RESUMO
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.
Assuntos
Grafite/química , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Barreira Hematoencefálica/química , Barreira Hematoencefálica/efeitos dos fármacos , Western Blotting , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/metabolismo , Grafite/toxicidade , Imuno-Histoquímica , Masculino , Nanoestruturas/química , Estresse Oxidativo/fisiologia , RatosRESUMO
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.
Assuntos
Grafite/farmacologia , Hipocampo/efeitos dos fármacos , Rim/efeitos dos fármacos , Fígado/efeitos dos fármacos , Nanopartículas/administração & dosagem , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/ultraestrutura , Nitrogênio da Ureia Sanguínea , Esquema de Medicação , Índices de Eritrócitos , Grafite/química , Grafite/farmacocinética , Hipocampo/ultraestrutura , Injeções Intravenosas , Rim/ultraestrutura , Contagem de Leucócitos , Fígado/ultraestrutura , Masculino , Nanopartículas/química , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Óxidos , Ratos , Ratos Wistar , Superóxido Dismutase/metabolismo , Testes de ToxicidadeRESUMO
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.
Assuntos
Barreira Hematoencefálica/metabolismo , Grafite/farmacologia , Óxidos/farmacologia , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/ultraestrutura , Western Blotting , Capilares/ultraestrutura , Hipocampo/ultraestrutura , Masculino , Microscopia Confocal , Modelos Biológicos , Oxirredução , Ratos Wistar , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrofotometria Ultravioleta , Frações Subcelulares/metabolismo , Distribuição Tecidual/efeitos dos fármacosRESUMO
We previously demonstrated that N-acetylcysteine (NAC) could reduce the toxicity of silver (Ag) materials (nanoparticles (NPs) and Ag nitrate) to the soil invertebrate Enchytraeus crypticus (Oligochaeta). It remains however, unclear whether the antitoxic mechanism of NAC was caused by NAC-Ag binding in the soil or inside the organisms. This study aimed at determining the bioavailability of Ag in the soil in a 21-day toxicity test as well as the Ag uptake and elimination kinetics in E. crypticus exposed to AgNPs in LUFA 2.2 standard soil amended with low (100â¯mg/kg dry soil) and high (600â¯mg/kg dry soil) NAC concentrations. The addition of NAC to the soil alleviated the toxicity of AgNPs by decreasing the internal Ag concentration of E. crypticus in a dose-dependent manner. Indeed, NAC reduced the binding of Ag to the soil, which probably was due to the formation of soluble but biologically unavailable Ag-cysteine complexes. The reduced Ag uptake in the enchytraeids was explained from an increased elimination at high NAC levels. These findings reinforce the view that metal complexing-compounds like NAC play a key role in the modulation of AgNP toxicity and bioavailability in terrestrial environments. Further, it may inform on the potential of NAC as a remediation solution for Ag or other metal-contaminated soils.
Assuntos
Nanopartículas Metálicas , Oligoquetos , Acetilcisteína , Animais , Disponibilidade Biológica , Prata , Solo , Poluentes do Solo , ToxicocinéticaRESUMO
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.
Assuntos
Picada de Aranha , Venenos de Aranha/toxicidade , Animais , Masculino , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Aranhas , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Cationic solid lipid nanoparticles (cSLNs) are considered as one of the most effective lipid nanocarriers for delivery of low water-solubility compounds and genetic materials. As the excipients used in the cSLN production are generally regarded as safe (GRAS), the formulations are granted as non-toxic. However, the toxicological profile of new SLN-based formulations should always be performed to confirm that the delivery systems themselves may not impose risks to the human health. Therefore, in this study, we delineate the toxicological profile of the cSLN formulation at 24 and 72 h after single intravenous injection to male Wistar rats. Hematological, biochemical, and histopathological evaluations of the spleen, lungs, liver, and kidneys indicated short-lived alterations including neutrophilia. We found increases in the population of macrophages in the lungs, liver, and spleen and also migration of circulating neutrophils into inflamed tissue and a decrease in blood urea nitrogen. We also observed the presence of cSLNs within the brain parenchyma without any sign of damage to the blood-brain barrier. These side effects appeared to be mild and transitory (< 72 h). These findings reinforce the importance of investigating the toxicity of SLN-based formulations before the incorporation of drugs/genetic material to the formulation and its translation to the clinic.
