scholarly journals Effects of Immunomodulatory and Organism-Associated Molecules on the Permeability of an In Vitro Blood-Brain Barrier Model to Amphotericin B and Fluconazole

2009 ◽  
Vol 54 (3) ◽  
pp. 1305-1310 ◽  
Author(s):  
Vasilios Pyrgos ◽  
Diane Mickiene ◽  
Tin Sein ◽  
Margaret Cotton ◽  
Andrea Fransesconi ◽  
...  
Keyword(s):  
Amphotericin B ◽  
Blood Brain Barrier ◽  
Fungal Infections ◽  
Interleukin 1 ◽  
Brain Barrier ◽  
Blood Brain ◽  
Tnf Α ◽  
Junction Protein ◽  
The Mean

ABSTRACT Amphotericin B (AMB) is used to treat fungal infections of the central nervous system (CNS). However, AMB shows poor penetration into the CNS and little is known about the factors affecting its permeation through the blood-brain barrier (BBB). Therefore, we studied immunomodulatory and organism-associated molecules affecting the permeability of an in vitro BBB model to AMB. We examined the effects of interleukin-1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), lipopolysaccharide (LPS), lipoteichoic acid (LTA), zymosan (ZYM), dexamethasone (DEX), cyclosporine, and tacrolimus on transendothelial electrical resistance (TEER); endothelial tight junctions; filamentous actin; and permeability to deoxycholate AMB (DAMB), liposomal AMB (LAMB), and fluconazole. Proinflammatory cytokines and organism-associated molecules significantly decreased the mean TEER by 40.7 to 100% (P ≤ 0.004). DEX increased the mean TEER by 18.2 to 26.4% (P ≤ 0.04). TNF-α and LPS increased the permeability to AMB by 8.2 to 14.5% compared to that for the controls (1.1 to 2.4%) (P ≤ 0.04). None of the other molecules affected the model's permeability to AMB. By comparison, the BBB model's permeability to fluconazole was >78% under all conditions studied, without significant differences between the controls and the experimental groups. LPS and TNF-α decreased tight-junction protein zona occludens 1 (ZO-1) between endothelial cells. In conclusion, IL-1β, ZYM, and LTA increased the permeability of the BBB to small ions but not to AMB, whereas TNF-α and LPS, which disrupted the endothelial layer integrity, increased the permeability to AMB.

scholarly journals In VitroandIn VivoEvidence for Amphotericin B as a P-Glycoprotein Substrate on the Blood-Brain Barrier

2014 ◽  
Vol 58 (8) ◽  
pp. 4464-4469 ◽  
Author(s):  
Ji-Qin Wu ◽  
Kun Shao ◽  
Xuan Wang ◽  
Rui-Ying Wang ◽  
Ya-Hui Cao ◽  
...  
Keyword(s):  
Amphotericin B ◽  
Blood Brain Barrier ◽  
Fungal Infections ◽  
Brain Barrier ◽  
Content Type ◽  
P Glycoprotein ◽  
Blood Brain ◽  
The Impact ◽  
The Brain

ABSTRACTAmphotericin B (AMB) has been a mainstay therapy for fungal infections of the central nervous system, but its use has been limited by its poor penetration into the brain, the mechanism of which remains unclear. In this study, we aimed to investigate the role of P-glycoprotein (P-gp) in AMB crossing the blood-brain barrier (BBB). The uptake of AMB by primary brain capillary endothelial cellsin vitrowas significantly enhanced after inhibition of P-gp by verapamil. The impact of two model P-gp inhibitors, verapamil and itraconazole, on brain/plasma ratios of AMB was examined in both uninfected CD-1 mice and those intracerebrally infected withCryptococcus neoformans. In uninfected mice, the brain/plasma ratios of AMB were increased 15 min (3.5 versus 2.0;P< 0.05) and 30 min (5.2 versus 2.8;P< 0.05) after administration of verapamil or 45 min (6.0 versus 3.9;P< 0.05) and 60 min (5.4 versus 3.8;P< 0.05) after itraconazole administration. The increases in brain/plasma ratios were also observed in infected mice treated with AMB and P-gp inhibitors. The brain tissue fungal CFU in infected mice were significantly lower in AMB-plus-itraconazole or verapamil groups than in the untreated group (P< 0.005), but none of the treatments protected the mice from succumbing to the infection. In conclusion, we demonstrated that P-gp inhibitors can enhance the uptake of AMB through the BBB, suggesting that AMB is a P-gp substrate.

