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EIGER is a single-photon-counting hybrid pixel detector developed at the Paul Scherrer Institut, Switzerland. It is designed for applications at synchrotron light sources with photon energies above 5â keV. Features of EIGER include a small pixel size (75â µm × 75â µm), a high frame rate (up to 23â kHz), a small dead-time between frames (down to 3â µs) and a dynamic range up to 32-bit. In this article, the use of EIGER as a detector for electrons in low-energy electron microscopy (LEEM) and photoemission electron microscopy (PEEM) is reported. It is demonstrated that, with only a minimal modification to the sensitive part of the detector, EIGER is able to detect electrons emitted or reflected by the sample and accelerated to 8-20â keV. The imaging capabilities are shown to be superior to the standard microchannel plate detector for these types of applications. This is due to the much higher signal-to-noise ratio, better homogeneity and improved dynamic range. In addition, the operation of the EIGER detector is not affected by radiation damage from electrons in the present energy range and guarantees more stable performance over time. To benchmark the detector capabilities, LEEM experiments are performed on selected surfaces and the magnetic and electronic properties of individual iron nanoparticles with sizes ranging from 8 to 22â nm are detected using the PEEM endstation at the Surface/Interface Microscopy (SIM) beamline of the Swiss Light Source.
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JUNGFRAU (adJUstiNg Gain detector FoR the Aramis User station) is a two-dimensional hybrid pixel detector for photon science applications at free-electron lasers and synchrotron light sources. The JUNGFRAUâ 0.4 prototype presented here is specifically geared towards low-noise performance and hence soft X-ray detection. The design, geometry and readout architecture of JUNGFRAUâ 0.4 correspond to those of other JUNGFRAU pixel detectors, which are charge-integrating detectors with 75â µm × 75â µm pixels. Main characteristics of JUNGFRAUâ 0.4 are its fixed gain and r.m.s. noise of as low as 27â e(-) electronic noise charge (<100â eV) with no active cooling. The 48 × 48 pixels JUNGFRAUâ 0.4 prototype can be combined with a charge-sharing suppression mask directly placed on the sensor, which keeps photons from hitting the charge-sharing regions of the pixels. The mask consists of a 150â µm tungsten sheet, in which 28â µm-diameter holes are laser-drilled. The mask is aligned with the pixels. The noise and gain characterization, and single-photon detection as low as 1.2â keV are shown. The performance of JUNGFRAUâ 0.4 without the mask and also in the charge-sharing suppression configuration (with the mask, with a `software mask' or a `cluster finding' algorithm) is tested, compared and evaluated, in particular with respect to the removal of the charge-sharing contribution in the spectra, the detection efficiency and the photon rate capability. Energy-dispersive and imaging experiments with fluorescence X-ray irradiation from an X-ray tube and a synchrotron light source are successfully demonstrated with an r.m.s. energy resolution of 20% (no mask) and 14% (with the mask) at 1.2â keV and of 5% at 13.3â keV. The performance evaluation of the JUNGFRAUâ 0.4 prototype suggests that this detection system could be the starting point for a future detector development effort for either applications in the soft X-ray energy regime or for an energy-dispersive detection system.
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OBJECTIVE: To evaluate the Caiman vessel-sealing device for peripheral lung biopsy and total lung lobectomy in cadaveric canine lung lobes. MATERIAL AND METHODS: Twelve lung lobes were randomly assigned to peripheral lung biopsy (n=6) or total lung lobectomy (n=6) with the 12-mm Caiman vessel-sealing device. Lungs were connected to a ventilator set at 10 breaths per minute with an initial pressure of 5 cm H2 O during the procedure. The lungs were submerged in water for leak testing and the pressure increased until leakage occurred. RESULTS: Mean airway pressure at which leakage occurred was 39.17 ±13.20 cm H2 O for peripheral lung biopsies and 38.33 ±13.67 cm H2 O for total lung lobectomies. None of the samples leaked below 25 cm H2 O, which is well above the physiologic airway pressure. Histologically, the largest bronchial diameter at the sealed area was 8.84 mm and the extent of collateral damage was approximately 2.7 mm in all specimens. CLINICAL SIGNIFICANCE: The Caiman vessel-sealing device was successfully used for peripheral lung biopsy and total lung lobectomy in cadaveric canine lung lobes. All sealed lung lobes tolerated supra-physiologic airway pressure, displayed minimal collateral damage, and were of good diagnostic quality. Further experimental studies are needed to evaluate the clinical safety of the device for peripheral lung biopsy or total lung lobectomy.
