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To evaluate changes in concentrations of selected biomarkers, neurotrophic factors, and growth factors in the cerebrospinal fluid during pregnancy. A prospective observational study was conducted in 32 pregnant women undergoing gynecological and obstetrical surgery under spinal anesthesia in a university hospital. Beta-amyloid(1-42) and beta-amyloid(1-40) peptides, brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, and vascular endothelial growth factor were analyzed in cerebrospinal fluid using an enzyme-linked immunosorbent assay. Eight women in second trimester pregnancy who underwent spinal anesthesia for gynecological or obstetrical surgery were compared with 24 matched women in third trimester pregnancies. CSF concentrations of beta-amyloid(1-42) were significantly higher in third trimester pregnancies (p = 0.025). During third trimester, the beta-amyloid ratio correlated with the vascular endothelial growth factor (rs = 0.657; p = 0.008). Higher concentrations of beta-amyloid(1-42) in cerebrospinal fluid of third trimester pregnancies and correlations between the beta-amyloid ratio and the vascular endothelial growth factor support the hypothesis that beta-amyloid peptides are involved in complex adaptive brain alterations during pregnancy.
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Péptidos beta-Amiloides/líquido cefalorraquídeo , Fragmentos de Péptidos/líquido cefalorraquídeo , Embarazo/líquido cefalorraquídeo , Biomarcadores , Factor Neurotrófico Derivado del Encéfalo/líquido cefalorraquídeo , Femenino , Factor Neurotrófico Derivado de la Línea Celular Glial/líquido cefalorraquídeo , Humanos , Segundo Trimestre del Embarazo , Estudios Prospectivos , Factor A de Crecimiento Endotelial Vascular/líquido cefalorraquídeoRESUMEN
In vitro cell cultures are an important tool for obtaining insights into cellular processes in an isolated system and a supplement to in vivo animal experiments. While primary dissociated cultures permit a single homogeneous cell population to be studied, there is a clear need to explore the function of brain cells in a three-dimensional system where the main architecture of the cells is preserved. Thus, organotypic brain slice cultures have proven to be very useful in investigating cellular and molecular processes of the brain in vitro. This review summarizes (1) the historical development of organotypic brain slices focusing on the membrane technology, (2) methodological aspects regarding culturing procedures, age of donors or media, (3) whether the cholinergic neurons serve as a model of neurodegeneration in Alzheimer's disease, (4) or the nigrostriatal dopaminergic neurons as a model of Parkinson's disease and (5) how the vascular network can be studied, especially with regard to a synthetic blood-brain barrier. This review will also highlight some limits of the model and give an outlook on future applications.
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Encéfalo/citología , Neuronas Colinérgicas/fisiología , Neuronas Dopaminérgicas/fisiología , Técnicas de Cultivo de Órganos , Animales , Técnicas In VitroRESUMEN
BACKGROUND: Spontaneous intracerebral hemorrhage (ICH) is associated with high morbidity and mortality. Cortical spreading depolarizations (CSDs) increase brain matrix metalloproteinase (MMP)-9 activity leading to perihematomal edema expansion in experimental ICH. METHODS: The purpose of this report is to describe cerebral metabolic changes and brain extracellular MMP-9 levels in a patient with CSDs and perihematomal edema expansion after ICH. RESULTS: We present a 66-year-old male patient with ICH who underwent craniotomy for hematoma evacuation. Multimodal neuromonitoring data of the perihematomal region revealed metabolic distress and increased MMP-9 levels in the brain extracellular fluid during perihematomal edema progression. At the same time, subdural electrocorticography showed clusters of CSDs, which disappeared after ketamine anesthesia on day six. Perihematomal edema regression was associated with decreasing cerebral MMP-9 levels. CONCLUSIONS: This novel association between clusters of CSDs, brain metabolic distress, and increased MMP-9 levels expands our knowledge about secondary brain injury after ICH. The role of ketamine after this devastating disorder needs further studies.
