RESUMEN
The vast majority of stroke cases are classified as ischemic stroke, but effective pharmacotherapy strategies to treat brain infarction are still limited. Glutamate, which is a primary mediator of excitotoxicity, contributes to neuronal damage in numerous pathologies, including ischemia. The aim of this study was to investigate the effect of the hydrogen sulfide donor AP39 on excitotoxicity. AP39 was administered as a single dose of 100 nmol/kg b.w. i.v. 10 min after the restoration of blood flow and 100 min after middle cerebral artery occlusion (MCAO) in male Sprague-Dawley rats. Neurological deficits by Phillips's score, and infarct volume by TTC staining were evaluated (n = 8). LC-MS was used to determine the extracellular glutamate concentration in microdialysates collected intrasurgically and from freely moving animals 24 h and 3 days after reperfusion (n = 6). The expression of proteins involved in the regulation of glutamatergic transmission was investigated 24 h after reperfusion by Western-blot analysis (n = 6). The results were verified by double-immunostaining of brain cryosections (n = 6). The results showed a significant longitudinal decrease in extracellular glutamate concentrations in the motor cortex and hippocampus in MCAO + AP39 rats compared to MCAO rats. Moreover, the administration of AP39 increased the content of the GLT-1 transporter and reduced the content of VGLUT1 in the ischemic core. Upregulation of the GLT-1 transporter responsible for glutamate reuptake from the synaptic cleft, and downregulation of VGLUT1, which regulates glutamate transport to synaptic vesicles, indicate that these are important mechanisms by which AP39 reduces extracellular glutamate concentrations and, consequently, excitotoxicity after ischemia.
Asunto(s)
Isquemia Encefálica , Sulfuro de Hidrógeno , Ratas , Masculino , Animales , Ácido Glutámico/metabolismo , Sulfuro de Hidrógeno/farmacología , Ratas Sprague-Dawley , Isquemia Encefálica/metabolismo , Infarto de la Arteria Cerebral Media/tratamiento farmacológicoRESUMEN
Benzophenone-3 (BP-3) is the most commonly used UV filter in cosmetics, which is absorbed through the skin and crosses the blood-brain barrier. This compound increases extracellular glutamate concentrations, lipid peroxidation, the number of microglia cells and induces process of apoptosis. The aim of this study was to determine the effect of BP-3 on the activation and polarization of microglial cells in the frontal cortex and hippocampus of adult male rats exposed to BP-3 prenatally and then for two weeks in adulthood. It has been found, that exposure to BP-3 reduced the expression of the marker of the M2 phenotype of glial cells in both examined brain structures. An increase in the CD86/CD206 microglial phenotype ratio, expression of transcription factor NFκB and activity of caspase-1 were observed only in the frontal cortex, whereas BP-3 increased the level of glucocorticoid receptors in the hippocampus. The in vitro study conducted in the primary culture of rat frontal cortical microglia cells showed that BP-3 increased the LPS-stimulated release of pro-inflammatory cytokines IL-1α, IL-1ß, TNFα, but in cultures without LPS there was decreased IL-1α, IL-6 and TNFα production, while the IL-18 and IP-10 was elevated. The obtained results indicate that differences in the level of immunoactivation between the frontal cortex and the hippocampus may result from the action of this compound on glucocorticoid receptors. In turn, changes in cytokine production in microglial cells indicate that BP-3 aggravates the LPS-induced immunoactivation.
Asunto(s)
Microglía , Factor de Necrosis Tumoral alfa , Ratas , Animales , Masculino , Microglía/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Lipopolisacáridos/farmacología , Receptores de Glucocorticoides/metabolismo , Citocinas/metabolismoRESUMEN
Brain ischemia is one of the leading causes of death and long-term disability in the world. Interruption of the blood supply to the brain is a direct stimulus for many pathological events. The massive vesicular release of glutamate (Glu) after ischemia onset induces excitotoxicity, which is a potent stress on neurons. Loading of presynaptic vesicles with Glu is the first step of glutamatergic neurotransmission. Vesicular glutamate transporters 1, 2, and 3 (VGLUT1, 2, and 3) are the main players involved in filling presynaptic vesicles with Glu. VGLUT1 and VGLUT2 are expressed mainly in glutamatergic neurons. Therefore, the possibility of pharmacological modulation to prevent ischemia-related brain damage is attractive. In this study, we aimed to determine the effect of focal cerebral ischemia on the spatiotemporal expression of VGLUT1 and VGLUT2 in rats. Next, we investigated the influence of VGLUT inhibition with Chicago Sky Blue 6B (CSB6B) on Glu release and stroke outcome. The effect of CSB6B pretreatment on infarct volume and neurological deficit was compared with a reference model of ischemic preconditioning. The results of this study indicate that ischemia upregulated the expression of VGLUT1 in the cerebral cortex and in the dorsal striatum 3 days after ischemia onset. The expression of VGLUT2 was elevated in the dorsal striatum and in the cerebral cortex 24 h and 3 days after ischemia, respectively. Microdialysis revealed that pretreatment with CSB6B significantly reduced the extracellular Glu concentration. Altogether, this study shows that inhibition of VGLUTs might be a promising therapeutic strategy for the future.
