Cytotoxicity profile of pristine graphene on brain microvascular endothelial cells.

Graphene-based nanomaterials hold the potential to be used in a wide variety of applications, including biomedical devices. Pristine graphene (PG) is an un-functionalized, defect-free type of graphene that could be used as a material for neural interfacing. However, the neurotoxic effects of PG, particularly to the blood-brain barrier (BBB), have not been fully studied. The BBB separates the brain tissue from the circulating substances in the blood and is essential to maintain the brain homeostasis. The principal components of the BBB are brain microvascular endothelial cells (BMVECs), which maintain a protectively low permeability due to the expression of tight junction proteins. Here we analyzed the effects of PG on BMVECs in an in vitro model of the BBB. BMVECs were treated with PG at 0, 10, 50 and 100 µg/mL for 24 hours and viability and functional analyses of BBB integrity were performed. PG increased lactate dehydrogenase release at 50 and 100 µg/mL, suggesting the induction of necrosis. Surprisingly, 2,3,-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium (XTT) conversion was increased at 10 and 50 µg/mL. In contrast, XTT conversion was decreased at 100 µg/mL, suggesting the induction of cell death. In addition, 100 µg/mL PG increased DNA fragmentation, suggesting induction of apoptosis. At the same time, 50 and 100 µg/mL of PG increased the endothelial permeability, which corresponded with a decrease in the expression of the tight junction protein occludin at 100 µg/mL. In conclusion, these results suggest that PG negatively affects the viability and function of the BBB endothelial cells in vitro.

Characterization of uniaxial high-speed stretch as an in vitro model of mild traumatic brain injury on the blood-brain barrier.

Traumatic brain injury (TBI) occurs when external mechanical forces induce brain damage as result of impact, penetration or rapid acceleration/deceleration that causes deformation of brain tissue. Depending on its severity, TBI can be classified as mild, moderate or severe and can lead to blood-brain barrier (BBB) dysfunction. In the present study, we evaluated the effects of uniaxial high-speed stretch (HSS) at 0, 5, 10 and 15% on a pure culture of primary rat brain endothelial cells as an in vitro model of TBI to the BBB. LDH release, viability and apoptosis analysis, expression of tight junction proteins and endothelial permeability were evaluated 24 h after a single stretch episode. HSS slightly increased cell death and apoptosis at 10 and 15%, while LDH release was increased only at 15% stretch. Occludin expression was increased at 10% stretch, while claudin-5 expression was increased at 5% stretch, which also decreased the endothelial permeability. In summary, 15% HSS induced low levels of cell death, consistent with mild TBI and very low percentages of HSS (5%) enhanced the BBB properties, promoting the formation of a stronger barrier. These data support the use of 15% HSS as valuable tool in the study of mild TBI to the BBB in vitro.

Characterization of Biaxial Stretch as an In Vitro Model of Traumatic Brain Injury to the Blood-Brain Barrier.

Traumatic brain injury (TBI) is one of the major causes of disability in the USA. It occurs when external mechanical forces induce brain damage that causes deformation of brain tissue. TBI is also associated with alterations of the blood-brain barrier (BBB). Using primary rat brain microvascular endothelial cells as an in vitro BBB model, the effects of biaxial stretch were characterized at 5, 10, 15, 25, and 50% deformation using a commercially available system. The results were compared to the effects of mild and moderate TBI in vivo, induced by the weight-drop method in mice. In vitro, live/dead cells, lactate dehydrogenase (LDH) release, caspase 3/7 staining, and tight junction (TJ) protein expression were evaluated 24 h after a single stretch episode. In vivo, Evans blue extravasation, serum levels of S100ß, and TJ protein expression were evaluated. Stretch induced a deformation-dependent increase in LDH release, cell death, and activation of caspase 3/7, suggesting the induction of apoptosis. Interestingly, low magnitudes of deformation increased the expression of TJ proteins, likely in an attempt to compensate for stretch damage. High magnitudes of deformation decreased the expression of TJ proteins, suggesting that the damage was too severe to counteract. In vivo, mild TBI did not affect BBB permeability or the expression of TJ proteins. However, moderate TBI significantly increased BBB permeability and decreased the expression of these proteins, similar to the results obtained with a high magnitude deformation. These data support the use biaxial stretch as valuable tool in the study of TBI in vitro.

Isolation and Culture of Brain Microvascular Endothelial Cells for In Vitro Blood-Brain Barrier Studies.

The blood-brain barrier (BBB) is essential to maintain the proper microenvironment for brain function. Although formed by different cell types, the endothelial cells (ECs) of the brain microvessels provide the BBB with its selective permeability. To study the BBB in vitro, EC lines as well as primary isolated ECs have been used. In this chapter, we will provide a detailed protocol on how to isolate and culture primary brain microvascular endothelial cells from different species for use as in vitro models of the BBB. When performed properly, this protocol will allow one to obtain a pure culture of brain microvascular endothelial cells with which to analyze the effects of therapeutic and toxic agents on BBB functions.

Changes in the metabolome and microRNA levels in biological fluids might represent biomarkers of neurotoxicity: A trimethyltin study.

