Monique Culturato Padilha Mendonça

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

BackgroundThe 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.ResultsIn 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.ConclusionsrGO 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.

Expression of VEGF and Flk-1 and Flt-1 Receptors during Blood-Brain Barrier (BBB) Impairment Following Phoneutria nigriventer Spider Venom Exposure

Apart from its angiogenic and vascular permeation activity, the vascular endothelial growth factor (VEGF) has been also reported as a potent neuronal protector. Newborn rats with low VEGF levels develop neuron degeneration, while high levels induce protective mechanisms in several neuropathological conditions. Phoneutria nigriventer spider venom (PNV) disrupts the blood-brain barrier (BBB) and causes neuroinflammation in central neurons along with excitotoxic signals in rats and humans. All these changes are transient. Herein, we examined the expression of VEGF and its receptors, Flt-1 and Flk-1 in the hippocampal neurons following envenomation by PNV. Adult and neonatal rats were evaluated at time limits of 2, 5 and 24 h. Additionally, BBB integrity was assessed by measuring the expression of occludin, β-catenin and laminin and neuron viability was evaluated by NeuN expression. VEGF, Flt-1 and Flk-1 levels increased in PNV-administered rats, concurrently with respective mRNAs. Flt-1 and Flk-1 immunolabeling was nuclear in neurons of hippocampal regions, instead of the VEGF membrane-bound typical location. These changes occurred simultaneously with the transient decreases in BBB-associated proteins and NeuN positivity. Adult rats showed more prominent expressional increases of the VEGF/Flt-1/Flk-1 system and earlier recovery of BBB-related proteins than neonates. We conclude that the reactive expressional changes seen here suggest that VEGF and receptors could have a role in the excitotoxic mechanism of PNV and that such role would be less efficient in neonate rats.

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.

Environmental enrichment attenuates the blood brain barrier dysfunction induced by the neonatal hypoxia-ischemia.

Environmental enrichment (EE) is considered an efficient neuroprotector against neonatal hypoxia‐ischemia (HI). Nevertheless, the mechanisms involved are not yet clear. In this context, the aim of this study was to investigate the effects of neonatal HI and environmental stimulation in the hippocampus of rats at 3 different time points (PND 8, 22 and 60), evaluating some aspects of BBB structure and function. Seven‐day‐old Wistar rats were divided into four groups: a control group maintained in a standard environment (CTSE), a control group maintained in an enrichment environment (CTEE), an HI group maintained in a standard environment (HISE) and an HI group maintained in an enrichment environment (HIEE). At the 7th postnatal day (PND), rats were submitted to the Levine‐Rice model of neonatal HI. This method consists of permanent occlusion of the right common carotid artery with subsequent exposure to hypoxia. Rats from CTEE and HIEE were stimulated with environmental enrichment. The EE protocol started 24 h after HI, in which pup rats with their dams were stimulated in a maintained EE (PND 8–22). Subsequently, animals were submitted to daily EE (1 h/day, PND 23–60). The expression of some proteins involved in BBB structure (β‐catenin, occludin, connexin‐43, aquaporin‐4, glut‐1 and GFAP) were quantified by western blotting in the hippocampi of rats in three periods, at PND 8, 22 and 60. The BBB permeability and integrity was assessed by Evans blue staining and the immunohistochemistry for GFAP in the CA1 region of the hippocampus were also performed. The results showed an HI‐induced decreased occludin expression at PND 22 and low levels of occludin, β‐catenin and GFAP at PND 60 in the hippocampus of the hypoxic‐ischemic rats. Interestingly, in young and adult rats, EE reversed these effects. Evans blue extravasation into the brain parenchyma confirmed the BBB dysfunction brought on by HI. No differences were observed at PND 8, probably due to the immaturity of the BBB at this age. The present study makes an important contribution to understanding the mechanism of the hypoxic‐ischemic brain damage and also to presents, for the first time, the recovery of BBB dysfunction as a possible pathway for the protective effect of EE.

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.

Vascular Endothelial Growth Factor Increases during Blood-Brain Barrier-Enhanced Permeability Caused by Phoneutria nigriventer Spider Venom

Phoneutria nigriventer spider accidental envenomation provokes neurotoxic manifestations, which when critical, results in epileptic-like episodes. In rats, P. nigriventer venom (PNV) causes blood-brain barrier breakdown (BBBb). The PNV-induced excitotoxicity results from disturbances on Na+, K+ and Ca2+ channels and glutamate handling. The vascular endothelial growth factor (VEGF), beyond its angiogenic effect, also, interferes on synaptic physiology by affecting the same ion channels and protects neurons from excitotoxicity. However, it is unknown whether VEGF expression is altered following PNV envenomation. We found that adult and neonates rats injected with PNV showed immediate neurotoxic manifestations which paralleled with endothelial occludin, β-catenin, and laminin downregulation indicative of BBBb. In neonate rats, VEGF, VEGF mRNA, and Flt-1 receptors, glutamate decarboxylase, and calbindin-D28k increased in Purkinje neurons, while, in adult rats, the BBBb paralleled with VEGF mRNA, Flk-1, and calbindin-D28k increases and Flt-1 decreases. Statistically, the variable age had a role in such differences, which might be due to age-related unequal maturation of blood-brain barrier (BBB) and thus differential cross-signaling among components of the glial neurovascular unit. The concurrent increases in the VEGF/Flt-1/Flk-1 system in the cerebellar neuron cells and the BBBb following PNV exposure might imply a cytokine modulation of neuronal excitability consequent to homeostatic perturbations induced by ion channels-acting PNV neuropeptides. Whether such modulation represents neuroprotection needs further investigation.

