RESUMO
Neuroinflammation and the accompanying activation of glial cells is an important feature of many neurodegenerative conditions. It is known that factors such as peripheral infections and stress can influence immune processes in the brain. However, the effect of these stressors on astrocyte activation in vivo remains elusive. In this study, transgenic Gfap-luc mice expressing the luciferase gene under the transcriptional control of the glial fibrillary acidic protein promoter were used to quantify the kinetics of in vivo astrocyte activation following immune challenges relevant to clinical inflammation. It was found that astrocytes respond rapidly to peripheral immune activation elicited by either bacterial lipopolysaccharide (LPS) or the viral mimetic polyinosinic:polycytidylic acid (poly(I:C)). By measuring bioluminescence and 18-kDa translocator protein radioligand binding in the same animal it was observed that LPS induces both astrocyte as well as microglial activation at 6 h post-administration. Furthermore, the astrocyte response decreased upon repeated systemic LPS injections, indicating development of tolerance to the LPS challenge. Finally, restraining Gfap-luc mice for 1 h daily on 5 consecutive days did not affect brain bioluminescence, thereby indicating that sub-chronic stress does not influence astrocyte activation under unchallenged conditions. However, stressed animals showed a reduced response to a subsequent systemic LPS injection, suggesting that the immune system is compromised in these animals. Here, we demonstrate that Gfap-luc mice can be used to study astrocyte activation in response to stimuli relevant for clinical inflammation and that this approach may provide a more complete characterization of existing and novel models of neuroinflammation
Assuntos
Astrócitos/fisiologia , Encéfalo/imunologia , Inflamação/fisiopatologia , Neuroimunomodulação/fisiologia , Estresse Psicológico/imunologia , Animais , Modelos Animais de Doenças , Proteína Glial Fibrilar Ácida , Lipopolissacarídeos , Luciferases/genética , Luciferases/metabolismo , Medições Luminescentes , Masculino , Camundongos Transgênicos , Microglia/fisiologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Poli I-C , Distribuição Aleatória , Restrição FísicaRESUMO
Substantial evidence indicates an association between clinical depression and altered immune function. Systemic administration of bacterial lipopolysaccharide (LPS) is commonly used to study inflammation-associated behavioral changes in rodents. In these experiments, we tested the hypothesis that peripheral immune activation leads to neuroinflammation and depressive-like behavior in mice. We report that systemic administration of LPS induced astrocyte activation in transgenic GFAP-luc mice and increased immunoreactivity against the microglial marker ionized calcium-binding adapter molecule 1 in the dentate gyrus of wild-type mice. Furthermore, LPS treatment caused a strong but transient increase in cytokine levels in the serum and brain. In addition to studying LPS-induced neuroinflammation, we tested whether sickness could be separated from depressive-like behavior by evaluating LPS-treated mice in a panel of behavioral paradigms. Our behavioral data indicate that systemic LPS administration caused sickness and mild depressive-like behavior. However, due to the overlapping time course and mild effects on depression-related behavior per se, it was not possible to separate sickness from depressive-like behavior in the present rodent model.
Assuntos
Astrócitos/citologia , Depressão/imunologia , Comportamento de Doença , Inflamação/patologia , Animais , Comportamento Animal , Encéfalo/imunologia , Encéfalo/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Comportamento de Escolha , Citocinas/sangue , Citocinas/metabolismo , Modelos Animais de Doenças , Comportamento Alimentar , Imuno-Histoquímica , Lipopolissacarídeos/química , Luminescência , Masculino , Camundongos , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Microglia/metabolismo , Neurônios/metabolismo , Sacarose/químicaRESUMO
Phosphoinositide 3-kinases, delta (PI3Kδ) and gamma (PI3Kγ) are enriched in immune cells and regulate the development and function of innate and adaptive immunity. Dual PI3Kδγ inhibitors are considered high value targets for their potential to treat a variety of immune-mediated diseases, but their discovery has been challenging. Here we describe the preclinical pharmacology of HM5023507, an orally active dual inhibitor of δγ isoforms in immune signaling. HM5023507 inhibited PI3Kδ and PI3Kγ isoforms with greater than 100-fold selectivity against PI3Kα and PI3Kß in recombinant enzymatic assays and in primary human immune cells with an exquisite selectivity against other targets. HM5023507 attenuated the PI3Kδ/γ signaling in human basophils (IC50: 42/340 nmol/L; selectivity ratio ~1:8). HM5023507 attenuated the activation and function of human B and T cells, Th17 differentiation of CD4 T cells in the blood of healthy donors and rheumatoid arthritis patients, and cytokine and IgG production in human T and B cell cocultures, in vitro. Orally dosed HM5023507 attenuated PI3K δ/γ-mediated immune signaling in the rat in a dose-related manner. In addition, HM5023507 inhibited semiestablished collagen-induced arthritic inflammation in the rats (ED50 of 0.25mg/kg, p.o. BID or 0.5 mg/kg, QD, AUC: 1422 ng/mL*h), improved histopathology- and micro-computed tomography (µCT)-based indices of joint damage, bone destruction, and attenuated the levels of anti-collagen antibody, with an overall anti-inflammatory profile matching that of a TNFα neutralizing antibody. The PI3K δγ inhibitory profile of HM5023507 and its selectivity make it a useful tool to further delineate immunobiology of dual PI3K δγ targeting.
