RESUMEN
BACKGROUND AND PURPOSE: ADC as a marker of tumor cellularity has been promising for evaluating the response to therapy in patients with glioblastoma but does not successfully stratify patients according to outcomes, especially in the upfront setting. Here we investigate whether restriction spectrum imaging, an advanced diffusion imaging model, performed after an operation but before radiation therapy, could improve risk stratification in patients with newly diagnosed glioblastoma relative to ADC. MATERIALS AND METHODS: Pre-radiation therapy diffusion-weighted and structural imaging of 40 patients with glioblastoma were examined retrospectively. Restriction spectrum imaging and ADC-based hypercellularity volume fraction (restriction spectrum imaging-FLAIR volume fraction, restriction spectrum imaging-contrast-enhanced volume fraction, ADC-FLAIR volume fraction, ADC-contrast-enhanced volume fraction) and intensities (restriction spectrum imaging-FLAIR 90th percentile, restriction spectrum imaging-contrast-enhanced 90th percentile, ADC-FLAIR 10th percentile, ADC-contrast-enhanced 10th percentile) within the contrast-enhanced and FLAIR hyperintensity VOIs were calculated. The association of diffusion imaging metrics, contrast-enhanced volume, and FLAIR hyperintensity volume with progression-free survival and overall survival was evaluated by using Cox proportional hazards models. RESULTS: Among the diffusion metrics, restriction spectrum imaging-FLAIR volume fraction was the strongest prognostic metric of progression-free survival (P = .036) and overall survival (P = .007) in a multivariate Cox proportional hazards analysis, with higher values indicating earlier progression and shorter survival. Restriction spectrum imaging-FLAIR 90th percentile was also associated with overall survival (P = .043), with higher intensities, indicating shorter survival. None of the ADC metrics were associated with progression-free survival/overall survival. Contrast-enhanced volume exhibited a trend toward significance for overall survival (P = .063). CONCLUSIONS: Restriction spectrum imaging-derived cellularity in FLAIR hyperintensity regions may be a more robust prognostic marker than ADC and conventional imaging for early progression and poorer survival in patients with glioblastoma. However, future studies with larger samples are needed to explore its predictive ability.
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Neoplasias Encefálicas/diagnóstico por imagen , Imagen de Difusión por Resonancia Magnética/métodos , Glioblastoma/diagnóstico por imagen , Adulto , Neoplasias Encefálicas/clasificación , Neoplasias Encefálicas/patología , Progresión de la Enfermedad , Supervivencia sin Enfermedad , Femenino , Glioblastoma/clasificación , Glioblastoma/patología , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Modelos de Riesgos Proporcionales , Estudios RetrospectivosRESUMEN
BACKGROUND: Magnetic resonance imaging (MRI) is emerging as a robust, noninvasive method for detecting and characterizing prostate cancer (PCa), but limitations remain in its ability to distinguish cancerous from non-cancerous tissue. We evaluated the performance of a novel MRI technique, restriction spectrum imaging (RSI-MRI), to quantitatively detect and grade PCa compared with current standard-of-care MRI. METHODS: In a retrospective evaluation of 33 patients with biopsy-proven PCa who underwent RSI-MRI and standard MRI before radical prostatectomy, receiver-operating characteristic (ROC) curves were performed for RSI-MRI and each quantitative MRI term, with area under the ROC curve (AUC) used to compare each term's ability to differentiate between PCa and normal prostate. Spearman rank-order correlations were performed to assess each term's ability to predict PCa grade in the radical prostatectomy specimens. RESULTS: RSI-MRI demonstrated superior differentiation of PCa from normal tissue, with AUC of 0.94 and 0.85 for RSI-MRI and conventional diffusion MRI, respectively (P=0.04). RSI-MRI also demonstrated superior performance in predicting PCa aggressiveness, with Spearman rank-order correlation coefficients of 0.53 (P=0.002) and -0.42 (P=0.01) for RSI-MRI and conventional diffusion MRI, respectively, with tumor grade. CONCLUSIONS: RSI-MRI significantly improves upon current noninvasive PCa imaging and may potentially enhance its diagnosis and characterization.