Asunto(s)
Isquemia Encefálica , Proteínas de Transporte Vesicular de Glutamato , Ratas , Animales , Neuroprotección , Azul de Tripano/farmacología , Infarto CerebralRESUMEN
A proper reference gene (RG) is required to reliably measure mRNA levels in biological samples via quantitative reverse transcription PCR (RT-qPCR). Various experimental paradigms require specific and stable RGs. In studies using rodent models of brain ischaemia, a variety of genes, such as ß-actin (Actb), hypoxanthine phosphoribosyltransferase 1 (Hprt1), peptidyl-propyl isomerase A (Ppia) and glyceraldehyde-3-phosphate dehydrogenase (Gapdh), are used as RGs. However, most of these genes have not been validated in specific experimental settings. The aim of this study was to evaluate the time- and brain region-dependent expression of RG candidates in a rat model of transient middle cerebral artery occlusion (tMCAO). The following genes were selected: Actb, Hprt1, Ppia, Gapdh, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta (Ywhaz) and beta-2 microglobulin (B2m). Focal cerebral ischaemia was induced by 90 min of tMCAO in male Sprague-Dawley rats. Expression was investigated at four time points (12 and 24 h; 3 and 7 days) and in three brain areas (the frontal cortex, hippocampus and dorsal striatum) within the ischaemic brain hemisphere. The RT-qPCR results were analysed using variance analysis and the ΔCt, GeNorm, NormFinder and BestKeeper methods. Data from these algorithms were ranked using the geometric mean of ranks of each analysis. Ppia, Hprt1 and Ywhaz were the most stable genes across the analysed brain areas and time points. B2m and Actb exhibited the greatest fluctuations, and the results for Gapdh were ambiguous.
Asunto(s)
Isquemia Encefálica , Gliceraldehído-3-Fosfato Deshidrogenasas , Actinas/genética , Animales , Isquemia Encefálica/genética , Expresión Génica , Perfilación de la Expresión Génica/métodos , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Masculino , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Estándares de ReferenciaRESUMEN
Ischemic stroke is the third leading cause of death in the world, which accounts for almost 12% of the total deaths worldwide. Despite decades of research, the available and effective pharmacotherapy is limited. Some evidence underlines the beneficial properties of hydrogen sulfide (H2S) donors, such as NaSH, in an animal model of brain ischemia and in in vitro research; however, these data are ambiguous. This study was undertaken to verify the neuroprotective activity of AP39, a slow-releasing mitochondria-targeted H2S delivery molecule. We administered AP39 for 7 days prior to ischemia onset, and the potential to induce brain tolerance to ischemia was verified. To do this, we used the rat model of 90-min middle cerebral artery occlusion (MCAO) and used LC-MS/MS, RT-PCR, LuminexTM assays, Western blot and immunofluorescent double-staining to determine the absolute H2S levels, inflammatory markers, neurotrophic factor signaling pathways and apoptosis marker in the ipsilateral frontal cortex, hippocampus and in the dorsal striatum 24 h after ischemia onset. AP39 (50 nmol/kg) reduced the infarct volume, neurological deficit and reduced the microglia marker (Iba1) expression. AP39 also exerted prominent anti-inflammatory activity in reducing the release of Il-1ß, Il-6 and TNFα in brain areas particularly affected by ischemia. Furthermore, AP39 enhanced the pro-survival pathways of neurotrophic factors BDNF-TrkB and NGF-TrkA and reduced the proapoptotic proNGF-p75NTR-sortilin pathway activity. These changes corresponded with reduced levels of cleaved caspase 3. Altogether, AP39 treatment induced adaptative changes within the brain and, by that, developed brain tolerance to ischemia.
