PEGylation of Reduced Graphene Oxide Induces Toxicity in Cells of the Blood-Brain Barrier: An in Vitro and in Vivo Study.

Polyethylene glycol (PEG) coating has been frequently used to improve the pharmacokinetic behavior of nanoparticles. Studies that contribute to better unravel the effects of PEGylation on the toxicity of nanoparticle formulation are therefore highly relevant. In the present study, reduced graphene oxide (rGO) was functionalized with PEG, and its effects on key components of the blood-brain barrier, such as astrocytes and endothelial cells, were analyzed in culture and in an in vivo rat model. The in vitro studies demonstrated concentration-dependent toxicity. The highest concentration (100 µg/mL) of non-PEGylated rGO had a lower toxic influence on cell viability in primary cultures of astrocytes and rat brain endothelial cells, while PEGylated rGO induced deleterious effects and cell death. We assessed hippocampal BBB integrity in vivo by evaluating astrocyte activation and the expression of the endothelial tight and adherens junctions proteins. From 1 h to 7 days post-rGO-PEG systemic injection, a notable and progressive down-regulation of protein markers of astrocytes (GFAP, connexin-43), the endothelial tight (occludin), and adherens (ß-catenin) junctions and basal lamina (laminin) were observed. The formation of intracellular reactive oxygen species demonstrated by increases in the enzymatic antioxidant system in the PEGylated rGO samples was indicative of oxidative stress-mediated damage. Under the experimental conditions and design of the present study the PEGylation of rGO did not improve interaction with components of the blood-brain barrier. In contrast, the attachment of PEG to rGO induced deleterious effects in comparison with the effects caused by non-PEGylated rGO.

Reduced graphene oxide: nanotoxicological profile in rats.

BACKGROUND: We have previously demonstrated that reduced graphene oxide (rGO) administered intravenously in rats was detected inside the hippocampus after downregulation of the tight and adherens junction proteins of the blood-brain barrier. While down-regulators of junctional proteins could be useful tools for drug delivery through the paracellular pathway, concerns over toxicity must be investigated before clinical application. Herein, our purpose was to trace whether the rGO inside the hippocampus triggered toxic alterations in this brain region and in target organs (blood, liver and kidney) of rats at various time points (15 min, 1, 3 h and 7 days). RESULTS: The assessed rGO-treated rats (7 mg/kg) were clinically indistinguishable from controls at all the time points. Hematological, histopathological (neurons and astrocytes markers), biochemical (nephrotoxicity and hepatotoxicity assessment) and genotoxicological based tests showed that systemic rGO single injection seemed to produce minimal toxicological effects at the time points assessed. Relative to control, the only change was a decrease in the blood urea nitrogen level 3 h post-treatment and increases in superoxide dismutase activity 1 h and 7 days post-treatment. While no alteration in leukocyte parameters was detected between control and rGO-treated animals, time-dependent leukocytosis (rGO-1 h versus rGO-3 h) and leukopenia (rGO-3 h versus rGO-7 days) was observed intra-treated groups. Nevertheless, no inflammatory response was induced in serum and hippocampus at any time. CONCLUSIONS: The toxic effects seemed to be peripheral and transitory in the short-term analysis after systemic administration of rGO. The effects were self-limited and non-significant even at 7 days post-rGO administration.

Reduced graphene oxide induces transient blood-brain barrier opening: an in vivo study.

BACKGROUND: The blood-brain barrier (BBB) is a complex physical and functional barrier protecting the central nervous system from physical and chemical insults. Nevertheless, it also constitutes a barrier against therapeutics for treating neurological disorders. In this context, nanomaterial-based therapy provides a potential alternative for overcoming this problem. Graphene family has attracted significant interest in nanomedicine because their unique physicochemical properties make them amenable to applications in drug/gene delivery and neural interface. RESULTS: In this study, reduced graphene oxide (rGO) systemically-injected was found mainly located in the thalamus and hippocampus of rats. The entry of rGO involved a transitory decrease in the BBB paracellular tightness, as demonstrated at anatomical (Evans blue dye infusion), subcellular (transmission electron microscopy) and molecular (junctional protein expression) levels. Additionally, we examined the usefulness of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) as a new imaging method for detecting the temporal distribution of nanomaterials throughout the brain. CONCLUSIONS: rGO was able to be detected and monitored in the brain over time provided by a novel application for MALDI-MSI and could be a useful tool for treating a variety of brain disorders that are normally unresponsive to conventional treatment because of BBB impermeability.

VEGF/VEGFR-2 system exerts neuroprotection against Phoneutria nigriventer spider envenomation through PI3K-AKT-dependent pathway.

