RESUMO
Amyotrophic lateral sclerosis (ALS) is a devastating disorder characterized by progressive death of motoneurons. The Wobbler (WR) mouse is a preclinical model sharing neuropathological similarities with human ALS. We have shown that progesterone (PROG) prevents the progression of motoneuron degeneration. We now studied if allopregnanolone (ALLO), a reduced metabolite of PROG endowed with gabaergic activity, also prevents WR neuropathology. Sixty-day old WRs remained untreated or received two steroid treatment regimens in order to evaluate the response of several parameters during early or prolonged steroid administration. ALLO was administered s.c. daily for 5days (4mg/kg) or every other day for 32days (3, 3mg/kg), while another group of WRs received a 20mg PROG pellet s.c. for 18 or 60days. ALLO administration to WRs increased ALLO serum levels without changing PROG and 5 alpha dihydroprogesterone (5α-DHP), whereas PROG treatment increased PROG, 5α-DHP and ALLO. Untreated WRs showed higher basal levels of serum 5α-DHP than controls. In the cervical spinal cord we studied markers of oxidative stress or associated to trophic responses. These included nitric oxide synthase (NOS) activity, motoneuron vacuolation, MnSOD immunoreactivity (IR), brain derived neurotrophic factor (BDNF) and TrkB mRNAs, p75 neurotrophin receptor (p75NTR) and, cell survival or death signals such as pAKT and the stress activated kinase JNK. Untreated WRs showed a reduction of MnSOD-IR and BDNF/TrkB mRNAs, associated to high p75NTR in motoneurons, neuronal and glial NOS hyperactivity and neuronal vacuolation. Also, low pAKT, mainly in young WRs, and a high pJNK in the old stage characterized WRs spinal cord. Except for MnSOD and BDNF, these alterations were prevented by an acute ALLO treatment, while short-term PROG elevated MnSOD. Moreover, after chronic administration both steroids enhanced MnSOD-IR and BDNF mRNA, while attenuated pJNK and NOS in glial cells. Long-term PROG also increased pAKT and reduced neuronal NOS, parameters not modulated by chronic ALLO. Clinically, both steroids improved muscle performance. Thus, ALLO was able to reduce neuropathology in this model. Since high oxidative stress activates p75NTR and pJNK in neurodegeneration, steroid reduction of these molecules may provide adequate neuroprotection. These data yield the first evidence that ALLO, a gabaergic neuroactive steroid, brings neuroprotection in a model of motoneuron degeneration.
Assuntos
Degeneração Neural/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Pregnanolona/uso terapêutico , Esclerose Lateral Amiotrófica , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Colina O-Acetiltransferase/metabolismo , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/patologia , Degeneração Neural/genética , Degeneração Neural/metabolismo , Fármacos Neuroprotetores/sangue , Fármacos Neuroprotetores/farmacologia , Óxido Nítrico Sintase/metabolismo , Pregnanolona/sangue , Pregnanolona/farmacologia , Progesterona/sangue , Progesterona/farmacologia , Progesterona/uso terapêutico , Receptor trkB/genética , Receptores de Fator de Crescimento Neural/metabolismo , Medula Espinal/metabolismo , Medula Espinal/patologia , Superóxido Dismutase/metabolismoRESUMO
Neuroinflammation involves the activation of glial cells, which is associated to the progression of neurodegeneration in Parkinson's disease. Recently, we and other researchers demonstrated that dopamine receptor D3 (D3R)-deficient mice are completely refractory to neuroinflammation and consequent neurodegeneration associated to the acute intoxication with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In this study we examined the therapeutic potential and underlying mechanism of a D3R-selective antagonist, PG01037, in mice intoxicated with a chronic regime of administration of MPTP and probenecid (MPTPp). Biodistribution analysis indicated that intraperitoneally administered PG01037 crosses the blood-brain barrier and reaches the highest concentration in the brain 40 min after the injection. Furthermore, the drug was preferentially distributed to the brain in comparison to the plasma. Treatment of MPTPp-intoxicated mice with PG01037 (30 mg/kg, administrated twice a week for five weeks) attenuated the loss of dopaminergic neurons in the substantia nigra pars compacta, as evaluated by stereological analysis, and the loss of striatal dopaminergic terminals, as determined by densitometric analyses of tyrosine hydroxylase and dopamine transporter immunoreactivities. Accordingly, the treatment resulted in significant improvement of motor performance of injured animals. Interestingly, the therapeutic dose of PG01037 exacerbated astrogliosis and resulted in increased ramification density of microglial cells in the striatum of MPTPp-intoxicated mice. Further analyses suggested that D3R expressed in astrocytes favours a beneficial astrogliosis with anti-inflammatory consequences on microglia. Our findings indicate that D3R-antagonism exerts a therapeutic effect in parkinsonian animals by reducing the loss of dopaminergic neurons in the nigrostriatal pathway, alleviating motor impairments and modifying the pro-inflammatory phenotype of glial cells.
