Nifedipine toxicity is exacerbated by acetyl l-carnitine but alleviated by low-dose ketamine in zebrafish in vivo.

Calcium channel blocker (CCB) poisoning is a common and sometimes life-threatening emergency. Our previous studies have shown that acetyl l-carnitine (ALCAR) prevents cardiotoxicity and developmental toxicity induced by verapamil, a CCB used to treat patients with hypertension. Here, we tested whether toxicities of nifedipine, a dihydropyridine CCB used to treat hypertension, can also be mitigated by co-treatment with ALCAR. In the zebrafish embryos at three different developmental stages, nifedipine induced developmental toxicity with pericardial sac edema in a dose-dependent manner, which were surprisingly exacerbated with ALCAR co-treatment. Even with low-dose nifedipine (5 µm), when the pericardial sac looked normal, ALCAR co-treatment showed pericardial sac edema. We hypothesized that toxicity by nifedipine, a vasodilator, may be prevented by ketamine, a known vasoconstrictor. Nifedipine toxicity in the embryos was effectively prevented by co-treatment with low (subanesthetic) doses (25-100 µm added to the water) of ketamine, although a high dose of ketamine (2 mm added to the water) partially prevented the toxicity.As expected of a CCB, nifedipine either in the presence or absence of ketamine-reduced metabolic reactive oxygen species (ROS), a downstream product of calcium signaling, in the rapidly developing digestive system. However, nifedipine induced ROS in the trunk region that showed significantly stunted growth indicating that the tissues under stress potentially produced pathologic ROS. To the best of our knowledge, these studies for the first time show that nifedipine and the dietary supplement ALCAR together induce adverse effects while providing evidence on the therapeutic efficacy of subanesthetic doses of ketamine against nifedipine toxicity in vivo.

Cyclosporine exacerbates ketamine toxicity in zebrafish: Mechanistic studies on drug-drug interaction.

Cyclosporine A (CsA) is an immunosuppressive drug commonly used in organ transplant patients to prevent allograft rejections. Ketamine is a pediatric anesthetic that noncompetitively inhibits the calcium-permeable N-methyl-d-aspartic acid receptors. Adverse drug-drug interaction effects between ketamine and CsA have been reported in mammals and humans. However, the mechanism of such drug-drug interaction is unclear. We have previously reported adverse effects of combination drugs, such as verapamil/ketamine and shown the mechanism through intervention by other drugs in zebrafish embryos. Here, we show that ketamine and CsA in combination produce developmental toxicity even leading to lethality in zebrafish larvae when exposure began at 24 h post-fertilization (hpf), whereas CsA did not cause any toxicity on its own. We also demonstrate that acetyl l-carnitine (ALCAR) completely reversed the adverse effects. Both ketamine and CsA are CYP3A4 substrates. Although ketamine and CsA independently altered the expression of the hepatic marker CYP3A65, a zebrafish ortholog of human CYP3A4, both drugs together induced further increase in CYP3A65 expression. In the presence of ALCAR, however, CYP3A65 expression was normalized. ALCAR has been shown to prevent ketamine toxicity in mammal and zebrafish. In conclusion, CsA exacerbated ketamine toxicity and ALCAR reversed the effects. These results, providing evidence for the first time on the reversal of the adverse effects of CsA/ketamine interaction by ALCAR, would prove useful in addressing potential occurrences of such toxicities in humans. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

Acetyl L-carnitine targets adenosine triphosphate synthase in protecting zebrafish embryos from toxicities induced by verapamil and ketamine: An in vivo assessment.