This study was undertaken to elucidate why VEGF/VEGFR-2 is elevated in the hippocampus of rats injected with Phoneutria nigriventer spider venom (PNV). PNV delays Na+ channels inactivation; blocks Ca2+ and K+ channels, increases glutamate release, causes blood-brain breakdown (BBBb), brain edema and severe excitotoxicity. Analytical FT-IR spectroscopy showed profound alteration in molecular biochemical state, with evidences for VEGFR-2 (KDR/Flk-1) signaling mediation. By blocking VEGF/VEGFR-2 binding via pre-treatment with itraconazole we demonstrated that animals' condition was deteriorated soon at 1-2 h post-PNV exposure concurrently with decreased expression of VEGF, BBB-associated proteins, ZO-1, ß-catenin, laminin, P-gp (P-glycoprotein), Neu-N (neuron's viability marker) and MAPKphosphorylated-p38, while phosphorylated-ERK and Src pathways were increased. At 5 h and coinciding with incipient signs of animals' recuperation, the proteins associated with protection (HIF-1α, VEGF, VEGFR-1, VEGFR-2, Neu-N, occludin, ß-catenin, laminin, P-gp efflux protein, phosphorylated-p38) increased thus indicating p38 pathway activation together with paracellular route strengthening. However, the BBB transcellular trafficking and caspase-3 increased (pro-apoptotic pathway activation). At 24 h, the transcellular route reestablished physiological state but the pro-survival pathway PI3K/(p-Akt) dropped in animals underwent VEGF/VEGFR-2 binding inhibition, whereas it was significantly activated at matched interval in PNV group without prior itraconazole; these results demonstrate impaired VEGF' survival effects at 24 h. The inhibition of VEGF/VEGFR-2 binding identified 5 h as turning point at which multi-level dynamic interplay was elicited to reverse hippocampal damage. Collectively, the data confirmed VEGFR-2 signaling via serine-threonine kinase Akt as neuroprotective pathway against PNV-induced damage. Further studies are needed to elucidate mechanisms underlying PNV effects.

Aqueous Extract of Brazilian Berry (Myrciaria jaboticaba) Peel Improves Inflammatory Parameters and Modulates Lactobacillus and Bifidobacterium in Rats with Induced-Colitis.

Natural compounds could be a complementary alternative to inflammatory bowel disease (IBD) management. This study determined the effects of an aqueous extract of Myrciaria jaboticaba peel (EJP) (50 g L-1) on 2,4,6-trinitrobenzenesulfonic acid-induced colitis. Wistar rats were randomized into five groups: HC-healthy control, CC-colitis control, DC-drug control, SJ-short-term treatment with EJP, and LJ-long-term treatment with EJP. The EJP treatments reduced body weight loss, stool consistency score, and spleen enlargement. Gut microbiota was modulated through increased Lactobacillus and Bifidobacterium counts after EJP treatment. Short-chain fatty acids were also higher in the EJP treatment groups. The antioxidant enzyme activities were greater than CC or DC controls. Myeloperoxidase activity (LJ), inducible nitric oxide synthase (LJ/SJ), and intercellular adhesion molecule (SJ) levels were lower than in the CC group. EJP decreased histological scoring, mucosal thickness, and preserved the crypts and histological structure. Therefore, EJP showed beneficial effects and could be potentially used as an adjuvant in IBD treatment.

Inhibition of VEGF-Flk-1 binding induced profound biochemical alteration in the hippocampus of a rat model of BBB breakdown by spider venom. A preliminary assessment using FT-IR spectroscopy.

Phoneutria nigriventer spider venom (PNV) contains ion channels-acting neuropeptides that in rat induces transitory blood-brain barrier breakdown (BBBb) in hippocampus in parallel with VEGF upregulation. We investigated whether VEGF has a neuroprotective role by inhibiting its binding to receptor Flk-1 by itraconazole (ITZ). FT-IR spectroscopy examined the biochemical status of hippocampus and evaluated BBBb in rats administered PNV or ITZ/PNV at periods with greatest toxicity (1-2h), recovery (5h) and visual absence of symptoms (24h), and compared to saline and ITZ controls. The antifungal treatment before venom intoxication aggravated the venom effects and increased BBB damage. FT-IR spectra of venom, hippocampi of controls, PNV and ITZ-PNV showed a 1400 cm-1 band linked to symmetric stretch of carboxylate and 1467 cm-1 band (CH2 bending: mainly lipids) that were considered biomarker and reference bands, respectively. Inhibition of VEGF/Flk-1 binding produced marked changes in lipid/protein stability at 1-2h. The largest differences were observed in spectra regions assigned to lipids, both symmetric (2852 cm-1) and asymmetric (2924 and 2968 cm-1). Quantitative analyses showed greatest increases in the 1400 cm-1/1467 cm-1 ratio also at 1h. Such changes at period of rats' severe intoxication referred to wavenumber region from 3106 cm-1 to 687 cm-1 assigning for C-H and N-H stretching of protein, Amide I, C=N cytosine, N-H adenine, Amide II, CH2 bending: mainly lipids, C-O stretch: glycogen, polysaccharides, glycolipids, z-type DNA, C-C, C-O and CH out-of-plane bending vibrations. We conclude that VEGF has a neuroprotective role and can be a therapeutic target in PNV envenomation. FT-IR spectroscopy showed to be instrumental for monitoring biochemical changes in this model of P. nigriventer venom-induced BBB disruption.

