RESUMO
Under pathological conditions, microglia, the resident CNS immune cells, become reactive and release pro-inflammatory cytokines and neurotoxic factors. We investigated whether this phenotypic switch includes changes in the expression of the L-type voltage-gated calcium channel (VGCC) in a rat model of N-methyl-D-aspartate-induced hippocampal neurodegeneration. Double immunohistochemistry and confocal microscopy evidenced that activated microglia express the L-type VGCC. We then analyzed whether BV2 microglia express functional L-type VGCC, and investigated the latter's role in microglial cytokine release and phagocytic capacity. Activated BV2 microglia express the CaV1.2 and CaV1.3 subunits of the L-type VGCC determined by reverse transcription-polymerase chain reaction, Western blot and immunocytochemistry. Depolarization with KCl induced a Ca2+ entry facilitated by Bay k8644 and partially blocked with nifedipine, which also reduced TNF-α and NO release by 40%. However, no nifedipine effect on BV2 microglia viability or phagocytic capacity was observed. Our results suggest that in CNS inflammatory processes, the L-type VGCC plays a specific role in the control of microglial secretory activity.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Microglia/metabolismo , Animais , Canais de Cálcio Tipo L/genética , Linhagem Celular , Hipocampo/citologia , Hipocampo/metabolismo , Masculino , Camundongos , Óxido Nítrico/metabolismo , Fagocitose , Ratos , Ratos Wistar , Fator de Necrose Tumoral alfa/metabolismoRESUMO
With its capacity to survey the environment and phagocyte debris, microglia assume a diversity of phenotypes to respond specifically through neurotrophic and toxic effects. Although these roles are well accepted, the underlying energetic mechanisms associated with microglial activation remain largely unclear. This study investigates microglia metabolic adaptation to ATP, NADPH, H(+) , and reactive oxygen species production. To this end, in vitro studies were performed with BV-2 cells before and after activation with lipopolysaccharide + interferon-γ. Nitric oxide (NO) was measured as a marker of cell activation. Our results show that microglial activation triggers a metabolic reprogramming based on an increased glucose uptake and a strengthening of anaerobic glycolysis, as well as of the pentose pathway oxidative branch, while retaining the mitochondrial activity. Based on this energy commitment, microglial defense capacity increases rapidly as well as ribose-5-phosphate and nucleic acid formation for gene transcription, essential to ensure the newly acquired functions demanded by central nervous system signaling. We also review the role of NO in this microglial energy commitment that positions cytotoxic microglia within the energetics of the astrocyte-neuron lactate shuttle.
Assuntos
Adaptação Fisiológica/fisiologia , Metabolismo Energético/fisiologia , Glucose/metabolismo , Microglia/metabolismo , Transdução de Sinais/fisiologia , Animais , Linhagem Celular , Potencial da Membrana Mitocondrial/fisiologia , Camundongos , Fenótipo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The human intestinal cell lines: Caco-2 and HT29-MTX cells have been used extensively in 2D and 3D cell cultures as simple models of the small intestinal epithelium in vitro. This study aimed to investigate the potential of three hydrogel scaffolds to support the 3D culture of Caco-2 and HT29-MTX cells and critically assess their use as scaffolds to stimulate villi formation to model a small intestinal epithelium in vitro. Here, alginate, l-pNIPAM, and l-pNIPAM-co-DMAc hydrogels were investigated. The cells were suspended within or layered on these hydrogels and maintained under static or dynamic culture conditions for up to 21days. Caco-2 cell viability was increased when layered on the synthetic hydrogel scaffolds, but reduced when suspended within the synthetic hydrogels. In contrast, HT29-MTX cells remained viable when suspended within or layered on all 3D cultures. Interestingly, cells cultured in and on the alginate hydrogel scaffolds formed multilayer spheroid structures, whilst the cells layered on synthetic hydrogels formed villus-like structures. Immunohistochemistry staining demonstrated positive expression of enterocyte differentiation markers and goblet cell marker. In conclusion, l-pNIPAM hydrogel scaffolds supported both cell lines and induced formation of villus-like structures when cells were layered on and cultured under dynamic conditions. The ability of the l-pNIPAM to recapitulate the 3D structure and differentiate main cell types of human intestinal villi may deliver a potential alternative in vitro model for studying intestinal disease and for drug testing. STATEMENT OF SIGNIFICANCE: Forty percent of hospital referrals are linked to disorders of the digestive tract. Current studies have utilised animal models or simple cultures of isolated cells which do not behave in the same manner as human intestine. Thus new models are required which more closely mimic the behaviour of intestinal cells. Here, we tested a number of scaffolds and conditions to develop a cell culture model which closely represents the 3D environment seen within the human small intestine. We successfully created structures seen within the intestine which have not previously been possible with other culture models. These models could be used to investigate tissue engineering, drug discovery, and used asan alternative to in vivo animal models in drug toxicity studies.