Assuntos
Linfócitos B/citologia , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Inibidores Enzimáticos/administração & dosagem , Linfócitos T/citologia , Administração Oral , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Cultura Primária de Células , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismoRESUMO
The need to study dynamic biologic processes in intact small-animal models of disease has stimulated the development of high-resolution nuclear imaging methods. These methods are capable of clarifying molecular interactions important in the onset and progression of disease, assessing the biologic relevance of drug candidates and potential imaging agents, and monitoring therapeutic effectiveness of pharmaceuticals serially within a single-model system. Single-photon-emitting radionuclides have many advantages in these applications, and SPECT can provide 3-dimensional spatial distributions of gamma- (and x-) ray-emitting radionuclide imaging agents or therapeutics. Furthermore, combining SPECT with CT in a SPECT/CT system can assist in defining the anatomic context of biochemical processes and improve the quantitative accuracy of the SPECT data. Over the past decade, dedicated small-animal SPECT and SPECT/CT systems have been developed in academia and industry. Although significant progress in this arena has been realized through system development and biologic application, further innovation continues to address challenges in camera sensitivity, spatial resolution, and image reconstruction and quantification. The innumerable applications of small-animal SPECT and SPECT/CT in drug development, cardiology, neurology, and oncology are stimulating further investment in education, research, and development of these dedicated small-animal imaging modalities.
Assuntos
Radioisótopos/farmacologia , Cintilografia/métodos , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Tomografia Computadorizada por Raios X/métodos , Animais , Humanos , Imageamento por Ressonância Magnética/métodos , Camundongos , Miocárdio/patologia , Transplante de Neoplasias , Perfusão , Reprodutibilidade dos Testes , Imagem Corporal TotalRESUMO
This paper demonstrates the application of mutual information based coregistration of radionuclide and magnetic resonance imaging (MRI) in an effort to use multimodality imaging for noninvasive localization of stem cells grafted in the infarcted myocardium in rats. Radionuclide imaging such as single photon emission computed tomography (SPECT) or positron emission tomography (PET) inherently has high sensitivity and is suitable for tracking of labeled stem cells, while high-resolution MRI is able to provide detailed anatomical and functional information of myocardium. Thus, coregistration of PET or SPECT images with MRI will map the location and distribution of stem cells on detailed myocardium structures. To validate this coregistration method, SPECT data were simulated by using a Monte Carlo-based projector that modeled the pinhole-imaging physics assuming nonzero diameter and photon penetration at the edge. Translational and rotational errors of the coregistration were examined with respect to various SPECT activities, and they are on average about 0.50 mm and 0.82 degrees , respectively. Only the rotational error is dependent on activity of SPECT data. Stem cells were labeled with (111)Indium oxyquinoline and grafted in the ischemic myocardium of a rat model. Dual-tracer small-animal SPECT images were acquired, which allowed simultaneous detection of (111)In-labeled stem cells and of [(99m)Tc]sestamibi to assess myocardial perfusion deficit. The same animals were subjected to cardiac MRI. A mutual-information-based coregistration method was then applied to the SPECT and MRIs. By coregistration, the (111)In signal from labeled cells was mapped into the akinetic region identified on cine MRIs; the regional perfusion deficit on the SPECT images also coincided with the akinetic region on the MR image.
