RESUMEN
The neurotoxin MPTP is known to induce dopamine release and depletion of ATP in the striatum of rats. Therefore, we studied the changes induced by MPTP and pargyline protection both on striatal dopamine release and on extracellular energy metabolites in freely moving rats, using dual asymmetric-flow microdialysis. A dual microdialysis probe was inserted in the right striatum of rats. MPTP (25mg/kg, 15mg/kg, 10mg/kg) was intraperitoneally administered for three consecutive days. MAO-B inhibitor pargyline (15mg/kg) was systemically administered before neurotoxin administration. The first MPTP dose induced an increase in dialysate dopamine and a decrease of DOPAC levels in striatal dialysate. After the first neurotoxin administration, increases in striatal glucose, lactate, pyruvate, lactate/pyruvate (L/P) and lactate/glucose (L/G) ratios were observed. Subsequent MPTP administrations showed a progressive reduction of dopamine, glucose and pyruvate levels with a concomitant further increase in lactate levels and L/P and L/G ratios. At day 1, pargyline pre-treatment attenuated the MPTP-induced changes in all studied analytes. Starting from day 2, pargyline prevented the depletion of dopamine, glucose and pyruvate while reduced the increase of lactate, L/P ratio and L/G ratio. These in vivo results suggest a pargyline neuroprotection role against the MPTP-induced energetic impairment consequent to mitochondrial damage. This neuroprotective effect was confirmed by TH immunostaining of the substantia nigra.
Asunto(s)
Cuerpo Estriado/metabolismo , Dopamina/metabolismo , Metabolismo Energético/efectos de los fármacos , Intoxicación por MPTP/metabolismo , Inhibidores de la Monoaminooxidasa/uso terapéutico , Pargilina/uso terapéutico , Animales , Masculino , Ratas , Ratas WistarRESUMEN
A new telemetry system for simultaneous detection of extracellular brain glucose and lactate and motion is presented. The device consists of dual-channel, single-supply miniature potentiostat-I/V converter, a microcontroller unit, a signal transmitter, and a miniaturized microvibration sensor. Although based on simple and inexpensive components, the biotelemetry device has been used for accurate transduction of the anodic oxidation currents generated on the surface of implanted glucose and lactate biosensors and animal microvibrations. The device was characterized and validated in vitro before in vivo experiments. The biosensors were implanted in the striatum of freely moving animals and the biotelemetric device was fixed to the animal's head. Physiological and pharmacological stimulations were given in order to induce striatal neural activation and to modify the motor behavior in awake, untethered animals.
Asunto(s)
Encéfalo/metabolismo , Glucosa/metabolismo , Lactatos/metabolismo , Telemetría , Animales , Técnicas Biosensibles , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Ethyl alcohol may be considered one of the most widespread central nervous system (CNS) depressants in Western countries. Because of its toxicological and neurobiological implications, the detection of ethanol in brain extracellular fluid (ECF) is of great importance. In a previous study, we described the development and characterization of an implantable biosensor successfully used for the real-time detection of ethanol in the brain of freely-moving rats. The implanted biosensor, integrated in a low-cost telemetry system, was demonstrated to be a reliable device for the short-time monitoring of exogenous ethanol in brain ECF. In this paper we describe a further in-vitro characterization of the above-mentioned biosensor in terms of oxygen, pH and temperature dependence in order to complete its validation. With the aim of enhancing ethanol biosensor performance, different enzyme loadings were investigated in terms of apparent ethanol Michaelis-Menten kinetic parameters, viz. IMAX, KM and linear region slope, as well as ascorbic acid interference shielding. The responses of biosensors were studied over a period of 28 days. The overall findings of the present study confirm the original biosensor configuration to be the best of those investigated for in-vivo applications up to one week after implantation.
Asunto(s)
Técnicas Biosensibles/instrumentación , Química Encefálica , Etanol/análisis , Modelos Teóricos , Prótesis e Implantes , Animales , Técnicas Biosensibles/métodos , Enzimas/metabolismo , Concentración de Iones de Hidrógeno , Oxígeno , Ratas , TemperaturaRESUMEN
Ethanol is one of the most widespread psychotropic agents in western society. While its psychoactive effects are mainly associated with GABAergic and glutamatergic systems, the positive reinforcing properties of ethanol are related to activation of mesolimbic dopaminergic pathways resulting in a release of dopamine in the nucleus accumbens. Given these neurobiological implications, the detection of ethanol in brain extracellular fluid (ECF) is of great importance. In this study, we describe the development and characterization of an implantable biosensor for the amperometric detection of brain ethanol in real time. Ten different designs were characterized in vitro in terms of Michaelis-Menten kinetics (V(MAX) and K(M)), sensitivity (linear region slope, limit of detection (LOD), and limit of quantification (LOQ)), and electroactive interference blocking. The same parameters were monitored in selected designs up to 28 days after fabrication in order to quantify their stability. Finally, the best performing biosensor design was selected for implantation in the nucleus accumbens and coupled with a previously developed telemetric device for the real-time monitoring of ethanol in freely moving, untethered rats. Ethanol was then administered systemically to animals, either alone or in combination with ranitidine (an alcohol dehydrogenase inhibitor) while the biosensor signal was continuously recorded. The implanted biosensor, integrated in the low-cost telemetry system, was demonstrated to be a reliable device for the short-time monitoring of exogenous ethanol in brain ECF and represents a new generation of analytical tools for studying ethanol toxicokinetics and the effect of drugs on brain ethanol levels.