Verapamil is a Ca2+ channel blocker and is highly prescribed as an anti-anginal, antiarrhythmic and antihypertensive drug. Ketamine, an antagonist of the Ca2+ -permeable N-methyl-d-aspartate-type glutamate receptors, is a pediatric anesthetic. Previously we have shown that acetyl l-carnitine (ALCAR) reverses ketamine-induced attenuation of heart rate and neurotoxicity in zebrafish embryos. Here, we used 48 h post-fertilization zebrafish embryos that were exposed to relevant drugs for 2 or 4 h. Heart beat and overall development were monitored in vivo. In 48 h post-fertilization embryos, 2 mm ketamine reduced heart rate in a 2 or 4 h exposure and 0.5 mm ALCAR neutralized this effect. ALCAR could reverse ketamine's effect, possibly through a compensatory mechanism involving extracellular Ca2+ entry through L-type Ca2+ channels that ALCAR is known to activate. Hence, we used verapamil to block the L-type Ca2+ channels. Verapamil was more potent in attenuating heart rate and inducing morphological defects in the embryos compared to ketamine at specific times of exposure. ALCAR reversed cardiotoxicity and developmental toxicity in the embryos exposed to verapamil or verapamil plus ketamine, even in the presence of 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester, an inhibitor of intracellular Ca2+ release suggesting that ALCAR acts via effectors downstream of Ca2+ . In fact, ALCAR's protective effect was blunted by oligomycin A, an inhibitor of adenosine triphosphate synthase that acts downstream of Ca2+ during adenosine triphosphate generation. We have identified, for the first time, using in vivo studies, a downstream effector of ALCAR that is critical in abrogating ketamine- and verapamil-induced developmental toxicities. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Porcine brain microvessel endothelial cells show pro-inflammatory response to the size and composition of metallic nanoparticles.

The purpose of the current studies was to determine if systemic exposure of various metallic nanoparticles differing in size and composition [silver (Ag-NPs, 25, 40 and 80 nm), copper-oxide (Cu-NPs, 40 and 60 nm) or gold (Au-NPs, 3 and 5 nm)] can induce the release of pro-inflammatory mediators that influence the restrictive nature of the blood-brain barrier (BBB) in vitro. Confluent porcine brain microvessel endothelial cells (pBMECs) (8-12 days) were treated with various metallic nanoparticles (15 µg/ml). Extracellular concentrations of pro-inflammatory mediators (IL-1ß, TNFα and PGE2) were evaluated using ELISA. pBMECs were cultured in standard 12-well Transwell® inserts, and permeability was evaluated by measuring the transport of fluorescein across the pBMEC monolayers. PGE2 release following Cu-NP exposure was significantly increased when compared to the control. Similar results were observed for Ag-NPs but not Au-NPs. The secretion of TNFα and IL-1ß was observed for both Cu-NPs and Ag-NPs but not in response to Au-NPs. The post-treatment time profiles of TNFα and IL-1ß revealed that the IL-1ß response was more persistent. The permeability ratios (exposure/control) were significantly greater following exposure to Cu-NPs or Ag-NPs, compared to Au-NPs. Together, these data suggest that the composition and size of NPs can cause significant pro-inflammatory response that can influence the integrity of the BBB.

Impaired Amyloid Beta Clearance and Brain Microvascular Dysfunction are Present in the Tg-SwDI Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) pathology is characterized by amyloid plaques containing amyloid beta (Aß) peptides, neurofibrillary tangles containing hyperphosphorylated tau protein, and neuronal loss. In addition, Aß deposition in brain microvessels, known as cerebral amyloid angiopathy (CAA), increases blood-brain barrier (BBB) permeability and induces vascular dysfunction which aggravates AD pathology. The aim of the present study was to characterize neurovascular dysfunction in the Tg-SwDI mouse model of AD. Isolated brain capillaries from wild type (WT) and Tg-SwDI mice were used to evaluate the expression of monomeric and aggregated forms of Aß, P-glycoprotein (P-gp), the receptor for advance glycation end-products (RAGE) and the tight junction (TJs) proteins occludin and claudin-5. Cultured brain endothelial cells were used to analyze barrier function via fluorescein flux. Isolated capillaries from Tg-SwDI mice contained increased levels of aggregated and oligomeric Aß compared to WT animals. Isolated capillaries from Tg-SwDI had decreased levels of P-gp, which transports Aß from brain to blood, and increased levels of RAGE, which transports Aß from blood to brain. In addition, the TJ protein occludin was decreased in Tg-SwDI mice relative to WT mice, which correlated with an increase in BBB permeability in cultured brain endothelial cells. These findings demonstrated that Tg-SwDI mice exhibit Aß aggregation that is due, in part, to impaired Aß clearance driven by both a decrease in P-gp and increase in RAGE protein levels in brain capillaries. Aß aggregation promotes a decrease in the expression of the TJ protein occludin, and as consequence an increase in BBB permeability.