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Lipídeos/química , Macrófagos/metabolismo , Nanopartículas/toxicidade , Administração Intravenosa , Animais , Nitrogênio da Ureia Sanguínea , Cátions , Rim/efeitos dos fármacos , Rim/imunologia , Fígado/efeitos dos fármacos , Fígado/imunologia , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Macrófagos/efeitos dos fármacos , Masculino , Nanopartículas/química , Tamanho da Partícula , Ratos , Ratos Wistar , Baço/efeitos dos fármacos , Baço/imunologiaRESUMO
The increasing use of silver nanoparticles (AgNPs) in consumer products raises the risk of human toxicity. Currently, there are no therapeutic options or established treatment protocols in cases of AgNPs intoxication. We demonstrated previously that thiol antioxidants compounds can reverse the cytotoxicity induced by AgNPs in Huh-7 hepatocarcinoma cells. Here, we investigated the use of N-acetylcysteine (NAC) against the systemic toxic effects of AgNPs (79.3 nm) in rats. Biochemical, histopathological, hematological, and oxidative parameters showed that a single intravenous injection of AgNPs (5 mg/kg b.w.) induced deleterious effects such as hepatotoxicity, potentially as a result of AgNPs accumulation in the liver. Treatment with a single intraperitoneal injection of NAC (1 g/kg b.w.) one hour after AgNPs exposure significantly attenuated all toxic effects evaluated and altered the bioaccumulation and release patterns of AgNPs in rats. The findings show that NAC may be a promising candidate for clinical management of AgNPs intoxication.
Assuntos
Acetilcisteína/farmacologia , Antioxidantes/farmacologia , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Fígado/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Prata/toxicidade , Animais , Humanos , Injeções Intraperitoneais , Injeções Intravenosas , Masculino , Nanopartículas Metálicas/química , Ratos , Prata/químicaRESUMO
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 (15â¯mg/kg) and kefir was administered by daily gavage for 8 weeks (5â¯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.
Assuntos
Focos de Criptas Aberrantes/patologia , Neoplasias Colorretais/patologia , Kefir , Focos de Criptas Aberrantes/induzido quimicamente , Animais , Azoximetano/toxicidade , Carcinógenos/toxicidade , Neoplasias Colorretais/induzido quimicamente , Feminino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
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 1400â¯cm-1 band linked to symmetric stretch of carboxylate and 1467â¯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 (2852â¯cm-1) and asymmetric (2924 and 2968â¯cm-1). Quantitative analyses showed greatest increases in the 1400â¯cm-1/1467â¯cm-1 ratio also at 1h. Such changes at period of rats' severe intoxication referred to wavenumber region from 3106â¯cm-1 to 687â¯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.
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Venenos de Aranha/farmacologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Transporte Biológico/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Hipocampo/metabolismo , Masculino , Neuropeptídeos/metabolismo , Ratos Wistar , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
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.
Assuntos
Cavéolas/efeitos dos fármacos , Venenos de Aranha/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Cavéolas/ultraestrutura , Caveolina 1/metabolismo , Cerebelo/ultraestrutura , Relação Dose-Resposta a Droga , Dinamina II/metabolismo , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Microscopia Eletrônica de Transmissão , Ratos , Ratos Wistar , Fatores de Tempo , Quinases da Família src/metabolismoRESUMO
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.