Asunto(s)
Técnicas Biosensibles/instrumentación , Encéfalo/metabolismo , Electrodos Implantados , Etanol/metabolismo , Movimiento , Telemetría/instrumentación , Animales , Encéfalo/efectos de los fármacos , Electroquímica , Etanol/administración & dosificación , Etanol/farmacología , Límite de Detección , Masculino , Ranitidina/farmacología , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
Brain microdialysis is an analytical technique used for the dynamic monitoring of brain neurochemistry in awake, freely moving animals. This technique requires the insertion of a small dialysis catheter, called a microdialysis probe, into a specific brain region, and its perfusion with an artificial extracellular fluid. The microdialysate samples, obtained from the probe outlet, can be analysed using high-performance liquid chromatography with electrochemical detection for the quantification of oxidizable molecules recovered from the extracellular space. In this chapter, we describe a protocol for performing a microdialysis setup and experiment in freely moving rats and mice. Furthermore, the high-performance liquid chromatographic determination of ascorbic acid, uric acid, catecholamines, indolamines and derivatives is described in detail.
Asunto(s)
Química Encefálica , Técnicas de Química Analítica , Líquido Extracelular/química , Microdiálisis/métodos , Animales , Ácido Ascórbico/análisis , Catecolaminas/análisis , Cromatografía Líquida de Alta Presión/métodos , Electroquímica , Indoles/análisis , Ratones , Ratas , Ácido Úrico/análisisRESUMEN
Microdialysis is an extensively used technique for both in vivo and in vitro experiments, applicable to animal and human studies. In neurosciences, the in vivo microdialysis is usually performed to follow changes in the extracellular levels of substances and to monitor neurotransmitters release in the brain of freely moving animals. Catecholamines, such as dopamine and their related compounds, are involved in the neurochemistry and in the physiology of mental diseases and neurological disorders. It is generally supposed that the brain's energy requirement is supplied by glucose oxidation. More recently, lactate was proposed to be the metabolic substrate used by neurons during synaptic activity. In our study, an innovative microdialysis approach for simultaneous monitoring of catecholamines, indolamines, glutamate and energy substrates in the striatum of freely moving rats, using an asymmetric perfusion flow rate on microdialysis probe, is described. As a result of this asymmetric perfusion, two samples are available from the same brain region, having the same analytes composition but different concentrations. The asymmetric flow perfusion could be a useful tool in neurosciences studies related to brain's energy requirement, such as toxin-induced models of Parkinson's disease.
Asunto(s)
Encéfalo/metabolismo , Microdiálisis/métodos , Neurotransmisores/metabolismo , Animales , Metabolismo Energético , Masculino , Microdiálisis/instrumentación , Neostriado/metabolismo , Perfusión , Ratas , Reproducibilidad de los Resultados , Factores de TiempoRESUMEN
The classical animal models of Parkinson's disease (PD) rely on the use of neurotoxins, including 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 6-hydroxydopamine and, more recently, the agricultural chemicals paraquat and rotenone, to deplete dopamine (DA). These neurotoxins elicit motor deficits in different animal species although MPTP fails to induce a significant dopaminergic neurodegeneration in rats. In the attempt to better reproduce the key features of PD, in particular the progressive nature of neurodegeneration, alternative PD models have been developed, based on the genetic and neuropathological links between -synuclein ( -syn) and PD. In vivo microdialysis was used to investigate extracellular striatal DA dynamics in MPTP- and -syn-generated rodent models of PD. Acute and sub-acute MPTP intoxication of mice both induce prolonged release of striatal DA. Such DA release may be considered the first step in MPTP-induced striatal DA depletion and nigral neuron death, mainly through reactive oxygen species generation. Although MPTP induces DA reduction, neurochemical and motor recovery starts immediately after the end of treatment, suggesting that compensatory mechanisms are activated. Thus, the MPTP mouse model of PD may be unsuitable for closely reproducing the features of the human disease and predicting potential long-term therapeutic effects, in terms of both striatal extracellular DA and behavioral outcome. In contrast, the -syn-generated rat model of PD does not suffer from a massive release of striatal DA during induction of the nigral lesion, but rather is characterized by a prolonged reduction in baseline DA and nicotine-induced increases in dialysate DA levels. These results are suggestive of a stable nigrostriatal lesion with a lack of dopaminergic neurochemical recovery. The -syn rat model thus reproduces the initial stage and slow development of PD, with a time-dependent impairment in motor function. This article will describe the above experimental PD models and demonstrate the utility of microdialysis for their characterization.
