RESUMEN
BACKGROUND: Severe illness, disability and immobility increase the risk of pressure ulcer development. Pressure ulcers are localized injuries to the skin and/or underlying tissue as a result of long enduring pressure and shear. Little is known about the role of the stratum corneum and the upper skin layers in superficial pressure ulcer development. OBJECTIVES: To investigate possible effects of long enduring loading on the skin barrier function under clinical conditions at two pressure ulcer predilection sites. METHODS: Under controlled conditions 20 healthy females (mean age 69.9 (3.4) years) followed a standardized immobilization protocol of 90 and 150 min in supine position wearing hospital nightshirts on a standard hospital mattress. Before and immediately after the loading periods skin surface temperature, stratum corneum hydration, transepidermal water loss and erythema were measured at the sacral and heel skin. RESULTS: Prolonged loading caused increases of skin surface temperature and erythema at the sacral and heel skin. Stratum corneum hydration remained stable. Transepidermal water loss increased substantially after loading at the heel but not at the sacral skin. CONCLUSIONS: Skin functions change during prolonged loading at the sacral and heel skin in aged individuals. Accumulation of heat and hyperaemia seem to be primarily responsible for increasing skin temperature and erythema which are associated with pressure ulcer development. Increased transepidermal water loss at the heels indicate subclinical damages of the stratum corneum at the heel but not at the sacral skin during loading indicating distinct pathways of pressure ulcer development at both skin areas.
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Fenómenos Fisiológicos de la Piel , Anciano , Femenino , Humanos , Úlcera por Presión/etiologíaRESUMEN
In the framework of a project on the hygiene status of freshly shot game 289 samples were microbiologically analysed: 127 samples from wild boars, 95 from roe deer and 67 from red deer. The microbiological parameters evaluated were the mesophilic aerobic count (APC), which showed mean log10-counts of 2.6cfu/cm(2) for roe deer, 2.9cfu/cm(2) for red deer and 3.2cfu/cm(2) for wild boars and the numbers of Enterobacteriaceae, which gave mean log10-values of 2.1cfu/cm(2) for all three species with differing ranges. The concentrations of coagulase positive staphylococci were >2.0 log10cfu/cm(2) between 3.2 and 6.3%, according to species. Listeria was found in 14 samples and three samples gave a positive result for Campylobacter. Salmonella was not found in any of the samples analysed.
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Regulatory T cells are promising candidates for the modulation of inflammation and autoimmunity. To generate regulatory T cells in vitro, we have infected naïve CD4+CD25- T cells with a retrovirus encoding the transcription factor Foxp3. Foxp3-infected T cells are similar to naturally occurring regulatory T cells as evidenced by surface marker expression and function. To investigate the effects of Foxp3-infected T cells on contact hypersensitivity (CHS) responses, sensitized mice were injected with Foxp3- or control virus-infected T cells. Only injection of Foxp3-infected T cells into sensitized mice significantly inhibited CHS compared to controls, indicating that Foxp3-infected T cells are suppressive in vivo. These findings prompted treatment of autoimmune-prone CD40L transgenic (tg) mice, which develop a severe systemic autoimmune disease including autoreactive T cells and autoantibodies, with Foxp3-infected T cells. Interestingly, injections of Foxp3-infected T cells into CD40L tg mice inhibited the ongoing development of autoimmune dermatitis and activation of cytotoxic CD8+ T cells. Strikingly, treatment with Foxp3-infected T cells reduced serum concentrations of antinuclear antibodies in CD40L tg mice, which was paralleled with reduced renal immunoglobulin depositions and increased kidney function. Together, these findings indicate that newly in vitro-generated regulatory T cells can be successfully used to treat inflammatory and ongoing autoimmune disorders.