Asunto(s)
Isquemia Encefálica/prevención & control , Sulfuro de Hidrógeno/metabolismo , Infarto de la Arteria Cerebral Media/complicaciones , Mitocondrias/metabolismo , Compuestos Organofosforados/farmacología , Sustancias Protectoras/farmacología , Tionas/farmacología , Animales , Isquemia Encefálica/etiología , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Sulfuro de Hidrógeno/análisis , Masculino , Mitocondrias/efectos de los fármacos , Compuestos Organofosforados/administración & dosificación , Sustancias Protectoras/administración & dosificación , Ratas , Ratas Sprague-Dawley , Tionas/administración & dosificaciónRESUMEN
BACKGROUND: The bidirectional communication between neurons and microglia is fundamental for the homeostasis and biological function of the central nervous system. Maternal immune activation (MIA) is considered to be one of the factors affecting these interactions. Accordingly, MIA has been suggested to be involved in several neuropsychiatric diseases, including schizophrenia. The crucial regulatory systems for neuron-microglia crosstalk are the CX3CL1-CX3CR1 and CD200-CD200R axes. METHODS: We aimed to clarify the impact of MIA on CX3CL1-CX3CR1 and CD200-CD200R signalling pathways in the brains of male Wistar rats in early and adult life by employing two neurodevelopmental models of schizophrenia based on the prenatal challenge with lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (Poly I:C). We also examined the effect of MIA on the expression of microglial markers and the profile of cytokines released in the brains of young offspring, as well as the behaviour of adult animals. Moreover, we visualized the localization of ligand-receptor systems in the hippocampal regions (CA1, CA3 and DG) and the frontal cortex of young rats exposed to MIA. The differences between groups were analysed using Student's t test. RESULTS: We observed that MIA altered developmental trajectories in neuron-microglia communication in the brains of young offspring, as evidenced by the disruption of CX3CL1-CX3CR1 and/or CD200-CD200R axes. Our data demonstrated the presence of abnormalities after LPS-induced MIA in levels of Cd40, Il-1ß, Tnf-α, Arg1, Tgf-ß and Il-10, as well as IBA1, IL-1ß and IL-4, while after Poly I:C-generated MIA in levels of Cd40, iNos, Il-6, Tgf-ß, Il-10, and IBA1, IL-1ß, TNF-α, IL-6, TGF-ß and IL-4 early in the life of male animals. In adult male rats that experienced prenatal exposure to MIA, we observed behavioural changes resembling a schizophrenia-like phenotype. CONCLUSIONS: Our study provides evidence that altered CX3CL1-CX3CR1 and/or CD200-CD200R pathways, emerging after prenatal immune challenge with LPS and Poly I:C, might be involved in the aetiology of schizophrenia.
Asunto(s)
Encéfalo/efectos de los fármacos , Lipopolisacáridos/farmacología , Poli I-C/farmacología , Efectos Tardíos de la Exposición Prenatal/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Antígenos CD/metabolismo , Encéfalo/metabolismo , Receptor 1 de Quimiocinas CX3C/metabolismo , Quimiocina CX3CL1/metabolismo , Femenino , Masculino , Embarazo , Ratas , Ratas Wistar , Receptores Inmunológicos/metabolismo , Esquizofrenia/metabolismoRESUMEN
Benzophenone-3 (BP-3), the most widely used UV chemical filter, is absorbed well through the skin and gastrointestinal tract and can affect some body functions, including the survival of nerve cells. Previously, we showed that BP-3 evoked a neurotoxic effect in male rats, but since the effects of this compound are known to depend on gender, the aim of the present study was to show the concentration and potential neurotoxic action of this compound in the female rat brain. BP-3 was administered dermally to female rats during pregnancy, and then in the 7th and 8th weeks of age to their female offspring. The effect of BP-3 exposure on short-term and spatial memory, its concentrations in blood, the liver, the frontal cortex, and the hippocampus, and the effect on selected markers of brain damage were determined. Also, the impact of BP-3 on sex and thyroid hormone levels in blood and hematological parameters was examined. It has been found that this compound was present in blood and brain structures in females at a lower concentration than in males. BP-3 in both examined brain structures increased extracellular glutamate concentration and enhanced lipid peroxidation, but did not induce the apoptotic process. The tested compound also evoked hyperthyroidism and decreased the blood progesterone level and the number of erythrocytes. The presented data indicated that, after the same exposure to BP-3, this compound was at a lower concentration in the female brain than in that of the males. Although BP-3 did not induce apoptosis in the hippocampus and frontal cortex, the increased extracellular glutamate concentration and lipid peroxidation, as well as impaired spatial memory, suggested that this compound also had adverse effects in the female brain yet was weaker than in males. In contrast to the weaker effects of the BP-3 on females than the brain of males, this compound affected the endocrine system and evoked a disturbance in hematological parameters more strongly than in male rats.
