RESUMEN
UNLABELLED: Histopathologic validation of a PET tracer requires assessment of colocalization of the tracer with its intended biologic target. Using thin tissue section autoradiography, it is possible to visualize the spatial distribution of the PET tracer uptake and compare it with the distribution of the intended biologic target (as visualized with immunohistochemistry). The purpose of this study was to develop and evaluate an objective methodology for deformable coregistration of autoradiography and microscopy images acquired from a set of sequential tissue sections. METHODS: Tumor-bearing animals were injected with 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT), (14)C-FDG, and other markers of tumor microenvironment including Hoechst 33342 (blood-flow surrogate). After sacrifice, tumors were excised, frozen, and sectioned. Multiple stacks of sequential 8 µm sections were collected from each tumor. From each stack, the middle (reference) sections were used to obtain images of (18)F-FLT and (14)C-FDG uptake distributions using dual-tracer autoradiography. Sections adjacent to the reference were used to acquire all histopathologic data (e.g., images of cell proliferation, hematoxylin and eosin). Hoechst images were acquired from all sections. To correct for deformations and misalignments induced by tissue processing and image acquisition, the Hoechst image of each nonreference section was deformably registered to the reference Hoechst image. This transformation was then applied to all images acquired from the same tissue section. In this way, all microscopy images were registered to the reference Hoechst image. The Hoechst-to-autoradiography image registration was done using rigid point-set registration based on external markers visible in both images. RESULTS: The mean error of Hoechst to (18)F-FLT autoradiography registration (both images acquired from the same section) was 30.8 ± 20.1 µm. The error of Hoechst-based deformable registration of histopathologic images (acquired from sequential tissue sections) was 23.1 ± 17.9 µm. Total error of registration of autoradiography images to the histopathologic images acquired from adjacent sections was evaluated at 44.9 µm. This coregistration precision supersedes current rigid registration methods with reported errors of 100-200 µm. CONCLUSION: Deformable registration of autoradiography and histopathology images acquired from sequential sections is feasible and accurate when performed using corresponding Hoechst images.
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Autorradiografía/estadística & datos numéricos , Tomografía de Emisión de Positrones/estadística & datos numéricos , Interpretación de Imagen Radiográfica Asistida por Computador/métodos , Microambiente Tumoral , Animales , Radioisótopos de Carbono , Línea Celular Tumoral , Didesoxinucleósidos , Fluorodesoxiglucosa F18 , Humanos , Inmunohistoquímica/estadística & datos numéricos , Masculino , Ratones , Ratones Desnudos , Neoplasias Experimentales/diagnóstico por imagen , Neoplasias Experimentales/patología , Radiofármacos , Trasplante HeterólogoRESUMEN
PURPOSE: Alterations in dopamine neurotransmission in animal models of epilepsies have been frequently demonstrated using invasive neuroscience or ex vivo techniques. We aimed to test whether corresponding alterations could be detected by noninvasive in vivo brain imaging with positron emission tomography (PET) in the chronic phase of the rat pilocarpine model of temporal lobe epilepsy. METHODS: Six pilocarpine-treated Wistar rats exhibiting spontaneous recurrent seizures and nine control rats were studied with PET using [(18)F]-fallypride, a high-affinity dopamine D(2/3) receptor ligand. Parametric images of [(18)F]-fallypride specific binding were calculated using a reference tissue method, and the two groups were contrasted by whole-brain voxel-based analysis implemented in statistical parametric mapping (SPM5). RESULTS: Dopamine D(2/3) receptor availability was 27% lower in the bilateral anterior caudate-putamen of pilocarpine-treated rats as compared to controls (p < 0.05), but binding was unaffected in other striatal or extrastriatal regions. CONCLUSIONS: The finding of substantially reduced availability of dopamine D(2/3) receptors in the anterior caudate-putamen of rats during the chronic phase of the pilocarpine model is in agreement with results of invasive (microinjection, microdialysis) animal studies that have revealed increased dopamine tonus and a D(2/3) receptor-mediated anticonvulsant action of dopamine in the anterior segment of the rat striatum. The present PET approach could be prospectively applied for monitoring dopamine receptor changes longitudinally, that is, at different phases of the epileptogenic process, and opens perspectives for testing dopaminergic agents as potential antiepileptogenic drugs.