scholarly journals Amphotericin B Penetrates into the Central Nervous System through Focal Disruption of the Blood-Brain Barrier in Experimental Hematogenous Candida Meningoencephalitis

2019 ◽  
Vol 63 (12) ◽  
Author(s):  
Vidmantas Petraitis ◽  
Ruta Petraitiene ◽  
Jessica M. Valdez ◽  
Vasilios Pyrgos ◽  
Martin J. Lizak ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Amphotericin B ◽  
Blood Brain Barrier ◽  
Brain Barrier ◽  
Blue Dye ◽  
Bolus Administration ◽  
Blood Brain ◽  
Mri Scans

ABSTRACT Hematogenous Candida meningoencephalitis (HCME) is a life-threatening complication of neonates and immunocompromised children. Amphotericin B (AmB) shows poor permeation and low cerebrospinal fluid (CSF) concentrations but is effective in the treatment of HCME. In order to better understand the mechanism of CNS penetration of AmB, we hypothesized that AmB may achieve focally higher concentrations in infected CNS lesions. An in vitro blood-brain barrier (BBB) model was serially infected with Candida albicans. Liposomal AmB (LAMB) or deoxycholate AmB (DAMB) at 5 μg/ml was then provided, and the vascular and CNS compartments were sampled 4 h later. For in vivo correlation, rabbits with experimental HCME received a single dose of DAMB at 1 mg/kg of body weight or LAMB at 5 mg/kg and were euthanized after 1, 3, 6, and 24 h. Evans blue dye solution (2%, 2 ml/kg) administered intravenously (i.v.) at 1 h prior to euthanasia stained infected regions of tissue but not histologically normal areas. AmB concentrations in stained and unstained tissue regions were measured using ultraperformance liquid chromatography. For selected rabbits, magnetic resonance imaging (MRI) scans performed on days 1 to 7 postinoculation were acquired before and after i.v. bolus administration of gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) at 15-min intervals through 2 h postinjection. The greatest degree of penetration of DAMB and LAMB through the in vitro BBB occurred after 24 h of exposure (P = 0.0022). In vivo the concentrations of LAMB and DAMB in brain abscesses were 4.35 ± 0.59 and 3.14 ± 0.89 times higher, respectively, than those in normal tissue (P ≤ 0.019). MRI scans demonstrated that Gd-DTPA accumulated in infected areas with a disrupted BBB. Localized BBB disruption in HCME allows high concentrations of AmB within infected tissues, despite the presence of low cerebrospinal fluid concentrations.

Effects of selective hypothermia on blood-brain barrier integrity and tight junction protein expression levels after intracerebral hemorrhage in rats

2013 ◽  
Vol 394 (10) ◽  
pp. 1317-1324 ◽  
Author(s):  
Hongwei Sun ◽  
Ying Tang ◽  
Xiqin Guan ◽  
Lanfeng Li ◽  
Desheng Wang
Keyword(s):  
Intracerebral Hemorrhage ◽  
Tight Junction ◽  
Blood Brain Barrier ◽  
Brain Barrier ◽  
Edema Formation ◽  
Expression Levels ◽  
Blood Brain ◽  
Tnf Α ◽  
Junction Protein ◽  
Local Hypothermia