Asunto(s)
Apoptosis/efectos de los fármacos , Benzofenonas/toxicidad , Lóbulo Frontal/efectos de los fármacos , Hormonas Esteroides Gonadales/sangre , Hipocampo/efectos de los fármacos , Protectores Solares/toxicidad , Hormonas Tiroideas/sangre , Administración Cutánea , Animales , Proteínas Reguladoras de la Apoptosis/efectos de los fármacos , Benzofenonas/administración & dosificación , Femenino , Lóbulo Frontal/metabolismo , Ácido Glutámico/metabolismo , Hipocampo/metabolismo , Ratas Sprague-Dawley , Receptores de Hidrocarburo de Aril/metabolismo , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Protectores Solares/administración & dosificaciónRESUMEN
Benzophenone-3 is the most commonly used UV filter. It is well absorbed through the skin and gastrointestinal tract. Its best-known side effect is the impact on the function of sex hormones. Little is known about the influence of BP-3 on the brain. The aim of this study was to show whether BP-3 crosses the blood-brain barrier (BBB), to determine whether it induces nerve cell damage in susceptible brain structures, and to identify the mechanism of its action in the central nervous system. BP-3 was administered dermally during the prenatal period and adulthood to rats. BP-3 effect on short-term and spatial memory was determined by novel object and novel location recognition tests. BP-3 concentrations were assayed in the brain and peripheral tissues. In brain structures, selected markers of brain damage were measured. The study showed that BP-3 is absorbed through the rat skin, passes through the BBB. BP-3 raised oxidative stress and induced apoptosis in the brain. BP-3 increased the concentration of extracellular glutamate in examined brain structures and changed the expression of glutamate transporters. BP-3 had no effect on short-term memory but impaired spatial memory. The present study showed that dermal BP-3 exposure may cause damage to neurons what might be associated with the increase in the level of extracellular glutamate, most likely evoked by changes in the expression of GLT-1 and xCT glutamate transporters. Thus, exposure to BP-3 may be one of the causes that increase the risk of developing neurodegenerative diseases.
RESUMEN
Benzophenones, frequently used as UV chemical filters, are absorbed through the skin and can exert systemic adverse effects. So far, most of the data are related to their action on sex hormone receptors whereas potential neurotoxic effect is expected mainly on the basis of in vitro studies. The aim of the present study was to determine concentrations of BP-2, oxidative stress and apoptosis markers in the rat brain after topical administration of this compound. Male Wistar rats were treated dermally with BP-2 (100 mg/kg, 4 weeks), and next, blood and tissue BP-2 concentrations and oxidative stress and apoptotic markers in the frontal cortex and hippocampus were determined. After dermal BP-2 administration, blood level of this compound was about 300 ng/ml while in the liver and adipose tissue 1354 and 823 ng/g wt tissue, respectively. In the studied brain structures, the levels of the test compound were from 5 to 19 ng/g tissue. In the hippocampus, where BP-2 level was about 3.5-fold lower than in the frontal cortex, no significant changes in either oxidative stress and apoptosis markers were observed. There was also no change in apoptosis markers in the frontal cortex but unexpectedly the oxidative stress markers were reduced. The research showed that BP-2 passes through the blood-brain barrier but its concentration in the brain structures are much lower than in the blood. This compound did not exacerbate oxidative stress and apoptosis markers in the hippocampus and frontal cortex, and even lowered oxidative stress in the frontal cortex.
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Apoptosis/efectos de los fármacos , Benzofenonas/análisis , Benzofenonas/toxicidad , Lóbulo Frontal/efectos de los fármacos , Hipocampo/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Animales , Lóbulo Frontal/química , Lóbulo Frontal/metabolismo , Hipocampo/química , Hipocampo/metabolismo , Masculino , Ratas WistarRESUMEN
Benzophenone-3 (BP-3) is the most commonly used chemical UV filter. This compound can easily be absorbed through the skin and the gastrointestinal tract and can disturb sex hormone receptor function. BP-3 is lipophilic and should cross the blood-brain barrier and it may reduce the survival of neurons, although so far, its effects on nerve cells have been studied in only in vitro cultures. The aim of the present study was to determine the effects of BP-3 on apoptosis and the expression of oestrogen, androgen and arylhydrocarbon receptors (AhR) in the rat frontal cortex and hippocampus. This compound was administered dermally to female rats during pregnancy and next to their male offspring through 6 and 7 weeks of age. BP-3 in the frontal cortex induced the mitochondrial apoptosis pathway by increasing the active forms of caspase-3 and caspase-9, inducing the pro-apoptotic proteins Bax and Bak and increasing the number of cells with apoptotic DNA fragmentation. In the hippocampus, an increase in the caspase-9 level and a downward trend in the level of anti-apoptotic proteins were observed. In both brain regions, the contents of ERß in the nuclear fraction and GPR30 in the membrane fraction were significantly reduced. BP-3 significantly increased AhR in the cytosol of the frontal cortex but had no effect on the content of this receptor in the hippocampus. This is the