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Cuerpo Estriado/diagnóstico por imagen , Epilepsia del Lóbulo Temporal/diagnóstico por imagen , Receptores Dopaminérgicos/metabolismo , Animales , Autorradiografía/estadística & datos numéricos , Benzamidas/metabolismo , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Mapeo Encefálico , Cuerpo Estriado/metabolismo , Modelos Animales de Enfermedad , Dopamina/metabolismo , Epilepsia del Lóbulo Temporal/inducido químicamente , Epilepsia del Lóbulo Temporal/metabolismo , Humanos , Masculino , Pilocarpina , Tomografía de Emisión de Positrones/métodos , Tomografía de Emisión de Positrones/estadística & datos numéricos , Pirrolidinas/metabolismo , Ratas , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D3/metabolismoRESUMEN
BACKGROUND: In situ hybridisation (ISH) combined with autoradiography is a standard method of measuring the amount of gene expression in histological sections, but the methods used to quantify gene expression in the resulting digital images vary greatly between studies and can potentially give conflicting results. RESULTS: The present study examines commonly used methods for analysing ISH images and demonstrates that these methods are not optimal. Image segmentation based on thresholding can be subject to floor-effects and lead to biased results. In addition, including the area of the structure or region of interest in the calculation of gene expression can lead to a large loss of precision and can also introduce bias. Finally, converting grey level pixel intensities to optical densities or units of radioactivity is unnecessary for most applications and can lead to data with poor statistical properties. A modification of an existing method for selecting the structure or region of interest is introduced which performs better than alternative methods in terms of bias and precision. CONCLUSION: Based on these results, suggestions are made to reduce bias, increase precision, and ultimately provide more meaningful results of gene expression data.
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Autorradiografía/métodos , Perfilación de la Expresión Génica/métodos , Hibridación in Situ/métodos , Algoritmos , Animales , Autorradiografía/estadística & datos numéricos , Sesgo , Encéfalo/metabolismo , Radioisótopos de Carbono , Expresión Génica , Perfilación de la Expresión Génica/estadística & datos numéricos , Procesamiento de Imagen Asistido por Computador/métodos , Procesamiento de Imagen Asistido por Computador/estadística & datos numéricos , Hibridación in Situ/estadística & datos numéricos , Masculino , Modelos Estadísticos , Ratas , Ratas Sprague-DawleyRESUMEN
Quantitative whole-body autoradiography (QWBA) and liquid scintillation counting (LSC) have been conducted to determine the metabolic profiles and tissue distribution of [(14)C] labeled artesunate (AS) injection in rats. The QWBA technique showed more accurate results in the quantification of radioactivity in 40 organs and tissues, compared to 19 organs with the LSC technique. The benefit of QWBA was especially apparent on measurements of bile, bone marrow, and gland organs; however, the LSC method produced more relevant findings than QWBA. Particularly, the LSC method allowed access to the following distribution patterns that were unavailable via QWBA performance: such as pharmacokinetic evaluation of radiolabeled AS in blood and plasma, tissue/plasma partition coefficients, conversion pathway of AS to dihydroartemisinin (DHA, an active and major metabolite of AS), unchanged AS and DHA in plasma, mass balance assessment, urinary and faecal eliminations, drug pathway with conjugation, [(14)C] AS binding with RBC and plasma protein, and metabolites identification. Even though the each method has its own advantages, common profiles were obtained from the two processes as shown in the results of the biliary metabolism, long-lasting metabolites, tissue distribution profiles, and multiple concentration peaks, which indicate a [(14)C] AS enterohepatic circulation.
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Artemisininas/farmacocinética , Disección/métodos , Radiofármacos/farmacocinética , Animales , Área Bajo la Curva , Artesunato , Autorradiografía/métodos , Autorradiografía/estadística & datos numéricos , Calibración , Radioisótopos de Carbono/farmacocinética , Recolección de Datos/estadística & datos numéricos , Inyecciones Intravenosas/métodos , Masculino , Guías de Práctica Clínica como Asunto/normas , Ratas , Ratas Sprague-Dawley , Estándares de Referencia , Conteo por Cintilación/métodos , Equivalencia Terapéutica , Distribución TisularRESUMEN
PURPOSE: The objective of this investigation was to characterize quantitatively the time-dependent changes in midazolam (MDL) efficacy in the silent period after induction of status epilepticus (SE) in rats. The changes in MDL efficacy were correlated to changes in ex vivo GABA(A)-receptor expression. METHODS: MDL efficacy was quantified by pharmacokinetic-pharmacodynamic (PK-PD) modeling by using the beta-frequency of the EEG as PD end point. Two PK-PD experiments were performed in each animal: the first experiment before and the second experiment at either day 4 or day 14 after SE. SE was induced by repetitive intraperitoneal injections with kainate. GABA(A)-receptor expression was determined by ex vivo autoradiography with [(3)H]flumazenil. RESULTS: The concentration versus EEG effect relation of midazolam was successfully described by the sigmoidal E(max) model. The maximal effect on the beta-frequency of the EEG (E(max)) was reduced to 51.6 +/- 35.6% and 25.8 +/- 33.7% of the original value at 4 and 14 days after induction of SE. The ex vivo study with [(3)H]flumazenil showed that the observed reductions in E(max) were paralleled by a reduction in GABA(A)-receptor density. CONCLUSIONS: The efficacy of MDL is decreased in the silent period after SE, which can be partly accounted for by a reduction in GABA(A)-receptor density.