Assuntos
Espectroscopia de Ressonância Magnética/métodos , Infarto do Miocárdio/diagnóstico por imagem , Transplante de Células-Tronco/métodos , Células-Tronco/diagnóstico por imagem , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Animais , Linhagem Celular , Simulação por Computador/normas , Radioisótopos de Índio , Masculino , Radiografia , Ratos , Ratos Sprague-Dawley , Tecnécio/químicaRESUMO
Imaging the dopaminergic neurotransmitter system with positron emission tomography (PET) or single photon emission tomography (SPECT) is a powerful tool for the diagnosis of Parkinson's disease (PD). Previous studies have indicated that human observers have a diagnostic accuracy similar to conventional ROI analysis of SPECT imaging data. Consequently, it has been hypothesized that an artificial neural network (ANN), which can mimic the pattern recognition skills of human observers, may provide similar results. A set of patients with PD, and normal healthy control subjects, were studied using the dopamine transporter tracer [(99m)Tc]TRODAT-1 and SPECT. The sample was comprised of 81 patients (mean age +/- SD: 63.4 +/- 10.4 years; age range: 39.0-84.2 years) and 94 healthy controls (mean age +/- SD: 61.8 +/- 11.0 years; age range: 40.9-83.3 years). The images were processed to extract the striatum and the striatal pixel values were used as inputs to a three-layer ANN. The same set of data was used to both train and test the ANN, in a 'leave one out' procedure. The diagnostic accuracy of the ANN was higher than any previous analysis method applied to the same data (94.4% total accuracy, 97.5% specificity and 91.4% sensitivity). However, it should be stressed that, as with all applications of an ANN, it was difficult to interpret precisely what triggers in the images were being detected by the network.
Assuntos
Algoritmos , Interpretação de Imagem Assistida por Computador/métodos , Redes Neurais de Computação , Compostos de Organotecnécio , Doença de Parkinson/diagnóstico por imagem , Reconhecimento Automatizado de Padrão/métodos , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Tropanos , Adulto , Idoso , Idoso de 80 Anos ou mais , Inteligência Artificial , Análise por Conglomerados , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Compostos Radiofarmacêuticos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
The point spread function (PSF) of a pinhole collimator plays an important role in determining the resolution and characterizing the sensitivity of the accepted photons from a given point in the image space. The focus of this paper is to derive an analytical expression for the PSF of two different types of focusing pinhole collimators that are based on (1) right-circular double cones and (2) oblique-circular double cones. Conventionally, focusing pinhole collimators used in multi-pinhole SPECT were designed using right-circular double cones, as they were easier to fabricate. In this work, a novel focusing collimator consisting of oblique-circular double cones was designed and its properties were studied in detail with respect to right-circular double-cone based collimators. The main advantage of determining the PSF is the fact that they can be used to accurately model the PSF during the reconstruction, thereby improving the resolution of the reconstructed image. The PSF of the focusing collimators based on oblique-circular cones were found to be almost shift invariant for low and medium energy photons (below 200 keV). This property is very advantageous as algorithms such as slice-by-slice reconstruction can be used for resolution recovery thereby drastically reducing the reconstruction time. However, the PSF of focusing oblique-circular double cones (FOCDC) for higher energy photons were found to be asymmetric and hence need to be modelled more accurately during the reconstruction. On the other hand, the PSF for the right-circular cone based collimators were found to be asymmetric for all energy levels. However, due to the smaller acceptance angle used, the number of penetration photons was found to be far less than that observed for oblique-circular cones. This results in a smaller PSF making right-circular cone based collimators preferable for high-resolution small animal imaging, especially where very small pinhole diameters are used. The analytically derived PSF for both collimators were validated using a ray-tracing based Monte Carlo approach and found to agree well with a mean square error of less than 1%.