Caveolae as a target for Phoneutria nigriventer spider venom.

An important transcellular transport mechanism in the blood-brain barrier (BBB) involves caveolae, which are specialized delta-shaped domains of the endothelial plasma membrane that are rich in cholesterol, glycosphingolipids and the scaffolding protein Caveolina-1 (Cav-1). In this work, we investigated whether the increase in endocytosis and transendothelial vesicular trafficking in rat cerebellum after blood-brain barrier breakdown (BBBb) induced by Phoneutria nigriventer spider venom (PNV) was mediated by caveolae. The expression of Cav-1, phosphorylated Cav-1 (pCav-1), dynamin-2 (Dyn2), Src kinase family (SKF) and matrix-metalloproteinase-9 (MMP9), proteins involved in caveolar dynamics and BBB opening, was investigated. Immunofluorescence, western blotting (WB) and transmission electron microscopy were used to assess changes at 1, 2, 5, 24 and 72h post-venom. WB showed upregulation of Cav-1, Dyn2 and MMP9 at 1, 5 and 72h (corresponding, respectively, to intervals when intoxication was most evident, when signs of recovery were present, and when no intoxication was detectable). In contrast, pCav-1 and SKF, which are essential for internalization and transport, decreased when Cav-1 and Dyn2, proteins essential for caveolar formation, were increased. Overall, these changes indicated that vesicular trafficking across the endothelium (high pCav/SKF levels) coincided with lower numbers of caveolae (Cav-1/Dyn2 downregulation) and lower expression of MMP9. Thus, the internalization (disassembly) of caveolae alternates with caveolar neoformation (assembly), resulting in changes in caveolar density in the endothelium membrane. These caveolar dynamics imply tensional mechanical stress that is important in triggering key signaling mechanisms. We conclude that PNV-induced breakdown of transcellular transport in the BBB is caused by an increase in caveolae-mediated endocytosis; this effect was correlated with the progression of temporal signs of envenoming. Caveolar dynamics are probably involved in shear stress and BBBb regulatory mechanisms in this experimental model.

Are Synchronized Changes in Connexin-43 and Caveolin-3 a Bystander Effect in a Phoneutria nigriventer Venom Model of Blood-Brain Barrier Breakdown?

Upregulation of caveolin-3 (Cav-3) or connexin-43 (Cx43) in astrocytes has been associated with important brain pathologies. We used Phoneutria nigriventer spider venom (PNV), which induces blood-brain barrier breakdown in rats, in order to investigate Cav-3 and Cx43 expression in the cerebellum over critical periods of rat envenomation. By immunofluorescence, western blotting (WB), and transmission electron microscopy (TEM), we assessed changes at 1, 2, 5, 24, and 72 h post-venom. WB showed immediate increases in Cav-3 and Cx43 at 1 h (interval of greatest manifestations of envenomation) that persisted at 5 h (when there were signs of recovery) and peaked at 24 h when no signs of envenomation were detectable. At 2 and 72 h, Cav-3 was downregulated and Cx43 had returned to baseline. PNV markedly intensified Cx43 in molecular, Purkinje and granular layers and Cav-3 in astrocytes whose colocalization to increased GFAP suggests interaction between reactive astrogliosis and Cav-3 upregulation. TEM showed swollen perivascular astrocytic end-feet and synaptic contact alterations that had generally resolved by 72 h. It is uncertain whether such PNV-induced synchronized changes are an interactive effect between Cav-3 and Cx43, or a bystander effect. Evidences indicate that Cav-3 downregulation coupled to Cx43 return to baseline at 72 h when no signs of envenomation were visible, suggesting homeostasis reestablishment. This experimental model is relevant to studying mechanisms involved in neurological disorders associated with Cav-3 overexpression.

eNOS uncoupling in the cerebellum after BBB disruption by exposure to Phoneutria nigriventer spider venom.