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Dermatitis por Contacto/terapia , Desensibilización Inmunológica/métodos , Factores de Transcripción Forkhead/genética , Terapia Genética/métodos , Retroviridae/genética , Linfocitos T Reguladores/virología , Animales , Anticuerpos Antinucleares/sangre , Autoinmunidad , Antígenos CD4/inmunología , Ligando de CD40/genética , Dermatitis por Contacto/inmunología , Dermatitis por Contacto/metabolismo , Femenino , Citometría de Flujo , Riñón/inmunología , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Animales , Receptores de Interleucina-2/inmunología , Proteínas Recombinantes de Fusión/administración & dosificación , Infecciones por Retroviridae/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T Citotóxicos/inmunología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismoRESUMEN
With the recent development of new hybrid compounds having histamine H3 receptor antagonist with combined histamine Ntau-methyltransferase (HMT) inhibitory potency an innovative approach was described in the research of novel lead compounds modulating histaminergic neurotransmission. Several compounds containing an ether moiety derived from the recently published 4-(3-piperidinopropoxy)phenylaminoquinoline derivatives (like FUB 836), were synthesized in this study and tested for their affinity at cloned human histamine H3 (hH3) receptors and on the inhibition of rat HMT. Besides different heterocycles, e.g. nitro- or amino-substituted pyridines, quinolines, benzothiazole or pyrroline, three classes of compounds were produced: heteroaromatic 3-piperidinopropyl ethers, keto- or imino-substituted 4-(3-piperidinopropyl)phenyl ethers and 4-(3-piperidinopropyl)phenyl ethers with substituted (alkyl)aminopyridines. Whereas the (3-piperidinopropoxy)heterocycles showed only moderate activities on both test models, the 4-(3-piperidinopropoxy)phenyl derivatives were identified as potent histamine H3 receptor ligands and/or HMT inhibitors. Ki values up to 0.42 nM were found for the affinity to the hH3 receptor. HMT inhibitory potency was identified with IC50 values about 0.3 microM for the most potent compounds in this series. Comparison of the pyridine-containing derivatives to recently published quinoline analogues showed a decrease in potencies for the pyridines. The dual activity, H3 receptor affinity and HMT inhibition, was moderate to good. For all compounds affinities at hH3 receptors were higher than their inhibitory HMT potencies. The described new histamine H3 receptor antagonists with an ether moiety represent a further promising step in our investigations for a dual activity.
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Éteres/farmacología , Antagonistas de los Receptores Histamínicos/farmacología , Metiltransferasas/metabolismo , Receptores Histamínicos H3/efectos de los fármacos , Animales , Unión Competitiva , Células CHO , Fenómenos Químicos , Química Física , Cricetinae , Femenino , Antagonistas de los Receptores H2 de la Histamina/metabolismo , Humanos , Imidazoles/metabolismo , Indicadores y Reactivos , Riñón/efectos de los fármacos , Riñón/metabolismo , Ratas , Relación Estructura-ActividadRESUMEN
Traumatic brain injury (TBI) is a risk factor for the development of Alzheimer's disease (AD). After a traumatic brain injury depositions of amyloid beta (Abeta) in the brain parenchyma were found. In this study we investigated the expression pattern of beta-secretase (BACE-1) in ipsi- or contralateral hippocampus and cortex following controlled cortical TBI in rats. BACE-1 mRNA levels, estimated by real time RT-PCR, were elevated 24 h post injury, and persisting up to 72 h, in the ipsi- and contralateral hippocampus and cerebral cortex as compared to the sham-treated animals (p<0.01). The TBI-induced changes in BACE-1 mRNA are due to enhanced hippocampal and cortical expression of BACE-1 mRNA in neurons and reactive astrocytes as revealed by in situ hybridization. The alterations in hippocampal BACE-1 mRNA levels are accompanied by corresponding increases in BACE-1 protein levels in ipsi- and contralateral hippocampus and ipsilateral cortex as demonstrated by Western blot analysis. In contrast, in the contralateral cortex only a weak increase of traumatically induced BACE-1 protein production was found. The activity of BACE-1 as measured by the formation of the cleavage product of amyloid beta precursor protein, transiently increased up to 48 h after injury, but returned to basal level 7 days post injury. This study demonstrates that the beta-secretase is stimulated following TBI and may suggest a mechanism for the temporal increase of Abeta levels observed in patients with brain trauma.