Asunto(s)
1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacología , Modelos Animales de Enfermedad , Dopamina/fisiología , Microdiálisis , Neurotoxinas/farmacología , Enfermedad de Parkinson/metabolismo , Trastornos Parkinsonianos/metabolismo , alfa-Sinucleína/metabolismo , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Dopamina/metabolismo , Humanos , Ratones , Ratones Transgénicos/genética , Ratones Transgénicos/metabolismo , Trastornos Parkinsonianos/inducido químicamente , Ratas , Ratas Transgénicas/genética , Ratas Transgénicas/metabolismo , alfa-Sinucleína/fisiologíaRESUMEN
Ascorbic acid (AA), one of the principal micronutrients in horticultural crops, plays a key role in the human metabolism, and its determination in food products has a great significance. Citrus fruits are rich in AA, but its content is highly susceptible to change during postharvest processing and storage. We present a new ultralow-cost system, constituted of an amperometric microsensor composed of three rod carbon electrodes connected to a telemetric device, for online detection of AA in orange juice, as an alternative to conventional analytical methods. The in vitro calibration, ranged from 0 to 5 mM, and AA juice content was calculated by adding low volumes of sample into an acetate buffer solution at a constant potential of +120 mV vs carbon pseudoreference. This new approach, which is simple, expandable, and inexpensive, seems appropriate for large scale commercial use.
Asunto(s)
Ácido Ascórbico/análisis , Citrus/química , Técnicas Electroquímicas/instrumentación , Telemetría/instrumentación , Calibración , Técnicas Electroquímicas/economía , Diseño de Equipo , Telemetría/economíaRESUMEN
Alterations in developmental programming of neuroendocrine and immune system function may critically modulate vulnerability to various diseases. In particular, genetic factors, including gender, may interact with early life events such as exposure to hormones, endotoxins, or neurotoxins, thereby influencing disease predisposition and/or severity, but little is known about the role of the astroglial cell compartment and its mediators in this phenomenon. Indeed, in the context of innate inflammatory mechanisms, a dysfunction of the astroglial cell compartment is believed to contribute to the selective degeneration of dopaminergic (DA) neurons in the substantia nigra pars compacta in Parkinson's disease (PD) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of PD. Hence, in response to brain injury the roles of astrocytes and microglia are very dynamic and cell type-dependent, in that they may exert the known proinflammatory (harmful) effects, but in certain circumstances they can turn into highly protective cells and exert anti-inflammatory (beneficial) functions, thereby facilitating neuronal recovery and repair. Here, we summarize our work suggesting a chief role of hormonal programming of glial response to inflammation and oxidative stress in MPTP-induced loss of DA neuron functionality and demonstrate that endogenous glucocorticoids and the female hormone estrogen (E(2)) inhibit the aberrant neuroinflammatory cascade, protect astrocytes and microglia from programmed cell death, and stimulate recovery of DA neuron functionality, thereby triggering the repair process. The overall results highlight glia as a final common pathway directing neuroprotection versus neurodegeneration. Such recognition of endogenous glial protective pathways may provide a new insight and may contribute to the development of novel therapeutic treatment strategies for PD and possibly other neurodegenerative disorders.
Asunto(s)
Ambiente , Predisposición Genética a la Enfermedad , Hormonas/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/metabolismo , Animales , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Encéfalo/fisiología , Dopamina/metabolismo , Estrógenos/metabolismo , Glucocorticoides/metabolismo , Humanos , Neuroglía/fisiología , Neuronas/fisiología , Neurotoxinas/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Enfermedad de Parkinson/fisiopatologíaRESUMEN
Glucocorticoids (GCs) exert via glucocorticoid receptors (GRs) potent anti-inflammatory and immunosuppressive effects. Emerging evidence indicates that an inflammatory process is involved in dopaminergic nigro-striatal neuronal loss in Parkinson's disease. We here report that the GR deficiency of transgenic (Tg) mice expressing GR antisense RNA from early embryonic life has a dramatic impact in "programming" the vulnerability of dopaminergic neurons to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The GR deficiency of Tg mice exacerbates MPTP-induced toxicity to dopaminergic neurons, as revealed by both severe loss of tyrosine hydroxylase positive nigral neurons and sharp decreases in striatal levels of dopamine and its metabolites. In addition, the late increase in dopamine oxidative metabolism and ascorbic acid oxidative status in GR-deficient mice was far greater than in wild-type (Wt) mice. Inducible nitric oxide synthase (iNOS) was sharply increased in activated astrocytes, macrophages/microglia of GR-deficient as compared with Wt mice. Moreover, GR-deficient microglia produced three- to fourfold higher nitrite levels than Wt mice; these increases preceded the loss of dopaminergic function and were resistant to GR the inhibitory effect of GC, pointing to peroxynitrites as candidate neurotoxic effectors. The iNOS inhibitor N6-(1-iminoethyl)-L-lysine normalized vulnerability of Tg mice, thus establishing a novel link between genetic impairment of GR function and vulnerability to MPTP.