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Epilepsia del Lóbulo Temporal/inducido químicamente , Epilepsia del Lóbulo Temporal/tratamiento farmacológico , Ácido Kaínico , Midazolam/farmacología , Receptores de GABA-A/efectos de los fármacos , Animales , Autorradiografía/estadística & datos numéricos , Ritmo beta/efectos de los fármacos , Ritmo beta/estadística & datos numéricos , Modelos Animales de Enfermedad , Electroencefalografía/efectos de los fármacos , Electroencefalografía/estadística & datos numéricos , Epilepsia del Lóbulo Temporal/metabolismo , Flumazenil/metabolismo , Inyecciones Intraperitoneales , Midazolam/farmacocinética , Midazolam/uso terapéutico , Ratas , Receptores de GABA-A/metabolismo , Estado Epiléptico/inducido químicamente , Resultado del Tratamiento , Tritio/metabolismoRESUMEN
Sigma-1 receptors are widely expressed in the mammalian brain and also in organs of the immune, endocrine and reproductive systems. Based on behavioral and pharmacological assessments, sigma-1 receptors are important in memory and cognitive processes, and are thought to be involved in specific psychiatric illnesses, including schizophrenia, depression, and drug addiction. It is thought that specific neuroactive steroids are endogenous ligands for these sites. In addition, several sigma-1 receptor binding steroids including progesterone, dihydroepiandrosterone (DHEA), and testosterone are being examined clinically for specific therapeutic purposes; however, their mechanisms of action have not been clearly defined. We previously described the high affinity sigma-1 receptor selective PET tracer [(18)F]FPS. This study examines the effect of neuroactive steroids on [(18)F]FPS binding in vitro and in vivo. Inhibition constants were determined in vitro for progesterone, testosterone, DHEA, estradiol, and estriol binding to the [(18)F]FPS labeled receptor. The affinity order (K(i) values) for these steroids ranged from 36 nM for progesterone to >10,000 nM for estrodiol and estriol. Biodistribution studies revealed that i.v. coadministration of progesterone (10 mg/kg), testosterone (20 mg/kg), or DHEA (20 mg/kg) significantly decreased [(18)F]FPS uptake (%ID/g) by up to 50% in nearly all of eight brain regions examined. [(18)F]FPS uptake in several peripheral organs that express sigma-1 receptors (heart, spleen, muscle, lung) was also reduced (54-85%). These studies clearly demonstrate that exogenously administered steroids can occupy sigma-1 receptors in vivo, and that [(18)F]FPS may provide an effective tool for monitoring sigma-1 receptor occupancy of specific therapeutic steroids during clinical trials.
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Unión Competitiva/efectos de los fármacos , Membrana Celular/diagnóstico por imagen , Tomografía de Emisión de Positrones , Receptores sigma/metabolismo , Esteroides/farmacología , Animales , Autorradiografía/métodos , Autorradiografía/estadística & datos numéricos , Encéfalo/ultraestructura , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Relación Dosis-Respuesta a Droga , Radioisótopos de Flúor/farmacocinética , Técnicas In Vitro , Unión Proteica/efectos de los fármacos , Ratas , Factores de Tiempo , Distribución Tisular/efectos de los fármacos , Receptor Sigma-1RESUMEN
This study explored whether common rules exist for the distribution patterns across tissues in tissue distribution studies. To investigate this we tested whether tissue:plasma partition coefficients (PCs) of radioactivity are correlated with muscle:plasma PCs. The relationships between PCs of radioactivity in muscle and those in other tissues were investigated in 25 tissues for 20 structurally unrelated drug candidates. Tissue distribution data were obtained by quantitative whole-body autoradiography. Linear regression analysis was performed for each tissue. Radioactivity from basic and acidic/neutral compounds was analyzed separately. Results for acidic/neutral compounds: for the majority of the tissues investigated, the tissue:plasma PCs were well correlated with muscle:plasma PCs (R2 > 0.7). Correlations were worse (R2 < 0.7) in blood, white fat, excretory organs and tissues protected by a penetration barrier (e.g. brain). Slope factors for the regression ranged from 0.2 (blood) to 3.8 (Harderian gland) and were correlated with neutral lipid contents in tissues. Results for basic compounds: in most tissues, slope factors appeared to be higher than for acidic/neutral compounds. Correlations, however, were poorer than for acidic/neutral compounds. Overall, the present study demonstrates that muscle:plasma PCs are indicative of the overall tissue distribution of drug-related material, as they are well correlated with tissue:plasma PCs in most other tissues. Correlations for acidic/neutral compounds differ from those for basic compounds. The found PC relationships provide an explanation for the distribution pattern across tissues usually seen in tissue distribution studies.