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Ácido Aspártico Endopeptidasas/genética , Lesiones Encefálicas/enzimología , Lesiones Encefálicas/genética , Regulación Enzimológica de la Expresión Génica/genética , Enfermedad de Alzheimer/enzimología , Secretasas de la Proteína Precursora del Amiloide , Animales , Corteza Cerebral/enzimología , Modelos Animales de Enfermedad , Endopeptidasas , Lateralidad Funcional , Genes Reporteros , Proteína Ácida Fibrilar de la Glía/análisis , Hipocampo/enzimología , Hibridación in Situ , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Ratas , Transcripción GenéticaRESUMEN
Alzheimer's disease is associated with markedly impaired cerebral glucose metabolism as detected by reduced cortical desoxyglucose utilization, by altered activities of key glycolytic enzymes or by reduced densities of cortical glucose transporter subtypes. To determine whether formation and/or deposition of beta-amyloid plays a role in the pathology of glucose metabolism, transgenic Tg2576 mice that overexpress the Swedish mutation of the human amyloid precursor protein and demonstrate a progressive, age-related cortical and hippocampal deposition of beta-amyloid plaques, were used to study expression and activity of key enzymes of brain glycolysis (phosphofructokinase, PFK) and glyconeogenesis (fructose1,6-bisphosphatase; FbPase). Quantitative RT-PCR revealed high expression levels of both C- and M-type PFK mRNA in non-transgenic mouse cerebral cortex, whilst there was little expression of the L-type. In 24-month-old transgenic Tg2576 mouse cortex, but not in 7-, 13-, and 17-month-old mice, the copy number of PFK-C mRNA was significantly reduced in comparison to non-transgenic littermates, while the mRNA level of the other PFK isoforms and FbPase did not differ between transgenic and non-transgenic tissue samples. In situ hybridization in brain sections from aged Tg2576 mice revealed reduced PFK-C mRNA expression in beta-amyloid plaque-associated neurons and upregulation in reactive astrocytes surrounding beta-amyloid deposits. The decreased PFK-C protein level detected by Western analysis in cerebral cortical tissue from 24-month-old transgenic Tg2576 mice was accompanied by reduced enzyme activity of PFK in comparison to non-transgenic littermates. Our data demonstrate that impairment of cerebral cortical glucose metabolism occurs only due to the long-lasting high beta-amyloid burden. This results from a reduction in glycolytic activity in beta-amyloid plaque-associated neurons and a concomitant upregulation in reactive, plaque-surrounding astrocytes.
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Enfermedad de Alzheimer/metabolismo , Corteza Cerebral/metabolismo , Fructosa-Bifosfatasa/metabolismo , Glucosa/metabolismo , Fosfofructoquinasa-1/metabolismo , Placa Amiloide/metabolismo , Factores de Edad , Enfermedad de Alzheimer/enzimología , Precursor de Proteína beta-Amiloide/genética , Animales , Western Blotting , Corteza Cerebral/enzimología , Modelos Animales de Enfermedad , Fructosa-Bifosfatasa/genética , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Fosfofructoquinasa-1/genética , Placa Amiloide/enzimología , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
We studied properties of cloned BACE mRNA (beta-site of the enzyme cleaving amyloid precursor protein) and evaluated the possibility of using this clone for identification and/or prediction of neurodegenerative disorders associated with cholinergic deficiency. Wistar rats subjected to immunohistochemical destruction of the basal forebrain cholinergic system were used as the experimental model. Nonradioactive in situ hybridization and immunohistochemical visualization of the astroglia revealed strong hybridization signal of BACE mRNA in neurons of the cortex, hippocampus, and thalamus. Astrocytes remained unstained. Immunohistochemical destruction of the basal forebrain produced no significant changes in the distribution of BACE mRNA.