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.

Jaboticaba berry peel intake prevents insulin resistance-induced tau phosphorylation in mice

The hyperphosphorylation of microtubule-associated protein tau (tau) in the hippocampus can be caused by central and peripheral insulin resistance and these alterations are related to the development of tauopathies, such as Alzheimers disease. In this study, we used a high-fat diet to induce obesity and insulin resistance in adult Swiss mice and checked whether supplementation with Myrciaria jaboticaba berry peel for 10 weeks could improve insulin sensitivity, learning/memory performance, and prevent tau phosphorylation in the hippocampus. Furthermore, adipocytokines, inflammatory markers, and oxidative stress were assessed. Myrciaria jaboticaba peel has phenolic compounds (e.g., cyanidin, ellagic acid), dietary fiber and carotenoids, which contribute to great antioxidant capacity. Supplementation of the high-fat diet with 4% M. jaboticaba peel prevented fat weight gain and reduced peripheral insulin resistance. The treated group also showed lower tau phosphorylation in the hippocampus corroborating better learning/memory performance in the Morris water maze test. Maintenance of neuronal viability, lower levels of hippocampal inflammatory markers, and improved brain antioxidant defenses were also related to the consumption of M. jaboticaba peel. These findings contribute to a better understanding of how a high-fat diet supplemented with jaboticaba berry peel counteracts the impairment of cognitive functions caused by high-fat diet intake and diet-induced insulin resistance.

Age-Related Modulations of AQP4 and Caveolin-1 in the Hippocampus Predispose the Toxic Effect of Phoneutria nigriventer Spider Venom

We have previously demonstrated that Phoneutria nigriventer venom (PNV) causes blood–brain barrier (BBB) breakdown, swelling of astrocytes end-feet and fluid permeation into brain interstitium in rats. Caveolae and water channels respond to BBB alterations by co-participation in shear stress response and edema formation/resolution. Herein, we showed post-natal developmental-related changes of two BBB-associated transporter proteins: the endothelial caveolin-1 (Cav-1), the major scaffolding protein from caveolae frame, and the astroglial aquaporin-4 (AQP4), the main water channel protein expressed in astrocytic peri-vascular end-feet processes, in the hippocampus of rats intraperitoneally-administered PNV. Western blotting protein levels; immunohistochemistry (IHC) protein distribution in CA1, CA2, and CA3 subfields; and gene expression by Real Time-Polymerase Chain Reaction (qPCR) were assessed in post-natal Day 14 (P14) and 8–10-week-old rats over critical periods of envenomation. The intensity and duration of the toxic manifestations indicate P14 neonate rats more vulnerable to PNV than adults. Histologically, the capillaries of P14 and 8–10-week-old rats treated with PNV showed perivascular edema, while controls did not. The intensity of the toxic manifestations in P14 decreases temporally (2 > 5 > 24 h), while inversely the expression of AQP4 and Cav-1 peaked at 24 h when clinically PNV-treated animals do not differ from saline controls. IHC of AQP4 revealed that hippocampal CA1 showed the least expression at 2 h when toxic manifestation was maximal. Subfield IHC quantification revealed that in P14 rats Cav-1 peaked at 24 h when toxic manifestations were absent, whereas in 8–10-week-old rats Cav-1 peaked at 2 h when toxic signs were highest, and progressively attenuated such increases until 24 h, remaining though significantly above baseline. Considering astrocyte-endothelial physical and functional interactions, we hypothesize that age-related modulations of AQP4 and Cav-1 might be linked both to changes in functional properties of astrocytes during post-natal development and in the BBB breakdown induced by the venom of P. nigriventer.

The protective effects of fermented kefir milk on azoxymethane-induced aberrant crypt formation in mice colon

Kefir is a probiotic fermented milk product produced from grains with a complex composition of bacteria and yeasts that live in a symbiotic association. Anti-proliferative, anti-inflammatory, and anti-mutagenic effects are some of the health beneficial properties of kefir grains. The present study was conducted to evaluate whether regular consumption of kefir milk would be capable of preventing the development of pre-neoplastic lesions induced by azoxymethane (AOM). Aberrant crypt foci were induced in BALB-c mice via 2 subcutaneous injections of azoxymethane (15 mg/kg) and kefir was administered by daily gavage for 8 weeks (5 ml/kg). Additionally, bacterial growth was monitored in pasteurized and ultra-high temperature (UHT) treated milk to compare different fermentation conditions. Our results showed that UHT milk presented better growth of Lactobacillus acidophilus colonies. The aberrant crypt foci were attenuated by approximately 43% (height) and 20% (width) in the kefir group compared to AOM group, suggesting that kefir treatment may contribute to prevent and control the growth of intestinal neoplastic cells.