‐1 levels, restoring BBB integrity and also improving learning and memory. Our findings clearly suggest that GSNO could prevent hyperglycemia‐induced disruption of BBB by suppressing MMP‐9 activity.

     

Infection of human pericytes by HIV‐1 disrupts the integrity of the blood–brain barrier

Human immunodeficiency virus type 1 (HIV‐1) infection of the central nervous system (CNS) affects cross‐talk between the individual cell types of the neurovascular unit, which then contributes to disruption of the blood–brain barrier (BBB) and the development of neurological dysfunctions. Although the toxicity of HIV‐1 on neurons, astrocytes and brain endothelial cells has been widely studied, there are no reports addressing the influence of HIV‐1 on pericytes. Therefore, the purpose of this study was to evaluate whether or not pericytes can be infected with HIV‐1 and how such an infection affects the barrier function of brain endothelial cells. Our results indicate that human brain pericytes express the major HIV‐1 receptor CD4 and co‐receptors CXCR4 and CCR5. We also determined that HIV‐1 can replicate, although at a low level, in human brain pericytes as detected by HIV‐1 p24 ELISA. Pericytes were susceptible to infection with both the X4‐tropic NL4‐3 and R5‐tropic JR‐CSF HIV‐1 strains. Moreover, HIV‐1 infection of pericytes resulted in compromised integrity of an in vitro model of the BBB. These findings indicate that human brain pericytes can be infected with HIV‐1 and suggest that infected pericytes are involved in the progression of HIV‐1‐induced CNS damage.     

Microfluidic blood–brain barrier model provides in vivo‐like barrier properties for drug permeability screening

Efficient delivery of therapeutics across the neuroprotective blood–brain barrier (BBB) remains a formidable challenge for central nervous system drug development. High‐fidelity in vitro models of the BBB could facilitate effective early screening of drug candidates targeting the brain. In this study, we developed a microfluidic BBB model that is capable of mimicking in vivo BBB characteristics for a prolonged period and allows for reliable in vitro drug permeability studies under recirculating perfusion. We derived brain microvascular endothelial cells (BMECs) from human induced pluripotent stem cells (hiPSCs) and cocultured them with rat primary astrocytes on the two sides of a porous membrane on a pumpless microfluidic platform for up to 10 days. The microfluidic system was designed based on the blood residence time in human brain tissues, allowing for medium recirculation at physiologically relevant perfusion rates with no pumps or external tubing, meanwhile minimizing wall shear stress to test whether shear stress is required for in vivo‐like barrier properties in a microfluidic BBB model. This BBB‐on‐a‐chip model achieved significant barrier integrity as evident by continuous tight junction formation and in vivo‐like values of trans‐endothelial electrical resistance (TEER). The TEER levels peaked above 4000 Ω · cm² on day 3 on chip and were sustained above 2000 Ω · cm² up to 10 days, which are the highest sustained TEER values reported in a microfluidic model. We evaluated the capacity of our microfluidic BBB model to be used for drug permeability studies using large molecules (FITC‐dextrans) and model drugs (caffeine, cimetidine, and doxorubicin). Our analyses demonstrated that the permeability coefficients measured using our model were comparable to in vivo values. Our BBB‐on‐a‐chip model closely mimics physiological BBB barrier functions and will be a valuable tool for screening of drug candidates. The residence time‐based design of a microfluidic platform will enable integration with other organ modules to simulate multi‐organ interactions on drug response. Biotechnol. Bioeng. 2017;114: 184–194. © 2016 Wiley Periodicals, Inc.     