Assuntos
Pulmão , Instrumentos Cirúrgicos , Animais , Biópsia/veterinária , Cadáver , CãesRESUMO
PECAM-1/CD31 is a cell adhesion and signaling molecule that is enriched at the endothelial cell junctions. Alternative splicing generates multiple PECAM-1 splice variants, which differ in their cytoplasmic domains. It has been suggested that the extracellular ligand-binding property, homophilic versus heterophilic, of these isoforms is controlled by their cytoplasmic tails. To determine whether the cytoplasmic domains also regulate the cell surface distribution of PECAM-1 splice variants, we examined the distribution of CD31-EGFPs (PECAM-1 isoforms tagged with the enhanced green fluorescent protein) in living Chinese hamster ovary cells and in PECAM-1-deficient endothelial cells. Our results indicate that the extracellular, rather than the cytoplasmic domain, directs PECAM-1 to the cell-cell borders. Furthermore, coculturing PECAM-1 expressing and deficient cells along with transfection of CD31-EGFP cDNAs into PECAM-1 deficient cells reveal that this PECAM-1 localization is mediated by homophilic interactions. Although the integrin alphavbeta3 has been shown to interact with PECAM-1, this trans-heterophilic interaction was not detected at the borders of endothelial cells. However, based on cocapping experiments performed on proT cells, we provide evidence that the integrin alphavbeta3 associates with PECAM-1 on the same cell surface as in a cis manner.
Assuntos
Endotélio Vascular/fisiologia , Junções Intercelulares/fisiologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Receptores de Vitronectina/metabolismo , Processamento Alternativo , Animais , Células CHO , Capilares/citologia , Capilares/fisiologia , Linhagem Celular , Células Cultivadas , Circulação Cerebrovascular , Cricetinae , Citoplasma/fisiologia , Endotélio Vascular/ultraestrutura , Éxons , Proteínas de Fluorescência Verde , Proteínas Luminescentes/análise , Camundongos , Camundongos Knockout , Modelos Moleculares , Molécula-1 de Adesão Celular Endotelial a Plaquetas/química , Conformação Proteica , Receptores de Vitronectina/química , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , TransfecçãoRESUMO
OBJECTIVE: To describe the clinical workup and laparoscopic treatment of ovarian remnant syndrome in dogs and cats. MATERIAL AND METHODS: After confirming the diagnosis with some or all of the following tests - vaginoscopy with cytology, hormonal tests, and ultrasound - laparoscopic removal of the ovarian remnants was performed. A three-portal technique was used in the four dogs and a two-portal technique in the two cats. RESULTS: All patients recovered well and were discharged the same day. No post-operative complications occurred in any patient. CONCLUSION AND CLINICAL RELEVANCE: Overall, in the hands of an experienced laparoscopic surgeon, laparoscopic removal of ovarian remnants appears to be a safe procedure in dogs and cats. In addition, laparoscopy offers the advantages of excellent visualization and a reduced morbidity for the patient. Careful case selection and complete pre-operative workup to rule out co-morbidities or underlying neoplasia are important. As with any laparoscopy the surgeon should always be prepared to convert to an open laparotomy if necessary.
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Gatos/cirurgia , Cães/cirurgia , Laparoscopia/veterinária , Ovariectomia/veterinária , Ovário/fisiologia , Animais , Estro , Feminino , Laparoscopia/efeitos adversos , Laparoscopia/normas , Ovariectomia/efeitos adversos , Ovariectomia/normas , Ovário/diagnóstico por imagem , Reoperação/veterináriaRESUMO
A 21-month-old male castrated domestic short hair cat was presented due to suspected unilateral abdominal cryptorchidism. Unilateral abdominal cryptorchidism was confirmed with ultrasonography and laparoscopic-assisted cryptorchidectomy was performed. Laparoscopic-assisted cryptorchidectomy is a simple, fast and safe method for the treatment of abdominal cryptorchidism in dogs and cats, offering the benefits of minimal invasive surgery, which is still underreported in the veterinary literature.