first study showing that exposure to BP-3 induces the mitochondrial apoptosis pathway in the rat frontal cortex and this effect may result from a weakening of the neuroprotective effects of oestrogen and/or an intensification of AhR-mediated apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Benzofenonas/toxicidad , Lóbulo Frontal/metabolismo , Hormonas Esteroides Gonadales/metabolismo , Hipocampo/metabolismo , Protectores Solares/toxicidad , Animales , Caspasa 3/biosíntesis , Caspasa 9/biosíntesis , Femenino , Lóbulo Frontal/efectos de los fármacos , Hipocampo/efectos de los fármacos , Masculino , Embarazo , Efectos Tardíos de la Exposición Prenatal , Ratas , Ratas Sprague-Dawley , Receptores Androgénicos/biosíntesis , Receptores Androgénicos/efectos de los fármacos , Receptores de Hidrocarburo de Aril/efectos de los fármacos , Receptores de Estrógenos/biosíntesis , Receptores de Estrógenos/efectos de los fármacos , Proteína Destructora del Antagonista Homólogo bcl-2/biosíntesis , Proteína X Asociada a bcl-2/biosíntesisRESUMEN
Benzophenones used as UV filters, in addition to the effects on the skin, can be absorbed into the blood and affect the function of certain organs. So far, their effects on the sex hormone receptors and gonadal function have been studied, but not much is known about their potential action on other systems. The aim of the present study was to determine the effect of benzophenone-2 (BP-2) on immune system activity, hypothalamic-pituitary-thyroid (HPT) axis activity and hematological parameters. BP-2 was administered dermally, twice daily at a dose of 100â¯mg/kg for 4-weeks to male Wistar rats. Immunological and hematological parameters and HPT axis activity were assayed 24â¯h after the last administration. It was found that BP-2 did not change relative weights of the thymus and spleen and did not exert toxic effect on tymocytes and splenocytes. However, this compound increased proliferative activity of splenocytes, enhanced metabolic activity of splenocytes and thymocytes and nitric oxide production of these cells. In animals exposed to BP-2, the HPT axis activity was increased, as evidenced by reduction in the thyroid stimulating hormone (TRH) level and increase in free fraction of triiodothyronine (fT3) and thyroxin (fT4) in blood. BP-2 had no effect on leukocyte, erythrocyte and platelet counts or on morphology and hemoglobin content in erythrocytes. The conducted research showed that dermal, sub-chronic BP-2 administration evoked hyperthyroidism, increased activity or function of the immune cells but did not affect hematological parameters. We suggest that topical administration of BP-2 leading to a prolonged elevated BP-2 level in blood causes hyperthyroidism, which in turn may be responsible for the increased immune cell activity or function. However, only future research can explain the mechanism and functional importance of the changes in thyroid hormones and immunological parameters observed after exposure to BP-2.
Asunto(s)
Benzofenonas/toxicidad , Sistema Hipotálamo-Hipofisario/efectos de los fármacos , Sistema Hipotálamo-Hipofisario/inmunología , Hormonas Tiroideas/inmunología , Administración Cutánea , Animales , Benzofenonas/administración & dosificación , Sistema Hipotálamo-Hipofisario/metabolismo , Sistema Inmunológico/efectos de los fármacos , Sistema Inmunológico/inmunología , Sistema Inmunológico/metabolismo , Masculino , Ratas , Ratas Wistar , Hormonas Tiroideas/metabolismoRESUMEN
One of the major players in the pathophysiology of cerebral ischemia is disrupted homeostasis of glutamatergic neurotransmission, resulting in elevated extracellular glutamate (Glu) concentrations and excitotoxicity-related cell death. In the brain, Glu concentrations are regulated by Glu transporters, including Glu transporter-1 (GLT-1) and cystine/Glu antiporter (system xc-). Modulation of these transporters by administration of ceftriaxone (CEF, 200 mg/kg, i.p.) or N-acetylcysteine (NAC, 150 mg/kg, i.p.) for 5 days before focal cerebral ischemia may induce brain tolerance to ischemia by significantly limiting stroke-related damage and normalizing Glu concentrations. In the present study, focal cerebral ischemia was induced by 90-minute middle cerebral artery occlusion (MCAO). We compared the effects of CEF and NAC pretreatment on Glu concentrations in extracellular fluid and cellular-specific expression of GLT-1 and xCT with the effects of two reference preconditioning methods, namely, ischemic preconditioning and chemical preconditioning in rats. Both CEF and NAC significantly reduced Glu levels in the frontal cortex and hippocampus during focal cerebral ischemia, and this decrease was comparable with the Glu level achieved with the reference preconditioning strategies. The results of immunofluorescence staining of GLT-1 and xCT on astrocytes, neurons and microglia accounted for the observed changes in extracellular Glu levels to a certain extent. Briefly, after MCAO, the expression of GLT-1 on astrocytes decreased, but pretreatment with CEF seemed to prevent this downregulation. In addition, every intervention used in this study seemed to reduce xCT expression on astrocytes and neurons. The results of this study indicate that modulation of Glu transporter expression may restore Glu homeostasis. Moreover, our results suggest that CEF and NAC may induce brain tolerance to ischemia by influencing GLT-1 and system xc- expression levels. These transporters are presumably good targets for the development of novel therapies for brain ischemia.