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Edema Encefálico/fisiopatología , Corteza Cerebral/fisiopatología , Hemorragia Cerebral/fisiopatología , Metaloproteinasa 9 de la Matriz/metabolismo , Monitorización Neurofisiológica/métodos , Anciano , Edema Encefálico/etiología , Edema Encefálico/metabolismo , Corteza Cerebral/metabolismo , Hemorragia Cerebral/complicaciones , Hemorragia Cerebral/metabolismo , Hemorragia Cerebral/cirugía , Humanos , MasculinoRESUMEN
INTRODUCTION: Platelets store serotonin and brain-derived neurotrophic factor (BDNF) as well as amyloid precursor protein and nerve growth factor (NGF), thus platelets are of special interest in depression and Alzheimer's disease, respectively. Both diseases are associated with inflammation and release of NGF or BDNF from platelets may play a potent role. METHODS: Platelets were isolated from adult Sprague-Dawley rats and were incubated with anti-inflammatory drugs (ibuprofen and indomethacin) and antidepressants (citalopram, paroxetine and sertraline) (final concentration: 0.3 µM) with or without 2 mM calcium chloride. The release of NGF and BDNF was analyzed in comparison to serotonin release from rat platelets after 10 or 60 min. RESULTS: Spontaneous release of serotonin and BDNF was approximately 10-15% of total serotonin or BDNF content in platelets, but nearly all NGF was released within 10 min. All antidepressants increased the serotonin release from rat platelets. NGF release was reduced by sertraline, paroxetine and ibuprofen, but only when calcium was present, except for sertraline after 10 min. BDNF release was only reduced by ibuprofen when calcium was added. CONCLUSION: We conclude that antidepressants and anti-inflammatory drugs differentially influence the NGF and BDNF release, in a time-, dose- and calcium-specific pattern.
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Antiinflamatorios/farmacología , Antidepresivos/farmacología , Plaquetas/metabolismo , Factor Neurotrófico Derivado del Encéfalo/sangre , Factor de Crecimiento Nervioso/sangre , Animales , Plaquetas/efectos de los fármacos , Calcio/fisiología , Cromatografía Líquida de Alta Presión , Interpretación Estadística de Datos , Ensayo de Inmunoadsorción Enzimática , Ratas , Ratas Sprague-Dawley , Serotonina/sangreRESUMEN
There is strong evidence that vascular risk factors play a role in the development of Alzheimer's disease (AD) or vascular dementia (vaD). Ethanol (EtOH) and cholesterol are such vascular risk factors, and we recently showed that hypercholesterolemia causes pathologies similar to AD [Ullrich et al. (2010) Mol Cell Neurosci 45, 408-417]. The aim of this study was to investigate the effects of long-term (12 months) EtOH treatment (20% v/v in drinking water) alone or long-term 5% cholesterol diet alone or a combination (mix) in adult Sprague-Dawley rats. Long-term EtOH treatment (plasma EtOH levels 58±23 mg/dl) caused significant impairment of spatial memory, reduced the number of choline acetyltransferase- and p75 neurotrophin receptor-positive nucleus basalis of Meynert neurons, decreased cortical acetylcholine, elevated cortical monocyte chemoattractant protein-1 and tissue-type plasminogen activator, enhanced microglia, and markedly induced anti-rat immunoglobulin G-positive blood-brain barrier leakage. The effect of long-term hypercholesterolemia was similar. Combined long-term treatment of rats with 20% EtOH and 5% cholesterol (mix) did not potentiate treatment with EtOH alone, but instead counteracted some of the EtOH-associated effects. In conclusion, our data show that vascular risk factors EtOH and cholesterol play a role in cognitive impairment and possibly vaD.
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Trastornos del Sistema Nervioso Inducidos por Alcohol/inducido químicamente , Neuronas Colinérgicas/metabolismo , Demencia Vascular/inducido químicamente , Hipercolesterolemia/inducido químicamente , Mediadores de Inflamación/farmacología , Trastornos del Sistema Nervioso Inducidos por Alcohol/metabolismo , Trastornos del Sistema Nervioso Inducidos por Alcohol/fisiopatología , Animales , Neuronas Colinérgicas/efectos de los fármacos , Neuronas Colinérgicas/patología , Demencia Vascular/etiología , Demencia Vascular/fisiopatología , Modelos Animales de Enfermedad , Hipercolesterolemia/metabolismo , Hipercolesterolemia/fisiopatología , Masculino , Ratas , Ratas Sprague-Dawley , TiempoRESUMEN
Neuroinflammation results in dysregulation of serotonergic neurons in the dorsal raphe nucleus (doR) and is considered to play an important role in the pathophysiology of depression. The aim of the present study was to induce neuroinflammation in a simple doR brain slice model using lipopolysaccharide (LPS), interferon-gamma (IFNγ), beta-amyloid1â42 or tumor necrosis factor-alpha and to explore the survival of serotonergic neurons and the expression of the tryptophan degrading enzyme indoleamine 2,3-dioxygenase (IDO). Administration of pro-inflammatory stimuli reduced survival of serotonergic neurons in doR slices and increased IDO expression. IFNγ most potently induced IDO expression, which co-localized with neurons, including serotonergic neurons, but not with microglia or astrocytes. IFNγ did not induce PI-positive staining in slices, but increased the average nuclei size of IDO-positive cells. The inflammation-induced decline did not return to control levels, when slices were withdrawn from inflammation, pointing to neurodegeneration. The growth factors BDNF or GDNF did not counteract the inflammation-induced decrease in serotonergic neurons, except for LPS-induced neuronal decline. The inflammation-induced effect was not blocked by the NMDA-receptor antagonist MK-801. Further LPS, but not IFNγ increased inflammatory markers and microglia activity. In conclusion, our data show that a range of inflammatory stimuli decline serotonergic neurons in doR slices and upregulate IDO expression. The data suggest that IDO does not contribute to serotonergic decline, but may serve as a marker of neurodegeneration. Neuroinflammation may contribute to the development of depression.