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Doenças do Gato/cirurgia , Criptorquidismo , Laparoscopia , Orquiectomia , Animais , Gatos , Criptorquidismo/cirurgia , Criptorquidismo/veterinária , Laparoscopia/métodos , Laparoscopia/veterinária , Masculino , Orquiectomia/métodos , Orquiectomia/veterináriaRESUMO
Physicians specializing in dysphagia are needed in modern intensive care medicine. Long-term intubation is associated with aspiration and swallowing disorders. Early and standardised dysphagia management should be initiated during a patient's stay on intensive care unit. A clinically experienced, interdisciplinary team is required to provide optimal care for critically ill patients with dysphagia. Intensive care physicians should therefore know about basics in dysphagiology.
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Cuidados Críticos/métodos , Transtornos de Deglutição/terapia , Medicina Interna , Transtornos de Deglutição/etiologia , Transtornos de Deglutição/mortalidade , Endoscopia , Tecnologia de Fibra Óptica , Humanos , Comunicação Interdisciplinar , Colaboração Intersetorial , Intubação Intratraqueal/efeitos adversos , Equipe de Assistência ao Paciente , Pneumonia Aspirativa/etiologia , Pneumonia Aspirativa/prevenção & controle , Análise de SobrevidaRESUMO
JUNGFRAU (adJUstiNg Gain detector FoR the Aramis User station) is a two-dimensional hybrid pixel detector for photon science applications in free electron lasers, particularly SwissFEL, and synchrotron light sources. JUNGFRAU is an automatic gain switching, charge-integrating detector which covers a dynamic range of more than 10(4) photons of an energy of 12 keV with a good linearity, uniformity of response, and spatial resolving power. The JUNGFRAU 1.0 application-specific integrated circuit (ASIC) features a 256 × 256 pixel matrix of 75 × 75 µm(2) pixels and is bump-bonded to a 320 µm thick Si sensor. Modules of 2 × 4 chips cover an area of about 4 × 8 cm(2). Readout rates in excess of 2 kHz enable linear count rate capabilities of 20 MHz (at 12 keV) and 50 MHz (at 5 keV). The tolerance of JUNGFRAU to radiation is a key issue to guarantee several years of operation at free electron lasers and synchrotrons. The radiation hardness of JUNGFRAU 1.0 is tested with synchrotron radiation up to 10 MGy of delivered dose. The effect of radiation-induced changes on the noise, baseline, gain, and gain switching is evaluated post-irradiation for both the ASIC and the hybridized assembly. The bare JUNGFRAU 1.0 chip can withstand doses as high as 10 MGy with minor changes to its noise and a reduction in the preamplifier gain. The hybridized assembly, in particular the sensor, is affected by the photon irradiation which mainly shows as an increase in the leakage current. Self-healing of the system is investigated during a period of 11 weeks after the delivery of the radiation dose. Annealing radiation-induced changes by bake-out at 100 °C is investigated. It is concluded that the JUNGFRAU 1.0 pixel is sufficiently radiation-hard for its envisioned applications at SwissFEL and synchrotron beam lines.
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Artefatos , Eletrônica/instrumentação , Fotometria/instrumentação , Fótons , Radiometria/instrumentação , Semicondutores , Desenho de Equipamento , Falha de Equipamento , Análise de Falha de Equipamento , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Razão Sinal-Ruído , Eletricidade EstáticaRESUMO
Necroptosis is a form of regulated necrotic cell death mediated by receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3. Necroptotic cell death contributes to the pathophysiology of several disorders involving tissue damage, including myocardial infarction, stroke and ischemia-reperfusion injury. However, no inhibitors of necroptosis are currently in clinical use. Here we performed a phenotypic screen for small-molecule inhibitors of tumor necrosis factor-alpha (TNF-α)-induced necroptosis in Fas-associated protein with death domain (FADD)-deficient Jurkat cells using a representative panel of Food and Drug Administration (FDA)-approved drugs. We identified two anti-cancer agents, ponatinib and pazopanib, as submicromolar inhibitors of necroptosis. Both compounds inhibited necroptotic cell death induced by various cell death receptor ligands in human cells, while not protecting from apoptosis. Ponatinib and pazopanib abrogated phosphorylation of mixed lineage kinase domain-like protein (MLKL) upon TNF-α-induced necroptosis, indicating that both agents target a component upstream of MLKL. An unbiased chemical proteomic approach determined the cellular target spectrum of ponatinib, revealing key members of the necroptosis signaling pathway. We validated RIPK1, RIPK3 and transforming growth factor-ß-activated kinase 1 (TAK1) as novel, direct targets of ponatinib by using competitive binding, cellular thermal shift and recombinant kinase assays. Ponatinib inhibited both RIPK1 and RIPK3, while pazopanib preferentially targeted RIPK1. The identification of the FDA-approved drugs ponatinib and pazopanib as cellular inhibitors of necroptosis highlights them as potentially interesting for the treatment of pathologies caused or aggravated by necroptotic cell death.