Asunto(s)
Sistemas de Transporte de Aminoácidos Acídicos/genética , Isquemia Encefálica/tratamiento farmacológico , Transportador 2 de Aminoácidos Excitadores/genética , Ácido Glutámico/metabolismo , Acetilcisteína/administración & dosificación , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Isquemia Encefálica/genética , Isquemia Encefálica/patología , Ceftriaxona/administración & dosificación , Lóbulo Frontal/efectos de los fármacos , Lóbulo Frontal/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Humanos , Infarto de la Arteria Cerebral Media , Ratas , Transmisión Sináptica/efectos de los fármacosRESUMEN
Ethylene glycol ethers (EGEs) are compounds widely used in many branches of industry. Their toxicological profile in the peripheral tissues is relatively well described, but little is known about their action on the central nervous system (CNS). In this study, we evaluated the effect of 2-ethoxyethanol (EE), 2-butoxyethanol (BE), 2-phenoxyethanol (PHE) and their metabolites on necrotic (estimated by cell viability and lactate dehydrogenase release) and apoptotic (caspase-3 activity and mitochondrial membrane potential) processes and reactive oxygen species' (ROS) production in human neuroblastoma (SH-SY5Y) cells. We have shown that, similar to the peripheral tissues, EGE metabolites in most of the performed assays revealed greater potential to damage than the parent compounds in the CNS cells. Subsequently, we investigated the participation of some selected protein kinases in the degenerative activity of PHE and its main metabolite, phenoxyacetic acid (PHA). It has been found that a GSK3ß inhibitor weakened the damaging effects of PHE and PHA in each of the performed assays. Furthermore, the kinases, p38-MAPK, JNK-MAPK and PKC, had a significant role in the cytotoxic and proapoptotic effects of PHA. These results indicate that the neurotoxic effect of EGEs may stem from their impact on many intracellular signal transduction pathways.
Asunto(s)
Glicoles de Etileno/toxicidad , Proteínas Quinasas/metabolismo , Acetatos/toxicidad , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Necrosis/inducido químicamente , Inhibidores de Proteínas Quinasas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Solventes/toxicidadRESUMEN
Glutamate (Glu) plays a key role in excitotoxicity-related injury in cerebral ischemia. In the brain, Glu homeostasis depends on Glu transporters, including the excitatory amino acid transporters and the cysteine/Glu antiporter (xc-). We hypothesized that drugs acting on Glu transporters, such as ceftriaxone (CEF, 200 mg/kg, i.p.) and N-acetylcysteine (NAC, 150 mg/kg, i.p.), administered repeatedly for 5 days before focal cerebral ischemia in rats and induced by a 90-min middle cerebral artery occlusion (MCAO), may induce brain tolerance to ischemia. We compared the effects of these drugs on brain infarct volume, neurological deficits and the mRNA and protein expression of the Glu transporter-1 (GLT-1) and xc- with the effects of ischemic preconditioning and chemical preconditioning using 3-nitropropionic acid. Administration of CEF and NAC significantly reduced infarct size and neurological deficits caused by a 90-min MCAO. These beneficial effects were accompanied by changes in GLT-1 expression caused by a 90-min MCAO at both the mRNA and protein levels in the frontal cortex, hippocampus, and dorsal striatum. Thus, the results of this study suggest that the regulation of GLT-1 and xc- plays a role in the development of cerebral tolerance to ischemia and that this regulation may be a novel approach in the therapy of brain ischemia.
Asunto(s)
Acetilcisteína/farmacología , Acetilcisteína/uso terapéutico , Ceftriaxona/farmacología , Ceftriaxona/uso terapéutico , Transportador 2 de Aminoácidos Excitadores/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Análisis de Varianza , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Edema Encefálico/etiología , Edema Encefálico/prevención & control , Infarto Encefálico/etiología , Infarto Encefálico/prevención & control , Modelos Animales de Enfermedad , Esquema de Medicación , Ensayo de Inmunoadsorción Enzimática , Transportador 2 de Aminoácidos Excitadores/genética , Infarto de la Arteria Cerebral Media/complicaciones , Infarto de la Arteria Cerebral Media/patología , Masculino , Enfermedades del Sistema Nervioso/etiología , Enfermedades del Sistema Nervioso/prevención & control , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , ARN Mensajero/metabolismo , Ratas , Ratas WistarRESUMEN
BACKGROUND: Ethylene glycol ethers (EGEs) are compounds widely used in industry and household products, but their potential, adverse effect on brain is poorly understood, so far. The aim of the present study was to determine whether 4-week administration of 2-buthoxyethanol (BE), 2-phenoxyethanol (PHE), and 2-ethoxyethanol (EE) induces apoptotic process in the rat hippocampus and frontal cortex, and whether their adverse effect on the brain cells can result from disturbances in the glucose metabolism. METHODS: Experiments were conducted on 40 rats, exposed to BE, PHE, EE, saline or sunflower oil for 4 weeks. Markers of apoptosis and glucose metabolism were determined in frontal cortex and hippocampus by western blot, ELISA, and fluorescent-based assays. RESULTS: BE and PHE, but not EE, increased expression of the active form of caspase-3 in the examined brain regions. BE and PHE increased caspase-9 level in the cortex and PHE also in the hippocampus. BE and PHE increased the level of pro-apoptotic proteins (Bax, Bak) and/or reduced the concentration of anti-apoptotic proteins (Bcl-2, Bcl-xL); whereas, the effect of BE was observed mainly in the cortex and that of PHE in the hippocampus. It has also been found that PHE increased brain glucose level, and both BE and PHE elevated pyruvate and lactate concentration. CONCLUSIONS: It can be concluded that chronic treatment with BE and PHE induced mitochondrial pathway of apoptosis, and disturbed glucose metabolism in the rat brain.
