RESUMO
Previous studies have demonstrated that pioglitazone, a peroxisome proliferator-activated receptor gamma (PPARγ) agonist, inhibits ischemia-induced brain injury. The present study was conducted to examine whether pioglitazone can reduce impairment of behavioral deficits mediated by inflammatory-induced brain white matter injury in neonatal rats. Intraperitoneal (i.p.) injection of lipopolysaccharide (LPS, 2 mg/kg) was administered to Sprague-Dawley rat pups on postnatal day 5 (P5), and i.p. administration of pioglitazone (20 mg/kg) or vehicle was performed 5 min after LPS injection. Sensorimotor behavioral tests were performed 24 h after LPS exposure, and changes in biochemistry of the brain was examined after these tests. The results show that systemic LPS exposure resulted in impaired sensorimotor behavioral performance, reduction of oligodendrocytes and mitochondrial activity, and increases in lipid peroxidation and brain inflammation, as indicated by the increment of interleukin-1ß (IL-1ß) levels and number of activated microglia in the neonatal rat brain. Pioglitazone treatment significantly improved LPS-induced neurobehavioral and physiological disturbances including the loss of body weight, hypothermia, righting reflex, wire-hanging maneuver, negative geotaxis, and hind-limb suspension in neonatal rats. The neuroprotective effect of pioglitazone against the loss of oligodendrocytes and mitochondrial activity was associated with attenuation of LPS-induced increment of thiobarbituric acid reactive substances (TBARS) content, IL-1ß levels and number of activated microglia in neonatal rats. Our results show that pioglitazone prevents neurobehavioral disturbances induced by systemic LPS exposure in neonatal rats, and its neuroprotective effects are associated with its impact on microglial activation, IL-1ß induction, lipid peroxidation, oligodendrocyte production and mitochondrial activity.
Assuntos
Comportamento Animal , Encefalite/tratamento farmacológico , Mitocôndrias/patologia , Pioglitazona/uso terapêutico , Substância Branca/patologia , Animais , Animais Recém-Nascidos , Comportamento Animal/efeitos dos fármacos , Citocinas/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Encefalite/patologia , Feminino , Hipotermia Induzida , Lipopolissacarídeos , Microglia/efeitos dos fármacos , Microglia/patologia , Mitocôndrias/efeitos dos fármacos , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/patologia , Pioglitazona/farmacologia , Gravidez , Ratos Sprague-Dawley , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Redução de Peso/efeitos dos fármacos , Substância Branca/efeitos dos fármacosRESUMO
In this study, we investigated the effects of minocycline, a putative suppressor of microglial activation, on systemic lipopolysaccharide (LPS)-induced spinal cord inflammation, allodynia, and hyperalgesia in neonatal rats. Intraperitoneal (i.p.) injection of LPS (2 mg/kg) or sterile saline was performed in postnatal day 5 (P5) rat pups and minocycline (45 mg/kg) or vehicle (phosphate buffer saline; PBS) was administered (i.p.) 5 min after LPS injection. The von Frey filament and tail-flick tests were performed to determine mechanical allodynia (a painful sensation caused by innocuous stimuli, e.g., light touch) and thermal hyperalgesia (a condition of altered perception of temperature), respectively, and spinal cord inflammation was examined 24 h after the administration of drugs. Systemic LPS administration resulted in a reduction of tactile threshold in the von Frey filament tests and pain response latency in the tail-flick test of neonatal rats. The levels of microglia and astrocyte activation, pro-inflammatory cytokine interleukin-1ß (IL-1ß), cyclooxygenase-2 (COX-2), and prostaglandin E2 (PGE2) in the spinal cord of neonatal rats were increased 24 h after the administration of LPS. Treatment with minocycline significantly attenuated LPS-induced allodynia, hyperalgesia, the increase in spinal cord microglia, and astrocyte activation, and elevated levels of IL-1ß, COX-2, and PGE2 in neonatal rats. These results suggest that minocycline provides protection against neonatal systemic LPS exposure-induced enhanced pain sensitivity (allodynia and hyperalgesia), and that the protective effects may be associated with its ability to attenuate LPS-induced microglia activation, and the levels of IL-1ß, COX-2, and PGE2 in the spinal cord of neonatal rats.
Assuntos
Antibacterianos/uso terapêutico , Hiperalgesia/tratamento farmacológico , Minociclina/uso terapêutico , Animais , Antibacterianos/farmacologia , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Feminino , Hiperalgesia/etiologia , Inflamação , Interleucina-1beta/metabolismo , Lipopolissacarídeos/toxicidade , Masculino , Minociclina/farmacologia , Ratos , Ratos Sprague-Dawley , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Medula Espinal/patologiaRESUMO
Neonatal lipopolysaccharide (LPS) exposure-induced brain inflammation resulted in motor dysfunction and brain dopaminergic neuronal injury, and increased the risks of neurodegenerative disorders in adult rats. Our previous studies showed that intranasal administration of insulin-like growth factor-1 (IGF-1) protects against LPS-induced white matter injury in the developing rat brain. To further examine whether IGF-1 protects against LPS-induced brain neuronal injury and neurobehavioral dysfunction, recombinant human IGF-1 (rhIGF-1) at a dose of 50 µg/pup was administered intranasally 1 h following intracerebral injection of LPS (1 mg/kg) in postnatal day 5 (P5) Sprague-Dawley rat pups. Neurobehavioral tests were carried out from P7 to P21, and brain neuronal injury was examined at P21. Our results showed that LPS exposure resulted in disturbances of motor behaviors in juvenile rats. Moreover, LPS exposure caused injury to central catecholaminergic neurons, as indicated by a reduction of tyrosine hydroxylase (TH) immunoreactivity in the substantia nigra (SN), ventral tegmental area (VTA) and olfactory bulb (OB), and brain noradrenergic neurons, as indicated by a reduction of TH immunoreactivity in the locus coeruleus (LC) of the P21 rat brain. The LPS-induced reduction of TH+ cells was observed at a greater degree in the SN and LC of the P21 rat brain. Intranasal rhIGF-1 treatment attenuated LPS-induced central catecholaminergic neuronal injury and motor behavioral disturbances, including locomotion, beam walking test and gait analysis. Intranasal rhIGF-1 administration also attenuated LPS-induced elevation of IL-1ß levels and numbers of activated microglia, and cyclooxygenase-2+ cells, which were double labeled with TH+ cells in the SN, VTA, OB and LC of the P21 rat brain. These results suggest that IGF-1 may provide protection against neonatal LPS exposure-induced central catecholaminergic neuronal injury and motor behavioral disturbances, and that the protective effects are associated with the inhibition of microglia activation and the reduction of neuronal oxidative stress by the suppression of the neuronal cyclooxygenase-2 expression.