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Imagen por Resonancia Magnética/métodos , Neoplasias de la Próstata/diagnóstico por imagen , Neoplasias de la Próstata/patología , Anciano , Humanos , Procesamiento de Imagen Asistido por Computador , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Estadificación de Neoplasias , Neoplasias de la Próstata/cirugía , Curva ROC , Estudios Retrospectivos , Carga TumoralRESUMEN
Freshly extracted axoplasm from giant axons of the marine fan worm Myxicola infundibulum and the squid Loligo can be pulled into fibres that contain highly oriented cytoskeletal elements suitable for X-ray diffraction. A major advantage of studying axoplasmic components by this technique is that it allows essentially native structures and their interactions to be examined. We describe here the analyses of the X-ray diffraction patterns. We show that in Myxicola the pattern can be explained by diffraction from both neurofilaments and microtubules, whilst in Loligo the pattern arises solely from microtubules. At low resolution, X-ray patterns obtained from dehydrated axoplasmic microtubules resemble strongly the Fourier transforms generated from electron micrographs of negatively stained specimens. Hydration of axoplasmic fibres produced reversible changes in the X-ray pattern intensities, although the layer-line positions were unaltered. On the 4 nm layer-line, the intensity of the J3 reflection was dramatically reduced on hydration, though its position was unchanged. Hydration also affected the J10/J16 reflections, which increased in intensity, though here again the positions of the peaks were little altered. The X-ray patterns from our hydrated fibres resemble those produced by others from fibres of purified microtubules, though in our patterns contrast is generated towards the centre of the wall. We interpret our findings in the light of current ideas about microtubule structure as determined by X-ray diffraction and electron microscope techniques.
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Axones/ultraestructura , Citoesqueleto/ultraestructura , Filamentos Intermedios/ultraestructura , Microtúbulos/ultraestructura , Animales , Decapodiformes/ultraestructura , Electroforesis en Gel de Poliacrilamida , Microscopía Electrónica , Poliquetos/ultraestructura , Difracción de Rayos XRESUMEN
BACKGROUND AND PURPOSE: Robust, automated segmentation algorithms are required for quantitative analysis of large imaging datasets. We developed an automated method that identifies and labels brain tumor-associated pathology by using an iterative probabilistic voxel labeling using k-nearest neighbor and Gaussian mixture model classification. Our purpose was to develop a segmentation method which could be applied to a variety of imaging from The Cancer Imaging Archive. MATERIALS AND METHODS: Images from 2 sets of 15 randomly selected subjects with glioblastoma from The Cancer Imaging Archive were processed by using the automated algorithm. The algorithm-defined tumor volumes were compared with those segmented by trained operators by using the Dice similarity coefficient. RESULTS: Compared with operator volumes, algorithm-generated segmentations yielded mean Dice similarities of 0.92 ± 0.03 for contrast-enhancing volumes and 0.84 ± 0.09 for FLAIR hyperintensity volumes. These values compared favorably with the means of Dice similarity coefficients between the operator-defined segmentations: 0.92 ± 0.03 for contrast-enhancing volumes and 0.92 ± 0.05 for FLAIR hyperintensity volumes. Robust segmentations can be achieved when only postcontrast T1WI and FLAIR images are available. CONCLUSIONS: Iterative probabilistic voxel labeling defined tumor volumes that were highly consistent with operator-defined volumes. Application of this algorithm could facilitate quantitative assessment of neuroimaging from patients with glioblastoma for both research and clinical indications.
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Algoritmos , Neoplasias Encefálicas/patología , Glioblastoma/patología , Procesamiento de Imagen Asistido por Computador/métodos , Neuroimagen/métodos , Archivos , Humanos , Imagen por Resonancia Magnética/métodosRESUMEN
BACKGROUND: Standard magnetic resonance imaging (MRI) of the prostate lacks sensitivity in the diagnosis and staging of prostate cancer (PCa). To improve the operating characteristics of prostate MRI in the detection and characterization of PCa, we developed a novel, enhanced MRI diffusion technique using restriction spectrum imaging (RSI-MRI). METHODS: We compared the efficacy of our novel RSI-MRI technique with standard MRI for detecting extraprostatic extension (EPE) among 28 PCa patients who underwent MRI and RSI-MRI prior to radical prostatectomy, 10 with histologically proven pT3 disease. RSI cellularity maps isolating the restricted isotropic water fraction were reconstructed based on all b-values and then standardized across the sample with z-score maps. Distortion correction of the RSI maps was performed using the alternating phase-encode technique. RESULTS: 27 patients were evaluated, excluding one patient where distortion could not be performed. Preoperative standard MRI correctly identified extraprostatic the extension in two of the nine pT3 (22%) patients, whereas RSI-MRI identified EPE in eight of nine (89%) patients. RSI-MRI correctly identified pT2 disease in the remaining 18 patients. CONCLUSIONS: In this proof of principle study, we conclude that our novel RSI-MRI technology is feasible and shows promise for substantially improving PCa imaging. Further translational studies of prostate RSI-MRI in the diagnosis and staging of PCa are indicated.