Asunto(s)
Apoptosis/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Glicoles de Etileno/toxicidad , Glucosa/metabolismo , Animales , Apoptosis/fisiología , Éteres/toxicidad , Masculino , Ratas , Ratas WistarRESUMEN
Many herbs, and recently their biomass from in vitro cultures, are essential for the treatment of diseases. The aim of this study was to determine the optimal growth of Bacopa monnieri (water hyssop) in an in vitro culture and to examine if extracts of the B. monnieri biomass from the in vitro culture would affect hydrogen peroxide- and staurosporine-induced injury of the human neuroblastoma SH-SY5Y cell line. It has been found that B. monnieri at concentrations of 25, 50, and 100 µg/mL inhibited both hydrogen peroxide-induced efflux of lactate dehydrogenase from damaged cells to culture medium and increased cell viability determined by an MTT assay. Moreover, B. monnieri at concentrations of 10, 25, and 50 µg/mL decreased staurosporine-induced activity of an executive apoptotic enzyme-caspase-3 and protected mitochondrial membrane potential. The obtained data indicate that the biomass from the in vitro culture of B. monnieri prevented SH-SY5Y cell damage related to oxidative stress and had the ability to inhibit the apoptotic process. Thus, this study supports the traditional use of B. monnieri as a neuroprotective therapy, and further in vivo studies on the effects of this preparation on morphology and function of nerve cells could lead to its wider application.
Asunto(s)
Bacopa/química , Peróxido de Hidrógeno/antagonistas & inhibidores , Fármacos Neuroprotectores/farmacología , Extractos Vegetales/farmacología , Estaurosporina/antagonistas & inhibidores , Línea Celular Tumoral , Humanos , NeuroblastomaRESUMEN
Ethylene glycol ethers (EGEs) are components of many industrial and household products. Their hemolytic and gonadotoxic effects are relatively well known while their potential adverse effects on the central nervous system have not yet been clearly demonstrated. The aim of the present study was to examine the effects of 4-week administration of 2-buthoxyethanol (BE), 2-phenoxyethanol (PHE) and 2-ethoxyethanol (EE) on the total antioxidant capacity, activity of some antioxidant enzymes, such as the superoxide dismutase (SOD), catalase, glutathione peroxidase (GPX) and glutathione reductase and lipid peroxidation in the frontal cortex and hippocampus in the rat. These studies showed that BE and PHE decreased the total antioxidant activity, SOD and GPX activity, while increased lipid peroxidation in the frontal cortex. Like in the frontal cortex, also in the hippocampus BE and PHE attenuated the total antioxidant activity, however, lipid peroxidation was increased only in animals which received BE while reduction in GPX activity was present in rats administered PHE. The obtained data indicated that 4-week administration of BE and PHE, but not EE, reduced the total antioxidant activity and enhanced lipid peroxidation in the brain. In the frontal cortex, adverse effects of PHE and BE on lipid peroxidation probably depended on reduction in SOD and GPX activity, however, in the hippocampus the changes in the total antioxidant activity and lipid peroxidation were not connected with reduction of the investigated antioxidant enzyme activity.
Asunto(s)
Glicoles de Etileno/toxicidad , Lóbulo Frontal/efectos de los fármacos , Hipocampo/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Animales , Catalasa/metabolismo , Lóbulo Frontal/metabolismo , Glutatión Peroxidasa/metabolismo , Glutatión Reductasa/metabolismo , Hipocampo/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Peroxidación de Lípido/fisiología , Masculino , Estrés Oxidativo/fisiología , Distribución Aleatoria , Ratas Wistar , Superóxido Dismutasa/metabolismoRESUMEN
In this review, we briefly describe glutamate (Glu) metabolism and its specific transports and receptors in the central nervous system (CNS). Thereafter, we focus on excitatory amino acid transporters, cystine/glutamate antiporters (system xc-) and vesicular glutamate transporters, specifically addressing their location and roles in CNS and the molecular mechanisms underlying the regulation of Glu transporters. We provide evidence from in vitro or in vivo studies concerning alterations in Glu transporter expression in response to hypoxia or ischemia, including limited human data that supports the role of Glu transporters in stroke patients. Moreover, the potential to induce brain tolerance to ischemia through modulation of the expression and/or activities of Glu transporters is also discussed. Finally we present strategies involving the application of ischemic preconditioning and pharmacological agents, eg ß-lactam antibiotics, amitriptyline, riluzole and N-acetylcysteine, which result in the significant protection of nervous tissues against ischemia.