Assuntos
Neurônios Dopaminérgicos/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/farmacologia , Locus Cerúleo/efeitos dos fármacos , Substância Negra/efeitos dos fármacos , Administração Intranasal , Envelhecimento , Animais , Animais Recém-Nascidos , Comportamento Animal/efeitos dos fármacos , Encéfalo/metabolismo , Lesões Encefálicas/tratamento farmacológico , Lesões Encefálicas/metabolismo , Neurônios Dopaminérgicos/metabolismo , Feminino , Lipopolissacarídeos/farmacologia , Locus Cerúleo/metabolismo , Atividade Motora/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Ratos Sprague-Dawley , Substância Negra/metabolismoRESUMO
The hematopoietic growth factor erythropoietin (EPO) has been shown to be neuroprotective against hypoxia-ischemia (HI) in Postnatal Day 7 (P7)-P10 or adult animal models. The current study was aimed to determine whether EPO also provides long-lasting neuroprotection against HI in P5 rats, which is relevant to immature human infants. Sprague-Dawley rats at P5 were subjected to right common carotid artery ligation followed by an exposure to 6% oxygen with balanced nitrogen for 1.5 h. Human recombinant EPO (rEPO, at a dose of 5 units/g) was administered intraperitoneally one hour before or immediately after insult, followed by additional injections at 24 and 48 h post-insult. The control rats were injected with normal saline following HI. Neurobehavioral tests were performed on P8 and P20, and brain injury was examined on P21. HI insult significantly impaired neurobehavioral performance including sensorimotor, locomotor activity and cognitive ability on the P8 and P20 rats. HI insult also resulted in brain inflammation (as indicated by microglia activation) and neuronal death (as indicated by Jade B positive staining) in the white matter, striatum, cortex, and hippocampal areas of the P21 rat. Both pre- and post-treatment with rEPO significantly improved neurobehavioral performance and protected against the HI-induced neuronal death, microglia activation (OX42+) as well as loss of mature oligodendrocytes (APC-CC1+) and hippocampal neurons (Nissl+). The long-lasting protective effects of rEPO in the neonatal rat HI model suggest that to exert neurotrophic activity in the brain might be an effective approach for therapeutic treatment of neonatal brain injury induced by hypoxia-ischemia.
Assuntos
Eritropoetina/uso terapêutico , Hipocampo/fisiopatologia , Hipóxia-Isquemia Encefálica/tratamento farmacológico , Transtornos Motores/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Eritropoetina/farmacologia , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/crescimento & desenvolvimento , Humanos , Hipóxia-Isquemia Encefálica/complicações , Locomoção , Transtornos Motores/etiologia , Neurônios/metabolismo , Neurônios/patologia , Fármacos Neuroprotetores/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Serotonin (5-hydroxytryptamine, 5-HT) has been implicated to play critical roles in early neural development. Recent reports have suggested that perinatal exposure to selective serotonin reuptake inhibitors (SSRIs) resulted in cortical network miswiring, abnormal social behavior, callosal myelin malformation, as well as oligodendrocyte (OL) pathology in rats. To gain further insight into the cellular and molecular mechanisms underlying SSRIs-induced OL and myelin abnormalities, we investigated the effect of 5-HT exposure on OL development, cell death, and myelination in cell culture models. First, we showed that 5-HT receptor 1A and 2A subtypes were expressed in OL lineages, using immunocytochemistry, Western blot, as well as intracellular Ca(2+) measurement. We then assessed the effect of serotonin exposure on the lineage development, expression of myelin proteins, cell death, and myelination, in purified OL and neuron-OL myelination cultures. For pure OL cultures, our results showed that 5-HT exposure led to disturbance of OL development, as indicated by aberrant process outgrowth and reduced myelin proteins expression. At higher doses, such exposure triggered a development-dependent cell death, as immature OLs exhibited increasing susceptibility to 5-HT treatment compared to OL progenitor cells (OPC). We showed further that 5-HT-induced immature OL death was mediated at least partially via 5-HT2A receptor, since cell death could be mimicked by 5-HT2A receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride, (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride, but atten-uated by pre-treatment with 5-HT2A receptor antagonist ritanserin. Utilizing a neuron-OL myelination co-culture model, our data showed that 5-HT exposure significantly reduced the number of myelinated internodes. In contrast to cell injury observed in pure OL cultures, 5-HT exposure did not lead to OL death or reduced OL density in neuron-OL co-cultures. However, abnormal patterns of contactin-associated protein (Caspr) clustering were observed at the sites of Node of Ranvier, suggesting that 5-HT exposure may affect other axon-derived factors for myelination. In summary, this is the first study to demonstrate that manipulation of serotonin levels affects OL development and myelination, which may contribute to altered neural connectivity noted in SSRIs-treated animals. The current in vitro study demonstrated that exposure to high level of serotonin (5-HT) led to aberrant oligodendrocyte (OL) development, cell injury, and myelination deficit. We propose that elevated extracellular serotonin levels in the fetal brain, such as upon the use of selective serotonin reuptake inhibitors (SSRIs) during pregnancy, may adversely affect OL development and/or myelination, thus contributing to altered neural connectivity seen in Autism Spectrum Disorders. OPC = oligodendrocyte progenitor cell.