Hydrogen sulfide protects blood–brain barrier integrity following cerebral ischemia

By using two structurally unrelated hydrogen sulfide (H₂S) donors 5‐(4‐methoxyphenyl) ‐3H‐1, 2‐dithiole‐3‐thione (ADT) and sodium hydrosulfide (NaHS), this study investigated if H₂S protected blood–brain barrier (BBB) integrity following middle cerebral artery occlusion (MCAO). ICR mice underwent MCAO and received H₂S donors at 3 h after reperfusion. Infarction, neurological scores, brain edema, Evans blue (EB) extravasation, and tight junction protein expression were examined at 48 h after MCAO. We also investigated if ADT protected BBB integrity by suppressing post‐ischemic inflammation‐induced Matrix Metalloproteimase‐9 (MMP9) and Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX). ADT increased blood H₂S concentrations, decreased infarction, and improved neurological deficits. Particularly, ADT reduced EB extravasation, brain edema and preserved expression of tight junction proteins in the ischemic brain. NaHS also increased blood H₂S levels and reduced EB extravasation following MCAO. Moreover, ADT inhibited expression of pro‐inflammatory markers induced Nitric Oxide Synthase (iNOS) and IL‐1β while enhanced expression of anti‐inflammatory markers arginase 1 and IL‐10 in the ischemic brain. Accordingly, ADT attenuated ischemia‐induced expression and activity of MMP9. Moreover, ADT reduced NOX‐4 mRNA expression, NOX activity, and inhibited nuclear translocation of Nuclear Factor Kappa‐B (NF‐κB) in the ischemic brain. In conclusion, H₂S donors protected BBB integrity following experimental stroke possibly by acting through NF‐κB inhibition to suppress neuroinflammation induction of MMP9 and NOX4‐derived free radicals.

     

Identification and quantification of blood–brain barrier transporters in isolated rat brain microvessels

The blood–brain barrier (BBB ) maintains brain homeostasis by tightly regulating the exchange of molecules with systemic circulation. It consists primarily of microvascular endothelial cells surrounded by astrocytic endfeet, pericytes, and microglia. Understanding the make‐up of transporters in rat BBB is essential to the translation of pharmacological and toxicological observations into humans. In this study, experimental workflows are presented in which the optimization of (a) isolation of rat brain microvessels (b) enrichment of endothelial cells, and (c) extraction and digestion of proteins were evaluated, followed by identification and quantification of BBB proteins. Optimization of microvessel isolation was indicated by 15‐fold enrichment of endothelial cell marker Glut1 mRNA , whereas markers for other cell types were not enriched. Filter‐aided sample preparation was shown to be superior to in‐solution sample preparation (10251 peptides vs. 7533 peptides). Label‐free proteomics was used to identify nearly 2000 proteins and quantify 1276 proteins in isolated microvessels. A combination of targeted and global proteomics was adopted to measure protein abundance of 6 ATP‐binding cassette and 27 solute carrier transporters. Data analysis using proprietary Progenesis and open access MaxQuant software showed overall agreement; however, Abcb9 and Slc22a8 were quantified only by MaxQuant, whereas Abcc9 and Abcd3 were quantified only by Progenesis. Agreement between targeted and untargeted quantification was demonstrated for Abcb1 (19.7 ± 1.4 vs. 17.8 ± 2.3) and Abcc4 (2.2 ± 0.7 vs. 2.1 ± 0.4), respectively. Rigorous quantification of BBB proteins, as reported in this study, should assist with translational modeling efforts involving brain disposition of xenobiotics.

     

Effect of exposure to 50 Hz magnetic field with or without insulin on blood–brain barrier permeability in streptozotocin‐induced diabetic rats