Assuntos
Radioterapia/métodos , Tomografia Computadorizada de Emissão de Fóton Único/instrumentação , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Algoritmos , Elétrons , Humanos , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Modelos Estatísticos , Modelos Teóricos , Método de Monte Carlo , Fótons , Espalhamento de Radiação , Sensibilidade e EspecificidadeRESUMO
This study determined the relative accuracy of diagnosis of Parkinson's disease (PD) using SPECT imaging data, comparing a semi-quantitative region-of-interest (ROI) approach and human observers. A set of patients with PD and normal healthy control subjects were studied using the dopamine transporter tracer [(99m)Tc]TRODAT-1 and SPECT. The sample comprised 81 patients (mean age +/- SD, 63.4 +/- 10.4 years; age range, 39.0-84.2 years) and 94 healthy controls (mean age +/- SD, 61.8 +/- 11.0 years; age range, 40.9-83.3 years). A standardized template containing six ROIs was transposed onto subregions of the brain, and the ratio of striatal to background ROI values was used as a semi-quantitative outcome measure. All images were used in a human observer study, with four experienced investigators. The data from the observer and ROI studies were analysed using a receiver operating characteristic (ROC) analysis, where the area under the ROC curve (AUC) indicated the diagnostic accuracy. ROI analysis and human observers gave similar diagnostic performance (mean observer AUC = 0.89, best ROI AUC = 0.90). This suggested that the human observers are visually acquiring similar information from the images that are contained in the semi-quantitative striatal uptake.
Assuntos
Compostos de Organotecnécio/farmacologia , Doença de Parkinson/diagnóstico por imagem , Doença de Parkinson/diagnóstico , Compostos Radiofarmacêuticos/farmacologia , Tecnécio/farmacologia , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Tropanos/farmacologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Área Sob a Curva , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Grão Comestível/metabolismo , Humanos , Pessoa de Meia-Idade , Variações Dependentes do Observador , Curva ROC , Radiografia , Reprodutibilidade dos TestesRESUMO
UNLABELLED: The evaluation of stem cell-mediated cardiomyoplasty by noninvasive in vivo imaging is critical for its clinical application. We hypothesized that dual-tracer small-animal SPECT would allow simultaneous imaging of (99m)Tc-sestamibi to assess myocardial perfusion and of (111)In-labeled stem cells to delineate stem cell engraftment. METHODS: Three to 4 million rat embryonic cardiomyoblasts (H9c2 cells) were labeled with 11.1-14.8 MBq (0.3-0.4 mCi) of (111)In-oxyquinoline and then injected into the border zones of infarcted myocardium of rats. (111)In images were acquired with a SPECT scanner 2, 24, 48, 72, and 96 h after the stem cells were injected into the infarcted myocardium. To visualize the perfusion deficit in the infarcted myocardium, we injected 74 MBq (2 mCi) of (99m)Tc-sestamibi (Cardiolite) intravenously 48 h after grafting. Dual-isotope pinhole SPECT was used to image (99m)Tc-sestamibi uptake simultaneously with (111)In to delineate retention of (111)In-labeled stem cells. The presence of labeled stem cells was confirmed by autoradiography and histology. RESULTS: SPECT of (99m)Tc-sestamibi was used to delineate perfusion deficits and infarcted myocardium. Bull's-eye plots indicated that the (111)In signal from the labeled stem cells overlapped the perfusion deficits identified from the (99m)Tc-sestamibi images. The (111)In signal associated with the radiolabeled stem cells could be detected with SPECT of the heart for 96 h after engraftment. CONCLUSION: This study demonstrated the feasibility of using dual-isotope pinhole SPECT for high-resolution detection of perfusion deficits with (99m)Tc-sestamibi and with (111)In-labeled stem cells grafted into the region of the infarct.
Assuntos
Radioisótopos de Índio , Infarto do Miocárdio/diagnóstico por imagem , Infarto do Miocárdio/cirurgia , Transplante de Células-Tronco/métodos , Células-Tronco/diagnóstico por imagem , Tecnécio Tc 99m Sestamibi , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Animais , Aumento da Imagem/métodos , Masculino , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
This study simulates a multi-pinhole single-photon emission computed tomography (SPECT) system using the Monte Carlo method, and investigates different multi-pinhole designs for quantitative mouse brain imaging. Prior approaches investigating multi-pinhole SPECT were not often optimal, as the number and geometrical arrangement of pinholes were usually chosen empirically. The present study seeks to optimize the number of pinholes for a given pinhole arrangement, and also for the specific application of quantitative neuroreceptor binding in the mouse brain. An analytical Monte Carlo simulation based method was used to generate the projection data for various count levels. A three-dimensional ordered-subsets expectation-maximization algorithm was developed and used to reconstruct the images, incorporating a realistic pinhole model for resolution recovery and noise reduction. Although artefacts arising from overlapping projections could be a major problem in multi-pinhole reconstruction, the cold-rod phantom study showed minimal loss of spatial resolution in multi-pinhole systems, compared to a single-pinhole system with the same pinhole diameter. A quantitative study of neuroreceptor binding sites using a mouse brain phantom and low activity (37 MBq) showed that the multi-pinhole system outperformed the single-pinhole system by maintaining the mean and lowering the variance in the measured uptake ratio. Multi-pinhole collimation can be used to reduce the injected dose and thereby reduce the radiation exposure to the animal. Results also suggest that the nine-pinhole configuration shown in this paper is a good choice for mouse brain imaging.