Characterization of Serum Exosomes from a Transgenic Mouse Model of Alzheimer's Disease.

BACKGROUND: Alzheimer's Disease (AD) is the most common type of dementia characterized by amyloid plaques containing Amyloid Beta (Aß) peptides and neurofibrillary tangles containing tau protein. In addition to neuronal loss, Cerebral Amyloid Angiopathy (CAA) commonly occurs in AD. CAA is characterized by Aß deposition in brain microvessels. Recent studies have suggested that exosomes (cell-derived vesicles containing a diverse cargo) may be involved in the pathogenesis of AD. OBJECTIVE: Isolate and characterize brain-derived exosomes from a transgenic mouse model of AD that presents CAA. METHODS: Exosomes were isolated from serum obtained from 13-month-old wild type and AD transgenic female mice using an exosome precipitation solution. Characterization of exosomal proteins was performed by western blots and dot blots. RESULTS: Serum exosomes were increased in transgenic mice compared to wild types as determined by increased levels of the exosome markers flotillin and alix. High levels of neuronal markers were found in exosomes, without any difference any between the 2 groups. Markers for endothelial-derived exosomes were decreased in the transgenic model, while astrocytic-derived exosomes were increased. Exosome characterization showed increased levels of oligomeric Aß and oligomeric and monomeric forms tau on the transgenic animals. Levels of amyloid precursor protein were also increased. In addition, pathological and phosphorylated forms of tau were detected, but no difference was observed between the groups. CONCLUSION: These data suggest that monomeric and oligomeric forms of Aß and tau are secreted into serum via brain exosomes, most likely derived from astrocytes in the transgenic mouse model of AD with CAA. Studies on the implication of this event in the propagation of AD are underway.

The time course of blood brain barrier leakage and its implications on the progression of methamphetamine-induced seizures.

The initial goals of these experiments were to determine: 1) if blood-brain barrier (BBB) breakdown was a cause or an effect of METH-induced seizures; 2) all the brain regions where BBB is disrupted as seizures progress; and 3) the correlations between body temperature and vascular leakage and neurodegeneration. A fourth objective was added after initial experimentation to determine if sub-strain differences existed in adult male C57 B6 J (Jackson laboratories, JAX) versus C57 B6N (Charles River, CR) mice involving their susceptibility to BBB breakdown and seizure severity. With the 1st "maximal" intensity myoclonic-tonic seizure (MCT) varying degrees of IgG infiltration across the BBB (≤1 mm2) were prominent in olfactory system (OS) associated regions and in thalamus, hypothalamus and neocortex. IgG infiltration areas in the OS-associated regions of the bed nucleus of the stria terminalis, septum and more medial amygdala nuclei, and the hypothalamus were increased significantly by the time continuous behavioral seizures (CBS) developed. Mice receiving METH that had body temperatures of ≥40 °C had IgG infiltration along with MCT or CBS but peak body temperatures above 40 °C did not significantly increase IgG infiltration. Neurodegeneration seen at ≥6 h was restricted to the OS in both JAX and CR mice and was most prominent in the posteromedial cortical amygdaloid nucleus. Neurodegeneration in the anterior septum (tenia tecta) was seen only in the JAX mice. We hypothesize that METH-induced hypertension and hyperthermia lead to BBB breakdown and other vascular dysfunctions in the OS brain regions resulting in OS hyperexcitation. Excitation of the OS neural network then leads to the development of seizures. These seizures in turn exacerbate the energy depletions and the reactive oxygen stress produced by hyperthermia further damaging the BBB and vascular function. These events form a recurrent cycle that results in ever increasing seizure activity and neurotoxicity.

Mechanistic studies on ketamine-induced mitochondrial toxicity in zebrafish embryos.