We investigated the effect of long‐term exposure to modulation magnetic field (MF), insulin, and their combination on blood–brain barrier (BBB) permeability in a diabetic rat model. Fifty‐three rats were randomly assigned to one of six groups: sham, exposed to no MF; MF, exposed to MF; diabetes mellitus (DM), DM induced with streptozotocin (STZ); DM plus MF (DMMF); DM plus insulin therapy (DMI); and DM plus insulin therapy plus MF (DMIMF). All the rats underwent Evans blue (EB) measurement to evaluate the BBB 30 days after the beginning of experiments. The rats in MF, DMMF, and DMIMF groups were exposed to MF (B = 5 mT) for 165 min every day for 30 days. Mean arterial blood pressure (MABP), body mass, and serum glucose level of the study rats were recorded. The extravasation of brain EB of the MF, DM, DMMF, DMI, and DMIMF groups was higher than that of the sham group and the extravasation of right hemisphere of the DMIMF group was highest (P < 0.05). The post‐procedure body mass of the sham and MF groups were significantly higher than those of the DM and DMMF groups (P < 0.05). In the DM, DMMF, DMI, and DMIMF groups, the baseline glucose was significantly lower than the post‐procedure glucose (P < 0.05). DM and MF increase BBB permeability; in combination, they cause more increase in BBB permeability, and insulin decreases their effect on BBB. Improved glucose metabolism may prevent body mass loss and the hypoglycemic effect of MF. DM increases MABP but MF causes no additional effect. Bioelectromagnetics 31:262–269, 2010. © 2009 Wiley‐Liss, Inc.     

Induction of P‐glycoprotein and Bcrp at the rat blood–brain barrier following a subchronic morphine treatment is mediated through NMDA/COX‐2 activation

Subchronic morphine treatment induces P‐glycoprotein (P‐gp) up‐regulation at the blood–brain barrier. This study investigates the rate and extent to which P‐gp and breast cancer‐resistance protein (Bcrp) increase at the rat blood–brain barrier following subchronic morphine treatment. Rats were given increasing doses of morphine (10–40 mg/kg) or saline i.p. twice daily for 5 days. The brain cortex large vessels and microvessels were then mechanical isolated 6, 9, 12, 24, and 36 h after the last injection. The gene and protein expression of P‐gp and Bcrp in morphine‐treated and control rats were compared by qRT‐PCR and western blotting. The levels of Mdr1a and Bcrp mRNAs were not significantly modified 6 h post morphine, but the Mdr1a mRNA increased 1.4‐fold and Bcrp mRNA 2.4‐fold at 24 h. P‐gp and Bcrp protein expression in brain microvessels was unchanged 6 h post morphine and increased 1.5‐fold at 24 h. This effect was more pronounced in large vessels than in microvessels. However, extracellular morphine concentrations of 0.01–10 μM did not modify the expressions of the MDR1 and BCRP genes in hCMEC/D3 human endothelial brain cells in vitro. MK‐801 (NMDA antagonist) and meloxicam (cyclo‐oxygenase‐2 inhibitor) given after morphine treatment completely blocked P‐gp and Bcrp up‐regulation. Interestingly, misoprostol and iloprost, two well‐known agonists of prostaglandin E2 receptors induced both MDR1 and BCRP mRNA levels in hCMEC/D3. Thus, morphine does not directly stimulate P‐gp and Bcrp expression by the brain endothelium, but glutamate released during morphine withdrawal may do so by activating the NMDA/cyclo‐oxygenase‐2 cascade.     

Platelet activating factor induces transient blood‐brain barrier opening to facilitate edaravone penetration into the brain

The blood–brain barrier (BBB) greatly limits the efficacy of many neuroprotective drugs' delivery to the brain, so improving drug penetration through the BBB has been an important focus of research. Here we report that platelet activating factor (PAF) transiently opened BBB and facilitated neuroprotectant edaravone penetration into the brain. Intravenous infusion with PAF induced a transient BBB opening in rats, reflected by increased Evans blue leakage and mild edema formation, which ceased within 6 h. Furthermore, rat regional cerebral blood flow (rCBF) declined acutely during PAF infusion, but recovered slowly. More importantly, this transient BBB opening significantly increased the penetration of edaravone into the brain, evidenced by increased edaravone concentrations in tissue interstitial fluid collected by microdialysis and analyzed by Ultra‐performance liquid chromatograph combined with a hybrid quadrupole time‐of‐flight mass spectrometer (UPLC‐MS/MS). Similarly, incubation of rat brain microvessel endothelial cells monolayer with 1 μM PAF for 1 h significantly increased monolayer permeability to ¹²⁵I‐albumin, which recovered 1 h after PAF elimination. However, PAF incubation with rat brain microvessel endothelial cells for 1 h did not cause detectable cytotoxicity, and did not regulate intercellular adhesion molecule‐1, matrix‐metalloproteinase‐9 and P‐glycoprotein expression. In conclusion, PAF could induce transient and reversible BBB opening through abrupt rCBF decline, which significantly improved edaravone penetration into the brain.