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Caveolina 3/metabolismo , Conexina 43/metabolismo , Neurotoxinas/farmacologia , Venenos de Aranha/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/ultraestrutura , Caveolina 3/genética , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Cerebelo/ultraestrutura , Conexina 43/genética , Masculino , Neurotoxinas/toxicidade , Ratos , Venenos de Aranha/toxicidade , Sinapses/efeitos dos fármacos , Sinapses/metabolismoRESUMO
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 24h 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 (1h/day, PND 23-60). The expression of some proteins involved in BBB structure (ß-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, ß-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.
Assuntos
Barreira Hematoencefálica/patologia , Meio Ambiente , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Hipóxia-Isquemia Encefálica/patologia , Hipóxia-Isquemia Encefálica/fisiopatologia , Fatores Etários , Análise de Variância , Animais , Animais Recém-Nascidos , Aquaporina 4/metabolismo , Barreira Hematoencefálica/metabolismo , Conexinas/metabolismo , Modelos Animais de Doenças , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Transportador de Glucose Tipo 1/metabolismo , Masculino , Ocludina/metabolismo , Ratos , Ratos WistarRESUMO
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.
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Cerebelo/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/metabolismo , Venenos de Aranha/toxicidade , Aranhas/metabolismo , Animais , Barreira Hematoencefálica/metabolismo , Calbindina 1/genética , Calbindina 1/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Cerebelo/metabolismo , Eletroforese em Gel de Poliacrilamida , Endotélio/efeitos dos fármacos , Endotélio/metabolismo , Masculino , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Ratos , Ratos Wistar , Picada de Aranha/patologiaRESUMO
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.
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Cavéolas/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Neuropeptídeos/farmacologia , Neurotoxinas/farmacologia , Venenos de Aranha/farmacologia , Animais , Western Blotting , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Caveolina 1/biossíntese , Masculino , Células de Purkinje/efeitos dos fármacos , Células de Purkinje/metabolismo , Células de Purkinje/patologia , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , AranhasRESUMO
This study characterizes the distribution and quantifies the expression of the tyrosine kinase receptor for the vascular endothelial growth factor (VEGF), Flt-1, in the rat hippocampus following intra-peritoneal injection of Phoneutria nigriventer venom (PNV). Post-natal day 14 (P14) and 8-10 weeks (adult) old rats were used and analyses were done at 1, 2, 5 and 24 h after venom exposure and compared with saline-injected counterparts. PNV-injected animals showed hippocampal venules with perivascular edema indicating blood-brain barrier (BBB) dysfunction. This was accompanied by significant overexpression of Flt-1 which though was not the same for CA1, CA2, CA3 and dentate gyrus (DG) hippocampal regions, neither for P14 and adult rats. Regional analysis using GIMP methodology showed that Flt-1 was constitutively distributed more densely in neurons of DG, followed by CA1/CA2 and CA3 of both control P14 and adult animals, without variation over time, but significantly more expressed in P14 than in adults. A time-course analysis showed that Flt-1 upregulation was progressive and that neurons VEGFR1/Flt-1+ of PNV-exposed animals are timely and regionally modulated depending on the hippocampal region, being CA2 the least responsive region regardless animal's age, whilst DG was the most susceptible with adult animals having higher upregulation than neonates. Since VEGF has been reported to confer protection in several pathological processes we suggest that VEGF may be involved in hippocampal neurons response via Flt-1 mediation following PNV envenoming; its higher upregulation in adult envenomed rats may be an indication that Flt-1 neuroprotective mediation is more efficient with age. The Flt-1 upregulation and the incidence of perivascular edema in young animals may indicate a pro-inflammatory role of the receptor.