Assuntos
Linhagem da Célula/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Oligodendroglia/efeitos dos fármacos , Serotonina/efeitos adversos , Animais , Morte Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Relação Dose-Resposta a Droga , Embrião de Mamíferos , Células-Tronco Embrionárias/efeitos dos fármacos , Feminino , Proteínas da Mielina/genética , Proteínas da Mielina/metabolismo , Bainha de Mielina/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Oligodendroglia/metabolismo , Gravidez , Ratos , Ratos Sprague-Dawley , Receptor 5-HT1A de Serotonina/metabolismo , Receptor 5-HT2A de Serotonina/metabolismoRESUMO
Our previous study showed that a single lipopolysaccharide (LPS) treatment to neonatal rats could induce a long-lasting neuroinflammatory response and dopaminergic system injury late in life. This is evidenced by a sustained activation of microglia and elevated interleukin-1ß (IL-1ß) levels, as well as reduced tyrosine hydroxylase (TH) expression in the substantia nigra (SN) of P70 rat brain. The object of the current study was to test whether co-administration of IL-1 receptor antagonist (IL-1ra) protects against LPS-induced neurological dysfunction later in life. LPS (1 mg/kg) with or without IL-1ra (0.1 mg/kg), or sterile saline was injected intracerebrally into postnatal day 5 (P5) Sprague-Dawley male rat pups. Motor behavioral tests were carried out from P7 to P70 with subsequent examination of brain injury. Our results showed that neonatal administration of IL-1ra significantly attenuated LPS-induced motor behavioral deficits, loss of TH immunoreactive neurons, as well as microglia activation in the SN of P70 rats. These data suggest that IL-1ß may play a pivotal role in mediating a chronic neuroinflammation status by a single LPS exposure in early postnatal life, and blockading IL-1ß might be a novel approach to protect the dopaminergic system against perinatal infection/inflammation exposure.
Assuntos
Neurônios Dopaminérgicos/efeitos dos fármacos , Proteína Antagonista do Receptor de Interleucina 1/farmacologia , Lipopolissacarídeos/farmacologia , Fármacos Neuroprotetores/farmacologia , Transtornos Psicomotores/prevenção & controle , Animais , Animais Recém-Nascidos , Neurônios Dopaminérgicos/imunologia , Complexo I de Transporte de Elétrons/metabolismo , Locomoção , Masculino , Microglia/imunologia , Microglia/metabolismo , Transtornos Psicomotores/imunologia , Ratos Sprague-Dawley , Substância Negra/efeitos dos fármacos , Substância Negra/imunologia , Substância Negra/patologiaRESUMO
Neonatal brain inflammation produced by intraperitoneal (i.p.) injection of lipopolysaccharide (LPS) results in long-lasting brain dopaminergic injury and motor disturbances in adult rats. The goal of the present work is to investigate the effect of neonatal systemic LPS exposure (1 or 2 mg/kg, i.p. injection in postnatal day 5, P5, male rats)-induced dopaminergic injury to examine methamphetamine (METH)-induced behavioral sensitization as an indicator of drug addiction. On P70, subjects underwent a treatment schedule of 5 once daily subcutaneous (s.c.) administrations of METH (0.5 mg/kg) (P70-P74) to induce behavioral sensitization. Ninety-six hours following the 5th treatment of METH (P78), the rats received one dose of 0.5 mg/kg METH (s.c.) to reintroduce behavioral sensitization. Hyperlocomotion is a critical index caused by drug abuse, and METH administration has been shown to produce remarkable locomotor-enhancing effects. Therefore, a random forest model was used as the detector to extract the feature interaction patterns among the collected high-dimensional locomotor data. Our approaches identified neonatal systemic LPS exposure dose and METH-treated dates as features significantly associated with METH-induced behavioral sensitization, reinstated behavioral sensitization, and perinatal inflammation in this experimental model of drug addiction. Overall, the analysis suggests that the implementation of machine learning strategies is sensitive enough to detect interaction patterns in locomotor activity. Neonatal LPS exposure also enhanced METH-induced reduction of dopamine transporter expression and [3H]dopamine uptake, reduced mitochondrial complex I activity, and elevated interleukin-1ß and cyclooxygenase-2 concentrations in the P78 rat striatum. These results indicate that neonatal systemic LPS exposure produces a persistent dopaminergic lesion leading to a long-lasting change in the brain reward system as indicated by the enhanced METH-induced behavioral sensitization and reinstated behavioral sensitization later in life. These findings indicate that early-life brain inflammation may enhance susceptibility to drug addiction development later in life, which provides new insights for developing potential therapeutic treatments for drug addiction.