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Imidazóis/farmacologia , Necrose/tratamento farmacológico , Piridazinas/farmacologia , Pirimidinas/farmacologia , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Sulfonamidas/farmacologia , Células 3T3 , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proteína de Domínio de Morte Associada a Fas/genética , Células HEK293 , Células HT29 , Humanos , Indazóis , Células Jurkat , Células L , MAP Quinase Quinase Quinases/antagonistas & inibidores , MAP Quinase Quinase Quinases/metabolismo , Camundongos , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Chronic inhibition of protein phosphatases 1 and 2A in vivo was induced by infusion of okadaic acid into lateral ventricles of rat brain for up to 4 months. Cytoskeletal pathology, alterations of the amyloid precursor protein, and apoptotic cell death induced by this treatment followed a certain sequence and spatial distribution. Changes in the expression, phosphorylation, and subcellular distribution of neurofilament proteins and tau, as well as first signs of apoptotic cell death, occurred already after about 2 weeks. The distribution of apoptotic cells, however, was different from those revealing a high accumulation of hyperphosphorylated tau, indicating that those cytoskeletal pathology had no obvious sequelae for the viability of these neurones. A continuation of treatment for longer than 2 weeks induced diffuse deposits of both hyperphosphorylated tau and A beta-amyloid-immunoreactive material in white matter areas that increased in size and number over time. Because tau-phosphorylation is a regulator of the dynamic stability of microtubules, the pathology observed in the present experimental paradigm in the white matter might be viewed as an indication of a disturbed axonal transport. It is hypothesized that perturbations of the axonal transport might also be critically involved in the formation of paired helical filaments and amyloid deposits in Alzheimer's disease.
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Peptídeos beta-Amiloides/biossíntese , Apoptose/fisiologia , Doenças Neurodegenerativas/metabolismo , Fosfoproteínas Fosfatases/biossíntese , Proteínas tau/metabolismo , Peptídeos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide/biossíntese , Animais , Apoptose/genética , Western Blotting , Morte Celular , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/toxicidade , Imuno-Histoquímica , Doenças Neurodegenerativas/induzido quimicamente , Doenças Neurodegenerativas/patologia , Proteínas de Neurofilamentos/metabolismo , Ácido Okadáico/toxicidade , Fosfoproteínas Fosfatases/genética , Fosforilação , Ratos , Ratos Wistar , Proteínas tau/genéticaRESUMO
Changes in the dendritic arborisation of Golgi-impregnated basal forebrain neurones with respect to size, shape, orientation, and topology of branching were quantitatively investigated in ageing, Alzheimer's disease (AD), Korsakoff's disease (KD), and Parkinson's disease (PD). A reorganisation of the whole dendritic tree characterized by an increase in both the total dendritic length and the degree of dendritic arborisation as well as by changes in the shape of the dendritic field was found during ageing, in KD, PD, and AD. Dendritic growth under these conditions was related to the extent of cell loss in basal forebrain nuclei. There appeared to be major differences, however, with respect to the overall pattern of dendritic reorganisation between AD on one side and ageing, KD, and PD on the other side. In both ageing and KD, dendritic growth was largely restricted to the terminal dendritic segments, resulting in an increase of the size of the dendritic field (pattern of "extensive growth") In AD, however, dendritic growth mainly resulted in an increase of the dendritic density within the dendritic field without being accompanied by an increase in the size of the volume occupied by the dendritic tree (pattern of "intensive growth"). In AD, aberrant growth processes were frequently observed in the perisomatic area or on distal dendritic segments of basal forebrain neurones of the reticular type. Neurones with aberrant growth profiles were typically located in the direct vicinity of deposits of beta/A4 amyloid. Perisomatic growth profiles were covered by the low-affinity receptor of nerve growth factor p75NGFR. Aberrant growth processes were not present in ageing, KD, and PD. On the basis of the present study, it is concluded that under certain degenerative conditions, reticular basal forebrain neurones undergo a compensatory reorganisation of their dendritic arborisation, a process that has become defective in AD, thereby converting a physiological signal into a cascade of events contributing to the pathology of the disease.