Asunto(s)
Dopamina/metabolismo , Lisina/análogos & derivados , Intoxicación por MPTP/etiología , Neostriado/metabolismo , Neuroglía/enzimología , Óxido Nítrico/fisiología , Receptores de Glucocorticoides/fisiología , Sustancia Negra/metabolismo , Animales , Corticosterona/farmacología , Inhibidores Enzimáticos/farmacología , Lisina/farmacología , Intoxicación por MPTP/metabolismo , Intoxicación por MPTP/patología , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Ratones , Ratones Transgénicos , Neuroglía/efectos de los fármacos , Neuroglía/patología , Neuronas/enzimología , Neuronas/metabolismo , Neuronas/fisiología , Óxido Nítrico Sintasa/análisis , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Estrés Oxidativo , Receptores de Glucocorticoides/genética , Tirosina 3-Monooxigenasa/análisisRESUMEN
The effects of either intraperitoneally (i.p.) or intrastriatally administered sufentanil on the release and metabolism of dopamine (DA) in the rat striatum were evaluated using in vivo microdialysis. Dialysate concentrations of DA and its acidic metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were increased following i.p. administration of either clinical anesthetic (20 microg/kg) or clinical analgesic (1 microg/kg) sufentanil doses. In addition, sufentanil also increased uric acid concentrations. In contrast, dialysate ascorbic acid and glutamate concentrations were unaffected. Intrastriatal infusion of sufentanil (250 nM) induced only a short lasting decrease in dialysate DA. Subcutaneous naloxone (1.0 mg/kg) abolished sufentanil-induced increases in dialysate DA, DOPAC+HVA and uric acid; however, naloxone (0.1 mM) failed to affect these increases when infused intrastriatally. These results demonstrate that sufentanil, at clinical doses, increases striatal DA release and oxidative metabolism of both DA and xanthine acting at extrastriatal sites with a mu-receptor-mediated mechanism.
Asunto(s)
Analgésicos Opioides/farmacología , Ácido Ascórbico/metabolismo , Cuerpo Estriado/efectos de los fármacos , Dopamina/metabolismo , Ácido Glutámico/metabolismo , Sufentanilo/farmacología , Ácido 3,4-Dihidroxifenilacético/análisis , Ácido 3,4-Dihidroxifenilacético/metabolismo , Analgésicos Opioides/administración & dosificación , Animales , Ácido Ascórbico/análisis , Cuerpo Estriado/metabolismo , Dopamina/análisis , Ácido Glutámico/análisis , Ácido Homovanílico/análisis , Ácido Homovanílico/metabolismo , Inyecciones Intraperitoneales , Inyecciones Intraventriculares , Masculino , Microdiálisis , Movimiento , Naloxona/farmacología , Antagonistas de Narcóticos/farmacología , Ratas , Ratas Wistar , Sufentanilo/administración & dosificación , Ácido Úrico/análisis , Ácido Úrico/metabolismoRESUMEN
Swiss mice were given 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 25 mg/kg/day, for 5 consecutive days and killed at different days after MPTP discontinuance. Decreases in striatal tyrosine hydroxylase activity and levels of dopamine and its metabolites were observed 1 day after MPTP discontinuance. Ascorbic acid and glutamate levels had increased, dehydroascorbic acid and GSH decreased, whereas catabolites of high-energy phosphates (inosine, hypoxanthine, xanthine, and uric acid) were unchanged. In addition, gliosis was observed in both striatum and substantia nigra compacta (SNc). Sections of SNc showed some terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling (TUNEL)-positive cells. Neurochemical parameters of dopaminergic activity showed a trend toward recovery 3 days after MPTP discontinuance. At this time point, TUNEL-positive cells were detected in SNc; some of them showed nuclei with neuronal morphology. A late (days 6-11) increase in striatal dopamine oxidative metabolism, ascorbic acid oxidative status, and catabolites of high-energy phosphates were observed concomitant with nigral neuron and nigrostriatal glial cell apoptotic death, as revealed by TUNEL, acridine orange, and Hoechst staining, and transmission electron microscopy. These data suggest that MPTP-induced activation/apoptotic death of glial cells plays a key role in the sequential linkage of neurochemical and cellular events leading to dopaminergic nigral neuron apoptotic death.