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Imagen por Resonancia Magnética/métodos , Próstata/diagnóstico por imagen , Neoplasias de la Próstata/diagnóstico por imagen , Anciano , Gadolinio , Humanos , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Próstata/patología , Antígeno Prostático Específico , Prostatectomía , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/cirugía , RadiografíaRESUMEN
Cisplatin has become one of the most commonly prescribed cytotoxic chemotherapeutic agents. Unfortunately, the cure rate is low due to the development or outgrowth of cisplatin-resistant cells which repopulate tumors, resulting in patient death. We reported previously that the calcium channel blocker nifedipine enhances the antitumour actions of cisplatin (cis-diamminedichloroplatinum (II] against murine tumors which are inherently cisplatin-sensitive (B16a) or inherently cisplatin-resistant (3LL). We have developed an induced cisplatin-resistant tumor variant (B16a-Pt) that is 30 times more resistant to cisplatin than its cisplatin-sensitive parent line. In short-term studies, we report that nifedipine significantly enhanced the cytotoxicity of cisplatin against primary B16a-Pt tumors and their spontaneous pulmonary metastases. In long term studies, we report that combination therapy with nifedipine and cisplatin results in significantly enhanced survival.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Cisplatino/uso terapéutico , Neoplasias Pulmonares/secundario , Melanoma/tratamiento farmacológico , Nifedipino/uso terapéutico , Animales , Supervivencia Celular/efectos de los fármacos , Cisplatino/administración & dosificación , Cisplatino/farmacología , Resistencia a Medicamentos , Sinergismo Farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Masculino , Ratones , Ratones Endogámicos , Nifedipino/administración & dosificación , Nifedipino/farmacología , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
This study investigates the optical effects observed from uncoated and protein vaccine coated gold microparticles while imaging with two-photon excitation in the Mie scattering regime. When observed with time correlated single photon counting fluorescence lifetime microscopy, the emission from the gold microparticles appeared as an intense instrument-limited temporal response. The intensity of the emission showed a second-order dependence on the laser power and frequency doubling of the emitted light was observed for fundamental light between 890 and 970 nm. The optical effect was attributed to two-photon induced second harmonic generation. The vaccine coated gold microparticles had a much weaker second harmonic signal than the uncoated gold microparticles. Chemical analysis of the surface of the gold microparticles revealed that the vaccine coating decreases the surface charge thereby diminishing the observed second harmonic signal. These optical properties can be exploited to identify both the location of the protein vaccine coating as well as the gold microparticles in vitro and potentially to investigate the vaccine delivery kinetics in vivo.
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Oro , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Microesferas , Vacunas , Sistemas de Liberación de Medicamentos , Microanálisis por Sonda Electrónica , Electrones , Oro/análisis , Humanos , Cinética , Microscopía Confocal , Microscopía Electrónica de Rastreo , Fotones , Proteínas/administración & dosificación , Proteínas/química , Dispersión de Radiación , Factores de Tiempo , Tomografía Computarizada de Emisión de Fotón Único/métodos , Rayos XRESUMEN
BACKGROUND AND PURPOSE: Restriction spectrum imaging is a sensitive DWI technique for probing separable water diffusion compartments in tissues. Here, we evaluate RSI-CMs derived from the spherically-restricted water compartment for improved tumor conspicuity and delineation from nontumor tissue and reduced sensitivity to edema compared with high-b-value DWI and ADC. MATERIALS AND METHODS: RSI was performed in 10 presurgical patients: 4 with glioblastoma, 3 with primary CNS lymphoma, and 3 with metastatic brain tumors. Multidirectional DWI data were collected at b = 500, 1500, and 4000 s/mm(2). Quantification of tumor conspicuity, edema conspicuity, and relative sensitivity to edema for RSI-CMs; DWI at b = 4000 (DWI-4000); and ADC were compared in manually drawn VOIs. Receiver operating characteristic curves were used to evaluate the sensitivity and specificity of each method for delineating tumor from normal-appearing WM. RESULTS: Significant TC was seen with both RSI-CMs and DWI-4000, but not ADC. Significant EC was seen with ADC, but not RSI-CMs or DWI-4000. Significantly greater TC was seen with RSI-CMs compared with DWI-4000. Significantly reduced RSE was seen with RSI-CMs compared with both DWI-4000 and ADC. Greater sensitivity and specificity for delineating tumor from normal-appearing WM were seen with RSI-CMs (AUC = 0.91) compared with both DWI-4000 (AUC = 0.77) and ADC (AUC = 0.66). CONCLUSIONS: RSI-CMs offer improved conspicuity and delineation of high-grade primary and metastatic brain tumors and reduced sensitivity to edema compared with high-b-value DWI and ADC.