Assuntos
Algoritmos , Encéfalo/diagnóstico por imagem , Método de Monte Carlo , Tomografia Computadorizada de Emissão de Fóton Único , Animais , Camundongos , Imagens de Fantasmas , RadiografiaRESUMO
RATIONALE AND OBJECTIVES: Introduction of suicide genes, such as herpes simplex virus type1 thymidine kinase (HSV1-tk), in tumor cells has provided a useful method for tumor gene therapy. Several L-nucleosides, such as Lamivudine (3TC) and Clevudine (L-FMAU), have been successfully tested as high-potency antiviral agents. To investigate the potential differences between D- and L-isomers of nucleosides, [(125/123)I]-2'-fluoro-2'-deoxy-1beta-D/L-arabino-furanosy-5-iodo-uracil (D/L-FIAU) have been synthesized and evaluated as potential SPECT agents for imaging HSV1-tk gene expression. MATERIALS AND METHODS: [(125/123)I]D- and L-FIAU were prepared by iododestannylation of the respective tin precursors with (125/123)I-sodium iodide. In vitro cell uptake studies were performed by incubation of [(125)I]D- and L-FIAU in RG2 cells expressing HSV1-tk (RG2TK+). In vivo studies including biodistribution and SPECT were performed in RG2TK+ and RG2TK- tumor-bearing nude mice using [(123)I]D- and L-FIAU. RESULTS: Cell uptake and biodistribution studies indicated that [(125/123)I]L-FIAU did not show any high accumulation (sensitivity) or uptake ratios (selectivity) in HSV1-TK-positive (RG2TK+) tumors as compared to control tumors. In contrast, [(125/123)I]D-FIAU displayed both sensitivity and selectivity to RG2TK+ tumors. The selective in vivo accumulation of [(123)I]D-FIAU increased with time and the tumor uptake ratios (RG2TK+/RG2TK-) for 2, 4, and 24 hours averaged 6.2, 22.7, and 58.8, respectively. High-resolution SPECT of four nude tumor-bearing mice demonstrated a very high uptake of [(123)I]D-FIAU in the RG2TK+ tumor, while no significant tracer accumulation was observed in the RG2TK- tumor and other organs. CONCLUSION: The data suggest that only the D-isomer of [(123)I]FIAU is useful for imaging HSV1-tk gene expression in mice by high-resolution SPECT imaging.
Assuntos
Arabinofuranosiluracila/análogos & derivados , Glioma/diagnóstico por imagem , Nucleosídeos , Simplexvirus/enzimologia , Timidina Quinase/genética , Tomografia Computadorizada de Emissão de Fóton Único , Animais , Expressão Gênica , Terapia Genética , Glioma/enzimologia , Radioisótopos do Iodo , Masculino , Camundongos , Camundongos Nus , Compostos Radiofarmacêuticos , RatosRESUMO
Clinical observations indicate that activation of the TNF-α system may contribute to the development of inflammation-associated depression. Here, we tested the hypothesis that systemic upregulation of TNF-α induces neuroinflammation and behavioral changes relevant to depression. We report that a single intraperitoneal injection of TNF-α in mice increased serum and brain levels of the proinflammatory mediators TNF-α, IL-6, and MCP-1, in a dose- and time-dependent manner, but not IL-1ß. Protein levels of the anti-inflammatory cytokine IL-10 increased in serum but not in the brain. The transient release of immune molecules was followed by glial cell activation as indicated by increased astrocyte activation in bioluminescent Gfap-luc mice and elevated immunoreactivity against the microglial marker Iba1 in the dentate gyrus of TNF-α-challenged mice. Additionally, TNF-α-injected mice were evaluated in a panel of behavioral tests commonly used to study sickness and depressive-like behavior in rodents. Our behavioral data imply that systemic administration of TNF-α induces a strong sickness response characterized by reduced locomotor activity, decreased fluid intake, and body weight loss. Depressive-like behavior could not be separated from sickness at any of the time points studied. Together, these results demonstrate that peripheral TNF-α affects the central nervous system at a neuroimmune and behavioral level.