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Supervivencia Celular/efectos de los fármacos , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Inflamación/metabolismo , Neuronas/metabolismo , Núcleos del Rafe/metabolismo , Serotonina/metabolismo , Animales , Astrocitos/efectos de los fármacos , Astrocitos/inmunología , Astrocitos/metabolismo , Supervivencia Celular/inmunología , Indolamina-Pirrol 2,3,-Dioxigenasa/inmunología , Inflamación/inmunología , Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Microglía/efectos de los fármacos , Microglía/inmunología , Microglía/metabolismo , Neuronas/efectos de los fármacos , Neuronas/inmunología , Núcleos del Rafe/efectos de los fármacos , Núcleos del Rafe/inmunología , Ratas , Ratas Sprague-Dawley , Serotonina/inmunologíaRESUMEN
Alzheimer's disease (AD) is a severe chronic neurodegenerative disease. During aging and neurodegeneration, misfolded proteins accumulate and activate the ubiquitin-proteasome system. The aim of the present study is to explore whether ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, ubiquitin or proteasome activity are affected in peripheral blood mononuclear cells (PBMC) of AD, mild cognitive impairment (MCI) and Parkinson's disease (PD) patients compared to healthy subjects. PBMCs were isolated from EDTA blood samples and extracts were analyzed by Western Blot. Proteasome activity was measured with fluorogenic substrates. When compared to healthy subjects, the concentration of enzyme E1 was increased in PBMCs of AD patients, whereas the concentration of the enzyme E2 was decreased in these same patients. Ubiquitin levels and proteasome activity were unchanged in AD patients. No changes in enzyme expression or proteasome activity was observed in MCI patients compared to healthy and AD subjects. In PD patients E2 levels and proteasomal activity were significantly reduced, while ubiquitin and E1 levels were unchanged. The present investigation demonstrates the differences in enzyme and proteasome activity patterns of AD and PD patients. These results suggest that different mechanisms are involved in regulating the ubiquitin-proteasomal system in different neurodegenerative diseases.
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Enfermedad de Alzheimer/patología , Células Sanguíneas/metabolismo , Trastornos del Conocimiento/patología , Enfermedad de Parkinson/patología , Complejo de la Endopetidasa Proteasomal/metabolismo , Enzimas Activadoras de Ubiquitina/metabolismo , Ubiquitina/metabolismo , Anciano , Animales , Quimotripsina/metabolismo , Femenino , Humanos , Masculino , Escala del Estado Mental , Persona de Mediana Edad , Pruebas Neuropsicológicas , Ratas , Estadísticas no Paramétricas , Enzimas Activadoras de Ubiquitina/clasificaciónRESUMEN
BACKGROUND: In this pilot study we examined circulating concentrations of nerve growth factor (NGF) in controls and patients suffering from primary or secondary Raynaud's syndrome and determined their relation to digital vasospasm. PATIENTS AND METHODS: Eighteen controls, 16 patients with primary RP and 19 patients with systemic sclerosis (SScl) were included. Degree of vasospasm was graduated according to the degree of plethysmographically measured vasospastic reaction after cold test. A diary was handed out for documentation of the daily number and duration of RP during a period of 16 days. Circulating NGF levels were analysed by a commercial ELISA (Promega Inc., USA). RESULTS: SScl-patients were significantly older (p < 0.0001) and more severely affected by spontaneously occurring RP (p = 0.03), whereas the severity of the vasospastic reactions after a standard cold test were not significantly different between the groups (p = 0.09). Within each study group and between the study groups elevated NGF levels were observed only in SScl-patients after thermal provocations (p = < 0.05). In a correlation analysis restricted to patients with PRP or SRP, the degree of vasospasm after cold testing as well as the intensity of Raynaud's symptoms were not correlated with NGF-levels (p = n.s.). CONCLUSIONS: Our results do not support the hypothesis that NGF plays a major role in the generation of vasospasm in Raynaud's phenomenon.