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Envelhecimento/patologia , Transtorno Amnésico Alcoólico/patologia , Doença de Alzheimer/patologia , Dendritos/fisiologia , Doença de Parkinson/patologia , Prosencéfalo/patologia , Adulto , Idoso , Benzotiazóis , Contagem de Células , Feminino , Corantes Fluorescentes , Histocitoquímica , Humanos , Masculino , Pessoa de Meia-Idade , Degeneração Neural/fisiologia , Plasticidade Neuronal/fisiologia , Coloração e Rotulagem , TiazóisRESUMO
In the present study, the dendritic organisation of neurones in the normal human basal forebrain was analysed as a prerequisite for the evaluation of pathological changes occurring in Alzheimer's disease and related conditions (see other Arendt et al. papers in this issue). Neurones in the basal nucleus of Meynert (NbM), the nucleus of the vertical limb of the diagonal band, and the medial septal nucleus were examined after Golgi impregnation. Cells were classified according to the dendritic branching pattern and soma shape as either reticular neurones or multipolar giant neurones. The reticular type of neurones constitutes more than 90% of neurones in the BnM. Cholinergic neurones also belong to this cell type. Reticular neurones were further subdivided into four subtypes. Morphological features and arrangement of reticular basal forebrain neurones were identical to those described for "reticular formation cells" or "isodendritic" neurones. Dendritic trees of reticular neurones show a spatial orientation perpendicular to passing fibres as well as a high degree of overlap, both of which are hallmarks of "open nuclei." The qualitative classification of Golgi-impregnated basal forebrain neurones was substantiated by a computer-based three-dimensional analysis. Topologic and metric parameters of the dendritic tree were calculated for each type of neurone to characterise the degree of dendritic branching, the shape and orientation of the dendritic arborisation, the spatial extension of the dendritic tree, and soma size. The classification criteria were evaluated according to their power of discrimination between different cell types by means of a discriminant analysis. The quantitative approach applied in the present study not only provides an objective measure for the description and comparison of the structure of various types of neurones but also makes it possible to elucidate fine structural changes that might occur under pathologic conditions and that are not evident during qualitative studies alone.
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Doença de Alzheimer/patologia , Dendritos/fisiologia , Degeneração Neural/fisiologia , Prosencéfalo/patologia , Adulto , Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/metabolismo , Feminino , Histocitoquímica , Humanos , Masculino , Pessoa de Meia-Idade , Emaranhados Neurofibrilares/patologia , Plasticidade Neuronal/fisiologia , Formação Reticular/patologia , Substância Inominada/patologia , Fixação de TecidosRESUMO
The distribution of the reticular neuronal type in the human brain and its involvement in both degeneration and dendritic reorganisation under the conditions of ageing, Korsakoff's disease (KD), Alzheimer's disease (AD), and Parkinson's disease (PD) was comparatively investigated after Golgi impregnation. Reticular neurones are distributed throughout different areas along the brain axis. The cholinergic basal forebrain nuclei, i.e., the basal nucleus of Meynert, the nucleus of the diagonal band, and the medial septal nucleus form the most rostral part of this network of "open nuclei," which is collectively referred to as the "reticular core." Reticular neurones of the following areas were quantitatively investigated by a computer-based three-dimensional analysis: caudate nucleus, globus pallidus, medial septal nucleus, nucleus of the vertical limb of the diagonal band, basal nucleus, medial amygdaloid nucleus, reticular thalamic nucleus, lateral hypothalamic area, subthalamic nucleus, substantia nigra, locus coeruleus, pedunculopontine tegmental nucleus, and raphe magnus nucleus. There are three major findings. First, neurones that were found to be susceptible to degeneration in AD were largely part of the same neuronal populations prone to degeneration during ageing, in KD and PD. Thus, areas could be classified according to their overall degree of vulnerability under the present degenerative conditions as being highly vulnerable (basal forebrain nuclei, caudate nucleus, locus coeruleus), moderately vulnerable (medial amygdaloid nucleus, raphe magnus nucleus, lateral hypothalamic area, substantia nigra, pedunculopontine tegmental nucleus), or marginally vulnerable (globus pallidus, subthalamic nucleus, reticular thalamic nucleus). Second, neuronal populations that are particularly vulnerable to degenerative changes show a high degree of structural plasticity. Third, the degree of this dendritic plasticity is inversely related to the complexity of dendritic arborisation of the neurone. It is concluded that the sparsely ramified reticular type of neurone forms a pool of pluripotent neurones that have retained their plastic capacity throughout life, which makes them vulnerable to a variety of perturbations.