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Edema Encefálico/etiología , Edema Encefálico/patología , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/secundario , Imagen de Difusión por Resonancia Magnética/métodos , Aumento de la Imagen/métodos , Interpretación de Imagen Asistida por Computador/métodos , Anciano , Anciano de 80 o más Años , Algoritmos , Neoplasias Encefálicas/complicaciones , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
BACKGROUND AND PURPOSE: DTI is being increasingly used to visualize critical white matter tracts adjacent to brain tumors before neurosurgical resection. However, brain tumors, particularly high-grade gliomas, are typically surrounded by regions of FLAIR hyperintensity that include edema, which increase isotropic diffusion, degrading the ability of standard DTI to uncover orientation estimates within these regions. We introduce a new technique, RSI, which overcomes this limitation by removing the spherical, fast diffusion component introduced by edema, providing better analysis of white matter architecture. MATERIALS AND METHODS: A total of 10 patients with high-grade gliomas surrounded by FLAIR-HI that at least partially resolved on follow-up imaging were included. All patients underwent RSI and DTI at baseline (FLAIR-HI present) and at follow-up (FLAIR-HI partially resolved). FA values obtained with RSI and DTI were compared within regions of FLAIR-HI and NAWM at both time points. RESULTS: RSI showed higher FA in regions of FLAIR-HI and NAWM relative to DTI, reflecting the ability of RSI to specifically measure the slow, restricted volume fraction in regions of edema and NAWM. Furthermore, a method by time interaction revealed that FA estimates increased when the FLAIR-HI resolved by use of standard DTI but remained stable with RSI. Tractography performed within the region of FLAIR-HI revealed the superior ability of RSI to track fibers through severe edema relative to standard DTI. CONCLUSIONS: RSI improves the quantification and visualization of white matter tracts in regions of peritumoral FLAIR-HI associated with edema relative to standard DTI and may provide a valuable tool for neurosurgical planning.
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Edema Encefálico/patología , Neoplasias Encefálicas/patología , Imagen de Difusión Tensora/métodos , Glioma/patología , Fibras Nerviosas Mielínicas/patología , Adulto , Anciano , Astrocitoma/patología , Astrocitoma/cirugía , Edema Encefálico/cirugía , Neoplasias Encefálicas/cirugía , Femenino , Estudios de Seguimiento , Glioblastoma/patología , Glioblastoma/cirugía , Glioma/cirugía , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Cuidados PreoperatoriosRESUMEN
Confocal laser scanning optical microscopy (CLSM) in the reflection contrast mode has been used to image single 40 nm gold particles, and to study changes in the distribution of gold label associated with capping of the leukocyte sialoglycoprotein (LSGP) antigen on the surface of fixed rat thymocytes, labelled with the mouse monoclonal antibody W3/13 and a goat anti-mouse IgG immunogold conjugate. This imaging method has also been applied to live thymocytes labelled with gold-conjugated antibodies, to study the dynamics of the capping process.