Numerous studies have shown that the venom of Phoneutria nigriventer (PNV) armed-spider causes excitotoxic signals and blood-brain barrier breakdown (BBBb) in rats. Nitric oxide (NO) is a signaling molecule which has a role in endothelium homeostasis and vascular health. The present study investigated the relevance of endothelial NO synthase (eNOS) uncoupling to clinical neurotoxic evolution induced by PNV. eNOS immunoblotting of cerebellum lysates processed through low-temperature SDS-PAGE revealed significant increased monomerization of the enzyme at critical periods of severe envenoming (1-2 h), whereas eNOS dimerization reversal paralleled to amelioration of animals condition (5-72 h). Moreover, eNOS uncoupling was accompanied by increased expression in calcium-sensing calmodulin protein and calcium-binding calbindin-D28 protein in cerebellar neurons. It is known that greater eNOS monomers than dimers implies the inability of eNOS to produce NO leading to superoxide production and endothelial/vascular barrier dysfunction. We suggest that transient eNOS deactivation and disturbances in calcium handling reduce NO production and enhance production of free radicals thus contributing to endothelial dysfunction in the cerebellum of envenomed rats. In addition, eNOS uncoupling compromises the enzyme capacity to respond to shear stress contributing to perivascular edema and it is one of the mechanisms involved in the BBBb promoted by PNV.

Evidences of endocytosis via caveolae following blood-brain barrier breakdown by Phoneutria nigriventer spider venom.

Spider venoms contain neurotoxic peptides aimed at paralyzing prey or for defense against predators; that is why they represent valuable tools for studies in neuroscience field. The present study aimed at identifying the process of internalization that occurs during the increased trafficking of vesicles caused by Phoneutria nigriventer spider venom (PNV)-induced blood-brain barrier (BBB) breakdown. Herein, we found that caveolin-1α is up-regulated in the cerebellar capillaries and Purkinje neurons of PNV-administered P14 (neonate) and 8- to 10-week-old (adult) rats. The white matter and granular layers were regions where caveolin-1α showed major upregulation. The variable age played a role in this effect. Caveolin-1 is the central protein that controls caveolae formation. Caveolar-specialized cholesterol- and sphingolipid-rich membrane sub-domains are involved in endocytosis, transcytosis, mechano-sensing, synapse formation and stabilization, signal transduction, intercellular communication, apoptosis, and various signaling events, including those related to calcium handling. PNV is extremely rich in neurotoxic peptides that affect glutamate handling and interferes with ion channels physiology. We suggest that the PNV-induced BBB opening is associated with a high expression of caveolae frame-forming caveolin-1α, and therefore in the process of internalization and enhanced transcytosis. Caveolin-1α up-regulation in Purkinje neurons could be related to a way of neurons to preserve, restore, and enhance function following PNV-induced excitotoxicity. The findings disclose interesting perspectives for further molecular studies of the interaction between PNV and caveolar specialized membrane domains. It proves PNV to be excellent tool for studies of transcytosis, the most common form of BBB-enhanced permeability.

Temporal relationship between aquaporin-4 and glial fibrillary acidic protein in cerebellum of neonate and adult rats administered a BBB disrupting spider venom.

Two astrocyte markers, the glial water channel aquaporin-4 (AQP4) and the glial fibrillary acidic protein (GFAP), have been implicated in several physiological and pathological conditions in the central nervous system (CNS) as well as in blood-brain barrier breakdown (BBBb). By color segmentation the immunoreactivity of both proteins, we demonstrate that the expression of AQP4 and GFAP was increased in the cerebellum of neonate (14-day-old, P14) and adult (8-week-old) rats administered Phoneutria nigriventer spider venom (PNV) known to cause perivascular edema, BBBb and convulsion. In the cerebellum's gray matter, PNV produced a major response, especially in the granular layer. Parallel increases in AQP4 and GFAP expression occurred 24 h after envenomation in the white matter of P14 and in the molecular layer of adults, as well as in the granular layer 2 h after envenomation. In the Purkinje layer there was a tendency of increased AQP4, for both, neonates (5 h), and adults (2 and 24 h). Moreover, PNV also provoked nonparallel upregulation of both markers with prevalence of upregulation of AQP 4 for P14 rats, and GFAP for adults. The major expression of both proteins was in the gray matter. The data indicates a venom effect in water/electrolyte balance in the cerebellum and the participation of AQP4 in these effects. Age-related and time-related regional differences probably reflect specificity in AQP4 distribution in different astrocytic membrane domains as well as its participation in K(+) buffering and neural activity. This study is the first to associate astrocytic AQP4 expression and reactive gliosis in a model of BBB permeability promoted by P. nigriventer venom. Our data provide compelling evidence that AQP4 expression was increased in the cerebellum of rats administered PNV.