Assuntos
Antígenos de Diferenciação/biossíntese , Regulação da Expressão Gênica , Hidrogéis/química , Mucosa Intestinal/metabolismo , Modelos Biológicos , Alicerces Teciduais/química , Células CACO-2 , Humanos , Mucosa Intestinal/citologiaRESUMO
Diazoxide, a well-known mitochondrial KATP channel opener with neuroprotective effects, has been proposed for the effective and safe treatment of neuroinflammation. To test whether diazoxide affects the neurogenesis associated with excitotoxicity in brain injury, we induced lesions by injecting excitotoxic N-methyl-d-aspartate (NMDA) into the rat hippocampus and analyzed the effects of a daily oral administration of diazoxide on the induced lesion. Specific glial and neuronal staining showed that NMDA elicited a strong glial reaction associated with progressive neuronal loss in the whole hippocampal formation. Doublecortin immunohistochemistry and bromo-deoxyuridine (BrdU)-NeuN double immunohistochemistry revealed that NMDA also induced cell proliferation and neurogenesis in the lesioned non-neurogenic hippocampus. Furthermore, glial fibrillary acidic protein (GFAP)-positive cells in the injured hippocampus expressed transcription factor Sp8 indicating that the excitotoxic lesion elicited the migration of progenitors from the subventricular zone and/or the reprograming of reactive astrocytes. Diazoxide treatment attenuated the NMDA-induced hippocampal injury in rats, as demonstrated by decreases in the size of the lesion, neuronal loss and microglial reaction. Diazoxide also increased the number of BrdU/NeuN double-stained cells and elevated the number of Sp8-positive cells in the lesioned hippocampus. These results indicate a role for KATP channel activation in regulating excitotoxicity-induced neurogenesis in brain injury.
Assuntos
Diazóxido/farmacologia , Hipocampo/efeitos dos fármacos , N-Metilaspartato/toxicidade , Doenças Neurodegenerativas/tratamento farmacológico , Neurogênese/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Administração Oral , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Astrócitos/fisiologia , Modelos Animais de Doenças , Proteína Duplacortina , Hipocampo/patologia , Hipocampo/fisiopatologia , Canais KATP/metabolismo , Masculino , Microglia/efeitos dos fármacos , Microglia/patologia , Microglia/fisiologia , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/fisiopatologia , Neurogênese/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Neurônios/fisiologia , Ratos WistarRESUMO
The study aimed to assess the effects of polyphenols when used in combination with doxorubicin and etoposide, and to determine whether polyphenols sensitised leukaemia cells, causing inhibition of cell proliferation, cell cycle arrest and induction of apoptosis. This study is based on findings in solid cancer tumours, which have shown that polyphenols can sensitize cells to chemotherapy, and induce apoptosis and/or cell-cycle arrest. This could enable a reduction of chemotherapy dose and off-target effects, whilst maintaining treatment efficacy. Quercetin, apigenin, emodin, rhein and cis-stilbene were investigated alone and in combination with etoposide and doxorubicin in two lymphoid and two myeloid leukaemia cells lines. Measurements were made of ATP levels (using CellTiter-Glo assay) as an indication of total cell number, cell cycle progression (using propidium iodide staining and flow cytometry) and apoptosis (NucView caspase 3 assay and Hoechst 33342/propidium iodide staining). Effects of combination treatments on caspases 3, 8 and 9 activity were determined using Glo luminescent assays, glutathione levels were measured using the GSH-Glo Glutathione Assay and DNA damage determined by anti-γH2AX staining. Doxorubicin and etoposide in combination with polyphenols synergistically reduced ATP levels, induced apoptosis and increased S and/or G2/M phase cell cycle arrest in lymphoid leukaemia cell lines. However, in the myeloid cell lines the effects of the combination treatments varied; doxorubicin had a synergistic or additive effect when combined with quercetin, apigenin, emodin, and cis-stilbene, but had an antagonistic effect when combined with rhein. Combination treatment caused a synergistic downregulation of glutathione levels and increased DNA damage, driving apoptosis via caspase 8 and 9 activation. However, in myeloid cells where antagonistic effects were observed, this was associated with increased glutathione levels and a reduction in DNA damage and apoptosis. This study has demonstrated that doxorubicin and etoposide activity were enhanced by polyphenols in lymphoid leukaemia cells, however, differential responses were seen in myeloid cells with antagonistic responses seen in some combination therapies.