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Doença de Alzheimer/patologia , Dendritos/fisiologia , Degeneração Neural/fisiologia , Prosencéfalo/patologia , Adulto , Idoso , Transtorno Amnésico Alcoólico/patologia , Córtex Cerebral/patologia , Feminino , Histocitoquímica , Humanos , Masculino , Pessoa de Meia-Idade , Emaranhados Neurofibrilares/patologia , Plasticidade Neuronal/fisiologia , Neurônios/fisiologia , Doença de Parkinson/patologia , Formação Reticular/patologiaRESUMO
The sequential activation of the mitogen-activated protein kinase kinase and its substrate, the mitogen-activated protein kinase is involved in a cascade of protein kinases which link a number of cell surface signals to intracellular changes in enzyme activity and gene expression. In vitro, mitogen-activated protein kinase is able to phosphorylate the microtubule-associated protein tau at Ser-Pro and Thr-Pro sites, thereby generating abnormally hyperphosphorylated tau species that are similar to paired helical filament-tau found in Alzheimer's disease. In the present study, we analysed the levels of immunoreactive mitogen-activated protein kinase kinase and mitogen-activated protein kinase in the temporal cortex (area 22) of patients with Alzheimer's disease by means of enzyme-linked immuno-sorbent assays and compared these changes with the content of abnormally phosphorylated paired helical filament-tau. The levels of immunochemically detected mitogen-activated protein kinase kinase and mitogen-activated protein kinase were both increased in Alzheimer's disease by between 35 and 40% compared with age-matched controls. Elevation of mitogen-activated protein kinase kinase was most pronounced during early stages of Alzheimer's disease and was inversely related to the tissue content of abnormally phosphorylated paired helical filament-tau. Pronounced immunoreactivity of mitogen-activated protein kinase kinase and mitogen-activated protein kinase was present in both tangle bearing neurons and unaffected neurons of the temporal cortex. Immunoreactive neurons were most often localized in the direct vicinity of neuritic plaques. In Alzheimer's disease, the subcellular distribution of mitogen-activated protein kinase kinase and mitogen-activated protein kinase showed a striking translocation from the cytoplasmic to the nuclear compartment. It is suggested that the activation of the mitogen-activated protein kinase cascade which appears to be an early feature of Alzheimer's disease might be critically involved in self-stimulating processes of neurodegeneration and aberrant repair under these conditions.
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Doença de Alzheimer/enzimologia , Proteínas Quinases/biossíntese , Frações Subcelulares/enzimologia , Lobo Temporal/enzimologia , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/patologia , Western Blotting , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática/fisiologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Quinases de Proteína Quinase Ativadas por Mitógeno , Degeneração Neural/fisiologia , Emaranhados Neurofibrilares/enzimologia , Emaranhados Neurofibrilares/patologia , Lobo Temporal/patologia , Proteínas tau/metabolismoRESUMO
Alzheimer's disease is histopathologically characterized by neurofibrillary tangles, formed by the abnormally high phosphorylated tau protein, and senile plaques which largely consist of the beta/A4-amyloid peptide. Metabolism of the amyloid precursor protein and its processing into beta/A4-amyloid is regulated by protein phosphorylation. Thus, an imbalance between protein phosphorylation and dephosphorylation might be crucial for the development of the molecular hallmarks of Alzheimer's disease. We report here that chronic infusion into rat brain ventricles of okadaic acid, a specific inhibitor of the serine/threonine protein phosphatases 1 and 2A, results in a severe memory impairment, accompanied by a paired helical filament-like phosphorylation of tau protein and the formation of beta/A4-amyloid containing plaque-like structures in gray and white matter areas.