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Autorradiografía/estadística & datos numéricos , Radiofármacos/farmacocinética , Distribución Tisular , Animales , Fenómenos Químicos , Química Farmacéutica , Química Física , Concentración de Iones de Hidrógeno , Masculino , Valor Predictivo de las Pruebas , Ratas , Ratas Sprague-Dawley , Análisis de RegresiónRESUMEN
The benefits of a mouse model are efficiency and availability of transgenics/ knockouts. Quantitation of cerebral blood in small animals is difficult because the cannulation procedure may introduce errors. The [14C]-iodoantipyrine autoradiography (IAP) method requires both the tissue concentration and the time course of arterial concentration of the [14C] radioactive tracer. A single point-analysis technique was evaluated for measuring blood flow in mice (30 g +/- 0.3 g; n = 11) by using computational models of sensitivity analysis, which quantitates relationships between the predictions of a model and its parameters. Using [14C]-IAP in conjunction with mathematical algorithms and assumed arterial concentration-versus-time profiles, cortical blood flow was deduced from single-point measurements of the arterial tracer concentration. The data showed the arterial concentration profile that produced the most realistic blood flows (1.6 +/- 0.4; mean +/- SD, ml/g/min) was a profile with a ramp time of 30 sec followed by a constant value over the remaining time period of 30 sec. Sensitivity analysis showed that the total experimental time period was a more important parameter than the lag period and the ramp period. Thus, it appears that the accuracy of the assumption of linearly increasing arterial concentration depends on the experimental time period and the final arterial [14C]-iodoantipyrine concentration.
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Circulación Cerebrovascular , Algoritmos , Animales , Antipirina/análogos & derivados , Autorradiografía/métodos , Autorradiografía/estadística & datos numéricos , Radioisótopos de Carbono , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Flujo Sanguíneo Regional , Sensibilidad y EspecificidadRESUMEN
Whole-body autoradioluminography (WBAL) has evolved as the preferred method for conducting tissue distribution studies that are required for regulatory filings of a new drug entity (DE) and for projecting tissue dosimetry in human mass balance studies. Four experiments were designed to assess WBAL utility using tritium as early as lead development in the drug discovery process. The objective of experiment 1 was to determine the minimum amount of tritium to administer to rats required for obtaining widespread distribution into most tissues at concentrations greater than quantification limits. Experiments 2, 3, and 4 were conducted to identify a tissue compartment responsible for observed triphasic pharmacokinetics, to characterize the distribution of a [3H]DE into brain tissue, and to compare tissue distribution patterns between two rat strains, respectively. The minimum amount of tritium necessary to investigate the tissue distribution of [3H]DE in rats was 865 microCi/kg. Results from experiments 2, 3, and 4 illustrated A) the identification of adipose as the tissue compartment responsible for an extended terminal elimination phase, B) sustained penetration of a DE into brain tissues for at least 24 h, and C) tissue distribution differences between two DEs of the same therapeutic class, respectively. These experiments exemplify the value of WBAL as a screening tool to assist with the selection of a drug candidate. WBAL utilization in drug discovery provides insightful data toward designing pharmacology and toxicology experiments. Substituting the use of tritium for carbon-14 is of crucial importance in drug discovery since [3H]DEs are more readily obtainable than [14C]DEs.
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Preparaciones Farmacéuticas/análisis , Tecnología Farmacéutica/métodos , Tritio/metabolismo , Recuento Corporal Total/métodos , Recuento Corporal Total/estadística & datos numéricos , Animales , Autorradiografía/métodos , Autorradiografía/estadística & datos numéricos , Relación Dosis-Respuesta en la Radiación , Masculino , Preparaciones Farmacéuticas/metabolismo , Ratas , Ratas Endogámicas SHR , Ratas Long-Evans , Ratas Sprague-DawleyRESUMEN
Autoradiographs are conventionally analyzed by a region-of-interest (ROI) analysis. However, definition of ROIs on an image set is labor intensive, is subject to potential inter-rater bias, and is not well suited for anatomically variable structures that may not consistently correspond to specific ROIs. Most importantly, the ROI method is poorly suited for whole-brain analysis, where one wishes to detect all activations resulting from an experimental paradigm. A system developed for analysis of imaging data in humans, Statistical Parametric Mapping (SPM), avoids some of these limitations but has not previously been adapted as a tool for the analysis of autoradiographs. Here, we describe the application of SPM to an autoradiographic data set mapping cerebral activation in rats during treadmill walking. We studied freely moving, non-tethered rats that received injections of the cerebral blood flow tracer [14C]-iodoantipyrine, while they were performing a treadmill task (n = 7) or during a quiescent control condition (n = 6). Results obtained with SPM were compared to those previously reported using a standard ROI-based method of analysis [J. Cereb. Blood Flow Metab. 23(2003) 925]. The SPM method confirmed most areas detected as significant using the ROI approach. However, in the subcortex, SPM detected additional significant regions that, because of their irregular structures, fell short of statistical significance when analyzed by ROI. The SPM approach offers the ability to perform a semi-automated whole-brain analysis, and coupled with autoradiography, provides an effective means to globally localize functional activity in small animals.