Neurotoxicity has been linked with exposure to a number of common drugs and chemicals, yet efficient, accurate, and minimally invasive methods to detect it are lacking. Fluid-based biomarkers such as those found in serum, plasma, urine, and cerebrospinal fluid have great potential due to the relative ease of sampling but at present, data on their expression and translation are lacking or inconsistent. In this pilot study using a trimethyl tin rat model of central nervous system toxicity, we have applied state-of-the-art assessment techniques to identify potential individual biomarkers and patterns of biomarkers in serum, plasma, urine or cerebral spinal fluid that may be indicative of nerve cell damage and degeneration. Overall changes in metabolites and microRNAs were observed in biological fluids that were associated with neurotoxic damage induced by trimethyl tin. Behavioral changes and magnetic resonance imaging T2 relaxation and ventricle volume changes served to identify animals that responded to the adverse effects of trimethyl tin. Impact statement These data will help design follow-on studies with other known neurotoxicants to be used to assess the broad applicability of the present findings. Together this approach represents an effort to begin to develop and qualify a set of translational biochemical markers of neurotoxicity that will be readily accessible in humans. Such biomarkers could prove invaluable for drug development research ranging from preclinical studies to clinical trials and may prove to assist with monitoring of the severity and life cycle of brain lesions.

Monoaminergic toxicity induced by cathinone phthalimide: An in vitro study.

Bath salts, or synthetic cathinones, have cocaine-like or amphetamine-like properties and induce psychoactive effects via their capacity to modulate serotonin (5-HT) and dopamine (DA). Structurally distinct synthetic cathinones are continuously being generated to skirt existing drug laws. One example of these modified compounds is cathinone phthalimide (CP), which has already appeared on the global market. The lack of toxicological studies on the effects of CP on monoaminergic systems led to the development of the present study in order to generate an acute toxicity profile for CP, and to clarify whether it primarily affects both dopamine and serotonin, like the synthetic cathinones mephedrone and methylone, or primarily affects dopamine, like 3, 4-methylenedioxypyrovalerone (MDPV). For the first time, the toxicity profile of CP (10µM-1000µM) is reported. In pheochromocytoma cells, exposure to CP induced cell death, and altered mitochondrial function, as well as intracellular DA and 5-HT levels; at the same time, reduced glutathione (GSH) levels remained unaffected. This seems to indicate that CP functions like mephedrone or methylone. The role of CP metabolites, the effect of CP induced hyperthermia on neurotoxicity, and its ability to traverse the blood-brain barrier warrant further consideration.

Protein Kinases and Parkinson's Disease.

Currently, the lack of new drug candidates for the treatment of major neurological disorders such as Parkinson's disease has intensified the search for drugs that can be repurposed or repositioned for such treatment. Typically, the search focuses on drugs that have been approved and are used clinically for other indications. Kinase inhibitors represent a family of popular molecules for the treatment and prevention of various cancers, and have emerged as strong candidates for such repurposing because numerous serine/threonine and tyrosine kinases have been implicated in the pathobiology of Parkinson's disease. This review focuses on various kinase-dependent pathways associated with the expression of Parkinson's disease pathology, and evaluates how inhibitors of these pathways might play a major role as effective therapeutic molecules.

Methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxypyrovalerone (MDPV) induce differential cytotoxic effects in bovine brain microvessel endothelial cells.

Designer drugs such as synthetic psychostimulants are indicative of a worldwide problem of drug abuse and addiction. In addition to methamphetamine (METH), these drugs include 3,4-methylenedioxy-methamphetamine (MDMA) and commercial preparations of synthetic cathinones including 3,4-methylenedioxypyrovalerone (MDPV), typically referred to as "bath salts." These psychostimulants exert neurotoxic effects by altering monoamine systems in the brain. Additionally, METH and MDMA adversely affect the integrity of the blood-brain barrier (BBB): there are no current reports on the effects of MDPV on the BBB. The aim of this study was to compare the effects of METH, MDMA and MDPV on bovine brain microvessel endothelial cells (bBMVECs), an accepted in vitro model of the BBB. Confluent bBMVEC monolayers were treated with METH, MDMA and MDPV (0.5mM-2.5mM) for 24h. METH and MDMA increased lactate dehydrogenase release only at the highest concentration (2.5mM), whereas MDPV induced cytotoxicity at all concentrations. MDMA and METH decreased cellular proliferation only at 2.5mM, with similar effects observed after MDPV exposures starting at 1mM. Only MDPV increased reactive oxygen species production at all concentrations tested whereas all 3 drugs increased nitric oxide production. Morphological analysis revealed different patterns of compound-induced cell damage. METH induced vacuole formation at 1mM and disruption of the monolayer at 2.5mM. MDMA induced disruption of the endothelial monolayer from 1mM without vacuolization. On the other hand, MDPV induced monolayer disruption at doses ≥0.5mM without vacuole formation; at 2.5mM, the few remaining cells lacked endothelial morphology. These data suggest that even though these synthetic psychostimulants alter monoaminergic systems, they each induce BBB toxicity by different mechanisms with MDPV being the most toxic.