RESUMO
Distribution and age-related changes of MAO in BL/C57 mouse were studied by quantitative enzyme radioautography with [3H]Ro41-1049 and [3H]Ro19-6327. In the brain, MAO-A was highest in locus coeruleus and interpeduncular nucleus, and MAO-B in raphe nuclei, paraventricular thalamic nucleus, and ependyma of ventricles. Extremely high MAO-B levels were also measured in the choroid plexus in contrast to the very low MAO-B levels in rat choroid plexus. With aging, brain MAO-A showed a clear decrease between 4 and 9 weeks, followed by no change between 9 weeks and 19 months, and a slight increase between 19 and 25 months. On the other hand, all brain structures showed age-related increases in MAO-B. Peripheral organs showed different patterns of MAO age-related changes. Particularly interesting was the marked MAO-B increase in heart, parallel to the MAO-A increase in rat heart. Also of interest is the decrease of liver MAO-B in old animals, which, together with the increase of MAO-B in the brain, might underlie the high sensitivity of old BL/C57 mice to MPTP.
Assuntos
Envelhecimento/metabolismo , Encéfalo/enzimologia , Encéfalo/crescimento & desenvolvimento , Monoaminoxidase/metabolismo , Animais , Autorradiografia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Inibidores da Monoaminoxidase/farmacologia , Ácidos Picolínicos , TiazóisRESUMO
Variations of monoamine oxidases (MAO) A and B were studied during aging in 27 human subjects (age range 17-93 years) in 18 brain structures of temporal cortex, frontal gyrus, hippocampal formation, striatum, cerebellum, and brainstem. [3H]Ro41-1049 and [3H]lazabemide were used as selective radioligands to image and quantify MAO-A and MAO-B respectively by enzyme autoradiography. Postmortem delay or time of tissue storage did not affect MAO-A or MAO-B levels. There was, moreover, no evidence of sexual dimorphism. A marked age-related increase in MAO-B was observed in most structures. This increase started at the age of 50-60 years. Before this age, MAO-B levels were constant in all structures studied. MAO-B-rich senile plaques were observed in some cortical areas but they did not significantly influence the age-related MAO-B increase. Surprisingly, no age-related MAO-B changes were observed in the substantia nigra. In contrast to MAO-B, no clear age-related changes in MAO-A were observed, indicating an independent regulation of the two isoenzymes, also suggested by the cross-correlation analysis of these data.