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Dedos/irrigación sanguínea , Factor de Crecimiento Nervioso/sangre , Enfermedad de Raynaud/sangre , Esclerodermia Sistémica/complicaciones , Vasoconstricción , Adulto , Anciano , Estudios de Casos y Controles , Frío , Ensayo de Inmunoadsorción Enzimática , Humanos , Persona de Mediana Edad , Proyectos Piloto , Pletismografía , Enfermedad de Raynaud/etiología , Enfermedad de Raynaud/fisiopatología , Esclerodermia Sistémica/sangre , Esclerodermia Sistémica/fisiopatología , Índice de Severidad de la Enfermedad , Factores de Tiempo , Adulto JovenRESUMEN
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder which primarily affects motor neurons. Eight cases of ALS and seven control cases were studied with semiquantitative immunocytochemistry for chromogranin A, chromogranin B and secretogranin II that are soluble constituents of large dense core vesicles, synaptophysin as a membrane protein of small synaptic vesicles and superoxide dismutase 1. Among the chromogranin peptides, the number and staining intensity of motor neurons was highest for chromogranin A. In ALS, the staining intensity for chromogranin peptides and synaptophysin was significantly lower in the ventral horn of ALS patients due to a loss in immunoreactive motor neurons, varicose fibers and varicosities. For all chromogranins, the remaining motor neurons displayed a characteristic staining pattern consisting of an intracellular accumulation of immunoreactivity with a high staining intensity. Confocal microscopy of motor neurons revealed that superoxide dismutase 1-immunopositive intracellular aggregates also contained chromogranin A, chromogranin B and secretogranin II. These findings indicate that there is a loss of small and large dense core vesicles in presynaptic terminals. The intracellular co-occurrence of superoxide dismutase 1 and chromogranins may suggest a functional interaction between these proteins. This study should prompt further experiments to elucidate the role of chromogranins in ALS patients.
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Esclerosis Amiotrófica Lateral/metabolismo , Cromograninas/metabolismo , Anciano , Estudios de Casos y Controles , Cromogranina A/análisis , Cromogranina A/metabolismo , Cromogranina B/análisis , Cromogranina B/metabolismo , Cromograninas/análisis , Femenino , Humanos , Masculino , Microscopía Confocal , Persona de Mediana Edad , Péptidos/análisis , Péptidos/metabolismo , Secretogranina II/análisis , Secretogranina II/metabolismo , Sinaptofisina/análisis , Sinaptofisina/metabolismoRESUMEN
Alzheimer's disease (AD) is characterized by beta-amyloid plaques, tau pathology, cholinergic cell death and inflammation. The aim of this study was to investigate whether beta-amyloid is generated, released and extracellularly deposited in organotypic brain slices. In developing slices, no amyloid-precursor protein (APP) was detectable; however, there was a strong upregulation in aging slices. In such slices, rat beta-amyloid(1-42) and -(1-40) peptides were found using four sequence-specific antibodies. APP and beta-amyloid were expressed in neurons and to a lesser extent in astrocytes. Beta-amyloid was secreted into the medium. Beta-amyloid was located extracellularly when aging slices were incubated with medium at pH 6.0 including apolipoprotein E4 (ApoE4). It is concluded that aging organotypic brain slices express beta-amyloid and that acidosis induces cell death with efflux of beta-amyloid and extracellular depositions, which is triggered by ApoE4. This novel in vitro model may enable us to investigate further the pathological cascade for AD and may be useful to explore future therapeutics.
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Envejecimiento , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Neuronas/metabolismo , Fragmentos de Péptidos/metabolismo , Anciano , Anciano de 80 o más Años , Envejecimiento/patología , Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Apolipoproteína E4/metabolismo , Encéfalo/patología , Colina O-Acetiltransferasa/genética , Colina O-Acetiltransferasa/metabolismo , Técnicas de Cocultivo , Ensayo de Inmunoadsorción Enzimática/métodos , Expresión Génica/fisiología , Humanos , Concentración de Iones de Hidrógeno , Indoles , Neuronas/patología , Técnicas de Cultivo de Órganos , Fosfopiruvato Hidratasa/metabolismo , Cambios Post Mortem , ARN Mensajero/metabolismo , Ratas , Factores de TiempoRESUMEN
The human vagal/nucleus solitary complex is a primary visceral relay station and an integrative brain stem area which displays a high density of chromogranin B- and secretoneurin-like immunoreactivity. In this study, we localized and biochemically identified these proteins during prenatal development. At prenatal week 11, 15, 20 and 37, we performed a chromatographic analysis to identify the molecular forms of PE-11, a peptide within the chromogranin B sequence, and secretoneurin, a peptide within secretogranin II. Their localization was studied with immunocytochemistry, and was compared to that of substance P which is well established as a functional neuropeptide in the vagal/nucleus solitary complex. At prenatal week 11, chromogranin B-, secretoneurin- and substance P-like immunoreactivities were detected consisting of varicosities, varicose fibers and single cells. At the same time, PE-11 and secretoneurin appeared as a single peak in chromatographic analysis. Prohormone convertases PC1- and PC2-like immunoreactivities were also present at week 11. In general, the density for each peptide increased during later fetal stages with the highest density at week 37. These results demonstrate that each chromogranin peptide is expressed during human fetal life in neurons of the vagal/nucleus solitary complex indicating that these peptides could be important during prenatal development.