Ketamine, a phencyclidine derivative, is an antagonist of the Ca2+-permeable N-methyl-d-aspartate (NMDA)-type glutamate receptors. It is a pediatric anesthetic and has been implicated in developmental neurotoxicity. Ketamine has also been shown to deplete ATP in mammalian cells. Our previous studies showed that acetyl l-carnitine (ALCAR) prevented ketamine-induced cardiotoxicity and neurotoxicity in zebrafish embryos. Based on our finding that ALCAR's protective effect was blunted by oligomycin A, an inhibitor of ATP synthase, we further investigated the effects of ketamine and ALCAR on ATP levels, mitochondria and ATP synthase in zebrafish embryos. The results demonstrated that ketamine reduced ATP levels in the embryos but not in the presence of ALCAR. Ketamine reduced total mitochondrial protein levels and mitochondrial potential, which were prevented with ALCAR co-treatment. To determine the cause of ketamine-induced ATP deficiency, we explored the status of ATP synthase. The results showed that a subunit of ATP synthase, atp5α1, was transcriptionally down-regulated by ketamine, but not in the presence of ALCAR, although ketamine caused a significant upregulation in another ATP synthase subunit, atp5ß and total ATP synthase protein levels. Most of the ATP generated by heart mitochondria are utilized for its contraction and relaxation. Ketamine-treated embryos showed abnormal heart structure, which was abolished with ALCAR co-treatment. This study offers evidence for a potential mechanism by which ketamine could cause ATP deficiency mediated by mitochondrial dysfunction.

Effects of L-carnitine pretreatment in methamphetamine and 3-nitropropionic acid-induced neurotoxicity.

Adult, male Sprague-Dawley rats were injected with 3-ni-tropropionic acid (3-NPA) at 30 mg/kg or methamphetamine (METH) at 20 mg/kg alone or following pretreatment with L-cartnitine (LC) at 100 mg/kg. Rectal temperature was measured before and 4 h following treatment. Animals were sacrificed at 4 h posttreatment. Monoamine neurotransmitters, dopamine (DA) and serotonin (5-HT), and their metabolites were analyzed in the striatum using high-performance liquid chromatography method coupled with electrochemical detection (HPLC/ED). Transcripts of several genes related to DA metabolism were quantified using real time reverse transciption polymerase chain reaction (RT-PCR). Core temperature decreased significantly after 3-NPA acid and increased in METH-treated rats (P < 0.05). Temperature change at 4 h exhibited a significant LC effect for 3-NPA, preventing hypothermia (P < 0.05) and no effect for METH. Concentration of DA and 5-HT, and their metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA), increased significantly in 3-NPA and decreased in METH-treated rats. An increase in DOPAC/DA turnover and serotonin observed after 3-NPA was abolished in LC-/3-NPA-treated rats. In both 3-NPA- and METH-treated rats, LC prevented an increase in DA receptor D(1) gene expression. It appears that carnitine effect preventing hypothermia after 3-NPA treatments may be related not only to its mitochondriotropic actions but also to inhibitory effect on the DA and 5-HT systems activated after the exposure to 3-NPA. The same effect observed at the transcriptional level, at least for the DA receptor D(1), may account for protection against METH toxicity.

Distinct effects of ketamine and acetyl L-carnitine on the dopamine system in zebrafish.

Ketamine, a noncompetitive N-methyl-D-aspartic acid (NMDA) receptor antagonist is commonly used as a pediatric anesthetic. We have previously shown that acetyl L-carnitine (ALCAR) prevents ketamine toxicity in zebrafish embryos. In mammals, ketamine is known to modulate the dopaminergic system. NMDA receptor antagonists are considered as promising anti-depressants, but the exact mechanism of their function is unclear. Here, we measured the levels of dopamine (DA) and its metabolites, 3, 4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), in the zebrafish embryos exposed to ketamine in the presence and absence of 0.5 mM ALCAR. Ketamine, at lower doses (0.1-0.3 mM), did not produce significant changes in DA, DOPAC or HVA levels in 52 h post-fertilization embryos treated for 24 h. In these embryos, tyrosine hydroxylase (TH) mRNA expression remained unchanged. However, 2 mM ketamine (internal embryo exposure levels equivalent to human anesthetic plasma concentration) significantly reduced DA level and TH mRNA indicating that DA synthesis was adversely affected. In the presence or absence of 2 mM ketamine, ALCAR showed similar effects on DA level and TH mRNA, but increased DOPAC level compared to control. ALCAR reversed 2 mM ketamine-induced reduction in HVA levels. With ALCAR alone, the expression of genes encoding the DA metabolizing enzymes, MAO (monoamine oxidase) and catechol-O-methyltransferase (COMT), was not affected. However, ketamine altered MAO mRNA expression, except at the 0.1 mM dose. COMT transcripts were reduced in the 2 mM ketamine-treated group. These distinct effects of ketamine and ALCAR on the DA system may shed some light on the mechanism on how ketamine can work as an anti-depressant, especially at sub-anesthetic doses that do not affect DA metabolism and suppress MAO gene expression.