Prolactin and blood-brain barrier permeability.

The blood-brain barrier (BBB) consists in part of a highly specialized set of cells which separates the brain from the vascular system. The BBB controls the entry and exit of substances from the brain tissue through tight junctions (TJs) between endothelial cells. It is known that the hormone prolactin (PRL) is able to regulate endothelial-dependent processes, like the balance between proliferation and apoptosis and the mammary epithelial permeability. However, the effects of PRL and the role it plays in the BBB permeability are still not well understood. A primary culture of bovine brain microvessel endothelial cells was used as in vitro model of BBB. Cells were treated with PRL (0.1, 1, 10 and 100 nM) for 24 hours. PRL significantly increased cellular proliferation at 10 and 100 nM, but did not modify basal apoptosis. These effects were dependent on the production of the mitogenic factor nitric oxide (NO). PRL significantly decreased the permeability and promoted an increase in trans-endothelial electrical resistance in a NO-independent way. PRL also increased the expression of the TJs proteins claudin-5 and occludin. The short form of the PRL receptor was detected in these cells but its expression was not modified by PRL. Together, these results suggest that PRL has the ability to increase cellular proliferation associated with a decrease on BBB permeability by increasing the expression of TJs proteins.

Prolactin protects against the methamphetamine-induced cerebral vascular toxicity.

Methamphetamine (Meth) is a highly addictive drug of abuse which alters the dopaminergic system and damages the blood-brain barrier (BBB), structure that protects the brain tissue from the circulating substances in the blood, keeping a low permeability through the presence of tight junctions (TJs) between endothelial cells. Meth increases BBB permeability by decreasing the TJs proteins claudin-5 and occludin and by decreasing the viability of endothelial cells. Individuals abused of Meth have increased blood concentrations of prolactin (PRL); hormone related with milk production, but able to increase the expression of TJs proteins and to decrease permeability on the mammary epithelium and brain endothelial cells. However, the effects of PRL on the permeability of the BBB in the presence of Meth have not been studied. Here, we report Meth-induced apoptosis and decreased cellular proliferation as well as the trans-endothelial electrical resistance (TEER), related to a decrease of claudin-5 and occludin in primary cultured bovine brain microvessel endothelial cells. The expression of the PRL receptor was not altered. Administration of PRL prevented a decrease in cellular proliferation, an increase in apoptosis and restored the TEER and TJs proteins to basal levels. This protection was absent at high Meth concentrations. These data suggest that PRL protects brain endothelial cells against the Meth-induced toxicity. Further investigation is required to study the mechanisms involved and to confirm these effects in vivo.

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.

Mechanistic toxicity evaluation of uncoated and PEGylated single-walled carbon nanotubes in neuronal PC12 cells.

We investigated and compared the concentration-dependent cytotoxicity of single-walled carbon nanotubes (SWCNTs) and SWCNTs functionalized with polyethylene glycol (SWCNT-PEGs) in neuronal PC12 cells at the biochemical, cellular, and gene expressional levels. SWCNTs elicited cytotoxicity in a concentration-dependent manner, and SWCNT-PEGs exhibited less cytotoxic potency than uncoated SWCNTs. Reactive oxygen species (ROS) were generated in both a concentration- and surface coating-dependent manner after exposure to these nanomaterials, indicating different oxidative stress mechanisms. More specifically, gene expression analysis showed that the genes involved in oxidoreductases and antioxidant activity, nucleic acid or lipid metabolism, and mitochondria dysfunction were highly represented. Interestingly, alteration of the genes is also surface coating-dependent with a good correlation with the biochemical data. These findings suggest that surface functionalization of SWCNTs decreases ROS-mediated toxicological response in vitro.

On the in vivo early toxic properties of A-beta 25-35 peptide in the rat hippocampus: involvement of the Receptor-for-Advanced Glycation-End-Products and changes in gene expression.

Amyloid-beta peptide (Aß) deposition is assumed to play a pathogenic role in the brain of Alzheimer's disease patients. To date, the precise mechanisms underlying Aß toxicity are not fully understood. A recent hypothesis suggesting that the Receptor-for-Advanced-Glycation-End-Products (RAGE)-a trans-membrane protein signaling for oxidative stress-is involved in Aß toxicity is gaining attention. Early Aß toxicity could indeed help to explain the deleterious events further produced by this molecule in the brain. In this work, we evaluated the pattern of early expression of RAGE in the toxic model induced by Aß25₋35 in rat CA1 region. Intrahippocampal injections of Aß25₋35 in rats increased the RAGE expression at 24 h post-injection; this event was accompanied by increased components of RAGE downstream signaling in hippocampal cells, such as enhanced expression of the pro-apoptotic factor NF-κB, increased nitric oxide production, LDH leakage, mitochondrial dysfunction, increased TNF-α expression, antioxidant genes down-regulation, and augmented neurodegeneration. Our findings support an active role of RAGE during the early stages of Aß25₋35 toxicity in the hippocampus.

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.