Abstract Hypothermia has neuroprotective effects on global cerebral ischemic injuries. However, its efficacy after intracerebral hemorrhage (ICH) is inconclusive. In this study, bacterial collagenase was used to induce ICH stroke in male Wistar rats. We assessed the effects of normothermia and 4 h of local hypothermia (∼33.2°C) initiated 1, 6, or 12 h after collagenase infusion on hemorrhage volume and neurological outcomes. Following early cooling initiated after 1 h, blood-brain barrier (BBB) disruption and brain water content were tested. Furthermore, the expression levels of tight junction (TJ) proteins (claudin 5 and occludin) and the proinflammatory cytokines interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) were determined using Western blotting, real-time quantitative PCR, and immunohistochemical staining at 1 and 3 d after ICH. Early local hypothermia tends to reduce hemorrhagic volume and neurological deficits, but the difference is not statistically significant compared with other groups. However, early hypothermia significantly reduces BBB disruption, edema formation, the expression levels of IL-1β and TNF-α, and the loss of TJ proteins. Together, these data suggest that local hypothermia is an effective treatment for edema formation and BBB disruption via the upregulation of TJ proteins and the suppression of TNF-α and IL-1β.

Abstract T P250: Role of Autophagy in Blood-Brain Barrier Disruption and Tight Junction Degradation Under Ischemic Condition

Stroke ◽  
2015 ◽  
Vol 46 (suppl_1) ◽  
Author(s):  
Kyeong-A Kim ◽  
Young-Jun Shin ◽  
Eun-Sun Kim ◽  
Muhammad Akram ◽  
Dabi Noh ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Tight Junction ◽  
Cell Viability ◽  
Blood Brain Barrier ◽  
Neuronal Damage ◽  
Cell Damage ◽  
Brain Barrier ◽  
Blood Brain ◽  
Junction Protein

During ischemic stroke, the integrity of blood-brain barrier (BBB), which shows selective permeability for substances to brain, is significantly damaged amplifying ischemic neuronal damage. There have been attempts to identify the exact mechanism ischemic BBB disruption to minimize brain damage under ischemic stroke. Autophagy is catabolic process which involves degradation and recycling of damaged or unnecessary organelles. However, excessive autophagy can induce cell damage and death under pathological conditions such as ischemia. In this study, we evaluated if autophagy is a key mechanism of BBB dysfunction under ischemic stroke. In vitro BBB model of bEnd.3 cells were exposed to oxygen-glucose deprivation (OGD), an ischemic mimic condition. After exposure to OGD for 18 hours, cell viability was significantly decreased and cellular permeability was impaired. The conversion of LC3-I to LC3-II and puncta of LC3 in bEnd.3 were increased, demonstrating that autophagy is induced under ischemic condition. Modulation of autophagy by 3-methyladenine, an autophagy inhibitor, reversed the conversion of LC3 as well as decreased cell viability, suggesting that autophagy involves in ischemic BBB damage. The level of occludin, a tight junction protein in BBB, was decreased after OGD, and this was reversed by inhibition of autophagy. Our findings showed that induction of autophagy might contribute to increased permeability through occludin degradation in brain endothelial cells under ischemia, providing a new mechanism of BBB disruption in ischemic stroke.

scholarly journals Hydralazine is a Suitable Mimetic Agent of Hypoxia to Study the Impact of Hypoxic Stress on In Vitro Blood-Brain Barrier Model

2017 ◽  
Vol 42 (4) ◽  
pp. 1592-1602 ◽  
Author(s):  
Morgane Chatard ◽  
Clémentine Puech ◽  
Nathalie Perek ◽  
Frédéric Roche
Keyword(s):  
Blood Brain Barrier ◽  
Brain Barrier ◽  
Hypoxic Stress ◽  
Cellular Mechanisms ◽  
Blood Brain ◽  
Junction Protein ◽  
Set Up ◽  
In Vitro Bbb Model ◽  
The Impact