Assuntos
Animais Recém-Nascidos , Lipopolissacarídeos , Aprendizado de Máquina , Metanfetamina , Animais , Metanfetamina/farmacologia , Metanfetamina/toxicidade , Ratos , Masculino , Lipopolissacarídeos/toxicidade , Comportamento Animal/efeitos dos fármacos , Estimulantes do Sistema Nervoso Central/farmacologia , Encefalite/induzido quimicamente , Encefalite/metabolismo , Doenças Neuroinflamatórias/tratamento farmacológico , Doenças Neuroinflamatórias/induzido quimicamente , Doenças Neuroinflamatórias/metabolismo , Locomoção/efeitos dos fármacos , Locomoção/fisiologia , Feminino , Ratos Sprague-Dawley , Atividade Motora/efeitos dos fármacosRESUMO
Brain inflammation via intracerebral injection with lipopolysaccharide (LPS) in early life has been shown to increase risks for the development of neurodegenerative disorders in adult rats. To determine if neonatal systemic LPS exposure has the same effects on enhancement of adult dopaminergic neuron susceptibility to rotenone neurotoxicity as centrally injected LPS does, LPS (2 µg/g body weight) was administered intraperitoneally into postnatal day 5 (P5) rats and when grown to P70, rats were challenged with rotenone, a commonly used pesticide, through subcutaneous minipump infusion at a dose of 1.25 mg/kg/day for 14 days. Systemically administered LPS can penetrate into the neonatal rat brain and cause acute and chronic brain inflammation, as evidenced by persistent increases in IL-1ß levels, cyclooxygenase-2 expression and microglial activation in the substantia nigra (SN) of P70 rats. Neonatal LPS exposure resulted in suppression of tyrosine hydroxylase (TH) expression, but not actual death of dopaminergic neurons in the SN, as indicated by the reduced number of TH+ cells and unchanged total number of neurons (NeuN+) in the SN. Neonatal LPS exposure also caused motor function deficits, which were spontaneously recoverable by P70. A small dose of rotenone at P70 induced loss of dopaminergic neurons, as indicated by reduced numbers of both TH+ and NeuN+ cells in the SN, and Parkinson's disease (PD)-like motor impairment in P98 rats that had experienced neonatal LPS exposure, but not in those without the LPS exposure. These results indicate that although neonatal systemic LPS exposure may not necessarily lead to death of dopaminergic neurons in the SN, such an exposure could cause persistent functional alterations in the dopaminergic system and indirectly predispose the nigrostriatal system in the adult brain to be damaged by environmental toxins at an ordinarily nontoxic or subtoxic dose and develop PD-like pathological features and motor dysfunction.
Assuntos
Encéfalo/patologia , Neurônios Dopaminérgicos/patologia , Inflamação/complicações , Lipopolissacarídeos/toxicidade , Rotenona/toxicidade , Animais , Animais Recém-Nascidos , Comportamento Animal/efeitos dos fármacos , Western Blotting , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Contagem de Células , Neurônios Dopaminérgicos/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Imuno-Histoquímica , Inflamação/induzido quimicamente , Inflamação/metabolismo , Masculino , Síndromes Neurotóxicas/metabolismo , Síndromes Neurotóxicas/patologia , Síndromes Neurotóxicas/fisiopatologia , Ratos , Ratos Sprague-Dawley , Desacopladores/toxicidadeRESUMO
BACKGROUND: Cyclooxygenase-2 (COX-2) is induced in inflammatory cells in response to cytokines and pro-inflammatory molecules, suggesting that COX-2 has a role in the inflammatory process. The objective of the current study was to examine whether celecoxib, a selective COX-2 inhibitor, could ameliorate lipopolysaccharide (LPS)-induced brain inflammation, dopaminergic neuronal dysfunction and sensorimotor behavioral impairments. METHODS: Intraperitoneal (i.p.) injection of LPS (2 mg/kg) was performed in rat pups on postnatal Day 5 (P5), and celecoxib (20 mg/kg) or vehicle was administered (i.p.) five minutes after LPS injection. Sensorimotor behavioral tests were carried out 24 h after LPS exposure, and brain injury was examined on P6. RESULTS: Our results showed that LPS exposure resulted in impairment in sensorimotor behavioral performance and injury to brain dopaminergic neurons, as indicated by loss of tyrosine hydroxylase (TH) immunoreactivity, as well as decreases in mitochondria activity in the rat brain. LPS exposure also led to increases in the expression of α-synuclein and dopamine transporter proteins and enhanced [3H]dopamine uptake. Treatment with celecoxib significantly reduced LPS-induced sensorimotor behavioral disturbances and dopaminergic neuronal dysfunction. Celecoxib administration significantly attenuated LPS-induced increases in the numbers of activated microglia and astrocytes and in the concentration of IL-1ß in the neonatal rat brain. The protective effect of celecoxib was also associated with an attenuation of LPS-induced COX-2+ cells, which were double labeled with TH + (dopaminergic neuron) or glial fibrillary acidic protein (GFAP) + (astrocyte) cells. CONCLUSION: Systemic LPS administration induced brain inflammatory responses in neonatal rats; these inflammatory responses included induction of COX-2 expression in TH neurons and astrocytes. Application of the COX-2 inhibitor celecoxib after LPS treatment attenuated the inflammatory response and improved LPS-induced impairment, both biochemically and behaviorally.