     

Mycobacteria employ two different mechanisms to cross the blood–brain barrier

Central nervous system (CNS) infection by Mycobacterium tuberculosis is one of the most devastating complications of tuberculosis, in particular in early childhood. In order to induce CNS infection, M. tuberculosis needs to cross specialised barriers protecting the brain. How M. tuberculosis crosses the blood–brain barrier (BBB) and enters the CNS is not well understood. Here, we use transparent zebrafish larvae and the closely related pathogen Mycobacterium marinum to answer this question. We show that in the early stages of development, mycobacteria rapidly infect brain tissue, either as free mycobacteria or within circulating macrophages. After the formation of a functionally intact BBB, the infiltration of brain tissue by infected macrophages is delayed, but not blocked, suggesting that crossing the BBB via phagocytic cells is one of the mechanisms used by mycobacteria to invade the CNS. Interestingly, depletion of phagocytic cells did not prevent M. marinum from infecting the brain tissue, indicating that free mycobacteria can independently cause brain infection. Detailed analysis showed that mycobacteria are able to cause vasculitis by extracellular outgrowth in the smaller blood vessels and by infecting endothelial cells. Importantly, we could show that this second mechanism is an active process that depends on an intact ESX‐1 secretion system, which extends the role of ESX‐1 secretion beyond the macrophage infection cycle.     

β‐sheet breaker peptide inhibitor of Alzheimer's amyloidogenesis with increased blood–brain barrier permeability and resistance to proteolytic degradation in plasma

Short synthetic peptides homologous to the central region of Aβ but bearing proline residues as β‐sheet blockers have been shown in vitro to bind to Aβ with high affinity, partially inhibit Aβ fibrillogenesis, and redissolve preformed fibrils. While short peptides have been used extensively as therapeutic drugs in medicine, two important problems associated with their use in central nervous system diseases have to be addressed: (a) rapid proteolytic degradation in plasma, and (b) poor blood–brain barrier (BBB) permeability. Recently, we have demonstrated that the covalent modification of proteins with the naturally occurring polyamines significantly increases their permeability at the BBB. We have extended this technology to iAβ11, an 11‐residue β‐sheet breaker peptide that inhibits Aβ fibrillogenesis, by covalently modifying this peptide with the polyamine, putrescine (PUT), and evaluating its plasma pharmacokinetics and BBB permeability. After a single intravenous bolus injection in rats, both ¹²⁵I‐YiAβ11 and ¹²⁵I‐PUT‐YiAβ11 showed rapid degradation in plasma as determined by trichloroacetic acid (TCA) precipitation and paper chromatography. By switching to the all d ‐enantiomers of YiAβ11 and PUT‐YiAβ11, significant protection from degradation by proteases in rat plasma was obtained with only 1.9% and 5.7% degradation at 15 min after intravenous bolus injection, respectively. The permeability coefficient × surface area product at the BBB was five‐ sevenfold higher in the cortex and hippocampus for the ¹²⁵I‐PUT‐d ‐YiAβ11 compared to the ¹²⁵I‐d ‐YiAβ11, with no significant difference in the residual plasma volume. In vitro assays showed that PUT‐d ‐YiAβ11 retains its ability to partially inhibit Aβ fibrillogenesis and dissolve preformed amyloid fibrils. Because of its five‐ to sevenfold increase in permeability at the BBB and its resistance to proteolysis in the plasma, this polyamine‐modified β‐sheet breaker peptide may prove to be an effective inhibitor of amyloidogenesis in vivo and, hence, an important therapy for Alzheimer's disease. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 371–382, 1999