Background/Aims: Understanding cellular mechanisms induced by hypoxia is fundamental to reduce blood-brain barrier (BBB) disruption. Nevertheless, the investigation of hypoxia on cellular pathways is complex with true hypoxia because HIF-1α has a short lifetime and rapidly reverts back to a normoxic state. To overcome this difficulty, mimetic agents of the hypoxia pathway have been developed, including the gold standard CoCl2. In this study, we proposed to compare CoCl2 and hydralazine in order to determine a suitable mimetic agent of hypoxia for the study on the BBB. Methods: We studied the cytotoxicity and the impact of these molecules on the integrity of an in vitro BBB model by comparing them to hypoxia controls. Results: We showed that the impact of hypoxic stress in our in vitro BBB model is rather similar between hydralazine and CoCl2. Chemical hypoxic stress led to an increase of BBB permeability either with CoCl2 or hydralazine. Tight junction protein expressions showed that this chemical hypoxic stress decreased ZO-1 but not occluding expressions, and cells had set up a defence mechanism by increasing expression and activity of their efflux transporters. Conclusion: Our results demonstrated that hydralazine is a better mimetic agent and more suitable than CoCl2 because it had the same effect but without the cytotoxic effect on in vitro BBB cells.

scholarly journals Activation of PKC Isoform βI at the Blood–Brain Barrier Rapidly Decreases P-Glycoprotein Activity and Enhances Drug Delivery to the Brain

2010 ◽  
Vol 30 (7) ◽  
pp. 1373-1383 ◽  
Author(s):  
Robert R Rigor ◽  
Brian T Hawkins ◽  
David S Miller
Keyword(s):  
Rat Brain ◽  
Blood Brain Barrier ◽  
Brain Barrier ◽  
Brain Capillaries ◽  
P Glycoprotein ◽  
Blood Brain ◽  
Tnf Α ◽  
The Brain

P-glycoprotein is an ATP (adenosine triphosphate)-driven drug efflux transporter that is highly expressed at the blood–brain barrier (BBB) and is a major obstacle to the pharmacotherapy of central nervous system diseases, including brain tumors, neuro-AIDS, and epilepsy. Previous studies have shown that P-glycoprotein transport activity in rat brain capillaries is rapidly reduced by the proinflammatory cytokine, tumor necrosis factor-α (TNF-α) acting through protein kinase C (PKC)-dependent signaling. In this study, we used isolated rat brain capillaries to show that the TNF-α-induced reduction of P-glycoprotein activity was prevented by a PKCβI/II inhibitor, LY333531, and mimicked by a PKCβI/II activator, 12-deoxyphorbol-13-phenylacetate-20-acetate (dPPA). Western blotting of brain capillary extracts with phospho-specific antibodies showed that dPPA activated PKCβI, but not PKCβII. Moreover, in intact rats, intracarotid infusion of dPPA potently increased brain accumulation of the P-glycoprotein substrate, [3H]-verapamil without compromising tight junction integrity. Thus, PKCβI activation selectively reduced P-glycoprotein activity both in vitro and in vivo. Targeting PKCβI at the BBB may prove to be an effective strategy for enhancing the delivery of small molecule therapeutics to the brain.

scholarly journals Penetration of the Blood-Brain Barrier by Bacillus anthracis Requires the pXO1-Encoded BslA Protein

2009 ◽  
Vol 191 (23) ◽  
pp. 7165-7173 ◽  
Author(s):  
Celia M. Ebrahimi ◽  
Justin W. Kern ◽  
Tamsin R. Sheen ◽  
Mohammad A. Ebrahimi-Fardooee ◽  
Nina M. van Sorge ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Blood Brain Barrier ◽  
Molecular Mechanisms ◽  
Human Infection ◽  
Brain Barrier ◽  
Brain Infection ◽  
Blood Brain ◽  
Junction Protein