Assuntos
Comportamento Animal/efeitos dos fármacos , Encéfalo/metabolismo , Encefalite/metabolismo , Fator de Necrose Tumoral alfa/efeitos adversos , Animais , Biomarcadores/metabolismo , Encéfalo/patologia , Proteínas de Ligação ao Cálcio/metabolismo , Quimiocina CCL2/metabolismo , Depressão/metabolismo , Depressão/patologia , Encefalite/induzido quimicamente , Encefalite/patologia , Inflamação/induzido quimicamente , Inflamação/metabolismo , Inflamação/patologia , Interleucina-10/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Systems aimed at detecting gene expression noninvasively in vivo are desirable for evaluating the outcome of gene transfer in clinical trials. Several approaches have been exploited using magnetic resonance imaging and spectroscopy ((31)P MRS), positron emission tomography (PET), single-photon emission tomography (SPECT), and detection of bioluminescent signals. An ideal system is based on transfer of a marker gene, the activity of which can be detected against a background from the target tissue without interfering with normal physiology or eliciting an immune response. The majority of approaches described to date use genes encoding a nonmammalian protein that can elicit immune responses or a transmembrane receptor as a marker gene whose ectopic expression may cause aberrant signaling in the target cell through binding to endogenous ligands. The dopamine transporter (DAT) is normally expressed at high levels, mainly in the dopaminergic neurons of the central nervous system. We previously synthesized a radioactive ligand, [(99m)Tc]TRODAT-1, that binds with high affinity to the dopamine transporter, allowing for SPECT imaging of the striatum in normal control subjects and individuals affected with Parkinson's disease. Here we describe a strategy to monitor gene transfer based on adeno-associated viral vector (AAV)-mediated transduction of DAT in murine muscle followed by [(99m)Tc]TRODAT-1 imaging by SPECT of cells expressing the transgene. We show that quantitative, noninvasive imaging of gene transfer is successfully achieved in vivo, using a single-photon computed tomography camera. This system employs a reporter gene encoding a mammalian protein that is absent in most tissues, has no enzymatic activity, and does not activate intracellular pathways. This should be useful to monitor gene transfer in the settings of gene therapy.
Assuntos
Técnicas de Transferência de Genes , Glicoproteínas de Membrana , Proteínas do Tecido Nervoso , Tomografia Computadorizada de Emissão de Fóton Único , Animais , Proteínas da Membrana Plasmática de Transporte de Dopamina , Feminino , Imunofluorescência , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Músculos/metabolismo , CoelhosRESUMO
BACKGROUND: Structural deficiencies within limbic and prefrontal regions may contribute to the characteristic drug-seeking and drug-taking behaviors that prevail in persons dependent on cocaine. To date, a focal structural analysis of the brains of cocaine patients has not been undertaken. METHODS: We used voxel based morphometry in conjunction with statistical parametric mapping on the structural magnetic resonance images of cocaine-dependent (n = 13) and cocaine-naive individuals (n = 16) to assess differences between the two groups in gray and white matter concentration. RESULTS: We report a decrease in gray matter concentration in the ventromedial orbitofrontal, anterior cingulate, anteroventral insular, and superior temporal cortices of cocaine patients in comparison to controls (p <.01 corrected for multiple comparisons). The average percentage decrease in gray matter concentration within a region ranged from 5% to 11%. White matter concentration did not differ between groups. CONCLUSIONS: We conclude that the brains of cocaine patients are structurally dissimilar from those of nondrug-using controls. The differences were detected in regions involved in decision-making, behavioral inhibition and assignation of emotional valence to environmental stimuli and, hence, may contribute to some of the behavioral deficits characteristic of chronic cocaine users.