Assuntos
Envelhecimento/metabolismo , Encéfalo/enzimologia , Monoaminoxidase/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Autorradiografia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Inibidores da Monoaminoxidase/farmacocinética , Inibidores da Monoaminoxidase/farmacologia , Ácidos Picolínicos/farmacocinética , Ácidos Picolínicos/farmacologia , Caracteres Sexuais , Tiazóis/farmacocinética , Tiazóis/farmacologiaRESUMO
The aim of this study was to determine possible differences in peripheral blood mononuclear cells (PBMC) proliferation of healthy donors and Hashimoto's thyroiditis patients and whether a statistical approach to cell proliferation analysis might be used to discern the differences. The effect of a wide range of 2'-deoxyguanosine (dGuo) concentrations (0-1250 microM) on the mitogen-induced proliferation of PBMC was studied in healthy donors and Hashimoto's thyroiditis patients. Activity levels of purine nucleoside phosphorylase (PNP) and adenosine deaminase (ADA) in PBMC were also measured. For the first time in a study of these models of dGuo toxicity in vitro, the analysis of polynomial trends of orders from 1 to 7 was applied to evaluate cell proliferation. A dose-dependent inhibition of mitogen-induced PBMC proliferation was observed in both groups. Data for linear trend established that PBMC from Hashimoto's thyroiditis patients were more sensitive to dGuo toxicity than PBMC from healthy donors. A positive quadratic trend at low dGuo doses was found in the cell proliferation of Hashimoto's thyroiditis patients. A decrease in PNP activity (P < 0.025) and an increase in ADA activity (P < 0.005) was observed in PBMC of Hashimoto's thyroiditis group. The differences in PBMC proliferation subjected to dGuo toxicity between the two groups could be related with the distinct pattern of purine salvage enzymes observed.
Assuntos
Desoxiguanosina/farmacologia , Leucócitos Mononucleares/citologia , Tireoidite Autoimune/sangue , Adenosina Desaminase/metabolismo , Adulto , Idoso , Divisão Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Humanos , Leucócitos Mononucleares/enzimologia , Masculino , Pessoa de Meia-Idade , Purina-Núcleosídeo Fosforilase/metabolismoRESUMO
To investigate the long-term effects of excitatory amino acid microinjections into the basal forebrain and its correlation with a possible Ca2+ imbalance associated with the excitotoxic process, ibotenic acid, mainly an N-methyl-D-aspartate receptor agonist, and quisqualic acid, an agonist of non-N-methyl-D-aspartate receptors, were injected into two regions rich in cholinergic neurons, namely the medial septal nucleus and the ventral globus pallidus. Within the globus pallidus but not within the medial septal nucleus, 13 days and one year postlesion, nerve cell death was associated with the appearance of calcium deposits within the large putative GABAergic pallidal neurons, being more pronounced in ibotenic acid than quisqualic acid-lesioned rats. An intermediate two month post-lesion study with alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and ibotenic acid microinjections in globus pallidus demonstrated that the AMPA subtype of glutamate receptor may also be involved in this Ca2+ imbalance, together with the N-methyl-D-aspartate and metabotropic subtype receptors. Quisqualic acid lesions in globus pallidus and medial septum were associated with a substantial disappearance of cholinergic cell bodies and their nerve terminal networks within the cerebral cortex and hippocampal formation respectively, as assessed by choline acetyltransferase and acetylcholine esterase immunocytochemistry. Ibotenic acid lesions resulted in a lower reduction of cholinergic markers. One year after septal lesions induced either by ibotenic or quisqualic acid, a marked atrophy of the entire dorsolateral septal nucleus was observed. Our results support the hypothesis that brief and intense glutamate exposure can induce long-term neurodegenerative processes and give evidence that long-term excitotoxic lesions of the two areas studied result in marked differences in neuronal damage, including intracellular calcium deposits which do not correlate with the cholinergic deficits produced by multiple glutamate receptor subtypes.
Assuntos
Dano Encefálico Crônico/induzido quimicamente , Neurotoxinas/toxicidade , Animais , Cálcio , Imuno-Histoquímica , Masculino , Degeneração Neural , Prosencéfalo , Ácido Quisquálico/toxicidade , Ratos , Ratos Sprague-Dawley , Núcleos Septais , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologiaRESUMO
Aged rats may be behaviorally classified as either cognitively impaired or unimpaired based upon their performance in the Morris water maze task. In aged Long-Evans rats, emergence of functional deficits has been related to the increase in the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subtype in most hippocampal subfields, not observed in other brain structures. As AMPA receptors expressed in astrocytes may participate in the delayed and long-term glial response to injury, we investigated whether astrocytes participate in the increase of AMPA receptor observed in these aged rats. To this end, distribution of monoamine oxidase B, used as an astroglial marker, was characterized by quantitative autoradiography in the hippocampus and septum of young adults (six months) and aged (24-25 months) rats using [3H]lazabemide. Specific binding to brain sections of young, aged unimpaired, and aged impaired animals were calculated densitometrically. Compared to young animals, all hippocampal subfields in the aged unimpaired group showed a significant age-related increased labeling, which was not present in the aged impaired group. This contrasts with the increased glial transcription described in this last group. We propose that increase in AMPA receptors in the aged memory-impaired animals may be related to an atypic astrocytic reactivity.