ABSTRACT Anthrax is a zoonotic disease caused by the gram-positive spore-forming bacterium Bacillus anthracis. Human infection occurs after the ingestion, inhalation, or cutaneous inoculation of B. anthracis spores. The subsequent progression of the disease is largely mediated by two native virulence plasmids, pXO1 and pXO2, and is characterized by septicemia, toxemia, and meningitis. In order to produce meningitis, blood-borne bacteria must interact with and breach the blood-brain barrier (BBB) that is composed of a specialized layer of brain microvascular endothelial cells (BMEC). We have recently shown that B. anthracis Sterne is capable of penetrating the BBB in vitro and in vivo, establishing the classic signs of meningitis; however, the molecular mechanisms underlying the central nervous system (CNS) tropism are not known. Here, we show that attachment to and invasion of human BMEC by B. anthracis Sterne is mediated by the pXO1 plasmid and an encoded envelope factor, BslA. The results of studies using complementation analysis, recombinant BslA protein, and heterologous expression demonstrate that BslA is both necessary and sufficient to promote adherence to brain endothelium. Furthermore, mice injected with the BslA-deficient strain exhibited a significant decrease in the frequency of brain infection compared to mice injected with the parental strain. In addition, BslA contributed to BBB breakdown by disrupting tight junction protein ZO-1. Our results identify the pXO1-encoded BslA adhesin as a critical mediator of CNS entry and offer new insights into the pathogenesis of anthrax meningitis.

scholarly journals Intermittent Hypoxia and Its Impact on Nrf2/HIF-1α Expression and ABC Transporters: An in Vitro Human Blood-Brain Barrier Model Study.

2020 ◽  
Vol 54 (6) ◽  
pp. 1231-1248
Keyword(s):  
Blood Brain Barrier ◽  
Abc Transporters ◽  
Intermittent Hypoxia ◽  
Brain Barrier ◽  
Upper Airways ◽  
Whole Cell ◽  
Blood Brain ◽  
Junction Protein ◽  
The Impact

BACKGROUND/AIMS: Obstructive sleep apnea (OSA) is characterized by repeated episodes of complete or partial obstruction of the upper airways, leading to chronic intermittent hypoxia (IH). OSA patients are considered at high cerebrovascular risk and may also present cognitive impairment. One hypothesis explored is that disturbances may be linked to blood-brain barrier (BBB) dysfunction. The BBB is a protective barrier separating the brain from blood flow. The BBB limits the paracellular pathway through tight and adherens junctions, and the transcellular passage by efflux pumps (ABC transporters). The aims of this study were to evaluate the impact of IH and sustained hypoxia (SH) on a validated in vitro BBB model and to investigate the factors expressed under both conditions. METHODS: Exposure of endothelial cells (HBEC-5i) in our in vitro model of BBB to hypoxia was performed using IH cycles: 1% O2-35 min/18% O2-25 min for 6 cycles or 6 h of SH at 1% O2. After exposure, we studied the cytotoxicity and the level of ROS in our cells. We measured the apparent BBB permeability using sodium fluorescein, FITC-dextran and TEER measurement. Whole cell ELISA were performed to evaluate the expression of tight junctions, ABC transporters, HIF-1α and Nrf2. The functionality of ABC transporters was evaluated with accumulation studies. Immunofluorescence assays were also conducted to illustrate the whole cell ELISAs. RESULTS: Our study showed that 6 h of IH or SH induced a BBB disruption marked by a significant decrease in junction protein expressions (claudin-5, VE-cadherin, ZO-1) and an increase in permeability. We also observed an upregulation in P-gp protein expression and functionality and a downregulation in BCRP. Hypoxia induced production of ROS, Nrf2 and HIF-1α. They were expressed in both sustained and intermittent conditions, but the expression and the activity of P-gp and BCRP were different. CONCLUSION: Understanding these mechanisms seems essential in order to propose new therapeutic strategies for patients with OSA

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