Assuntos
Córtex Cerebral/patologia , Transtornos Relacionados ao Uso de Cocaína/patologia , Imageamento por Ressonância Magnética , Adulto , Mapeamento Encefálico , Feminino , Lobo Frontal/patologia , Giro do Cíngulo/patologia , Humanos , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Masculino , Córtex Pré-Frontal/patologia , Valores de Referência , Lobo Temporal/patologiaRESUMO
Imaging serotonin transporters (SERT) is an emerging research tool potentially useful to cast light on the mechanisms of drug action as well as to monitor the treatment of depressed patients. We have prepared two new derivatives of 3, 2-(2-(dimethylaminomethyl)phenoxy)-5-iodophenylamine (4) and 2-(2-(dimethylaminomethyl)benzyl)-5-iodophenylamine (5) (K(i) for SERT = 0.37 and 48.6 nM, respectively). Both [(125)I]4 and [(125)I]5 displayed excellent brain uptakes in rats, and they showed a highest uptake in hypothalamus (between 60 and 240 min), a region populated with the highest density of SERT. The specific uptake of [(125)I]4 in the hypothalamus resulted in a target to nontarget ratio ([hypothalamus-cerebellum]/cerebellum) of 4.3 at 2 h. Autoradiography of rat brain sections (ex vivo at 2 h) of [(125)I]4 showed an excellent regional distribution pattern consistent with known SERT localization. These data suggest that [(123)I]4 may be useful for imaging SERT binding sites in the brain by single photon emission computed tomography (SPECT).
Assuntos
Compostos de Anilina/síntese química , Benzilaminas/síntese química , Proteínas de Transporte/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras , Proteínas do Tecido Nervoso/metabolismo , Compostos Radiofarmacêuticos/síntese química , Serotonina/metabolismo , Compostos de Anilina/química , Compostos de Anilina/farmacocinética , Animais , Autorradiografia , Benzilaminas/química , Benzilaminas/farmacocinética , Encéfalo/metabolismo , Linhagem Celular , Técnicas In Vitro , Radioisótopos do Iodo , Marcação por Isótopo , Ensaio Radioligante , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Ratos , Proteínas da Membrana Plasmática de Transporte de Serotonina , Relação Estrutura-Atividade , Distribuição Tecidual , Tomografia Computadorizada de Emissão de Fóton ÚnicoRESUMO
UNLABELLED: Methylphenidate (MPH) is an effective symptomatic treatment of attention deficit hyperactivity disorder (ADHD), but the mechanisms of its therapeutic action have not been fully elucidated. To address this issue, we assessed the effects of discontinuation of chronic MPH treatment on regional cerebral blood flow (rCBF) in ADHD patients. METHODS: Twenty-two prepubescent boys with ADHD (age range, 8.2-11.5 y) and 7 healthy volunteers were studied with SPECT on and off MPH. Their rCBF data were automatically normalized to whole-brain counts and coregistered with standard anatomic space. rCBF changes were evaluated with statistical parametric mapping based on voxel-by-voxel ANOVA. RESULTS: When the subjects were not taking MPH, rCBF was higher in the motor, premotor, and the anterior cingulate cortices (Brodmann's areas 4, 6, and 32). CONCLUSION: Brief discontinuation of MPH treatment is associated with increased motor and anterior cingulate cortical activity. Our findings suggest that MPH treatment modulates motor and anterior cingulate cortical activity directly or indirectly. Alternatively, our findings may be related to MPH withdrawal. These data provide novel information on the potential mechanisms of the therapeutic action of MPH. Furthermore, they are clinically relevant to the commonly occurring brief interruptions in MPH treatment.
Assuntos
Transtorno do Deficit de Atenção com Hiperatividade/tratamento farmacológico , Encéfalo/diagnóstico por imagem , Circulação Cerebrovascular/efeitos dos fármacos , Metilfenidato/uso terapêutico , Tomografia Computadorizada de Emissão de Fóton Único , Transtorno do Deficit de Atenção com Hiperatividade/diagnóstico por imagem , Transtorno do Deficit de Atenção com Hiperatividade/fisiopatologia , Criança , Humanos , MasculinoRESUMO
UNLABELLED: There has been considerable interest in the development of PET radioligands that are useful for imaging serotonin transporter (SERT) in the living human brain. For the last decade, (11)C-(+)McN5652 has been the most promising PET agent for studying SERT in humans. However, this agent has some limitations. Recently, a new promising SERT PET radioligand, 3-(11)C-amino-4-(2-dimethylaminomethylphenylsulfanyl)benzonitrile, has been reported. We recently reported the synthesis of a new (18)F-labeled SERT PET radioligand, N,N-dimethyl-2-(2-amino-4-(18)F-fluorophenylthio)benzylamine (4-(18)F-ADAM), which may have advantages over (11)C-labeled radioligands. The purpose of this study was to evaluate this newly developed (18)F-labeled PET radioligand as a promising agent for studying SERT in the living human brain. METHODS: This agent was evaluated