Assuntos
Envelhecimento/metabolismo , Hipocampo/metabolismo , Transtornos da Memória/metabolismo , Monoaminoxidase/metabolismo , Receptores de AMPA/metabolismo , Animais , Transtornos da Memória/genética , Ácidos Picolínicos/metabolismo , Ratos , Ratos Long-Evans/genética , Valores de ReferênciaRESUMO
Previous data from our laboratory indicate that 25 mM ibotenic acid induces intracellular calcifications in the rat basal forebrain. Because of the lack of specificity of ibotenic acid for a glutamate receptor subtype, a dose-response study with alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate was undertaken and calcified areas (identified with Alizarin Red staining) as well as astro- and microglial reactions (by autoradiography with [3H]lazabemide and [3H]Ro 5-4864) were quantified at one month post-lesion. alpha-Amino-3-hydroxy-5-methyl-4-isoxazole propionate administered into the globus pallidus induced, in a dose-dependent manner, the formation of calcium deposits and the activation of both glial cells, the microglial reaction being particularly robust. From this study, a dose of 5.4 mM alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate was selected for further experiments. [3H]alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate, [3H]dizocilpine maleate and [3H]PN 200-110 binding in vitro were performed to assess autoradiographically whether the tissue damage was associated with changes in glutamate receptors and calcium channel binding sites. In the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate-treated animals, the specific binding of [3H]alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate was significantly reduced by 28% in the lesioned ventral pallidum, whereas it was unchanged in the globus pallidus and substantia innominata. In these three nuclei, calcifications developed and an increase in both glial markers was measured. In contrast, the binding of [3H]PN 200-110 and [3H]dizocilpine maleate were unaffected. Co-injection of 15 mM 6-nitro-7-sulphamoylbenzo(f)quinoxaline-2,3-dione, a selective alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate/kainate receptor antagonist, prevented the formation of calcium concretions, the microglial reaction and the decrease in [3H]alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate binding but it failed to inhibit totally the astroglial reaction induced by alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate. This may suggest that the microglial reaction and calcification take place through different mechanisms from the astrogliosis associated with the neuronal loss. In conclusion, acute administration of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate in the rat globus pallidus elicits a dose-dependent calcification process associated with a chronic reaction of astrocytes and microglia. alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate-induced injury is accompanied by a slight reduction of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors in the ventral pallidum, whereas the binding of N-methyl-D-aspartate and L-type calcium channels receptors remains unchanged in any lesioned nucleus.