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Recubrimiento Inmunológico , Sialoglicoproteínas/metabolismo , Timo/inmunología , Animales , Células Cultivadas , Técnicas Inmunológicas , Ratas , Timo/citologíaRESUMEN
The cytoplasmic pH of growing pollen tubes of Lilium longiflorum Thunb. was measured using the pH-sensitive fluorescent dye 2',7'-bis-(carboxyethyl)-5(6')-carboxyfl uorescein and confocal fluorescence ratio imaging. The average cytoplasmic pH in the clear zone of the pollen tube tip was pH 7.11, and no consistent pH gradients were detected in the clear zone, averaging around -1.00 milli pH unit microm(-1), or along the first 50 microm of the tube (3.62 milli pH units microm[-1]). In addition, no correlation was observed between the absolute tip cytoplasmic pH or the pH gradient and the pollen tube growth rates. Shifts of external pH to more acidic pH values (pH 4.5) caused a relatively small acidification by 0.18 pH units, whereas a more alkaline external pH >7.0 caused a dramatic increase in cytoplasmic pH and growth stopped immediately. Stimulation of the plasma membrane H+-ATPase by fusicoccin, resulted in an increase of tube growth but no change in cytoplasmic pH. On the other hand, vanadate (250-500 microM), a putative inhibitor of the pump, stopped tube growth and a slight cytoplasmic alkalinisation of 0.1 pH units was observed. Vanadate also arrested fusicoccin-stimulated growth and stimulated an increased alkalinisation of around 0.2 pH units. External application of CaCl2 (10 mM) caused a small acidification of less than 0.1 pH units in the clear zone, whilst LaCl3 (250 microM) caused slight and rather variable perturbations in cytoplasmic pH of no more than 0.1 pH units. Both treatments stopped growth. It was inferred from these data that tip-acid cytoplasmic pH gradients do not play a central role in the organisation or maintenance of pollen tube tip growth.
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Desarrollo de la Planta , Cloruro de Calcio/farmacología , Membrana Celular/enzimología , Citoplasma/metabolismo , Fluoresceínas , Colorantes Fluorescentes , Glicósidos/farmacología , Concentración de Iones de Hidrógeno , Lantano/farmacología , Microscopía Confocal , Plantas/efectos de los fármacos , Plantas/metabolismo , Polen , ATPasas de Translocación de Protón/antagonistas & inhibidores , ATPasas de Translocación de Protón/metabolismo , Vanadatos/farmacologíaRESUMEN
Using electron microscopy (EM), optical diffraction and image reconstruction techniques, we have demonstrated polymorphism of neurofilamentous network (NFN) in vitro based on phase transitions of the protein assemblies. The specific polymorphic appearances depended upon a number of factors, such as K+, Mg2+, Ca2+ ions, as well as the charge and hydration state of the molecules. Furthermore, modifications initiated by the state of phosphorylation of the sidearm proteins played an important role, especially in determining the sidearm disposition of the NFN. The Ca2+-activated protease removed the sidearms. Other enzymes activated by Ca2+ may initiate new association patterns of the peptide remnants and the intercoiling of two smooth neurofilaments (NFs) into paired helical filament-like (PHF-like) strands. Prolonged storage of the isolated NFs in Rubinson-Baker solution resulted in autocrosslinking and intercoiling of modified NFN components. The in vitro polymorphism and phase transitions of squid NFN induced under controlled conditions have been compared to modifications of cytoskeleton observed by EM in frontal lobe biopsies of Alzheimer patients. We conclude that similar processes, as induced in vitro, do occur in neurons of Alzheimer patients.
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Axones/ultraestructura , Citoesqueleto/fisiología , Filamentos Intermedios/fisiología , Red Nerviosa/fisiología , Fenómenos Fisiológicos del Sistema Nervioso , Polimorfismo Genético , Enfermedad de Alzheimer/patología , Animales , Calcio/farmacología , Decapodiformes , Modelos Animales de Enfermedad , Proteínas de Filamentos Intermediarios/análisis , Filamentos Intermedios/ultraestructura , Proteínas de Neurofilamentos , Neuronas/fisiología , FosforilaciónRESUMEN
Dual-excitation confocal laser scanning microscopy (CLSM) was used to image the pH-indicator, BCECF, iontophoretically microinjected into stomatal guard cells of Vicia faba during challenge with peptides derived from hydrophilic domains of the maize auxin-binding protein. Only the peptide corresponding to the C-terminal end (Pz151-163) caused significant changes in cytosolic pH, stimulating rapid alkalinisation of 0.4 +/- 0.1 pH units. Cytosolic pH was clamped using the permeant weak acid, butyrate, and this treatment buffered the peptide evoked alkalinisation. In concert with the electrical events monitored at the plasma membrane using whole-cell voltage clamp, this provides strong evidence for a role of [H+] as a signal intermediate in the guard cell transduction network. In preliminary experiments using single-wavelength imaging of the calcium-indicator, Fluo-3, Pz151-163 also stimulated rapid, reversible increases in cytosolic calcium, whilst two other peptides tested had no effect.