Opposing effects of ketamine and acetyl L-carnitine on the serotonergic system of zebrafish.

Ketamine, a pediatric anesthetic, is a noncompetitive N-methyl-D-aspartic acid (NMDA) receptor antagonist. Studies show that ketamine is neurotoxic in developing mammals and zebrafish. In both mammals and zebrafish, acetyl L-carnitine (ALCAR) has been shown to be protective against ketamine toxicity. Ketamine is known to modulate the serotonergic system in mammals. Here, we measured the levels of serotonin (5-HT) and its metabolite, 5-hydroxyindoleacetic acid (5-HIAA) in the embryos exposed to ketamine in the presence and absence of ALCAR. Ketamine, at lower doses, did not produce significant changes in the 5-HT or 5-HIAA levels in 3 dpf (day post-fertilization) embryos. However, 2 mM ketamine (internal embryo exposure levels comparable to human anesthetic plasma concentration) significantly reduced 5-HT level, and 5-HIAA was not detectable indicating that 5-HT metabolism was abolished. In the presence or absence of 2 mM ketamine, ALCAR by itself did not significantly alter 5-HT or 5-HIAA levels compared to the control. Ratios of metabolite/5-HT indicated that 2 mM ketamine inhibited 5-HT metabolism to 5-HIAA whereas lower doses (0.1-0.3 mM) of ketamine did not have any effect. ALCAR reversed the effects of 2 mM ketamine not only by restoring 5-HT and 5-HIAA levels but also 5-HT turnover rate to control levels. Whole mount immunohistochemical studies showed that 2 mM ketamine reduced the serotonergic area in the brain whereas ALCAR expanded it with increased axonal sprouting and branching. These results indicate that ketamine and ALCAR have opposing effects on the zebrafish serotonergic system.

Effects of adolescent treatment with nicotine, harmane, or norharmane in male Sprague-Dawley rats.

The initiation of tobacco use occurs most often in adolescence and may be especially detrimental as the adolescent brain is undergoing substantial development. In addition to nicotine, there are over 9000 other compounds present in tobacco products, including the ß-carbolines harmane and norharmane. The present study aimed to determine the long-term effects of adolescent exposure to nicotine (NIC), harmane (HAR), or norharmane (NOR) on locomotor activity, learning and memory, anxiety-like behavior, motor coordination, and monoamine/metabolite concentrations in the striatum and nucleus accumbens of male Sprague-Dawley rats. Beginning on postnatal day (PND) 27 and continuing through PND 55, subjects received twice daily intraperitoneal injections of 1ml/kg saline (CON), 0.5mg NIC/kg, 0.5mg HAR/kg, or 0.5mg NOR/kg. Body weight, food, and water intake were measured daily (PNDs 27-96). Locomotor activity was assessed on PND 40 or 41, PND 55, and PNDs 81 and 82. Other behaviors (anxiety-like behavior, motor coordination, and spatial learning and memory) were assessed at least 25 days after drug exposure ended (PNDs 80-91). On PND 97, subjects were decapitated and the striatum and nucleus accumbens were dissected and frozen for analysis. NIC treatment significantly decreased food intake, but did not alter locomotor activity during or after treatment. HAR and NOR treatment, however, caused significant open field hypoactivity. Motor coordination, water maze performance, and concentrations of monoamines and metabolites in the striatum and nucleus accumbens were unaltered by any drug treatment. These results indicate a long-lasting effect on activity levels from adolescent HAR or NOR treatment; however, there were few long-lasting NIC effects. Given the paucity of data describing effects of HAR or NOR exposure, these data should encourage additional studies of these tobacco constituents as well as constituent combination studies.