Assuntos
Benzamidas/administração & dosagem , Neurônios Dopaminérgicos/efeitos dos fármacos , Encefalite/prevenção & controle , Fármacos Neuroprotetores/administração & dosagem , Transtornos Parkinsonianos/prevenção & controle , Piridinas/administração & dosagem , Receptores de Dopamina D3/antagonistas & inibidores , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Benzamidas/sangue , Benzamidas/farmacologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/patologia , Neurônios Dopaminérgicos/patologia , Encefalite/etiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Atividade Motora/efeitos dos fármacos , Fármacos Neuroprotetores/sangue , Fármacos Neuroprotetores/farmacologia , Doença de Parkinson/complicações , Doença de Parkinson/prevenção & controle , Transtornos Parkinsonianos/complicações , Parte Compacta da Substância Negra/efeitos dos fármacos , Parte Compacta da Substância Negra/patologia , Piridinas/sangue , Piridinas/farmacologia , Receptores de Dopamina D3/metabolismoRESUMO
A rapid, sensitive and specific method for quantifying piracetam in human plasma using Piracetam d-8 as the internal standard (IS) is described. The analyte and the IS were extracted from plasma by one-step precipitation of protein using an acetonitrile (100%). The extracts were analyzed by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry (HPLC-MS/MS). The method had a chromatographic run time of 3.8 min and a linear calibration curve over the range 0.5-50 µg/mL (r > 0.99). This LC-MS-MS procedure was used to assess the bioavailability of two piracetam formulations: piracetam + l-carnitine (Piracar®; 270/330 mg tablet) and piracetam (Nootropil®; 800 mg tablet) in healthy volunteers of both sexes. The geometric means with corresponding 90% confidence interval (CI) for test/reference percentage ratios were 88.49% (90% CI = 81.19 - 96.46) for peak concentration/dose and 102.55% (90% CI = 100.62 - 104.51) for AUCinf /dose. The limit of quantitation of 0.5 µg/mL is well suited for pharmacokinetic studies in healthy volunteers. It was concluded that piracetam (Piracar®; 270/330 mg tablet) has a bioavailability equivalent to the piracetam (Nootropil®; 800 mg tablet) formulation with regard to both the rate and the extent of absorption.
Assuntos
Carnitina/sangue , Fármacos Neuroprotetores/sangue , Piracetam/sangue , Administração Oral , Adolescente , Adulto , Disponibilidade Biológica , Carnitina/administração & dosagem , Cromatografia Líquida de Alta Pressão/métodos , Feminino , Voluntários Saudáveis , Humanos , Limite de Detecção , Masculino , Pessoa de Meia-Idade , Fármacos Neuroprotetores/administração & dosagem , Piracetam/administração & dosagem , Espectrometria de Massas por Ionização por Electrospray/métodos , Comprimidos , Espectrometria de Massas em Tandem/métodos , Adulto JovemRESUMO
Flavonoids have known anti-inflammatory and antioxidative actions, and they have been described as neuroprotective and able to reduce damage in CNS diseases. We evaluated the action of the flavonoid rutin in an animal model of focal cortical ischemia induced by unilateral thermocoagulation of superficial blood vessels of motor (M1) and somatosensory (S1) primary cortices. Ischemic rats were submitted to daily injections (i.p.) for five days, starting immediately after induction of ischemia. We tested two doses: 50mg/kg or 100mg/kg of body weight. Sensorimotor tests were used to evaluate functional recovery. Bioavailability in plasma was done by chromatographic analysis. The effect of treatment in lesion volume and neurodegeneration was evaluated 48 h and 72 h after ischemia, respectively. We observed significant sensorimotor recovery induced by rutin, and the dose of 50mg/kg had more pronounced effect. Thus, this dose was used in further analyses. Plasma availability of rutin was detected from 2h to at least 8h after ischemia. The treatment did not result in reduction of lesion volume but reduced the number of degenerated neurons at the periphery of the lesion. The results suggest rutin as an efficient drug to treat brain ischemia since it was able to promote significant recovery of sensorimotor loss, which was correlated to the reduction of neurodegeneration in the periphery of cortical injury. Increasing studies with rutin and other flavonoids might give support for further clinical trials with these drugs.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/patologia , Córtex Cerebral/patologia , Fármacos Neuroprotetores/uso terapêutico , Recuperação de Função Fisiológica/efeitos dos fármacos , Rutina/uso terapêutico , Análise de Variância , Animais , Cromatografia Líquida de Alta Pressão , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Fluoresceínas , Masculino , Força Muscular/efeitos dos fármacos , Fármacos Neuroprotetores/sangue , Fármacos Neuroprotetores/farmacologia , Desempenho Psicomotor/efeitos dos fármacos , Ratos , Ratos Wistar , Rutina/sangue , Rutina/farmacologia , Fatores de TempoRESUMO