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Calcio/metabolismo , Membrana Celular/metabolismo , Fabaceae/metabolismo , Péptidos/metabolismo , Reguladores del Crecimiento de las Plantas , Proteínas de Plantas , Plantas Medicinales , Receptores de Superficie Celular/metabolismo , Citosol/metabolismo , Concentración de Iones de Hidrógeno , Procesamiento de Imagen Asistido por Computador , Microscopía Confocal , Microscopía FluorescenteRESUMEN
BACKGROUND: As the unitary theory of anesthesia gives way to the "multiple sites, multiple mechanisms" concept, the sites involved in mediating the components of anesthesia must be identified. In the current study, we test the hypothesis that the basolateral amygdala complex (BLAC) is a brain site involved with mediating propofol-induced amnesia. METHODS: Male Sprague-Dawley rats were divided into two groups, sham-operated control animals and rats given bilateral excitotoxic N-methyl-D-aspartate lesions of the BLAC. For each group, animals were given intraperitoneal saline or propofol (25 mg/kg) 5 min before inhibitory avoidance learning. Rats were given a foot shock (0.4 mA) upon entering the dark side of a two-sided apparatus. Rats could escape additional shock by returning to and staying in the light side. Training ended after shock avoidance for greater than 60 s. Memory was tested at 24 h. Longer latencies to enter the dark side 24 h after training imply better memory. RESULTS: Sham-saline-treated animals had a robust memory latency (median latency [interquartile range] = 300 [163-567] s). Sham-propofo-treated animals exhibited a significant anterograde amnesia (latency = 63 [14-111] s) (P < 0.05 vs. sham-saline-treated animal). Both the saline-injected and propofol-injected animals with BLAC lesions showed robust memory (latency = 300 [264-485] and 323 [143480] s, respectively). These latencies did not differ from performance in the sham-saline-treated group and were significantly higher than the latency of the sham-propofol-treated group (both P < 0.05). CONCLUSIONS: Discrete BLAC lesions blocked the amnestic effect of propofol. BLAC activity appears to be a requirement for propofol-induced amnesia. This finding suggests that the BLAC is a key brain site mediating anesthetic-induced amnesia.
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Amnesia/inducido químicamente , Amígdala del Cerebelo/efectos de los fármacos , Anestésicos Intravenosos/farmacología , Reacción de Prevención/efectos de los fármacos , Propofol/farmacología , Amígdala del Cerebelo/fisiología , Animales , Masculino , Memoria/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
Levels of glutathione were measured for different cell types in roots of intact Arabidopsis seedlings after labelling with monochlorobimane to give fluorescent glutathione S-bimane (GSB) and imaging using confocal laser scanning microscopy with excitation at 442 nm. Labelling increased to a plateau in most cell types after about 15-20 min and the GSB accumulated rapidly in the vacuole. Formation of GSB in the cytoplasm was not affected by treatment with sodium azide; however, vacuolar transport of GSB was substantially inhibited under these conditions. We infer that vacuolar sequestration was mediated by a tonoplast glutathione S-conjugate pump. Quantitative estimates of the cytoplasmic glutathione concentration involved correction for the loss in fluorescence signal with depth into the specimen using an empirically determined model derived in situ from a permeabilized root. Correction for the dilution experienced on transport into the vacuole also required an estimate of the amount of cytoplasm present in each cell type. This was achieved in two stages: first, the levels of protein were mapped after fixation, permeabilization and labelling with fluroescein isothiocyanate. Second, the corresponding cytoplasmic volume was determined as 40% for epidermal cells in the elongation zone by manual segmentation of the cytoplasm in serial optical sections. Values of relative cytoplasmic volume for other cells were extrapolated in proportion to their protein content. Using this approach, cytoplasmic glutathione concentrations were found to be 2-3 mM in most cell types. There was a marked difference between the central cells and the neighbouring, rapidly dividing initials, and between the columella cells and the outermost cells of the root cap. In the latter case, the difference was equalized in the presence of azide. This might indicate that additional cell-cell movement and preferential sequestration of GSB can occur during the detoxification process in an intact system.