Asunto(s)
Sistemas de Transporte de Aminoácidos Acídicos/efectos de los fármacos , Isquemia Encefálica/terapia , Encéfalo/efectos de los fármacos , Ácido Glutámico/metabolismo , Precondicionamiento Isquémico , Fármacos Neuroprotectores/uso terapéutico , Proteínas de Transporte Vesicular de Glutamato/efectos de los fármacos , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/patología , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Proteínas de Transporte de Glutamato en la Membrana Plasmática/efectos de los fármacos , Proteínas de Transporte de Glutamato en la Membrana Plasmática/metabolismo , Humanos , Transmisión Sináptica/efectos de los fármacos , Resultado del Tratamiento , Proteínas de Transporte Vesicular de Glutamato/metabolismoRESUMEN
BACKGROUND: Ethylene glycol ethers (EGEs) are widely used as mixtures in various industrial processes and in many household products. 2-Methoxyethanol and 2-ethoxyethanol primarily exert gonadotoxic effect, while 2-butoxyethanol and 2-isopropoxyethanol have potent hemolytic activity. EGEs can cross the blood-brain barrier, but their potential neurodegenerative action in vivo has not been investigated, yet. In the present work, we examined potential adverse effects of EGEs on some selected brain structures. METHODS: A mixture of two compounds: one with stronger hydrophilic properties (2-methoxyethanol or 2-ethoxyethanol) and the second more lipophilic (2-butoxyethanol or 2-isopropoxyethanol) were administered sc for 4 weeks. Total antioxidant capacity, lipid peroxidation and caspase-3 activity were determined in the frontal cortex and hippocampus. RESULTS: It has been found that 4-week administration of a mixture of two EGEs, with various intensity, decreased total antioxidant capacity, enhanced lipid peroxidation and increased caspase-3 activity in the frontal cortex and hippocampus of Wistar rat. CONCLUSION: The obtained results suggested that EGEs exerted adverse effects on the CNS cells and may contribute in pathogenesis of neurodegenerative disorders.
Asunto(s)
Corteza Cerebral/efectos de los fármacos , Éteres/toxicidad , Glicoles de Etileno/toxicidad , Lóbulo Frontal/efectos de los fármacos , Hipocampo/efectos de los fármacos , Síndromes de Neurotoxicidad/etiología , Animales , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Corteza Cerebral/metabolismo , Lóbulo Frontal/metabolismo , Lóbulo Frontal/patología , Hipocampo/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Degeneración Nerviosa , Síndromes de Neurotoxicidad/metabolismo , Síndromes de Neurotoxicidad/patología , Estrés Oxidativo/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
The growing body of evidence implicates the significance of oxidative stress in the pathophysiology of depression. The aim of this paper was to examine N-acetylcysteine (NAC) - a putative precursor of the most important tissue antioxidant glutathione - in an animal model of depression and in ex vivo assays to detect oxidative stress parameters. Imipramine (IMI), a classical and clinically-approved antidepressant drug was also under investigation. Male Wistar rats which underwent either bulbectomy (BULB; removal of the olfactory bulbs) or sham surgery (SHAM; olfactory bulbs were left undestroyed) were treated acutely or repeatedly with NAC (50-100mg/kg, ip) or IMI (10mg/kg, ip). Following 10-daily injections with NAC or IMI or their solvents, or 9-daily injections with a corresponding solvent plus acute NAC or acute IMI forced swimming test on day 10, and locomotor activity were performed; immediately after behavioral tests animals were decapitated. Biochemical tests (the total antioxidant capacity - TAC and the superoxide dismutase activity - SOD) were performed on homogenates in several brain structures. In behavioral studies, chronic (but not acute) administration of NAC resulted in a dose-dependent reduction in the immobility time seen only in BULB rats while chronic IMI produced a significant decrease in this parameter in both SHAM and BULB animals. On the other hand, chronic administration of NAC and IMI resulted in a significant increase in cellular antioxidant mechanisms (SOD activity) that reversed the effects of BULB in the frontal cortex, hippocampus and striatum. Our study further supports the antidepressant-like activity of NAC and links its effect as well as IMI actions with the enhancement of brain SOD activity.