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Ácido Aspártico Endopeptidasas/biosíntesis , Encéfalo/metabolismo , Prosencéfalo/metabolismo , ARN Mensajero/metabolismo , Secretasas de la Proteína Precursora del Amiloide , Animales , Endopeptidasas , Hipocampo/metabolismo , Inmunohistoquímica , Hibridación in Situ , Microscopía por Video , Oligonucleótidos Antisentido/metabolismo , Prosencéfalo/patología , Ratas , Ratas Wistar , Distribución TisularRESUMEN
The reference compounds for histamine H(3)-receptor antagonists carry as a common feature an imidazole moiety substituted in the 4-position. Very recently novel ligands lacking an imidazole ring have been described possessing a N-containing non-aromatic heterocycle instead. In this study we investigated whether imidazole replacement, favourably by a piperidine moiety, is generally applicable to different structural classes of reference compounds, e.g., thioperamide, carboperamide, clobenpropit, FUB 181, ciproxifan, etc. While replacement led to a loss of affinity for many of the compounds, it was successfully applied to some ether derivatives. The piperidine analogues of FUB 181 and ciproxifan, 3-(4-chlorophenyl)propyl 3-piperidinopropyl ether hydrogen oxalate (6) and cyclopropyl 4-(3-piperidinopropyloxy)phenyl methanone hydrogen maleate (7), almost maintained in vitro affinities, pK(i) values of 7.8 and 8.4, respectively, and showed high potency in vivo after p.o. administration (ED(50) values of 1.6 and 0.18 mg/kg, respectively).
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Antagonistas de los Receptores Histamínicos/farmacología , Imidazoles/farmacología , Receptores Histamínicos H3/efectos de los fármacos , Animales , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Cobayas , Antagonistas de los Receptores Histamínicos/química , Íleon/efectos de los fármacos , Íleon/metabolismo , Imidazoles/química , Técnicas In Vitro , Indicadores y Reactivos , Ratones , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Ratas , Relación Estructura-Actividad , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismoRESUMEN
We measured tissue distribution and expression pattern of the beta-site amyloid precursor protein (APP)-cleaving enzyme (BACE) in the brains of transgenic Tg2576 mice that show amyloid pathology. BACE protein was expressed at high levels in brain; at lower levels in heart and liver; and at very low levels in pancreas, kidney, and thymus and was almost absent in spleen and lung when assayed by Western blot analysis. We observed strictly neuronal expression of BACE protein in the brains of nontransgenic control mice, with the most robust immunocytochemical labeling present in the cerebral cortex, hippocampal formation, thalamus, and cholinergic basal forebrain nuclei. BACE protein levels did not differ significantly between control and transgenic mice or as a result of aging. However, in the aged, 17-month-old Tg2576 mice there was robust amyloid plaque formation, and BACE protein was also present in reactive astrocytes present near amyloid plaques, as shown by double immunofluorescent labeling and confocal laser scanning microscopy. The lack of astrocytic BACE immunoreactivity in young transgenic Tg2576 mice suggests that it is not the APP overexpression but rather the amyloid plaque formation that stimulates astrocytic BACE expression in Tg2576 mice. Our data also suggest that the neuronal overexpression of APP does not induce the overexpression of its metabolizing enzyme in neurons. Alternatively, the age-dependent accumulation of amyloid plaques in the Tg2576 mice does not require increased neuronal expression of BACE. Our data support the hypothesis that neurons are the primary source of beta-amyloid peptides in brain and that astrocytic beta-amyloid generation may contribute to amyloid plaque formation at later stages or under conditions when astrocytes are activated.