Iron Oxide Nanoparticles Induce Dopaminergic Damage: In vitro Pathways and In Vivo Imaging Reveals Mechanism of Neuronal Damage.

Various iron-oxide nanoparticles have been in use for a long time as therapeutic and imaging agents and for supplemental delivery in cases of iron-deficiency. While all of these products have a specified size range of ∼ 40 nm and above, efforts are underway to produce smaller particles, down to ∼ 1 nm. Here, we show that after a 24-h exposure of SHSY-5Y human neuroblastoma cells to 10 µg/ml of 10 and 30 nm ferric oxide nanoparticles (Fe-NPs), cellular dopamine content was depleted by 68 and 52 %, respectively. Increases in activated tyrosine kinase c-Abl, a molecular switch induced by oxidative stress, and neuronal α-synuclein expression, a protein marker associated with neuronal injury, were also observed (55 and 38 % percent increases, respectively). Inhibition of cell-proliferation, significant reductions in the number of active mitochondria, and a dose-dependent increase in reactive oxygen species (ROS) were observed in neuronal cells. Additionally, using a rat in vitro blood-brain barrier (BBB) model, a dose-dependent increase in ROS accompanied by increased fluorescein efflux demonstrated compromised BBB integrity. To assess translational implications, in vivo Fe-NP-induced neurotoxicity was determined using in vivo MRI and post-mortem neurochemical and neuropathological correlates in adult male rats after exposure to 50 mg/kg of 10 nm Fe-NPs. Significant decrease in T 2 values was observed. Dynamic observations suggested transfer and retention of Fe-NPs from brain vasculature into brain ventricles. A significant decrease in striatal dopamine and its metabolites was also observed, and neuropathological correlates provided additional evidence of significant nerve cell body and dopaminergic terminal damage as well as damage to neuronal vasculature after exposure to 10 nm Fe-NPs. These data demonstrate a neurotoxic potential of very small size iron nanoparticles and suggest that use of these ferric oxide nanoparticles may result in neurotoxicity, thereby limiting their clinical application.

Effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on retinal dopaminergic system in mice.

The neurotoxins methamphetamine (METH) and MPTP are well-known for their effects on the nigrostriatal dopaminergic system and use in modeling neurodegenerative disorders such as Parkinson's disease. It is not well-known though, how METH or MPTP affects the visual system and specifically the retinal dopaminergic system. This study was designed to examine acute effects of multiple doses of METH and MPTP on the retinal dopaminergic system. Mice were exposed to either low- (LD) 10 mg/kg total dose or high-dose (HD) 30 mg/kg total dose, of METH or MPTP and the retinal catecholaminergic system was analyzed by HPLC. METH produced no significant changes in dopamine (DA), its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) or DA usage in the retina. LD-MPTP produced no change in DA level, but significantly decreased DOPAC and HVA. LD-MPTP also significantly decreased DA usage as measured by the DOPAC/DA and HVA/DA ratios. HD-MPTP significantly decreased DA, DOPAC and HVA, but did not affect DA usage. Taken together these results suggest that inhibition of the DA metabolizing enzymes monoamine oxidase A (MAO) or catechol-O-methyl transferase (COMT) may take place at lower doses of MPTP treatment; conversely, higher doses of MPTP may cause decreases in DA, DOPAC and HVA through another mechanism.

Effects of copper nanoparticles on rat cerebral microvessel endothelial cells.