Assuntos
Hipocampo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Venenos de Aranha/toxicidade , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Fatores Etários , Animais , Biomarcadores/metabolismo , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Edema Encefálico/induzido quimicamente , Edema Encefálico/metabolismo , Edema Encefálico/patologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Hipocampo/metabolismo , Hipocampo/patologia , Processamento de Imagem Assistida por Computador , Masculino , Neurônios/metabolismo , Neurônios/patologia , Permeabilidade/efeitos dos fármacos , Ratos , Ratos Wistar , Aranhas/fisiologia , Regulação para CimaRESUMO
O fator de crescimento endotelial vascular (VEGF), o principal regulador da angiogênese e da permeabilidade vascular, foi recentemente reconhecido como neurotrófico, neurogênico e neuroprotetor, sendo, portanto, regulado positivamente em muitos processos neuropatológicos. Neste modelo experimental de quebra da barreira hematoencefálica (BHE) pelo veneno da aranha Phoneutria nigriventer (PNV), a expressão do VEGF e seus receptores tirosina-quinase, Flt-1 e Flk-1 e de seus RNAs mensageiros foi investigada no hipocampo e cerebelo de ratos Wistar (Rattus norvegicus) por imunohistoquímica (IHQ), western blotting (WB) e reação em cadeia da polimerase em tempo real (qPCR). Paralelamente, a integridade da BHE foi avaliada através da expressão das proteínas da via paracelular, Ocludina e β-catenina, e da principal proteína da membrana basal, a Laminina, que estão presentes no endotélio na interface sangue-cérebro. O estudo foi realizado em ratos de 14 dias (neonatos) e de 8-10 semanas (adultos jovens) para avaliar diferenças em função da idade na funcionalidade da BHE e na possível mediação dos efeitos neurotóxicos do PNV pelo VEGF. A via escolhida para administração de PNV (1,7 mg/kg em 0,5ml de salina 0,9%) foi intraperitoneal, devido sua administração mais favorável nos animais neonatos. Os tempos de 2, 5 e 24 horas após a administração de PNV visaram investigar a expressão das proteínas, RNAs mensageiros e uma possível mediação pelo VEGF na fase aguda do envenenamento. A administração do PNV provocou sinais imediatos de intoxicação nos animais, os quais foram mais severos e imediatos nos neonatos do que nos adultos.
Vascular endothelial growth factor (VEGF), a major regulator of developmental angiogenesis and vascular permeability, was recently recognized as neurotrophic, neurogenic and neuroprotector, hence being upregulated in many neuropathological processes. In this experimental model of blood brain barrier (BBB) breakdown by the Phoneutria nigriventer spider venom (PNV), the expression of VEGF and its receptor tyrosine kinases, Flt-1 and Flk-1 and their mRNAs was investigated in the hippocampus and cerebellum of Wistar rats (Rattus norvegicus) by immunohistochemistry (IHC), western blotting (WB) and real time polymerase chain reaction (qPCR). Simultaneously, the BBB integrity was assessed through expression of paracellular pathway proteins, β- catenin and Occludin, and the main basement membrane protein, Laminin, which are present in the endothelium blood-brain interface. The study was performed in rats by 14 days (neonates) and 8-10 weeks (young adults) to assess differences related to age in the BBB functionality and the possible mediation of the PNV neurotoxic effects by VEGF. The via chosen for PNV administration (1.7 mg/kg in 0.5 ml of 0.9% saline) was intraperitoneally, due to more favorable application in neonate animals. The times of 2, 5 and 24 hours after PNV administration aimed to investigate the expression of proteins, mRNAs, and possible mediation by VEGF in acute envenomation. The PNV administration provoked immediate signs of intoxication in animals, which were more severe and immediate in neonates than in adults. In hippocampus, the WB data showed increased expression of VEGF, Flt-1 and Flk-1 and their mRNAs, which were concomitant with the development of perivascular edema, and decreased expression of Occludin, β-catenin and Laminin. IHC data show that VEGF immunoreactivity occurred in the bodies and dendrites of pyramidal neurons in the subfield CA1, CA2, CA3 and dentate gyrus of the hippocampus, in contrast with nuclear staining of Flt-1 and Flk-1.