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Cromograninas/análisis , Neuropéptidos/análisis , Núcleo Solitario/embriología , Nervio Vago/embriología , Cromogranina B , Cromograninas/fisiología , Femenino , Feto/química , Edad Gestacional , Humanos , Inmunohistoquímica , Masculino , Proproteína Convertasa 1/análisis , Proproteína Convertasa 2/análisis , Secretogranina II , Núcleo Solitario/química , Sustancia P/análisis , Nervio Vago/químicaRESUMEN
Cholinergic neurons degenerate in Alzheimer's disease and dementia and neuroprotective substances are of high interest to counteract this cell death. The aim of the present study was to test the effect of urea and the nitric oxide synthetase inhibitor l-thiocitrulline on the survival of cholinergic neurons. Organotypic brain slices of the basal nucleus of Meynert were cultured for 2 weeks in the presence of 1-100 microM urea with or without NGF or other growth factors or with or without 1-10 microM of the NOS inhibitor L-thiocitrulline. A high number of cholinergic neurons survived in the presence of 0.1-100 ng/ml NGF. Urea or L-thiocitrulline alone did not exhibit neuroprotective activity; however, when brain slices were incubated with urea or L-thiocitrulline together with NGF there was a significant potentiating survival effect. Incubation of brain slices with NGF + urea + L-thiocitrulline did not further enhance the number of cholinergic neurons. NGF as well as urea did not stimulate expression of the enzyme choline acetyltransferase pointing to survival promoting effects. Urea did not modulate the NGF binding in PC12 cells indicating that this effect was indirect. It is concluded that urea may play a role as an indirect survival promoting molecule possibly involving the nitric oxide pathway.
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Núcleo Basal de Meynert/efectos de los fármacos , Fibras Colinérgicas/efectos de los fármacos , Citrulina/análogos & derivados , Factor de Crecimiento Nervioso/agonistas , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Tiourea/análogos & derivados , Urea/farmacología , Animales , Animales Recién Nacidos , Núcleo Basal de Meynert/citología , Núcleo Basal de Meynert/metabolismo , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Colina O-Acetiltransferasa/efectos de los fármacos , Colina O-Acetiltransferasa/metabolismo , Fibras Colinérgicas/metabolismo , Fibras Colinérgicas/ultraestructura , Citrulina/farmacología , Sinergismo Farmacológico , Inhibidores Enzimáticos/farmacología , Factor de Crecimiento Nervioso/farmacocinética , Factor de Crecimiento Nervioso/farmacología , Neuronas/citología , Neuronas/metabolismo , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa/antagonistas & inhibidores , Técnicas de Cultivo de Órganos , Células PC12 , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Ratas , Tiourea/farmacologíaRESUMEN
Nerve growth factor (NGF) is a potent growth factor for cholinergic neurons. The aim of the present study was to investigate if NGF affects cholinergic neurons of the basal nucleus of Meynert (nBM) in organotypic brain slices. In single nBM slices cholinergic neurons rapidly degenerated when incubated without NGF. The number of remaining neurons was rescued by NGF application at any time point. When nBM slices were co-cultured with a cortex slice the number of cholinergic neurons was significantly increased pointing to a trophic influence of the cortex. Incubation with acetylcholine precursors did not affect the survival of cholinergic neurons. There was no significant difference when postnatal day 3 or day 10 nBM slices were cultured. In conclusion, NGF is the most potent growth factor for cholinergic neurons and is a promising candidate for treating Alzheimers disease, however, the delivery of NGF to the brain must the solved.