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Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Éteres Cíclicos/farmacologia , Transtornos da Memória/induzido quimicamente , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Proteínas tau/metabolismo , Animais , Feminino , Injeções Intraventriculares , Ácido Okadáico , Fosforilação , Ratos , Ratos Wistar , Fatores de TempoRESUMO
The process of degeneration and dendritic reorganization of cholinergic neurons was investigated in the rat basal forebrain under the conditions of chronic neurotoxic injury induced by long-term consumption of ethanol. After 28 weeks of ethanol treatment (20% v/v), both the number of choline acetyltransferase-immunoreactive basal forebrain neurons and levels of biochemical measures of cholinergic neurons, such as the activity of choline acetyltransferase and the synthesis and content of acetylcholine, were decreased by about 60-80%. The number of cholinergic neurons showing a positive hybridization signal to choline acetyltransferase messenger RNA was decreased to a similar extent. On the contrary, the reduction in the number of neurons immunoreactive for nerve growth factor receptor p75, which in control brains is highly co-localized with the expression of choline acetyltransferase, was much less pronounced and reached only 20-30%. The loss of choline acetyltransferase expression was associated with a cellular hypertrophy. Neurons which had survived the neurotoxic damage, furthermore, showed a remodelling of the dendritic organization which was quantitatively investigated after Golgi impregnation. This process of dendritic reorganization was mainly characterized by an increase in number and length of terminal dendritic segments. The results indicate that under the conditions of the present paradigm of chronic neurodegeneration, a certain number of cholinergic neurons persists in a form where they lost their ability to express detectable amounts of choline acetyltransferase messenger RNA and the enzyme protein. Persisting neurons, however, show both expression of nerve growth factor receptor p75 and signs of perikaryal and dendritic growth. It might, therefore, be hypothesized that chronic degeneration of cholinergic basal forebrain neurons triggers reactive attempts of repair which involve the action of trophic factors such as nerve growth factor.
Assuntos
Colina O-Acetiltransferase/genética , Degeneração Neural , Fatores de Crescimento Neural/genética , Prosencéfalo/patologia , Consumo de Bebidas Alcoólicas , Intoxicação Alcoólica , Animais , Autorradiografia , Contagem de Células , Imuno-Histoquímica , Masculino , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Substância Negra , Fatores de TempoRESUMO
The formation of neurofibrillary tangles in Alzheimer's disease shows a preferential involvement of certain cytoarchitecturally defined cortical areas suggesting systematic differences in regional neuronal vulnerability. The cellular and molecular nature of this selective neuronal vulnerability that follows a certain hierarchy of structural brain organization is largely unknown. In the present study, we compared the regional pattern of tangle density in Alzheimer's disease with systematic regional differences in neuronal plasticity that can be observed both during ageing and in Alzheimer's disease. Changes in dendritic length and arborization of Golgi-impregnated pyramidal neurons were analysed after three-dimensional reconstruction in 12 cortical areas. The intensity of dendritic remodelling that was observed during ageing as well as in Alzheimer's disease was regionally different and decreased in the following order: transentorhinal region > limbic areas (entorhinal region, hippocampus) > non-primary association areas (37, 40, 46) > primary sensory association areas (7, 18, 22) > primary sensory and motor cortex (17, 41, 4). These regional differences of neuronal plasticity follow the same pattern as the regional vulnerability to tangle formation in Alzheimer's disease. The results of the present study provide evidence that a high degree of structural neuronal plasticity might predispose neurons to tangle formation.