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Enfermedad de Alzheimer/enzimología , Ácido Aspártico Endopeptidasas/metabolismo , Encéfalo/enzimología , Neuroglía/enzimología , Neuronas/enzimología , Placa Amiloide/enzimología , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/fisiopatología , Secretasas de la Proteína Precursora del Amiloide , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Ácido Aspártico Endopeptidasas/genética , Encéfalo/metabolismo , Encéfalo/fisiopatología , Modelos Animales de Enfermedad , Endopeptidasas , Regulación Enzimológica de la Expresión Génica/fisiología , Humanos , Inmunohistoquímica , Ratones , Ratones Transgénicos/genética , Ratones Transgénicos/metabolismo , Neuroglía/patología , Neuronas/patología , Placa Amiloide/patología , Vísceras/enzimologíaRESUMEN
Transgenic mice (Tg2576) that express the Swedish double mutation of human amyloid precursor protein and develop Alzheimer-like beta-amyloid deposits in the aged brain, were used to study the effect of beta-amyloid deposition on expression of both neuronal (nNOS) and inducible nitric oxide synthase (iNOS) in cells surrounding beta-amyloid plaques. Nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry and double immunofluorescent labeling revealed that most of the fibrillary, thioflavine-S-positive cortical beta-amyloid deposits in 13-, 17-, and 21-month-old transgenic animals were closely associated with dystrophic nNOS-positive neurons, while nNOS-bearing neurons located more distal to plaques appeared to be unaffected. There was no significant expression of iNOS in transgenic mouse brain. The data suggest enhanced vulnerability of nNOS-containing neocortical neurons to beta-amyloid toxicity. Alternatively, expression of nNOS may also be a response to plaque-mediated damage of neurons, consistent with a neuroprotective role of nitric oxide.
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Enfermedad de Alzheimer/enzimología , Péptidos beta-Amiloides/metabolismo , Atrofia/enzimología , Encéfalo/enzimología , Degeneración Nerviosa/enzimología , Neuronas/enzimología , Óxido Nítrico Sintasa/metabolismo , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/fisiopatología , Animales , Atrofia/patología , Atrofia/fisiopatología , Encéfalo/patología , Encéfalo/fisiopatología , Modelos Animales de Enfermedad , Inmunohistoquímica , Ratones , Ratones Transgénicos/anatomía & histología , Ratones Transgénicos/metabolismo , NADPH Deshidrogenasa/metabolismo , Degeneración Nerviosa/patología , Degeneración Nerviosa/fisiopatología , Neuronas/patología , Placa Amiloide/enzimología , Placa Amiloide/patologíaRESUMEN
To elucidate the mechanisms involved in beta-amyloid-mediated inflammation in Alzheimer's disease, transgenic Tg2576 mice containing as transgene the Swedish double mutation of human amyloid precursor protein 695, were examined for the expression pattern of various cytokines using double immunocytochemistry and laser scanning microscopy. Tg2576 mice studied at postnatal ages of 13, 16 and 19 months demonstrated an age-related accumulation of both senile and diffuse beta-amyloid plaques in neocortex and hippocampus. Reactive interleukin (IL)-1beta-immunoreactive astrocytes were found in close proximity to both fibrillary and diffuse beta-amyloid deposits detectable at very early stages of plaque development, while activated microglia appeared in and around fibrillary beta-amyloid plaques only. Subpopulations of reactive astrocytes also demonstrated immunolabeling for transforming growth factor (TGF)-beta1, TGF-beta3, and IL-10, already detectable in 13-month-old transgenic mouse brain, while a few IL-6-immunoreactive astrocytes were observed only at later stages of plaque development. The early beta-amyloid-mediated upregulation of IL-1beta, TGF-beta, and IL-10 in surrounding reactive astrocytes indicates the induction of both pro- and anti-inflammatory mechanisms. The transgenic approach used in this study may thus provide a useful tool to further disclose the in vivo mechanisms by which pro- and anti-inflammatory cytokines interact and/or contribute to the progression of Alzheimer's disease.