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Enfermedad de Alzheimer/tratamiento farmacológico , Núcleo Basal de Meynert/citología , Corteza Cerebral/citología , Fibras Colinérgicas/química , Factor de Crecimiento Nervioso/farmacología , Acetilcoenzima A/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Colina/farmacología , Colina O-Acetiltransferasa/análisis , Fibras Colinérgicas/enzimología , Degeneración Nerviosa/patología , Factor de Crecimiento Nervioso/análisis , Nootrópicos/farmacología , Técnicas de Cultivo de Órganos , RatasRESUMEN
Repetitive transcranial magnetic stimulation is a novel non-invasive method with antidepressant properties, where electromagnetic fields are applied via an electrode. The aim of the present study was to investigate in an in vitro model if magnetic stimulation may activate the transcription factor c-fos. Organotypic brain slices of the parietal cortex were cultured for 2 weeks and then treated with a magnetic stimulator. Immunohistochemistry was used to detect c-fos like immunoreactivity. We show that magnetic stimulation (1 Hz, 10 min, 75% machine output/magstim 200 rapid stimulator) transiently enhanced c-fos 3-6 h after stimulation. Co-localization experiments revealed that c-fos was expressed in neurons but not astroglia. The activation of c-fos by magnetic stimulation was inhibited by the sodium-channel blocker tetrodotoxin (TTX) (10 microM). It is concluded that magnetic stimulation induces neuronal c-fos via TTX-sensitive sodium channels in organotypic cortex slices.
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Corteza Cerebral/metabolismo , Magnetismo , Neuronas/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Canales de Sodio/metabolismo , Animales , Corteza Cerebral/citología , Estimulación Eléctrica , Técnicas de Cultivo de Órganos , Ratas , Bloqueadores de los Canales de Sodio , Tetrodotoxina/farmacologíaRESUMEN
Chromogranin A, chromogranin B and secretogranin II belong to the chromogranin family which consists of large protein molecules that are found in large dense core vesicles. Chromogranins are endoproteolytically processed to smaller peptides. This study was designed to elucidate the regulation of chromgranin expression by acute and subchronic phencyclidine administration. The behavioral syndrome produced by phencyclidine represents a pharmacological model for some aspects of schizophrenia [Jentsch and Roth (1999) Neuropsychopharmacology 20, 201-225]. Tissue concentrations of chromogranins were measured with specific radioimmunoassays. Alterations in secretogranin II gene expression were investigated by in situ hybridization. A single dose of phencyclidine (10mg/kg) led to a transient decrease in secretoneurin tissue levels in the prefrontal cortex after 4h followed by an increase in secretoneurin tissue levels after 12h. Repeated phencyclidine treatment (10mg/kg/day) for five days resulted in elevated secretoneurin levels in cortical areas whereas chromogranin A and chromogranin B tissue levels were unchanged. After the same treatment, a significant increase in the number of secretoneurin containing neurons was found in cortical layers II-III, and V-VI as revealed by immunocytochemistry. The increases in secretoneurin levels were paralleled by an increased number of secretogranin II messenger RNA containing neurons as well as by an increased expression of secretogranin II by individual neurons. The present study shows that secretoneurin II tissue concentration and secretogranin II messenger RNA expression is distinctly altered after acute and subchronic phencyclidine application. From these results we suggest that phencyclidine may induce synaptic alterations in specific brain areas and may contribute to a better understanding of synaptic dysfunction which may also occur in schizophrenia.
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Encéfalo/efectos de los fármacos , Cromograninas/efectos de los fármacos , Antagonistas de Aminoácidos Excitadores/farmacología , Neuronas/efectos de los fármacos , Fenciclidina/farmacología , Proteínas/efectos de los fármacos , Psicosis Inducidas por Sustancias/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/fisiopatología , Cromogranina A , Cromogranina B , Cromograninas/genética , Cromograninas/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Inmunohistoquímica , Masculino , Plasticidad Neuronal/efectos de los fármacos , Plasticidad Neuronal/fisiología , Neuronas/metabolismo , Neuropéptidos/efectos de los fármacos , Neuropéptidos/genética , Neuropéptidos/metabolismo , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/farmacocinética , Proteínas/genética , Proteínas/metabolismo , Psicosis Inducidas por Sustancias/fisiopatología , ARN Mensajero/metabolismo , Radioinmunoensayo , Ratas , Ratas Sprague-Dawley , Esquizofrenia/metabolismo , Esquizofrenia/fisiopatología , Secretogranina II , Vesículas Secretoras/efectos de los fármacos , Vesículas Secretoras/metabolismo , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiologíaRESUMEN
Loss of cholinergic neurons is found in the medial septum and nucleus basalis of Meynert in Alzheimer's disease. Recent observations suggest that cholinergic neurons down-regulate their phenotype and that growth factors may rescue cholinergic neurons. The aim of this study was to investigate whether cholinergic neurons of the basal nucleus of Meynert can be cultured in rat organotypic slices, and if nerve growth factor and glial cell line-derived neurotrophic factor can rescue the cholinergic phenotype. In the organotypic slices, glial cells, GABAergic and cholinergic neurons were visualized using immunohistochemistry. The number of cholinergic neurons was found to be very low in slices cultured without exogenous nerve growth factor. Analysis of nerve growth factor tissue levels by enzyme-linked immunosorbent assay revealed very low endogenous tissue levels. When slices were incubated with 100ng/ml nerve growth factor during the initial phase of culturing, a stable expression of choline acetyltransferase was found for up to several weeks. After eight weeks in culture with nerve growth factor or two to three weeks after nerve growth factor withdrawal, numbers of detected cholinergic neurons decreased. Neurons incubated with nerve growth factor displayed a significantly enlarged cell soma compared to neurons without growth factors. In cultures incubated for up to nine weeks, it was also found that glial cell line-derived neurotrophic factor was capable of restoring the cholinergic phenotype. The low-affinity p75 and high-affinity trkA receptors, as well as the glial cell line-derived neurotrophic factor receptor GFRalpha-1, could be visualized in slices using immunohistochemistry. In conclusion, it is shown that, in the axotomized organotypic slice model, the number of cholinergic neurons is decreased, but can be partly restored by nerve growth factor and glial cell line-derived neurotrophic factor.