Assuntos
Astrócitos/patologia , Gânglios da Base/efeitos dos fármacos , Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Quinoxalinas/farmacologia , Receptores de AMPA/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologia , Animais , Astrócitos/química , Autorradiografia , Gânglios da Base/enzimologia , Gânglios da Base/patologia , Química Encefálica/efeitos dos fármacos , Calcinose/induzido quimicamente , Cálcio/análise , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/análise , Canais de Cálcio Tipo L/metabolismo , Relação Dose-Resposta a Droga , Proteína Glial Fibrilar Ácida/análise , Isradipino/metabolismo , Isradipino/farmacologia , Masculino , Microglia/química , Microglia/patologia , Monoaminoxidase/análise , Inibidores da Monoaminoxidase/metabolismo , Inibidores da Monoaminoxidase/farmacologia , Nimodipina/metabolismo , Nimodipina/farmacologia , Ácidos Picolínicos/metabolismo , Ácidos Picolínicos/farmacologia , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/análise , Receptores de GABA-A/metabolismo , Receptores de N-Metil-D-Aspartato/análise , Receptores de N-Metil-D-Aspartato/metabolismo , TrítioRESUMO
Semicarbazide-sensitive amine oxidase (SSAO), widely distributed in highly vascularized mammalian tissues, metabolizes endogenous and xenobiotic aromatic and aliphatic monoamines. To assess whether its physiological role in humans is restricted to oxidation, we used an immunohistochemical approach to examine the cellular localization of SSAO in human peripheral tissues (adrenal gland, duodenum, heart, kidney, lung, liver, pancreas, spleen, thyroid gland, and blood vessels) and also analyzed its subcellular localization. The results are in agreement with the specific activities also determined in the same samples and are discussed with reference to the tissue distribution of monoamine oxidase A and B. Together with the oxidative deamination of monoamines, SSAO cellular localization indicates that, in most human peripheral tissues, it might participate in the regulation of physiological processes via H(2)O(2) generation. (J Histochem Cytochem 49:209-217, 2001)
Assuntos
Amina Oxidase (contendo Cobre)/metabolismo , Inibidores Enzimáticos/farmacologia , Semicarbazidas/farmacologia , Amina Oxidase (contendo Cobre)/antagonistas & inibidores , Linhagem Celular , Humanos , Immunoblotting , Imuno-Histoquímica , Especificidade de ÓrgãosRESUMO
We studied monoamine oxidase (MAO) A and B localization in human pancreas, thyroid gland, and adrenal gland by immunohistochemistry. The primary antibodies used were mouse monoclonal anti-human MAO-A (6G11/E1) and anti-human MAO-B (3F12/G10/2E3). Samples were obtained from six routine autopsy cases and fixed in 2% paraformaldehyde. Exocrine pancreas showed a widespread distribution of MAO-A, whereas MAO-B was present only in centroacinar cells and epithelial cells of pancreatic ducts. In endocrine pancreas, MAO-A was observed in around 50% of islet cells, whereas MAO-B was less abundant and was restricted to the periphery of islets. Thyroid gland showed strong MAO-A immunoreactivity in all cell types and was MAO-B-negative. In adrenal gland, the capsule displayed MAO-A but not MAO-B immunoreactivity, whereas the cortex showed widespread MAO-A staining but was MAO-B-negative in interstitial cells. Finally, in the medulla only a few scattered cells showed either MAO-A or MAO-B immunoreactivity. To our knowledge, these data represent the first study of the cellular distribution of MAO-A and MAO-B in the three human tissues included.
Assuntos
Glândulas Suprarrenais/enzimologia , Monoaminoxidase/isolamento & purificação , Pâncreas/enzimologia , Glândula Tireoide/enzimologia , Idoso , Idoso de 80 Anos ou mais , Especificidade de Anticorpos , Feminino , Humanos , Imuno-Histoquímica , Ilhotas Pancreáticas/enzimologia , Isoenzimas/imunologia , Isoenzimas/isolamento & purificação , Masculino , Pessoa de Meia-Idade , Monoaminoxidase/imunologiaRESUMO
In order to obtain further insight into the interactions between the purinergic and dopaminergic pathways in the striatum, we studied both metabolisms simultaneously, using a microdialysis technique in 1-methyl-1,4-phenylpyridinium ion (MPP+) unilaterally-denervated conscious rats. In these rats the contralateral side was used as control. The perfusates were collected every 20-25 min using 4 mm dialysis probes, implanted in each striatum, and assayed for dopamine and purine metabolites. After MPP+ administration, all adenosine metabolites - with the exception of uric acid - and dopamine levels were significantly increased in the extracellular medium. However, the time-course change in dopamine level did not correlate with the adenosine and inosine time-courses, suggesting a different mechanism of liberation in response to MPP+ administration.