Assuntos
Inibidores de Ciclo-Oxigenase 2/farmacologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Lipopolissacarídeos/toxicidade , Atividade Motora/efeitos dos fármacos , Desempenho Psicomotor/efeitos dos fármacos , Pirazóis/farmacologia , Sulfonamidas/farmacologia , Animais , Animais Recém-Nascidos , Western Blotting , Celecoxib , Complexo I de Transporte de Elétrons/fisiologia , Ensaio de Imunoadsorção Enzimática , Feminino , Imuno-Histoquímica , Inflamação/induzido quimicamente , Inflamação/psicologia , Interleucina-1beta/análise , Interleucina-1beta/metabolismo , Masculino , Mitocôndrias/metabolismo , Equilíbrio Postural/efeitos dos fármacos , Gravidez , Ratos , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Neuroblastoma is a type of malignant extracranial tumor that occurs in children. Advanced neuroblastoma, and tumors with MYCN amplification in particular, have poor prognoses. Therefore, it is important to find an effective cure for this disease. Small interfering RNA (siRNA) disrupts gene function by specifically binding to target mRNA. In this study, we used siRNA against telomerase to treat neuroblastoma, to evaluate any anti-proliferative effect on these cells. We evaluated cell viability by WST-1 assay on neuroblastoma cells treated with or without telomerase siRNA. Nuclear condensation, an indicator for apoptotic cells, was determined by DAPI labeling following siRNA treatment. The effectiveness of telomerase siRNA on altering the neuroblastoma cell cycle was detected by flow cytometry. Our results indicated that telomerase siRNA reduces the viability of neuroblastoma cells and increases the percentage of cells in the cell cycle's sub-G1 phase. We found that telomerase siRNA increases the percentage of condensed DNA in neuroblastoma cells. In conclusion, using siRNA against telomerase could be further developed as a therapy for the treatment of neuroblastoma.
Assuntos
Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Nucléolo Celular/patologia , Interferência de RNA/fisiologia , RNA Interferente Pequeno/farmacologia , Telomerase/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Citometria de Fluxo , Humanos , Neuroblastoma/patologia , Telomerase/genética , TransfecçãoRESUMO
PURPOSE: Retinoblastoma is a malignant tumor of the retina usually occurring in young children. To date, the conventional treatments for retinoblastoma have been enucleation, cryotherapy, external beam radiotherapy, or chemotherapy. Most of these treatments, however, have possible side effects, including blindness, infections, fever, gastrointestinal toxicity, and neurotoxicity. More effective treatments are therefore imperative. Gossypol has been reported as a potential inhibitor of cell proliferation in various types of cancers, such as prostate cancer, breast cancer, leukemia, and lung cancer. This study investigates the possible antiproliferative effect of gossypol on retinoblastoma. METHODS: Human retinoblastoma cells were cultured with various concentrations of gossypol and checked for cell viability with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Nuclear condensation caused by cell apoptosis was detected by staining retinoblastoma cells with 4',6-diamidino-2-phenylindole (DAPI), counting those with condensed nuclei, and determining the percentage of apoptotic cells. In addition, the stages of apoptosis and phases in cell cycles were examined with flow cytometry. The possible signal transduction pathways involved were examined with a protein array assay and western blot analysis. RESULTS: After incubation, the cell survival rate was significantly lower after treatment with 5, 10, and 20 µM of gossypol. The maximum antisurvival effect of gossypol was observed at 20 µM, and the number of apoptotic cells was higher in the preparations cultured with 10 and 20 µM of gossypol. The results in flow cytometry indicated that at concentrations of 10 and 20 µM, gossypol increased the proportion of early- and late-apoptotic retinoblastoma cells and induced cell arrest of retinoblastoma cells at the same concentrations. This antiproliferative effect was later confirmed by upregulating the expression of death receptor 5 (DR5), caspase 8, caspase 9, caspase 3, cytochrome C, tumor protein 53 (p53), and second mitochondria-derived activator of caspases (Smac) in the signal transduction pathways. CONCLUSIONS: We concluded that gossypol has an antiproliferative effect on retinoblastoma cells.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Caspases/metabolismo , Gossipol/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Mitocondriais/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose , Western Blotting , Caspases/genética , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocromos c/genética , Citocromos c/metabolismo , Relação Dose-Resposta a Droga , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Mitocondriais/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Neoplasias da Retina/tratamento farmacológico , Neoplasias da Retina/metabolismo , Neoplasias da Retina/patologia , Retinoblastoma/tratamento farmacológico , Retinoblastoma/metabolismo , Retinoblastoma/patologia , Transdução de Sinais/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
Little is known about genes that induce stem cells differentiation into astrocytes. We previously described that heat shock protein 27 (HSP27) downregulation is directly related to neural differentiation under chemical induction in placenta-derived multipotent stem cells (PDMCs). Using this neural differentiation cell model, we cross-compared transcriptomic and proteomic data and selected 26 candidate genes with the same expression trends in both omics analyses. Those genes were further compared with a transcriptomic database derived from Alzheimer's disease (AD). Eighteen out of 26 candidates showed opposite expression trends between our data and the AD database. The mRNA and protein expression levels of those candidates showed downregulation of HSP27, S100 calcium-binding protein A16 (S100A16) and two other genes in our neural differentiation cell model. Silencing these four genes with various combinations showed that co-silencing HSP27 and S100A16 has stronger effects than other combinations for astrocyte differentiation. The induced astrocyte showed typical astrocytic star-shape and developed with ramified, stringy and filamentous processes as well as differentiated endfoot structures. Also, some of them connected with each other and formed continuous network. Immunofluorescence quantification of various neural markers indicated that HSP27 and S100A16 downregulation mainly drive PDMCs differentiation into astrocytes. Immunofluorescence and confocal microscopic images showed the classical star-like shape morphology and co-expression of crucial astrocyte markers in induced astrocytes, while electrophysiology and Ca2+ influx examination further confirmed their functional characteristics. In conclusion, co-silencing of S100A16 and HSP27 without chemical induction leads to PDMCs differentiation into functional astrocytes.