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Nivel de Alerta/fisiología , Autorradiografía/estadística & datos numéricos , Encéfalo/fisiología , Procesamiento de Imagen Asistido por Computador/estadística & datos numéricos , Cómputos Matemáticos , Caminata/fisiología , Animales , Encéfalo/anatomía & histología , Mapeo Encefálico , Cerebelo/anatomía & histología , Cerebelo/fisiología , Corteza Cerebral/anatomía & histología , Corteza Cerebral/fisiología , Estudios de Factibilidad , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Comparison of brain imaging data requires the exact matching of data sets from different individuals. Warping methods, used to optimize matching of data sets, can exploit either local gray value distribution or identifiable reference points within the images to be compared. Gray value-based warping, which is more comfortable, cannot be used if gray values include functional information that should be compared between images. A major drawback in the use of point-based warping methods is the lack of methods for efficient and precise definition of reference points (landmarks) within comparable data sets. Here, we present a novel approach to automatically detect sufficient numbers of landmarks, which is based on 3D differential operators. In addition, we have developed a new distance-weighted warping method, which optimizes individual local weighting factors of displacement vectors. The quality of the methods was evaluated using a set of autoradiographs documenting the metabolic activity of gerbil brains after acoustic stimulation. The new warping method was compared with known methods of landmark-based warping, i.e., warping with radial basis functions and with distance-weighted methods. For the data sets presented in this study our new optimized warping method produced an increase in linear cross correlation of 4.44%, an increase in volume overlap index of 1.55%, and a decrease in the registration error of 36.2%. In addition, the detection of functional differences was improved after warping. Therefore, the new method is a powerful tool, which enhances the comparison of complex biological structures and the quantitative evaluation of functional imaging data.
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Autorradiografía/estadística & datos numéricos , Encéfalo/fisiología , Aumento de la Imagen/métodos , Procesamiento de Imagen Asistido por Computador/estadística & datos numéricos , Imagenología Tridimensional/estadística & datos numéricos , Modelos Lineales , Cómputos Matemáticos , Algoritmos , Animales , Autorradiografía/métodos , Encéfalo/anatomía & histología , Mapeo Encefálico/métodos , Simulación por Computador , Fluorodesoxiglucosa F18 , Gerbillinae , Procesamiento de Imagen Asistido por Computador/métodosRESUMEN
We present the first images of beta autoradiography obtained with the high-resolution hybrid pixel detector consisting of the Medipix2 single photon counting read-out chip bump-bonded to a 300 microm thick silicon pixel detector. This room temperature system has 256 x 256 square pixels of 55 microm pitch (total sensitive area of 14 x 14 mm2), with a double threshold discriminator and a 13-bit counter in each pixel. It is read out via a dedicated electronic interface and control software, also developed in the framework of the European Medipix2 Collaboration. Digital beta autoradiograms of 14C microscale standard strips (containing separate bands of increasing specific activity in the range 0.0038-32.9 kBq g(-1)) indicate system linearity down to a total background noise of 1.8 x 10(-3) counts mm(-2) s(-1). The minimum detectable activity is estimated to be 0.012 Bq for 36,000 s exposure and 0.023 Bq for 10,800 s exposure. The measured minimum detection threshold is less than 1600 electrons (equivalent to about 6 keV Si). This real-time system for beta autoradiography offers lower pixel pitch and higher sensitive area than the previous Medipix1-based system. It has a 14C sensitivity better than that of micro channel plate based systems, which, however, shows higher spatial resolution and sensitive area.