Assuntos
Corpo Estriado/metabolismo , Dopamina/metabolismo , Purinas/metabolismo , Compostos de Piridínio/farmacologia , 1-Metil-4-fenilpiridínio , Animais , Corpo Estriado/efeitos dos fármacos , Masculino , Ratos , Ratos Endogâmicos , Fatores de TempoRESUMO
Activation of excitatory amino acid (EAA) receptors can induce neurodegeneration by two major mechanisms of excitotoxicity, one related to the influx of Na(+), Cl(-) and water, and the other to the increase in intracellular calcium concentration ([Ca(2+)](i)). Thus, acute microinjection of EAAs in several areas of the central nervous system (CNS) has been used to produce neurodegenerative models. We studied the excitotoxic pattern associated with acute microinjection of AMPA in rat hippocampus, medial septum-diagonal band of Broca (MS-DBB), prefrontal cortex and retina. In all cases progressive neuronal loss, glial reaction and development of intra- and extracellular calcium concretions were observed. However, a CNS-area differential vulnerability was revealed, as shown by the specific atrophy of MS-DBB and its limited calcification. Whether calcium deposits are a defensive mechanism against the massive increment of free cytoplasmatic calcium is discussed on the basis of ultrastructural data and previous results.
Assuntos
Calcinose/metabolismo , Cálcio/metabolismo , Agonistas de Aminoácidos Excitatórios/farmacologia , Degeneração Neural/induzido quimicamente , Degeneração Neural/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologia , Animais , Calcinose/induzido quimicamente , Cálcio/análise , Microanálise por Sonda Eletrônica , Aminoácidos Excitatórios/metabolismo , Ácido Glutâmico/metabolismo , Hipocampo/metabolismo , Hipocampo/patologia , Homeostase/efeitos dos fármacos , Ácido Láctico/metabolismo , Masculino , Microscopia Eletrônica , Neuroglia/metabolismo , Neuroglia/ultraestrutura , Neurônios/química , Neurônios/metabolismo , Neurônios/ultraestrutura , Córtex Pré-Frontal/metabolismo , Córtex Pré-Frontal/patologia , Ratos , Ratos Sprague-Dawley , Retina/metabolismo , Retina/patologiaRESUMO
Ibotenic acid-induced lesion of the basal forebrain resulted after 13 days in a 90% reduction of octopamine (OA) in the frontoparietal cortex of adult rats, whereas dihydroxyphenylacetic (DOPAC), homovanillic (HVA) and 5-hydroxyindoleacetic (5-HIAA) acids were not modified as measured by microdialysis and high-performance liquid chromatography (HPLC) with electrochemical detection. At this time, cortical choline acetyltransferase (ChAT) activity was decreased by 34%. The results are discussed with respect to possible octopamine involvement in reduced age-associated performance in neurodegenerative processes.
Assuntos
Lobo Frontal/metabolismo , Octopamina/metabolismo , Lobo Parietal/metabolismo , Prosencéfalo/efeitos dos fármacos , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Animais , Colina O-Acetiltransferase/metabolismo , Cromatografia Líquida de Alta Pressão , Diálise , Ácido Homovanílico/metabolismo , Ácido Hidroxi-Indolacético/metabolismo , Ácido Ibotênico/toxicidade , Masculino , Microquímica , Prosencéfalo/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Excitotoxic lesion of the medial septum with ibotenic acid leading to partial disappearance of the septal cholinergic nerve cells was used to investigate the role of cholinergic mechanisms in the control of trophic factors for hippocampal plasticity, namely brain-derived neurotrophic factor (BDNF) and glucocorticoid receptor (GR). Their mRNA levels were tested by in situ hybridization 13 days after the lesion. A persistent and widespread increase of BDNF mRNA was found in all parts of the dorsal hippocampal formation that was not accompanied by a significant modification in GR expression. The present data suggest that subcortical excitotoxic lesions at the septal level have long-term consequences for the adaptive trophic responses occurring in the dorsal hippocampus.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/genética , Agonistas de Aminoácidos Excitatórios/farmacologia , Hipocampo/efeitos dos fármacos , Ácido Ibotênico/farmacologia , Núcleos Septais/efeitos dos fármacos , Animais , Sobrevivência Celular/efeitos dos fármacos , Colina O-Acetiltransferase/metabolismo , Fibras Colinérgicas/efeitos dos fármacos , Hipocampo/citologia , Hipocampo/enzimologia , Hibridização In Situ , Masculino , Microinjeções , Neurotoxinas/farmacologia , Sondas RNA , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Núcleos Septais/citologia , Núcleos Septais/fisiologia , Radioisótopos de Enxofre , Uridina Trifosfato/genéticaRESUMO