Assuntos
Astrócitos , Proteínas de Choque Térmico HSP27 , Células-Tronco Multipotentes , Astrócitos/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/farmacologia , Feminino , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico HSP27/farmacologia , Humanos , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/metabolismo , Placenta/citologia , Placenta/metabolismo , Gravidez , Proteômica , Proteínas S100/genética , Proteínas S100/metabolismoRESUMO
Brain inflammation in early life has been proposed to play important roles in the development of neurodegenerative disorders in adult life. To test this hypothesis, we used a neonatal rat model of lipopolysaccharide (LPS) exposure (1000 EU/g body weight, intracerebral injection on P5) to produce brain inflammation. By P70, when LPS-induced behavioral deficits were spontaneously recovered, animals were challenged with rotenone, a commonly used pesticide, through subcutaneous mini-pump infusion at a dose of 1.25 mg/kg per day for 14 days. This rotenone treatment regimen ordinarily does not produce toxic effects on behaviors in normal adult rats. Our results show that neonatal LPS exposure enhanced the vulnerability of nigrostriatal dopaminergic neurons to rotenone neurotoxicity in later life. Rotenone treatment resulted in motor neurobehavioral impairments in rats with the neonatal LPS exposure, but not in those without the neonatal LPS exposure. Rotenone induced losses of tyrosine hydroxylase immunoreactive neurons in the substantia nigra and decreased mitochondrial complex I activity in the striatum of rats with neonatal LPS exposure, but not in those without this exposure. Neonatal LPS exposure with later exposure to rotenone decreased retrogradely labeled nigrostriatal dopaminergic projecting neurons. The current study suggests that perinatal brain inflammation may enhance adult susceptibility to the development of neurodegenerative disorders triggered later on by environmental toxins at an ordinarily non-toxic or sub-toxic dose. Our model may be useful for studying mechanisms involved in the pathogenesis of nonfamilial Parkinson's disease and the development of potential therapeutic treatments.
Assuntos
Neurônios Dopaminérgicos/efeitos dos fármacos , Inseticidas/toxicidade , Lipopolissacarídeos/farmacologia , Rotenona/toxicidade , Substância Negra/citologia , Fatores Etários , Animais , Animais Recém-Nascidos , Peso Corporal/efeitos dos fármacos , Antígeno CD11b/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Neurônios Dopaminérgicos/ultraestrutura , Sinergismo Farmacológico , Complexo I de Transporte de Elétrons/metabolismo , Feminino , Infusões Subcutâneas/métodos , Masculino , Proteínas dos Microfilamentos/metabolismo , Microscopia Eletrônica de Transmissão , Atividade Motora/efeitos dos fármacos , Movimento/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Gravidez , Desempenho Psicomotor/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Comportamento Estereotipado/efeitos dos fármacos , Vibrissas/inervaçãoRESUMO
BACKGROUND: Repeated exposure to methamphetamine (METH) can cause not only neurotoxicity but also addiction. Behavioral sensitization is widely used as an animal model for the study of drug addiction. We previously reported that the µ-opioid receptor knockout mice were resistant to METH-induced behavioral sensitization but the mechanism is unknown. METHODS: The present study determined whether resistance of the µ-opioid receptor (µ-OR) knockout mice to behavioral sensitization is due to differential expression of the stimulatory G protein α subunit (Gαs) or regulators of G-protein signaling (RGS) coupled to the dopamine D1 receptor. Mice received daily intraperitoneal injections of saline or METH (10 mg/kg) for 7 consecutive days to induce sensitization. On day 11(following 4 abstinent days), mice were either given a test dose of METH (10 mg/kg) for behavioral testing or sacrificed for neurochemical assays without additional METH treatment. RESULTS: METH challenge-induced stereotyped behaviors were significantly reduced in the µ-opioid receptor knockout mice when compared with those in wild-type mice. Neurochemical assays indicated that there is a decrease in dopamine D1 receptor ligand binding and an increase in the expression of RGS4 mRNA in the striatum of METH-treated µ-opioid receptor knockout mice but not of METH-treated wild-type mice. METH treatment had no effect on the expression of Gαs and RGS2 mRNA in the striatum of either strain of mice. CONCLUSIONS: These results indicate that down-regulation of the expression of the dopamine D1 receptor and up-regulation of RGS4 mRNA expression in the striatum may contribute to the reduced response to METH-induced stereotypy behavior in µ-opioid receptor knockout mice. Our results highlight the interactions of the µ-opioid receptor system to METH-induced behavioral responses by influencing the expression of RGS of dopamine D1 receptors.