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Autorradiografía/métodos , Autorradiografía/instrumentación , Autorradiografía/estadística & datos numéricos , Partículas beta , Fenómenos Biofísicos , Biofisica , Radioisótopos de Carbono , Procesamiento de Imagen Asistido por Computador , Fotones , SilicioRESUMEN
Galanin receptor (GALR) expression is increased in various areas of the limbic system in end stage Alzheimer's disease (AD). The amygdaloid complex is a key component of the limbic circuit, is involved in homeostatic and cognitive functions, is impacted in AD and contains the peptide and receptor for galanin. Although GALR expression occurs in the amygdala in end stage AD, it remains to be determined whether a plasticity response occurs early or late in the disease. Therefore, we analyzed the distribution and associated changes in GALR binding in the amygdala during the progression of AD using an in vitro receptor autoradiographic method. Human galanin ([125I]hGAL) receptor binding was performed on brain sections from early and late AD cases, as well as normal age-matched control subjects. In aged controls, densities of [125I]hGAL binding sites were found in the central and the corticomedial nuclei. Relative to controls, possible/early AD cases displayed significantly greater numbers of [125I]hGAL binding sites in the central nucleus and cortico-amygdaloid transition area. In contrast, we found a decrease in the number of binding sites for [125I]hGAL in late as compared to early AD cases. The over-expression of GALRs in subfields of the amygdaloid early in AD suggests that galaninergic systems play a key role in limbic related behavioral changes during the disease process.
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Enfermedad de Alzheimer/metabolismo , Amígdala del Cerebelo/metabolismo , Receptores de Neuropéptido/metabolismo , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/patología , Amígdala del Cerebelo/patología , Animales , Autorradiografía/métodos , Autorradiografía/estadística & datos numéricos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Ratas , Receptores de Galanina , Receptores de Neuropéptido/biosíntesis , Porcinos , Regulación hacia Arriba/fisiologíaRESUMEN
(1) Disturbances of mesolimbic and mesocortical dopamine (DA) function have been implicated in the pathophysiology of several psychiatric disorders, including major depressive disorder. (2) Utilizing the learned helplessness (LH) animal model of clinical depression and quantitative autoradiography, the authors studied the densities of D1 and dopamine-2-like receptors (D2-like receptors) in medial prefrontal cortex, septum, nucleus accumbens and caudate nucleus in rats that received inescapable stress and were subsequently tested for LH behavior. (3) Dopamine-1 receptor (D1 receptor) densities were significantly higher in the core and shell of the nucleus accumbens and in the medial caudate nucleus of rats that did not become helpless after stress, compared to rats that developed LH. (4) Densities of D2-like receptors were significantly lower in the core of the nucleus accumbens in both the LH and the nonhelpless (NH) rats compared to controls. Densities of D2-like receptors were also lower in the medial and lateral caudate nuclei in LH rats compared to the other groups. (5) Increased D1 receptor densities in NH rats in the nucleus accumbens may be associated with an adaptive or protective role of this brain region in the prevention of escape deficits after exposure to inescapable stress. (6) Decreased D2-like receptor densities in the caudate nucleus in helpless rats may reflect a motor deficit associated with LH behavior, while decreases of D2-like receptor densities in the core of the nucleus accumbens may reflect a generalized effect of exposure to inescapable stress. (7) This study highlights the importance of the mesolimbic/nigrostriatal dopaminergic systems in mediating behavioral responses to inescapable stress.
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Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Desamparo Adquirido , Receptores Dopaminérgicos/metabolismo , Animales , Autorradiografía/métodos , Autorradiografía/estadística & datos numéricos , Regulación hacia Abajo/fisiología , Masculino , Radiografía , Ratas , Ratas Sprague-Dawley , Regulación hacia Arriba/fisiologíaRESUMEN
The present study employed standard peroxidase immunohistochemistry to map the distribution of P2Y(1) receptors in the rat brainstem and nodose ganglia and characterised the binding profile of [alpha(33)P]dATP. Binding of [alpha(33)P]dATP was fully displaceable by adenosine 5'-triphosphate (ATP), and was found on both human and rat nodose ganglia, and throughout the rat brainstem, including the nucleus tractus solitarius and ventrolateral medulla. [Alpha(33)P]dATP binding in the human nodose ganglia was significantly displaced by both 2-methylthio ATP and alpha,beta-methylene ATP, but not by uridine 5'-triphosphate, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid, 8,8'-(carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino))bis(1,3,5-naphtalenetrisulfonic) acid (NF279) or N-ethylcarboxamidoadenosine. [Alpha(33)P]dATP binding in the rat nodose ganglia and brainstem was significantly displaced by only 2-methylthio ATP, suggesting that [alpha(33)P]dATP is binding to P2Y receptors in the rat. Binding of [alpha(33)P]dATP was also significantly displaced by alpha,beta-methylene adenosine 5'-diphosphate, suggesting a component of the binding is to endogenous ecto-5'-nucleotidase, however, almost all binding could be displaced by a combination of receptor agonists (2-methylthio ATP, uridine 5'-triphosphate and alpha,beta-methylene ATP), suggesting preferential binding to receptors. Immunoreactivity to P2Y(1) receptor (P2Y(1)-IR) exhibited similar distribution patterns to [alpha(33)P]dATP binding, with a clear topographic profile. Particularly dense P2Y(1)-IR labeling was evident in cells and fibres of the dorsal vagal complex. Immunolabeling was also present in the dorsal motor nucleus of the vagus and nucleus ambiguus, indicating the possibility of P2Y(1) receptors on vagal efferents. Unilateral vagal ligation was also performed to examine the transport of P2Y(1) receptor, using both immunohistochemistry and [alpha(33)P]dATP autoradiography. Accumulations of both P2Y(1)-IR and [alpha(33)P]dATP binding were apparent adjacent to both ligatures, suggesting bi-directional transport of P2Y(1) receptors along the rat vagus nerve. This current study represents the first description of P2Y(1) receptor distribution within the rodent brainstem and nodose ganglion and also characterises [alpha(33)P]dATP binding to P2Y receptors.