AIM: The purpose of the current study was to determine whether copper nanoparticles (Cu-NPs) can induce the release of proinflammatory mediators that influence the restrictive characteristics of the blood-brain barrier. MATERIAL & METHODS: Confluent rat brain microvessel endothelial cells (rBMECs) were treated with well-characterized Cu-NPs (40 or 60 nm). Cytotoxicity of the Cu-NPs was evaluated by cell proliferation assay (1.5-50 µg/ml). The extracellular concentrations of proinflammatory mediators (IL-1ß, IL-2, TNF-α and prostaglandin E(2)) were evaluated by ELISA. RESULTS: The exposure of Cu-NPs at low concentrations increases cellular proliferation of rBMECs, by contrast, high concentrations induce toxicity. Prostaglandin E(2) release was significantly increased (threefold; 8 h) for Cu-NPs (40 and 60 nm). The extracellular levels of both TNF-α and IL-1ß were significantly elevated following exposure to Cu-NPs. The P-apparent ratio, as an indicator of increased permeability of rBMEC was approximately twofold for Cu-NPs (40 and 60 nm). CONCLUSION: These data suggest that Cu-NPs can induce rBMEC, proliferation at low concentrations and/or induce blood-brain barrier toxicity and potential neurotoxicity at high concentrations.

Brain microvessel endothelial cells responses to gold nanoparticles: In vitro pro-inflammatory mediators and permeability.

This report examined blood-brain barrier (BBB) related proinflammatory mediators and permeability changes in response to various sized gold nanoparticles (Au-NPs) (3, 5, 7, 10, 30 and 60 nm) in vitro using primary rat brain microvessel endothelial cells (rBMEC). The Au-NPs were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS) and laser Doppler velocimetry (LDV). The accumulation of Au-NPs was determined spectrophotometrically. The rBMEC cytotoxicity of Au-NPs was evaluated by cell proliferation assay (XTT) (concentration range 0.24-15.63 µg/cm², for 24 h). The time-dependent changes (0, 2, 4 and 8 h) of several proinflammatory mediators (IL-1ß, IL-2, TNFα and PGE2) were evaluated by ELISA. The smaller Au-NPs (3-7 nm) showed higher rBMEC accumulation compared to larger Au-NPs (10-60 nm), while only moderate decreased cell viability was observed with small Au-NPs (3 nm) at high concentrations (≥ 7.8 µg/cm²). Even though slight changes in cell viability were observed with small Au-NPs, the basal levels of the various proinflammatory mediators remained unchanged with all treatments except LPS (positive control). rBMEC morphology appeared unaffected 24 h after exposure to Au-NPs with only mild changes in fluorescein permeability indicating BBB integrity was unaltered. Together, these data suggest the responses of the cerebral microvasculature to Au-NPs have a significant relationship with the Au-NPs unique size-dependent physiochemical properties.

Silver nanoparticle induced blood-brain barrier inflammation and increased permeability in primary rat brain microvessel endothelial cells.

The current report examines the interactions of silver nanoparticles (Ag-NPs) with the cerebral microvasculature to identify the involvement of proinflammatory mediators that can increase blood-brain barrier (BBB) permeability. Primary rat brain microvessel endothelial cells (rBMEC) were isolated from adult Sprague-Dawley rats for an in vitro BBB model. The Ag-NPs were characterized by transmission electron microscopy (TEM), dynamic light scattering, and laser Doppler velocimetry. The cellular accumulation, cytotoxicity (6.25-50 µg/cm(3)) and potential proinflammatory mediators (interleukin [IL]-1ß, IL-2, tumor necrosis factor [TNF] α, and prostaglandin E(2) [PGE(2)]) of Ag-NPs (25, 40, or 80 nm) were determined spectrophotometrically, cell proliferation assay (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) and ELISA. The results show Ag-NPs-induced cytotoxic responses at lower concentrations for 25 and 40 nm when compared with 80-nm Ag-NPs. The proinflammatory responses in this study demonstrate both Ag-NPs size and time-dependent profiles, with IL-1B preceding both TNF and PGE(2) for 25 nm. However, larger Ag-NPs (40 and 80 nm) induced significant TNF responses at 4 and 8 h, with no observable PGE(2) response. The increased fluorescein transport observed in this study clearly indicates size-dependent increases in BBB permeability correlated with the severity of immunotoxicity. Together, these data clearly demonstrate that larger Ag-NPs (80 nm) had significantly less effect on rBMEC, whereas the smaller particles induced significant effects on all the end points at lower concentrations and/or shorter times. Further, this study suggests that Ag-NPs may interact with the cerebral microvasculature producing a proinflammatory cascade, if left unchecked; these events may further induce brain inflammation and neurotoxicity.