by studying its in vitro binding to different monoamine transporters, its in vivo biodistributions in rats, its integrity and pharmacologic profiles in rat brain, and its distribution in a female baboon brain. RESULTS: In vitro binding assays showed that 4-F-ADAM displayed high affinity to SERT sites (inhibition constant = 0.081 nmol/L, using membrane preparations of LLC-PK1 cells expressing the specific transporter) and showed more than 1,000- and 28,000-fold selectivity for SERT over norepinephrine transporter and dopamine transporter, respectively. Biodistribution of 4-(18)F-ADAM in rats showed a high initial uptake and slow clearance in the brain (2.13%, 1.90%, and 0.95% injected dose per organ at 2, 30, and 60 min after intravenous injection, respectively), with the specific binding peaking at 2 h after injection (hypothalamus/cerebellum = 12.49). The uptake in blood, muscle, lung, kidney, and liver was also initially high but cleared rapidly. The radioactivity in the femur increases with time for 4-(18)F-ADAM, indicating that in vivo defluorination may occur. In vivo metabolism studies in rats showed that 4-(18)F-ADAM was not metabolized in rat brain (>96% of radioactivity was recovered as parent compound at 1 h after injection). However, it metabolized rapidly in the blood. Less than 7% of the radioactivity recovered from plasma was the parent compound, with the majority of radioactivity in the plasma not extractable by ethyl acetate. Blocking studies showed significant decreases in the uptake of 4-(18)F-ADAM in the brain regions (hypothalamus, hippocampus, and striatum) where SERT concentrations are high when rats were pretreated with (+)McN5652 (2 mg/kg 5 min before intravenous injection of 4-(18)F-ADAM). However, changes in the uptake of 4-(18)F-ADAM in these brain regions were less significant when rats were pretreated with either methylphenidate or nisoxetine. The baboon study showed that uptake of 4-(18)F-ADAM in the midbrain peaked at approximately 1 h after injection and then declined slowly. The ratios of the radioactivity in the midbrain to that in the cerebellum (where the concentration of SERT is low) at 2 and 3 h after injection were 3.2 and 4.2, respectively. CONCLUSION: 4-(18)F-ADAM is suitable as a PET radioligand for studying SERT in the living brain. Further characterization of this new radioligand in humans is warranted.
Assuntos
Benzilaminas/farmacocinética , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Proteínas de Transporte/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras , Proteínas do Tecido Nervoso , Tomografia Computadorizada de Emissão/métodos , Animais , Estudos de Viabilidade , Feminino , Células LLC-PK1 , Ligantes , Masculino , Taxa de Depuração Metabólica , Especificidade de Órgãos , Papio , Ligação Proteica , Compostos Radiofarmacêuticos/farmacocinética , Ratos , Ratos Sprague-Dawley , Proteínas da Membrana Plasmática de Transporte de Serotonina , Especificidade da Espécie , Suínos , Distribuição TecidualRESUMO
Quantification provides the link between the concentrations of radioactivity measured in tissue and the underlying physiologic processes occurring in the organ. It relates the rate at which radioactivity levels in the body change over time to quantitative parameters such as absolute rate glucose metabolism, regional blood flow, or concentrations of receptors or other binding sites. Absolute measurement of physiologic parameters generally requires accurate measurement of activity concentrations in arterial blood, which provides the input function to the kinetic model. Although absolute quantification can be a difficult process, simplifications of these invasive techniques, involving reference tissues or normalization approaches (eg, SUV), have been applied with some success. Any simplified model of tracer behavior must be validated against the full model to test for bias and systematic errors.
Assuntos
Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos , Algoritmos , Humanos , Aumento da Imagem/métodos , Modelos Químicos , Compostos Radiofarmacêuticos/farmacocinética , Reprodutibilidade dos Testes , Distribuição TecidualRESUMO
Molecular imaging of small animals in vivo is vital in the study of mouse and rat models of human diseases, and will provide important clues to the pathogenesis, progression and treatment of many disorders. Functional imaging of small animals using ultra-high resolution single photon emission tomography (SPECT) should be a valuable tool in the molecular imaging armamentarium. SPECT has been used to study cerebral binding sites, to image the expression of reporter genes, and in applications in cardiology and oncology. In this review, we summarize the most recent developments in SPECT imaging of small animals, with particular reference to the types of systems available, their application, and some of the potential limitations.