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Núcleo Basal de Meynert/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Fibras Colinérgicas/efectos de los fármacos , Proteínas de Drosophila , Factor de Crecimiento Nervioso/farmacología , Factores de Crecimiento Nervioso , Regeneración Nerviosa/efectos de los fármacos , Proteínas del Tejido Nervioso/farmacología , Neuronas/efectos de los fármacos , Acetilcolina/metabolismo , Animales , Núcleo Basal de Meynert/citología , Núcleo Basal de Meynert/metabolismo , Supervivencia Celular/fisiología , Colina O-Acetiltransferasa/metabolismo , Fibras Colinérgicas/metabolismo , Fibras Colinérgicas/ultraestructura , Factor Neurotrófico Derivado de la Línea Celular Glial , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial , Inmunohistoquímica , Regeneración Nerviosa/fisiología , Neuronas/citología , Neuronas/metabolismo , Fenotipo , Proteínas Proto-Oncogénicas/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-ret , Ratas , Ratas Sprague-Dawley , Proteínas Tirosina Quinasas Receptoras/efectos de los fármacos , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor de Factor de Crecimiento Nervioso/efectos de los fármacos , Receptor de Factor de Crecimiento Nervioso/metabolismo , Receptor trkA/efectos de los fármacos , Receptor trkA/metabolismoRESUMEN
In Alzheimer's disease cholinergic neurons degenerate, resulting in loss of hippocampal acetylcholine. The fimbria-fornix aspiration is a well-known animal model mimicking hippocampal cholinergic deficiency. The aim of the present study was to use in vivo lipid-mediated gene transfer to introduce an expression vector coding for the acetylcholine synthesizing enzyme choline acetyltransferase into the hippocampus to replace the loss of enzyme activity after unilateral fimbria-fornix aspiration. Our data show that the lipid FuGene is useful to transfer DNA in vitro into 3T3 fibroblasts, C6 glioma cells, and primary astroglia and to express the respective enzyme. Lipid-mediated gene transfer in vivo resulted in a marked but transient expression of green fluorescent protein below the injection site peaking 5 days after the injection. Unilateral fimbria-fornix aspiration led to a marked reduction in the activity of choline acetyltransferase in the hippocampus, which was completely replaced 5 days after lipid-mediated gene transfer of the choline acetyltransferase vector. In conclusion, our data provide evidence that lipid-mediated gene transfer using FuGene is a useful tool to replace loss of choline acetyltranseferase activity in the hippocampus after fimbria-fornix aspiration; however, the lack of good gene transfer efficiency and the transient nature of expression limit its use for clinical applications.