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Péptidos beta-Amiloides/farmacología , Interleucina-10/metabolismo , Interleucina-1/metabolismo , Interleucina-6/metabolismo , Neuroglía/efectos de los fármacos , Factor de Crecimiento Transformador beta/efectos de los fármacos , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Citocinas/efectos de los fármacos , Citocinas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/efectos de los fármacos , Microglía/metabolismo , Neuroglía/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
On the basis of the recent cloning of the beta-secretase, the beta-site amyloid precursor protein (APP)-cleaving enzyme (BACE), (Science, 286 (1999) 735), digoxigenin-labelled riboprobes were generated to localize the cellular expression pattern of BACE mRNA in brain sections of transgenic Tg2576 mice, overexpressing the Swedish mutation of the APP695 isoform. Non-radioactive in situ hybridization in combination with immunohistochemistry to identify the cell types and beta-amyloid deposits revealed strong BACE mRNA hybridization signals in neurons of the cerebral cortex, hippocampal formation, thalamus and cholinergic basal forebrain nuclei, while astrocytes did not display any labeling. Neurons surrounding beta-amyloid deposits did not demonstrate altered expression level of BACE mRNA as compared to neurons in cortical areas that are free of beta-amyloid deposits, and the regional expression pattern of BACE mRNA did not correlate with the distribution of beta-amyloid deposits. These data suggest that high level of expression of BACE mRNA is not necessarily related to enhanced deposition of beta-amyloid plaques. To elucidate those factors that contribute to beta-amyloid plaque deposition in a particular region, the transgenic Tg2576 mouse may represent an appropriate tool.
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Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Ácido Aspártico Endopeptidasas/genética , Prosencéfalo/enzimología , Prosencéfalo/patología , Secretasas de la Proteína Precursora del Amiloide , Precursor de Proteína beta-Amiloide/análisis , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Ácido Aspártico Endopeptidasas/metabolismo , Digoxigenina , Endopeptidasas , Regulación Enzimológica de la Expresión Génica , Humanos , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Placa Amiloide/patología , Sondas ARN , ARN Mensajero/análisis , Transgenes/fisiologíaRESUMEN
The insulin-sensitive glucose transporter (GLUT) 4, expressed primarily in peripheral tissue, has recently been detected also in the brain, demonstrating a region-specific distribution. To identify the chemical nature of neurons expressing GLUT4 and to disclose whether GLUT4-containing neurons also express the GLUT3 isoform, combined in situ hybridization for GLUT3 mRNA and double-labeling immunocytochemistry for GLUT4 and different cellular markers was performed in brain sections through rat basal forebrain, cerebral cortex, hippocampus, and cerebellum. In all brain regions examined, GLUT4 immunoreactivity was exclusively found in neurons, and GLUT4-immunoreactive cells were colocalized with neurons expressing GLUT3 mRNA. In rat basal forebrain, cholinergic and parvalbumin-containing gamma-aminobutyric acid-ergic cells demonstrated GLUT4 immunoreactivity, whereas calretinin-, calbindin-D-, and neuronal nitric oxide synthase-containing neurons did not express GLUT4 protein. Because brain GLUT4 transporters have been suggested to play a role in rapidly providing additional glucose to neurons under conditions of high-energy demand, the selective presence of GLUT4 in basal forebrain cholinergic cells may explain the specific vulnerability of these cells to a lack of glucose supply.