It is concluded from immunohistochemical that all four types of prostaglandin-E(2) (PGE(2)) (EP1, EP2, EP3 and EP4) receptors are associated with specific cell-types in primary rat retinal cultures. Analysis specifically of EP2 receptor immunoreactivity shows it to coexist with some neurones expressing Thy-1 and calbindin immunoreactivities as well as with vimentin-positive Müller cells. Moreover, exposure of cultures to the EP2 specific agonist butaprost (100 nM) for a period of 24h results in a generation of cAMP thus providing support for the functionality of EP2 receptors. Cell survival was significantly affected in cultures where the serum concentration was reduced from 10 to 1% for 24h. This was reflected by a reduction in the number of GABA-positive neurons and an elevation of released lactate dehydrogenase (LDH) into the culture medium. Moreover, a number of cells displayed a clear generation of reactive oxygen species (ROS) and a staining for the breakdown of DNA by the TUNEL procedure as an indicator for apoptosis. These negative effects were attenuated when butaprost (100 nM) was present during the serum reduction and 30 min before the insult. The present studies provide evidence to show that all PGE(2) receptor types exist in the retina of rat pups, remain functional when the retinal cells are cultured and that specific activation of EP2 receptors with butaprost can attenuate a detrimental insult caused by insufficient serum that may occur in situ by reduced trophic support.
Assuntos
Alprostadil/análogos & derivados , Dinoprostona/agonistas , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Receptores de Prostaglandina E/agonistas , Retina/efeitos dos fármacos , Alprostadil/sangue , Alprostadil/farmacologia , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Calbindinas , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Técnicas de Cocultura , Meios de Cultura Livres de Soro/farmacologia , AMP Cíclico/metabolismo , Dano ao DNA/efeitos dos fármacos , Dinoprostona/metabolismo , Marcação In Situ das Extremidades Cortadas , L-Lactato Desidrogenase/metabolismo , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Fármacos Neuroprotetores/sangue , Ratos , Espécies Reativas de Oxigênio/metabolismo , Receptores de Prostaglandina E/metabolismo , Retina/citologia , Retina/metabolismo , Proteína G de Ligação ao Cálcio S100/metabolismo , Antígenos Thy-1/metabolismo , Vimentina/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
Glucose is the main substrate that fulfills energy brain demands. However, in some circumstances, such as diabetes, starvation, during the suckling period and the ketogenic diet, brain uses the ketone bodies, acetoacetate and beta-hydroxybutyrate, as energy sources. Ketone body utilization in brain depends directly on its blood concentration, which is normally very low, but increases substantially during the conditions mentioned above. Glutamate neurotoxicity has been implicated in neurodegeneration associated with brain ischemia, hypoglycemia and cerebral trauma, conditions related to energy failure, and to elevation of glutamate extracellular levels in brain. In recent years substantial evidence favoring a close relation between glutamate neurotoxic potentiality and cellular energy levels, has been compiled. We have previously demonstrated that accumulation of extracellular glutamate after inhibition of its transporters, induces neuronal death in vivo during energy impairment induced by glycolysis inhibition. In the present study we have assessed the protective potentiality of the ketone body, acetoacetate, against glutamate-mediated neuronal damage in the hippocampus of rats chronically treated with the glycolysis inhibitor, iodoacetate, and in hippocampal cultured neurons exposed to a toxic concentration of iodoacetate. Results show that acetoacetate efficiently protects against glutamate neurotoxicity both in vivo and in vitro probably by a mechanism involving its role as an energy substrate.