Asunto(s)
Colina O-Acetiltransferasa/genética , Fórnix/cirugía , Hipocampo/enzimología , Lípidos , Transfección/métodos , Células 3T3 , Animales , Biotecnología , Colina O-Acetiltransferasa/metabolismo , Expresión Génica , Vectores Genéticos , Proteínas Fluorescentes Verdes , Indicadores y Reactivos/análisis , Liposomas , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Ratones , Enfermedades Neurodegenerativas/terapia , Ratas , Succión , Células Tumorales CultivadasRESUMEN
Human NTera-2/clone D1 teratocarcinoma cells are induced by retinoic acid (RA) to differentiate into postmitotic cells with morphological and biochemical characteristics of embryonic human neurones. Currently only limited information concerning peptide-contents and neurotransmitter pools of these cells is available. Zeller and Strauss [Int. J. Dev. Neurosci. 1995;13(5):437] described an increase in choline acetyltransferase (ChAT) activity in RA-treated, but not in untreated NTera-2 cells, suggesting the induction of a cholinergic phenotype during treatment with RA. In the present study we investigated the effect of RA-differentiation on the amount of the neurotransmitters acetylcholine (ACh), and dopamine in NTera-2 in order to specify the transmitter phenotype induced by RA-differentiation. We found that a 4-week treatment of NTera-2 cells with 10 microM RA markedly increased the ACh-content of these cells, while dopamine levels were unchanged. Depolarisation with potassium (60 mM) enhanced ACh-outflow in the differentiated cells in a Ca(++) dependent way. Also neuropeptides like substance P and NPY were detectable in the undifferentiated NTera-2 cells, while vasointestinal peptide (VIP) could not be found in either precursor or RA-differentiated cells. Differentiation was accompanied by a marked reduction of neutral endopeptidase enzyme activity and aminopeptidase activity. From these observations it was concluded that RA induces a cholinergic neurochemical differentiation of this human teratocarcinoma cell line, and that these cells might provide a model system to investigate cholinergic properties of human origin.
Asunto(s)
Acetilcolina/metabolismo , Teratocarcinoma/metabolismo , Tretinoina/farmacología , Antígenos CD13/metabolismo , Calcio/farmacología , Diferenciación Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Dopamina/metabolismo , Humanos , Neprilisina/metabolismo , Neuropéptido Y/metabolismo , Fenotipo , Cloruro de Potasio/farmacología , Sustancia P/metabolismo , Teratocarcinoma/patología , Células Tumorales Cultivadas , Péptido Intestinal Vasoactivo/metabolismoRESUMEN
Growth factors play an important role in proliferation and differentiation of malignant brain gliomas in humans. Glial cell line-derived neurotrophic factor (GDNF) has been shown recently to be highly expressed in human glioblastomas and in rat glial cell lines B49 and C6. The aim of the present study was to knockdown GDNF, its receptor GFR-alpha1, and the related family member persephin by using antisense oligonucleotides and to observe the effects on cell proliferation. To enhance cellular uptake into C6 glioma cells, 15-mer phosphorothioate oligonucleotides were complexed with the cationic lipid Lipofectamine. The complex was applied for 3 x 12 hours to C6 glioma cells, and cells were allowed to recover for 24 hours after each transfection and then analyzed. This protocol markedly reduced GDNF and GFR-alpha1 protein levels in C6 glioma cells compared with control oligonucleotides. Knockdown of C6 cells with GDNF and GFR-alpha1 but not with persephin antisense oligonucleotides significantly decreased the number of C6 glioma cells and also inhibited the incorporation of bromodeoxyuridine as a sign of reduced DNA synthesis. In conclusion, it is shown that GDNF but not persephin is a potent proliferation factor for rat glioma cells. Knockdown of GDNF using antisense oligonucleotides complexed with lipids as carriers may be useful in gene therapeutic approaches in vitro and possibly also in vivo.
Asunto(s)
Proteínas de Drosophila , Glioma/patología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Oligonucleótidos Antisentido/genética , Animales , Western Blotting , Bromodesoxiuridina/metabolismo , Resinas de Intercambio de Catión/metabolismo , Recuento de Células , División Celular/efectos de los fármacos , Supervivencia Celular , Sistema Nervioso Central/metabolismo , Ensayo de Inmunoadsorción Enzimática , Terapia Genética , Factor Neurotrófico Derivado de la Línea Celular Glial , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial , Glioma/genética , Glioma/metabolismo , Metabolismo de los Lípidos , Lípidos , Liposomas/química , Liposomas/metabolismo , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/metabolismo , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/farmacología , Oligonucleótidos Antisentido/metabolismo , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-ret , Ratas , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transfección , Azul de Tripano/metabolismo , Células Tumorales CultivadasRESUMEN
The isolated single organotypic slice model allows to investigate the effects of drugs and toxins on the expression of transcription factors in the striatum without dopaminergic and glutamatergic interactions. In this study the effects of margatoxin and iberiotoxin on the expression of c-fos mRNA by in situ hybridization as well as on c-fos like protein by immunohistochemistry in isolated dorsal striatum after 10 days in culture were investigated. C-fos mRNA dose-dependently increased 30 min after incubation with margatoxin and iberiotoxin. Expression of c-fos like protein was transiently detected 3h afterwards. This effect is independent from extrinsic neuronal circuitry as dopamine neurons were found to be absent in the cultured slices. It is concluded that inhibition of voltage-gated as well as calcium-activated (Slo) potassium channels leads to activation of gene transcription in striatal neurons which may trigger long-term changes in transmitter plasticity.