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Encéfalo/fisiología , Insulina/farmacología , Proteínas de Transporte de Monosacáridos/genética , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Neuronas/fisiología , Transcripción Genética , Animales , Calbindina 2 , Calbindinas , Transportador de Glucosa de Tipo 3 , Transportador de Glucosa de Tipo 4 , Proteínas de Transporte de Monosacáridos/efectos de los fármacos , Proteínas del Tejido Nervioso/metabolismo , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo I , Especificidad de Órganos , Parvalbúminas/metabolismo , ARN Mensajero/genética , Ratas , Ratas Wistar , Proteína G de Unión al Calcio S100/metabolismo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
To address the question whether the changes in cortical glucose metabolism observed in patients with Alzheimer's disease are interrelated with, or consequences of, basal forebrain cholinergic cell loss, an experimental approach was employed to produce cortical cholinergic dysfunction in rat brain by administration of the cholinergic immunotoxin 192IgG-saporin. [14C]D-glucose utilization in brain homogenates, D-glucose-displaceable [3H]cytochalasin B binding to glucose transporters (GLUT). Northern and Western analyses, as well as in vivo [14C]2-deoxyglucose autoradiography were used to quantify the regional glucose metabolism. Basal forebrain cholinergic lesion resulted in transient increases in glucose transporter binding in cortical regions displaying reduced acetylcholinesterase activity, already detectable seven days after lesion with peak values around 30 days post lesion. Western analysis revealed that the changes in total glucose transporter binding are mainly due to changes in the GLUT3 subtype only, while the levels of GLUT1 and GLUT3 mRNA (Northern analysis) were not affected by cholinergic lesion. Both immunocytochemistry and in situ hybridization demonstrated preferential localizations of GLUT1 on brain capillaries and GLUT3 on neurons, respectively. A lesion-induced transient decrease in [14C]D-glucose utilization seven days post lesion was detected in the lesion site, whereas cholinoceptive cortical regions were not affected. In vivo [14C]deoxyglucose uptake was transiently increased in cholinoceptive cortical regions and in the lesion site being highest between three to seven days after lesion. The cholinergic lesion-induced transient up-regulation of cortical glucose transporters and deoxyglucose uptake reflects an increased glucose demand in regions depleted by acetylcholine suggesting functional links between cortical cholinergic activity and glucose metabolism in cholinoceptive target regions.
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Anticuerpos Monoclonales/toxicidad , Colinérgicos/toxicidad , Glucosa/metabolismo , Inmunotoxinas/toxicidad , Prosencéfalo/efectos de los fármacos , Receptores Colinérgicos/efectos de los fármacos , Animales , Autorradiografía , Northern Blotting , Western Blotting , Citocalasina B/metabolismo , Masculino , Proteínas de Transporte de Monosacáridos/metabolismo , N-Glicosil Hidrolasas , Prosencéfalo/metabolismo , Ensayo de Unión Radioligante , Ratas , Ratas Wistar , Receptores Colinérgicos/metabolismo , Proteínas Inactivadoras de Ribosomas Tipo 1 , SaporinasRESUMEN
Pathological processing of the amyloid precursor protein (APP) is assumed to be responsible for the amyloid deposits in Alzheimer-diseased brain tissue, but the physiological function of this protein in the brain is still unclear. The aim of this study is to reveal whether the expression of different splicing variants of APP transcripts in distinct brain regions is driven by postnatal maturation and/or regulated by cortical cholinergic transmission, applying quantitative in situ hybridization histochemistry using 35S-labeled oligonucleotides as specific probes to differentiate between APP isoforms. In cortical brain regions, the expression of both APP695 and APP751 is high at birth and exhibits nearly adult levels. The developmental expression pattern of cortical APP695 displays a peak value around postnatal day 10, while the age-related expression of APP751 demonstrates peak values on postnatal days 10 and 25, with the highest steady state levels of APP751 mRNA on day 25. During early development, the cortical laminar distribution of the APP695, but not APP751, mRNA transiently changes from a more homogeneous distribution at birth to a pronounced laminar pattern with higher mRNA levels in cortical layer III/IV detectable at the age of 4 days and persisting until postnatal day 10. The distinct age-related changes in cortical APP695 and APP751 mRNA levels reflect the functional alterations during early brain maturation and suggest that APP695 might play a role in establishing the mature connectional pattern between neurons, whereas APP751 could play a role in controlling cellular growth and synaptogenesis. Lesion of basal forebrain cholinergic system by the selective cholinergic immunotoxin 192IgG-saporin resulted in decreased levels of APP695 but not APP751 and APP770 transcripts by about 15-20% in some cortical (cingulate, frontal, parietal, piriform cortex), hippocampal regions (CA1, dentate gyrus), and basal forebrain nuclei (medial septum, vertical limb of diagonal band), detectable not earlier than 30 days after lesion and persisting until 90 days postlesion, suggesting that the nearly complete loss of cortical cholinergic input does not have any significant impact on the expression of APP mRNA isoforms in cholinoceptive cortical target regions.