Asunto(s)
Tronco Encefálico/metabolismo , Nucleótidos de Desoxiadenina/metabolismo , Ganglio Nudoso/metabolismo , Receptores Purinérgicos P2/metabolismo , Adolescente , Adulto , Anciano , Animales , Autorradiografía/métodos , Autorradiografía/estadística & datos numéricos , Tronco Encefálico/química , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Ganglio Nudoso/química , Radioisótopos de Fósforo/metabolismo , Ratas , Ratas Endogámicas WKY , Receptores Purinérgicos P2/análisis , Receptores Purinérgicos P2Y1RESUMEN
The 32P-postlabeling assay is widely used for the analysis of DNA adducts. Some adducts can be detected with very high sensitivity but quantification can be unreliable, particularly if it is based only on comparison with unmodified nucleotides (relative adduct labeling, RAL values). Furthermore, guidelines to calculate detection limits for adduct concentrations are lacking. This is particularly important for human biomonitoring studies of environmental exposures, where a low adduct level can remain undetected. Reports of null results of toxicity studies should always include a limit of detection, indicating the effect magnitude that would have produced, with a given probability of false negative (type II error), a statistically significant increase (type I error). Here, we report on a procedure based on t-statistics to calculate two types of detection limits, the "critical level (CL)" and the "detection level (DL)". The first is the size of the difference between exposed and controls required to achieve statistical significance. The second is the size of the difference that will be detected with a chosen probability of a false negative. For the degrees of freedom (d.f.) to be used for the t-values, a general formula is given so that different standard deviations and group sizes of control and exposed groups can be handled. A sample calculation of the whole procedure is shown, using the null data for the formation of a particular adduct in lung DNA of styrene-treated mice, analyzed by 32P-postlabeling. The procedure takes into account: (i) TLC-specific background radioactivity; (ii) variability within the control and exposed groups; and (iii) confidence limits for the factor to convert 32P-radioactivity to amounts of adduct. The latter step incorporates the variance of the differences between the samples and replicates spiked with adduct standard. A statement such as follows is the result: the concentration of the alpha-isomer adduct of styrene 7,8-oxide at the O(6)-position of guanine in mouse lung DNA would have to be at least 12 adducts per 10(8) nucleotides to be detected in the given experiment on a 5% level (type I error), with a probability of 5% to miss an existing effect (type II error).
Asunto(s)
Autorradiografía/estadística & datos numéricos , Cromatografía de Afinidad/estadística & datos numéricos , Aductos de ADN/análisis , Pruebas de Mutagenicidad/estadística & datos numéricos , Medición de Riesgo/estadística & datos numéricos , Animales , Autorradiografía/métodos , Cromatografía de Afinidad/métodos , Intervalos de Confianza , ADN/efectos de los fármacos , ADN/metabolismo , Aductos de ADN/efectos de los fármacos , Femenino , Pulmón/efectos de los fármacos , Ratones , Ratones Endogámicos , Pruebas de Mutagenicidad/métodos , Mutágenos/metabolismo , Mutágenos/toxicidad , Radioisótopos de Fósforo , Medición de Riesgo/métodos , Estirenos/metabolismo , Estirenos/toxicidadRESUMEN
The biological effect of a radiopharmaceutical depends heavily on the heterogeneity of the uptake in the various tissues. A comparative study of two radiopharmaceuticals should therefore include a comparison of the uptake patterns in different tissues. To eliminate the problems caused by variation in kinetics and tumour characteristics between individuals, such a comparison should be based on measured distributions of the radiopharmaceuticals in the same tissue sample. The excellent linearity between activity and counts in images obtained with a digital silicon strip detector allows such distributions to be derived from two autoradiographs acquired at different time points. This method was applied in a comparison of the uptake patterns of 153Sm-EDTMP and 89SrCl2 in sections obtained from a dog with spontaneous osteosarcoma, containing both tumour and normal bone tissues. As the areas of the section were larger than the detector area, the section had to be cut into smaller parts. Images of these were later merged by means of image processing techniques. There were significant differences in the uptake patterns of the two nuclides. In the primary tumour, the uptake of 153Sm was highly heterogeneous, while 89Sr was more uniformly distributed. In trabecular bone, the accumulation of 153Sm was higher than that of 89Sr. In solid cortical bone, 89Sr had the highest uptake.