Assuntos
Corpo Estriado/efeitos dos fármacos , Dopamina/metabolismo , Metanfetamina/farmacologia , Receptores de Dopamina D1/metabolismo , Receptores Opioides mu/genética , Animais , Comportamento Animal/efeitos dos fármacos , Sensibilização do Sistema Nervoso Central/efeitos dos fármacos , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas RGS/genética , Proteínas RGS/metabolismo , Receptores de Dopamina D1/genética , Receptores Opioides mu/metabolismo , Comportamento Estereotipado/efeitos dos fármacosRESUMO
Our previous studies have shown that neonatal exposure to lipopolysaccharide (LPS) resulted in motor dysfunction and dopaminergic neuronal injury in the juvenile rat brain. To further examine whether neonatal LPS exposure has persisting effects in adult rats, motor behaviors were examined from postnatal day 7 (P7) to P70 and brain injury was determined in P70 rats following an intracerebral injection of LPS (1 mg/kg) in P5 Sprague-Dawley male rats. Although neonatal LPS exposure resulted in hyperactivity in locomotion and stereotyped tasks, and other disturbances of motor behaviors, the impaired motor functions were spontaneously recovered by P70. On the other hand, neonatal LPS-induced injury to the dopaminergic system such as the loss of dendrites and reduced tyrosine hydroxylase immunoreactivity in the substantia nigra persisted in P70 rats. Neonatal LPS exposure also resulted in sustained inflammatory responses in the P70 rat brain, as indicated by an increased number of activated microglia and elevation of interleukin-1ß and interleukin-6 content in the rat brain. In addition, when challenged with methamphetamine (METH, 0.5 mg/kg) subcutaneously, rats with neonatal LPS exposure had significantly increased responses in METH-induced locomotion and stereotypy behaviors as compared to those without LPS exposure. These results indicate that although neonatal LPS-induced neurobehavioral impairment is spontaneously recoverable, the LPS exposure-induced persistent injury to the dopaminergic system and the chronic inflammation may represent the existence of silent neurotoxicity. Our data further suggest that the compromised dendritic mitochondrial function might contribute, at least partially, to the silent neurotoxicity.
Assuntos
Animais Recém-Nascidos/fisiologia , Encéfalo/patologia , Dopamina/fisiologia , Lipopolissacarídeos/farmacologia , Neurônios/patologia , Síndromes Neurotóxicas/patologia , Animais , Comportamento Animal/fisiologia , Estimulantes do Sistema Nervoso Central , Citocinas/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Feminino , Membro Anterior/fisiologia , Imuno-Histoquímica , Metanfetamina , Atividade Motora/efeitos dos fármacos , Destreza Motora/fisiologia , Estimulação Física , Gravidez , Ratos , Ratos Sprague-Dawley , Comportamento Estereotipado/efeitos dos fármacos , Vibrissas/inervação , Vibrissas/fisiologiaRESUMO
Intrauterine growth restriction (IUGR) is a major cause of morbidity and mortality and is worldwide associated with delayed neurodevelopment. The exact mechanism involved in delayed neurodevelopment associated with IUGR is still unclear. Reduced uterine perfusion (RUP) is among the main causes of placental insufficiency leading to IUGR, which is associated with increases in oxidative stress. This study investigated whether oxidative stress is associated with delayed neurodevelopment in IUGR rat pups. Pregnant rats were exposed to RUP surgery on gestational day 14 to generate IUGR rat offspring. We evaluated offspring's morphometric at birth, and neurodevelopment on postnatal day 21 (PD21) as well as markers of oxidative stress in plasma and brain. Offspring from dams exposed to RUP showed significant (p < 0.05) lower birth weight compared to controls, indicating IUGR. Motor and cognitive deficits, and levels of oxidative stress markers, were significantly (p < 0.05) elevated in IUGR offspring compared to controls. IUGR offspring showed significant (p < 0.05) negative correlations between brain lipid peroxidation and neurocognitive tests (open field and novel object recognition) in comparison with controls. Our findings suggest that neurodevelopmental delay observed in IUGR rat offspring is associated with increased levels of oxidative stress markers.
RESUMO
Repeated administration of psychostimulants to rodents can lead to behavioral sensitization. Previous studies, using nonspecific opioid receptor (OR) antagonists, revealed that ORs were involved in modulation of behavioral sensitization to methamphetamine (METH). However, the contribution of OR subtypes remains unclear. In the present study, using mu-OR knockout mice, we examined the role of mu-OR in the development of METH sensitization. Mice received daily intraperitoneal injection of drug or saline for 7 consecutive days to initiate sensitization. To express sensitization, animals received one injection of drug (the same as for initiation) or saline on day 11. Animal locomotor activity and stereotypy were monitored during the periods of initiation and expression of sensitization. Also, the concentrations of METH and its active metabolite amphetamine in the blood were measured after single and repeated administrations of METH. METH promoted significant locomotor hyperactivity at low doses and stereotyped behaviors at relative high doses (2.5 mg/kg and above). Repeated administration of METH led to the initiation and expression of behavioral sensitization in wild-type mice. METH-induced behavioral responses were attenuated in the mu-OR knockout mice. Haloperidol (a dopamine receptor antagonist) showed a more potent effect in counteracting METH-induced stereotypy in the mu-OR knockout mice. Saline did not induce behavioral sensitization in either genotype. No significant difference was observed in disposition of METH and amphetamine between the two genotypes. Our study indicated that the mu-opioid system is involved in modulating the development of behavioral sensitization to METH. (c) 2010 Wiley-Liss, Inc.