Adenosine has been shown to inhibit dopamine release from striatal slices and synaptosomes. Recently, a direct interaction between the adenosine A2 receptor and dopamine D2 receptor has been provided. Activation of striatal adenosine A1 receptors is known to partially inhibit the release of dopamine (DA), but some aspects of this mechanism remain unclear. We have studied the participation of adenosine A1 receptors in the control of DA release 'in vivo' in awake, freely moving rats using microdialysis. To this end, the effects of 2-chloroadenosine (2-CADO), a non-metabolizable adenosine A1 receptor agonist, were studied on basal and stimulated striatal DA release. Basal levels were found to be slightly decreased by a maximal concentration of 2-CADO without any changes in DA metabolites. Haloperidol stimulated DA release was fully counteracted by 2-CADO. However, high K+ (100 mM) or (+)-amphetamine stimulated DA release was not altered by 2-CADO. Altogether, these data suggest that adenosine acting through A1 receptors possibly localized on striatal dopaminergic nerve terminals can block an induced D2 receptor blockade, but not the releasing effects caused by (+)-amphetamine and high K+ concentration. It is postulated that the increase in DA release by haloperidol is mainly due to an increased firing rate of the DA neurons and that A1 receptor activation can block the DA release observed in response to the action potential activation of DA nerve terminals.
Assuntos
2-Cloroadenosina/farmacologia , Corpo Estriado/fisiologia , Dopamina/metabolismo , Receptores Purinérgicos P1/fisiologia , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Anfetamina/farmacologia , Animais , Haloperidol/farmacologia , Masculino , Neurônios/fisiologia , Potássio/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Effects of the parkinsonism inducing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on striatal dopamine metabolism and the influence of sex on the recovery were investigated in adult (2-month-old) male and female C57/BL mice. We present here evidence that MPTP treatment (2 doses of 30 mg/kg i.p., each at 24 h interval) produced a similar reduction (-65% to -70%) of striatal dopamine in both sexes 24 h after the last injection of MPTP, and a greater loss of the metabolites in the female group. In contrast to the partial recovery observed in the male group, an increased dopamine loss occurred in the female group within 10 days following the last injection of MPTP. This impairment in recovery appears to be different to the one already observed in aged (24-month-old) male mice treated in similar conditions. As the neurotoxic effects of MPTP depend on its conversion to the 1-methyl-4-phenylpyridinium ion (MPP+) by monoamine oxidase B (MAO B), the presence of a different peripheral or central MAO B type in female mice could be in part responsible for these sex related effects. To investigate this possibility, MAO A and B activities were characterized in liver and brain of adult female control mice during the different steps of the oestrous cycle and compared to those of adult control male mice. Significant differences in MAO A and MAO B activities could be detected during the oestrous cycle and between the adult male and female groups. It is concluded that MAO B may be involved in the sex related effects of MPTP.
Assuntos
1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologia , Monoaminoxidase/metabolismo , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/metabolismo , 1-Metil-4-fenilpiridínio/metabolismo , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Animais , Dopamina/metabolismo , Estro/fisiologia , Feminino , Ácido Homovanílico/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neostriado/enzimologia , Neostriado/metabolismo , Caracteres SexuaisRESUMO
In efforts to test the cholinergic hypothesis for Alzheimer's disease and to create an animal model for this disease, ibotenic acid has been used to lesion cholinergic neurons in the basal forebrain. In this study we have used in situ hybridization with oligonucleotide probes specific for mRNAs encoding choline acetyltransferase and glutamic acid decarboxylase, respectively, to study the effects of such a lesion. Our results show that lesion paradigms normally used to induce neuronal degeneration in nucleus basalis by ibotenic acid not only lesion the cholinergic neurons within this nucleus, but in addition, a major fraction of gamma-aminobutyric acid (GABA) neurons in nucleus basalis, substantia innominata, globus pallidus and ventral pallidum.