Asunto(s)
Autorradiografía/métodos , Neoplasias Óseas/veterinaria , Enfermedades de los Perros/diagnóstico por imagen , Compuestos Organometálicos , Compuestos Organofosforados , Osteosarcoma/veterinaria , Radioisótopos , Radiofármacos , Samario , Radioisótopos de Estroncio , Estroncio , Animales , Autorradiografía/estadística & datos numéricos , Neoplasias Óseas/diagnóstico por imagen , Perros , Compuestos Organometálicos/farmacocinética , Compuestos Organofosforados/farmacocinética , Osteosarcoma/diagnóstico por imagen , Radioisótopos/farmacocinética , Cintigrafía , Radiofármacos/farmacocinética , Samario/farmacocinética , Estroncio/farmacocinética , Radioisótopos de Estroncio/farmacocinéticaRESUMEN
Statistical analysis of neuroimages is commonly approached with intergroup comparisons made by repeated application of univariate or multivariate tests performed on the set of the regions of interest sampled in the acquired images. The use of such large numbers of tests requires application of techniques for correction for multiple comparisons. Standard multiple comparison adjustments (such as the Bonferroni) may be overly conservative when data are correlated and/or not normally distributed. Resampling-based step-down procedures that successfully account for unknown correlation structures in the data have recently been introduced. We combined resampling step-down procedures with the Minimum Variance Adaptive method, which allows selection of an optimal test statistic from a predefined class of statistics for the data under analysis. As shown in simulation studies and analysis of autoradiographic data, the combined technique exhibits a significant increase in statistical power, even for small sample sizes (n = 8, 9, 10).
Asunto(s)
Procesamiento de Imagen Asistido por Computador/estadística & datos numéricos , Algoritmos , Autorradiografía/estadística & datos numéricos , Simulación por Computador , Leucina/metabolismo , Modelos Estadísticos , Análisis Multivariante , Distribución AleatoriaRESUMEN
Two methods have been developed for the separation of contributions from two different nuclides in an autoradiography picture. Both approaches, a modified least squares (LS) and a maximum likelihood (ML) algorithm, are based on the position and energy information available from a digital detector. Tests and comparisons, using artificially as well as measured data, demonstrate that the ML approach performs slightly better, but is much more computationally demanding than the LS method.
Asunto(s)
Autorradiografía/métodos , Algoritmos , Animales , Autorradiografía/estadística & datos numéricos , Fenómenos Biofísicos , Biofisica , Procesamiento de Imagen Asistido por Computador/métodos , Procesamiento de Imagen Asistido por Computador/estadística & datos numéricos , Análisis de los Mínimos Cuadrados , Funciones de Verosimilitud , Radioisótopos de Fósforo , Silicio , Radioisótopos de AzufreRESUMEN
Whole-body autoradiography has been widely used in the investigation of the distribution of radiolabeled compounds in animals. The newly introduced radioluminography offers a reliable way of quantifying the radioactivity distribution within whole-body sections. Since the radioactivity is distributed over the entire depth of the section, self-absorption of beta-radiation in tissues is supposed to relevantly affect the detection of radioactivity at the section surface. The self-absorption of radiation energy ((14)C) was investigated in 28 organs/tissues of routinely produced lyophilized rat sections. Nonradioactive whole-body sections with different thickness between 20 and 120 microm were placed between a homogeneous (14)C source and the imaging plates to detect the transmitted radioactivity. The self-absorption was expressed in terms of percentage of transmission of the radioactivity through the sections. Transmission decreased with increasing section thickness, e.g., from 44% (20 microm) to 28% (120 microm) for blood. Comparison of three complete sets of data disclosed intertissue variations of up to about 30% (i.e., +/-15%) disregarding bone. A defined bandwidth of +/-15% around the blood transmission would cover the transmission of almost all tissues. Thus, for most organs radioactivity can be quantified by direct comparison with radioactive blood calibration samples.