Assuntos
Acatisia Induzida por Medicamentos/metabolismo , Estimulantes do Sistema Nervoso Central/farmacologia , Metanfetamina/farmacologia , Receptores Opioides mu/metabolismo , Acatisia Induzida por Medicamentos/sangue , Acatisia Induzida por Medicamentos/tratamento farmacológico , Anfetamina/sangue , Animais , Estimulantes do Sistema Nervoso Central/administração & dosagem , Estimulantes do Sistema Nervoso Central/sangue , Antagonistas de Dopamina/farmacologia , Relação Dose-Resposta a Droga , Haloperidol/farmacologia , Masculino , Metanfetamina/administração & dosagem , Metanfetamina/sangue , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Receptores Opioides mu/deficiência , Receptores Opioides mu/genética , Comportamento Estereotipado/efeitos dos fármacosRESUMO
Perinatal inflammation-induced reduction in pain threshold may alter pain sensitivity to hyperalgesia or allodynia which may persist into adulthood. In this study, we investigated the anti-inflammatory protective effect of interleukin-1 receptor antagonist (IL-1ra), an anti-inflammatory cytokine, on systemic lipopolysaccharide (LPS)-induced spinal cord inflammation and oxidative stress, thermal hyperalgesia, and mechanical allodynia in neonatal rats. Intraperitoneal (i.p.) injection of LPS (2 mg/kg) or sterile saline was performed in postnatal day 5 (P5) rat pups, and IL-1ra (100 mg/kg) or saline was administered (i.p.) 5 min after LPS injection. Pain reflex behavior, spinal cord inflammation and oxidative stress were examined 24 h after LPS administration. Systemic LPS exposure led to a reduction of tactile threshold in the von Frey filament tests (mechanical allodynia) and pain response latency in the tail-flick test (thermal hyperalgesia) of P6 neonatal rats. Spinal cord inflammation was indicated by the increased numbers of activated glial cells including microglia (Iba1+) and astrocytes (GFAP+), and elevated levels of pro-inflammatory cytokine interleukin-1ß (IL-1ß), cyclooxygenase-2 (COX-2), and prostaglandin E2 (PGE2) 24 h after LPS treatment. LPS treatment induced spinal oxidative stress as evidenced by the increase in thiobarbituric acid reactive substances (TBARS) content in the spinal cord. LPS exposure also led to a significant increase in oligodendrocyte lineage population (Olig2+) and mature oligodendrocyte cells (APC+) in the neonatal rat spinal cord. IL-1ra treatment significantly reduced LPS-induced effects including hyperalgesia, allodynia, the increased number of activated microglia, astrocytes and oligodendrocytes, and elevated levels of IL-1ß, COX-2, PGE2, and lipid peroxidation (TBARS) in the neonatal rat spinal cord. These data suggest that IL-1ra provides a protective effect against the development of pain hypersensitivity, spinal cord inflammation and oxidative stress in the neonatal rats following LPS exposure, which may be associated with the blockade of LPS-induced pro-inflammatory cytokine IL-1ß.
Assuntos
Hiperalgesia/tratamento farmacológico , Proteína Antagonista do Receptor de Interleucina 1/uso terapêutico , Lipopolissacarídeos/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Receptores de Interleucina-1/antagonistas & inibidores , Medula Espinal/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Feminino , Hiperalgesia/induzido quimicamente , Hiperalgesia/metabolismo , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Proteína Antagonista do Receptor de Interleucina 1/farmacologia , Masculino , Estresse Oxidativo/fisiologia , Medição da Dor/efeitos dos fármacos , Medição da Dor/métodos , Gravidez , Ratos , Ratos Sprague-Dawley , Receptores de Interleucina-1/metabolismo , Medula Espinal/metabolismoRESUMO
PURPOSE: The cytotoxicity of single and repeated administration of bevacizumab to retinal ganglion cells was studied in vivo and in vitro. METHODS: Rats received single or repeated injections of 125 microg of bevacizumab into the vitreous cavity of the left eye, while saline was injected into the right eye as a control. In the repeated injection group, bevacizumab was injected at the same concentration once per week for 4 weeks. Retinal ganglion cells were retrogradely labeled with Fluorogold dye, and counted after kill at 0 and 6 months after treatment. In the in vitro study, PC12 cells were cultured in various concentrations of bevacizumab (0, 1, 2, 5, and 10 mg/mL) to model retinal ganglion cells exposure. Cell viability in each group was assessed by an MTT assay. RESULTS: There was no significant difference in retinal ganglion cell numbers between control and bevacizumab-treated eyes following either single or repeated injection, or did bevacizumab have any significant influence on PC12 cell viability in vitro. CONCLUSION: These results suggest that intravitreal injection of bevacizumab poses no risk to retinal ganglion cells, even after repeated application.
Assuntos
Inibidores da Angiogênese/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Células Ganglionares da Retina/efeitos dos fármacos , Animais , Anticorpos Monoclonais Humanizados , Bevacizumab , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Feminino , Seguimentos , Injeções , Ratos , Ratos Wistar , Células Ganglionares da Retina/citologia , Fator A de Crescimento do Endotélio Vascular , Corpo VítreoRESUMO
Neonatal exposure to infectious agents may result in long-term neurological disability, and is particularly associated with the subsequent development of motor and cognitive disturbances. Our previous studies have shown that treatment with alpha-phenyl-n-tert-butyl-nitrone (PBN) following exposure to lipopolysaccharide (LPS) reduces LPS-induced brain injury in the neonatal rat. To examine whether PBN has long-lasting protective effects and ameliorates LPS-induced motor and cognitive dysfunction, PBN (100 mg/kg) was administered intraperitoneally 5 min after an LPS (1 mg/kg) intracerebral injection in postnatal day 5 (P5) Sprague-Dawley rat pups. Neurobehavioral tests were carried out from P3 to P21, and brain injury was examined at 24 h and 16 days after LPS injection. Neonatal LPS exposure resulted in hyperactivity from P13 to P17 in the open field task as compared with the control rat. Neurobehavioral deficits that were still observable at P21 included dysfunction in the beam-walking and pole tests, learning and memory deficits in the passive avoidance task, and less anxiety-like response in the elevated plus-maze task. These behavioral findings were matched by LPS-induced axonal injury in the CA1 region of the middle dorsal hippocampus (HP), reduction in the size of the HP and the number of neurons in the CA1 region of the middle dorsal HP, and loss of tyrosine hydroxylase immunoreactivity in neurons in the substantia nigra and ventral tegmental areas. Treatment with PBN provided long-lasting protection against the LPS-induced axonal injury and neuronal loss, and improved the associated neurological dysfunctions in juvenile rats.