Assuntos
Doença de Alzheimer/patologia , Encéfalo/patologia , Córtex Cerebral/patologia , Emaranhados Neurofibrilares/patologia , Neurônios/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Doença de Alzheimer/genética , Apolipoproteínas E/genética , Encéfalo/citologia , Córtex Cerebral/citologia , Dendritos/patologia , Córtex Entorrinal/citologia , Córtex Entorrinal/patologia , Feminino , Complexo de Golgi/patologia , Humanos , Sistema Límbico/citologia , Sistema Límbico/patologia , Masculino , Pessoa de Meia-Idade , Plasticidade Neuronal , Células Piramidais/patologia , Valores de ReferênciaRESUMO
The distribution of abnormally phosphorylated tau proteins was investigated in a number of cortical areas and in the basal nucleus of Meynert of 12 patients with Alzheimer's disease. Quantification was performed with a sensitive sandwich enzyme-linked immunosorbent assay employing the monoclonal antibody B5-2, which immunoreacts with neurofibrillary tangles, dystrophic neurites surrounding amyloid cores of plaques and neuropil threads. On western blots, the monoclonal antibody B5-2 specifically labels the abnormally phosphorylated paired helical filament-tau species in a phosphorylation-dependent manner but is not cross-reactive to normal tau proteins. The preferential involvement of cytoarchitecturally defined cortical areas showed marked individual differences in Alzheimer's disease, and no unique distribution pattern of abnormally phosphorylated tau proteins could be established. While regional heterogeneities along the rostrocaudal extension of the brain declined with the progression of the disease, lateral differences which were largely non-directional appeared to be more stable over time. The mean content of abnormally phosphorylated tau proteins of individual cases was significantly related to the severity of the disease. On a regional scale, this correlation was highest for the basal nucleus. The formation of abnormally phosphorylated tau was associated with a loss of normal soluble tau proteins. Those cortical areas which in normal brain showed the highest amount of normal soluble tau proteins appeared to be particularly prone to deposition of abnormally phosphorylated tau proteins. The present results indicate that the formation of abnormally phosphorylated tau proteins can be initiated in the neuropil at more than one brain area. The spreading of the pathology appears to involve both intracortical and subcortical pathways but largely spares interhemispheric pathways. It is hypothesized that regional differences in the compartmentation and metabolism of tau proteins might reflect the molecular basis for the regionally different vulnerability in Alzheimer's disease.
Assuntos
Doença de Alzheimer/metabolismo , Proteínas tau/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/psicologia , Anticorpos Monoclonais , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Fosforilação , Escalas de Graduação Psiquiátrica , Distribuição TecidualRESUMO
Until now guinea-pigs have been rarely used to investigate formation and deposition of Alzheimer's disease-associated amyloid beta peptides despite the sequence identity of human and guinea-pig amyloid beta peptides being known, and the overall similarity of human and guinea-pig amyloid precursor protein. We now describe a primary cell culture system of mixed fetal guinea-pig brain cells, which we have applied to characterize endogenous amyloid precursor protein processing and amyloid beta formation. These cell cultures were established at embryonic day 24 of guinea-pigs after comparison of selected stages of guinea-pig ontogenetic development with the known ontogeny of rats, and were characterized by immunocytochemical detection of neuronal and glial marker proteins. Amyloid precursor protein expression, processing and amyloid beta formation increased in parallel with cellular maturation during cultivation and reached a stable phase after approximately 14 days in vitro therefore providing a suitable time for analysis. Aged cultures display strong neuronal amyloid precursor protein immunoreactivity and an altered profile of amyloid precursor protein isoform messenger RNA expression due to glial proliferation as single neurons were shown to retain their typical pattern of amyloid precursor protein expression. We show that amyloid precursor protein in guinea-pig cells is processed by different protease activities which most likely represent alpha- and beta-secretase, leading to the generation of soluble amyloid precursor protein derivatives. Furthermore, endogenous amyloid precursor protein processing leads to production of substantial amounts of amyloid beta-peptides which accumulate in conditioned culture medium. Amyloid beta was readily detectable by western blot analysis and was shown to consist of approximately 80-90% amyloid beta(1-40). We suggest that primary guinea-pig cell cultures provide a valuable tool in amyloid research that resembles amyloid precursor protein processing under physiological concentrations and, therefore, the situation in humans more closely than current rodent models. It should be especially useful in screening experiments for secretase inhibiting compounds.