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Arabidopsis/química , Glutatión/química , Raíces de Plantas/química , Arabidopsis/anatomía & histología , Colorantes Fluorescentes , Meristema/química , Microscopía Confocal , Pirazoles/farmacologíaRESUMEN
A lateral compression clamp system has been used in the treatment of fractures of the mandible. It provides rigid fixation and promotes earlier osteogenesis at the fracture site. Intermaxillary fixation can often be eliminated in edentulous persons and greatly reduced in duration when necessary as an adjunct to fixation in patients with teeth. These significant qualities make the compression clamp quite beneficial in the treatment plan and postoperative management of many otherwise difficult cases. Having evaluated our experiences with compression clamps, we believe that this approach deserves further attention and may contribute to a solution of the controversy that often exists regarding the management of mandibular fractures in both edentulous patients and those with a compromised complement of teeth.
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Fijación de Fractura/instrumentación , Fracturas Mandibulares/cirugía , Accidentes de Tránsito , Anciano , Anodoncia/complicaciones , Antibacterianos/uso terapéutico , Antidepresivos/uso terapéutico , Ansiedad/tratamiento farmacológico , Atrofia/complicaciones , Femenino , Humanos , Masculino , Mandíbula/diagnóstico por imagen , Enfermedades Mandibulares/complicaciones , Fracturas Mandibulares/complicaciones , Fracturas Mandibulares/etiología , Boca Edéntula/complicaciones , Radiografía PanorámicaRESUMEN
Regulation of cell volume is a fundamental cellular homeostatic mechanism in the face of osmotic stress. In normal articular cartilage, chondrocytes are exposed to a changing osmotic environment. We present a comprehensive protocol for studying the volume regulatory behavior of chondrocytes within intact cartilage tissue using confocal laser-scanning microscopy. Our data acquisition regime optimizes both signal-to-noise and cell viability during time-lapsed three-dimensional (3-D) (x, y, z, t) imaging. The porcine cartilage is treated as an integrated component of the imaging system, and we demonstrate methods for the direct assessment of tissue-induced axial attenuation and image distortion. Parameterized functions describing these two components of image degradation are used to correct experimental data. The current study also highlights the problems associated with the analysis and visualization of four-dimensional (4-D) images. We have devised two new types of data reconstruction. The first compresses each 3-D time point into a single quantitative view, termed a coordinate view. From these reconstructions we are able to simultaneously view and extract cell measurements. A second type, a 4-D reconstruction, uses color to represent relative changes in cell volume, again while maintaining the morphological and spatial information. Both these approaches of image analysis and visualization have been implemented to study the morphology, spatial distribution, and dynamic volume behavior of chondrocytes after osmotic perturbation. We have mapped chondrocyte shape, arrangement, and absolute volume in situ, which vary significantly from the tissue surface through to the underlying bone. Despite the rigid nature of the extracellular matrix, cartilage cells are osmotically sensitive and respond to stimulation of volume regulatory mechanisms. The combined techniques of confocal laser-scanning microscopy and vital cell labeling have enabled us to study, for the first time, the response of chondrocytes in situ to changes in interstitial osmotic pressure.
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Cartílago/citología , Microscopía Confocal/métodos , Equilibrio Hidroelectrolítico , Animales , Tamaño de la Célula , Procesamiento de Imagen Asistido por Computador , Técnicas In Vitro , PorcinosRESUMEN
HeLa cells synchronized at different stages of the cell cycle were permeabilized and incubated with analogues of nucleotide triphosphates; then sites of incorporation were immunolabeled with the appropriate fluorescent probes. Confocal microscopy showed that sites of replication and transcription were not diffusely spread throughout nuclei, reflecting the distribution of euchromatin; rather, they were concentrated in 'foci' where many polymerases act together. Transcription foci aggregated as cells progressed towards the G1/S boundary; later they dispersed and became more diffuse. Replication was initiated only at transcription sites; later, when heterochromatin was replicated in enlarged foci, these remained sites of transcription. This illustrates the dynamic nature of nuclear architecture and suggests that transcription may be required for the initiation of DNA synthesis.